Protection Studies - Infection and Immunity

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IAN A. HOLDER,l'2* ROBERT WHEELER,2 AND THOMAS C. MONTIE3. Departments ofMicrobiology and Surgery, University ofCincinnati College ofMedicine, Cincinnati, Ohio. 45267,1 Shriners ..... In J. R. McGhee, J. Mes- tecky, and J.L. ...
Vol. 35, No. 1

INFECTION AND IMMUNITY, Jan. 1982, p. 276-280

0019-9567/82/010276-05$02.00/0

Flagellar Preparations from Pseudomonas aeruginosa: Animal Protection Studies IAN A. HOLDER,l'2* ROBERT WHEELER,2 AND THOMAS C. MONTIE3 Departments of Microbiology and Surgery, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267,1 Shriners Burns Institute, Cincinnati, Ohio 45219,2* and Department of Microbiology, University of Tennessee, Knoxville, Tennessee 379163

Received 21 May 1981/Accepted 28 August 1981

Recent reports have suggested that motility is associated with virulence in Pseudomonas aeruginosa. We have confirmed this observation by showing that groups of mice immunized with P. aeruginosa flagellar-antigen preparations display enhanced survival when they are subsequently burned and challenged locally in the burned area with strains of P. aeruginosa. The protection appears to be due to the immobilization of the microorganisms in the burned skin tissue. Liver elongation factor 2 is also protected. The protection afforded by immunization with flagellar-antigen preparations is independent of the somatic antigenic type of the challenging strain but is flagellar-antigen specific. These data suggest that vaccination with flagellar-antigen preparations may provide a viable prophylactic or therapeutic alternative to antibiotic therapy for use in compromised patient populations in which P. aeruginosa poses a serious infection threat. M-2, originally isolated from the small intestines of CF-1 mice (16), were used, as well as several clinical isolates from patients at the Shriners Bums Institute. Strain GNB-1, an isolate from a nonburned patient, was obtained from the microbiology laboratory of the Cincinnati General Hospital. FAg preparations. FAg preparations were prepared from motile strains RM-46 and M-2 by the methods of Montie et al. (11). The same procedures were used to make a preparation from the nonflagellated strain PA103. This preparation, non-FAg, was used to control for any immunogenic materials, other than flagella, which might have been released from the organisms by the preparative procedures. Immunization procedure and burned mouse model. Female CF-1 mice (22 to 24 g; Carworth Farms, New York, N.Y.) were injected intramuscularly in a hind limb with 0.1 ml of an immunogen (10 ,ug/ml in 0.9%o NaCl solution). After 14 days, the immunized mice were either sacrificed to obtain serum for serological procedures or bumed and challenged according to the bumed mouse model of Stieritz and Holder (16). In this procedure, anesthetized mice received a partialthickness (10-s) alcohol flame bum covering 30% of the total body surface. Immediately after being bumed, they received 0.5 ml of sterile 0.9%o NaCl solution intraperitoneally as fluid replacement therapy. Such a bum followed by this type of therapy is not lethal. Viable P. aeruginosa cells (100 to 250 colonyforming units in 0.1 ml of 0.9%o NaCI solution) were then injected subcutaneously (s.c.) into the bumed site or 108 colony-forming units of P. aeruginosa cells was MATERLIL AND METHODS "painted" on the surface of the bum wound. Survival Microorganisms. Laboratory strains RM-46, a me- was monitored for 5 days. Data presented for s.c. thionine auxotroph of P. aeruginosa PAO1 (9), PA- challenge represent the combined results from three 103, originally supplied by P. V. Liu, University of separate experiments. Serological procedures: 0 antigen typing. All strains Louisville School of Medicine, Louisville, Ky., and

