Killing of Neisseria gonorrhoeae by Human

Transcription

1 INFECTION AND IMMUNITY, Aug. 1979, p /79/ /06$02.00/0 Vol. 25, No. 2 Killing of Neisseria gonorrhoeae by Human Polymorphonuclear Neutrophil Granule Extracts RICHARD F. REST Department ofmicrobiology, College of Medicine, Arizona Health Sciences Center, Tucson, Arizona Received for publication 23 May 1979 Neisseria gonorrhoeae was grown in vitro (on agar and in broth) and in vivo (in 10-day chicken embryos) and tested for its sensitivity to the bactericidal action of human neutrophil granule extracts. Under all conditions studied, type 1 and type 4 N. gonorrhoeae were killed equally well by dialyzed extracts of neutrophil granules (containing both azurophil and specific granule contents) and by the myeloperoxidase-cl--h202 bactericidal system. However, sensitivity to the bactericidal activity of granule extracts depended upon growth conditions and growth phase. Log-phase, egg-grown gonococci were the most sensitive; they were killed 100% by 250 to 300,ug of granule extract (60 min, 3700) per ml. N. gonorrhoeae grown on agar for 20 h (to stationary phase) were the least sensitive, being killed only 80 to 90% with 500,Ag of granule extract per ml. Thus, susceptibility to granule extract of gonococci grown under the four conditions studied in this report decreased in the order: log phase, egg grown; log phase, broth grown; stationary phase, egg grown; and stationary phase, agar grown. Killing was time and temperature dependent; little killing occurred when incubations were done at 100C. Boiled granule extract had only minimal effects on N. gonorrhoeae viability. Addition of catalase (500 U/ml) to the granule extract bactericidal system did not protect; however, the same concentration of catalase completely inhibited the bactericidal activity of the myeloperoxidase-cl--h202 system. Many hypotheses have been proposed to explain the persistence ofn. gonorrhoeae infection within the human urethra despite the presence of massive quantities of polymorphonuclear neutrophils. Two recent hypotheses, which are not necessarily mutually exclusive, suggest that virulent gonococci (GO) resist neutrophil killing (i) by avoiding phagocytosis, or (ii) by resisting intraphagosomal bactericidal mechanisms. Support for the first hypothesis comes from a number of laboratories (1, 2, 9, 18-21) showing that virulent (types 1 or 2) agar-grown GC are phagocytosed by human neutrophils to a lesser degree than are avirulent GC (types 3 or 4). Others, observing postphagocytic (i.e., intracellular) events, showed that both virulent and avirulent agar-grown GC are killed to the same degree (1, 3, 4). Swanson et al., however, have shown that some type 4 GC are actually killed to a lesser degree than are type 2 by human neutrophils (17). In these and other systems serum has little opsonic effect on phagocytosis of type 1 but appears to aid in phagocytosis of type 4 (1, 4, 16) Ṡupport for the second hypothesis comes mostly from laboratories in England, which have 574 been studying phagocytosis of GC grown in fluid in subcutaneous chambers implanted in the backs of guinea pigs (11, 21, 22). They showed that phagocytosed type 1 GC survive within human neutrophils substantially longer than do type 4. These authors observed, as did others, that type 4 were phagocytosed better than were type 1. The major difference in experimental approach between the group of investigators supporting the first hypothesis above and those supporting the second is the conditions used to grow the GC. It has been demonstrated by many that bacteria grown under various conditions or to various growth phases react differently to bactericidal agents. Novotny et al. (9) and Ward et al. (24) were among the first to suggest such differences in the interaction of GC with human neutrophils. An approach to the problem of interpreting GC/neutrophil interactions that has only been briefly studied is to observe how GC grown under various conditions are affected by neutrophil bactericidal components. In this communication I report the effects of neutrophil granule extracts on type 1 and type 4 GC that have been grown under various conditions.

