Serum Resistance in an Invasive, NontypeableHaemophilus influenzaeStrain

Abstract

A common feature of many different organisms causing bacteremia is the ability to avoid the bactericidal effects of normal human serum. In Haemophilus influenzae encapsulated strains are particularly serum resistant; however, we found that a nonencapsulated strain (R2866) isolated from the blood of an immunocompetent child with meningitis who had been successfully immunized with H. influenzae type b conjugate vaccine was serum resistant. Since serum resistance usually involves circumventing the action of the complement system, we defined the deposition of various complement components on the surfaces of this H. influenzae strain (R2866), a nonencapsulated avirulent laboratory strain (Rd), and a virulent type b encapsulated strain (Eagan). Membrane attack complex (MAC) accumulation correlated with the loss of bacterial viability; correspondingly, the rates of MAC deposition on the serum-sensitive strain Rd and the serum-resistant strains differed. Analysis of cell-associated immunoglobulin G (IgG), C1q, C3b, and C5b indicated that serumresistant H. influenzae prevents MAC accumulation by delaying the synthesis of C3b through the classical pathway. Among the initiators of the classical pathway, IgG deposition contributes most of the C3 convertase activity necessary to start the cascade ending with MAC deposition. Despite similar IgG binding, strain R2866 delays C3 convertase activity compared to strain Rd. We conclude that strain R2866 can persist in the bloodstream, in part by inhibiting or delaying C3 deposition on the cell surface, escaping complement mediated killing.

lgtC Expression Modulates Resistance to C4b Deposition on an Invasive Nontypeable Haemophilus influenzae

“…These data suggest that complement inhibition in R2866 occurs at or before the step of C4 binding. We were unable to detect differences in IgG (total IgG and IgG subclasses) or IgM binding to both strains Rd KW20 and R2866, which suggests that differences in the amount of Ab binding are not responsible for differences in C4b deposition (16). Furthermore, we did not observe in either strain binding to lectin pathway activators (mannose binding lectin and ficolins) or recruitment of complement regulatory proteins C4b-binding protein and C1-esterase inhibitor (data not shown).…”

“…Our initial studies suggested that delayed C3 deposition mediates serum resistance in strain R2866 and that a functional CP is required for complement activation (16). We hypothesized that complement inhibition on R2866 occurred between the initiation of CP activation and C3 deposition.…”

“…Using this approach, we have shown that C3 deposition on the surface of strain R2866 is delayed despite the binding of multiple Ab classes and C1 to a similar extent as the serum-susceptible strain Rd KW20. This phenotype is independent of alternative pathway activity, factor H recruitment, and sialylation or phosphorylcholine decoration of lipooligosaccharide (LOS) (16).…”

“…By ELISA analysis, we were unable to detect capsular polysaccharide production in R2866. Subsequent PCR and Southern analysis using serotypespecific primers revealed the lack of capsule biosynthetic genes (types a-f) and the bexA gene (required for capsule export), genetically corroborating the observed nontypeable phenotype (2,16). Because bloodstream survival and serum resistance usually require evasion of Ab and complement-mediated host defenses, we used kinetic flow cytometry to characterize the deposition of various complement components on the surface of R2866 and an avirulent, serum-sensitive laboratory strain, Rd KW20.…”

Adhesin genes and serum resistance in Haemophilus influenzae type f isolates

“…Unencapsulated strains, such as Rd KW20 (R652), are relatively serum sensitive compared with capsule-producing strains (e.g. Eagan), which can replicate in 50 % NHS and whole blood, or the rare serum-resistant non-typable strain INT-1 (R2866) ( Table 3) (Williams et al, 2001). In the serumresistance assay, the reported IC 50 was the mean (±SD) concentration of serum required to kill 50 % of the inoculum within a 30 min incubation period (Table 3).…”