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The role of staphylococcal Hsp60 in the internalization of S. aureus inside immortalized human keratinocytes Godin, Patrice 2006

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T H E R O L E OF S T A P H Y L O C O C C A L HSP60 I N T H E I N T E R N A L I Z A T I O N O F S . AUREUS INSIDE I M M O R T A L I Z E D H U M A N K E R A T L N O C Y T E S by P A T R I C E G O D L N B.Sc. Microbiology, Sherbrooke University, 2003 A THESIS S U B M I T T E D I N P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R OF S C I E N C E in T H E F A C U L T Y OF G R A D U A T E S T U D I E S (Experimental Medicine) T H E U N I V E R S I T Y OF B R I T I S H C O L U M B I A December 2005 © Patrice Godin, 2005 A B S T R A C T S. aureus is a gram positive commensal which can be found on the skin and in the nose of the majority of healthy individuals. Its abilities to cause a wide range of life threatening diseases, to cause recurrent infections and to easily acquire antibiotic resistance have made S. aureus undeniably one of the most important pathogens everywhere in the world. Although this pathogen is certainly not new to the scientific community, S. aureus recently became increasingly important as we realized that we are quickly running out of effective therapies to treat these infections. Interestingly, it has been found within the last few years that S. aureus is able to invade and survive within a variety of human cells. In vitro studies have shown that this intracellular location allows S. aureus to resist to most known antibiotics in vitro. Further studies revealed intracellular reservoirs of S. aureus have been found in patients with chronic infections. These suggest that this intracellular phenomenon is an important neglected aspect of S. aureus pathogenesis, and could partly explain how S. aureus evades the immune system and causes recurrent infections. A s this pathogen is predominantly associated with skin infections, we decided to study the capacity of S. aureus to internalize inside human skin keratinocytes (HaCaT). We have examined the dynamics of infection as well as the role of S. aureus Hsp60 protein, which has been shown to mediate the adherence and internalization of various other pathogens. Our study has helped to further our understanding of S. aureus internalization by showing that this pathogen can survive at least 76 hrs inside HaCaT cells, and that the infection can easily be monitored by flow cytometry using fluorescein isothyocyanate (FITC) labelled bacteria. We have also shown that this internalization is proportional to the starting multiplicity of infection up to 100 C F U per cell. A t this infection ratio, we have demonstrated using fluorescence activated cell sorter ( F A C S ) and confocal microscopy analyses that nearly up to 8% of HaCaT cells were found to be infected. Although the internalization could be blocked using the actin polymerization inhibitor cytochalasin D , we did not notice any significant decrease in the internalization levels of S. aureus after the addition of exogenous staphylococcal Hsp60 proteins or anti-Hsp60 antisera and F(ab) 2 fragments as an attempt to block this internalization. Our studies on the membrane localization of Hsp60 on the surface of S. aureus using antibody staining and membrane separation also failed to prove that this protein is associated on the membrane of this bacterium to a high extent. We therefore showed that staphylococcal Hsp60 does not appear to play a role in the internalization of S. aureus inside human HaCaT keratinocytes. i i TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS iii LIST OF TABLES v LIST OF FIGURES vi ABBREVIATIONS viii ACKNOWLEDGEMENTS ix 1.0 INTRODUCTION 1 1.1 CHARACTERISTICS OF STAPHYLOCOCCUS A UREUS 1 1.2 S.A UREUS AS AN INTRACELLULAR PATHOGEN 2 1.3 BASICS OF HEAT-SHOCK PROTEIN 60 5 1.4 IN VITRO INTERNALIZATION MODELS OF S. A UREUS 8 2.0 WORKING HYPOTHESIS & SPECIFIC AIMS 10 2.1 HYPOTHESIS 10 2.2 SPECIFIC AIMS 10 2.3 SUMMARY 10 3.0 EXPERIMENTAL 11 3.1 MATERIALS AND METHODS 11 3.1 CULTURE OF STAPHYLOCOCCUS A UREUS 11 3.2 TRANSFORMATION OF S. A UREUS 11 3.3 LABELLING OF S. A UREUS WITH FLUORESCEIN ISOTHIOCYANATE 12 3.4 INFECTION ASSAY OF HACAT KERATINOCYTES 12 3.5 DETERMINATION OF INTERNALIZED BACTERIA NUMBERS 13 3.6 DETERMINATION OF THE PERCENTAGE OF INFECTED CELLS ....13 3.7 DETECTION OF INTERNALIZED BACTERIA BY MICROSCOPY 14 3.8 KILLING CURVE OF S. AUREUS INSIDE HACAT KERATINOCYTES14 3.9 EFFECT OF CYTOCHALASIN D PRE-TREATMENT ON THE INTERNALIZATION LEVELS 15 3.10 INHIBITION OF INTERNALIZATION USING ANTI-HSP60 ANTIBODIES OR RECOMBINANT HSP60 PROTEINS ...15 3.11 PREPARATION OF RECOMBINANT HSP60 15 3.12 GENERATION OF ANTI-HSP60 AND ANTI-GROEL ANTISERA 16 3.13 DETERMINATION OF SERUM ANTIBODY TITERS 16 3.14 GENERATION OF F(ab)2 ANTIBODY FRAGMENTS 17 3.15 MEMBRANE SEPARATION OF S. AUREUS 17 iii 3.16 WESTERN BLOT OF MEMBRANE FRACTIONS 17 3.17 STATISTICS 18 4.0 RESULTS 19 4.1 INTERNALIZATION OF S. A UREUS INSIDE HACAT CELLS 19 4.1.1 DOSE DEPENDANT INTERNALIZATION 19 4.1.2 VISUAL DETERMINATION OF BACTERIA INSIDE CELLS 22 4.1.3 DETERMINATION OF THE PERCENTAGE OF INFECTED CELLS .... 24 4.1.4 SURVIVAL CURVE OF S. A UREUS INSIDE HACAT CELLS 26 4.1.5 EFFECT OF CYTOCHALASIN D ON THE INTERNALIZATION 28 4.2 MEMBRANE LOCALIZATION OF S. A UREUS HSP60 30 4.2.1 EXTRACELLULAR STAINING OF HSP60 30 4.2.2 MEMBRANE SEPARATION OF S. A UREUS 37 4.3 BLOCKING THE INTERNALIZATION USING ANTI-HSP60 41 ANTIBODIES 41 4.4 BLOCKING THE INTERNALIZATION USING EXOGENOUS S. AUREUS HSP60 PROTEINS 43 5.0 DISCUSSION 45 5.1 INTERNALIZATION OF S. A UREUS INTO HUMAN HACAT KERATINOCYTES 45 5.2 ROLE OF HSP60 IN THE INTERNALIZATION OF S. A UREUS 49 6.0 FUTURE WORK 53 7.0 REFERENCES 55 i v LIST OF TABLES Table Title Page Percentage of the initial bacterial numbers which were found to be 21 internalized by plate count. Increase in the lethality of infected cells compared to an uninfected 27 control as assessed by trypan blue and 7-AAD staining. v LIST OF FIGURES Figure Title Page 1 Effect of varying the multiplicity of infection on the amounts of internalized bacteria 20 2 Intracellular localization of S. aureus inside HaCaT cells 23-24 3 Determination of the percentage of infected cells by microscopy and F A C S 25 4 Time course of the intracellular survival of S. aureus inside keratinocytes (MOI100) 26 5 Effect of cytochalasin D pre-treatment on the numbers of internalized S .aureus 28 6 Effect of cytochalasin D pre-treatment on the percentage of infected cells found by F A C S 29 7 Purity of the purified Hsp60 used for immunization 30 8 E L I S A results of the 1 s t and 2 n d bleed antisera titers against S. aureus Hsp60 31 9 E L I S A results of the 1 s t and 2 n d bleed antisera titers against E. coli G r o E L 32 10 E L I S A results of the 3 r d bleed antisera titers against S. aureus Hsp60 proteins 32 11 Purity of the F(ab)2 preparations before and after the protein A purification step 34 12 Localization of Hsp60 on the surface of S. aureus using F(ab)2 antibody staining. 36 13 Comparison of mean fluorescence results from membrane localization experiments using 20 ug/ml F(ab)2 fragments. 36 14 Western blot showing the detection of membrane, cytosolic and whole-cell fractions (2 fig) of S. aureus using our anti-Hsp60 and pre-immune sera 37 v i 15 Western blot showing the detection of membrane, cytosolic and whole-cell fractions of S. aureus using commercial anti-GroEL antibody preparations 38 16 Overexposition of the previous western blot using anti-Hsp60 and pre-immune sera 40 17 Effect of various dilutions of anti-Hsp60 antiserum on the internalization level of S. aureus as assessed by plate count. 41 18 Effect of adding various concentrations of anti-Hsp60 F(ab)2 antibody fragments on the internalization level of S. aureus as assessed by plate count. 42 19 Effect of adding recombinant S. aureus Hsp60 on the internalization levels as assessed by plate count 43 20 Effect of adding recombinant S. aureus Hsp60 on the internalization by assessed by F A C S analysis 44 v i i A B B R E V I A T I O N S Abbreviations Definitions BHI Brain heart infusion CFU Colony forming unit CNISP Canadian nosocomial infection surveillance program DEAE Diethylaminoethyl DMEM Dulbeco's modified eagle's medium DTT Dithiothreitol ECL Enhanced chemoluminescence (reagent) EDTA Ethylenediaminetetraacetic acid ELISA Enzyme-linked immuno-sorbent assay FACS Fluorescence activated cell sorter FCS Fetal calf serum FITC Fluorescein isothyocyanate GFP Green fluorescent protein GST Glutathione-S-transferase HRP Horseradish peroxidase HUVEC Human umbilical vein endothelial cells MOI Multiplicity of infection, ratio of CFU per cell MRSA Methicillin resistance Staphylococcus aureus NCTC National collection of type culture (London, UK) PMSF Phenylmethylsulphonylfluoride S. aureus Staphylococcus aureus SCV Small colony variant TBS Tris buffered saline TBST Tris buffered saline containing 0.05% Tween-20 TMB Tetramethylbenzidine VRSA Vancomycin resistant Staphylococcus aureus V l l l A C K N O W L E D G E M E N T S This thesis was made possible due to the following people... D r . Anthony W C h o w - for allowing me in his laboratory, for his trust, support, and guidance in this project. D r . Z a k a r i a H m a m a & D r . Rachel Fernandez - for their invaluable suggestions and agreeing to be supervisory committee members despite their busy schedules Past & present members of the Chow lab - for teaching me about the wonderful world of T-regs. Sabine Ivison - for her help and indispensable efforts without which the purification of S. aureus Hsp60 would not have been possible Kor ine U n g - For absolutely everything else T H A N K S E V E R Y O N E ! ! ! i x 1.0 INTRODUCTION 1.1 CHARACTERISTICS OF STAPHYLOCOCCUS AUREUS Staphylococcus aureus is a gram-positive bacterium of great importance for the developed world, being a predominant cause of nosocomial infections while also causing hundreds of millions of dollars in losses yearly in agricultural industries (Wall et al, 2005). It is literally present everywhere, being the subject of hundreds of scientific papers each year and even being routinely found in the nares of up to 60% of healthy adults (Solberg, 2000). But above all, S. aureus is now becoming especially important due to the increased prevalence of antibiotic resistant strains and their propagation in the community. S. aureus is an opportunistic pathogen which is commonly found on the skin of healthy individuals and causes an amazingly wide array of infections. Most of the time these represent skin-related diseases such as atopic dermatitis, impetigo or scalded skin syndrome, but in many cases S. aureus may also cause more severe deep-tissue infections including bacteremia, endocarditis, toxic shock syndrome and osteomyelitis (Archer, 1998). The ease of S. aureus in causing this diverse range of diseases is due to the presence o f its just as diverse weaponry of virulence factors. For example, S. aureus possesses various toxins (toxic shock syndrome toxin-1 [TSST-1], enterotoxins, exfoliating toxins, cytolytic toxins, etc.), adhesins (fibronectin, fibrinogen, collagen and other binding proteins [FnBPs, Cna, C l f respectively]), various matrix-destroying enzymes (hyaluronidase, coagulases, lipases and other proteases, etc.), other factors to escape host defenses such as protein A (IgG binding protein) and chemotaxis inhibitory proteins as well as a robust capsule (Archer, 1998;Foster, 2004;Lowy, 1998). Despite the impressive research efforts directed against S. aureus, its pathogenesis remains incompletely understood. Although S. aureus can be found as a commensal inhabitant in a variety of flora (in the vagina, skin, nares, pharynx, axilla, etc), the triggers that differentiate between colonization and infection remain, in most cases, unclear. However, as expected, the incidence of disease depends on the delicate balance between S. aureus and the integrity of host defenses, as the incidence of disease appears related to the genetic background of the infecting strains (Day et al, 2001) and also increases in immunocompromised patients (Weinke et al, 1992;Tumbarello et al, 1996). This is in agreement with the fact that S. aureus is found to be particularly present in hospital environments where patients' natural defenses are frequently already compromised. Notably, S. aureus infections are particularly associated with patients 1 undergoing surgery or using intravascular devices that cause a breach in the integrity of the skin barrier (Steinberg, Clark, and Hackman, 1996). One of the hallmarks of S. aureus as a pathogen is its abilities to cause recurrent infections and easily acquire antibiotic resistance. This antibiotic resistance is particularly problematic as we notice the worldwide increase in the incidence of methicilin-resistant S. aureus ( M R S A ) strains with multiple resistance to various antibiotics classes such as fluoroquinolones, macrolides, lincosamides and aminoglycosides (Foster, 2004). These M R S A strains are now estimated to represent up to 50 % of S. aureus clinical isolates in the United States and up to 10% in Canadian hospitals (Farr, 2004;CNISP, 2005). It was also recently shown that these M R S A strains can be propagated in the community and that once patients develop M R S A infections, they could continue to carry these resistant strains for a few years after successful treatment (Sanford et al., 1994). Presently, vancomycin is the only widely available drug for the treatment of these resistant strains. However, some V R S A strains have been recently isolated in Japan and the United States ( C D C , 2005) and more w i l l undoubtedly be found i f vancomycin remains the drug of choice for M R S A infections. It is unclear how S. aureus acquires antibiotic resistance so easily. It has been shown that this may occur through horizontal gene transfer between bacterial species, such as transfer of the vancomycin resistance VanA gene from enterococci (Ruef, 2004). In other cases, strains of S. aureus with intermediate resistance to vancomycin were shown to have increased numbers of peptidoglycan layers (Hiramatsu, 2001). Moreover, recent evidence suggested that part of its innate resistance to antibiotics may be due to the recently discovered ability of S. aureus to enter and survive inside a variety of human cells. This intracellular survival is a very interesting phenomenon as it could also explain how S. aureus is able to evade immune defenses and also cause recurrent infections. 