Phosphorylcholine Allows for Evasion of Bactericidal Antibody by Haemophilus influenzae

The human pathogen Haemophilus influenzae has the ability to quickly adapt to different host environments through phase variation of multiple structures on its lipooligosaccharide (LPS), including phosphorylcholine (ChoP). During colonization with H. influenzae, there is a selection for ChoP+ phase variants. In a murine model of nasopharyngeal colonization, this selection is lost in the absence of adaptive immunity. Based on previous data highlighting the importance of natural antibody in limiting H. influenzae colonization, the effect of ChoP expression on antibody binding and its bactericidal activity was investigated. Flow cytometric analysis revealed that ChoP+ phase variants had decreased binding of antibody to LPS epitopes compared to ChoP− phase variants. This difference in antibody binding correlated with increased survival of ChoP+ phase variants in the presence of antibody-dependent, complement-mediated killing. ChoP+ phase variants were also more resistant to trypsin digestion, suggesting a general effect on the physical properties of the outer membrane. Moreover, ChoP-mediated protection against antibody binding correlated with increased resilience of outer membrane integrity. Collectively, these data suggest that ChoP expression provides a selective advantage during colonization through ChoP-mediated effects on the accessibility of bactericidal antibody to the cell surface

The fbpABC Operon Is Required for Ton-Independent Utilization of Xenosiderophores by Neisseria gonorrhoeae Strain FA19▿

Neisseria gonorrhoeae produces no known siderophores but can employ host-derived, iron-binding proteins, including transferrin and lactoferrin, as iron sources. Given the propensity of this pathogen to hijack rather than synthesize iron-sequestering molecules, we hypothesized that the ability to use siderophores produced by other bacteria, or xenosiderophores, may also play a role in the survival of the gonococcus. Among a panel of diverse siderophores, only the catecholate xenosiderophores enterobactin and salmochelin promoted growth of gonococcal strain FA19. Surprisingly, the internalization pathway was independent of TonB or any of the TonB-dependent transporters. Xenosiderophore-mediated growth was similarly independent of the pilin-extruding secretin formed by PilQ and of the hydrophobic-agent efflux system composed of MtrCDE. The fbpABC operon encodes a periplasmic-binding-protein-dependent ABC transport system that enables the gonococcus to transport iron into the cell subsequent to outer membrane translocation. We hypothesized that the FbpABC proteins, required for ferric iron transport from transferrin and lactoferrin, might also contribute to the utilization of xenosiderophores as iron sources. We created mutants that conditionally expressed FbpABC from an IPTG-inducible promoter. We determined that expression of FbpABC was required for growth of gonococcal strain FA19 in the presence of enterobactin and salmochelin. The monomeric component of enterobactin, dihydroxybenzoylserine (DHBS), and the S2 form of salmochelin specifically promoted FbpABC-dependent growth of FA19. This study demonstrated that the gonococcal FbpABC transport system is required for utilization of some xenosiderophores as iron sources and that growth promotion by these ferric siderophores can occur in the absence of TonB or individual TonB-dependent transporters

Type IV Secretion Machinery Promotes Ton-Independent Intracellular Survival of Neisseria gonorrhoeae within Cervical Epithelial Cells▿

Survival of Neisseria gonorrhoeae within host epithelial cells is expected to be important in the pathogenesis of gonococcal disease. We previously demonstrated that strain FA1090 derives iron from a host cell in a process that requires the Ton complex and a putative TonB-dependent transporter, TdfF. FA1090, however, lacks the gonococcal genetic island (GGI) that is present in the majority of strains. The GGI in strain MS11 has been partially characterized, and it encodes a type IV secretion system (T4SS) involved in DNA release. In this study we investigated the role of iron acquisition and GGI-encoded gene products in gonococcal survival within cervical epithelial cells. We demonstrated that intracellular survival of MS11 was dependent on acquisition of iron from the host cell, but unlike the findings for FA1090, expression of the Ton complex was not required. Survival was not dependent on a putative TonB-like protein encoded in the GGI but instead was directly linked to T4SS structural components in a manner independent of the ability to release or internalize DNA. These data suggest that expression of selected GGI-encoded open reading frames confers an advantage during cervical cell infection. This study provides the first link between expression of the T4SS apparatus and intracellular survival of gonococci

ChoP expression affects outer membrane accessibility and barrier function.