2 VOL. 25, 1979 MATERIALS AND METHODS Neutrophils. Neutrophils were obtained from healthy adult human volunteers by leukapheresis and were sedimented first through hydroxy-ethyl starch or Plasmagel and then through Ficoll-Hypaque, as described previously (14, 15). Leukapheresis yielded approximately 1010 purified neutrophils from each donor. Final cell suspensions were 292% neutrophils, with eosinophils the major contaminating cell population. Granules and granule extract. As described previously, granules were pelleted (20,000 x g, 20 min) from postnuclear supernatants (250 x g, 15 min) of neutrophil homogenates (90% breakage in 0.34 M sucrose). Granule pellets, containing both azurophil and specific granule populations, were extracted overnight at 30C with 0.2 M sodium acetate buffer (ph 4.0) containing 10 mm CaCl2. Extracts were clarified by centrifugation (20,000 x g, 20 min) and dialyzed against phosphate-buffered saline (PBS; ph 7.4; containing, per liter of distilled, deionized water: NaCl, 8 g; KCl, 0.2 g; Na2HPO4, 1.15 g; and KH2PO4, 0.2 g) using dialysis tubing with an average molecular weight cut-off of 3,500, as described previously (14, 15). Extracts of granules from 1.2 x 10' neutrophils yielded approximately 10 mg of total protein. Bacteria. N. gonorrhoeae F62 was obtained from P. Fred Sparling, University of North Carolina, Chapel Hill. Strain GC7 was a cervical isolate from a patient at the Arizona Health Sciences Center and was identified to species by colony morphology, Gram stain, oxidase reaction, and carbohydrate fermentation. Colony types were identified as described by Kellogg et al. (6, 7). GC were grown one of three ways, as follows. (i) GC were grown on GC agar supplemented with iron, glucose, and cocarboxylase as described by White and Kellogg (26). Freshly inoculated plates were incubated at 36 C for 18 to 22 h in a humidified incubator containing 5 to 7% CO2 in air. (ii) GC were grown in GC broth with the above-mentioned supplements. Cultures were started in 25 ml of GC broth with 0.5 ml of a suspension (optical density at 550 nm, 0.3) of 16- to 20-h GC agar-grown cells and were shaken at 36 C for 3 to 5 h in 10% CO2 in air to mid-log phase (Klett with green filter = 40 to 50 units, -6 x 107 colonyforming units [CFU] per ml). (iii) GC were also grown in 10-day chicken embryos, as described by Gibbs and Roberts (4). Egg-grown GC were harvested in log phase at 5 h and in stationary phase at 18 to 22 h. GC obtained by any of the above methods were diluted appropriately in GC broth. For the myeloperoxidase(mpo)-cl--h202 assays, bacteria were diluted in PBS or minimal Davis broth. Bactericidal assays. Assays for granule extract bactericidal activity were performed in wells of plastic Microtiter plates (Flow Labs, Inc.), each well containing a total of 0.2 ml (14, 15). At zero time, assay mixtures contained -6 x 103 CFU of GC per ml diluted in GC broth and any test substances. When possible, test substances were suspended or diluted in singlestrength GC broth before addition to the assay mixture. When not possible, controls were run with the buffer used to resuspend the test substance. Incubations were static for 1 h at 36'C at ph 7.4 in a KILLING OF GONOCOCCI BY NEUTROPHIL EXTRACTS 575 humidified incubator containing 5 to 7% CO2 in air, unless otherwise indicated. After appropriate incubation, 50 to 100 yd of assay mixture (containing 0 to 600 CFU) was spread on fresh GC agar plates, and CFU were counted 24 to 36 h later. Type 1 GC plates containing >10% type 4 GC were not included in results. Assays for the MPO-Cl--H202 bactericidal system were performed in microtiter wells containing 6 x 102 to 12 x 102 GC, H202, MPO, test substances, and PBS in a total of 0.2 ml. MPO was purified from human leukemic neutrophils as described previously (14) and had an R2 (ratio of absorbency at 430 nm to absorbancy at 280 nm) of Concentrations of assay constituents are described for individual experiments, and incubation conditions were as above except incubations were for 30 min. Results are described as percentage of test bacteria viable at 60 min compared to percentage of control bacteria viable at 60 min. In bactericidal assays performed with granule extracts, where GC broth was used as the incubation medium, 60-min control plates had the same