1.2 S. A UREUS AS A N I N T R A C E L L U L A R P A T H O G E N Staphylococcus aureus has up t i l l recently always been classified as an extracellular pathogen. However, the high rates of relapse occurring after S. aureus infection despite the use of proper antibiotic treatment and the discovery that many typically extracellular pathogens also have a secret intracellular lifestyle suggested that S. aureus may also possess the ability to internalize inside cells. These controversial findings are now widely accepted, as S. aureus has been shown by several groups to internalize inside a variety of different cell types, most of which 2 are not commonly considered professional phagocytes. For example, this was observed in vitro using human osteoblasts, fibroblasts, enterocytes as well as various epithelial and endothelial cells (Nair et al, 2003;Haggar et al, 2003;Hess et al, 2003;Mempel et al, 2002;Menzies and Kourteva, 1998). This internalization has also been observed in a few experiments in vivo using mouse mastitis and embryo chick osteoblast models (Brouillette et al, 2003;Reilly et al, 2000). Moreover, the intracellular localization of S. aureus has also been observed using electron microscopy in nasal epithelial cells taken from biopsy of chronic rhinosinusitis patients, which constitutes the first discovered reservoir of intracellular S. aureus (Clement et al, 2005). Although the observations of S. aureus intracellular presence in vivo are increasing, it remains very difficult to directly address the importance of intracellular survival in diseases due to the low bacterial density at sites of invasion and to extensive tissue damage in S. aureus infections. Being a relatively recent discovery, the mechanisms of S. aureus internalization and intracellular survival are still unclear. However, it appears that internalization might occur through a "zipper type" mechanism in which the bacteria would bind, directly or indirectly, to a host cell receptor with enough specificity to trigger the internalization of this receptor and the bound bacteria. This phenomenon has been observed for various other "facultative" intracellular pathogens such as Listeria monocytogenes (Mengaud et al, 1996). Similarly, the adherence of S. aureus to host cell has been studied in many different models. S. aureus possesses many adhesins which appear to have a role in this adherence amongst which the fibronectin binding proteins (FnBPs) are the most studied. These proteins play a major role in S. aureus internalization in vitro and several groups have found that FnBPs mutation caused over >90% decrease in the internalization levels of S. aureus inside human embryonic kidney epithelial 293 cells (Sinha et al, 1999), human corneal epithelial cells ( H C E C ) (Jett and Gilmore, 2002) and bovine mammary epithelial cells (Mac-T cells)(Dziewanowska et al, 1999). This last group found that one of the cell targets for FnBP would be the host 60kDa heat-shock protein (Hsp60) as internalization could be blocked using monoclonal antibodies specific for human Hsp60 (Dziewanowska et al, 2000). Interestingly, recent in vivo studies and studies using primary oral keratinocyte cells presented conflicting results for the role of F n B P in the internalization process suggesting that other adhesins of S. aureus may also be important in the internalization in vivo (Kintarak et al, 2004b;Brouillette et al, 2003). Many other surface proteins of S. aureus have also been investigated for their role in internalization, such as protein A , Eap (Extracellular adherence protein) and C l f A (Clumping factor, which provides fibrinogen binding capacity). Unlike C l f A , both protein A and Eap are linked to the internalization of S. aureus inside human 3 oral epidermal carcinoma cells and fetal lung fibroblasts, respectively (Jung et al, 2001;Kintarak et al, 2004b;Haggar et al, 2003). However, the implied role of protein A in internalization is still controversial. Overall, it is still unclear what proteins mediate the internalization of S. aureus. After entrance into human cells, S. aureus has been shown to escape the phagosome compartment which has been observed in osteoblasts (Hudson et al, 1995) as well as neutrophils (Gresham et al, 2000) and is estimated to occur as early as 3 hrs after infection for mammary epithelial cells (Shompole et al, 2003). It is still unclear as to which factors allow this escape from the phagosome, however accumulating evidence suggests that the agr gene (accessory gene regulator) of S. aureus is involved in this release (Wesson et al, 1998), l ikely through the control of alpha toxin expression (Haslinger-Loffler et al, 2005). It has also been shown that internalized bacteria, once inside the cytoplasm, replicate and eventually cause the lysis of the host cell (Qazi et al, 2004). It is interesting to note that the induction of cell lysis is contradictory to the hypothesis that internalization provides a long term persistence mechanism. However, it has been hypothesized that a subset of S. aureus known as S C V (Small colony variants) is able to persist longer inside human cells. These S C V would have greater intracellular survival capacity or be selected in the intracellular milieu (Vesga et al, 1996). This would be due to a defect in their electron transport chain (HemB deletion) resulting in a slow growth rate as well as an increased antibiotic resistance caused by a decrease in their membrane potential leading to reduced antibiotic import (Chuard et al, 1997). It has recently been shown that S C V s isolated from cystic fibrosis patients have considerably altered gene expression compared to wi ld type and interestingly would have increased SigB and decreased agr and alpha-toxin gene expression which would lead to an increased expression of adhesins and reduced exotoxin production (Moisan et al, 2006). These results are very interesting as they correlate perfectly with the ability of these strains to colonize the host while avoiding the extensive tissue destruction which is characteristic of wi ld type S. aureus infections. Accordingly, these S C V s were shown to be present in persistent infections such as osteomyelitis and cystic fibrosis (Looney, 2000) and are therefore the best current example of the importance of internalized S. aureus in chronic infections. Nevertheless, although in vivo examples of persistent S. aureus infections have been observed, they cannot be so easily attributed to intracellular persistence. It remains unclear how long staphylococci can survive inside cells. Several groups have tried to assess the length of survival, claiming this could last over 48 hrs in human vein endothelial cells (Matussek et al, 4 2005), 120 hrs in enterocytes (Hess et al, 2003), and even up to 6 days in mouse enterocytes ( m K S A ) despite antibiotic treatment (Krut, Sommer, and Kronke, 2004). However, these numbers remains relatively uncertain and it is unclear whether this long survival time could be due to re-infection caused by bacteria released through cell lysis of neighboring cells. Nevertheless, it does appear that S. aureus can persist intracellularly for a few days and thus might be sheltered against antimicrobial therapy and acclimatize the bacteria to lower concentrations of antibiotics which could allow the development of resistance. Indeed, it has been observed that antibiotics have different inhibitory effect against internalized bacteria in vitro; this has been observed in various cell types such as mammary epithelial cells ( M A C - T cells)(Qazi et al, 2004) as well as murine keratinocytes (PAM212) , fibroblasts (mKSA)(Krut, Sommer, and Kronke, 2004) and macrophages (J774) (Serai, V a n Bambeke, and Tulkens, 2003). This intracellular survival phenomenon appears to be an important aspect to take into account when developing new anti-staphylococcal treatments and some researchers have already started to evaluate the efficacy of novel antibacterial compounds on the small colony variants (Malouin et al, 2005). Therefore it appears that intracellular survival represents an important aspect of S. aureus pathogenesis, which would account for the persistence and resistance characteristics of staphylococcal infections. This remains a relatively new phenomenon which is far from being totally understood, however, there are some indications that the Hsp60 protein from S. aureus may be involved in this process. 1.3 BASICS OF HEAT-SHOCK PROTEIN 60 The 60kDa heat shock protein (Hsp60) is a well conserved protein necessary for the growth of every known living eukaryotic and prokaryotic organism where it is expressed constitutively. It belongs to the class 1 type of molecular chaperones whose main function is to allow the correct folding of damaged and newly synthesized proteins. In vivo, seven Hsp60 proteins binds together to form a ring shaped subunit which associate in dimers to form the active cylindrical shaped Hsp60 chaperone. It is known that various proteins can bind inside this complex. Their refolding would happen through changes in the chaperone complex shaped which would occur after association with the HsplO chaperone and A T P hydrolysis. Moreover, as its name indicates, Hsp60 is involved in the response to heat stress damage, as its expression can be increased during heat shock (Hightower, 1991). The fact that Hsp60 is highly conserved 5 throughout species of different kingdoms has allowed the creation of universal primers able to amplify Hsp60 homolog of virtually any genome (Hi l l et al, 2004). This great degree of conservation has made Hsp60 a very interesting target both as a diagnostic and a phylogenetic tool (Mikkonen, Karenlampi, and Hanninen, 2004;Wong and Chow, 2002). Human Hsp60 also draws researcher's interest as it was recently found to act as a cell signaling molecule, stimulating immune T cells and B cells to produce various cytokine responses through the binding of T L R 2 and T L R 4 receptors (Cohen-Sfady et al, 2005). Similarly, Hsp60 from Actinobacillus actinomycetemcomitans have been shown to induce TNF-alpha production in HaCaT keratinocytes, Hsp60 from Helicobacter pylori induced the production of IL-8 cytokine by gastric epithelial cells (Zhang, Pelech, and Uitto, 2004;Woods, 2003;Lin et al, 2005). These findings suggest that Hsp60 most likely binds to a receptor on the cell surface and may therefore be involved in the internalization of bacteria inside cells through a similar "zipper type" mechanism. On the other side, the cytokine production response triggered by microbial Hsp60 suggests that immune cells may be using this molecule as a danger signal which would help the host by promoting immune system activation. Alternately, the fact that Hsp60 from Histoplasma capsulatum binds to human macrophages integrin receptor (Woods, 2003) suggests that in some cases, immune cells may also be able to use Hsp60 as a bridging molecule allowing for phagocytosis and clearing of bacterial infections. Hsp60 has long been considered uniquely a cytosolic protein. Although it is still proposed to be predominantly located in the cytosol of bacteria and cells, there is accumulating evidence to show that it can be surface associated and even secreted. This cell surface association has been observed repeatedly in the case of human cells. The membrane association of human Hsp60 is suspected to happen in response to various stresses and was notably observed after heat shock of Huvec cells (Pfister et al, 2005). Interestingly, this membrane association has also been reported in many different microorganisms such as Legionella pneumophila (Garduno et al, 1998), Helicobacter pylori (Yamaguchi et al, 1996), Clostridium difficile (Hennequin et al, 2001a), Histoplasma capsulatum (Woods, 2003), Actinobacillus actinomycetemcomitans (Hara et al, 2000), Haemophilus ducreyi (Frisk, Ison, and Lagergard, 1998), Brucella abortus, to a small extent in Leptospira species (Cullen et al, 2005), and on the surface of Bacillus cereus endospores (Charlton et al, 1999). Although this cell surface localization is still controversial in some of these species, it has been repeatedly demonstrated using electron microscopy, cell fractionation techniques, as well as extracellular staining, and is well accepted for L. pneumophila and H. pylori (Hoffman and Garduno, 1999). The mechanism on how Hsp60 would 6 be directed to the surface of cells is currently unknown and is quite intriguing due to the fact that this protein does not contain any leader peptide sequence. Nevertheless, it has been shown that human Hsp60 have affinity to E . coli lipopolysaccharide suggesting that some Hsp60 homolog would have affinity for bacterial membranes (Habich et al, 2005). Moreover, E . coli Hsp60 injected in liposome was shown to integrate inside the l ipid membrane on its own (Torok et al, 1997). Nevertheless, although the insertion mechanisms are not completely understood, it has now become clear that surface localization of Hsp60 is possible. Bacterial Hsp60 is well known to be a highly immunogenic protein and most bacterial infections appear to induce high levels of host antibody response against it (Zugel and Kaufmann, 1999). Although, its role in bacterial pathogenesis remains unclear, it is currently believed that bacterial Hsp60 may play an important role in the development of host auto-immune diseases, such as atherosclerosis, type-1 diabetes, hepatitis and arthritis (Prakken et al, 2003;Raz, Eldor, and Naparstek, 2005;Wick, Knoflach, and X u , 2004;Zanin-Zhorov et al, 2005). In the case of atherosclerosis, bacterial Hsp60 is expected to be an effector of the disease through both direct activation of the immune system and antibody cross-reactivity with the host Hsp60 (Wick, Knoflach, and X u , 2004). However, in diabetes, arthritis, as well as the concanavalin A-induced hepatitis models, immunization with host Hsp60 has been found to have a protective effect against these diseases in mice, most likely due to a shift from a T h l to a Th2 polarization of the immune response (Raz et al, 2001;Elias et al, 1997;Zanin-Zhorov et al, 2005). Importantly, Hsp60 also appears to be implicated in bacterial diseases. Immunization with bacterial Hsp60 has been found to increase host resistance against infection for different bacteria such as Helicobacter pylori, Histoplasma capsulatum and Mycobacterium tuberculosis, suggesting a role for Hsp60 in the abilities of these pathogens to cause disease (Zugel and Kaufmann, 1999). Therefore, both human and bacterial Hsp60 appear to play important roles in various diseases. The observations that bacterial Hsp60 can bind to host cell receptors, its apparent importance in various diseases and its surface localization suggests that this protein could also be involved in the internalization of bacteria. Although this hypothesis may appear far-fetched at first, several investigators found that Hsp60 is linked to the internalization of many other bacterial and fungal pathogens. For the gram-negative pathogen L. pneumophila, the addition of exogenous Hsp60 was demonstrated to reduce the internalization levels of this pathogen inside H e L a cells (Garduno, Garduno, and Hoffman, 1998). In the same study, Garduno et al. also demonstrated that beads coated with L. pneumophila's Hsp60 could be internalized in enhanced levels by H e L a cells compared to B S A coated beads, and also do not appear to fuse with 7 lysosomal compartments. Similarly, studies using anti-Hsp60 antibodies suggest that Hsp60 mediates the internalization of the facultative intracellular pathogen H. pylori inside gastric epithelial cells (Yamaguchi et al, 1997). Other studies have also used electron microscopy to document the role of H. pylori Hsp60 in the adhesion of this pathogen to inflamed gastric tissue (Hoffman and Garduno, 1999). Antibodies against autologous Hsp60 as well as addition of exogeneous protein were shown to decrease the adherence of Clostridium difficile to human kidney cells (Vero) (Hennequin et al, 2001b). In a similar way, autologous Hsp60 was found to inhibit the binding of the facultative intracellular yeast Histoplasma capsulatum to human primary macrophages (Long et al, 2003). Overall, these results suggest that bacterial Hsp60 could be involved in the internalization of various pathogens, and it may potentially be the case for S. aureus as well . 