The rickettsial OmpB β-peptide of Rickettsia conorii is sufficient to facilitate factor H-mediated serum resistance

Abstract

Pathogenic species of the spotted fever group Rickettsia are subjected to repeated exposures to the host complement system through cyclic infections of mammalian and tick hosts. The serum complement machinery is a formidable obstacle for bacteria to overcome if they endeavor to endure this endozoonotic cycle. We have previously demonstrated that that the etiologic agent of Mediterranean spotted fever, Rickettsia conorii, is susceptible to complement-mediated killing only in the presence of specific monoclonal antibodies. We have also shown that in the absence of particular neutralizing antibody, R. conorii is resistant to the effects of serum complement. We therefore hypothesized that the interactions between fluid-phase complement regulators and conserved rickettsial outer membrane-associated proteins are critical to mediate serum resistance. We demonstrate here that R. conorii specifically interacts with the soluble host complement inhibitor, factor H. Depletion of factor H from normal human serum renders R. conorii more susceptible to C3 and membrane attack complex deposition and to complement-mediated killing. We identified the autotransporter protein rickettsial OmpB (rOmpB) as a factor H ligand and further demonstrate that the rOmpB β-peptide is sufficient to mediate resistance to the bactericidal properties of human serum. Taken together, these data reveal an additional function for the highly conserved rickettsial surface cell antigen, rOmpB, and suggest that the ability to evade complement-mediated clearance from the hematogenous circulation is a novel virulence attribute for this class of pathogens.

Fig 1

R. conorii interacts with complement factor H. (A) R. conorii was incubated with PBS or 50% NHS. After washing, interacting serum proteins were eluted from the bacteria with 1 M NaCl. The cell-free elutions were subjected to SDS-PAGE and anti-fH immunoblotting. A 155-kDa fH-reactive species is present after NHS incubation with R. conorii. Sizes are indicated in kilodaltons (kDa). (B) R. conorii were incubated with PBS, 10% NHS, or 10 μg of purified factor H/ml. After washing, the fH deposition on the bacteria was detected by flow cytometric analysis of fluorescent anti-fH deposition. All events shown were contingent upon the presence of DNA (as demonstrated by DAPI staining), which represented rickettsiae. Red, PBS; green, 10% NHS; blue, 50 μg of fH/ml.

Fig 2

Association with factor H promotes R. conorii resistance to complement-mediated killing. (A) R. conorii was incubated in 50% NHS or factor H-depleted human serum (fHDplHS), followed by quantitation of viable bacteria remaining. Values are expressed as the percentage of bacteria recovered compared to the bacteria incubated in PBS. The data represent three reactions for each condition and a minimum of three repetitions. P values are derived from unpaired Student t test. (B and C) Bacteria incubated with PBS (red), NHS (blue), or fHDplHS (green) were analyzed by flow cytometry for deposition of C3 (B) or the bactericidal MAC (C5b-9) (C) using fluorescently conjugated antibodies. Incubation of the bacteria in fHDplHS (green) results in more deposition of C3 and C5b-9 than NHS (blue) or PBS (red). Complement deposition is also demonstrated by an increase in the percentage of bacteria that have a fluorescence signal higher than the PBS-treated control. A total of 100,000 bacteria were analyzed for each histogram. The bars in panels B and C demarcate the data used for the area-under-the-curve statistical analysis. Sizes are indicated in panels A and C in kilodaltons (kDa).

Fig 3

rOmpB β-peptide binds factor H. (A) The indicated proteins were incubated, immunoprecipitated with goat anti-human fH antibody, separated on SDS-PAGE, and stained with Coomassie blue (upper panel). The arrow indicates the band that only coimmunoprecipitated when both fH and rickettsial lysate were present and was excised for analysis by mass spectrometry. The efficacy of the fH immunoprecipitation was verified by Western blotting (lower panel). (B) Protein sequencing of the excised band yielded peptide sequences (indicated in boldface) corresponding to the β-peptide (βp) of the autotransporter protein rOmpB. Amino acid sequence coverage of the rOmpB βp was 43.3%. No peptides were recovered from the remaining portion of rOmpB. Sizes are indicated in kilodaltons (kDa). (C) E. coli BL21 harboring the empty vector pEt22b, plasmid encoding for the rOmpB βp (pYC6), or control plasmid encoding for the Sca2 βp (pMC014) was induced with IPTG. Total detergent soluble lysates from these bacteria were incubated with purified fH and subsequently subjected to anti-fH immunoprecipitation. fH immunoprecipitated equally from the indicated samples (upper panel). A His₆-reactive species only coimmunoprecipitated with fH upon expression of the rOmpB βp (middle panel). Total protein lysates from the indicated samples were analyzed by Western immunoblotting to confirm recombinant protein expression (bottom panel, input).

Fig 4

Expression rOmpB βp is sufficient to mediate survival in human serum. (A) E. coli BL21(DE3) harboring empty vector (pEt22b) or pYC6 (rOmpB βp) was incubated in PBS or NHS and analyzed for serum survival. Uninduced bacteria (−IPTG) do not survive in NHS. (B) Western immunoblot analysis with anti-His₆ antibody reveals that expression of rOmpB βp (arrow) at the OM correlates with the ability to survive in NHS.

Fig 5

Analysis of rOmpB βp conservation and structure. (A) Alignment of rOmpB βp from diverse pathogenic Rickettsia spp. Indicated below the protein sequences are the predicted transmembrane β-sheets (yellow arrows) and six surface-exposed peptide loops (bracketed). (B) The model of rOmpB βp in the bacterial OM clearly demonstrates the β-barrel structure with 12 transmembrane β-sheets (yellow), a central α-helix that is the precleavage link to the passenger domain (red), and an approximation of the membrane localization (blue). The six extracellular loops protrude away from the OM and are readily exposed to the extracellular environment.