Jun 14, 1983 - for strain Ul, and 5 min for strain S2. pathogenic determinant of capsule-deficient H. influenzae, was then examined by comparing the.
INFECTION
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Vol. 42, No. 2
IMMUNITY, Nov. 1983, P. 708-715
0019-9567/83/110708-08$02.00/0 Copyright C 1983, American Society for Microbiology
Participation of Complement in Host Defense Against Capsule-Deficient Haemophilus influenzae ANDR1 ZWAHLEN,l* JERRY A. WINKELSTEIN,2 AND E. RICHARD MOXON1 The Department of Pediatrics, The Subdepartment of Immunology, The Eudowood Divisions of Infectious Diseases' and Immunology,2 The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 Received 14 June 1983/Accepted 25 August 1983
To investigate the role of complement in immunity to capsule-deficient Haemophilus influenzae, rats were depleted of C3 with cobra venom factor and challenged with three different strains of capsule-deficient H. influenzae. Two of them (Rd and U1) did not elaborate type b capsular antigen, whereas the other (S2) elaborated 0.16% of the amount made by its type b parent strain. Depletion of C3 significantly enhanced early intravascular bacterial survival after intravenous inoculation and strikingly increased the susceptibility of rats to infection with capsule-deficient H. influenzae. After intraperitoneal inoculation with strain Rd or Ut, C3-depleted rats developed bacteremia, whereas control rats did not; challenge with strain S2 resulted in transient bacteremia in normal rats and in death in C3-depleted animals. To determine whether the greater virulence of strain S2, as compared with strain Rd or Ut, was accounted for by the small amounts of capsular antigen it elaborated, we also compared its relative virulence to that of three genetically closely related capsule-deficient variants elaborating either small amounts of type b capsule or producing no detectable b antigen. No difference in virulence was observed among these four variants; all C3-depleted rats inoculated developed bacteremia of similar magnitude followed by similar mortality rates. These studies demonstrate a significant role for complement in host defense mechanisms against capsule-deficient H. influenzae and suggest that interstrain differences of virulence are not attributable to residual elaboration of small amounts of type b capsule.
Although the elaboration of a type b capsule is a major determinant of Haemophilus influenzae virulence, nontypable strains have been occasionally isolated from patients with systemic infections. Thus, a recent study of bacteremia due to H. influenzae in adults indicated that more than 40% of the isolates were nontypable with antisera to the six known capsular antigens (types a through f) (31). Similarly, nontypable strains have been isolated from the blood and cerebrospinal fluid of neonates (5) and children (7, 11). Wallace et al. have also reported a high proportion of nontypable strains among H. influenzae causing septicemia in neonates or in parturient women (32). Experimental infection of rats provides a biologically relevant model of
H. influenzae systemic disease (19, 27). Previ-
ous studies have shown that although unencapsulated strains may colonize the upper respiratory tract of rats, they are relatively avirulent as compared with type b or other encapsulated
organisms (20, 33). The designation of a given strain as typable or nontypable is somewhat arbitrary and depends on the sensitivity and specificity of the typing
assay (31). Strains lacking a type-specific capsule do not exhibit the iridescent colonial morphology characteristic of encapsulated H. influenzae (23). Similarly, capsule-deficient strains spontaneously derived from encapsulated organisms have a noniridescent phenotype and are nontypable by slide agglutination, although they may still elaborate detectable but markedly reduced amounts of capsular antigen (3, 7). Such capsule-deficient strains elaborating residual type b capsular polysaccharide (PRP) have been identified among clinical isolates (7, 34). These observations raise the question of whether the elaboration of the capsular antigen in reduced quantities might still confer a degree of virulence upon such capsule-deficient strains as suggested by May (17). Alternately, capsuledeficient strains arising from a type b ancestor might retain virulence determinants distinct from the capsule. Previous investigations have shown that complement plays an important role in host defense
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against H. influenzae type b and encapsulated H. influenzae of other serotypes (a, c, d, and f) (8, 9) (Zwahlen, Winkelstein, and Moxon, J.
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CAPSULE-DEFICIENT H. INFLUENZAE
Infect. Dis., in press). Several studies have demonstrated that complement has the potential to mediate bacteriolysis and opsonization of nontypable H. influenzae strains (3, 6, 21, 26, 28, 29). The current studies were performed to determine whether the complement system plays a role in vivo in the host defense against capsule-deficient H. influenzae. The results indicate a biologically significant role for complement in the host defense against capsule-deficient H. influenzae and emphasize the importance of virulence determinants distinct from PRP elaboration.