1.4 IN VITRO INTERNALIZATION MODELS OF S. A UREUS Various internalization models have been developed to study the internalization of S. aureus inside a variety of cell types in vitro. Most of them utilize an indirect approach relying on the elimination of the extracellular bacteria using cell-impermeable bactericidal agents. Amongst the most widely used is lysostaphin, a peptidoglycan hydrolase produced by Staphylococcus simulans, which cleaves glycyl-glycine bonds in S. aureus peptidoglycan (MIC 0.064 (xg/ml)(Wu et al, 2003). Other studies commonly use gentamicin, which binds to the bacterial ribosome, causing inhibition of protein synthesis. However, a recent study by Qazi et al showed that gentamicin treatment appears to be able to k i l l intracellular bacteria to some extent (Qazi et al, 2004), suggesting that lysostaphin may be a better choice. After the ki l l ing of extracellular bacteria, the level of internalization is generally assessed by plate count after lysis of the cell. This is typically done in distilled water with the detergent Triton-X 100 which is usually used at concentrations between 0.025 and 0.2 % (Ahmed et al, 2001;Kahl et al, 2000;Bayles et al, 1998a). The strains of bacteria used are usually the prototypical strains 8325-4 and RN6390, and are generally used in multiplicities of infection (MOIs) of 0.1 to 1000 C F U per cell. Iri at least one case, pre-staining of the bacteria followed by F A C S has been used to monitor the level of internalization (Krut, Sommer, and Kronke, 2004). Transformants have also been made to express luciferases genes from Photorabdhus luminescens as wel l as eGFP (enhanced green fluorescent protein) to allow monitoring of bacteria inside cells (Qazi et al, 2004). In almost every case, microscopy analyses are used as a tool to confirm the intracellular localization of the 8 bacteria. Similarly, cytochalasin D , an actin polymerization inhibitor, is frequently used to further verify this localization by blocking cell endocytosis (Qazi et al, 2001b). 9 2.0 W O R K I N G H Y P O T H E S I S & S P E C I F I C A I M S 2.1 H Y P O T H E S I S Hsp60 is involved in the internalization process of Staphylococcus aureus into human keratinocyte cell line HaCaT. 2.2 S P E C I F I C A I M S 1) Develop an in vitro model of infection of Staphylococcus aureus inside HaCaT keratinocytes 2) Characterize the internalization of S. aureus in this model. 2.1) Verify the intracellular localization of S. aureus 2.2) Characterize the model with respect to the levels of infection (numbers of infected cells and internalized bacteria) and the dose dependency of the initial inoculum on these internalization levels. 2.3) Determine the length of survival of internalized bacteria inside HaCaT keratinocytes 3) Verify the localization of Hsp60 on the surface of S. aureus 4) Attempt to block the internalization of S. aureus using antibody against Hsp60 and the addition of exogeneous protein 2.3 S U M M A R Y We showed that: 1) The internalization of S. aureus inside HaCaT keratinocytes is dose dependant up to the initial ratio of 100 C F U / cell. 2) This internalization can be blocked by the actin polymerization inhibitor cytochalasin D . 3) The intracellular presence of S. aureus was confirmed using fluorescence microscopy by quenching extracellular fluorescence using trypan blue. 4) S. aureus is able to survive at least 76 hrs inside HaCaT keratinocytes. 5) F A C S analyses can be used to determine the percentage of infected cells, which averages to nearly 8% for the M O I of 100 C F U per cell. 6) Hsp60 appears to be associated to the surface of S. aureus, but only to a small extent. 7) Staphylococcal Hsp60 does not appear to be involved in the internalization of S. aureus inside HaCaT cells. 10 3.0 E X P E R I M E N T A L 3.1 M A T E R I A L S A N D M E T H O D S 3.1 C U L T U R E O F STAPHYLOCOCCUS AUREUS Staphylococcus aureus strains used in this study are RN6390 (a widely used variant of the N C T C 8325-4 strain, cured of known prophages), RN4220 (a naturally competent derivative of N C T C 8325-4 (Novick, 1967)), and its transformant RN4220:pSB2035 (transformed with the double G F P / L u x reporter plasmid pSB2035 (Qazi et al, 2001a). RN4220 and its transformant were used for the dose-dependent internalization experiments while RN6390, which internalized in similar levels, was used for other experiments. Fresh bacterial cultures were grown from 1:50 dilutions of overnight cultures at 37°C 225 rpm in brain heart infusion (BHI) media for approximately 2 hrs, and then adjusted, to ODsyonm ~0.5 (estimated at 2x10 bacteria/ml) with B H I medium. Subsequent washings of bacteria were done in ice cold P B S with centrifugation at 4°C for 5 minutes at 2500 g. 3.2 T R A N S F O R M A T I O N O F S. A UREUS Transformation of S. aureus with the pSB2035 plasmid (containing a chloramphenicol resistance gene) was done using RN4220. Electrocompetent bacteria were prepared from fresh OD570nm 0.5 culture using serial washings with 10% glycerol in distilled water. Pellets from 200 ml cultures were washed in 200, 100, 10 and 4 ml volumes, then resuspended in 1 ml 10% glycerol. Aliquots of 125 ul (approximately 5 x l 0 9 C F U ) were then quickly frozen and stored at -70°C until use for up to 3 months. For the electroporation, the aliquots were quickly thawed on ice and 50 ul of bacteria was mixed for 1 minute on ice with up to 10 ul (50 ug) of plasmid preparation. Bacteria and plasmid preparations were then transferred to a 0.1 cm electroporation cuvette and electroporated at 25 uFD, 100 Ohms and 1 volt for 2 msec. 1 ml of S M M P regeneration medium (2x antibiotic medium #3 [Difco]) was added with 0.5 M sucrose, 20 m M maleic acid and 20 m M MgCh) was added immediately to the cuvette. The bacteria were then incubated at 37°C 225 rpm for 1.5 hrs and 150 ul of culture was plated on 10 ug/ml chloramphenicol B H I agar plates. Transformants were then cultured overnight at 37°C in B H I with 100 ug/ml chloramphenicol and 1 ml aliquots were resuspended in 500 ul of 15% glycerol in B H I and stored at -70°C. Unfortunately, the transformants could not be accurately detected inside HaCaT keratinocytes either by fluorescence or luminescence detection. This was likely 11 due to the fact that the G F P expression was controlled by the promoter of the Agr gene which likely would not have been highly induced in the intracellular milieu. Another reporter plasmid under the control of the growth dependent promoter xyl gene was also used but did not yield any successful transformant. 3.3 L A B E L L I N G O F S. AUREUS W I T H F L U O R E S C E I N I S O T H I O C Y A N A T E A s the fluorescent transformants could not be used to monitor the presence of the bacteria inside the cells, I decided to use fluorescein isothiocyanate (FITC) pre-staining. Fluorescein isothiocyanate is well known to react with lysine residue allowing for the staining of proteins at the surface of bacteria. This was done for every experiment requiring the detection of intracellular bacteria by fluorescence microscopy or for F A C S analysis of the percentage of infected cells. Fresh S. aureus cultures were divided into 1.5 m l aliquots and washed 2 times in 1 ml cold P B S (pH 7.2). Bacteria were incubated in 200 ul of 50 ug/ml F I T C in minimal medium for 30 minutes at 37°C and then washed 3 times in cold minimal medium. The fluorescence intensity of the bacteria was verified by F A C S analysis and demonstrated an increase of 2.5 logs in fluorescence compared to the unstained control, (treated with minimal medium without fluorescein). Similarly, the viability of the stained bacteria was assessed by plate count and was found to be similar to the unstained control. Washed bacteria were also allowed to replicate in B H I medium for an additional 2.5 h at 37°C and were still found to harbor high levels of fluorescence intensity as assessed by F A C S analyses. The ability of stained bacteria to internalize inside HaCaT keratinocytes was also found to be comparable to the unstained samples. 3.4 INFECTION ASSAY O F H A C A T K E R A T I N O C Y T E S Spontaneously immortalized human skin HaCaT keratinocytes (Boukamp et al, 1988) were seeded at 3 x l 0 4 cells/cm 2 and cultured to confluence in 6 or 24 well plates (BD Falcon) in D M E M (Dulbecco's Modified Eagle's Medium) with 4.5 g/L glucose (Stemcell Technologies), 50 U / m l penicillin/streptomycin and 10% F C S (fetal calf serum) at 37°C, 5% C 0 2 . Cel l culture medium was changed every two days. Infection of keratinocytes was performed using normal and FITC-labelled bacteria prepared as previously described and resuspended in cell culture 12 medium without antibiotics in volumes of 0.3 and 1 ml per wel l for 24 and 6 well plates, respectively. On the day of the experiment, cells were counted in 3 of the wells and appropriate amounts of bacteria (OD 5 70nm 0.5, estimated at 2x10 8 CFU/ml ) were added to achieve the desired M O I from 1 to 1000 C F U per cell. Plates were then centrifuged at 1000 g for 10 minutes to synchronize the infection, and further incubated for 50 minutes at 37°C, 5% C O 2 for a total of 1 hr of infection. The cells were then washed twice with 1 m l P B S , and cell culture medium containing 10 U / m l lysostaphin was added to k i l l the extracellular bacteria, and incubated for another 1 hr at 37°C, 5% C O 2 . The efficacy of the treatment was routinely confirmed by plate counting samples of the medium from different wells. Lysostaphin medium was then removed and the cells were washed 2 times with 1 m l P B S . Thereafter, cells were incubated with 0.05% E D T A for 10 minutes at 37°C to allow cell-cell dissociation followed by 0.025% trypsin treatment for 5 minutes at 37°C to detach the cells from the wells. Trypsinization was then stopped with the addition of 10% F C S and samples were processed for plate count, microscopy and F A C S analysis. 3.5 D E T E R M I N A T I O N OF I N T E R N A L I Z E D B A C T E R I A N U M B E R S The numbers of internalized bacteria were determined by plate count. A sample of the detached cells was diluted 1:10 in ice-cold sterile H 2 0 plus 0.025%) Triton X-100 and vortexed thoroughly to ensure lysis of the cells, which was confirmed by microscopy. Samples were then further serially diluted and 20 ul of the different dilutions were spread on B H I agar plates. Internalized bacterial numbers shown in this study were determined after overnight incubation at 37°C, and represent the total numbers of bacteria in the wel l taking into account the dilution factor, the volume plated and the original volume of cells in the well . 3.6 D E T E R M I N A T I O N OF T H E P E R C E N T A G E OF I N F E C T E D C E L L S The percentage of infected cells was determined by F A C S analysis. Trypsinized cells were washed once more in 200 ul P B S and fixed in the same volume of P B S plus 4% paraformaldehyde 30 minutes at room temperature in the dark. Fixed cells were then washed twice with 200 ul P B S containing 1% F C S and resuspended in 50 ul 1% F C S - P B S . Five ul of the cells were taken for microscopy analysis and the remaining cells were analysed by F A C S (FACScalibur, B D bioscience) in a final volume of 400 ul 1% F C S - P B S . The cell population 13 was identified by the forward scatter and side scatter parameters and cells were easily discriminated from debris, dead cells and free bacteria. The fluorescence of samples was then assessed by F L 1 and results were analyzed using the W i n M D I 2.8 software. Fluorescence quenching experiments were done in a similar manner by taking different readings of the samples before and after the addition of 200 ul of 4 mg/ml trypan blue. 3.7 DETECTION OF INTERNALIZED BACTERIA BY MICROSCOPY The detection of intracellular localization of S. aureus was done using trypsinized cells as well as using cells seeded on 12 mm autoclaved glass coverslips contained inside 24 well plates which were prepared as previously described until the trypsin treatment step. A t that point, cells were fixed using 300 ul 4% paraformaldehyde for 30 mins at room temperature in the dark and then washed twice with 500 ul 1% F C S - P B S . Trypsinized cells were prepared as described above and 5ul of infected cells were placed on coverslips. In both cases, coverslips were placed on glass microscope slide and mounted with 10 ul Fluorsave reagent (Calbiochem). Slides were allowed to dry in the dark, were fixed using clear nail polish and observed using an epifluorescence microscope (Zeiss Axioplan II) equipped with the F I T C filter. Samples were observed using a 63x objective and images were recorded using a C C D camera and the Northern Eclipse software (Empix imaging). For the trypan blue quenching study, trypsinized cells were premixed with an equal volume of a 1 mg/ml trypan blue solution and mounted on a slide without Fluorsave reagent. Red and green fluorescence intensities were read and the images were merged using the Northern Eclipse software. For the intracellular staining study, slides were prepared in the same conditions with the exception that cells were fixed using Cytofix/Cytoperrn buffer (BD Biosciences) and stained using rabbit anti-S. aureus antibodies followed by Texas Red conjugated anti-rabbit IgG at 1:200 and 1:400 dilutions, respectively, for 20 minutes at room temperature followed by washes in Perm/Wash buffer (BD Biosciences). 3.8 KILLING CURVE OF S. A UREUS INSIDE HACAT KERATINOCYTES The ki l l ing curve of S. aureus inside HaCaT keratinocytes was determined as previously described. Bacteria were left in contact with cells for 1 hr, and then lysostaphin was added and incubated at 37°C 5% CO2 for various times representing overall infection periods of 2, 8, 28, 55, 76 or 96 hrs; lysostaphin medium was changed every day. A t this point, cells were harvested as 14 usual and analysed by plate count. The viability of the cells was assessed immediately after the trypsinization step using trypan blue as well as using 5 ug/ml 7 - A A D (7-amino-actinomycin D) staining for 15 minutes at 4°C immediately before fixing the cells. 3.9 E F F E C T O F C Y T O C H A L A S I N D P R E - T R E A T M E N T O N T H E I N T E R N A L I Z A T I O N L E V E L S The effect of cytochalasin D pre-treatment was analysed as previously described, with the exception that cells were pretreated with cytochalasin D at 0.5 ug/ml in cell culture medium for 2 hrs before infection. Cells were then washed twice with P B S and bacteria were added, resuspended in cell culture medium containing cytochalasin D . Samples of bacteria were taken at the beginning and end of the treatment and were analysed by plate count. Similarly, cell viability was assessed by trypan blue exclusion before and after treatment to verify the effect of cytochalasin D on cell viability. 3 . 1 0 I N H I B I T I O N O F I N T E R N A L I Z A T I O N U S I N G A N T I - H S P 6 0 A N T I B O D I E S O R R E C O M B I N A N T H S P 6 0 P R O T E I N S For the antibodies inhibition studies, FITC-labelled bacteria were incubated with various concentrations of either rabbit anti-Hsp60 serum which was decomplemented at 56°C for 35 minutes, or incubated with F(ab)2 antibody fragments for 1.5 hr at 4°C in cell culture medium. The mixtures were then warmed (37°C) and used to infect the cells. Alternatively, 250 and 50 ug of recombinant Hsp60 (or bacterial membrane fraction) (see sections 3.11 and 3.15 for details of preparation) were added to the cells 15 minutes before the infection. Appropriate amounts of bacteria were then resuspended directly in the wells containing these proteins and the infection was allowed to progress as usual. 3.11 P R E P A R A T I O N O F R E C O M B I N A N T H S P 6 0 The preparation of the recombinant Hsp60 protein used was done in our laboratory by Susan Findlay under the supervision of Dr. Sabine Ivison. Briefly, Hsp60 amplified from S. aureus strain Rosenbach ( A T C C 27217) was expressed using the p G E X G S T fusion system (Amersham) in E. coli Top 10 cells (Invitrogen). Protein expression was induced from 250 ml of bacteria (OD 5 70nm 0.7) using 1 m M I P T G at 28°C for 3 hrs. Bacteria were then sonicated on ice 3 15 times for 30 seconds in the presence of 10 m M E D T A , 1 m M D T T and 1 m M P M S F . Cel l debris were pelleted and Triton-X 100 was added to supernatant to a final concentration of 0.1% and left on ice for 5 minutes. Proteins were then purified using a GST-sepharose column (Sigma), eluted with 10 m M glutathione and dialysed overnight. A t that point proteins were further purified by ion exhange using DEAE-sepharose (Sigma) and again dialysed overnight. Finally, the GST-tag was cleaved using Precision protease (Amersham), dialysed again overnight and concentrated the following day using an Amicon concentrator (Millipore). A l l dialyses were done at 4°C in the presence of I m M D T T . Protein purity was confirmed by S D S - P A G E followed by silver staining. The integrity of the Hsp60 D N A sequence used was confirmed by sequencing. 