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Interaction of macrophage cationic proteins with the outer membrane of Pseudomonas Aeruginosa Sawyer, Janet Gail 1987

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INTERACTION OF MACROPHAGE CATIONIC PROTEINS WITH THE OUTER MEMBRANE OF PSEUDOMONAS AERUGINOSA By JANET GAIL SAWYER B.Sc, Un i v e r s i t y of Guelph, 1985 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF MICROBIOLOGY We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA July 1987 © Janet G a i l Sawyer, 1987 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of M ^ B O S ' 6 U J £ M The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date OU.L4 2i ABSTRACT Purified macrophage oationlo proteins were used ln functional assays to determine their interactions with the outer membrane and lipopolysaocharide of Pseudomonas aeruginosa. A fluorescent derivative of polymyxin B (dansyl-polymyxin) was found to bind to saturation to purified lipopolysaocharide,with similar a f f i n i t y for the aminoglycoside supersensitive strain H215 and wild type strain H103 lipopolysaocharide. MCP-1 could displace more dansyl-polymyxln bound to the lipopolysaocharide of both strains, and bound with greater a f f i n i t y than MCP-2. When whole oells were used, MCPs also displaced bound dansyl-polymyxin. Effects on the outer membrane of whole oells were examined by determining the i n i t i a l rate of uptake of the hydrophobic fluorescent probe 1-N-phenylnaphthylamine. Uptake was enhanced in the presence of MCPs, indicating permeabilization of the outer membrane. MCP-1 caused maximal uptake of the probe at 40 ug/ml, MCP-2 at 70 ug/ml, and crude extraot at only 20 ug/ml. Uptake of the probe was found to be enhanced at add pH, with maximal uptake occurring with only 7.5 jog/ml MCP-1 at pH 6.5. The data suggested that MCPs aot to permeablllze the outer membranes of P_j_ aeruginosa in a manner analagous to that defined for other polycationic agents. i i TABLE OF CONTENTS Page. ABSTRACT i i TABLE OF CONTENTS i i i List of Figures i v List of Tables v ACKNOWLEDGEMENTS v i INTRODUCTION 1 MATERIALS AND METHODS Macrophage/granulocyte procurement 5 Peptide purification 6 Bacterial strains and growth conditions 7 Antibacterial assay. 7 LPS isolation 8 Dansyl-polymyxin binding experiments 8 Binding inhibition experiments 9 Permeabilization of whole ce l l s to 1-N-phenyl-naphthylamin e 10 Enhancement of phagocytosis by MCPs 10 RESULTS Purification of MCPs 12 Antibacterial assay 13 Dansyl-polymyxin binding to purified LPS 18 Inhibition of dansyl-polymyxin binding to LPS by polycations 27 Dansyl-polymyxin binding and inhibition to whole ce l l s 32 Enhancement of 1-N-phenylnaphthylamine uptake by MCPs 32 Enhancement of phagocytosis 46 DISCUSSION 49 LITERATURE CITED 54 i i i LIST OF FIGURES Page 1. P-10 column fractionation 14 2. MCP purification by reversed phase FPLC 16 3. LPS-dependent fluorescence of dansyl-polymyxin 21 4. H i l l plot of dansyl-polymyxin binding to LPS..23 5. Inhibition of dansyl-polymyxin binding to LPS by MCPs 28 6. Binding of dansyl-polymyxin to intact cells...33 7. Inhibition of dansyl-polymyxin binding to whole c e l l s by MCPs 37 8. Enhancement of NPN uptake by intact cells 39 9. MCP-1 promoted enhancement of NPN fluor-escence in intact c e l l s at varying pH 44 iv LIST OF TABLES Page 1. Examples of bactericidal cationio proteins of phagocytes 2 2. K i l l i n g of P. aeruginosa strains by crude macrophage extract and purified peptides 19 3. Kinetics of binding of dansyl-polymyxin to P. aeruginosa LPS 25 4. Inhibition by various polycations of dansyl-polymyxin binding to P_j_ aeruginosa LPS 30 5. Inhibition by various polycations of dansyl-polymyxin binding to whole c e l l s 35 6. H i l l numbers and S Q 's for interactions of MCPs with P^ aeruginosa 42 7. Phagocytosis of untreated and MCP-coincubated P. aeruginosa by unelicited rabbit alveolar macrophages 47 v ACKNOWLEDGEMENTS I wish to thank Dr. Joe Lam and Dr. Bob Hancock for their understanding and encouragement during my studies. Special thanks also to Nancy Martin and Niamh Kelly for their constant help, support and friendship. I gratefully acknowledge the support of a Medical Research Council Studentship 1 9 8 6 - 8 7 . v i INTRODUCTION Although phagocytosis by macrophages constitutes the major mechanism by which non-specific resistance to infection by aspirated bacteria i s achieved (8), our understanding of the biochemical mechanisms of bacterial k i l l i n g i s s t i l l far from complete (29). Mechanisms of macrophage antimicrobial k i l l i n g f a l l into two distinct categories, namely, oxygen-dependent and oxygen-independent. Oxygen-dependent bactericidal mechanisms include the production, during the respiratory burst, of highly toxic oxygen derivatives such as superoxide anions, hydrogen peroxide, hydroxyl radicals, and singlet oxygen (50). Oxygen-independent mechanisms comprise a more diverse group of antimicrobial functions. These include lysosome acidification (38), iron-binding proteins (13), production of arginase (18), synthesis of certain components of complement (50), lysosomal hydrolases including lysozyme (7), and macrophage cationic proteins (36). In recent years, several cationic proteins with antibacterial activity have been identified from several different types of phagocytes (Table 1) . In 1980, the f i r s t report