A wide variety of Pseudomonas aeruginosa products which may play a role in the virulence of this organism have been identified (8). In the past few years, studies have placed an emphasis on the contribution of proteolytic enzymes and exotoxin A to the pathogenesis of infections caused by this microorganism (5, 12, 13, 15). However, the pathogenic potential of P. aeruginosa is very complex, and all of its mechanisms of pathogenesis are still not fully understood. More recently, reports which demonstrate that motility or chemotactic ability or both are also associated with the virulence of P. aeruginosa strains have appeared. Strains which were highly chemotactic or motile or both had lower 50% lethal doses when tested in a burned mouse model (2), and motility-deficient mutants were found to be less virulent than their wild-type parents were in a burned rat model (8). Based on these associations, it seemed reasonable to hypothesize that antiflagellum immunization might enhance survival in burned animals infected with P. aeruginosa. We decided to test the effect of immunization with flagellar-antigen (FAg) preparations on mice which were subsequently burned and challenged with various strains of P. aeruginosa. This report describes the results of such experiments.

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typed for their 0 antigens according to the This response was nullified when heat-killed antigenic schema recommended by the Serotyping cells were used (flagella destroyed). On the Committee of the Japan Pseudomonas aeruginosa other hand, agglutination occurred when both Society (6). Antisera used in this slide agglutination live and heat-killed cells of strain PA-103 were procedure were prepared by Toshiba Kagaku Kogyo tested with antiserum prepared against the PACo. Ltd., Tokyo, Japan, and were kindly supplied by 103 non-FAg preparation. No cross-reactivity J. Y. Homma, The Kitasato Institute, Tokyo, Japan. was observed. No agglutination occurred when Agglutination of pseudomonas strains with anti-FAg normal mouse serum was used. Strains RM-46 sera. Bacterial strains to be tested against pooled antiFAg sera obtained from immunized mice were grown and M-2 are 0 serotype B, whereas strain PAfor 18 h on brain heart infusion agar, after which a 103 is 0 serotype G. small amount of the fresh growth was mixed with the The results when FAg preparations were used sera in a slide agglutination procedure. After their to immunize mice which were subsequently flagella were destroyed by heat, the same strains were burned and challenged with P. aeruginosa strain tested with the slide agglutination procedure. In this M-2 are given in Table 2. Mortality (90 to 100%) case, the organisms were grown in shake cultures overnight in brain heart infusion broth, centrifuged, occurred by day 3 postburn and postchallenge in washed twice with sterile 0.9o NaCl solution, and all control groups regardless of the route of then heated to 90°C for 1 h. P. aeruginosa strains challenge; a 5-day mortality of only 0 to 20o exposed to heat in excess of 75°C lose their H antigens occurred in the groups of mice immunized with either RM-46 or M-2 FAg preparations. Quanti(7). Quantitation of bacteria in tissues. At 25 h after s.c. tative bacterial counts of liver and skin tissues inoculation of the burn site with P. aeruginosa strain from FAg-immunized and control groups taken M-2, animals were sacrificed by cervical dislocation. 25 h postburn and postchallenge (Table 3) reLiver tissue and full-thickness specimens of burned vealed that the number of P. aeruginosa cells skin were immediately removed, rinsed in cold, sterile 0.9%o NaCl, and diced, weighed, and homogenized in per wet weight of skin tissue exceeded 7 logs in cold 0.99o NaCl (14). The number of P. aeruginosa all cases; however, in the liver tissue, the counts in the FAg-immunized groups (3.1 to 3.7 logs) cells per gram of tissue (wet weight) was determined by serial dilution plating with duplicate brain heart were more than 2 logs lower than the counts in infusion agar plates. Results are expressed as the mean the control groups (6.1 to 6.6 logs). The effects + standard error of the mean from at least four of burn and challenge on the liver EF-2 level in animals. FAg-immunized and control groups of mice 25 h Quantitation of iver EF-2. Elongation factor (EF-2) postburn and postchallenge are presented in quantitations were carried out 25 h postburn and post- Table 4. EF-2 levels were reduced 77.5 and 85% infection. The active EF-2 content of mouse liver tissue was assayed by the method of Gill and Dinius in the saline and PA-103 non-FAg-immunized (3). Tissues from four mice were pooled, rinsed, diced, control groups, respectively; however, little, if and homogenized in 3 ml of cold 0.25 M sucrose. any, reduction in the liver EF-2 level was obEndogenous NAD was removed from homogenates, served in the FAg-immunized mice. Since liver and the homogenates were centrifuged and assayed for EF-2 levels are reflections of the protein synactive EF-2 content in the presence or absence of 100 thetic capacity of this tissue, these data show Fg of diphtheria toxin. The reaction mixture contained that the protein synthetic process in the livers of 0.07 M dithiothreitol, 1 mg of bovine serum albumin control mice was severely impaired, although it per ml, 50 mM Tris-hydrochloride (pH 8.2), 0.1 mg of appeared to be essentially intact in the FAgdiphtheria toxin per ml, and 0.12 ,uCi of [14C]NAD immunized animals. (uniformly labeled in the adenine moiety; specific Table 5 presents data which show that the activity, 302 mCi/mmol; Amersham Corp., Arlington Heights, Ill.). After 15 min of incubation at 37°C, the protection afforded by immunization with FAg reaction was stopped by the addition of an equal preparations was independent of the 0 serotype volume of 12% trichloroacetic acid. Precipitates were but appeared to be H antigen specific. A 5-day collected and counted (13). All assays were performed survival of between 40 and 100% of the mice was in triplicate. The difference between the radioactive observed when three clinical isolates of P. aerucounts incorporated with and without diphtheria toxin ginosa with different 0 serotypes were used as represents the ADP-ribose-EF-2 complex formed. the challenge organisms. In control groups, 80 to Data are expressed as the percent inhibition of liver EF-2 relative to burned, uninfected controls and repre- 100% mortality occurred within 3 days. For each sent the mean + standard error of the mean of pooled case in which immunization provided enhancement of survival, the cells of the challenge strain livers from four mice. agglutinated with both RM-46 and M-2 anti-FAg RESULTS sera. For the one situation in which immunizaThe data presented in Table 1 indicate that tion was not protective, the challenge strain, live cells of P. aeruginosa strains RM-46 and GNB-1, did not agglutinate with either RM-46 or

