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Stress response of porphyromonas gingivalis Lu, Biqing 1993

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STRESS RESPONSE OF PORPHYROMONAS GINGIVALISbyBIQING LUB.Sc., Hubei Medical University, 1988A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIES(Department of Microbiology)We accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAFebruary 1993© Biqing Lu, 1993In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature) Department of MicrobiologyThe University of British ColumbiaVancouver, CanadaDate ^(1/4-1 ar OA 013 / 1 913 DE-6 (2/88)ABSTRACTThe heat shock response of Porphyromonas gingivalis was examined by one-and two-dimensional polyacrylamide gel electrophoresis after metabolic labelingwith ( 14C)-amino acids. When P. gingivalis cells were shifted from 37°C to 42 °C,elevated synthesis of four proteins with the apparent molecular weight of 92, 80, 74,and 62kDa was observed, whereas synthesis of a 50kDa protein decreased duringheat shock. The 74 and 62kDa proteins were identified as homologs of E. coli DnaKand GroEL respectively by Western immunoblotting. On two-dimensional Westernblots, two forms of DnaK and four forms of GroEL were identified due to their slightlydifferent isoelectric points. dnaK and groEL gene homologs were identified inseveral P. gingivalis and some other black-pigmented Bacteroides strains bySouthern hybridization. DnaK and GroEL homolog proteins and some other proteinswere also induced when P. gingivalis cells were challenged by ethanol. Exposure tooxidative stress and an elevation or decrease in pH did not show a discernibleinduction of these heat shock proteins. GroEL and DnaK homolog proteins were notinduced in P. gingivalis cells recovered from a guinea-pig infection model.TABLE OF CONTENTSAbstract  ^iiList of tables  ^vList of figures ^  vi,viiAcknowledgments  ^viiiIntroduction  ^1The heat shock response  ^1Regulation of the heat shock response ^2The function of HSPs  ^4Immunodominance and pathogenicity of HSPs  ^5The HSP7O/DnaK family  ^6The DnaJ and GrpE proteins  ^7The HSP60/GroEL family  ^7Microbiology of periodontal disease  ^8Classification of black-pigmented oral anaerobic rods  ^14Pathogenicity of P. gingivalis ^15Virulence factors of P. gingivalis ^17Materials and methods ^20Bacterial strains and culture conditions  ^20Purification of E. coil recombinant GroEL protein  ^20Purification of E. coli recombinant DnaK protein  ^21Protein determination  ^22Immunological procedures  ^22Affinity purification of anti-GroEL antibodies  ^22Heat shock and other stress responses ^23Electrophoretic analysis  ^24Western immunoblotting  ^24Preparation of gene probes  ^25Southern hybridization  ^25In vivo infection studies  ^26Results  ^28Purification of GroEL and DnaK proteins  ^28Affinity purification of anti-GroEL antibodies  ^33Heat shock response of P. gingivalis ^33Pulse-chase labeling study of the 50kDa protein  ^39Southern hybridization with heat shock gene probes  ^39Effect of different stress stimuli on P. gingivalis  ^44In vivo infection ^52Discussion  ^54Bibliography  ^60LIST OF TABLESTABLE PAGE1 Nomenclature of black-pigmented anaerobic rods ^ 162 Virulence factors of P. gingivalis ^ 183 Viability of P. gingivalis W50 cells exposed todifferent stress conditions ^ 474 Induction of GroEL and DnaK homolog proteinsby different stress stimuli^ 51LIST OF FIGURESFIGURE^ PAGE1^The speculative role of DnaK in heat shock response  ^32^A model for the action of the GroEL/GroES chaperonins  ^93 a The periodontium in health ^10b Established gingivitis lesion ^11c Periodontitis lesion ^124^Microbe-associated periodontal diseases  ^135^Purification of GroEL and DnaK proteins  ^296 Chromatography of GroEL on DEAE-Sepharose  ^307 Chromatography of GroEL protein on a MonoQ column  ^318 Affinity chromatography of DnaK on an ATP-agarose column ^329^Affinity purification of anti-GroEL antibodyon an Affi-gel 10 column  ^3410 SDS-PAGE autoradiogram of the heat shock responseof P. gingivalis  ^3511 2D PAGE autoradiogram of the heat shockresponse of P. gingivalis  ^3612 Time course study of the heat shock response of P. gingivalis ^3813 Western blot of P. gingivalis cells treated withdifferent stress stimuli  ^4014 2D Western blots of P. gingivalis before and after heat shock ^4115 Pulse-chase labeling study of the 50 kDa protein  ^4316 Southern hybridization of P. gingivalis and other black-pigmentedBacteroides chromosomal DNA with an E. coli dnaK/J probe^ 4517 Southern hybridization of P. gingivalis and other black-pigmentedBacteroides chromosomal DNA with an E. co/i groEL/ES probe^ 4618 SDS-PAGE autoradiogram of P. gingivalis grown underdifferent stress conditions  ^4919 SDS-PAGE autoradiogram of P. gingivalis grown at different pH...^5020 Western blot of P. gingivalis W50 grown in vivo and in vitro  ^53ACKNOWLEDGMENTSI am deeply grateful to Dr. B.C. McBride for his understanding and guidance,and for his continued encouragement and support of this research project, and forthe financial support of the University of British Columbia and Medical ResearchCouncil of Canada.My sincere thanks to Drs. R.E.W. Hancock and J.T. Beatty for serving on mycommittee and for their guidance and support. I wish to give my special thanks toDrs. P. Hannam, K. Widenhorn, K.H. Mueller and Barbara Waters for sharing theirknowledge and experience and for their constant encouragement.I am sincerely thankful to my fellow students, Angela Joe, Yoonsuk Park andKeung Wei Leung for consistently sharing their experience and knowledge.I also wish to thank Bruce McCaughey and Harold Traeger for theirphotography in the preparation of this work.I take this opportunity to express my gratitude to my family and friends for theirconstant encouragement and support.INTRODUCTIONThe heat shock responseProkaryotic and eucaryotic cells respond to potentially damaging stimuli such aselevated temperature by increasing the synthesis of a family of proteins collectivelyknown as stress proteins. One to two dozen proteins are induced in response to arange of different stresses, including heat shock, nutrition deprivation, oxygenradicals, and metabolic disruption. The best studied of these stresses is heat shock,in which a sudden increase in temperature induces the increased synthesis of heatshock proteins (HSPs). The heat shock response is amongst the most highlyconserved genetic systems known. Because many of the heat shock proteins arealso induced by other stresses, they are some times referred to as stress proteins.Heat shock response (HSR) was originally described by Ritossa in 1962 whoshowed that upon a temperature shift from 20 to 37 °C, as well as treatment withdinitrophenol or sodium salicylate, several new puffs appeared in the salivary glandpolytene chromosomes in Drosophila melanogaster (90). Over the next severalyears it became clear that the puffs were the sites of vigorous RNA transcription andthat a number of these RNAs were translated into the heat shock proteins. Discoveryof the E. coli heat shock response helped make it evident that there is a universalcellular response to a shift up in temperature, and soon it became established thatcomponents of the response have been highly conserved in bacteria, fungi, plants,and animals. The heat shock response has been intensively investigated bynumerous laboratories around the world. The continued interest in heat shockresponse rests on (a). the highly conserved nature of the structure and function ofmany HSPs. (b). the regulation of the HSR as a paradigm of gene expression. (c).the important role the HSPs play in protein folding, oligomerization, translocationand degradation. (d). the role of the major HSPs in infection and autoimmunity.1Regulation of the heat shock responseThe mechanism of HSR regulation is not completely clear. The HSR is positivelyregulated at the transcriptional level by the a 32 polypeptide, the product of the rpoH(htpR) gene (23, 45). In E. coli, one of the most intensively studied microorganisms,heat shock gene expression requires the heat shock-specific a subunit of RNA-polymerase, a32 , which confers on core RNA polymerase the specificity to transcribeheat shock genes (20, 46). Regulation of heat shock gene expression is mediated bycontrolling the cellular concentration as well as the activity (109) of a 32 . The cellularconcentration of a32 is controlled by regulation of the transcription of rpoH and thetranslation of its message (31, 47, 80), and regulation of the stability of a 32 (47, 108-110, 117). In fact, one of the most remarkable features of a 32 is its extremely shorthalf-life (about one minute) at steady state growth conditions (47, 108). Geneticevidence indicates key negative regulatory functions for the HSPs DnaK, DnaJ, andGrpE at the levels of synthesis, activity, and degradation of a32 (Figure 1).Mutations in dnaK, dnaJ, and grpE cause partial stabilization of a32 (116), loss ofrepression of heat shock gene transcription after temperature downshift(109, 116),and deficiencies in post-transcriptional regulation of a 32 synthesis after heat shock(47, 110). The mechanism by which DnaK, DnaJ, and GrpE regulate the activity andstability of a32 is assumed to rely on their concerted activity as chaperones. Thisactivity involves the ATP-dependent binding to substrates of DnaK and thestimulation of hydrolysis of DnaK-bound ATP by DnaJ and GrpE (58, 59, 