of macrophage cationic proteins (MCPs) isolated from rabbit alveolar macrophages appeared (36). These peptides, named MCP-1 and MCP-2, are rich i n arginine and cysteine (37). They are 33 amino acids each in length and d i f f e r by only a single 1 Table 1_. Examples of bactericidal cationic proteins of phagocytes. Source Size Activity References Human polymorphs- 37 kD nuclear leuko-cytes (PMNs) 57 kD Rat PMNs Rabbit PMNs 55 kD 25 kD <3.5 kD 4-6 kD 50 kD Gram negative bacteria, 26,47,48 including Salmonella typhimurium, Escherichia coli, Pseudomonas aeruginosa E. coli, 48,52,55 S. typhimurium, Neisseria gonorrhoeae P. aeruginosa 15,16 Staphylococcus aureus, 32,33 E. coli S. aureus, 5,6,9,43 E. coli, P. aeruginosa, Acinetobacter calcoaceticus (note: family of three distinct peptides) S. typhimurium, 14,23 A. calcoaceticus E. coli, 4,53 S. typhimurium 4 kD Gram negative and Gram positive bacteria, various fungi (note: family of six distinct peptides) Rabbit alveolar 4 kD macrophages (as above) (note: family of two distinct peptides) 2 substitution (42). Under conditions of relatively low ionic strength and at near-neutral pH, MCPs have been demonstrated to exhibit powerful antimicrobial effects against various fungal, Gram positive and Gram negative organisms, including P3eudomonas  aeruginosa (19,20,22,41). P. aeruginosa, being an opportunistic pathogen, i s of great interest to this laboratory. Indeed, accummulated evidence on the mechanism of polycation interaction with the outer membrane of P_^  aeruginosa led Hancock et a l (11) to propose the s e l f -promoted uptake model. That i s , polycations, such as polymyxins and aminoglycosides, interact with the outer membrane of P.  aeruginosa by displacing the divalent cations which serve to cross-link adjacent lipopolysaocharide (LPS) molecules. Displacement by large polycations results in the disruption of the outer membrane, and consequently, increased permeability to agents l i k e lysozyme, the hydrophobic fluorophor 1-N-phenylnaph-thylamine, and the ^ -lactam, nitrocefin (10,11,12,24). It has been proposed that, as a consequence of this perturbation of outer membrane permeability, that the uptake of the polycation i t s e l f i s promoted (11). The main objective of this study was to determine whether MCPs behaved like typical polycations when mediating the k i l l i n g of P_^  aeruginosa. To test this hypothesis, two different fluorescent probes were used, which have previously been shown to be effective in studying the interactions of polycations with P.  aeruginosa (24,27,28). Furthermore, three different strains of 3 P. aeruginosa were used in order to examine possible differences in the interaction of MCPs with P_;_ aeruginosa mutants. These strains were: 1) H103: PA01, wild type, previously described (30) 2) H215: PA01715, aminoglycoside super-sensitive, previously described (25) and 3) H234: AK1012, rough strain, previously described (17). In this thesis, the results of a study on the interaction of macrophage cationic proteins with the outer membrane of P.  aeruginosa are reported. Evidence was obtained that suggests that MCPs may function as an important mechanism of bacterial ki l l ing by macrophages. 4 MATERIALS AND METHODS Macrophage/granulocyte procurement. White, female New Zealand rabbits were injected intravenously with 1 ml of complete Freund's adjuvant (CFA) to e l i c i t alveolar macrophages. Three weeks later, rabbits were sacrificed using 360 mg sodium pentobarbital (Euthanyl, M.T.C. Pharmaceuticals, Mississauga, Ont.). The lungs and trachea were then removed to f a c i l i t a t e harvesting of c e l l s . Alveolar macrophages were collected by pulmonary lavage, introducing phosphate-buffered saline (PBS), pH 7.4, via a tracheal cannula. Approximately 250 ml of buffer was used to recover c e l l s . To harvest granulocytes, s t e r i l e peritoneal exudates were obtained from 8-9 week old female New Zealand white rabbits by intraperitoneal i n s t i l l a t i o n of 250 ml s t e r i l e saline containing 0.2$ glycogen (Type VIII, Sigma Chemical Company, St. Louis, Mo.) and 5 ug LPS. Exudates were collected approximately 16 hours later. Rabbits were sacrificed as before, and the peritoneum was lavaged with a total of 600 ml PBS. Cells collected either way were centrifuged at 200 x g for 10 minutes at room temperature and washed twice with PBS. Contaminating erythrocytes, i f present, were removed by hypotonic l y s i s . Cells were then counted in a haemocytometer, and slides were prepared with a cytocentrifuge (Cytospin 2, Shandon Southern Instruments, Inc., Sewickley, Pa.) and stained with Diff-quik (Canlab, Vancouver, B.C.) to determine c e l l yield and purity. 