were

M-2 agglutinated when tested with antisera prepared against RM-46 and M-2 FAg preparations. There was cross-reactivity between the strains.

M-2 anti-FAg sera. The fact that immunized mice challenged with strain SBI-S, whose 0 serotype was the same as that of strain GNB-1,

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TABLE 1. Slide agglutination assays of P. aeruginosa strains with serum prepared against FAg preparations Agglutination with test organisms

Live

Immunogen

M-2b -

Normal mouse serum (control) PA-103 non-FAg (control) M-2 FAg RM-46 FAg a 900C for 1 h. b Somatic serotype B. c Somatic serotype G.

RMp46b

PA-103c

+ +

+ -

+ +

protected by the immunization emphasizes the fact that protection is associated with H antigens but not 0 antigens. DISCUSSION The factors involved in the virulence of P. aeruginosa and in the pathogenesis of infections caused by this microorganism are complex. At a minimum, elaboration of exotoxin A and protease(s) appears to be essential for strains to be considered fully virulent (5, 12, 13, 15). Recent reports suggest that additional factors such as motility or chemotactic ability or both are also associated with the virulence of P. aeruginosa strains (9). The results of the experiments presented in this report support this suggestion. The fact that antisera prepared against RM-46 and M-2 FAg preparations agglutinated live cells, but that this capacity was lost when the cells were heat killed at a temperature which destroys flagella, indicates that these preparations generated anti-FAg antibodies (Table 1). Immunization with the strain PA-103, non-FAg preparation, on the other hand, generated antibodies which agglutinated both live and heat-killed PA103 cells. This suggests that antibodies are produced and directed against other surface structures cleaved from the organisms in the course of the preparation procedure. One might speculate that these same materials would have been