or more CFU than did zero-time plates. In bactericidal assays performed with MPO-Cl--H202, where PBS or minimal Davis broth was used as the incubation medium, 60-min controls had the same CFU as zero-time controls. Occasionally, 60-min PBS controls had fewer CFU than zero-time PBS controls, i.e., the GC were dying in PBS alone, and these experiments were not used. The bactericidal assay systems gave similar results with initial concentrations of GC from 103 to 107 per ml. To avoid time-consuming and often error-prone dilutions, lower GC concentrations were used. Protein. Protein was measured by the method of Lowry et al. (8) with chicken egg-white lysozyme as a standard. Materials. GC broth, minimal Davis broth, protease peptone no. 3, soluble starch, and agar were from Difco Laboratories, Detroit, Mich. Plasmagel was obtained from HTI Corp., Buffalo, N.Y. Ficoll, catalase (bovine liver, thymol free), and egg-white lysozyme (3x crystallized) were purchased from Sigma Chemical Co., St. Louis, Mo. Hypaque (50%, sodium) was from Winthrop Laboratories, Menlo Park, Calif. All chemicals were of analytical reagent grade if available, and other materials were of the highest quality commercially available. RESULTS Killing of agar-, broth-, and egg-grown GC by granule extract. GC were killed in a dose-dependent manner by dialyzed acetate extracts of purified human neutrophil granules (Fig. 1). Under no conditions were any significant differences observed between the effects of extract on type 1 and type 4 GC. However, growth conditions significantly altered the response of GC to extract. Log-phase, egg-grown GC (Fig. lc) showed the steepest response to increasing concentrations of extract and were

3 576 REST INFECT. IMMUN 'O 0~~~() (4) --ot *ao 14,S 0 ~~~~~~~(3) (4 a) (4) (4) s(4 0 i (3) b U V X s260 SW 4> A (7) 7 wt S~~~~~ 20- C (7) (2) An (3) 0) (5) d (11) 0'?- d (11) ' 7I O DX.9 0 GRANULE EXTRACT, 4ftnm 3112ACT. S GRANULE EXTRACT. Alf FIG. 1. Bactericidal activity of increasing amounts of granule extract on (a) broth-grown, log-phase, (b) agar-grown, stationary-phase, (c) egg-grown, log-phase, and (d) egg-grown, stationary-phase N. gonorrhoeae strains F62 and GC7. Ti, Type 1; T4, type 4. Points represent means. Half-bars above or below points represent 1 standard deviation. Numbers in parentheses represent the number of times a point was experimentally obtained. most sensitive to high concentrations of extract. GC grown in this manner were killed 100% by 250 to 300,pg of extract per ml. Stationary-phase, egg-grown (Fig. ld), and agar-grown GC (Fig. lb) showed the most gradual response to increasing concentrations of extract and were least sensitive to high concentrations of extract. GC grown in either manner were consistently killed only 80 to 90% by 500,jg of extract per ml. Logphase, broth-grown GC (Fig. la) were intermediate in their response to extract. Strains GC-7 and F62 reacted similarly to extract. Effects of time, temperature, and ph. In none of the following experiments were there any significant differences between the responses of type 1 and type 4 GC. GC appeared to be killed exponentially over time. The results of a representative group of experiments for one particular extract concentration, i.e., 125,Ag/ml, are shown in Fig. 2. This concentration was chosen so that 100% killing would occur over a reasonable period of time. The kinetics can be changed by changing extract concentration (data not shown). The bactericidal activity of extract was temperature dependent with little killing observed at 100C (Fig. 3). Values (percent viable) for each temperature were obtained by comparing experimental values with control values at their respective temperatures. No experimental values are shown below 100C, because sustained temperatures below 100C sustantially killed or clumped GC in the absence of extract, making controls difficult to interpret. -J 60 i TIME, min. FIG. 2. Bactericidal activity of granule extract (125 yig/ml) over time on broth-grown, log-phase N. gonorrhoeae GC7, types I and 4. Data are expressed as in Fig ' 60- > 40- (4) Ti m T4 TEMPERATURE, C FIG. 3. Effect of incubation temperature on the bactericidal activity ofgranule extract (250,g/ml) on broth-grown, log-phase N. gonorrhoeae GC7. Ti, Type 1; T4, type 4. Representative experiment.

