Role of Lipopolysaccharide Phase Variation in Susceptibility of Haemophilus influenzae to Bactericidal Immunoglobulin M Antibodies in Rabbit Sera

Abstract

The effect of phase variation of lipopolysaccharide (LPS) structure on the susceptibility of Haemophilus influenzae to complement-dependent killing by normal human sera and normal rat sera has been described previously. The phase-variable structure phosphorylcholine (ChoP) confers susceptibility to human serum, since ChoP on the bacterial cell surface binds to serum C-reactive protein and activates complement. In contrast, expression of gal␣1,4gal, a second phase-variable epitope that is also found on human glycoconjugates, confers resistance to human serum. We studied the role of phase variation of these structures in the susceptibilities of H. influenzae KW20 (Rd) and a clinical isolate of nontypeable H. influenzae to killing by rabbit sera, which often possess naturally acquired complement-dependent bactericidal activity for unencapsulated H. influenzae. Expression of ChoP increased the resistance of strain KW20 to killing by bactericidal rabbit sera. In contrast, the serum resistance of a clinical isolate, H233, was unaffected by ChoP expression but was reduced by gal␣1,4gal expression. The rabbit sera with bactericidal activity (but not the nonbactericidal sera) all contained immunoglobulin M (IgM) antibodies able to bind to the surface of H. influenzae bacteria, as detected by flow cytometry, and contained IgM antibodies to LPS purified from strain KW20. Preincubation of sera with LPS reduced their bactericidal activity. Bactericidal activity was recovered quantitatively in an IgM-enriched fraction of sera. It is concluded that naturally occuring bactericidal activity for unencapsulated H. influenzae is largely due to IgM antibodies directed against phase-variable structures of the LPS.

HumanAntibodies Specific for the High-Molecular-Weight Adhesion Proteins ofNontypeable Haemophilus influenzae Mediate OpsonophagocyticActivity

“…Because nontypeable H. influenzae is susceptible not only to opsonophagocytic killing but also to complement-dependent bactericidal killing, if the complement source is capable of mediating both sorts of activities, it would be difficult in an assay such as this to distinguish between the two. It is well known that rabbit complement can support complement-dependent bacteriolysis of nontypeable H. influenzae and, in fact, often has naturally occurring bactericidal activity for these organisms (18). In contrast, guinea pig serum as a complement source is unable to support complement-dependent bactericidal activity with human antibodies (34).…”

“…The explanation for these differences can only be speculated upon at this point. The bacterial surface of nontypeable H. influenzae is a very dynamic structure and a number of surface molecules, some of which may undergo antigenic and phase variation, are known to influence the interactions of bacteria with the host immune system (13,16,18,46,47). In the case of the HMW proteins, variation in expression level may directly influence the ability of the immune system to clear infection in vivo (5) and similar differences in levels of HMW protein expression could influence the ability of antibodies directed against these proteins to mediate opsonophagocytic activity in vitro.…”

Phosphorylcholine Expression by NontypeableHaemophilus influenzaeCorrelates with Maturation of Biofilm Communities In Vitro and In Vivo

“…Factors that undergo phase variation are commonly thought to confer some fitness advantage on the bacterial population only during a discrete phase of the infectious cycle, outside of which they may confer no benefit or even be detrimental (34,37). PCho, for example, is known to confer a number of advantages on NTHI (22,32,35,38,39) while also (in at least some structural settings) targeting bacteria for the binding of C-reactive protein or antibody and subsequent clearance (6)(7)(8)21). Consistent with this idea, Humphries and High showed that constitutive expression of the lic1 locus (and thus, presumably, expression of PCho) resulted in attenuation of encapsulated H. influenzae in an infection model of invasive disease (15).…”

Microbial Modulation of Host Immunity with the Small Molecule Phosphorylcholine

“…When CRP is readily available, such as in rat or human serum during infection, ChoP-expressing H. influ-enzae strains are more sensitive to complement-mediated killing than bacteria not expressing ChoP (2,91). In contrast, ChoPexpressing bacteria have the advantage in mouse and rabbit models of bloodstream infection, in which CRP levels remain low (92,93). The pentraxin serum amyloid protein (SAP) can also bind ChoP, although no bactericidal effect has been demonstrated (94).…”