were

M-2b -

Heat killeda RM.46b -

PA-103C + -

generated in the RM-46 and M-2 preparations as well. However, these substances should have constituted only a very small percentage of the total weight of the RM-46 and M-2 preparations, and immunization with only 1 ,ug of immunogen may not deliver enough of these contaminants for antibody production. This would explain the nonreactivity of these sera with heat-killed cells. By coincidence, the two motile strains chosen for preparing the FAg preparations are of the same 0 antigenic type (type B), and since they cross-reacted when tested against the anti-FAg sera, they appear to be of a similar H antigenic type. (P. aeruginosa has only two major antigenic types of flagella.) In any case, when RM-46 and M-2 FAg-immunized mice were burned and challenged with P. aeruginosa strain M-2, almost no mortality was observed after 5 days, as opposed to greater than 90% mortality after 3 days in the controls (Table 2). This was true regardless of whether the challenge was a low dose (102 cells) injected s.c. or a high dose (108 cells) painted on the burn wound. The fact that RM-46 FAg immunization protected as well as M-2 FAg immunization against M-2 challenge reinforces the serological data on the antigenic similarity of the flagella of these two strains. Although immunization with the PA-103 nonFAg generated antibodies which can be demonstrated in agglutination reactions (Table 1), the

TABLE 2. Mortality in burned, M-2-infected mice after immunization with P. aeruginosa FAg preparations

Immunogen' Saline (control) PA-103 non-FAg (control) M-2 FAg RM-46 FAg

Mortalityb on day:

Challenge

dose and route

102 s.c.

102 s.c. 102 s.c. 102 s.c.

1

0/15 (0) 1/15 (7)

0/I5 (0) 0/15 (0)

2

14/15 (93) 14/15 (93) 0/15 (0) 0/15 (0)

5/5 (100) 108 topically 0/5 (0) Saline (control) 2/5 (40) PA-103 non-FAg (control) 108 topically 0/5 (0) 1/5 (20) 108 topically 0/5 (0) M-2 FAg 0/5 (0) 108 topically 0/5 (0) RM-46 FAg a One microgram of the immunogen was injected on day 0; challenge b No. dead/no. in group (%).

3

4

5

15/15 (100) 14/15 (93) 1/15 (7) 0/15 (0)

14/15 (93) 1/15 (7)

14/15 (93)

0/15 (0)

1/15 (7) 0/15 (0)

5/5 (100) 1/5 (20) 1/5 (20) 0/5 (0) 0/5 (0) took place on day 14.

1/5 (20) 0/5 (0)

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TABLE 3. Quantitative bacterial counts of skin and liver tissues in burned mice immunized with FAg preparations and challenged with P. aeruginosa strain M-2

Immunogena

No. of mice tested

Quantitative count (log1o ± SEM) in: Skin

Liver

6.1 ± 0.23 4 7.8 ± 0.38 Saline (control) 6.6 ± 0.11 8.2 ± 0.06 4 PA-103 non-FAg (control) 3.7 ± 0.30 5 7.5 ± 0.13 RM-46 FAg 7.3 ± 0.11 3.1 ± 0.24 5 M-2 FAg a One microgram of the immunogen was injected on day 0; burn and challenge (102 strain M-2 cells s.c.) took place on day 14; quantitative counts were made 25 h postchallenge.