4 VOL. 25, 1979 Measuring the effects of incubation ph on killing by extract was also very difficult, since GC were very sensitive to acid ph. At ph 6, controls were similar to those described above for low temperatures. Those experiments that were well controlled showed that GC were equally susceptible between ph 6 and 8 to a range of extract concentrations (data not shown). Heating extract for 20 min at 100'C abolished 90% of its bactericidal activity. Heating extract for shorter times or at lower temperatures only partially inhibited such activity (Table 1). MPO-C1--H202 bactericidal system. MPO-C1--H202 assays were performed in PBS or minimal David broth instead of GC broth, because GC broth (and other bacteriological media) dramatically inhibits the bactericidal activity of this system. Both strains and both colony types of GC reacted similarly to MPO-Cl -H202, with 90 to 100% killing observed in 30 min (Table 2). Decreasing or increasing incubation time or the concentration of MPO respectively decreased or increased killing in the complete system. The H202 concentration was optimal. It could not be increased without increased death of controls nor decreased without decreased killing in the complete system. Effects of catalase on bactericidal activity. Catalase (500 U/ml) inhibited the bactericidal activity of the MPO-Cl--H202 system almost completely (Table 2), whereas it had no significant effect on killing by extract (Fig. 4). DISCUSSION Critical evaluation of the present literature on the interaction of virulent (type 1) versus avirulent (type 4) GC with human neutrophils is difficult because of the various methods used to grow GC and the different phagocytosis assays TABLE 1. Effect of heated granule extract on N. gonorrhoeaea Heating Viable bacteria (%) Time (mm) Temp ("C) Type 1 Type 4 10 Ice <1 <1 50 <1 < Ice 0 <1 50 < abroth-grown, log-phase N. gonorrhoeae F62. Granule extract concentration was 225,g/ml. Representative experiment of three. KILLING OF GONOCOCCI BY NEUTROPHIL EXTRACTS 577 TABLE 2. Bactericidal activity ofmpo-clu-h202 on N. gonorrhoeaea Viable bacteria Assay contents (%) Type 1 Type 4 PBS control MPO (0.67 jtg/ml) H202 (15 IM) 88 + MPO + H MPO + H202+ catalase ( U/mi). MDB control MPO (2.7,tg/ml) H202 (48 ttm) MPO + H "Broth-grown, log-phase N. gonorrhoeae GC7. Representative experiment of three. MDB, Minimal Davis broth Ui~~~~ O 80 > \ 20-0-, TIME, min. FIG. 4. Effect of catalase on the bactericidal activity of granule extract (250 Ag/ml) on broth-grown, log-phase N. gonorrhoeae F62, types 1 and 4. Heated catalase was placed in a boiling-water bath for 15 min. Representative experiment. 0, Control; U, catalase (500 U/ml), 0, heated catalase; A, granule extract; (l, granule extract plus catalase; A, granule extract plus heated catalase. employed. Data presented in this communication support certain aspects of current GC/neutrophil literature, i.e., that virulence is determined more by resistance to phagocytosis than

5 578 REST by resistance to intraleukocytic bactericidal activity. No differences were observed between the killing of type 1 and type 4 GC by human neutrophil granule extract or by MPO-ClO-H202. Ismail et al. observed no differences in killing of type 1 and type 4 and by 0j or by H202 (generated by the action of xanthine oxidase on purine) and observed no differences in killing of stationary versus log-phase GC (5). To my knowledge, there are no other reports in the literature describing other oxidative or nonoxidative bactericidal activity of human neutrophil constituents toward GC. It is evident from the data presented herein, however, that GC grown by various methods show substantially different susceptibilities to the bactericidal activities of human neutrophil granule contents. Log-phase GC, both type 1 and type 4, were more susceptible to the bactericidal activity of extract than were stationaryphase cells. Susceptibility of GC to extract decreased in the order: log phase, egg grown; log phase, broth grown; stationary phase, egg grown; and stationary phase, agar grown. This lends support to data showing that GC grown under certain circumstances (for instance, in vivo, from patients with acute gonorrhea, or in guinea pig chamber fluid) show markedly different interactions with human neutrophils than do GC grown in vitro on agar (9, 28). Changes have been observed with