5 Peptide purification. Peptides were purified by the method of Selsted et a l (45) with modifications. Washed ce l l s (10 8 macrophages or 10^ granulocytes) were resuspended in 10 ml of 10$ acetic acid and homogenized with a tissue homogenizer for 3 minutes at 4,000 rpm. Cells were then sonicated to ensure maximal disruption of c e l l membranes. To remove unbroken c e l l s and nuclei, the homogenate was centrifuged at 250 x g at 4 C for 10 minutes. The supernatant, i.e., the lysosome-enriched fraction, was then extracted for 4-6 hours in the presence of 25 ug/ml Pepstatin A (Sigma Chemical Comp.) at 4 C. Lysosomes and cellular debris were then removed by centrifugation at 27,000 x g. The pellet was reextracted overnight in fresh 10$ aoetic acid to ensure complete recovery of peptides. The 27,000 x g supematants were then dialyzed against 0.1$ acetic acid at 4 C for 48 hours in Spectrapor 3 (MW cutoff 3,500) dialysis tubing (Spectrum Medical Industries, Inc., Los Angeles, Ca.). Following dia l y s i s , the supematants were concentrated by lyophilization. The lyophilized retentate was referred to as "crude extract" which was used i n some experiments. Approximately 10 mg of crude extract was resusupended in 1 ml of 1$ acetic acid and applied to a column (2.5 by 30 cm) containing Bio-Gel P-10, 50-100 mesh (BioRad Laboratories, Richmond, Ca) that had been equilibrated at room temperature in 1$ acetic acid. Proteins were eluted at a constant flow rate of 5 ml per hour, and 1 ml fraotions were collected. The effluent was monitored by measuring i t s absorbance at 214 and 280 nm i n a model PU8600 spectrophotometer (Pye Unicam Ltd., Cambridge, Eng.). Samples of fractions were 6 also examined on acid-urea slab gels, run by the method of Panyim and Chalkley (35). Fractions containing only low molecular weight basio peptides (MCPs or neutrophil peptides) were collected, pooled, and lyophilized. Final purification and separation of the peptides was achieved by reversed-phase Fast Protein Liquid Chromatography (Pharmacia, Uppsala, Sweden) using a Pro-RPC 5/10 column. Water-acetonitrile gradients containing 0.1% trifluoroacetic acid were used in elution. Protein concentration was assessed by the BioRad Protein Assay using Standard I (Bovine Plasma Gamma Globulin). Bacterial strains and growth conditions. Pseudomonas aeruginosa PA01 strain H103 was used in a l l experiments. A gentamioin super-sensitive strain (H215) and a rough mutant (H234) were used for certain experiments. A l l strains were grown in 1% (w/v) proteose peptone no. 2 medium (Difco Laboratories, Detroit, Mich.). Experimental cultures were started from an overnight broth culture, diluted 1:50 into fresh medium, and grown at 37 C (or 30 C for H234) with vigorous shaking to an optical density of 0.4 to 0.6 at 600 nm. Antibacterial assay. The bactericidal assay was performed exactly as described by Lenrer et. al_ (20). Briefly, oells were washed three times in 10 mM sodium phosphate buffer, pH 7 . ^ . Assay mixtures, in a total volume of 100 u l , consisted of 105 colony forming units in buffer, to which d i s t i l l e d water (control), 10 ug crude extraot, or 5 ug purified peptide was 7 added. After incubation at 37 C for 60 minutes, s e r i a l dilutions were made in the same buffer and viable counts determined. LPS isolation. LPS was isolated as desoribed by Darveau and Hancock (3). The isolated LPS was extracted twice with an equal volume of ehloroform-methanol (2:1) to remove trace amounts of sodium dodecyl sulfate and phospholipids which resulted from the isolation procedure (3). Residual chloroform was removed by purging with nitrogen gas in a fume hood for about 30 minutes. A 10 mg/ml stock suspension of H103 and H215 LPS was prepared on a dry weight basis. Dansyl-polymyxin binding experiments. Dansyl-polymyxin (DPX) was prepared as described by Sohindler and Teuber (39) and quantitated by dinitrophenylation (1 ) . DPX binding to LPS or whole ce l l s was monitored by measuring the fluorescence intensity on a model 650-10S fluorescenoe spectrophotometer (The Perkin-Elmer Corp., Norwalk, Conn.) set with an excitation wavelength of 340 nm and an emission wavelength of 485 nm with s l i t widths of 5 nm. Binding assays were performed as described by Moore et a l (27). Briefly, aliquots of DPX were titrated into a cuvette containing 3 ug/ml (0.3 uM) LPS in 1 ml of 5 mM HEPES (N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid) buffer, pH 7.2, and the fluorescence intensity was recorded on a Perkin-Elmer Coleman strip chart recorder. The amount of DPX bound was determined by using the equation: amount of DPX bound = ( f 0/F m a x) x concentration of DPX, where f Q is the observed 8 fluorescence at a given DPX concentration added to 3 ug/ml LPS (subsaturating LPS concentration) and F i s the observed ID £UC fluorescence when the same concentration of DPX i s added to excess LPS ( 3 0 0 ug/ml) ( 2 7 ) . For binding to whole c e l l experiments, DPX was titrated into a cuvette containing 9 9 0 ul of 5 mM HEPES buffer with 1 0 mM sodium azide (to inhibit respiration), pH 7 . 2 , and 1 0 u l of c e l l s resuspended to an optical density at 6 0 0 nm of 0 . 5 in the same buffer. Binding inhibition experiments. Inhibition of DPX binding to LPS was performed as previously described ( 2 7 ) . Briefly, inhibitors of DPX binding, including MCPs, were titrated into a cuvette containing 3 ug/ml LPS and 2 . 5 uM DPX (resulting in 8 5 to 9 0 $ saturation of the LPS by the DPX) in 1 ml of 5 mM HEPES buffer, pH 7 . 2 , and the decrease in the observed fluorescenoe (peroent inhibition) was recorded. Maximum inhibition by a given compound was calculated as the extrapolated y intercept of a plot of 1/percent inhibition versus 1/inhibitor concentration. The x intercept gave - 1 / I 5 Q , where was the concentration of inhibitor giving 50% maximal inhibition at the LPS and DPX concentrations used. For inhibition of DPX binding to whole o e l l s , inhibitors were titrated into a cuvette containing 9 9 0 u l of 5 mM HEPES buffer/ 1 0 mM sodium azide, pH 7 . 