which, however, did not agglutinate with antiFAg sera. The degree of protection afforded by Immunization with RM-46 and M-2 FAg prep- FAg immunization varied; 5-day survivals rangarations affected not only the survival, but also ing from 40 (SBI-S-challenged, RM-46 FAgthe distribution of the challenge strain in the immunized group) to 100%o (SBI-H-challenged, tissues of the infected mice. Although the num- RM-46 and M-2 FAg-immunized groups) of the ber of organisms in the skin tissues of all of the mice were seen. This difference was probably due to the variation in the innate virulence of the groups was in excess of 7 logs and although the organisms should have had a chance to spread to different challenging strains. Virulence in P. the major organs, this was not the case (Table 3). aeruginosa is very complex, and although our The saline and PA-103 non-FAg-immunized data show that motility is important, it is only mice had approximately a 3-log-higher number one of a variety of factors which contribute to of organisms per gram of wet weight of liver tis- the overall virulence of this microorganism. With Vibrio cholerae, in which motility is also sue than did the RM-46 and M-2 FAg-immunized mice. This may have occurred by the anti-FAg associated with virulence (4, 18), flagella appear antibody immobilizing the flagellated pseudomo- to be necessary for the organism to attach to the intestinal mucosa so that toxin can be delivered nas challenge organisms growing in the burned skin tissue. Perhaps only after the numbers of most efficiently (17). Immunization with crude newly generated organisms exceeded the capaci- V. cholerae flagellum vaccines causes a dety of the anti-FAg antibody pool to immobilize crease in the attachment of radiolabeled vibrios them could the organisms enter the circulation to the mucosa of challenged rabbits (19). It and be filtered by the liver. This restriction would seem less likely that this should occur in a would not be true for the controls and might pseudomonas burn wound infection, however. The following conclusions may be drawn from explain the higher liver counts in these groups. At the same time, reduction of liver EF-2 levels, our data. Motility does appear to be associated with the virulence of P. aeruginosa strains in a reflection of the action of pseudomonas exotoxin A, was also prevented (Table 4). This was burned rodent models. Immunization with FAg probably due to the fewer numbers of challeng- preparations provides enhanced protection to burned mice which are then infected with P. ing organisms reaching the liver (Table 3). The data presented in Table 5 show that, aeruginosa; the protection is associated with the although protection afforded by FAg immuniza- antigenic type of the flagellum, but not with the tion was unrelated to the 0 serotype, there was a relationship between the H serotype and protec4. Reduction of liver EF-2 levels in burned tion. Enhanced survival was observed when TABLE mice immunized with FAg preparations and mice immunized with FAg preparations prewith P. aeruginosa strain M-2 challenged pared from the 0 serotype B strains RM-46 and % Reduction' Immunogena 0 M-2 were challenged with clinical isolates of serotypes H, B, and G. On the other hand, mice Saline (control) .77.5 were protected only when challenged with PA-103 non-FAg (control) .85.0 strains which agglutinated with the RM-46 and RM-46 FAg .5.5 0 M-2 anti-FAg sera. Thus, FAg-immunized mice M-2 FAg showed enhanced survival when challenged with a One microgram of the immunogen was injected on strain SBI-S, a somatic antigen type G strain day 0; burn and challenge (102 strain M-2 cells s.c.) which agglutinated with the anti-FAg sera, but took place on day 14; EF-2 levels were measured 25 h no enhanced survival was observed when FAg- postchallenge. immunized mice were challenged with strain b Values were derived from pools of four individual GNB-1, also a somatic antigen type G strain livers. mortality data show that these antibodies do not

protect against bacterial challenge.

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INFECT. IMMUN.

TABLE 5. Protection by immunization with FAg preparations of burned mice challenged with various 0 serotypes of clinical isolates of P. aeruginosa Challenge strain (O serotype)

Agglutination with antiserum to: RM-46 FAg3 M-2 FAga PA-103 non-FAgb

Immunogen

1

% Mortality' on day: 3 4 5 2

SBI-1 (H)

+

+

-

M-2 FAg RM-46 FAg PA-103 non-FAg Saline

0 0 0 0

0 0 0 0 60 80 80 100

0 0 80

0 0 80

SBI-H (B)

+

+

-

M-2 FAg RM-46 FAg PA-103 non-FAg Saline

0 0 0 0

20 20 0 0 60 100 80 100

40 0

40 0

SBI-S (G)

+

+

-

M-2 FAg RM-46 FAg PA-103 non-FAg Saline

0 0 0 0

40 40 20 60 100 80 100

40 60

40 60

GNB-1 (G)

-

-

-

M-2 FAg RM-46 FAg PA-103 non-FAg Saline

0 0 0 0

60 80 60 100 40 100 80 100

80 100

a 0 serotype B. b O serotype G. c Five mice per group.

somatic antigens. Since P. aeruginosa only contains two major antigenic types of flagella (1, 7) we suggest that a bivalent flagellum vaccine may be useful. This vaccine would have potential use by itself or in combination with other antigenic substances shown to play a role in the pathogenesis of P. aeruginosa infections. 1.