Animals. Five-day-old suckling COBS/CD SpragueDawley albino rats (natural litters of 10) were purchased from Charles River Breeding Laboratories, Wilmington, Mass., pooled, randomly reassigned to a nursing mother, and maintained as previously described (19, 27). Forty-day-old female rats (average weight, 120 g) of the same strain were used for intravascular clearance studies. Complement depletion. Purified CoVF obtained from Naja naja venom was purchased from Cordis Laboratories, Miami, Fla. Animals were injected intraperitoneally (i.p.) with either CoVF or saline, using the same techniques, dosage, timing, and controls as before (8). The serum C3 functional hemolytic activity measured by the assay of Shin and Meyer (25) fell to less than 5% of the control values within 3 h after CoVF treatment and remained under 10o through day 4. Animal inoculation and cultures. Infant rats were challenged i.p. (27) with similar inocula of the different
MATERIALS AND METHODS
Bacteria. H. influenzae, strain Garfaceous, designated Rd and kindly donated by G. Leidy (Columbia University, New York, N.Y.), is a spontaneous capsule-deficient mutant of a type d strain originally isolated from the respiratory tract of a child (1). Strain Ul, also obtained from G. Leidy, is a nontypable H. influenzae strain isolated from the cerebrospinal fluid of a child. Strain Eag type b (streptomycin-resistant mutant) and its spontaneous laboratory-derived capsule-deficient mutant, S2 (33), were provided by P. Anderson (University of Rochester, Rochester, N.Y.). Three other capsule-deficient variant strains (Secl, Rt43, and Rt2O), directly derived from strain Eag and strain S2 as described in other studies (E. R. Moxon, R. A. Deich, and C. Connelly, submitted for publication), were selected for their differences in quantities of PRP elaborated. The methods of storage, growth, quantitation, identification, serotyping, and biotyping of H. influenzae were as previously described (19, 20, 27). Phenotypic characterization of the capsule-deficient strains. The elaboration of type b capsular antigen was assessed by enzyme-linked immunosorbent assay (ELISA) (22) to quantitate PRP in the cell-free supernatant of log-phase cultures (brain heart infusion supplemented with Levinthal base) at an optical density of 0.15 at 490 nm. The results were expressed as nanograms per 109 cells. Cell-associated PRP was assessed by immunofluorescence, using fluorescein-conjugated anti-PRP rabbit antibodies kindly provided by R. Schneerson and J. B. Robbins (Bureau of Biologics, Bethesda, Md.). The elaboration of capsular polysaccharides other than PRP was not assessed quantitatively. Outer membrane protein (OMP) was prepared from the strains as described by Barenkamp et al. (4) and analyzed by sodium dodecyl sulfate-polyacrylamide
(11% acrylamide) gel electrophoresis (SDS-PAGE) using the methods of Lugtenberg et al. (15); the running buffer, pH 8.3, contained 0.025 M Tris, 0.19 M glycine, and 0.1% SDS. H. influenzae lipopolysaccharides (LPS) extracted by a modification of the hot phenol water method (T. J. Inzana, J. Infect. Dis., in press) were compared by using SDS-PAGE (15% acrylamide, 3 M urea; same running buffer as for protein gels) and stained by silver (30); the controls and techniques were as previously (Zwahlen et al., in press). Purified LPS from Salmonella typhimurium SH 4809, used as a reference LPS, wsa kindly provided by S. E. Griesman (University of Maryland, Baltimore).
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strains of H. influenzae. In each randomized group of 10 animals, one-half had received CoVF, and one-half received saline. The organisms were grown, stored, and prepared for inoculation as described previously (19, 27). The virulence studies in which strains Rd, Ut, and S2 were compared used organisms that had been passed once through infant rats before being stored, thus reproducing the experimental conditions of our previous studies on capsular transformants of strain Rd (Zwahlen et al., in press). The relative virulence of the genetically related strains (Secl, S2, Rt43, and Rt2O) was assessed with organisms subcultured onto artificial medium. Serial 0.01-ml blood samples were obtained for quantitative culture at 12, 24, 48, 72, and 96 h after challenge, using the techniques previously described (19, 20, 27). Early intravascular clearance of
H. influenzae was assessed after intravenous (i.v.) inoculation of 105 to 106 organisms into five CoVF-
treated and five saline-treated 40-day-old rats as previously described (Zwahlen et al., in press). From the final blood sample, obtained by cardiac puncture at 5 or 10 min, 0.1- and 0.01-ml volumes were plated immediately onto solid media, and 0.5 ml was mixed in 4.5 ml of brain heart infusion supplemented with 10% Levinthal base and containing 0.03% polyanethole sulfonic acid (Sigma Chemical Co., St. Louis, Mo.) to inhibit in vitro coagulation, killing, and phagocytosis. Statistical analysis. Comparison of the incidence of bacteremia or mortality was performed by the twotailed Fisher exact test, and magnitude of bacteremia were compared by the Mann-Whitney rank test and two-tailed t test (18).
RESULTS Phenotypic analysis of the capsule-deficient strains. Table 1 summarizes the origins of the strains, their biotypes, and their phenotypic characteristics relating to elaboration of type b capsular antigen. Strains Rd, Ut, and Rt2O did not elaborate detectable amounts of PRP, whereas the variants Secl, S2, and Rt43 respectively elaborated 1.70, 0.16, and 0.04% of the amount of PRP made by their parent strain, Eag type b. Immunofluorescence studies confirmed the absence of cell-associated PRP in strain Ut
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TABLE 1. Origins and phenotypic characteristics of the capsule-deficient (noniridescent) H. influenzae strains Elaboration of PRPZ (ng/109 cells)
Immunofluorescence for PRP1b
Origin
Biotype
Rd
Spontaneous mutant of a type d respiratory isolate
IV