2 , 1 0 u l of c e l l s resuspended to an optical density of 0 . 5 at 6 0 0 nm i n the same buffer, and the DPX concentration required to give 8 5 - 9 0 $ saturation. Maximal inhibition and I 5 Q values were read off of a graph of the 9 Inhibition curves. Permeabilization of whole ce l l s to 1-N-phenylnaphthylamine. Uptake of 1-N-phenylnaphthylamine (NPN) assays were performed as previously described (24). Briefly, c e l l s were prepared by washing twice in 5 mM HEPES buffer, pH 7.2, containing 1 mM KCN (to inhibit respiration), and resuspended to an optical density of 0.5 at 600 nm i n the same buffer. The c e l l suspension was then allowed to s i t at room temperature for 30 to 60 minutes before use. NPN (Sigma) was dissolved in acetone at a concentration of 500 uM and added to 1 ml of c e l l suspension to a f i n a l concentration of 10 uM. MCPs and crude macrophage extract were tested for the a b i l i t y to permeabilize c e l l s to NPN, and the increase i n NPN fluorescence intensity was continuously monitored. Excitation and emission wavelengths were set at 350 and 420 nm, respectively, with s l i t widths of 5 nm. To determine the effects of pH on enhancement of NPN uptake by MCPs, a separate experiment was done in which bacterial c e l l s were prepared as before, except that oells were resuspended i n 5 mM HEPES buffer plus 1 mM KCN at pH 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0, and allowed to s i t 30 to 60 minutes at room temperature before use. The permeability assay was then done exactly as described. Enhancement of phagocytosis by MCPs. The phagocytosis visual assay was done as previously desoribed by Battershill et a l (in press) with some modifications. Unelicited rabbit alveolar 10 macrophages were collected and washed as before, and resuspended to 5 x 10 5 cells/ml in RPMI-1640 (Gibco, Burlington, Ont.) supplemented with 44 mM sodium bicarbonate (Fisher Scientific, Vancouver, B.C.), 10$ (v/v) f e t a l calf serum (Gibco), 10 mM HEPES buffer (Terochem Laboratories, Vancouver, B.C.), 0.04$ (v/v) 2-mercaptoethanol (BioRad), 2 mM L-glutamine (Sigma), 40 units per ml p e n i c i l l i n and 40 ug/ml streptomycin (Giboo), pH 7.2. Two ml aliquots of the o e l l suspension were incubated in 35 x 10 mm NuncIon tissue culture dishes at 37 C in 10$ COg overnight. Just prior to the assay, the macrophage monolayer was gently washed twice with phagocytosis assay medium (RPMI 1640 medium supplemented with 10 mM HEPES buffer only), then one ml of this medium was placed over the monolayer. Bacteria were washed and resuspended to 10° cells/ml, and added to the monolayer to give a f i n a l bacteria to macrophage ratio of 20:1. Equal concentrations of MCP-1 and MCP-2 were added to the system to a f i n a l peptide concentration of 50 ug/ml or an equal volume of s t e r i l e water was added (control). The system was incubated for 90 minutes at 37 C in 10$ COg, at which time the macrophages were scraped from the dish using a rubber policeman and gently resuspended. Slides were prepared by cytocentrifugation of 100 u l aliquots followed by staining. To assess phagocytosis, the number of bacteria in each of 120 macrophages was recorded by visual inspection. 11 RESULTS Purification of MCPs. Peptides were originally sought from three macrophage or macrophage-like cel l l ines, inoluding: P388D.J, a DBA/2 murine macrophage tumour ce l l l ine; PU51.8, a BALB/c murine macrophage-like tumour ce l l line; and DC7, a macrophage-T lymphocyte fusion produot with macrophage properties, such as adherence and phagocytosis. However, after extensive screening, no small basic peptides were found in any of the cel l lines used, as judged by the lack of proteins migrating more cathodal than lysozyme on acid-urea gels (data not shown). To date, small cationic proteins from rodent phagocytes have been described in rabbits, guinea pigs and rats, but not mice (Table 1). Because no peptides were present in any of the cel l lines used, i t was decided to turn to the original source of macrophages from which MCPs had originally been isolated, ie , rabbit alveolar macrophages. Later, i t was learned that rabbit peritoneal granulocytes also produced peptides identical in structure and function to MCP-1 and -2, called NP (for neutrophil peptide) -1 and -2 (45). As granulocytes were faster and more easily harvested, much of the peptides used in this report were obtained from these ce l ls . Obtained from either source, cel l purity was high. From CFA-elicited rabbits, which were shown to yield cells of higher purity with increased peptide content (21), 12 approximately 10 alveolar macrophages could be recovered with >98$ purity and v i a b i l i t y (as judged by trypan blue exclusion); and from s t e r i l e peritoneal exudates, approximately 10^ granulocytes of >96$ purity were routinely recovered. Electrophoresis on acid-urea gels of extracts prepared from these sources revealed the presence of two bands migrating past lysozyme (which were not present i n c e l l l i n e preparations), i e , MCPs. In the original protocol (42), preparative acid-urea gels were used to isolate the peptides by elution of excised gel s t r i p s . However, recovering the peptide free of 5 M urea proved time-consuming and wasteful. Alternatively, gel f i l t r a t i o n was used. A single column run proved most effective i n separating the desired family of peptides (two from macrophages or six from granulocytes) from other larger molecular weight proteins in the sample (Fig. 1). The f i n a l separation and purification of the peptides was achieved by Fast Protein Liquid Chromatography (FPLC) (Fig. 2). Peptide recovery was estimated to be approximately 70%, with small losses occurring due to d i f f e r e n t i a l centrifugation, incomplete extraction, molecular sieving, and monitoring samples during the purification. Each preparation yielded approximately 200 pg of each of the purified 8 Q peptide from 10 alveolar macrophages or 10* granulocytes. Antibacterial assay. MCPs were previously found to be most effective under conditions of relatively low ionic strength and near-neutral pH (20), hence, the buffer used. Although the effects of k i l l i n g were not as dramatic as those seen with other 13 Figure 1_. P-10 column fractionation. Ten mg of crude extract from granulocytes was applied to the column equilibrated with 1$ acetic acid. One ml fractions were collected, and samples analyzed for protein content. Neutrophil peptide (NP) -1 and -2 are identical to MCP-1 and -2, respectively. Samples were electrophoresed on an acid-urea polyacrylamide gel containing 15$ acrylamide and 5 M urea. Symbols: L, hen egg white lysozyme; CE, 100 ug crude granulocyte extract. 14 60 75 L CE Figure 2_. MCP purification by reversed phase FPLC. Approximately 250 ug pooled neutrophil peptides were applied to a Pro-RPC 5/10 column. A linear water-acetonitrile gradient containing 0.1$ (v/v) trifluoroacetic acid at a flow rate of 0.3 ml/min was used to elute peptides. Fractions (0.6 ml) were collected and read spectrophotometrically. 16 7 9 11 13 15 Fraction number 17 bacterial species, i t was found that the rough strain (H23M was more susceptible to the k i l l i n g effects of MCPs and crude extract than the wild type smooth strain H103 or the gentamicin super-sensitive strain H215 (Table 2). MCP-1 was sl i g h t l y more bactericidal than MCP-2 for a l l strains tested. Despite the crude extract being very efficacious in the k i l l i n g assay, no additional k i l l i n g effects were seen when bacteria and peptide were co-incubated with 20 ug/ml hen egg white lysozyme (data not shown). DPX binding to purified LPS. DPX binds to purified LPS resulting in enhancement of fluorescence (27). When DPX was titrated into a solution containing LPS, an increase in fluorescence was seen u n t i l a l l of the sites which bind DPX on the LPS were f i l l e d (Fig. 3, ref. 27). However, H23M LPS would not saturate, even when up to 12 uM DPX was added and consequently was not used in these experiments. The data for H103 and H215 LPS-DPX binding was analyzed on a H i l l plot (Fig. 4). The slope of the H i l l plot (n) was a straight li n e with a slope greater than one, suggesting cooperative interaction of DPX with LPS. The value for n was the H i l l number, which provided a minimal estimate of the number of cooperative interaction sites. The H i l l number for H215 LPS was higher than that for H103 LPS (Table 3), suggesting higher oooperativity which might have resulted from the presence of more DPX binding s i t e s . The x intercept of the H i l l plot (S Q 5 ) f a measure of the a f f i n i t y of LPS for DPX, were similar for both strains. The information derived from these experiments was 18 Table 2. K i l l i n g of P_j_ aeruginosa strains by crude macrophage extract and purified peptides. The k i l l i n g assay was performed exactly as described by Lehrer et a l (20), exposing approximately 10^ bacteria for 60 minutes to 100 ug/ml crude extract, 50 ug/ml purified peptide, or s t e r i l e d i s t i l l e d water (control). 19 % SURVIVORS Crude Strain Control Extract MCP-1 MCP-2 H103 100 10 19 28 H215 100 3 19 22 H234 100 <1 1 5 20 Figure 3_. LPS-dependent fluorescence of dansyl-polymyxin. Dansyl-polymyxin was titrated into a cuvette containing 1 ml of 5 mM HEPES buffer, pH 7.2, with or without 3 ug/ml (0.3 uM) LPS. Symbols: • , strain H103 LPS; O , strain H215 LPS; x , no LPS. 21 Figure 4_. H i l l plot of dansyl-polymyxin binding to LPS. Values were derived from data in Fig. 3, where f i s the observed fluorescence at a given dansyl-polymyxin concentration and F i s the maximum fluorescence (extrapolated from the graph) when LPS is completely saturated with dansyl-polymyxin, as revealed by a lack of further fluorescence increase when more dansyl-polymyxin is added. Data (shown for H215) are averages of three independent experiments. The correlation coefficient for the linear regression of the data for this and a l l other H i l l plots was greater than 0.99. 23 Table 3.. Kinetics of binding of dansyl-polymyxin to P.  aeruginosa strain H103 and H215 LPS. Values are derived from H i l l plots (eg. Fig. 4). The S Q is the concentration of dansyl-polymyxin at which one half of the binding sites on the LPS molecule are saturated. The H i l l coefficient indicates the degree of binding cooperativity. The maximum number of binding sites was calculated as amount dansyl-polymyxin divided by number of LPS moleoules present (27). 