<p>Flow cytometric analysis of the MFI of Cy5 binding to outer membrane surface proteins following trypsin digestion for phase variants of NTHi strain H233 and revertants of the originally selected phase variants and constitutive ChoP+ and ChoP− mutants in Rd (A). Rate of ethidium bromide (EtBr) uptake, expressed in relative fluorescent units per second (RFU/s), in the presence of polymyxin B for phase variants of NTHi strain H233 and revertants and constitutive ChoP+ and ChoP− mutants in Rd (B). ChoP+ variants are shown in black bars, ChoP− variants are shown in grey bars. Values are derived from at least three independent experiments ± SD, *p<.05, **p<.01, ***p<.001.</p

ChoP expression protects against bactericidal antibody.

<p>Percent survival in C-reactive protein (CRP)-depleted NHS (relative to complement-inactivated control) is shown for phase variants of NTHi strain H233 and revertants of the originally selected phase variants (A). Percent survival in purified IgG from NHS with BRS (complement source) for phase variants of H233 (B). Percent survival in CRP-depleted NHS for constitutive ChoP+ and ChoP− mutants in Rd, phase variants of type b strain Eagan, and multiple NTHi strains (C). Percent survival of NTHi strain H233 ChoP− phase variant in NHS, IgG-depleted NHS, or in IgG-depleted NHS supplemented with purified IgG, is shown as indicated (D). ChoP+ variants are shown in black bars, ChoP− variants are shown in grey bars. Values are derived from at least three independent experiments ± SD, *p<.05, **p<.01, ***p<.001.</p

List of strains used in this study.

a<p>Sm, spontaneous streptomycin resistant mutant,</p>b<p>COPD, chronic obstructive pulmonary disease,</p>c<p>Km, contains kanamycin resistance cassette,</p>d<p>H3, ChoP attached to hexose extension from Heptose_III,</p>e<p>H1, ChoP in natural position (for Rd) attached to hexose extension from Heptose_I,</p>f<p>type b- spontaneous mutant lacking both copies of the <i>cap</i> locus.</p

ChoP expression increases outer membrane integrity.

<p>Percent survival following incubation in EDTA was determined for ChoP+ (black solid), ChoP− (grey solid), and ChoP−,Galα1-4Gal+ (grey dashed) phase variants of NTHi strain H233 (A). Percent survival in EDTA for Rd constitutive ChoP+ strain H491 grown in CDM alone (grey solid), or supplemented with choline (black solid) or with dimethylethanolamine (grey dashed) (B). Percent survival in EDTA for Rd <i>lic1D</i> exchange mutants, Rd <i>lic1D</i> (H1) (C) and Eagan <i>lic1D</i> (H3) (D). Flow cytometric analysis of the MFI of NHS IgG binding in the presence of 0–50 mM Mg²⁺ for ChoP+ (black bars) and ChoP− (grey bars) phase variants of NTHi strain H233 (E). Values are representative of three independent experiments performed in duplicate ± SD (A–D), or are derived from three independent experiments ± SD *p<.05, **p<.01 (E).</p

Position of ChoP affects ChoP-mediated protection against antibody binding.

<p>Flow cytometric analysis of the MFI of IgG binding to phase variants of Rd <i>lic1D</i> exchange mutants; H491 with Rd <i>lic1D</i> (Heptose_I, or H1), and H457 with Eagan <i>lic1D</i> (Heptose_III, or H3), from BALB/c serum (A) or NHS (B). Percent survival (relative to complement-inactivated control) was determined following incubation in CRP-depleted NHS (C). ChoP+ variants are shown in black bars, ChoP− variants are shown in grey bars. Values are derived from at least three independent experiments ± SD, *p<.05, **p<.01.</p

Adaptive immunity is required for selection of ChoP+ phase variants during colonization.

<p>BALB/c or BALB/c (severe combined immune deficiency, or SCID) mice were intranasally inoculated with 10⁷ CFU/mL of a 3∶1 mixture of ChoP−∶ChoP+ phase variants of NTHi strain H632. The percentage of ChoP+ phase variants was determined by colony immunoblotting of the inoculum (input, white bars) and the nasal lavage fluid following three days of colonization (output, stippled bars). Values are derived from five mice per group ± SD, ***p<.0001.</p