2. 3. 4.

LITERATURE CITED Ansorg, R. 1978. Flagella specific H antigenic schema of Pseudomonas aeruginosa. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe A 242:228238. Craven, R. C., and T. C. Monffe. 1981. Motility and chemotaxis of three strains of P. aeruginosa used for virulence studies. Can. J. Microbiol. 27:458-460. Gill, D. M., and L. L. Dinius. 1973. The elongation factor 2 of mammalian cells assay method and relation to ribosome number. J. Biol. Chem. 248:654-658. Guentzel, M. N., and L. J. Berry. 1975. Motility as a virulence factor for Vibrio cholerae. Infect. Immun.

11:890-897.

5. Holder, I. A., and C. G. Haidarls. 1979. Experimental studies of the pathogenesis of infections due to Pseudomonas aeruginosa: extracellular protease and elastase as in vivo factors. Can. J. Microbiol. 25:593-599. 6. Honuma, J. Y. 1976. A new antigenic schema and live-cell slide-agglutination procedure for the infrasubspecific, serologic classification of Pseudomonas aeruginosa. Jpn. J. Exp. Med. 46:329-336. 7. Lanyi, B. 1970. Serological properties of Pseudomonas aeruginosa. II. Type-specific thermolabile (flagella) antigens. Acta Microbiol. Acad. Sci. Hung. 17:35-48. 8. Lin, P. V. 1974. Extracellular toxins of Pseudomonas aeruginosa. J. Infect. Dis. 130:594-599. 9. McManus, A. T., E. E. Moody, and A. D. Mason. 1980. Bacterial motility: a component in experimental Pseudo-

monas aeruginosa burn wound sepsis. Burns 6:235-239. 10. Miller, R. V., and J. M. Becker. 1978. Peptide utilization in Pseudomonas aeruginosa: evidence for membraneassociated peptidase. J. Bacteriol. 133:165-171. 11. Montie, T. C., R. C. Craven, and I. A. Holder. 1982. Flagellar preparations from Pseudomonas aeruginosa: isolation and characterization. Infect. Immun. 35:291298.

12. Pavlovskis, 0. R., and B. Wretllnd. 1979. Assessment of

13.

14.

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18. 19.

protease (elastase) as a Pseudomonas aeruginosa virulence factor in experimental mouse burn infection. Infect. Immun. 24:181-187. Saelinger, C. B., K. Snell, and I. A. Holder. 1977. Experimental studies on the pathogenesis of infections due to Pseudomonas aeruginosa: direct evidence for toxin production during pseudomonas infection of burned skin tissue. J. Infect. Dis. 136:555-561. Saymen, D. B., P. Nathan, I. A. Holder, E. 0. Mill, and B. G. MacMllan. 1972. Infected surface wound: an experimental model and a method for the quantitation of bacteria in infected tissues. Appl. Microbiol. 23:509-524. Snell, K., I. A. Holder, S. A. Leppla, and C. B. Seelinger. 1978. Role of exotoxin and protease as possible virulence factors in experimental infections with Pseudomonas aeruginosa. Infect. Immun. 19:839845. Stkerltz, D. D., and I. A. Holder. 1975. Experimental studies of the pathogenesis of infections due to Pseudomonas aeruginosa: description of a burned mouse model. J. Infect. Dis. 131:688-691. Yancy, R. J., and L. J. Berry. 1978. Motility of the pathogen and intestinal immunity of the host in experimental cholera, p. 447-455. In J. R. McGhee, J. Mestecky, and J. L. Babb (ed.), Secretory immunity and infection. Plenum Publishing Corp., New York. Yancy, R. J., D. L. Willis, and L. J. Berry. 1978. Role of motility in experimental cholera in adult rats. Infect. Immun. 22:387-392. Yancy, R. J., D. L. Willis, and L. J. Berry. 1979. Flagellainduced immunity against experimental cholera in adult rabbits. Infect. Immun. 25:220-228.