INFECTION AND IMMUNITY, Aug. 1979, p. 574-579 0019-9567/79/08-0574/06$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 85724 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 ex- been studying phagocytosis of GC grown in fluid plain the persistence of N. gonorrhoeae infection in subcutaneous chambers implanted in the within the human urethra despite the presence backs of guinea pigs (11, 21, 22). They showed of massive quantities of polymorphonuclear neu- that phagocytosed type 1 GC survive within trophils. Two recent hypotheses, which are not human neutrophils substantially longer than do necessarily mutually exclusive, suggest that vir- type 4. These authors observed, as did others, ulent gonococci (GO) resist neutrophil killing (i) that type 4 were phagocytosed better than were by avoiding phagocytosis, or (ii) by resisting type 1. The major difference in experimental intraphagosomal bactericidal mechanisms. Sup- approach between the group of investigators port for the first hypothesis comes from a num- supporting the first hypothesis above and those ber of laboratories (1, 2, 9, 18-21) showing that supporting the second is the conditions used to virulent (types 1 or 2) agar-grown GC are phag- grow the GC. It has been demonstrated by many ocytosed by human neutrophils to a lesser de- that bacteria grown under various conditions or gree than are avirulent GC (types 3 or 4). Others, to various growth phases react differently to observing postphagocytic (i.e., intracellular) bactericidal agents. Novotny et al. (9) and Ward events, showed that both virulent and avirulent et al. (24) were among the first to suggest such agar-grown GC are killed to the same degree (1, differences in the interaction of GC with human 3, 4). Swanson et al., however, have shown that neutrophils. some type 4 GC are actually killed to a lesser An approach to the problem of interpreting degree than are type 2 by human neutrophils GC/neutrophil interactions that has only been (17). In these and other systems serum has little briefly studied is to observe how GC grown opsonic effect on phagocytosis of type 1 but under various conditions are affected by neutroappears to aid in phagocytosis of type 4 (1, 4, phil bactericidal components. In this communi16). cation I report the effects of neutrophil granule Support for the second hypothesis comes extracts on type 1 and type 4 GC that have been mostly from laboratories in England, which have grown under various conditions. 574

VOL. 25, 1979

KILLING OF GONOCOCCI BY NEUTROPHIL EXTRACTS

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 16to 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

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 0.85. 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

576

INFECT. IMMUN.

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FIG. 1. Bactericidal activity of increasing amounts 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. agar-grown,

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. i 60 lb) showed the most gradual response to in(4) creasing 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 interme60 90 120 0 15 30 diate in their response to extract. Strains GC-7 TIME, min. and F62 reacted similarly to extract. FIG. 2. Bactericidal activity of granule extract Effects of time, temperature, and pH. In none of the following experiments were there (125 yIg/ml) over time on broth-grown, log-phase N. any significant differences between the re- gonorrhoeae GC7, types I and 4. Data are expressed sponses of type 1 and type 4 GC. GC appeared as in Fig. 1. to be killed exponentially over time. The results 100 of a representative group of experiments for one Ti m particular extract concentration, i.e., 125 ,Ag/ml, T43 2 80 are shown in Fig. 2. This concentration was chosen so that 100% killing would occur over a 60reasonable period of time. The kinetics can be changed by changing extract concentration > 40(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 reTEMPERATURE, C spective temperatures. No experimental values are shown below 100C, because sustained temFIG. 3. Effect of incubation temperature on the peratures below 100C sustantially killed or bactericidal activity of granule extract (250 ,g/ml) on clumped GC in the absence of extract, making broth-grown, log-phase N. gonorrhoeae GC7. Ti, Type 1; T4, type 4. Representative experiment. controls difficult to interpret. -J

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KILLING OF GONOCOCCI BY NEUTROPHIL EXTRACTS

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

577

TABLE 2. Bactericidal activity of MPO-ClU-H202 on N. gonorrhoeaea Viable bacteria (%) Type 1 Type 4

Assay contents

PBS control

..................

+ MPO (0.67 jtg/ml) ......... +

83

H202 (15 IM)

+ MPO + H202

100

88 ..............

0

+ MPO + H202+ catalase (500

95

U/mi). MDB control .........

.....

+ MPO (2.7 ,tg/ml)

+ H202 (48 ttM) + MPO + H202 ..............

100

100

100

87

91

79

3

12

"Broth-grown, log-phase N. gonorrhoeae GC7. Representative experiment of three. MDB, Minimal Davis broth.

140

120

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20TABLE 1. Effect of heated granule extract on N.

gonorrhoeaea

Heating Time (mm) 10

Viable bacteria (%)

Temp ("C)

Ice 50 75 100

20

Type 1