3.12 G E N E R A T I O N O F A N T I - H S P 6 0 A N D A N T I - G R O E L A N T I S E R A Immunization were done at the U B C Animal Care Facility using female New Zealand rabbits. 1 m l of inoculum containing 300 ug of recombinant staphylococcal Hsp60 or commercial E. coli G r o E L (Stressgen) proteins, as well as 500 u.1 Freud Incomplete Adjuvant (Sigma) were emulsified and injected subcutaneously at 4 different sites. Two more boosters of 175 ug were given every 21 days and 10 ml bleeds were taken 10 days after every injection. Blood was allowed to coagulate at room temperature and serum was collected and stored at -70°C. 3.13 D E T E R M I N A T I O N O F S E R U M A N T I B O D Y T I T E R S The antisera titers were determined by E L I S A . Immunolon polystyrene plates ( V W R ) were coated overnight at 4°C with 4 p.g/ml of either purified Hsp60 or commercial G r o E L proteins. After one 10 minutes wash in 0.05% Tween 20, plates were blocked with 10% F C S for 1 hr at room temperature and washed once. Different dilutions of the various sera were made and incubated for 2 hrs at room temperature. Wells were washed 3 times and secondary H R P conjugated antibody against rabbit IgG was used at a dilution of 1:1000 for 1 hr at room temperature. After another 3 washes, T M B detection substrate (Pierce) was added and O D was read at 450 nm following the addition of 1 M H3PO4 stop reagent. 16 3.14 G E N E R A T I O N O F F(ab) 2 A N T I B O D Y F R A G M E N T S Rabbit antibodies used for F(ab)2 antibody purification were taken from sera prepared as described above, except for normal rabbit serum which was a pool of different pre-immune sera and polyclonal anti-S. aureus serum which was raised against supernatant proteins of S. aureus, made by Dr Winnie K u m . In every case, IgG was first purified using a protein A / G column (Pierce), then digested with pepsin (Sigma) with a 1:100 weight to weight ratio at 37°C, 350 rpm for 24 hrs. A t this point, remaining uncleaved IgG and Fc fragments were removed using a protein A-agarose column. (Bio-Rad) and purity was confirmed by S D S - P A G E under non-denaturing conditions followed by silver staining. 3.15 M E M B R A N E S E P A R A T I O N O F S. A UREUS The membrane fraction was isolated from a 20 ml OD 5 70nm ~ l - 0 fresh culture of S. aureus RN6390 grown at 37°C 250 rpm. The bacteria were washed twice in P B S and resuspended in Tris buffer containing 50 ug/ml lysozyme, 20 U / m l lysostaphin, 10 m M E D T A , 10 m M D T T , 1 m M P M S F and 1 m M pepstatin. Bacteria were then sonicated on ice 6 times 30 seconds and the remaining bacterial debris was pelleted for 10 minutes at 4°C, 10 000 g. The rest of the proteins was centrifuged at 100 000 g 4°C for 1 hr and the supernatant representing the cytosolic fraction was collected. The pellet was then further washed once in 5 m l T B S at 100 OOOg (4°C) and collected as the membrane fraction. Whole cell, cytosolic and membrane fractions samples were kept at -70°C in 10% glycerol in the presence of 1 m M pepstatin, l O m M E D T A and 10 m M D T T . Protein concentrations were determined by U V and verified by S D S - P A G E . 3.16 W E S T E R N B L O T O F M E M B R A N E F R A C T I O N S 2 ug of crude protein extracts and 0.2 ug of purified proteins were run on 12% SDS-P A G E and the proteins were blotted on a nitrocellulose membrane using a semi-dry blotting apparatus for 35 minutes at 1.25 V per cm 2 . Membranes were then rinsed briefly with water, blocked for 30 minutes at room temperature with 5% skim milk and washed once for 10 minutes in T B S T . Anti-Hsp60, pre-immune sera (~5 mg/ml IgG concentration) or commercial anti-G r o E L (SPA-875 [Stressgen, Victoria], 1 mg/ml) were used at a 1:1500 dilution and incubated overnight at 4°C. After three washes, secondary H R P conjugated anti-Rabbit-IgG antibodies 17 were used at a 1:2000 dilution and incubated for 1 hr at room temperature. The membranes were then washed another 3 times, incubated with chemiluminescent E C L detection substrate (Pierce) for 1 minute and developed on film. 3.17 STATISTICS A l l graphs were done using the Prism software version 4 (GraphPad Software Inc., San Diego) and displayed with means and S E M . Statistical analyses were done with the same software using two-tailed unpaired t-test unless stated otherwise. 18 4.0 R E S U L T S 4.1 I N T E R N A L I Z A T I O N O F S. A UREUS INSIDE H A C A T C E L L S 4.1.1 DOSE D E P E N D A N T I N T E R N A L I Z A T I O N In this study I investigated the internalization of S. aureus inside HaCaT keratinocytes by the lysostaphin exclusion model in which lysostaphin treatment and extensive washing are employed to remove bacteria outside of the cells, therefore allowing differentiation between intracellular and extracellular bacteria. The level of internalization is then determined by various methods but predominantly using plate count numbers. Naturally, these numbers only represent the amount of bacteria which survived the lysostaphin treatment and the several washings of the cells. However, even though the method of plate count does not reveal direct information regarding the localization of the C F U , I w i l l assume that these bacteria likely survived lysostaphin treatment as they were inside the cells and from now on w i l l refer to them as numbers of internalized bacteria for simplicity. In all of the experiments the efficacy of the lysostaphin treatment was verified by plate count. The intracellular localization of S. aureus w i l l be demonstrated later on in section 4.1.2. A s a first step in characterizing my internalization model, I evaluated the effect of varying multiplicity of infections on the numbers of internalized bacteria found. This was done using two different strains of bacteria. The first is RN4220, a commonly used laboratory strain derived from NCTC-8325-4, which has been cured of known prophages (Novick, 1967) and is highly competent due to a mutation in a restriction endonuclease. The second, RN4220:pSB2035, is the same strain transformed with the plasmid pSB2035 encoding the green fluorescent protein (GFP) and the l u x A B C D luciferase from Photorhabdus luminescens under the control of the Agr promoter (Qazi et al, 2001a). This transformant was developed at the beginning of the project to facilitate the monitoring of internalized bacteria but was later abandoned as I could not detect appreciable levels of fluorescence and luminescence when the bacteria were inside the cells. This is l ikely due to the fact that the G F P expression was under the control of the agr promoter which likely was not induced in high levels in the intracellular milieu. We also tried to use another reporter plasmid under the house-keeping xyl promoter regulation but failed to obtain any successful transformant. Nevertheless, both strains exhibited similar levels of internalization at the various multiplicities of infection. It is important to note that the multiplicity of infection was determined based on the estimated numbers of bacteria at OD57o nm 0.5, predetermined by plate counts. However, these 19 numbers truly represent colony forming units and may therefore be an underestimation of the true numbers of bacteria present for S. aureus which is arranged in clusters. Therefore, the term M O I , as used in this study, truly refers to the initial ratio of C F U per cell. Figure 1 shows the effect of varying the initial M O I on the number of internalized bacteria. We can see by these results that the number of internalized bacteria increases with the number of C F U used and appears to plateau at a M O I of nearly 100 C F U / c e l l . This increase is more easily seen with the transformant strain and also represents a similar trend observed for 2 of the 3 replicates of the RN4220 strain. The other replicate had a higher amount of internalized bacteria found at M O I 10 which explains the high standard error o f this sample. It is interesting to note that the numbers of bacteria found at M O I 10 in both cases were not significantly different than those at M O I 100 or higher which suggests that these numbers were relatively similar and that internalization is likely to be saturated at a ratio of C F U / c e l l between 10 and 100. I decided to use M O I 100 for future experiments to maximize the numbers of infected cells and internalized bacteria without over saturating the model which could result in increased cell lethality. Figure 1: Effect of varying the multiplicity of infection on the total amounts of internalized bacteria found by plate count. Log phase S. aureus cultures (RN4220 and its pSB2035 dual transformant) were added to confluent keratinocytes at indicated M O I s and allowed to internalize for 1 hr. Extracellular bacteria were removed by extensive washings and lysostaphin treatment at 10 U / m l for 1 hr. Cells were then harvested with trypsin, lysed in cold 0.025% Triton X-100 and the numbers of internalized bacteria were determined by plate count. Shown are mean and S E M of 3 replicates for each S. aureus strain. Multiplicities of infection (MOI) 2 0 Figure 1 also shows a relatively high variation in the numbers of internalized bacteria found for each sample. This is l ikely due to the fact that these results represent only 3 experiments but also because the MOIs were estimated from a predetermined concentration of bacteria at OD 5 70nm 0.5 which generally varied slightly between experiments (0.45-0.55). This experiment also required several washing steps for the bacteria and cells which, although were made in the same conditions, likely influenced both the true M O I used and the numbers of internalized bacteria found. Nevertheless, the fact that none of the strains had consistently higher numbers of bacteria than the other for all MOIs suggests that further repeats of these experiments would likely yield similar results between both strains. B y comparing the amounts of internalized bacteria found with the initial numbers of bacteria used (Table 1), it appears that on average between 1 to 7% of the initial numbers of bacteria were being internalized and survived for M O I up to 100 CFU/ce l l . Again the standard variations of the different samples are quite high, however at M O I of 1000, consistently only 0.2% of the initial bacteria survived through the infection assay in both strains, suggesting once again that at M O I 1000 the model is obviously saturated. Table 1: Relative numbers of the initial bacteria which were found to be internalized by plate count, normalized to the M O I of 1. Listed are the average percentages and standard deviations for 3 replicates of both S. aureus clones. These numbers were taken from the experiments shown in figure 1. M O I RN4220:2035 RN4220 1 4.2 ±3 .1 1.9 ± 0 . 6 10 24 ± 11 68 ± 7 0 100 180±140 110 ± 3 0 1000 200 ± 2 3 0 200 ± 70 21 4.1.2 V I S U A L D E T E R M I N A T I O N O F B A C T E R I A I N S I D E C E L L S Although we routinely verified the efficacy of the lysostaphin treatment in our model through plate counting, this remains only an indirect sign that the extracellular bacteria were really kil led and that the bacterial numbers found represent intracellular events only. We therefore verified the intracellular localization of the bacteria using confocal fluorescence microscopy. This was first done using intracellular staining with antibodies directed against S. aureus proteins. Comparison of infected and uninfected cells showed that the antibody bearing the Texas-Red probe almost did not bind to anything in the absence of the bacteria suggesting that this antibody staining recognized S. aureus quite specifically. These results showed that S. aureus indeed appeared to be localized inside the HaCaT cells and was verified at a M O I of 1000 (Figure 2a) and 100. The localization of S. aureus was also investigated by using bacteria labeled with fluorescein isothyocyanate. This method was used primarily as an attempt to increase the fluorescence signal of the bacteria to allow for F A C S detection. Using this method, I was able to confirm the intracellular localization of S. aureus for the M O I s of 100 and 10 (Figure 2b). In all cases, the localization of the bacteria could easily be seen and controls infected with unstained bacteria did not harbor any comparable fluorescence. Nevertheless, in order to further confirm its internalization the samples were treated with the cell impermeable dye trypan blue in an attempt to quench the fluorescence of extracellular bacteria. Figure (2c) clearly shows that although the HaCaT auto-fluorescence was efficiently quenched, we could still perceive bacteria with intact fluorescence inside the cell. 22 Figure 2 : Intracellular localization of S. aureus inside HaCaT cells. Arrows point to internalized S. aureus inside HaCaT keratinocytes. In all cases, infections were done at designated M O I and allowed to proceed for 1 hr at which point extracellular bacteria were removed using lysostaphin. HaCaT cells used for microscopy were either grown directly on glass coverslips (B and C) or removed from the wells after infection using trypsin treatment (A) . Images were taken with a magnification of 630x. A ) : Localization o f internalized bacteria using intracellular staining at a M O I of 1000. Shown are representative images of 2 different experiments. Infected cells were harvested using trypsin then fixed and permeabilized using Cytofix/Cytoperm buffer. Intracellular bacteria were labelled using anti-.S'. aureus rabbit serum and stained with goat-anti-rabbit IgG antibodies conjugated to Texas Red. MO11000 —w Uninfected B): Localization of FITC-labelled S. aureus inside HaCaT keratinocytes, shown are representative images of at least 3 different experiments. S. aureus labelled with 50 ug/ml FITC were used at a M O I of 100 and 10. MOM 00 Mono 23 C): Presence of intracellular S. aureus despite quenching with 0.5 mg/ml trypan blue ( M O I 100). Shown are different examples representative of two different slides (N=l) . HaCaT cells were infected with FITC-labelled S. aureus as previously described and mounted on slides with 0.5 mg/ml trypan blue in P B S . Negative controls were done with unstained bacteria and did not harbor any comparable green fluorescence. I ft 4.1.3 D E T E R M I N A T I O N O F T H E P E R C E N T A G E O F I N F E C T E D C E L L S To further characterize the internalization model we have quantified the proportion of infected cells. This was performed using F A C S as well as microscopy analyses as described previously. The results showed that at a M O I of 100 C F U / c e l l , the numbers of infected cells ranged from 7.0% to 11.5% on average depending of the method used (Figure 3). For the M O I of 10, the percentage of infected cells resided around 3.5% (judging mostly by microscopy analyses). It is difficult to assess which of the two methods used is the more reliable. Even though F A C S is a more quantitative and accurate method, microscopy analyses remain necessary to verify the intracellular localization. Microscopy also gives the advantage of being able to discern between infected cells and uninfected cells which appear to present residual fluorescence on their surface. However, for similar reasons, the microscopy results are likely to be underestimations as only clearly infected cells were counted whereas cells containing only few bacteria or some too close to their membrane were generally considered uninfected. Nevertheless, as seen for the two M O I s studied, both methods gave similar results, which strengthen the validity o f these numbers and suggest that the "true" values are l ikely to be somewhere in the middle. 