25 H i l l coefficient Maximum no. of binding sites/ Strain S (uM) (n) LPS molecule H103 H215 1.02 1.12 2.01 3.53 4.32 5.21 26 useful for interpreting DPX inhibition results. Inhibition of DPX binding to LPS by polycations. Acoording to the self-promoted uptake hypothesis, polycations disrupt the permeability barrier by displacing Mg 2 + which serves to cross-link adjacent LPS molecules on the surface of the outer membrane. Further, certain polycationic compounds have previously been shown to be capable of competing with DPX for binding to LPS (27). MCPs were also found to be capable of competing with DPX for binding to LPS to some extent (Fig. 5). The competitive displacement of DPX by MCPs suggested that at least some of the binding sites on LPS which bound DPX were also capable of binding MCPs. Similar displacement was observed using gentamicin or Mg 2 + as inhibitors (Table 4). From the graph, i t might appear that MCP-1 displaced DPX from H103 LPS more effectively than from H215 LPS. However, i t should be noted that although more DPX was displaced from H103 LPS (a higher MI value), H215 had more binding sites on i t s LPS for DPX. Further, less MCP-1 was required to displace half the maximal possible inh i b i t i b l e DPX bound from H215 LPS (a lower I5Q value), indicating a higher a f f i n i t y for MCP-1 to H215 LPS. Similar results were seen with MCP-2. As the inhibition curve for MCP-2 and H215 LPS was not amenable to similar analysis (inhibition appeared to be cooperative), the values reported were extrapolated from the graph in Fig. 5, panel B, and must not be considered definitive. The data indicated that MCPs were very efficient competitors for LPS binding, as indicated by the low I R n values (Table 4). 27 Figure 5_. Inhibition of dansyl-polymyxin binding to LPS by MCPs. MCPs were titrated into a cuvette containing 3 ug/ml (0.3 uM) LPS and 2.5 uM dansyl-polymyxin in 1 ml of 5 mM HEPES buffer, pH 7.2, and the decrease in fluorescence was recorded. Symbols: • , strain H103 LPS; O , strain H215 LPS. 28 % Fluorescence o o o o o o VO Table 4.* Inhibition by various polycations of dansyl-polymyxin binding to P. aeruginosa strain H103 and H215 LPS. The I C A and 5U MI values were determined as described in Materials and Methods, except for H215 LPS and MCP-2, which was extrapolated from a graph (Fig. 5, panel B). 30 H103 H215 Maximal Maximal Inhibitor I 5 Q Inhibition I 5 Q Inhibition MCP-1 2.30 uM 62.5$ 1.63 uM 45.5$ MCP-2 2.98 42.7 1.75 25.0 polymyxin 6.10 100 8.20 100 gentamicin 71.9 42.7 60.6 44.8 Mg 2 + 1,840 45.5 1,900 46.9 3 1 DPX binding and Inhibition to whole o e l l s . To show that MCPs could Interact with LPS in native outer membranes, Inhibition of DPX binding was studied using intact c e l l s . Because of the difference i n binding curves for each of the strains (Fig. 6) and the complexity of inhibition curves for some of the polycations for some strains, and MI values (Table 5) were read directly off of graphs, such as the one i n Figure 7. As with purified LPS, MCPs were capable of displacing DPX from whole c e l l s of H215 and H103. However, greater displacement of DPX ocourred from H215 oells, showing that in intaot c e l l s , MCPs interacted with this strain more readily than with H103 c e l l s . DPX was not readily removed from H234. Recalling that H231* was best k i l l e d by the peptides, i t i s probable that this reflects the relatively higher a f f i n i t y for the probe DPX than for MCPs. Enhancement of NPN uptake by MCPs. NPN had previously been shown to be an excellent probe to study the interactions of aminoglycosides with aeruginosa, as uptake was presumably due to the permeabilization of the outer membrane by the added antibiotic (24). A series of experiments were performed in which various concentrations of purified peptides or crude extraot were added to cyanide-pretreated c e l l s , and the i n i t i a l rates of fluorescence increase, reflecting NPN uptake, were recorded. A plot of the i n i t i a l rates of fluorescence increase versus the concentration of peptide or crude extract, such as that in Fig. 8, yielded a sigmoidal curve in a l l cases. Such a curve 32 Figure 6. Binding of dansyl-polymyxin to intact c e l l s . Dansyl-polymyxin was titrated into a cuvette containing 1 ml of 5 mM HEPES buffer/10 mM sodium azide, pH 7.2, and 10 u l of cel l s washed twice and resuspended in the same buffer to an optical density of 0.5 at 600 nm. Symbols: • , H103 c e l l s ; O , H215 ce l l s ; O , H234 c e l l s . 33 Table 5_. Inhibition by various polycations of dansyl-polymyxin binding to whole aeruginosa c e l l s . The I^Q and maximal inhibition (MI) values were read directly off of graphs, such as that i n Fig. 7. 35 H103 H215 H234 Inhibitor I... MI I,.. MI I c n MI polymyxin 0.55 uM 55$ 0.29 uM 76$ 0.80 uM 60$ MCP-2 2.50 30 1.13 65 2.50 5 MCP-1 2.81 50 2.25 54 5.63 15 gentamicin 36 50 20 70 40 55 Mg 2 + 2,400 75 1,200 84 2,000 80 36 Figure 7_. Inhibition of dansyl-polymyxin binding to whole c e l l s by MCPs. MCPs were titrated into a cuvette containing 1 ml of 5 mM HEPES buffer/10 mM sodium azide, pH 7.2, 10 p i of c e l l s washed twice and resuspended to an optical density of 0.5 at 600 nm, and the dansyl-polymyxin concentration required to cause 85-90$ saturation of whole ce l l s (1.87 uM for H215 or 3.75 uM for H103 and H234). Symbols: • , strain H103 c e l l s ; O , strain H215 c e l l s ; O , strain H234 c e l l s . 37 Figure 8^  Enhancement of NPN uptake by intact P_j_ aeruginosa c e l l s . Varying concentrations of MCP-1 were titrated into a cuvette containing 1 ml of c e l l s , washed and resuspended to an optical density of 0.5 at 600 nm in 5 mM HEPES buffer/1 mM KCN, pH 7.2, and 10 uM NPN. I n i t i a l rate of uptake of NPN, as reflected by fluorescence increase, were recorded. Symbols: • , H103 c e l l s ; O , H215 c e l l s . 