GC even within a single growth system. Gibbs and Roberts (4), using egg-grown GC, showed that log-phase type 1 GC resist phagocytosis by human neutrophils whereas stationary-phase type 1 do not. Although the granule extract used in these studies contains MPO, for at least four reasons killing of GC by extract is probably H202 independent and thus not influenced by the MPO- Cl--H202 system. (i) The MPO-Cl--H202 bactericidal system is inhibited by normal-strength bacteriological media. MPO-Cl--H202 assays done in GC broth instead of PBS showed no killing of GC (unpublished data). (ii) The addition of catalase to extract bactericidal assays did not inhibit killing (Fig. 4). (iii) No H202 was added to the extract bactericidal assays. (iv) GC are strongly catalase positive and thus would likely decompose any small amounts of H202 possibly produced by components of the assay system itself. The actual intraphagolysosomal concentrations of granule components can only be estimated. As an example, 109 neutrophils contain approximately 3 mg of MPO and 0.3 ml of water. Assuming that less than 10% of the cytoplasmic volume is occupied by granules or phagolysosomes, the concentration of MPO within these INFECT. IMMUN. organelles would be at least 300 mg/ml, i.e., far greater than concentrations used in the present studies. In spite of their tendency to be sensitive to ph, temperature, drying, buffer, autolysis, etc., GC were killed by extract to the same degree as are intermedite rough Escherichia coli and Salmonella typhimurium (15). As smooth E. coli or S. typhimurium lose their outer membrane polysaccharides, i.e., as they become rough, they become more sensitive to granule extract (15). Both type 1 and type 4 GC are reported to be intermediately rough, possessing a core polysaccharide, with little or no 0 antigen (27). Killing of GC by extract was also similar to that of E. coli and S. typhimurium in regard to the effects of boiled extract, assay incubation temperature, and the fact that log-phase GC were killed more easily than stationary phase. It appears that logphase bacteria are commonly more sensitive to bactericidal agents than are stationary-phase bacteria. The heat lability of the extract bactericidal activity is similar to that discussed in a recent paper from Elsbach's and Olsson's laboratories in regard to a nonenzymatic cationic bactericidal protein purified from human neutrophil granules (25). The extract bactericidal activity does not appear to be due to the heat-stable, proteolytic, cationic bactericidal proteins described by Odeberg and Olsson (10). Densen and Mandell recently showed, among other things, that type 1 and type 3 GC are iodinated to the same degree by lactoperoxidase-i--h202 and that type 1 GC are not phagocytized as well as type 3 by human neutrophils. What happens to those type 1 GC that are phagocytized, and what would the results of such experiments be if GC growth conditions were varied? Data from a number of British laboratories suggest that virulent type 1 GC grown in guinea pig chambers are phagocytized by human neutrophils in an in vitro system, although certainly to a lesser extent than type 4 (22-24, 28). The important observation is that intracellular type 1 GC resist neutrophil bactericidal activities and grow. These observations, however, are not universally accepted. Drutz, also using an in vitro phagocytosis assay but using 14- to 16-h, agar-grown GC, showed that "gonococci do not survive to a significant degree within human PMN, monocytes, or macrophages when care is taken to selectively eliminate extracellular, cell-adherent gonococci from the system" (3). The complex interactions of azurophil and specific granule contents and of 02-dependent and 02-independent microbicidal systems within