24 Figure 3: Percentage o f infected cells at M O I 100 and 10 as determined by microscopy and F A C S . Microscopy analyses were done as previously described. For F A C S analyses, cells were harvested after infection using trypsin and fixed with paraformaldehyde. After washing, cells were resuspended in 1% F C S and read by F A C S . For trypan blue quenching, samples were incubated in 2 mg/ml trypan blue 5 minutes before reading. Shown are the mean and the S E M of the different experiments. Numbers of replicates are written beside each column. 13 12 = m a) ° 10 TJ 4= 9 O 35 * 8H c O <u CL • Microscopy • FACS • FACS with trypan blue quenching (2 mg/ml) N=4 3.4% N=1 3.9% Mono Multipl icit ies o f in fec t ion MOI100 In order to further verify whether the cells with higher fluorescence intensity found by F A C S analyses truly represented internalization events, we tried to quench the extracellular fluorescence using trypan blue at a final concentration o f about 2 mg/ml. The results showed a decrease in the percentages of infected cells from 15.8% to 10.1%, which again suggests that the percentages of infected cells found by F A C S are likely to be slightly overestimated. I unfortunately did not try using other concentrations of trypan blue to optimize the quenching efficiency. However, due to the high concentration of dye used and judging by the very high red fluorescence of the trypan blue treated cells it is likely that the trypan blue treatment was able to quench most of the extracellular fluorescence at that concentration. Similarly, I do not believe that this high concentration may have caused nonspecific fluorescence attenuation as the cells were treated for only a few minutes. It is important to point out that these numbers of infected cells do not necessarily represent cells which contain live bacteria. Although it was usually possible to see the distinct round shapes of the bacteria, suggestive of the integrity o f their membranes, it is not necessarily a sign of their viability. B y comparing the numbers of infected cells with the numbers of 25 internalized bacteria found by plate count, MOI10 and MOI100 would have an average infection load of about 2.9 and 4.8 C F U per cell, respectively. Overall, microscopy analyses revealed that the numbers of bacteria per infected cell appeared to average between 5 and 10 bacteria per cell for the MOIs of 10 and 100 respectively. These results are in accordance with each other which would suggest that most of the infected cells found by microscopy likely contain live bacteria. 4 . 1 . 4 S U R V I V A L C U R V E O F S. A UREUS I N S I D E H A C A T C E L L S Figure 4: K i l l i n g curve of internalized S. aureus inside HaCaT keratinocytes. Shown are the mean and S E M representing the total numbers of internalized bacteria per well found by plate count as well as the percentage of live HaCaT cells found compared to an uninfected control at various timepoints ( N = 3). The infection was done as previously described with a M O I of 100. In all instances, fresh medium containing lysostaphin (10 U/ml) was added after 1 hr of infection and changed every 24 hrs. Plate count of the supernatant confirmed the ki l l ing of extracellular bacteria for every timepoint. Statistical analyses were done using two-tailed student t-test, * represents a significant decrease compared to the previous timepoints (p < 0.05). 0 10 20 30 40 50 60 70 80 90 100 Number of hours inside the cells Having shown the intracellular localization of S. aureus in our model, we decided to verify how long these bacteria could survive inside the host cells. The infection process was therefore allowed to continue up to 96 hrs, with constant presence of fresh medium containing 26 lysostaphin (10 U/ml) and daily monitoring of intracellular bacterial numbers. The results (Figure 4) showed that between 2 and 28 hrs the numbers of internalized bacteria appear to remain relatively constant. B y comparing the trend of the 2 and 8 hrs timepoints it appears as i f the total numbers of bacteria are increasing. Although this increase is not significant, it suggests that more repetition of the experiment may reveal a slight but "true" increase in the intracellular numbers, suggestive of intracellular replication. After 28 hrs of infection, the numbers decreased rapidly up to 96 hrs at which point only a small amount of internalized bacteria could still be found. However at 96 hrs, these numbers were barely at the limit of detection, and two times out of three no bacterium could be found. For this reason, we cannot conclude that S. aureus survives inside keratinocytes for 96 hrs, although we can say that it did consistently survived for at least 76 hrs. Table 2 : Increase in the lethality of infected cells compared to an uninfected control as assessed by trypan blue and 7 - A A D staining. Shown are the mean ± standard deviation for 7 - A A D (N = 2) and trypan blue (N = 3) analyses. The experiments were done as previously described using a M O I of 100. After trypsinization, cells were either diluted in equal volume of 0.25% trypan blue or stained with 5 ug/ml 7 - A A D . Trypan blue results represent the percentage of decrease in live cell numbers of infected samples compared to the uninfected control. 7 - A A D results represent the increase in the percentage of highly fluorescent cells compared to the uninfected control. Statistical analyses were done using unpaired two-tailed student t-test. Timepoints 2hrs 8hrs 28hrs 52hrs Trypan blue 7.9 ± 4 . 8 26.1 ± 8 . 1 * 45.0 ± 12.3** 59.6 ± 12.5** 7 - A A D 4.4 ± 6 . 3 4.5 ± 4 . 4 2.8 ± 2 . 4 9.9 ± 7 . 8 * Significant increase compared to the 2 hrs timepoint, p < 0.05; ** p < 0.01 We do not know from these results whether the decrease of live bacteria is caused by cell defense mechanisms or simply because the bacteria were released into the medium and killed by the lysostaphin. We therefore analyzed the viability of the cells during this time course for up to 52 hrs by trypan blue and 7-amino-actinomycin D ( 7 - A A D ) staining (Table 2). We can see that the results from these two methods are very different from each other. This could be explained by the fact that, while trypan blue analyses measured the variation in the cumulative numbers of live cells left, 7 - A A D staining only measured the amounts of dead cells present at a specific time. These amounts are likely to be underestimated as a good proportion of the dead cells destroyed by the bacteria would not accumulate throughout the time course. Nevertheless, trypan blue results show that the percentage of dead cells significantly increased from 7.9 % at 2 hrs to 26.1% as early as 8 hrs and reaching up to 59.6 % 52 hrs after infection. Since only 8-10% of the 27 cells were found to be infected at 2 hrs (Figure 3) and since the percentage of dead cells continues to increase despite the presence of lysostaphin, these results suggest that intracellular bacteria not only caused the death of initially infected cells, but may also have been able to infect neighbouring cells or cause their death through the release of diverse toxins. Overall, even though our results do not provide much information on the nature o f this cell lethality, they still suggest that cell death l ikely caused some of the decrease in the intracellular bacterial numbers observed after 8 hrs. 4.1.5 E F F E C T O F C Y T O C H A L A S I N D O N T H E I N T E R N A L I Z A T I O N In order to assess whether the internalization is dependent o f an active cell mechanism, inhibition studies were performed using cytochalasin D , a cell permeable toxin from Zygosporium mansonii, which acts as an actin polymerization inhibitor. Figure 5: Effect of cytochalasin D treatment on the numbers of internalized bacteria found by plate count. Shown are mean and S E M . 0.5 ug/ml cytochalasin D was added to the cells 2 hrs before and during the infection, which was done at MOIs of 100 ( N = 4) and 10 ( N = 2). Cells from the untreated controls were prepared in the same manner using P B S . Plate count of the supernatant after infection as well as trypan blue analyses confirmed that cytochalasin D treatment did not appear to affect bacterial or cell viability. Statistical analyses were done using student two tailed t-test. MOI 100, N=4 MOI 10, N=2 Figure 5 shows the effect of pre-treating cells with cytochalasin D on the numbers of internalized bacteria found by plate count. These results clearly demonstrate that cytochalasin D treatment decreased the amount of internalized bacteria found by over 2 fold, and was observed 28 at both MOIs of 10 and 100 CFU/ce l l . This decrease was found to be significant only at the M O I of 100, mostly because only 2 replicates were done for M O I 10. Cytochalasin D pre-treatment also caused the decrease in the number of infected cells as assessed by F A C S analyses (Figure 6). This decrease was also observed using fluorescence microscopy where cytochalasin D pre-treated samples were found to contain almost no infected cells (<0.005%, data not shown). Figure 6: Effect of cytochalasin D treatment on the percentage of infected cells found by F A C S . Shown are mean and S E M of the percentage of infected cells representing 2 replicates. The cytochalasin D treatment and infection process were made as previously described using FITC-labelled or unstained S. aureus at the M O I of 100. After the infection, cells were harvested with trypsin and fixed prior to F A C S analysis. The percentages of infected cells were determined based on samples infected with unstained S. aureus. The statistical analysis was made using paired two tailed t-test (* p < 0.05) Untreated Treated Untreated Treated To rule out the possibility that cytochalasin D treatment may have affected the viability of the cells, trypan blue viability studies were done in parallel, and revealed that the numbers of dead cells as well as the total numbers of cells were similar for both treated and untreated samples (5.0 ± 0.1% and 5.4 ± 1.4% respectively, N = 2). Similarly, the effect of cytochalasin D on the viability of the bacteria was assessed by plate count. B y comparing the numbers of bacteria in the supernatant at the beginning and the end of the 1 hr infection period, we could see that there was no significant change in bacterial numbers for treated and untreated samples (2.2 ± 1.9 and 3.9 ± 2.6 fold increase respectively, N = 4). 29 Overall these results demonstrate that the internalization of S. aureus inside HaCaT keratinocytes involve an actin-dependent mechanism. This also further suggests that most of the bacteria found at the end of the infection in our model survived within the host cell. 4.2 M E M B R A N E L O C A L I Z A T I O N O F S. A UREUS H S P 6 0 4.2.1 E X T R A C E L L U L A R S T A I N I N G O F HSP60 P U R I F I C A T I O N O F R E C O M B I N A N T S. AUREUS H S P 6 0 In order to assess the membrane localization of Hsp60, we generated antibodies directed against staphylococcal Hsp60. The recombinant protein used for the antibody generation was expressed in E. coli and purified using a G S T column and anion exchange chromatography. The purified protein was then concentrated and analyzed by silver staining, showing that overall the purity of the protein appears relatively good (Figure 7). We can also notice that the purified protein appears to migrate around 65 kDa, slightly higher than the commercial G r o E L protein (Hsp60 of E. coli). This is certainly unexpected as both proteins should have a size of approximately 57.5 kDa and therefore should have a similar migration pattern. I believe that this difference in size could be due to a fragment of the G S T tag sequence which may not have been completely removed after enzymatic cleavage. Figure 7: Purity of the recombinant staphylococcal Hsp60 used for immunization. This silver stain shows the protein content of the commercial E. coli Hsp60 (B) (Stressgen, Victoria) as well as our recombinant S. aureus Hsp60 preparations before (C) and after (A) cleavage of the GST tag. Samples were boiled 5 mins in SDS loading buffer and 1 ug was loaded on 10% S D S - P A G E along with 5 ul of PageRuler prestained protein marker (Fermentas). A B C 30 We therefore proceeded to the immunization of one rabbit using recombinant S. aureus Hsp60 and another using commercial GroEL. We then analyzed the different antibody titers against both Hsp60 (Figure 8) and G r o E L (Figure 9) proteins by E L I S A for the pre-immune as well as the first and second bleed sera. Figure 8: Titers of the 1 s t and 2 n d bleed antisera against S. aureus Hsp60 as found by E L I S A . Rabbits were immunized with 300 u.g of either commercial G r o E L or recombinant S. aureus Hsp60 and challenged every 21 days with 175 ug of the same protein. Bleed were taken before (pre-immune) and 10 days after each immunization. The E L I S A plates were coated with 4 ug/ml recombinant S. aureus Hsp60. Sample sera were used at the following dilutions and detected using HRP-l inked goat antibodies raised against rabbit IgGs at a 1:1000 dilution. These results represent the mean and S E M of the experiment done in duplicate. Dilution factors 2.00 o LO Q O 0) o o o Q O 1.50-1.25 0.00 100k 25k 6400 1600 400 100 25 1 i i i • . . . A . . Pre-imm. Anti-Hsp60 Anti-Hsp IstBleed i L — » i r , — T • Anti-Hsp 2nd Bleed Pre-imm. Anti-Gro^L jj[ \ ,~Jk—— A I Jr~~ • • • Anti-GroEL IstBleed Anti-GroEL 2nd Bleed \ / j / T i / / i / / X / ux • Q • i — * — i 1 10- 10 4 10"3 10"2 Log of concentration (1 / dilution factor) 10"1 3 1 Figure 9: Titers of the 1 s t and 2 n d bleed antisera against E. coli G r o E L as found by E L I S A . This experiment was done as described above using plates coated with 4 ug/ml of E. coli GroEL. These results represent the mean and S E M of the experiment done in duplicate. Figure 10: E L I S A results of the 2 n d and 3 r d bleed antisera titers against S. aureus Hsp60 proteins. This experiment was done as described previously based on estimated antibody concentrations of 5 mg/ml for all samples. Shown are the mean and S E M of the two duplicates. Dilution factors 25k 6400 1600 400 10"4 10-3 10-: Log of concentration (1 / dilution factor) 32 The results illustrated on Figure 8 show that there was a strong increase in antibody titers against Hsp60 for both S. aureus Hsp60 and G r o E L immunized rabbits. This cross-reactivity was expected due to the high sequence homology between those proteins (52% identity at the protein level and 62% identity at the nucleotide level, based on E. coli K12 and S. aureus N315 Hsp60 sequences), which was also confirmed by Dr Sabine Ivison, a post-doctoral fellow in our lab. The opposite cross-reactivity for anti-Hsp60 against G r o E L is also true as shown in Figure 9 and appears to be even stronger when comparing the second bleed sera. This could be explained by the fact that the rabbit used for Hsp60 immunization appeared to have had exposure to E. coli G r o E L or other similar Hsp60 proteins before the procedure (Figure 9). This is entirely expected due to the fact that the rabbits were not maintained in a pathogen-free environment and therefore were likely exposed to various bacterial species which could have led to a potent bacterial Hsp60 antibody response. In a different experiment, antibody titers against Hsp60 were determined by E L I S A for the 3 r d bleed sera. These results (Figure 10) showed that there did not appear to have been much of an increase in the antibody titers after the 2 n d immunization. We therefore concluded at that point that it was unnecessary to continue the immunization process any further. Overall, even though the pre-immune sera for the Hsp60 rabbit