39 INITIAL RATE suggested positive cooperativity. Further, similar curves were obtained for both H103 and H215, but uptake of the probe by H215 occurred at slig h t l y lower concentrations, suggesting that H215 was sl i g h t l y more easily permeabilized than was H103. H234 could not be used in this assay, perhaps due to a less stable outer membrane. When the data was reanalyzed by H i l l plots, H i l l numbers were obtained whioh indicated the minimal number of cooperative binding sites . These numbers were similar for both strains (Table 6). MCP-1 was a better permeabilizer than MCP-2, causing maximal uptake of NPN at 40 ug/ml relative to 70 ug/ml, respectively, for both strains. The S Q values were less for MCP-1, indicating a higher a f f i n i t y of MCP-1 for the c e l l s than MCP-2. However, maximal uptake for both strains occurred at only 20 ug/ml when crude macrophage extract was used, indicating the possible presenoe of other lysosomal contents with permeabilizing ac t i v i t y . The most interesting results obtained using the NPN assay were observed when the same assay was conducted using oells resuspended in buffer of varying pH. The buffers used ranged in pH from 5.5 to 8.0, the presumed range of lysosomal pH (2,40). At pH 7.5 and 8.0, MCP-1 exhibited relatively poor permeabilizing aotivity. However, below pH 7.0, the peptides caused remarkable permeabilization, with maximal NPN uptake occurring with the addition of 7.5 ug/ml MCP-1 or less (Fig. 9 ) . At pH 5.5, however, the effects of MCP-1 addition could not be observed as the c e l l s reached maximal fluorescence in the absence of any peptide addition. This was presumably due to the protonation of 41 Table 6. H i l l numbers and SQ5'a for interactions of MCPs with P. aeruginosa. Values were derived from H i l l plots drawn from plots of i n i t i a l rate of uptake versus MCP concentration, suoh as the one in Fig. 8. A H i l l number with a value of greater than one indicates positive cooperativity. 42 MCP-1 MCP-2 Strain n S Q > 5 n S q % 5 H103 2.77 5.00 uM 1.15 24.55 uM H215 3.00 4.26 1.46 17.7 43 Figure 9_. MCP-1 promoted enhancement of NPN fluorescence in intact P_^  aeruginosa strain H103 at varying pH. Cells were washed and resuspended in 5 mM HEPES buffer/1 mM KCN, pH as shown. The assay was then performed exactly as before. Curve a, pH 6.0; curve b, pH 6.5; curve c, pH 7.0; curve d, pH 7.5; curve e, pH 8.0. 44 phosphate groups in LPS, resulting In decreased cross-bridging and hence, a decrease in outer membrane s t a b i l i t y . Enhancement of phagocytosis. When bacteria and macrophages were co-incubated in the presence of peptides, significant enhancement of phagocytosis by unelicited rabbit alveolar macrophages occurred for a l l bacterial strains (Table 7). It should also be noted that uptake of the rough strain was significantly higher than that of H 1 0 3 and H215 in the absence of peptide. 46 Table £. Phagocytosis of untreated and MCP-ooincubated P.  aeruginosa by unelicited rabbit alveolar macrophages. Twenty bacteria per macrophage were incubated for 90 minutes in the presence of 50 ug/ml peptides or s t e r i l e d i s t i l l e d water (control). Values shown are means + standard deviations. By two-tailed student's t test, phagocytosis i s significantly enhanced in the presence of MCPs (p<0.002). 47 Number of bacteria phagocytosed per macrophage Strain Control H103 2 . 5 9 + 1 .62 H215 3 . 53 + 2 . 3 2 H234 6 . 2 5 + 2 . 9 4 MCP-coincubated 5 . 5 9 + 2 . 8 4 5 . 5 4 + 3.16 8 . 8 1 + 4 . 8 2 48 DISCUSSION Polycationio antibiotics, such as polymyxins and aminoglycosides, have been shown to cause enhancement of outer membrane permeability of P. aeruginosa (12,21,30). The uptake of these polycations has been hypothesized to occur via the s e l f -promoted uptake pathway, in which polycations displace divalent cations from sites where they noncovalently cross-bridge adjacent lipopolysaccharide molecules (30,31). Once the integrity of the outer membrane i s disrupted, c e l l s become permeable to agents l i k e lysozyme and NPN (12,24). In accord with this theory, i t was found that MCPs, being strongly polycationio, enhanced the uptake of NPN in a manner typical of other known permeabilizers (12). Further, MCPs were shown to interact with high a f f i n i t y to the LPS of P^ aeruginosa (the f i r s t requirement of the self-promoted uptake model), as demonstrated by competitive displacement of DPX bound to purified LPS or whole ce l l s (Tables 4 and 5). The exception to this was the displacement of DPX from H234 whole c e l l s , in which DPX was poorly displaced (Fig. 7). Strain H234 i s known to possess only six phosphate groups per LPS molecule, compared with 12 to 15 phosphate groups in smooth strain LPS (E.J. MoGroarty, unpublished data). Thus, i t i s possible that the LPS of this strain had lost i t s MCP binding s i t e (or that the a f f i n i t y of H234 LPS for MCPs was decreased relative to i t s a f f i n i t y for the 49 probe DPX). The greater susceptibility of H231* to MCPs (Table 2) might be due to a generally less stable outer membrane (see Hancock, ref. 10) as suggested for other rough strains. MCPs did not mediate cellular l y s i s i n the presence of lysozyme at the concentrations tested, nor did the co-incubation with lysozyme in the k i l l i n g assay have any effect (data not shown). This was atypical, as many polycationic compounds are capable of enhancing the passage of lysozyme across the outer membrane, resulting in cellular l y s i s (12). A possible explanation for this phenomenon could be that sterio hindrance protected the c e l l from the action of lysozyme. The permeabilizers that have been used in the lysozyme l y s i s assay were in general smaller molecules (eg. gentamioin, MW 500; polymyxin, MW 1000) relative to MCPs (MW 4000). Secondly, hen egg white lysozyme was used in these assays. It i s possible that rabbit lysozyme, had i t been available, may have been effective. Thirdly, there was a limitation i n the amounts of MCPs available for these studies, and the lysozyme l y s i s assay i s known to require much larger amounts of permeabilizer, such that an effect might have been seen at higher concentrations. However, due to the effectiveness of the crude extract in the k i l l i n g and permeabilization of c e l l s , i t i s l i k e l y that lysozyme, and/or other lysosomal constituents, take an active, possibly synergistic, role i n bacterial k i l l i n g . On the other hand, the role of lysozyme in the phagolysosome may be purely degradative. It was clear from the DPX and NPN experiments that the surface of the bacterial o e l l was somehow being modified, since 50 the fluorescence intensity of the probes changed in a manner dependent on their environment (Figs. 5,7, and 8). Furthermore, preliminary electron microscopic investigations (in collaboration with Nancy Martin) revealed surface changes of MCP-treated ce l l s compared with untreated c e l l s . Finally, enhancement of phagocytosis of MCP-coincubated c e l l s by rabbit alveolar macrophages also suggested that surface changes had occurred. The p o s s i b i l i t y was raised that these changes could have been the result of inoreased hydrophobicity due to MCP interaction (D.P.Speert, personal communication). To investigate this possibility, partitioning of MCP-treated and untreated c e l l s in a biphasic system of polyethylene glycol and dextran was attempted. However, no significant changes in partitioning behaviour were seen (data not shown). The most plausible explanation for this was that due to the negatively-charged nature of the dextran phase, any increase in surface hydrophobicity was abrogated by ionic interactions with dextran. However, the fact that l i t t l e or none of the hydrophobic probe NPN was taken up by whole ce l l s before MCP addition i s strongly in favour of Increased surface hydrophobicity due to MCP interaction with the bacterial c e l l surface. Indeed, the role of hydrophobic interactions between other phagocyte bactericidal proteins and the surface of c o l i and S.  typhimurium has been documented (46,51,55). Other studies have investigated the role of LPS chain length, finding that susceptibility of Gram negative bacteria to various bactericidal proteins increased in Inverse proportion to 0-antigen chain 51 length, and that deep rough mutants are more susceptible than their isogenic smooth strain parents (34,48,49,51,54). This conclusion can now be extended to include the interaction of MCPs with P_^  aeruginosa, as H234 was indeed more susceptible to the bactericidal effects of these peptides (Table 2). Hypotheses to explain increased susceptibility of LPS mutants to bactericial proteins have previously been proposed. For example, decreased length of core, or the absence of O-side chain, would tend to expose l i p i d A, making hydrophobic and electrostatic interactions with the proteins more l i k e l y (46,48,52,55). Alternatively, defects in Mg 2 + binding by rough LPS would destabilize the outer membrane (10), resulting in increased susceptibility to bactericidal proteins. The most interesting observation of this study was in finding that the permeabilizing effects of MCPs are greatly enhanced at acid pH (Fig. 9). This was intriguing, as purified MCPs were previously shown to exhibit maximal k i l l i n g of P.  aeruginosa between pH 7.0 and 8.0, with l i t t l e or no k i l l i n g occurring at or below pH 6.5 (20). Although this may appear contradictory, i t may define the role of MCPs during the acidification of the lysosome. I n i t i a l l y , during the brief transient rise in pH (2,40), MCPs could k i l l ingested baoteria. Then, as acidification proceeds, MCPs could permeabilize the c e l l s , making them more susceptible to other macrophage k i l l i n g systems, or to degradative enzymes. In the granules of human polymorphonuclear leukocytes (PMNs), several different bactericidal proteins have been 52 identified and characterized (Table 1). To date, a single 37 kD protein has been found to possess optimal k i l l i n g a ctivity at pH 5.5, while the optima for the other proteins i s pH 7.5. The presence of antimicrobial cationic proteins with different pH optima would allow human PMNs to exert antimicrobial aotivity for an extended amount of time after uptake (47). Suoh a complement of antibacterial proteins potentially exists in macrophages, but has yet to be eluoidated. 53 LITERATURE CITED 1. Bader, J. and M. Teuber. 1973. Binding of the O-antigenio lipopolysacoharide of Salmonella typhimurium. 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