98). It hasbeen proposed that DnaK interacts with a 32 and dissociates it from RNApolymerase, thereby rendering it accessible to cellular proteases (39). In a recentreport, Gamer et al. (36) presented evidence for the physical association of DnaK,DnaJ, and GrpE chaperones with a 32 in vivo.2-1 IP; Translation I IStabilitymRNAActivityPromoter hs genes[DnaK, DnaJ, and GrpE]^Figure 1. The speculative role of DnaK in heat shock response.A speculative model for the role of HSP7O(DnaK) in controlling expression ofHSPs, showing how DnaK, along with DnaJ and GrpE, could act to control HSPexpression in bacteria. Upon temperature upshift, depletion of the free pool of theseHSPs relieves their negative regulatory effects. Increased synthesis, stability, andactivity of 032 permits increased transcription of heat shock genes (adopted from(23)).3The signal transduction pathway that converts environmental stress to specificalterations of heat shock genes expression remains unclear. There is evidence tosuggest that the intracellular concentration of aberrant proteins is a majordeterminant of the cellular HSPs in E. co/i as well as in eucaryotic cells (5, 35, 41,53). Another suggested model is that by sequestering HSP70 through its binding toaberrant proteins the heat shock transcription factor 632 is prevented from interactingwith HSP70, which in turn allows activation of heat shock gene transcription (23).It was recently shown that there is another set of heat shock genes which arepositively regulated at the transcriptional level by the 624 (GE) polypeptide (45). Oneof the promoters of the rpoH (htpR) gene turned out to be transcribed by the E6 24RNA polymerase holoenzyme. The gene coding for 6 24 has not been discovered.The function of HSPsThe importance of many HSPs is based on their capacity to associate with otherproteins in a way that modifies the destiny and function of the proteins (21, 28, 60,61, 91). Anfinsen's classic experiments on the refolding of ribonuclease in vitro (6)established that all the information required to determine the final conformation of aprotein can reside in the polypeptide chain itself: the denatured protein can refoldinto its native conformation in the absence of other proteins. Such studies suggestthat refolding in vitro may be initiated by (1). collapse of hydrophobic regions into theinterior of the molecule., (2). formation of stable secondary structures that provide aframework for subsequent folding, and (3). formation of covalent interactions, such asdisulfide bonds, that stabilize the polypeptide in particular conformations. However,in vitro experiments do not accurately reflect the process of folding of nascentproteins in the interior of a cell. Refolding in vitro is frequently very inefficient incomparison to folding in vivo, and often requires proteins and physicochemicalconditions very different from those occurring intracellularly.4Under normal conditions, a polypeptide chain must be correctly folded,processed, localized, and in some cases, complexed with other polypeptides toproperly perform its biological functions. This is a very complex biological processwith many pitfalls along the way. For example, when the growing polypeptide chainemerges from the ribosome, it is subject to premature contact with other proteindomains, either intra or inter specific, because of the high cytosolic proteinconcentration. Such premature interaction among protein domains must be avoidedto prevent misfolding (23, 56, 78, 91). In the case of proteins crossing membranes, itis clear that only unfolded proteins can traverse biological membranes (84), againcreating the opportunity for inappropriate interactions among protein domains. Inboth cases, the need for some sort of "chaperoning' activity to maintain the protein inan unfolded state, and to prevent undesired interactions is clear.A large set of the protein chaperones are heat shock proteins, whose biologicalrole is to maintain and shield the unfolded state of newly synthesized proteins. Theirmain functions are (1). preventing proteins from misfolding or aggregating, (2).allowing proteins to traverse biological membranes, and (3). allowing them to foldproperly, thus leading to oligomerization. Under conditions of stress, chaperonesprotect other proteins from heat denaturation, or, once damage has occurred,disaggregate and allow them to refold back to an active form.Immunodominance and pathogenicity of HSPsHSPs are biologically complex proteins implicated not only in thermotolerancebut also in infection and autoimmunity (29, 50, 132). Studies have shown that 60kDaGroEL homologs from bacterial pathogens are major antigens, evoking both humoraland cellular immune response in infected hosts and immunized animals. A linkbetween stress protein synthesis and survival of bacterial pathogens within themammalian host during infection has been suggested by studies on Salmonellatyphimurium and Mycobacterium tuberculosis. Buchmeier and Heffron (14)5demonstrated that S. typhimurium DnaK and GroEL are among the most prominentproteins induced following entry into host macrophages in a tissue culture system,while Johnson et al. (49) reported that a periplasmic protease (HtrA) identified as aheat shock protein in E. coli plays an important role in in vivo survival andpathogenicity. Similarly, studies on the intracellular parasite Listeria monocytogeneshave led to the suggestion that factors important in host cell interactions may beinduced by heat shock (106).Since both DnaK and GroEL have 50% homology to their eucaryotic counterpartHSP70 and HSP60 at the amino acid sequence level and the fact that HSP60 andGroEL expression is increased in cells of both the host and pathogen during theprocess of infection (14, 51, 131), it has been hypothesized that these HSPs areinvolved in the development of autoimmunity. Evidence supporting this hypothesisincludes modulation of the immune response to GroEL homolog proteins fromMycobacterium tuberculosis and Chlamydia trachomatis, which resulted inimmunological damage in animal models (79, 129, 131).The HSP7O/DnaK FamilyThis class of proteins has been universally conserved, its members being at least50% identical to each other at the amino acid sequence level. In E. coli this class isrepresented by the single copy dnaK gene, whereas yeast possesses at least eightgene copies (22). Part of the reason for the abundance of gene copies in eukaryotesis the fact that some of their gene products are found exclusively in specializedorganelles such as the endoplasmic reticulum, mitochondria, and chloroplasts (22,30). The HSp70 proteins are the "workhorses" of the chaperones, not only becauseof their promiscuity in binding to other unfolded polypeptides (78) but also due totheir relative abundance in the various cellular compartments.The E. coli dnaK gene was originally discovered because mutations in it block X,DNA replication (39). subsequently it was found that DnaK performs almost6indispensable bacterial functions, since deletion of the gene can be tolerated onlywithin a narrow temperature range, and even under this condition, extragenicsuppressers accumulate very rapidly (15). HSP70 proteins have a weak ATPaseactivity, and the ATPase domain lies in the amino-terminal portion of the protein.DnaK can bind to other polypeptides, such as GrpE, XP, p53, staphylococcal Aprotein, and unfold bovine pancreatic trypsin inhibitor (19, 48, 58, 59, 135). Suchbinding is inhibited in the presence of ATP.The DnaJ and GrpE proteinsThe dnaJ and grpE genes of E. coli were originally discovered becausemutations in them block bacteriophage X growth. The dnaJ gene forms an operonwith the dnaK gene, the order being promoter-dnaK-dnaJ, whereas grpE mapselsewhere and is monocistronic. Bacterial homologues to the dnaJ gene have beendiscovered (39). The dnaJ and grpE proteins are absolutely essential forbacteriophage X DNA replication as well as bacteriophage P1 plasmid replication invivo and in vitro (1, 126, 136). The presence of both GrpE and DnaJ stimulatesDnaK's ATPase activity many fold. DnaJ specifically accelerates the hydrolysis step,and GrpE specifically stimulates the nucleotide release step. These results suggestthat one role of DnaJ and GrpE is to facilitate the intracellular recycling of DnaK (58).It is interesting that in some organisms the three genes form an operon, grpE-dnaK-dnaJ (124).The HSP60/GroEL familyThe HSP60/GroEL family of proteins has been widely conserved acrossevolution, although its members have been found only in bacteria, chloroplasts, andmitochondria. The groES and groEL genes of E. coli were originally discoveredbecause mutations in them block bacteriophage growth at the level of assembly ofthe dodecameric head-tail connector structure (134). The amino acid sequence andfunction of GroEL protein has been widely conserved, being approximately 50%7identical at the amino acid sequence level to the HSP60 proteins of eukaryotes (30).The GroES protein also appears to be universally conserved since its homologshave been found in mitochondria (65). The groES and groEL genes areindispensable for growth of E. coli at all temperatures and under all conditions tested(39). The groES and groEL genes form an operon expressed from both a a32 - and aan-dependent promoter, resulting in a substantial rate of transcription of the groEoperon, even in the absence of a 32 (45). The GroES and GroEL proteins functionallyinteract, leading to an inhibition of GroEL's ATPase activity (134). GroEL protein canbind various unfolded polypeptides in vitro. Although it is not clear how GroELrecognizes unfolded polypeptides, the binding appears to be both promiscuous andamino acid sequence-independent (56). The binding of unfolded polypeptides byGroEL promotes their correct assembly by preventing premature inter- orintramolecular interactions that can lead to aggregation. The GroEL/GroESinteraction is necessary for the release of some unfolded polypeptides bound toGroEL. Figure 2 depicts a proposed model of interaction between polypeptide,GroEL and GroES (adopted from (7)).Microbiology of Periodontal DiseaseThe periodontium consists of gingiva, periodontal ligament, root cementum, andalveolar bone (figure 3a). Diseases that affect the periodontium are collectivelycalled periodontal diseases (figure 3b, 3c).Various microorganisms can cause virtually all forms of inflammatory periodontaldiseases which can be broadly grouped into gingivitis and periodontitis, and eachcan be further divided into subgroups according to disease activity and severity, ageof onset, related systemic disorders, and other factors (figure 4).Gingivitis is inflammation of the gingiva, but it does not affect the attachmentapparatus of teeth. Periodontitis affects connective tissue attachment and adjacentalveolar bone. The classic progression of inflammatory periodontitis is characterized8recycleADP+PiATPG roEL,ATPADP+PiN^folded polypcvidc)1(Figure 2. A model for the action of the GroEL/GroES chaperonins.The GroEL chaperonin binds to many, but perhaps not all, unfolded polypeptides,some of which may still be nascent. The hydrolysis of ATP allows the release ofsome polypeptides. ATP hydrolysis coupled with the "cogwheel" action of GroESresults in release of the rest.CN^folded polypcptidcFigure 3a. The periodontium in health. A, periodontal ligament; B, alveolar bone; C,cementum; D, oral epithelium; E, sulcular epithelium; F, junctional epithelium; G,gingival sulcus; H, cementoenamel junction; I, tooth enamel; J, dental plaquemicroflora.Figure 3b. Established gingivitis lesion. A, inflammatory cell infiltrates in gingivalconnective tissues, sulcular epithelium, and junctional epithelium; B, gingival tissueswelling leading to increased gingival sulcus depth; C, junctional epithelium atcementoenamel junction; D, dental plaque microflora.Figure 3c. Periodontitis lesion. A, loss of connective tissue attachment; B, loss ofcrestal alveolar bone; C, apical migration of junctional epithelium; D, ulceration ofperiodontal pocket epithelium; E, inflammatory cell infiltrates in gingival connectivetissues, sulcular epithelium, and junctional epithelium; F, deepened periodontalpocket and pathogenic microbial flora; G, cementoenamel junction.Gingivitis— Gingivitis with no systemic influenceGingivitis with systemicsteroid hormone influence—Gingivitis with systemic diseaseinfluence{Chronic marginal gingivitisAcute necrotizing ulcerativegingivitis (ANUG)Pregnancy gingivitisOral contraceptive/steroidassociated gingivitisPuberty gingivitisE Acute leukemia-associated gingivitisHIV-associated gingivitisOthers— Adult periodontitis(after 21 years of age) —Periodontitis in youngindividuals i PrepubertalperiodontitisJuvenileperiodontitislocalizedgeneralized_E localizedgeneralizedPeriodontitis^_—Periodontitis withsystemic diseases— Periimplantitis•-- "Refractory" periodontitis— HIV-associated periodontitis— Diabetes mellitus(typel)Myelosuppressed cancer patients— Neutropenia—OthersFigure 4.^Microbe-associated periodontal diseases1 3by a highly reproducible microbiological transition of the subgingival microflora from amainly facultative Gram-positive microbiota to highly pathogenic Gram-negative rodsand motile organisms. Although at least 300 different microbial species have beenidentified from the oral region, it appears that only a very limited number of microbialspecies are involved in the destruction of periodontal tissues. Among these putativeperiodontal pathogens are members of the genera Porphyromonas, Fusobacterium,Wolinella, Actinobacillus, Capnocytophaga, and Eikenella. For example,Porphyromonas gingivalis has been implicated in chronic and advanced adultperiodontitis (100), Actinobacillus actinomycetemcomitans in localized juvenileperiodontitis (104, 133), Prevotella intermedius and Treponema denticola areinvolved in acute necrotizing ulcerative gingivitis (18, 63). Capnocytophaga spp.appear to play an important role in advanced periodontitis in juvenile diabetes (69).There is a growing body of evidence that Wolinella recta, Eikenella corroders, andBacteroides forsythus may play a role in the progression of periodontal disease (25,66, 67, 93, 112).The subgingival plaque microbiota is of such complexity that, at present, it is notclear whether these periodontal diseases are the result of a pathogenic synergy, orare the result of a monoinfection by an invading or opportunistic member of theresident oral microbiota. It is more likely that the development of periodontal diseaseinvolves a consortium of the plaque microbiota that interacts in a cooperative orsynergistic manner (42, 70, 114).Classification of black-pigmented oral anaerobic rods:Studies have shown that anaerobic, black-pigmented gram-negative rods formerlyknown as black-pigmented Bacteroides species are associated with destructiveperiodontitis (reviews by (100, 103, 122)). Since Oliver and Wherry (87) described asmall anaerobic gram-negative rod isolated from various parts of human body andnamed them as "Bacterium melaninogenicum", there have been many changes in the14taxonomy of this group of bacteria. These taxonomic changes have been extensivelyreviewed (71, 103, 121, 122). Since this group of black-pigmented gram-negativeanaerobic rods is very heterogeneous, comprising both saccharolytic andasaccharolytic species, new genera have been proposed: genus Porphyromonas forthe asaccharolytic species(94) and Prevotella for the saccharolytic species (95) , seeTable 1 (adopted from (120)).Pathogenicity of Porphyromonas gingivalisP. gingivalis has been repeatedly implicated in the establishment and progressionof periodontal diseases (100, 103, 122). White and Mayrand (125) demonstrated thatP. gingivalis was present in the gingival sulcus of patients with severe inflammation,but absent from healthy sites. P. gingivalis has been recovered not only fromsubgingival cultures of adults diagnosed as having generalized advancedperiodontitis (62, 99, 125), but also from adult patients who have actively progressingperiodontitis (101, 113). Di Murro and co-workers (24) found P. gingivalis to beconsistently associated with the subgingival microflora in patients with rapidlyprogressive periodontitis. P. gingivalis has also been suggested to be involved insevere, recurrent adult periodontitis (118) and both generalized and localizedjuvenile periodontitis (52, 62, 77, 127). Immunological studies provide additionalevidence of the involvement of P gingivalis in periodontal diseases. Patientsdiagnosed with adult periodontitis and generalized juvenile periodontitis have higherserum antibody levels against P. gingivalis than other groups of individuals (27, 32).Studies also showed elevated antibody levels to P. gingivalis in gingival crevicularfluid of patients with periodontitis (26, 115). The pathogenicity of P. gingivalis has alsobeen demonstrated in animal model infection studies (37, 38, 43, 44, 111, 119).15Table 1. Nomenclature of black-pigmented anaerobic rodsFormer designation^ New designationBlack-pigmented Bacteroides^Black-pigmented anaerobic rodsB. gingivalis^ Porphyromonas gingivalisB. asaccharolyticus^ Porphyromonas asaccharolyticusB. endodontalis Porphyromonas endodontalisB. salivosus^ Possibly related to PorphyromonasB. macacae Possibly related to PorphyromonasB. levii^Possibly related to PorphyromonasB. intermedius^ Prevotella intermediaB. corporis Prevotella corporisB. melaninogenicus^ Prevotella melaninogenicaB. denticola^ Prevotella denticolaB. loescheii Prevotella loescheii16Virulence factors of P. gingivalisA variety of putative pathogenic factors of P. gingivalis has been identified whichmay contribute to the colonization and virulence of this oral pathogen. These factorshave been shown to have wide-ranging effects on tissue comprising the periodontiumas well as on host immune mechanisms. These determinants include the ability toadhere to other bacterial species or epithelial surfaces, the ability to invade hosttissues, the production of toxins or enzymes which are destructive to the host tissue,as well as the bacterial surface components such as lipopolysaccharides or capsularmaterial which can induce infection or protect the bacteria from host immuneresponses, see Table 2 (adapted from (120)). These studies have usually beenconducted in vitro and the possible virulence remains to be determined in vivo.Aim of this studyPorphyromonas gingivalis has been implicated in the etiology of adultperiodontitis (102, 105). The virulence of P. gingivalis is associated with its ability toevade host defenses and produce a range of cytotoxins and enzymes with tissuedamaging potential (102). The expression of such virulence components may begreatly influenced by the environmental conditions in the periodontal pocket. Studieshave shown that expression of many virulence factors of P. gingivalis such as outermembrane proteins (11-13, 88), fimbriae, capsule, outer membrane vesicles (44, 74),and proteolytic activity (68, 73, 92) are regulated by the environmental conditionssuch as pH change, hemin-limitation and in vivo infections. It has been shown that inperiodontitis, the subgingival temperature was in general higher at diseased sitesthan at healthy sites (54), the pH in the periodontal pocket increases with its depthand also with the severity of the inflammatory host response (10). Phagocytosis ofbacteria by polymorphonuclear leukocytes (PMNLs) is accompanied byenhancement of PMNL oxidative metabolism (2, 76). All these17Table 2. Virulence factors of P. gingivalisFactors^ Possible effectfimbriaeouter membrane vesiclescolonizationactivate host immune responsecolonizationcolonizationresistance to phagocytosisinhibits growth of fibroblasts,induces bone resorption,inhibits bone collagen formation,activates inflammatory responseproteolytic and collagenolytic activities,hemagglutinate erythrocytes,promote adherence betweennoncoaggregating species,impede host immune defencedegradation of host tissuecollaborate with collagenase in tissuedestruction,degrade complement, immunoglobulinand serum Fe-binding proteinshemagglutininother surface binding moleculescapsulelipopolysaccharidecollagenaseproteasesbutyric addproprionic acidindole and ammoniavolatile sulphur compoundscytotoxiccytotoxicinhibition of matrix formationcytotoxiccytotoxic 18situations could inflict a heat shock and/or other stress on P. gingivalis. Very little isknown about the regulation of the heat shock response in obligate anaerobicbacteria. The heat shock response studies conducted in Clostridium acetobulylicum(81, 82) and several Spirochetes (107) have suggested the regulation of theexpression of some major heat shock proteins in strict anaerobes is different fromthat of E. coli. I therefore investigated the heat shock response and some other stressresponses of P. gingivalis as a convenient model for studying environmentallyregulated gene expression and anticipate that amongst the proteins induced by heatshock or other stresses we will be able to find some stress responding proteinswhich are also involved in the process of infection.19MATERIALS AND METHODSBacterial strains and culture conditionsP. gingivalis W50 and W83 were grown in peptone-yeast medium which contains1.7% trypticase peptone, 0.3% yeast extract, 0.25% K2HPO4, 0.5% NaCI, 1µg/mlvitamin K and 511.g/m1 hemin. The cultures were incubated in a Coy anaerobicchamber in a 5%CO2-10%H2-85%N2 atmosphere at 37°C. Recombinant E. colistrain MC4100 (kindly provided by Dr. McCarty (72)) contains plasmid pBB1 that hasbeen constructed by inserting a BamHl fragment containing E. coli dnaK/J genes intopBR322. Recombinant E. coli strain with plasmid pOF39 which contains an E. coligroEL/ES gene insert was kindly provided by Dr. Fayet (33). All recombinant E. colistrains were maintained in LB broth (1% tryptone, 0.5% yeast extract, 1% NaCI,pH7.2) with 10014/m1 ampicillin.Purification of E. coil recombinant GroEL proteinA single colony of the recombinant E. coli strain containing plasmid pOF39 wasinoculated into a 10m1 LB broth with 1004/m1 ampicillin. The culture was incubatedat 37°C overnight with vigorous shaking. 10m1 of this culture was inoculated into 1liter of LB broth with 20014/ml ampicillin and incubated at 37°C with vigorous shakingfor 24 hours. The bacterial culture was centrifuged in a Beckman JA-10 rotor at7,000rpm for 10 minutes at 0°C, the cell pellet was washed twice in 100m1 of10mMTris-HCI, pH7.5, resuspended in 50m1 10mMTris-HCI (pH7.5) with 0.02mg/m1DNAse A, 0.02mg/m1RNAse I, 10mMMgC12. The cell suspension was then sonicated(discontinuous sonication, five 3-minute periods, with cooling in an ice bath, and 2-minute resting periods between each sonication; 40% duty cycle, output 3, Sonifier,Cell Disrupter 350; Branson Sonic Power Co.). The sonicated mixture wascentrifuged for 20 minutes at 8,000xg. Unbroken cells and large cell debris werediscarded and the supernatant was further centrifuged for 2 hours at 100,000xg.20The semi-transparent, brownish colored pellet containing most of membranefraction was discarded and the supernatant was subjected to ammonium sulfateprecipitation. Ammonium sulfate precipitates were taken for 0-20%, 20-30%, 30-40%,40-50% and 50-60% saturation. Precipitates were resuspended in 10mM Tris-HCI,pH7.5 and the protein profile of each sample was analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The fractions containingmost of the GroEL protein were pooled, dialyzed against 4 liters of 10mM Tris-HCI(pH7.5), 50mM NaCI for 48 hours. The dialyzed solution containing GroEL and otherproteins was loaded onto a 25m1 DEAE-Sepharose column equilibrated with thesame buffer. The column was first washed with 5 bed-volumes of 10mM Tris-HCI,50mM NaCI (pH7.5) and then a salt gradient of 50mM to 0.5M NaCI. All fractions werecollected by an Automated Econo System (Bio-Rad) and the protein profile of allfractions was determined on a SDS-PAGE gel. The fractions containing most of theGroEL protein were pooled, dialyzed against 4 liters of 10mM Tris-HCI (pH7.5), and1/3 was loaded onto a 1m1 pre-equilibrated Mono() ion-exchange column connectedto an FPLC unit (Pharmacia Fine Chemicals AB.). The column was washed with10mM Tris-HCI (pH7.5) and a 50mM to 0.5M NaCI salt gradient. Fractions werecollected and protein contents were checked on SDS-PAGE. Fractions containingGroEL were pooled, dialyzed against 4 liters of 10mM Tris-HCI (pH7.5) and preparedfor immunization (see Immunological procedures).Purification of E. coli recombinant DnaK proteinAmmonium sulfate precipitates of recombinant E. coli strain MC4100 wereprepared the same way as described above. The precipitate fractions containing mostof the DnaK protein were pooled, dialyzed against 4 liters of 20mM Tris-HCI, pH7.5for 48 hours. The dialyzed sample was then loaded onto a 5 ml ATP-agarose affinitycolumn (C-8 linkage, Sigma A-2767) equilibrated with the same buffer. The columnwas washed with 20mM Tris-HCI and 0.5M NaCI. After re-equilibration of the column21with 20mM Tris-HCI (pH7.5), the DnaK protein was eluted with 10mM ATP in 20mMTris-HCI (pH7.5). Sample fractions were checked on SDS-PAGE and the fractionscontaining DnaK were pooled and dialyzed against 10mM Tris-HCI (pH7.5).Protein determinationThe amount of protein in the purified fractions and in the whole cell lysates wasdetermined according to the Bradford (Bio-Rad) protein assay. A standard proteinconcentration curve was prepared by using bovine serum albumin (BSA) as thestandard. Aliquots of diluted standard solution of BSA, containing 0 to 2014 of protein,were reacted with 0.2m1 of the Bio-Rad color reagent and the absorbance measuredat A595.Immunological proceduresRabbit antiserum was raised against purified GroEL as described below: 40111(4014) of purified GroEL protein was mixed with equal volume of Hunter's TiterMaxTMResearch Adjuvant (CytRXR Co.) and injected intramuscularly into a New Zealandwhite rabbit. The rabbit was given a 1 mg purified GroEL injection every two week forfour weeks. One week before collecting blood, the rabbit was given a booster of 1 mgpurified GroEL protein.Rabbit anti-E. coli DnaK polyclonal antibodies provided by Dr. McCarty(Massachusetts Institute of Technology, Boston) were used in all the immunoblotstudies.Affinity purification of anti-GroEL antibodiesAffi-Gel 10 affinity support (Bio-Rad) was cross-linked to purified GroEL proteinaccording to the procedures described by Formosa et al. (34). Briefly, 20mg ofpurified GroEL protein was dialyzed against deionized water to remove Tris-HCI (Trisinterferes with ligand coupling reactions) and then mixed with 2m1 of Affi-Gel 10 withgentle agitation of the gel slurry for four hours at 4°C. 0.2m1 of 1M ethanolamine-HCI(pH8) was added to block any active esters which might remain. The GroEL protein22was bound with an efficiency of approximately 85%. Serum from the rabbitimmunized with GroEL protein was passed through the affinity column which hadbeen equilibrated with deionized water. The column was washed with 5 bed volumesof water and 5 bed volumes of 2M urea, and then the anti-GroEL antibody bound tothe column was eluted with 8M urea. The specific activity of the purified anti-GroELantibodies was checked by an immunoblot dot assay on an Easy-TiterTm ELIFASystem (PIERCE) as directed by the manufacturer. Anti-GroEL activity wasdetermined by measuring the absorbance of the colored reaction products at 495nmin a microplate reader (Model 3550, Bio-Rad).Heat shock and other stress responseFor heat shock experiments, P. gingivalis cultures were incubated in a Coyanaerobic chamber in a 5%CO2-10%H2-85%N2 atmosphere at 37 °C until theyreached the mid-exponential phase of growth. Sets of 1 ml samples were transferredto sterile glass tubes under anaerobic conditions and the tubes sealed with rubberstoppers. The samples were incubated in a water bath at 37 °C for 10 minutes beforethey were shifted to 42 °C. A control sample remained at 37°C. 1 min after thetemperature shift, 20.tCi of ( 14C)-amino acid mix (Amersham, 273mCi/mMol) wasinjected into each of the tubes and the incubation was continued for 4 hours. For thetime course study of the heat shock response, 204Ci of ( 14 C)-amino acids wasinjected into each culture tubes and incubated at 1-5 minute, 5-10 minute, 10-20minute, and 20-60 minutes periods after the temperature shift to 42°C. The sampleswere then chilled in ice and the cells were pelleted by centrifugation, washed 3 timesin ice-cold phosphate-buffered saline, resuspended in protease inhibitor TLCK (finalconcentration 20mM) and stored at -20 °C.P. gingivalis response to other stresses such as oxidative stress (H202), KNO3,ethanol and high or low pH was similarly analyzed by radio-labeling for 4 hours aftermid-exponential growth phase cultures were exposed to the stress conditions. For23pH changes, 1N HCI or 1N NaOH were added to the cultures (pH7.3) until the pHreached 5.0 or 8.3 respectively. After each experiment, viable cells were counted byplating samples on blood agar plates. All heat shock and other stress experimentswere repeated three times. The amount of radio-labeling incorporated in eachsample was determined by a Beckman LS7500 Liquid Scintillation Counter(Beckman Instruments, Inc.)Electrophoretic analysisFor SDS-PAGE, bacterial extracts or purified protein samples were mixed with anequal volume of sample buffer (55) and incubated in a boiling water bath for 10minutes. Proteins were separated on 12% (wt/vol.) polyacrylamide resolving gelswith 4% (wt/vol.) polyacrylamide stacking gels by the method of Ames (4).Electrophoresis was carried out at room temperature at a constant 200V until the dyefront reached the bottom of the gels. The gels were stained with Coomassie BrilliantBlue or silver nitrate (86). For radio-labeled samples, the gels were stained inCoomassie Brilliant Blue, treated with fluorography amplification reagent(Amersham) and dried at 80°C under vacuum before exposure to Kodak X-Omat filmat -70°C.For two-dimensional PAGE, bacterial pellets were boiled in 5% Nonidet P-40 for5 minutes before solubilization in lysis buffer containing urea and Nonidet P-40 asdescribed by O'Farrell (85). Samples were separated initially by isoelectric focusingin mini-tube gels containing 4% ampholytes in the pH range of 5 to 8 and 1%ampholytes in the pH range of 3 to 10, and then by SDS-PAGE using 12%separation gels as described in the Bio-Rad mini-2D instruction manual.Western ImmunoblottingP. gingivalis W50 whole cell lysate proteins separated by SDS-PAGE or two-dimensional PAGE were transferred to nitrocellulose membranes in 25mM Tris-HCI,192mM glycine and 20% methanol buffer (pH8.3). Transfer was carried out first at2425V overnight then at 60V for 2 hours in a Bio-Rad Trans-Blot Cell. After blocking theunreactive sites with bovine serum albumin, the sheets were washed twice withTTBS (20mM Tris, 500mM NaCI, 0.05% Tween-20) and then incubated with rabbitantibodies against E. coli DnaK and GroEL. The unbound antibodies were removedby washing with TTBS. The sheets were then incubated with goat anti-rabbit IgGcoupled to alkaline phosphatase for one hour. Antigen-antibody reaction bands werevisualized by following the procedures described in the Bio-Rad technical bulletinsupplied with the Bio-Rad lmmuno-Blot (GAR-AP) assay kit.Preparation of gene probesAlkaline lysis miniprep of plasmid pBB1 and pOF39 was conducted as described(9). 5[II (214) of plasmid pBB1 DNA was mixed with 10U of BamHl and the reactionmixture was incubated at 37°C for 1 hour. Digested DNA fragments were separatedon a 0.8% agarose gel at 60 volts. The DNA bands were visualized by staining thegel in 0.51.1g/m1 ethidium bromide. pBB1 was digested by BamHl to 4 and 8kbfragments. The 8kb band which contained dnaK/J genes was cut out in a narrow stripof gel and the DNA was extracted from the gel following the QIAEX agarose gelextraction protocol (QIAGEN, Germany). The purified 8kb BamHl-BamHl internalfragment of pBB1 was labeled with biotin-14-dATP in the presence of DNAPolymerase I and DNAse I as described in the standard nick translation protocols(BluGENETm, Nonradioactive Nucleic Detection System, BRL). The 2kb EcoRl-Smalinternal fragment of plasmid pOF39 which contained the groEL/ES genes wassimilarly isolated and labeled with biotin-14-dATP.Southern hybridizationChromosomal DNA from P. gingivalis W50, W83, ATCC33277, W12, P.asaccharolyticus, Prevotella corporis, Prevotella denticola, Prevotella intermedius,Bacteroides levii, Prevotella loescheii, and Prevotella melaninogenicus was isolatedby the method of Ausubel (9). EcoR/-digested chromosomal DNA from the above25strains was separated by 0.8% agarose gel electrophoresis and transferred tonitrocellulose filters. The filters were baked at 80°C for 2 hours. After prehybridizationfor four hours at 42°C in 6X SSC (0.9M sodium acetate, 0.09M sodium citrate, pH7.0)containing 100µg/ml denatured sperm DNA, 0.5% SDS, 0.2% Denhardt's solution,the bound DNA was hybridized to the labeled probes at 55°C in 6X SSC, 100µg/mldenatured sperm DNA, 0.5% SDS, 0.2% Denhardt's solution, 254/m1 probe DNAfor 18 hours.Probes used were biotin-dATP-labeled BamHI-BamHI internal fragment ofplasmid pBB1 which contained the cloned E. coli dnaK/J genes or biotin-dATP-labeled EcoRl-Smal internal fragment of plasmid pOF39 which contained the clonedE. coli groEUES genes. The membranes were washed twice at room temperature in2X SSC buffer containing 0.1 % SDS for 3 minutes each followed by washing twicein 0.16X SSC-0.1% SDS at 55°C for 15 minutes each. The membranes wereincubated for 1 hour at 55°C in 3% BSA in 0.1M Tris-HCI, 0.15M NaCI (pH7.5) andthen reacted with streptavidin-alkaline phosphatase (SA-AP) conjugate for 20minutes. After washing in 0.1 M Tris-HCI buffer (pH7.5) for 30 min, the membraneswere incubated in nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolylphosphate (BCIP). The incubations were carried out at room temperature untilreactive bands appeared. Hybridization bands were detected by the BIuGENENonradioactive Nucleic Acid Detection System (BIuGENETM, BRL, Gaithersburg,MD). The reactions were stopped by washing the membranes in 20mM Tris-0.5mMNa2EDTA , pH7.5.In vivo infection studiesP. gingivalis W50 infections of guinea-pigs were conducted as described byGrenier and McBride (44). Briefly, P. gingivalis cells were cultivated to lateexponential growth phase and the cells were harvested by centrifugation. Thebacteria were resuspended in 0.05% sodium thioglycolate to a concentration of262x10 11 cells per ml. The bacterial suspension (0.5m1) was then injectedsubcutaneously into the abdomen region of each of two Hartley guinea-pigs. Afterthe development of a spreading infection, exudates (approximately 45 mls) from bothanimals were collected, 0.5m1 was injected into a second set of animals andexudates from the second animals were also collected. Exudates from the first andsecond infections were centrifuged twice at low speed (1,000xg for 5 min) to removered blood cells and leukocytes. The bacterial cells were then pelleted at 8,000xg for10 min, washed once in ice cold saline, resuspended in 20mM TLCK and stored at-20°C.27RESULTSPurification of GroEL and DnaK proteinsThe recombinant E. coli strain containing plasmid pOF39 overexpresses GroEL(apparent molecular weight 58kDa) and GroES (apparent molecular weight 16kDa)proteins at 37°C (Figure 5, lane 7). High concentrations of ampicillin (2004g/m1)which prevents the cells from losing plasmid pOF39 also enhances the yield ofGroEL protein. After ammonium sulfate precipitation, most of the GroEL protein wasfound in the 20-30% and 30-40% precipitates. These two fractions were pooled,dialyzed, and loaded on a pre-equilibrated DEAE-Sepharose column and elutedwith a gradient of 50mM-0.5M NaCI. Figure 6 shows several peaks were eluted fromthe DEAE-Sepharose column. SDS-PAGE analysis of the fractions revealed thatfractions eluting between 0.2-0.3M NaCI contained most of the GroEL protein. Thefractions containing most of the GroEL protein were pooled (Figure 5, lane 6),dialyzed, and 1/3 of the material was chromatographed on a Mono() column. Onemajor peak was eluted from the column as shown in Figure 7. SDS-PAGE analysisrevealed that this peak fraction contained highly purified GroEL protein (Figure 5,lane 5).Recombinant E. coli strain MC4100 expresses DnaK protein at a low level in thepresence of 2004/mlampicillin (Figure 5, lane 2), and heat shock does not enhancethe production of DnaK very much (data not shown). Most of the DnaK protein waspresent in the 0-20% and 20-30% ammonium sulfate precipitates. After dialysis, thesamples were chromatographed on an ATP-agarose column. Figure 8 shows thatseveral peaks were washed off the column by salt, the 69kDa DnaK protein waseluted by 10mM ATP as determined on SDS-PAGE (Figure 5. Lane 1). Becausesome other proteins also have affinity for ATP, we did not obtain a high purity DnaKsample. In a separate experiment, the DnaK fraction eluted by ATP was dialysed andchromatographed on a DEAE-Sepharose column. The DnaK protein was separated28Figure 5. Purification of GroEL and DnaK proteins.Protein samples were separated by SDS-PAGE and silver stained. Lanes: 1,partially purified DnaK (69kDa) protein after elution from an ATP-agarose column; 2,whole cell lysate of recombinant E. coli strain MC4100; 3, partially purified GroESprotein after elution from a MonoQ ion-exchange column; 4, partially purified GroESprotein after elution from a DEAE-Sepharose column; 5, purified GroEL proteineluted from a MonoQ ion-exchange column; 6, partially purified GroEL protein elutedfrom a DEAE-Sepharose column; 7, whole cell lysate of recombinant E. coli strainover-expressing GroEL, GroES proteins; 8, molecular weight markers. Molecularweight standards are indicated on the left in kilodaltons.1^2^3^4^5^6^7^866454.00,24^ 0.40.011.110•••361814 11111.••■•29Time(min)Figure 6. Chromatography of GroEL on DEAE-Sepharose.The column was eluted with a linear gradient of0.05-0.5M NaCI.30—4-- A2800^ NaCl(M)0^te-)^0^In^0^in,-, — C`I N0.6  0.5 —0.4 —0.3 —0.2 —0.1 —0.6—0.5—0.4—0.3—0.2—0.10 00M000Time(min)Figure 7. Chromatography of GroEL protein on a MonoQ column.Pooled and dialysed GroEL containing fractions fromDEAE-Sepharose chromatography were applied to a MonoQ columnand eluted with a gradient of 0.05-0.5M NaCI.31Fraction#Figure 8. Affinity chromatography of DnaK on anATP-agarose column. The 0-20% and 20-30% ammoniumsulphate precipitates of recombinant E. coil MC4100were dialysed and loaded on an ATP-agarose column. Thecolumn was first washed with a gradient of 0.05-0.5MNaCI. The DnaK protein bound to the column was elutedwith 10mM ATP. The high value of A280 after adding10mM ATP is due to the high absorbance of ATP at280nm.32from most of the other contaminating proteins. Due to the low yield of the DnaKprotein, we did not use this DnaK preparation to generate antibodies in animals.Affinity purification of anti-GroEL antibodiesSerum obtained from the rabbit immunized with purified E. coli GroEL protein hada high reactivity against a large number of P. gingivalis proteins. The serum wasaffinity purified by passing through an Affi-gel 10 column coupled with purified GroELprotein (Figure 9). Most of the anti-GroEL activity was found in the 8M urea eluate asdetermined by an immunoblot dot assay.Heat shock response of P. gingivalisWhen P. gingiva/is W50 cells were shifted from 37°C to 42°C and metabolicallylabeled with ( 14C)-amino acids, SDS-PAGE autoradiographic analysis of whole celllysates revealed elevated synthesis of five proteins with the apparent molecularweights of 92, 80, 74, 62, and 45kDa, a 50 and a 19 kDa protein diminished duringheat shock (Figure 10). Two-dimensional PAGE analysis of labeled proteins allowedbetter resolution of these heat responding proteins. Elevated synthesis of the 92, 80,74 and 62kDa proteins and decreased synthesis of the 50kDa protein were alsoobserved on 2D-PAGE (Figure 11, 37°C and 42°C). A 12kDa protein wasprominently induced and some other low-molecular weight proteins increased ordecreased during heat shock.Time course study of the heat shock response revealed that the 62kDa proteinwas induced within 5 minutes of temperature upshift and other heat shock proteinscould be seen within one hour of heat shock (Figure 12).33TIME(MIN)Figure 9. Affinity purification of anti-GroEL antibody on anAffi-gel 10 column coupled with purified GroEL.The column was washed with deionized water and 2M and 8Murea. Protein concentration was measured at A280. Anti-GroELactivity was determined by an ELIFA immunoblot dot assay andthe absorbance of the colored reaction product was measured atA495.34Figure 10. SDS-PAGE autoradiogram of the heat shock response of P. gingivalis.Mid-log phase P. gingivalis W50 cells were shifted from 37°C to 42°C andmetabolically labeled with ( 14 C)-amino acids followed by SDS-PAGE andautoradiography. Lanes: 1. P. gingivalis cells labeled at 37°C; 2. P. gingivalis cellslabeled at 42°C. Heat responding protein bands are indicated by arrows on the right.Molecular weight standards are indicated on the left in kilodaltons.35Figure 11. Two-dimensional PAGE autoradiogram of the heat shock response of P.gingivalis.Cell extracts from P. gingivalis W50 labeled with ( 14C)-amino acids at 37°C or42°C were analyzed by 2D-PAGE and autoradiography. The heat inducible proteinsare indicated by proteins decreased during heat shock are indicated by (1).Molecular weight standards are indicated on the left in kilodaltons.37Figure 12. Time course study of the heat shock response of P. gingivalis.P. gingivalis W83 cells were labeled with (14C)-amino acids for different timeintervals after heat shock, cell extracts were analyzed by SDS-PAGEautoradiography. Lanes: 1. control cells grown at 37°C radio-labeled for 4 hours; 2.cells radio-labeled at 1-5 min after heat shock at 42°C; 3. cells radio-labeled at 5-10min after heat shock; 4. cells labeled at 10-20 min after heat shock; 5. cells radio-labeled at 20-60 min after heat shock.38In order to identify some of the heat shock proteins, antibodies against E. coliDnaK and GroEL were applied in Western blots of heat shocked and control P.gingivalis whole cell lysates transferred from SDS-PAGE onto nitrocellulose filters.The 74kDa and the 62kDa proteins were recognized by anti-DnaK and anti-GroELantibodies respectively, therefore, they were identified as homologs of DnaK andGroEL (Figure 13, lane 1 and 2). On two-dimensional Western blots, two proteinspots with the apparent molecular weight of 74kDa reacted with anti-DnaK antibodyand at least four adjacent protein spots of 62kDa reacted with anti-GroEL antibodyboth before and after heat shock (Figure 14).Pulse-chase labeling study of the 50 kDa proteinIn order to determine whether the decreased radio-labeling of the 50 kDa proteinduring heat shock was due to specific degradation by proteases or due to decreasedsynthesis of this protein after heat shock, we pulse radio-labeled P. gingivalis W50cells at 37°C for 4 hours and then chased without labeling at 42°C for 2 hours (Figure15, lane 2). The level of radio-labeled 50kDa protein was similar to the control whichhad not been subjected to heat shock (Figure 15, lane 1). When P. gingivalis cellswere radio-labeled for 2 hours at 37°C and then shifted to 42°C and labeled foranother 2 hours, the intensity of radio-labeling of this 50 kDa protein band decreased(Figure 15, lane 3) when compared to the control. These results suggested that the50kDa protein synthesis was down-regulated by heat shock.Southern hybridization with heat shock gene probesTo determine whether dnaK and groEL gene homologs were conserved, EcoRl-digested genomic DNA from P. gingivalis W50, W83, ATCC33277, W12, P.asaccharolyticus, Prevotella corporis, Prevotella denticola, Prevotella intermedius,Bacteroides levii, Prevotella loescheii, and Prevotella melaninogenicus wereseparated on agarose gels, transferred to nitrocellulose filters and hybridized withbiotin-dATP-labeled E. coli dnaK/J or groEL/ES gene probes under conditions of low39Figure 13. Western blot of P. gingivalis cells treated with different stress stimuli.Solubilized cellular extracts from equal numbers of P. gingivalis W50 cells grownunder different stress conditions were separated by SDS-PAGE, transferred tonitrocellulose membrane, and incubated with a mixture of anti-DnaK and anti-GroELantibodies. Lanes: 1. control cells grown at 37°C; 2. cells heat shocked at 42°C; 3.cells treated with 3011M H202; 4. cells treated with 4% ethanol; 5. cells treated with10mM KNO3; 6. cells grown at pH5; 7. cells grown at pH8.3. The position of DnaKand GroEL homolog proteins are indicated by arrows on the right. Molecular weightstandards are indicated on the left in kilodaltons.1^2^3^4^5^6^7109724629181540Figure 14. Two-dimensional Western blots P. gingivalis before and after heat shock.Cellular extracts from equal numbers of P. gingivalis W50 cells grown at 37°C orheat shocked at 42°C were separated by 2D-PAGE, transferred to nitrocellulosemembranes and incubated with a mixture of anti-DnaK and anti-GroEL antibodies.The position of DnaK and GroEL homolog proteins are indicated by arrows on theright. Molecular weight standards are indicated on the left in kilodaltons.4142Figure 15. Pulse-chase labeling study of the 50 kDa protein.Lanes: 1. P. gingivalis W50 cells radio-labeled at 37°C for 4 hours; 2. Cells wereradio-labeled at 37°C for 4 hours and then chased without labeling at 42°C for 2hours; 3. Cells were radio-labeled at 37°C for 2 hours then shifted to 42°C and radio-labeled for 2 hours; 4. Cells radio-labeled at 42°C for 4 hours.43stringency. The E. coli dnaK/J probe hybridized to a discrete fragment in each of theP. gingivalis strains and all the black-pigmented Bacteroides strains tested(Figure16). The E. coli groEUES probe hybridized to a discrete DNA band in all the P.gingivalis strains and most of the black-pigmented Bacteroides strains except P.asaccharolyticus and Prevotella denticola(Figure 17). Among P. gingivalis strains,both dnaK/J and groEL/ES probes hybridized to similar size DNA fragments instrains W50, W83 and W12, but hybridized to much larger DNA fragments in strainATCC33277. Hybridization to heterogeneous DNA fragments was also observedamong the other black-pigmented Bacteroides strains.Effect of different stress stimuli on P. gingivalisSince we are interested in the heat shock proteins that may be induced under invivo conditions, we studied P. gingivalis response to stress stimuli such as H202,KNO3, high or low pH, and ethanol. The effect of stress stimuli on the viability of P.gingivalis W50 cells was studied by incubating P. gingivalis cells in different stressconditions for 4 hours and viable cells were determined by plating on blood-agarplates. The results are shown in Table 3. Growth at 42°C did not have any effect onthe viability of P. gingivalis cells. When the temperature increased to 45°C, morethan half of the cells lost their viability. 45% and 39% of P. gingivalis cells survived in4% ethanol and 30mM H202 respectively, but incubation of cells in 8% ethanol or600 H202 resulted in lost of most of the viable cells. 90% and 57% of the cellssurvived in 10mM and 20mM KNO3 respectively. About 20% and 50% of the cellsretained their viability when the culture pH was changed to pH5 and pH8.3respectively.44Figure 16. Southern hybridization of P. gingivalis and other black-pigmentedBacteroides chromosomal DNA with an E. coli dnaKIJ probe.Chromosomal DNA was digested to completion with EcoRl, electrophoresed onan agarose gel, and transferred to a nitrocellulose filter. Hybridization was performedat low stringency with a biotin-dATP-labeled BamHI-BamHI internal fragment ofplasmid pBB1 which contained the cloned dnaK/J genes. Lanes: 1. P. gingivalisW50; 2. P. gingivalis W83; 3. P. gingivalis ATCC33277; 4. P. gingivalis W12; 5. P.asaccharolyticus; 6. Prevotella corporis; 7. Prevotella denticola; 8. Prevotellaintermedius; 9. Bacteroides levii; 10. Prevotella loescheii; 11. Prevotellamelaninogenicus; 12. E. coliJM83; 13. plasmid pBB1.45Figure 17. Southern hybridization of P. gingivalis and a number of black-pigmentedBacteroides chromosomal DNA with an E. coli groEL/ES probe.Chromosomal DNA was digested to completion with EcoRl, electrophoresed onan agarose gel, and transferred to a nitrocellulose filter. Hybridization was performedat low stringency with a biotin-dATP-labeled EcoRI-Smal internal fragment ofplasmid pOF39 which contained the cloned groEL/ES genes. Lanes: 1. P. gingivalisW50; 2. P. gingivalis W83; 3. P. gingivalis ATCC33277; 4. P. gingivalis W12; 5. P.asaccharolyticus; 6. Prevotella corporis; 7. Prevotella denticola; 8. Prevotellaintermedius; 9. Bacteroides levii; 10. Prevotella loescheii; 11. Prevotellamelaninogenicus; 12. E. coli JM83; 13. plasmid pOF39.46Table 3. Viability of P.^gingivalis W50 cells exposed^to differentstress conditions.Stress condition Average number of viable cells/m la (%viability)bControl, no stress 2.30x109 (100)Heat shock at 42°C 2.50x109 (109)Heat shock at 45°C 1.03x109 (45)4% ethanol 1.02x109 (45)8% ethanol 0.06x109 (2.8)3011M H202 0.90x109 (39)60 uM H202 0.05x109 (2.2)10 mM KNO3 2.10x109 (90)20 mM KNO3 1.30x109 (57)pH 5 0.43x109 (19)pH8.3 1.10x109 (48)a. Average after three independent experiments. b. Control cells viability isexpressed as 100%.47Western immunoblots of P. gingivalis whole cell lysates with anti-DnaK and anti-GroEL antibodies have shown that both DnaK and GroEL homolog proteins areinduced by 4% ethanol (Figure 13, lane 4). Whereas 3011M H202 (Figure 13, lane 3),10mM KNO3 (Figure 13, lane 5) and pH8.3 (Figure 13, lane 7) did not induce GroELor DnaK. Because these HSPs are expressed under all growth conditions, threeindependent stress response experiments were conducted and the same resultswere obtained except variations of the expression of GroEL and DnaK homologproteins were observed in P. gingivalis cells grown at pH5 as checked by Westernblots. Two experiments showed these heat shock proteins were likely to be inducedby pH5 (Figure 13, lane 6), the other experiment did not show a discernible inductionof DnaK or GroEL homolog proteins (data not shown).SDS-PAGE autoradiographic analysis of the stress responses of P. gingivalis isshown in Figure 18 and Figure 19. The extent of radio-label incorporated into DnaKand GroEL homolog proteins was measured by densitometry as shown in Table 4.From the autoradiograph and densitometry analysis, it is clear that both DnaK andGroEL homolog proteins are induced by 4% ethanol (Figure 18, lane 4), nodiscernible induction of these HSPs was observed in 300 H202 (Figure 18, lane3), 10mM KNO3 (Figure 18, lane 5), pH5 (Figure 19, lane 2) and pH8.3 (Figure 19,lane 3). A 68kDa and a 60kDa protein was induced by 4% ethanol (Figure 18, lane4).One interesting observation was that when P. gingivalis cells were grown at pH5,the cell pellet had a much darker color than the control cells. SDS-PAGE analysisrevealed that two new proteins with the apparent molecular weight of 31 and 26kDawere induced at pH5 (Figure 19, lane 2).48Figure 18. SDS-PAGE autoradiogram of P gingivalis grown under different stressconditions.Solubilized cellular extracts from equal numbers of P. gingivalis W50 cells grownunder different stress conditions and labeled with ( 14C)-amino acids were separatedby SDS-PAGE, and autoradiographed. Lanes: 1. control cells grown at 37°C; 2. cellsheat shocked at 42°C; 3. cells treated with 300 H202; 4. cells treated with 4%ethanol; 5. cells treated with 10mM KNO3. The position of DnaK and GroEL homologproteins of 74 and 62kDa respectively are indicated by arrows on the right. Molecularweight standards are indicated on the left in kilodaltons.49Figure 19. SDS-PAGE autoradiogram of P. gingivalis grown at different pH values.Lanes: 1. control cells grown at 37°C, pH7; 2. cells grown at pH5; 3. cells grownat pH8.3. The position of DnaK and GroEL homolog proteins of 74 and 62kDarespectively are indicated by arrows on the right. Molecular weight standards areindicated on the left in kilodaltons.50Table 4. Induction of GroEL and Dnak homolog proteins by differentstress stimulia% incorporation in test sample/% incorporation incontrolstimulus^62 kDa (GroEL)^74 kDa (DnaK)42°C^ 7.8^ 2.33011M H202 1.2 1.34% Et0H^3.5^ 3.110mM KNO3 0.85 0.9pH5^ 1.6^ 1.4pH8.3 1.1 0.8a Labeling of DnaK and GroEL homolog proteins was estimated as a percentageof incorporation into total protein by densitometer scanning of SDS-PAGE as shownin Figure 18 and 19. Results are expressed as the percentage of incorporation in thetest sample divided by the percentage of incorporation into the same proteins in acontrol without addition of stress stimuli.51In vivo infectionThe subcutaneous injection of P. gingivalis W50 in guinea-pigs resulted in anacute infection throughout the abdominal region within 36 hours. The guinea-pigslost their appetite and movement. From each animal, about 25m1 of exudate wasrecovered. The injection of 0.5m1 of exudate into a second animal resulted inmassive infection within 24 hours. The pH of the exudates was between pH8.0-pH8.5. The first infection showed a 5 times increase in bacterial numbers (from 10 11to 5X10 11 ) and the second infection indicated that the bacterial number increasedabout 50 times (from 10 10 to 5X10 11 ). When observed under a microscope, theexudates contained a large number of P. gingivalis cells, a few red blood cells andvery few leukocytes and macrophages. Western immunoblotting studies of DnaKand GroEL homolog proteins showed that these HSPs were not induced during invivo infection of guinea-pigs (Figure 20).52Figure 20. Western blot of P. gingivalis W50 grown in vitro and in vivo.Cellular extracts from equal numbers of cells grown in the test tubes or recoveredfrom infected guinea-pig exudates were separated on SDS-PAGE, transferred to anitrocellulose membrane and incubated with a mixture of anti-DnaK and anti-GroELantibodies. Lanes; 1. cells grown in vitro; 2. cells from the second infection in guinea-pig. Molecular weight standards are indicated on the left in kilodaltons.53DISCUSSIONAlthough the heat shock response in many microorganisms has been studied,very little is known about the regulation of heat shock response in obligate anaerobicbacteria. In the study of heat shock response of Spirochetes, Stamm et al. (107)reported that Treponema pallidum failed to exhibit a heat shock response and theGroEL and DnaK homolog proteins in Treponema pallidum and Treponemadenticola were not thermoinducible. Because T. pallidum was cultured in rabbittissues, they suspected either T. pallidum HSP homologs were maximally expressedin vivo and could not be further induced in vitro or that T. pallidum lacks a regulatedstress response. Anzola et al. (8) provided evidence that the DnaK homolog proteinin Borrelia burgdorferi is an immunologically important Spirochetal antigen. Recentstudies by Narberhaus and co-workers (81, 82) found the dnaK locus of Clostridiumacetobutylicum contained four heat shock genes organized in an operon in the orderof orfA-grpE-dnaK-dnaJ. Analysis of the transcription start sites of these heat shockgenes suggested a32 factor was not involved in heat shock regulation. Therefore,they suggested that the chromosomal organization as well as the regulatorymechanism for the expression of major heat shock genes in C. acetobutylicum isdifferent from that in E. coli. There has been no report on heat shock response of P.gingivalis, the putative pathogen of adult periodontitis. In this study, I utilized thehighly conserved nature of the major heat shock proteins by using antibodies againstE. coli DnaK or GroEL and E. coli dnaK and groEL gene probes to study the heatshock proteins and genes in P. gingivalis.I have demonstrated a heat shock response in P. gingivalis. A 74kDa and a62kDa heat shock protein was identified as major HSPs DnaK and GroEL homologsrespectively by reacting with anti-E. coil DnaK and anti-E. coil GroEL antibodies.These are the most prominent proteins induced by heat shock in P. gingivalis. Byanalogy with other bacteria, the prominent high-molecular-weight 92kDa heat shock54protein may be related to the ATP-dependent protease encoded by the Ion gene inE. coli (41). The 45kDa heat shock protein observed by SDS-PAGE may be relatedto the E. coli DnaJ protein (41 kDa) which is coexpressed with DnaK. The low-molecular-weight 12kDa heat shock protein found on 2D-PAGE was suspected to bethe homolog of E. coli GroES protein.High affinity binding to ATP is a characteristic of the HSP70 family which hasbeen used for purification of the DnaK protein (123). But due to the ability of ATP tobind various proteins including some heat shock proteins (i.e. DnaJ and GrpE), myone-step purification of DnaK protein on the ATP-agarose column was not verysuccessful. In a separate experiment, further chromatography of the DnaK fraction ona DEAE-Sepharose column separated the DnaK protein from most of the othercontaminating proteins.During the immunization of rabbits with HSPs, I observed high immunity againstP. gingivalis in some of the animals. The serum from the rabbit immunized withpurified E. coli GroEL protein had a high reactivity against a large number of P.gingivalis proteins. Interestingly, the preimmune serum from the same rabbit had littlereactivity to P. gingivalis proteins. At the same time, several other rabbits exhibitedhigh immune responses to P. gingivalis proteins in their preimmune or immune seraeven though they had never been subjected to laboratory immunologicalprocedures. Anaerobic cultures of mouth and fecal samples from these rabbits didnot show black-pigmented colonies on blood-agar plates, but the possibility of aninfection by a bacterial strain close to P. gingivalis could not be excluded.My attempt to radio-label P. gingivalis with (35S)-methionine was not verysuccessful. This may due to the poor incorporation of methionine by P. gingivalis andalso to the fact that some heat shock proteins may not contain methionine residues,an example is the GroES-like protein in Mycobacterium tuberculosis (130). Bettersuccess was achieved when ( 14C)-amino acids were used. However, the relatively55low level of incorporation required a long radio-labeling time in order to obtainenough radio-activity to expose the X-ray film. The poor incorporation of amino acidsby P. gingivalis could be due to: (1). preference of P. gingivalis for peptides ratherthan amino acids (40, 96); (2). large pool of amino acids in the complex mediumused to culture P. gingivalis; (3). use of amino acids as a source of energy by P.gingivalis leading to degradation of many amino acids rather than incorporate theminto proteins.The time course study of the heat shock response of P. gingivalis showed that the62kDa GroEL homolog protein was induced within 5 minutes of an upshift intemperature. This heat shock response continued to exist throughout the one-hourheat shock experiment period. In E. coli, heat shock resulted in a rapid transientincrease in heat shock gene expression (maximal induction is about 5 minutes)followed by a rapid decrease that lead to a higher steady-state level of HSPs (110).The relatively slow kinetics of the heat shock response in P. gingiva/is may reflect theslower rate of protein synthesis in this organism. The doubling time of P. gingivalis isapproximately 8 times longer than that of E. co/i.Two-dimensional PAGE and 2D-Western blot analysis revealed that there were atleast two different isoelectric forms of DnaK and four isoelectric forms of GroELhomologs existing both at 37°C and 42°C. There have been reports that in E. coli,DnaK and GroEL are phosphorylated in the process of protein folding andoligomerization (57, 97), different levels of phosphorylation could result in differentisoelectric points of these HSPs on two-dimensional PAGE. The different forms ofDnaK and GroEL homologs found in P. gingivalis may represent differentphosphorylated forms of the same proteins or this microheterogeneity could occurdue to deamidation, acetylation or other modifications that result in an alteration ofcharge of these proteins.56In agreement with the finding that DnaK and GroEL homologs are conserved in P.gingivalis, we have identified a single copy of the dnaK gene homolog in all the P.gingivalis and black-pigmented Bacteroides strains we studied. We also found asingle copy of groEL gene homolog in all the P. gingivalis strains and most of theblack-pigmented Bacteroides strains except P. asaccharolyticus and Prevotelladenticola. A possible reason for this is that the groEL gene homologs in these twostrains have lower homology to E. coil groEL gene probe than the rest of the strainswe studied, therefore, they could not be detected by the low stringency hybridizationconditions we used. It was noticed that both dnaK and groEL gene probes hybridizedto similar sizes of DNA fragments in P. gingivalis W50, W83 and W12 strains but tomuch larger DNA fragments in the avirulent P. gingivalis strain ATCC33277. Whenstudying a protease gene cloned from P. gingivalis, Park and McBride (89) reportedthat the gene probe hybridized to a 5-kb DNA fragment in P. gingivalis ATCC33277but in P. gingivalis W50, W83, and W12, the gene probe hybridized to a 3.2-kb DNAfragment. Restriction endonuclease typing of genomic DNA from different P.gingivalis isolates has revealed extensive genetic heterogeneity within the species(64). This observation was supported by heterogeneous DNA fingerprints obtainedfrom the application of the polymerase chain reaction with arbitrary primer (AP-PCR)to different strains of P. gingivalis (75). This genetic heterogeneity may reflect thedifferent virulence levels of different P. gingivalis strains.In addition to the limited group of proteins induced by heat shock, we alsoobserved decreased radio-labeling of a prominent 50kDa protein and several minorlow-molecular-weight proteins during heat shock. Although in E. coli and manyeucaryotic cells, heat shock resulted in a slower rate of synthesis of a large number ofproteins, the specific decreased radio-labeling of the 50kDa protein is quite unique. Inorder to determine whether this protein was specifically degraded due to elevatedprotease activity such as Lon (or La) or clpP proteases (16, 17), or due to decreased57synthesis of this protein, we conducted a pulse-chase labeling study of theexpression of the 50 kDa protein upon heat shock. The results suggested that the50kDa protein synthesis was down-regulated by heat shock. The functionalsignificance of this protein during heat shock is not known.In E. coli, HSPs are not only induced by increased temperature, but also by avariety of stress insults, many of which affect protein structure and conformation (83).Therefore, we studied the expression of GroEL and DnaK homolog proteins in P.gingivalis grown under different stress conditions. Because of their importantintracellular functions, GroEL and DnaK homolog proteins are expressed under allgrowth conditions. This makes it difficult to determine the induction of these heatshock proteins under stress conditions that may have minor impact on the expressionof heat shock genes. Therefore, all the stress response experiments were conductedindependently three times and Western immunoblotting and ( 14C)-amino acidslabeling were performed to evaluate the expression of stress proteins. Our resultsshow the major heat shock protein homologs of DnaK and GroEL are significantlyinduced by ethanol, but not by the other stress stimuli. Ethanol also induced twoproteins of the molecular weight of 68 kDa and 60 kDa, these two proteins were notinduced by heat shock.When P. gingivalis was cultured at pH5, the cells had a much darker color than thecontrol cells. Since P. gingivalis stores hemin for growth, this black-pigmentationsuggests it was storing more hemin than normal. At the same time, two new proteinsof 26 kDa and 31 kDa were induced in these cells suggesting the function of theseproteins may be related to hemin uptake or storage. Bramanti and Holt (13) havereported a heat-modifiable, hemin-regulated 26 kDa surface protein of P. gingivaliswhich changes its location on the outer membrane in response to different stressstimuli. At this time the relationship between these two 26 kDa proteins is not known.The location of the stress proteins induced by pH5 has not been determined.58The finding that in P. gingivalis, DnaK and GroEL homolog proteins and someother stress proteins were induced by ethanol and low pH raised the questionwhether these stress proteins could be induced during infection. There has beengreat interest in the roles of some HSPs as immunodominant antigens inMycobacteria spp. (128, 129, 131, 132). Although our studies of P. gingivalisrecovered from a guinea-pig infection model did not show that DnaK or GroELhomolog proteins were induced, caution must be taken when interpreting theseresults. The guinea-pig infection model is quite different from what could behappening in human periodontitis. In the animal model, the infection is rapid insteadof chronic, the exudates recovered from the animal contained mostly of P. gingivaliscells with very few macrophages and leukocytes, showing that very little host immuneresponse was involved.In adult periodontitis, large numbers of polymorphonuclear leukocytes (PMNLs)accumulate in the area of inflammation. PMNLs produce oxygen metabolites such assuperoxide (02 -), hydrogen peroxide (H202), singlet oxygen ('02), and hydroxylradical ('OH) which could serve as stress protein inducing agents (2, 3). It is possiblethat P. gingivalis expresses elevated levels of stress proteins in order to survive in thehostile environment, or P. gingivalis may utilize protection mechanisms other thanheat shock response as suggested by our oxidative stress (H202) studies which didnot induce the major heat shock proteins in P. gingivalis. 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