was unusually high (compared to the other rabbit), it appeared to be possible to use dilutions at which the 3 r d bleed serum anti-Hsp60 would recognize S. aureus Hsp60 and the pre-immune serum would not. E X T R A C E L L U L A R S T A I N I N G U S I N G H S P 6 0 A N T I S E R U M Meanwhile, I first tried to assess the localization of S. aureus Hsp60 on the bacterial membrane by extracellular staining. This was done several times using whole antibodies with commercial anti-GroEL, anti-S. aureus supernatant proteins, commercial IgG isotype control, as well as an antibody against a Hsp60 peptide fragment generated in our lab. However, I could not see any difference between the different antibodies tested, which was predictable due to the fact that S. aureus harbors a high quantity of protein A on its surface which non-specifically binds Fc portion of antibodies (Results not shown). I therefore tried to use human IgG in an attempt to block nonspecific binding but preincubation using concentrations as high as 500 ug/ml did not allow differentiation between any of the antibodies used. I then decided to produce F(ab) 2 fragments of the newly generated sera and use them for extracellular staining. The fragments were generated by purification of the IgG using a protein A / G column followed by subsequent 33 pepsin digestion and removal of residual uncleaved IgG and Fc fragments using a protein A column. Figure 11: Purity of the F(ab) 2 preparations. For every sera, IgGs were purified using a protein A / G column (Pierce), cleaved using pepsin (at a proteimprotein ratio o f 1:100) and the F(ab) 2 fragments were purified using a protein A column (Bio-rad). A ) Shown is a silver stain representative of the protein content in the anti-Hsp60 (lanes 1, 2), pre-immune (3, 4) and normal rabbit sera (5, 6) F(ab) 2 preparations before (1, 3, 5) and after (2, 4, 6) the final protein A purification step. B) Shown is a silver stain of the normal rabbit sera showing a representative example of the protein content in all IgG preparations before (1) and after (2) pepsin treatment. In every case, -0.3 ug of the different antibody preparations were loaded on 10% S D S - P A G E in non-denaturing conditions. Arrows point to the bands representing the -100 kDa F(ab) 2 antibody fragment (broken arrow) and -150 kDa IgG (filled arrow). A) B) 1 2 3 4 5 6 1 2 Figure 11 . A shows the result of the last purification step with the protein A column and the purity representative of all the F(ab) 2 fragment preparations. A s we can see there does not appear to be any IgG left (150 kDa) in these preparations which suggests that the pepsin digestion was complete. However there does appear to be some lower molecular weight proteins left at around 40 kDa which likely represent remaining pepsin molecules. Although the pepsin digestion should have yielded complete digestion of the Fc fragments, this may not necessarily have been the case. We can see that, for all of the samples, the protein A column appears to have bound a fraction of these fragments, but not all o f it. Since the binding capacity of the column was greater than the total amount of protein loaded, it is possible that these fragments could have lost their protein A binding sites. Nevertheless, the amount o f F(ab) 2 is obviously much higher than the remaining putative Fc fragments thus I decided to use these preparations. 34 It is important to note that pepsin enzymes (35kDa) were not removed during the antibody preparation. I have previously repeatedly tried using size exclusion chromatography to isolate my F(ab)2 digest but unfortunately could not get any successful separation. Nevertheless, I do not think the presence of this enzyme affected any of the subsequent studies as, according to the manufacturer instructions, pepsin should have been permanently inactivated at a p H above 6.0 and all of the extracellular staining analyses were done at 4°C at p H 7.0. The efficacy of the pepsin treated antibody fragments was assessed by E L I S A (Figure 10). It is important to note that the secondary antibody raised against rabbit IgG used is more l ikely to recognize constant domains of the IgG molecules which are present mostly on the F c fragment of IgG. For this reason, we cannot compare the activity of pepsin-treated fragments to the activity of the untreated antisera. Nevertheless, we can observe that anti-Hsp60 antibody fragments recognize recombinant S. aureus Hsp60 much better than its pre-immune counterpart. E X T R A C E L L U L A R S T A I N I N G U S I N G F(ab) 2 F R A G M E N T S After having generated the F(ab)2 fragments, I proceeded to the extracellular staining of the bacteria to determine i f S. aureus Hsp60 appeared to be on the surface. This was done using different concentrations of the various anti-sera and the results are illustrated in Figure 12. Note that the double peaks observed likely represent the different recognition by the F(ab) and F(ab) 2 forms of antibodies which are expected to be found in our preparation. A s seen in Figure 12, it appears that extracellular staining using anti-Hsp60 antibodies led to higher fluorescence than pre-immune sera at concentrations of both 10 and 20 ug/ml. This increase in mean fluorescence was shown to be statistically significant at 20 ug/ml of F(ab) 2 fragments, p = 0.04; Figure 13). However this increase does not appear to be very high compared to staining with anti-S. aureus proteins, which is also statistically higher than anti-Hsp60 (p = 0.01). This could be due to the fact that anti-<S. aureus serum was raised against native proteins whereas anti-Hsp60 was raised against recombinant proteins which were likely denatured. This could also be due to the fact that the anti-S. aureus serum would l ikely recognize multiple proteins while the anti-Hsp60 serum is more specific and recognize only Hsp60, which may be present only to a small extent on the surface of the bacteria. It is important to note that the pre-immune serum also reacted to some extent to Hsp60 as seen by E L I S A (Figure 8), and likely caused the apparent increase in the fluorescence of the pre-immune serum compared to the 35 commercial rabbit IgG (Figure 12). Consequently, this unusually high pre-immune control could account for the reduced difference in staining compared to our anti-Ffsp60 serum. Figure 12: Staining of Hsp60 on the surface of S. aureus using F(ab)2 antibodies. Shown are FACS histograms representative of 3 different experiments showing the staining of S. aureus surface with various F(ab)2 antibody preparations. After fixation, stationary phase bacteria were labeled with 10 or 20 ng/ml of the different F(ab)2 fragments and stained with 2.5 ng/ml PE-conjugated donkey F(ab)2 anti-rabbit IgG for 45 minutes on ice. 10>g/ml F(ab)2 staining Anti-Hsp6Q (Filled) Pre Immune (Green) Normal Rabbit Serum (Blck) 20ug/ml F(ab)2 staining From Right to left: Anti-5. a., Anti-Hsp60, Pre-immune I » UJ 10' 10- 10' Anti-Rabbit IgG-PE 10* Figure 13: Comparison of the mean fluorescence intensity from the membrane staining experiments using 20 ng/ml F(ab)2 fragments. Shown are mean and S E M of the 3 different experiments. Statistical analyses were done using unpaired two-tailed student t-test. Hsp60 Pre-imm. Anti-S.a. F(ab)2 fragments of various sera Overall, these results suggest that Hsp60 is located on the surface of S. aureus. However, according to the small increase in fluorescence, this protein is likely present only to a small extent on the surface. 36 4.2.2 M E M B R A N E S E P A R A T I O N O F S. AUREUS In order to verify the membrane localization of Hsp60, I proceeded to the physical separation o f the membrane fraction of S. aureus, which is composed o f a lipidic cytoplasmic membrane surrounded by several layers of heavily cross-linked peptidoglycan chains. The separation was done by first using sonication to disrupt the membrane integrity and then using ultracentrifugation at 100 OOOg to collect the water insoluble membrane polymers as well as their associated proteins. For simplicity reason, I wi l l refer to the proteins present in this water insoluble fraction as membrane proteins even though these may not directly be associated to the cytoplasmic membrane. The membrane proteins were then analyzed by western blot, and detected using anti-Hsp60 and pre-immune sera, or commercial ant i -GroEL antibodies (E. coli Hsp60). Figure 14: Western blot showing the detection of S. aureus Hsp60 in the different bacterial cell fractions using anti-Hsp60 (4-8) and pre-immune (1-3) sera. L o g phase S. aureus were sonicated to produce whole cell lysate which was then centrifuged at 100 OOOg. The supernatant was collected as the cytosolic fraction while the insoluble proteins were washed, re-pelleted and collected as the membrane fraction. 2 ug of whole cell lysate (4), cytosolic (5) and membrane fractions (1, 6) as well as 0.2 ug of purified staphylococcal Hsp60 (2, 7) and commercial E . coli G r o E L (3, 8) proteins were loaded on a 10% S D S - P A G E gel which was transferred on a P V D F membrane. The membrane was labelled with our anti-<S. aureus Hsp60 and pre-immune sera (1:1500 dilution) and stained using HRP-l inked anti-rabbit IgG antibodies (1:2000 dilution). The blot was detected using enhanced chemiluminescent substrate ( E C L , Pierce) and developed on film. These results are representative of 3 experiments using different membrane preparations. Arrows point to a -57 kDa protein smear (top) and -48 k D a bands. The broken arrow points to a -43 kDa protein band which, unlike the others is not recognized by commercial anti-GroEL. 1 2 3 4 5 6 7 8 3 7 Figure 15: Detection of S. aureus Hsp60 in the different cell fractions using commercial anti-GroEL. The western blot was prepared as previously described using 0.6 ug/ml commercial anti-G r o E L (Stressgen). 2 ug of whole cell lysate (1), cytosolic (2) and membrane (3) fractions were loaded along with 0.2 ug of purified staphylococcal Hsp60 (4) and E . coli G r o E L (5). Arrows point to the -57 k D a protein smear and -48 kDa protein bands. 1 2 3 4 5 The results, shown in Figure 14, demonstrate that our anti-Hsp60 recognizes both our purified Hsp60 preparation and the commercial G r o E L . This cross-reactivity is of course expected and is in accordance to the previous E L I S A results. Anti-Hsp60 serum also recognizes a smear of bands around 57 kDa, a clear band around 48 kDa, as wel l as a band near 43 kDa in both the whole cell lysate ( W C L ) and cytosolic fractions, whereas only one band at 43 kDa is seen in the membrane fraction. A l l of these bands, except for the 43 k D a band also appear to be recognized by the commercial anti-GroEL antibody (Figure 15), which suggests that these proteins are likely to be homologous to Hsp60 or are its degradation products. It is of course very unusual to see that there isn't any clear protein band migrating close to our Hsp60 preparation i f not for a -57 kDa protein smear which is migrating even lower than the expected molecular weight of our recombinant S. aureus Hsp60. The presence of a smear could indicate the degradation of our protein and the difference in molecular weight is l ikely due to the fact that our recombinant Hsp60 protein has been expressed in E. coli and still possesses a small added peptide fragment which made the link to the G S T tag. A s seen previously, S. aureus Hsp60 should migrate at a molecular weight similar to E. coli G r o E L . This is nearly the position at which the upper 57 k D a protein smears is seen, suggesting that there may have been some Hsp60 3 8 protein left intact. I am still unsure at this point what the protein bands at around 48 kDa really are, but the fact that they are recognized by both anti-Hsp60 and anti-GroEL antibodies suggests that they could represent a stable degradation product or perhaps another form of Hsp60 which was post-translationally processed in a different way. Nevertheless, as seen by over-exposure of the membrane (Figure 16), these putative Hsp60 proteins (the 57kDa smear and 48kDa proteins) appear to be present to a very small extent in the membrane fraction o f S. aureus. Only the 43kDa protein band is present in the membrane fraction to considerable extent which suggests that this band could represent a differently processed Hsp60 molecule which could specifically be targeted for membrane-association. If this is true, this would explain why this protein would not be recognized by our anti-GroEL antibodies. The presence of this protein in the cytosolic fractions would however suggest that there was some contamination between the different fractions. This is of course inherent to the separation method used and further suggests that the amounts o f - 5 7 and ~48kDa Hsp60 proteins present in the membrane are even lower than what was observed. It is interesting to note that the pre-immune as well as the commercial anti-GroEL antibodies did not appear to recognize our Hsp60 preparation. This can be due to the fact that only 0.1 ug of the purified proteins were loaded and the antibody concentrations used were likely not enough to see cross-reactivity with such a small amount of protein. I am unsure as to why the commercial anti-GroEL antibodies did not react with the 43kDa protein, but I suspect that this may be due to the different processing of this Hsp60 homologous protein which may very well be linked to its apparent membrane association. 39 Figure 16: Over-exposure of the membrane detected with anti-S. aureus Hsp60 (4-8) and pre-immune (1-3) sera. This blot represents the same blot as shown in figure 14 which has been detected using a longer exposition time. 2 ug of whole cell lysate (4), cytosolic (5) and membrane (1, 6) fractions were loaded along with 0.2 ug of purified S. aureus Hsp60 (2, 7) and E. coli G r o E L (3, 8). 1 2 3 4 5 6 7 8 Overall, these results suggest that our anti-Hsp60 preparation is relatively specific compared with a commercial anti-GroEL antibody preparation. Although the Hsp60 proteins of our whole cell fractions were migrating at unusual positions, they appeared to be present in the membrane fraction of S. aureus to a very small extent, which agrees with our previous extracellular staining results. It is important to note that, due to the limitation of this method, we cannot tell from these results whether these membrane-associated proteins were really exposed on the outside of the bacteria as they could be hidden by the thick peptidoglycan layers or simply associated to the inside o f the cytoplasmic membrane. 4 0 4.3 B L O C K I N G T H E I N T E R N A L I Z A T I O N U S I N G A N T I - H S P 6 0 A N T I B O D I E S Figure 17: Effect o f various dilutions of anti-Hsp60 antiserum on the internalization levels of S. aureus as assessed by plate count. These results represent the mean and S E M of two different experiments done in duplicate. Infection assays were done as previously described using bacteria pre-incubated for 1.5 hr on ice with 1:10 or 1:100 dilutions o f the various decomplemented antisera. Infections were done at M O I 100 in the presence of the antisera. Statistical analyses using two-tailed student t-test demonstrated that anti-S. aureus treatment greatly decreased the internalization levels and was highly significant compared to the anti-Hsp60 sera (**, p < 0.01). 10 100 10 Dilution factors 100 Untreat Since it appears that Hsp60 is located on the surface of S. aureus, we investigated i f internalization of S. aureus Hsp60 inside HaCaT keratinocytes could be blocked using our anti-Hsp60 antibodies. This was performed using whole decomplemented serum and using F(ab) 2 antibody fragments. The results of whole serum inhibition suggest that no difference in the internalization level could be observed when comparing anti-Hsp60 with pre-immune sera treatments (Figure 17). On the other hand, it does appear that the anti-S. aureus serum treatment caused quite a decrease in the number of internalized bacteria compared to the other sera at a dilution of 1:10. This suggests that the overall method used was adequate and that the internalization simply may not be blocked using antibodies against Hsp60. Overall, it appears obvious from these results that i f Hsp60 is truly involved in the internalization of S. aureus, it is unlikely to be a major effector. It is interesting to note that both the anti-Hsp60 and pre-immune treated samples appeared inhibited to a greater extent at a 1:10 dilution compared to a 1:100 dilution. This was to 41 be expected as a 1:10 dilution represents a high amount of antibodies and other serum proteins, which can result in nonspecific inhibition. Figure 18: Effect of various dilutions of anti-Hsp60 F(ab)2 fragments on the internalization levels of S. aureus as assessed by plate count. These results represent the mean and S E M of two different experiments done in duplicate. The experiments were done as described above using 100 or 10 ug of F(ab)2 fragments from the various antisera. No significant difference could be observed between the different samples using student two-tailed t-test. 100 ug/ml 10 ug/ml It is also important to remember that S. aureus expresses a high amount of protein A on its surface, which could have diminished the effects of the different antibodies used. We therefore proceeded to inhibition studies using F(ab)2 antibody fragments to rule out this possibility. A s seen in Figure 18, there did not appear to have been any inhibition at all for each of the different sera used. This would suggest that the antibody concentration was not high enough to allow any difference to be detected. This is quite probable as a 1:100 dilution of the different whole sera, which did not cause much inhibition, would be comparable to the levels of purified F(ab)2 antibodies used which correspond to nearly 70 (xg/ml of antibodies. Nevertheless, these results suggest that 100 ug/ml of purified F(ab)2 antibody fragments cannot inhibit the internalization of S. aureus in HaCaT keratinocytes. 42 4.4 B L O C K I N G T H E I N T E R N A L I Z A T I O N U S I N G E X O G E N O U S S. AUREUS HSP60 P R O T E I N S In order to further elucidate the role of Hsp60 in the internalization of S. aureus, we investigated the effect o f adding exogenous staphylococcal Hsp60 proteins on the internalization levels in an attempt to block its putative cell receptor. This was done by pre-treating cells for 15 minutes with concentrations of 250 and 50 ng/ml of purified Hsp60 proteins as well as 50 ng/ml of S. aureus membrane fraction and subsequently proceeding to the infection as usual in the presence of these proteins. Figure 19: Effect o f adding exogenous S. aureus Hsp60 on the internalization levels as assessed by plate count. Shown are the mean and S E M of 2 different experiments. Cells were pre-incubated for 15 minutes with 250 and 50 ng of recombinant S. aureus Hsp60 or membrane fraction proteins. These proteins were kept throughout the infection process which was done at the M O I of 100. re Figure 19 shows that the addition of exogenous Hsp60 at a concentration of 50 ng/ml did not appear to have influenced the internalization levels of S. aureus at all . On the other hand, a control using 50 ng/ml o f S. aureus membrane proteins appears to have caused a high decrease in the percentages of internalized bacteria as compared to the uninfected control. This decrease was however not found to be statistically significant due to the small number of replicates and to the high variations in plate count numbers. Nevertheless, it appears that the experimental procedure in itself worked as expected and that the addition of exogenous S. aureus Hsp60 may simply not cause any inhibition at a concentration of 50 ng/ml. However, a higher concentration 43 of exogenous Hsp60 (250 ug/ml) appears to cause a decrease in the internalization levels of up to about 55% of the untreated control. Once again this decrease was not found to be statistically significant and still remains much lower than the decrease observed with the treatment of 50 ug/ml of membrane proteins. Figure 20: Effect of adding exogenous S. aureus Hsp60 on the percentage of infected cells as assessed by F A C S . Shown are mean and S E M for two different experiments. The infection was done as described previously using an M O I of 100 of FITC-labelled S. aureus. The various protein samples were added to the cells 15 minutes prior and during infection. Percentages of infected cells were determined based on the increase of the proportion o f highly fluorescent cells compared to an uninfected control. I800n Hsp60 250ug/ml_ Hsp60 50ug/ml_ Membrane 50ug/ml_ Untreated p<0.01 Hsp60-250ug Hsp60-50ug Membrane-50ug Untreated These results were verified by F A C S analyses (Figure 20) which showed once again that both concentrations of Hsp60 did not affect the mean fluorescence intensity of infected cells, whereas 50 ug/ml of membrane proteins significantly reduced it. Overall these results suggest that the internalization of S. aureus cannot be blocked by the addition of as much as 50 ug/ml of exogenous Hsp60 proteins. It is l ikely that further replicates may show an inhibitory effect at the concentration of 250 ug/ml of Hsp60 proteins. However, such a high concentration of proteins is likely to cause indirect effects through the nonspecific binding to receptors which may not necessarily bind to Hsp60 in normal conditions in vivo. 44 5.0 DISCUSSION 5.1 INTERNALIZATION OF S. A UREUS INTO HUMAN HACAT KERATINOCYTES In this study, we investigated the internalization of S. aureus inside human cells which was done using skin keratinocytes due to the well known ability of S. aureus to cause skin infections. A s we did not have access to a source of primary human keratinocytes, we decided to use the HaCaT keratinocyte cell line, which is a spontaneously immortalized cell line derived from the skin of human dorsal keratinocytes (Boukamp et al, 1988). This cell line has been used extensively in many studies and is believed to maintain a high genomic integrity despite repetitive passages (Fusenig and Boukamp, 1998). So far, only few studies have looked at S. aureus internalization using human skin keratinocytes and even fewer have used HaCaT cells. Our results clearly show that S. aureus is actively internalized inside HaCaT keratinocytes which was demonstrated by fluorescence microscopy and by the inhibition with cytochalasin D . This internalization was observed to be maximized at an initial M O I of between 10 and 100 CFU/ce l l . This however translated into only up to 8% ( M O I 100) of infected cells, which lays doubt as to whether or not the maximal infection level was truly reached. It is possible that a higher multiplicity of infection may allow higher internalization levels which may not have been observed due to faster and increased cell death. Moreover, increased numbers of bacteria used for infection could have resulted in increased sizes of bacterial clusters, which may therefore physically interfere with endocytosis. Similarly, although the bacteria used were initially taken from logarithmic phase, incubation with higher numbers of bacteria may lead to the induction of stationary phase related genes, most likely through the activation of the agr quorum sensing system, which leads to the decrease of surface adhesins and increased production of toxins (Yarwood and Schlievert, 2003). However, due to the short incubation time of 1 hr, I believe it is unlikely that the agr machinery, which requires accumulation of the auto-inducing peptide (AgrD) for activation, would have had a significant effect on the internalization process at the different MOIs . This study has found that the percentages of infected cells for initial MOIs of 10 and 100 were approximately 4 and 9% respectively. These low percentages raise the question of whether this internalization phenomenon is a specific virulence mechanism of S. aureus or is simply due to an accidental endocytosis phenomenon. Since raising the M O I from 10 to 100 only resulted in 45 a two-fold increase in the numbers of infected cells, and since an M O I of 100 represents a very high pathogen burden, it is quite probable that most of the cells would have had enough bacteria to potentially be infected. Thus, the low percentages of infected cells observed are more likely to be attributable to a difference in the capacity of these cells to endocytose bacteria, or clear them once they are inside as differences between different clones of a population is to be expected. One could also speculate that the host surface receptor used by S. aureus in this assay may simply be expressed on a small percentage of cells or may not have been readily available i f the keratinocytes, which are known to be polar cells, were not in the proper orientation. It could also be possible that the physical conditions of the cells may not have been uniformly the same, and may have been affected by the infection process itself in a manner so that some of the cells were not able to clear the infection. Our results however do not provide much evidence to shed light on why some of the cells may be more suitable than others to carry live bacteria. Interestingly, the percentage of infected cells in S. aureus internalization assays has not been definitively reported in the related literature. Nevertheless, it has been reported that the percentage of S. aureus-infected cells varies greatly between studies, reaching 6.8% for the M M - 3 9 tracheal epithelial cell line (MOI unknown) (da Silva et al, 2004), 15-20%) as assessed by electron microscopy using the 8325-4 strain in HaCaT keratinocytes ( M O I unclear) (Mempel et al, 2002), and even up to 80% in mammary epithelial cells ( M A C - T cells) (Bayles et al, 1998b) using the Novel strain isolated from cow mastitis at a M O I of 32. From these results, it would appear that the percentage of infected cells in our assay should have been higher, however the differences in the experimental procedures makes it impossible to compare our results with previous studies. We must however note that the percentage of infected cells were mostly determined by microscopy analyses in which only strikingly positively infected cells were counted and is thus likely to be an underestimate of the true numbers of infected cells. Alternately, our results do not suggest whether or not the infected cells all contained live bacteria. The numbers of live bacteria recovered at 2 hrs were however in accordance to the average numbers of bacteria seen in infected cells (about 5-10 bacteria per cell). Nevertheless, other studies using live/dead staining methods would be advised in order to properly address this issue. Another interesting result of this study is the confirmation that S. aureus was able to survive inside HaCaT keratinocytes for at least 76 hrs. The length of survival inside human cells has already been assessed in human enterocytes where S. aureus was shown to survive at least 5 days (Hess et al, 2003;Krut, Sommer, and Kronke, 2004). These results are in accordance with our studies as some bacteria could still be found at 96 hrs; however the numbers of bacteria 46 counted at that point were too small to make a strong interpretation. Interestingly, similar results have also been observed using primary human foreskin keratinocytes where clinical S. aureus isolates could internalize and survive at least 72 hrs under similar conditions (Nuzzo et al., 2000). It remains unclear as to how certain strains of S. aureus would be able to remain as long as 96 hrs inside cells while others would quickly be eliminated. Moreover, the nature of the bacterial clearance in itself is currently unknown. On one side, this bacterial clearance could be due to the intracellular replication of S. aureus inside cells and the expression of exotoxins such as alpha-toxin which could lead to the destruction of the cell membrane and the exposition to the lysostaphin medium. In this case, it can be expected that some variants of S. aureus clones present in the bacterial culture used would have had a decreased expression of these toxins or have a slower growth rate which would allow these strains to survive intracellularly for longer periods. This is in accordance with a recent study showing that clinical S C V s S. aureus strains isolated from cystic fibrosis patients with chronic S. aureus infection had a decreased toxin production and slower growth rate than wi ld type strain (Moisan et al, 2006). Alternately, the fact that only nearly 5% of the initial bacterial numbers were found to be internalized at 2 hrs further suggest that different bacterial clones were present with varying ability to enter and survive inside keratinocytes. On the other hand, the sharp decrease in the bacterial numbers observed could have also been caused by an active clearing of the bacteria by the cells. It is well known that keratinocytes have an important function in immune response, both through their cytokine secretion as well as their role in the skin where they undergo programmed apoptosis to create a physical barrier. It is therefore possible that this internalization observed could be an active immunological mechanism to remove infecting pathogens. Our results do not suggest whether the high percentage of decrease in live HaCaT cell numbers is due to programmed cell death or bacterial ki l l ing but it is very likely to be a combination of both. Other studies using HaCaT keratinocytes have reported that 24 hrs incubations with S. aureus at an M O I of nearly 50 induced cell death in 40% of the keratinocyte population (Mempel et al, 2002) as measured by trypan blue. It is interesting to find that these results are in accordance with ours as we also found a 45% decrease in the numbers of live cells after 28 hrs of. infection for the M O I of 100. The small difference between these results are likely to be attributable to the higher M O I used and to the slight variations in experimental procedures. It is difficult to assess i f this length of survival would be long enough to account for S. aureus's abilities to cause recurrent infections or allow for increase in antibiotic resistance in 47 vivo. However, the intracellular milieu appears to favor the selection of some slow growing strains of S. aureus with decreased virulence. This has been shown for the small colony variant (SCV) strains (Vesga et al, 1996) but could also hold true for other S. aureus mutants. These S C V s are believed to be associated with infections in vivo and were found in patients with persistent infections such as Darier's disease, osteomyelitis and cystic fibrosis (vonEiff et al, 1997;von E i f f et al, 2001;Kahl et al, 2003). Interestingly, Brouillette et al. demonstrated that these S C V s could internalize in vivo in a mouse mastitis model and showed that these strains had increased antibiotic resistance compared to the wi ld type (Brouillette et al, 2004). Unfortunately, we did not notice the formation of any S C V , which could be explained by the fact that these strains supposedly form very small colonies which are hard to notice. A novel aspect of our study comes from the fact that we used F A C S analysis to determine the percentages of infected cells and the effects of various treatments in a S. aureus internalization model. This has been reported very rarely in the literature in the case of S. aureus infections, but it has been reported at least once using mouse fibroblast m K S A cells infected with pretreated FITC-labeled S. aureus (Krut, Sommer, and Kronke, 2004). It is interesting to note that this group achieved improved detection of infected cells as seen by the higher increase in their fluorescence intensity which was even apparently detectable after 24 hrs of infection. This could be due to the fact that they were using a higher concentration of F I T C (100 ug/ml) and a higher p H for the staining. Nevertheless, we were still able to differentiate infected from uninfected cells by F A C S after at least 2 hrs of incubation. There was however great variation in the numbers found as seen by the large standard deviation. The numbers of infected cells found by F A C S were also generally higher than those found by microscopy analyses. This may be due to the fact that fluorescent bacterial debris were sometimes seen on the surface of otherwise uninfected cells by microscopy analyses. Nevertheless, our results suggest that it is possible to use quenching reagents such as trypan blue in order to remove some of the extracellular fluorescence and hopefully increase the reliability of F A C S analyses. This certainly would be a novel technique in the study of intracellular S. aureus and could therefore allow the use of F A C S as a rapid, reliable tool for measuring internalization levels. It is important to note that cytochalasin D dramatically reduced the levels of internalization as determined by F A C S , plate count and microscopy analyses. This 2-log decrease in the numbers of internalized bacteria corresponds to what has been repeatedly described in the literature (Donnarumma et al, 2004;Jett and Gilmore, 2002). Although this is no 48 longer a new phenomenon, this remains an important control which further verifies that the bacteria monitored are truly intracellular. 5.2 R O L E O F HSP60 I N T H E I N T E R N A L I Z A T I O N O F S. A UREUS We assessed the role of Hsp60 in the internalization process of S. aureus using different experimental methods. A s Hsp60 is a house-keeping gene necessary for almost every known organism, we could not rely on the use of mutagenesis to verify our hypothesis. For the same reason, as Hsp60 is necessary for normal survival and due to technical reasons, we decided to hypotheses on the role of Hsp60 in the penetration of human cells and not on the direct survival of the bacteria inside the cells. We first used antibodies directed against staphylococcal Hsp60 in an attempt to block the putative binding of this protein to its host receptor. This obviously relies on the premise that staphylococcal Hsp60 is associated to the membrane of S. aureus and, without necessarily being the only effector, mediates the binding of its host receptor. Alternatively, we also attempted to block the internalization with the addition of free exogenous Hsp60 proteins. In order to verify the membrane localization of staphylococcal Hsp60 on the surface of S. aureus, we generated antibodies against the recombinant S. aureus Ffsp60 whole protein and used those in western blots to analyze membrane and cytosolic fractions of the bacteria. The results from the membrane separation experiment were indeed very interesting. In all o f our experiments, Hsp60 migrated at a lower molecular weight than expected under denaturing conditions and appeared to migrate mostly at - 50 kDa. Interestingly, a smear of proteins was detected on the various western blots, migrating at around 57 kDa, only slightly lower than E. coli G r o E L (Figure 14). These protein localizations were detected by both our anti-Hsp60 antiserum as well as the commercial polyclonal antibody against G r o E L (Figure 15). This therefore suggests that the original Hsp60 (with an expected migrating pattern similar to E. coli GroEL) may have been degraded, despite the fact the proteins were kept at 4°C at all times in the presence of various protease inhibitors. This however poses a concern as to why this degradation would result in the formation of a major degradation product and not simply a smear of lower molecular weight products; our results are not conclusive in that aspect. Interestingly, another band could be detected at around ~43 kDa, which happened to be mainly present in the membrane fraction (Figure 14). This band possibly could represent another form of Hsp60 which would be differently processed to allow for membrane association. 49 However, although it is mostly present in the membrane fraction of the bacteria, it is still also found in some extent in the cytosolic fraction as well . This could be due to contamination between the different fractions which is inherent to the experimental method use. Interestingly, the commercial anti-GroEL antibodies do not appear to recognize this 43 k D a protein band. This could be due to the difference in the protein processing which could have made this membrane associated form of Hsp60 unrecognizable by anti-GroEL. However, more studies would be needed in order to determine what this 43kDa Hsp60 homolog truly is. Overall, the results of the western blots, although not as clear as one could have hoped to see, suggest that Hsp60 is present in or associated with the membrane fraction of S. aureus at the most only to a very small extent. The fact that only over-exposure of the blot revealed the presence of the putative Hsp60 protein bands in the membrane throws doubt on the notion that Hsp60 is truly associated with the membrane of S. aureus. However, due to the high protein adherence characteristic of molecular chaperones, to the quality of our pre-immune serum which was obviously already reacting to a G r o E L homolog, and finally due to the inherent limitations of this separation technique, the membrane localization of Hsp60 w i l l have to be resolved using other methods such as electron microscopy or biotinylation of surface proteins. We therefore attempted to verify this surface localization using extracellular staining with fluorescent antibodies. Due to the small size of the bacteria, the precise localization of the protein on the membrane could not be observed by fluorescence microscopy. We therefore used F A C S analysis and found that bacteria stained with anti-Hsp60 showed a small, constant but significant, increase in the mean fluorescence intensity in comparison to the pre-immune serum. These results therefore concurred with the previous findings that Hsp60 does appear to be associated with the membrane of S. aureus to some extent. It is important to note that once again, our anti-Hsp60 serum was compared to a pre-immune serum which was shown to also react, to a smaller degree, to Hsp60 homologs. It is therefore probable that this surface association may have been more obvious i f we had used another Hsp60 preparation. Unfortunately, we cannot rule out the fact that some of the bacteria may have been damaged during the staining, fixing and washing processes, which may have allowed some of the antibodies to recognize intracellular proteins. This is however very unlikely due to the presence of a thick peptidoglycan cell wall found in staphylococci. Nevertheless, although these are not strong evidence, these results suggest that membrane localization of Hsp60 in S. aureus is a possibility and it may therefore be possible that this protein may have a role to some extent in the internalization process. 50 It is important to note that for both of these membrane localization experiments, the results are highly dependent on the ability of our anti-Hsp60 preparation to recognize staphylococcal Hsp60. However, this antibody has been raised against a recombinant protein generated in E . coli which may not have been folded in the same way as the native staphylococcal Hsp60 protein. This is especially true as the purification procedure used for obtaining the recombinant protein may have caused its denaturation. It is therefore difficult to draw any strong conclusion from these results due to the quality of the antibody preparation. The use of another antibody preparation directed towards the native form of staphylococcal Hsp60 protein would be advised, but was not possible for this study. Further experiments using electron microscopy would also help to provide stronger evidence on Hsp60 membrane localization. We nevertheless proceeded to inhibition studies using antibody fragments to block the internalization. This was done using whole serum which was decomplemented by heat. The results from this experiment show that our anti-Hsp60 serum could not significantly inhibit the internalization of S. aureus in comparison to its pre-immune control at serum dilutions of 1:10 and 1:100. On the other hand, a 1:10 dilution of an antiserum directed against staphylococcal supernatant proteins was able to cause a two log decrease in the internalized numbers of bacteria. These results suggest that anti-Hsp60 whole serum failed to inhibit the internalization of S. aureus. It is worthy to note that at a dilution of 1:10, both pre-immune and anti-Hsp60 sera appear to block internalization to some extent, compared to the 1:100 dilution. This can be explained by the nonspecific effects of high protein concentrations which is l ikely attributable to nonspecific antibody recognition leading to the blocking of host cell receptor or staphylococcal adhesions. To rule out the possibility that protein A may have affected the extent of the inhibition, we repeated this experiment using up to 100 ug/ml of F(ab)2 fragments. Again, we could not see any difference in inhibition for our anti-Hsp60 compared to the pre-immune serum. However, this time, the antiserum against S. aureus supernatant protein did not cause any inhibition either. It is unclear as to why the inhibition was not observed, but this could be due to the process of the generation of these F(ab)2 fragments, which may have altered the ability of IgG to efficiently bind to its target. It is also possible that the concentration of F(ab)2 fragment used was not high enough to inhibit internalization. This however represents a relatively high concentration of antibodies (relative to an estimated 1:50 dilution in the crude serum preparation) and it is highly probable that raising the antibody concentration may lead to nonspecific recognition. 51 Nevertheless, we can conclude that 100 |J.g/ml F(ab)2 fragments for every type of sera used does not efficiently block internalization in our model to a very high extent. The effect of adding exogenous Hsp60 proteins on the internalization of S. aureus was assessed. Our results show that a concentration of 50 ug/ml of staphylococcal Hsp60 protein did not result in any inhibition of the internalization of S. aureus. This was observed both by plate count and by F A C S analyses. On the other hand, a control pre-treated with 50 ug of membrane proteins exhibited a major decrease in the internalization level, which was found to be significant by F A C S analyses. Similar inhibition assays were performed by other groups showing that as little as 10 ug/ml of a recombinant fragment of FnBP can cause a 80% decrease in the internalization of S. aureus by immortalized U P epidermal keratinocytes (Kintarak et al, 2004a). Therefore, further increasing the concentration of Hsp60 used is l ikely to result in nonspecific protein-protein interactions (which would be particularly expected due to the role of molecular chaperones in protein folding). Overall, due to the inherent limitations of the methods used in this study, the antibodies available, and due to the fact that it is impossible to "prove" the negative hypothesis that Hsp60 is not involved in the internalization, I w i l l conclude that, in our model, staphylococcal Hsp60 does not appear, to a high extent, to be present on the membrane and to be involved in the internalization of S. aureus inside human HaCaT keratinocytes. In the light of the results collected thus far, I no longer believe that Hsp60 is a major effector of the internalization of S. aureus inside HaCaT keratinocytes. However, it is important to keep in mind that other models and conditions could have allowed noticing a greater role for Hsp60 on the internalization or an increase in surface exposition. Our study only looked at a narrow range of conditions such as growth phase, temperature and even bacterial strains. Pathogens are constantly exposed to various stresses during infection in vivo and it is therefore possible that some of these stresses would be necessary in order to fully activate the pathogen's virulence determinants. For example, the expression or surface expression of Hsp60 is l ikely to be increased by the change in temperature occurring as the bacteria enters the host environment. This increase in Hsp60 production after heat-shock is well known and has been shown repeatedly in E . coli (Melkani, Zardeneta, and Mendoza, 2005). Conversely, many other factors found in vivo such as p H stress, which is known to be lower during abscess formation, or oxidative stress found inside host cells could potentially also induce Hsp60 surface localization. Moreover, the use of bacteria at different growth phase could have influenced Hsp60 membrane localization as growth phase as well as 52 population density play major roles in the expression of surface adhesion and virulence factors. Conversely, the use of freshly isolated clinical S. aureus strains as wel l as primary keratinocytes could have revealed a different role of Hsp60 in the internalization which would be more representative of in vivo infections. Therefore, our study is not completely discriminative for the implication of Hsp60 in the internalization of S. aureus in diseases. 6.0 F U T U R E W O R K Further investigations are necessary to verify i f Hsp60 may be playing a minor role in the internalization process. These experiments would however have to be repeated many times as the putative effect of this protein in the internalization levels is l ikely to be small. I believe it would be possible as an alternative approach to assess the effect of F£sp60-overproducing transformants on the internalization levels. However, this method is l ikely to be tedious and would bring unnecessary uncertainties as to the effect of Hsp60 overload on bacterial metabolism. Alternately, the role of Hsp60 in the internalization could be explored in other conditions which may be more representative of infections such as using, heat-shocked or pH-stressed bacteria, primary cells, clinical S. aureus isolates or even using an in vivo infection model. A s for the localization of Hsp60 on the membrane of S. aureus, I believe that the only way to further confirm the localization is to perform proper electron microscopy analyses. This method w i l l however require large investment in time and money, which we unfortunately could not afford in this study. Apart from the role of Hsp60, many other aspects in the internalization of S. aureus inside human cells remain unclear and would be of greater interest to elucidate. One would be to verify i f internalization events truly happen during S. aureus infections in vivo and importantly, i f these internalized bacteria would be able to survive antibiotic treatments or cause recurrent infections. Although it is possible to show that intracellular bacteria can be found even after antibiotic treatment, it would be difficult to directly associate the intracellular localization with S. aureus' ability to cause recurrent infections. This would have to be done by proving that antibiotic therapy is able to remove absolutely all extracellular bacteria in the system and that the recurrence of the infection was caused by the release of internalized bacteria. Alternatively, it may be possible to use cells which have been infected in vitro in a graft model to verify whether these cells, which we know are infected, can protect the bacteria from antibiotic treatment and induce recurrent infections. Although this would constitute a novel approach in addressing these 53 questions, it would also bring up other issues in regards to how in vitro infected cells would behave compared to in vivo. 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