6 VOL. 25, 1979 the phagolysosome remain to be elucidated. At the same time, the mechanisms) by which bacteria such as GC are killed or resist killing by leukocyte microbicidal systems is largely unknown. It is hoped that continuing work with bactericidal granule constituents will lead to a better understanding of such processes. ACKNOWLEDGMENTS This investigation was supported in part by Public Health Service biomedical research support grant S07 RR from the National Institutes of Health and by Public Health Service grant R01-AI from the National Institute of Allergy and Infectious Diseases. I thank Linda Mattson and Elizabeth Pretzer for expert and excellent technical assistance and for their extra measure of patience and dedication. LITERATURE CITED 1. Densen, P., and G. L Mandell Gonococcal interactions with polymorphonuclear neutrophils. Importance of the phagosome for bactericidal activity. J. Clin. Invest. 62: Dilworth, J. A., J. 0. Hendley, and G. L. Mandell Attachment and ingestion of gonococci by human neutrophils. Infect. Immun. 11: Drutz, D. J Intracellular fate of Neisseria gonorrhoeae, p In G. F. Brooks, E. C. Gotschlich, K. K. Holmes, W. D. Sawyer, and F. E. Young (ed.), Immunobiology of Neisseria gonorrhoeae. American Society for Microbiology, Washington, D.C. 4. Gibbs, D. L., and R. B. Roberts Interaction in vitro between human polymorphonuclear leukocytes and Neisseria gonorrhoeae cultivated in the chick embryo. J. Exp. Med. 141: Ismail, G., W. D. Sawyer, and W. S. Wegener Effect of hydrogen peroxide and superoxide radical on viability of Neisseria gonorrhoeae and related bacteria. Proc. Soc. Exp. Biol. Med. 155: Kellogg, D. S., Jr., I. R. Cohen, L C. Norins, A. L Schroeter, and G. Reising Neisseria gonorrhoeae. II. Colonial variation and pathogenicity during 35 months in vitro. J. Bacteriol. 96: Kellogg, D. S., Jr., W. L. Peacock, Jr., W. E. Deacon, L Brown, and C. I. Pirkle Neisseria gonorrhoeae. I. Virulence genetically linked to clonal variation. J. Bacteriol. 85: Lowry, 0. H., N. H. Rosebrough, A. L. Farr, and R. J. Randall Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: Novotny, P., J. A. Short, and P. D. Walker An electron microscope study of naturally occurring and cultured cells of Neisseria gonorrhoeae. J. Med. Microbiol. 8: Odeberg, H., and I. Olsson Mechanisms for the microbicidal activity of cationic proteins ofhuman granulcoytes. Infect. Immun. 14: Penn, C. W., D. Sen, D. R. Veale, N. J. Parsons, and H. Smith Morphological, biological and antigenic properties of Neisseria gonorrhoeae adapted to growth in guinea pig subcutaneous chambers. J. Gen. Microbiol. 97: Perry, M. B., V. Daoust, B. B. Diena, F. E. Ashton, and R. Wallace The lipopolysaccharides of Neisseria gonorrhoeae colony types 1 and 4. Can. J. Biochem. 53: Punsalang, A. P., and W. D. 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Immun. 11: Swanson, J., E. Sparks, B. Zeligs, M. Siam, and C. Parrott Studies on gonococcus infection. V. Observations on in vitro interactions of gonococci and human neutrophils. Infect. Immun. 10: Swanson, J., and B. Zeligs Studies on gonococcus infection. VI. Electron microscopic study on in vitro phagocytosis of gonococci by human leukocytes. Infect. Immun. 10: Thomas, D. W., J. C. Hill, and F. J. Tyeryar, Jr Interaction of gonococci with phagocytic leukocytes from man and mice. Infect. Immun. 8: Thongthai, C., and W. D. Sawyer Studies on the virulence of Neisseria gonorrhoeae. I. Relation of colonial morphology and resistance to phagocytosis by polymorphonuclear leukocytes. Infect. Immun. 7: Veale, D. R., C. W. Penn, and H. Smith Capacity of gonococci to survive and grow within human phagocytes, p In G. F. Brooks, E. C. Gotschlich, K. K. Holmes, W. D. Sawyer, and F. E. Young (ed.), Immunobiology of Neisseria gonorrhoeae. American Society for Microbiology, Washington, D.C. 23. Veale, D. R., H. Smith, K. A. Witt, and R. B. Marshall Differential ability of colonial types of Neisseria gonorrhoeae to produce infection and an inflammatory response in subcutaneous perforated plastic chambers in guinea pigs and rabbits. J. Med. Microbiol. 8: Ward, M. E., A. A. Glynn, and P. J. Watt The fate of gonococci in polymorphonuclear leukocytes: an electron microscope study of the natural disease. Br. J. Exp. Pathol. 53: Weiss, J., P. Elsbach, L Olsson, and J. Odeberg Purification and characterization of a potent bactericidal and membrane active protein from the granules of human polymorphonuclear leukocytes. J. Biol. Chem. 253: White, Li A., and D. S. Kellogg Neisseria gonorrhoeae identification in direct smears by a fluorescent antibody-counterstain method. Appl. Microbiol. 13: Wiseman, G. M., and J. D. Caird Composition of the lipopolysaccharide of Neisseria gonorrhoeae. Infect. Immun. 16: Witt, K., D. R. Veale, H. Finch, C. W. Penn, D. Sen, and H. Smith Resistance of Neisseria gonorrhoeae grown in vivo to ingestion and digestion by phagocytes of human blood. J. Gen. Microbiol. 96: