The Rickettsial OmpB �-Peptide of Rickettsia conorii Is Sufficient ToFacilitate Factor H-Mediated Serum Resistance

Sean P. Riley,a,b Jennifer L. Patterson,a* and Juan J. Martineza,b

University of Chicago, Department of Microbiology, Chicago, Illinois, USA,a and Howard T. Ricketts Laboratory, Argonne, Illinois, USAb

Pathogenic species of the spotted fever group Rickettsia are subjected to repeated exposures to the host complement systemthrough cyclic infections of mammalian and tick hosts. The serum complement machinery is a formidable obstacle for bacteriato overcome if they endeavor to endure this endozoonotic cycle. We have previously demonstrated that that the etiologic agentof Mediterranean spotted fever, Rickettsia conorii, is susceptible to complement-mediated killing only in the presence of specificmonoclonal antibodies. We have also shown that in the absence of particular neutralizing antibody, R. conorii is resistant to theeffects of serum complement. We therefore hypothesized that the interactions between fluid-phase complement regulators andconserved 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 humanserum 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 therOmpB �-peptide is sufficient to mediate resistance to the bactericidal properties of human serum. Taken together, these datareveal an additional function for the highly conserved rickettsial surface cell antigen, rOmpB, and suggest that the ability toevade complement-mediated clearance from the hematogenous circulation is a novel virulence attribute for this class ofpathogens.

Gram-negative alphaproteobacteria of the genus Rickettsia aresmall (0.3 to 0.5 �m by 0.8 to 1.0 �m), obligate intracellular

organisms. Spotted fever group (SFG) rickettsiae, including Rick-ettsia conorii (Mediterranean spotted fever [MSF]), are patho-genic organisms transmitted to humans through tick salivary con-tents during the blood meal. Commensurate mortality rates havebeen reported to be as high as 32% in Portugal in 1997 (17). Onceestablished in the host, SFG rickettsiae primarily infect the endo-thelial lining of the vasculature. Damage to target endothelial cells,especially in the lungs and brain, can result in the most severemanifestations of disease (77). Misdiagnosis of SFG Rickettsia in-fection is associated with severe disease outcomes, including acuterenal failure, pulmonary edema, interstitial pneumonia, neuro-logical pathology, and other multiorgan manifestations (63).

A major component of the rickettsial outer membrane (OM),rOmpB, has previously been demonstrated to mediate adherenceto and invasion of host cells (13, 26, 75). rOmpB belongs to afamily of Gram-negative proteins called autotransporters, whichhave a modular structure including a N-terminal secretion signal,central passenger domain, and C-terminal �-peptide (�p) (36).rOmpB is initially translated as a 168-kDa polypeptide and iscleaved at the OM to yield a 120-kDa surface-exposed passengerdomain that remains loosely associated with the OM and a 32-kDaintegral OM �p (27). The �p assumes a 12-stranded �-sheet-richbarrel confirmation that spans the bacterial OM, whereby the un-folded passenger domain is translocated from the periplasm to theextracellular milieu through the barrel pore (22). Despite a sharedtertiary barrel shape, �-peptides contain various numbers ofmembrane-spanning strands and have been demonstrated to per-form various functions, including porins, transporters, enzymes,and receptors (83).

Although Rickettsia species are exposed to blood during trans-mission both to and from the tick vector or potentially duringdissemination, little attention has been paid to the potential anti-

microbial effects of serum complement. Host complement is a keycomponent of the innate immune system that includes both anti-microbial and proinflammatory properties (78, 79, 85). Comple-ment consists of approximately 30 proteins, which togethercomplete a cascade-like process starting from recognition of im-munogens and results in direct microbial killing, inflammation,and enhancement of the adaptive immune reaction. Althoughcomplement can be initiated through various mechanisms, allmolecular pathways converge at C3 deposition on a target surfaceand its subsequent conversion to an unstable protease called C3convertase. This protease initiates the cascade for deposition of anti-microbial pore-like structure deemed the terminal complementcomplex (C5b-9 [TCC]) (35, 46, 48, 52, 59, 73). The direct microbialkilling mechanism is quite robust in the absence of microbial coun-termeasures (42, 43, 52). In addition, the latter steps in complementactivation produce major anaphlatoxins and result in opsonophago-cytosis of the pathogen (1, 21, 23, 84).

We have previously demonstrated that R. conorii is resistant toserum complement in the absence of specific neutralizing anti-bodies (14). Antibody recognition of its cognate ligand on a bac-terial surface and subsequent bacterial killing is likely attributableto the classical complement pathway, although other methods for

Received 2 April 2012 Returned for modification 26 April 2012Accepted 14 May 2012

Published ahead of print 21 May 2012

Editor: R. P. Morrison

Address correspondence to Juan J. Martinez, jmartine@bsd.uchicago.edu.

* Present address: Jennifer L. Patterson, Illinois State Police, Forensic ScienceCenter at Chicago, Chicago, Illinois, USA.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

doi:10.1128/IAI.00349-12

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complement activation cannot be completely eliminated. Inter-estingly, R. conorii incubated in nonimmune serum is not sensitiveto the spontaneous complement activation associated with thealternative pathway. This bacterial phenotype resembles the nat-ural host state where complement is intrinsically inhibited. Due tothe strong pathology associated with aberrant complement acti-vation, mammals encode for various fluid-phase and cell-associ-ated regulators of complement (11, 42, 50, 85).

Factor H (fH) is a single peptide of �155 kDa that consists of20 repetitive units of 60 amino acids that is described from struc-tural examination as “beads on a string” (5, 19, 41, 54, 56, 58).Factor H and the transcriptional splice variant, FHL-1, have threemajor functions: (i) accelerate the decay of the alternative pathway(AP) complement 3 (C3) convertase (C3bBb), (ii) compete withfactor Bb for access to C3b, and (iii) serve as a cofactor for factorI-mediated proteolytic inactivation of C3 (30, 55, 80, 82, 86). Thenormal host ligands of factor H include C-reactive protein (CRP),DNA, annexin II, and polyanionic polysaccharides (44, 57). Whilethese ligands serve to protect the host from aberrant complementdeposition, bacteria have frequently corrupted these host ligandsfor their own advantage. Streptococcus pyogenes (9, 34, 37, 39), S.pneumoniae (20, 53), Salmonella enterica (32), Yersinia spp. (6–8,15), Neisseria gonorrhoeae (62, 67), N. meningitidis (45, 61, 66),Haemophilus influenzae (28), Borrelia burgdorferi (3, 31, 40),Treponema denticola (49), Echinococcus granulosus (18), and thehuman immunodeficiency virus (70–72) all bind the soluble APinhibitors, factor H, or FHL-1. These bacteria therefore have anactive mechanism for preventing AP activation and subsequentdeleterious effects of complement.

MATERIALS AND METHODSPlasmid construction. R. conorii Malish7 ompB fragments were amplifiedby PCR from a chromosomal preparation. The region encoding therOmpB �-peptide was amplified using primers 5=-GGATCCACCTGAAGCTGGAGCAATACCG-3= and 5=-GGCTCGAGGAAGTTTACACGGACTTTTAG-3=, BamHI and XhoI digested (restriction sites underlined),and subsequently inserted into similarly digested pEt22b to constructpYC6. The pYC6-encoded protein contains the translational fusion of theN-terminal Escherichia coli PelB signal sequence, the R. conorii rOmpB�-peptide, and the C-terminal His6 tag under the control of an IPTG(isopropyl-�-D-thiogalactopyranoside)-inducible promoter. pMC014,which encodes for the R. conorii Sca2 �-peptide, was constructed similarlyto pYC6 as described above using the pET22b vector and the primers5=-AAGGATCCGGAAACTAGTATAACAGAGGGGTATGG-3= and 5=-AACTCGAGCAAATTGACTTTTAGTTTAATAAGCCCT-3=.

Bacterial growth. R. conorii Malish7 were propagated from Vero cellsand purified as described previously (4, 13, 14). Briefly, infected Vero cellmonolayers were lysed by needle and purified over a 20% sucrose cushion.Bacteria were stored at �80°C in 218 mM sucrose, 3.8 mM KH2PO4, and4.9 mM L-glutamate (pH 7.2). The yielded bacteria were pure and free ofhost contamination as visualized by microscopy. E. coli BL21(DE3)(pYC6,pMC014, or pET22b) was grown overnight in Luria-Bertani medium (LB)plus 50 �g of carbenicillin/ml at 37°C from a single colony. The bacteria werediluted 1:10 in fresh medium, grown to optical density at 600 nm (OD600) of0.5, and induced with 0.1 mM IPTG for 3 h at 37°C where appropriate.

Bacterial fractionation. Approximately 107 PFU of R. conorii or 1.0ml of a culture of E. coli BL21(DE3) at an OD600 of �1.0 harboring theappropriate plasmid was washed in phosphate-buffered saline (PBS), andthe total detergent soluble lysates were generated by incubating the bac-teria in 200 �l of BPER II bacterial protein extraction reagent (ThermoScientific/Pierce, Rockford, IL) containing 1� complete protease inhibi-tor cocktail (Complete, Inc.) according to the manufacturer’s directions.OM protein fractions were generated essentially as described previously

(74). Briefly, 5.0-ml portions of induced and noninduced E. coli harboringpET22b or pYC6 (OD600 of �1.0) were harvested, resuspended in 1.0 mlof 20 mM Tris (pH 8.0) containing 1� protease inhibitor cocktail, andthen disrupted by sonication. Unbroken cells were removed by centrifu-gation, and soluble lysates were incubated at room temperature for 5 minwith Sarkosyl (0.5% final concentration). The samples were then centri-fuged at 100,000 � g to isolate the OMs, and the OM fractions weresolubilized in 2� SDS-PAGE buffer. Proteins were separated by SDS-PAGE, transferred to nitrocellulose, and then immunoblotted with rabbitanti-His6 antibody (Covance) and goat anti-rabbit IgG conjugated tohorseradish peroxidase (HRP; Sigma-Aldrich, St. Louis, MO). His6-reac-tive species were revealed by chemiluminescence and exposure to film.

Detection of factor H interaction with R. conorii. Paraformalde-hyde-fixed R. conorii was incubated in PBS or in a 50% normal humanserum (NHS; human type AB; Lonza, Hopkinton, MA)-PBS mixture for 2h at room temperature with shaking. Each sample was washed twice bycentrifugation and suspension in PBS. In order to elute interacting pro-teins from the bacteria, the mixture was resuspended in a 1 M NaCl-PBSmixture, followed by incubation for 20 min. The remaining bacteria wereremoved by centrifugation. The final cell-free elution was subjected toSDS-PAGE for immunoblot determination of the presence of fH usinggoat anti-human fH (Complement Technology Inc., Tyler, TX) and don-key anti-goat-HRP (Sigma). For flow cytometric analysis, paraformalde-hyde-fixed R. conorii were incubated in PBS, 10% NHS, or 50 �g of puri-fied fH/ml for 1 h at 37°C with shaking. Each sample was washed twice bycentrifugation and resuspended in PBS. fH deposition was detected withgoat anti-fH (Complement Tech) and Alexa Fluor 488-conjugated rabbitanti-goat antibody (Molecular Probes). Then, 5 �g of DAPI (4=,6=-di-amidino-2-phenylindole)/ml was added to distinguish the bacteria frombackground events. The samples were analyzed with an LSR-II cytometer(BD Biosciences, San Jose, CA) using fluorescein isothiocyanate (FITC;488-nm excitation, 530/30-nm emission) and DAPI (325-nm excitation,450/50-nm emission) fluorescence settings. Analysis of fH deposition waspredicated upon DAPI positivity. All samples were analyzed with FlowJosoftware (Tree Star).

Serum sensitivity assay. R. conorii from Vero cell purified stocks wereresuspended in PBS and aliquoted to a final mixture of 100% PBS, 50%NHS-PBS, or 50% fH-depleted human serum (fHDplHS; Comptech)-PBS. Each of these mixtures was incubated for 1 h at 37°C with rotation,followed by centrifugation and recovery in ice-cold brain heart infusionmedium. These final suspensions were titered as described previously (14)to determine the viable R. conorii remaining. All values are expressed aspercentage of bacteria present after incubation with serum samples as afunction of those incubated only in PBS. E. coli BL21(DE3) harboringpET22b or pYC6 was grown as described above and induced with 0.1 mMIPTG (�IPTG) or left uninduced (�IPTG). Bacteria were diluted andwashed in PBS and then approximately 5 � 106 CFU were resuspended in200 �l of PBS or 30% NHS-PBS, followed by incubation for 1 h at 37°Cwith rotation. After incubation, the samples were serially diluted in PBSand then plated on LB agar plates to determine the recovered CFU. Thedata are presented as the number of bacteria recovered in PBS and 30%NHS-PBS after the 1-h incubation period and plotted on a logarithmicscale. Independent triplicate samples were processed for each experimen-tal condition, and the experiment was repeated a minimum of three times.

Complement component deposition on R. conorii. R. conorii fromVero cell purified stocks was resuspended in PBS and aliquoted into a finalmixture of PBS, 50% NHS-PBS, or 50% fHDplHS-PBS. Each of thesemixtures was incubated for 1 h at 37°C with rotation, followed by centrif-ugation, washing in PBS, and suspension in 4% paraformaldehyde. Sam-ples were split for flow cytometric analysis of C3 or C5b-9 deposition. C3was detected with FITC-conjugated goat (Fab=) anti-human C3 (ProtosImmunoresearch, Burlingame, CA). C5b-9 was detected with mouse anti-polyC9 (membrane attack complex [MAC]; Dako, Carpinteria, CA) andAlexa Fluor 488-conjugated goat anti-mouse antibody. Flow cytometricanalysis was performed as described above.

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fH immunoprecipitation. Whole-cell soluble protein lysates were in-cubated with 1.25 �g of purified human fH (Comptech) overnight at 4°C,and complexes were captured using goat anti-human fH sera (1:100 dilu-tion) and protein G-Sepharose. Immunoprecipitates were separated onSDS-PAGE and either stained with Coomassie blue or transferred to ni-trocellulose and immunoblotted with goat anti-human fH antibody (1:5,000) plus HRP-conjugated donkey anti-goat IgG (1:5,000) or with rab-bit anti-His6 antibody (1:1,000) plus HRP-conjugated goat anti-rabbitIgG (1:5,000). Reactive species were revealed by Super Signal West Picochemiluminescent substrates and exposure to film. Protein bands of in-terest were excised from the gel and analyzed by mass spectrometry.

Protein modeling. �-Peptide alignments from Rickettsia spp. wereperformed using the CLUSTAL W function in MacVector software(MacVector, Cary, NC) using the input accession sequences AAD39533(R. conorii), NP_221064 (R. prowazekii), YP_067640 (R. typhi),YP_001495174 (R. rickettsii), and AFC75259 (R. parkeri). The rOmpB �peptide consisting of amino acids 1335 to 1655 of the full-length peptidesequence was processed by the Phyre protein structural prediction server(38, 76). Secondary structure predictions were estimated using the Phyreoutput. The Phyre algorithm yielded multiple rOmpB �p models basedon crystal structures of autotransporter EstA from Pseudomonas aerugi-nosa (Protein Data Bank [PDB] no., 3KVN), the precleavage structure ofthe autotransporter EspP from Escherichia coli O157:H7 (PDB, 3SLJ), thetranslocator domain of autotransporter NalP from Neisseria meningitidis(PDB, 1UYN), and the beta domain of the Bordetella pertussis autotrans-porter BrkA (PDB, 3QQ2). The final model was based on the EstA struc-ture and was modeled with 100% confidence and 96% coverage. Likelymembrane localization and surface exposure was determined by model-ing the hydrophobic interfaces of the transmembrane �-strands based onJmol analysis (Jmol is an open-source Java viewer for chemical structuresin three dimensions [http://www.jmol.org/]).

RESULTSR. conorii binds complement factor H. Having previously shownthat R. conorii is resistant to complement-mediated killing inNHS (14), we sought to identify the bacterial mechanism of resis-tance. We hypothesized that rickettsiae would bind one of thesoluble complement regulatory proteins found in serum. Of themost abundant complement regulating proteins found in serum,factor H (fH) has previously been shown to interact with variousbacterial ligands (reviewed in reference 24). To determinewhether this interaction occurs in R. conorii, we incubated bacte-ria with NHS and queried for the association of complement fH.As shown in Fig. 1A, we detected a salt-sensitive interaction be-tween fH and R. conorii when incubated in NHS but not in PBS byimmunoblot. To further confirm this interaction, we incubated R.conorii with PBS, 10% NHS, or 50 �g of purified fH/ml and subse-quently queried for the presence of fH on the surface of the bacteria byflow cytometry. The fluorescence associated with fH binding to thesurface of the bacteria significantly increases when incubated witheither NHS or purified human fH (Fig. 1B). Taken together, theseexperiments confirm that R. conorii interacts with fH.

fH binding protects R. conorii from complement-mediatedkilling. To determine whether fH association can be a mechanismof resistance to alternative pathway (AP) complement, we incu-bated R. conorii with PBS, NHS, or factor H-depleted human se-rum (fHDplHS) and determined the titer of the surviving R. cono-rii. Depletion of factor H from NHS results in bacterial killing,since only 27% of the rickettsiae survive incubation in fHDplHScompared to incubation in NHS (Fig. 2A). These results demon-strate that association of R. conorii with human fH promotes in-hibition of AP and contributes to R. conorii survival in serum.

Factor H binding prevents C3 and MAC deposition. In mam-

mals, fH inhibits AP-complement by disrupting the C3 convertaseand by serving as a cofactor for factor I-mediated proteolysis (55,82). As such, the fH activity can be queried by examining the lattersteps of complement activation, including C3 and membrane at-tack complex (MAC; C5b-9) deposition on the bacteria. In orderto confirm that normal fH-mediated functions are occurring atthe rickettsial surface, we queried for complement activation inthe presence of serum. R. conorii was incubated with PBS, NHS, orfHDplHS, followed by washing and fixation. As shown in Fig. 2B,cytometric analysis of the fluorescence demonstrated that C3 de-position on the rickettsial surface was significantly increased infHDplHS compared to NHS or in the absence of any complement(PBS). The mean fluorescence values were as follows: PBS, 1,103;NHS, 1,936; and fHDplHS, 3,776, with a correlative increase in thepercentage of bacteria exhibiting a fluorescence reading largerthan the upper limit of the PBS control. Multicomponent analysisof 10,000 bins representing NHS and fHDplHS histograms indi-cates T(X) value of 795, whereby a value T(X) � 4 implies that thetwo distributions are different, with a P � 0.01. Similarly in Fig. 2C,the deposition of the MAC (C5b-9) occurred slightly more often inthe bacteria exposed to fHDplHS compared to rickettsiae incubatedin NHS. The mean fluorescence values were as follows: PBS, 1,250;NHS, 2,330; and fHDplHS, 2,709, with a correlative increase in thepercentage of bacteria that demonstrate fluorescence values largerthan the upper limit of the PBS control. In addition, the T(X) valuefor the comparison of NHS and fHDplHS is 125. Of note, only aportion of the bacteria treated with fHDplHS demonstrate an in-crease in MAC deposition. This is to be expected considering deple-tion of factor H does not result in complete bacterial killing (Fig. 2A).Taken together, these data indicate that R. conorii interact with hu-man fH to prevent complement activation and subsequent deposi-tion of the antibacterial MAC.

The rOmpB �-peptide interacts with factor H. To determinewhich rickettsial protein(s) interact with fH, we incubated whole-cell R. conorii detergent soluble lysate with purified fH, immuno-precipitated fH and any interacting proteins using anti-fH anti-body, separated the immune complexes by SDS-PAGE, andstained the gel using Coomassie blue. As shown in Fig. 3A (upper

FIG 1 R. conorii interacts with complement factor H. (A) R. conorii was incu-bated with PBS or 50% NHS. After washing, interacting serum proteins wereeluted from the bacteria with 1 M NaCl. The cell-free elutions were subjectedto SDS-PAGE and anti-fH immunoblotting. A 155-kDa fH-reactive species ispresent after NHS incubation with R. conorii. Sizes are indicated in kilodaltons(kDa). (B) R. conorii were incubated with PBS, 10% NHS, or 10 �g of purifiedfactor H/ml. After washing, the fH deposition on the bacteria was detected byflow cytometric analysis of fluorescent anti-fH deposition. All events shownwere contingent upon the presence of DNA (as demonstrated by DAPI stain-ing), which represented rickettsiae. Red, PBS; green, 10% NHS; blue, 50 �g offH/ml.

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panel), we identified a single prominent protein with an apparentmolecular mass of 32 kDa in immunoprecipitates containing R.conorii lysate and purified fH, but not in other control samples.Identical samples were immunoblotted with anti-fH antisera todetermine the efficacy and specificity of the immunoprecipitationreaction (lower panel). We excised the factor H coimmunopre-cipitating band and performed tandem mass spectrometry pro-tein sequencing. As shown in Fig. 3B, the protein sequence analy-sis yielded peptides (indicated in boldface) corresponding to theR. conorii rOmpB C-terminal � peptide domain with a 43.3%amino acid coverage (139/321 amino acids). To confirm thatrOmpB �p was truly interacting with factor H, we created a plas-mid, pYC6, which encodes for the rOmpB �p with a C-terminalHis6 tag. To control for putative nonspecific interactions withother rickettsial OM proteins, we also constructed a plasmid,pMC014, which encodes for the Sca2 �p. E. coli BL21(DE3) har-boring the empty vector pEt22b, pYC6, or pMC014, were inducedto make protein with the addition of IPTG. Whole-cell proteinlysates from these bacteria were incubated with fH, and subse-quently fH immunoprecipitation was performed. As shown in Fig.3C (middle panel), an anti-His6 reactive band of the appropriate

molecular mass coimmunoprecipitated when the rOmpB �p wasproduced (pYC6 � IPTG) but not when the Sca2 �p was made(pMC014 � IPTG). The efficacy of the anti-fH immunoprecipi-tation reaction and the expression of each indicated recombinantprotein was verified by immunoblot analysis (upper panel andlower panels, respectively, in Fig. 3C). These results demonstratethat the R. conorii rOmpB �p is sufficient to interact with factor H.

rOmpB �p mediates serum resistance. We had shown thatrOmpB can interact with fH; however, we still needed to deter-mine whether the expression of rOmpB �p is sufficient to mediateserum resistance. Serum-sensitive E. coli strain BL21(DE3) wastransformed with pYC6 or the empty parent vector, pEt22b. Bac-teria were left uninduced (�IPTG) or induced (�IPTG), sus-pended in PBS or in PBS containing 30% NHS, and assayed forsurvival after 1 h of incubation. As shown in Fig. 4A, E. coliBL21(DE3) containing pEt22b and uninduced (pYC6, �IPTG)were sensitive to NHS, experiencing �6-log10 killing (1:1,000,000survival). In contrast, expression of the rOmpB �p (pYC6,�IPTG) yields strong resistance to complement-mediated killing.We further confirmed the expression of the rOmpB �p at the OMof induced E. coli using anti-His6 antibody immunoblot analysis(Fig. 4B). Taken together, these data confirm that expression ofthe rOmpB �p is sufficient to mediate serum resistance in a man-ner that correlates with factor H binding.

Analysis of rOmpB �p conservation and structure. rOmpB�p is found in all pathogenic Rickettsia spp. sequenced to date(10). The �p amino acid sequence is remarkably well conserved,with an excess of 95% identity within the spotted fever group and�78% identity in the typhus group compared to the R. conoriisequence (Fig. 5A). Generally, conservation is seen throughoutthe amino acid sequence, with an exception being localized to the-helix that protrudes through the center of the �-barrel. Basedon Phyre modeling, we were able to predict the localization of the12 transmembrane �-sheets (yellow) and exposed peptide loopsof rOmpB �p (loops 1 to 6). The three-dimensional models gener-ated are based on the reported �-barrel crystal structures from otherGram-negative bacteria, and all rOmpB �p models were extremelysimilar. In Fig. 5B, we model rOmpB �p based on EstA from Pseu-domonas aeruginosa. This prediction had the highest confidence(100%) for the prediction of the rOmpB �p structure in the OM.Indeed, the hallmark membrane-spanning �-sheets form the barrel-like structure with lipophilic surfaces exposed to the bacterial OM.The six surface exposed cellular loops are highly polar and appear toprotrude away from the outer leaflet of the OM.

DISCUSSION

We have described here the first mechanism of serum resistance inthe highly pathogenic bacterium, Rickettsia conorii. We have dem-onstrated that R. conorii interacts with human factor H (fH) andthat this fH-R. conorii interaction is beneficial to the bacterium(Fig. 1 and 2). Thus, R. conorii recruitment of fH is advantageousto the bacterium and can be defined as an essential characteristicfor survival in serum. Moreover, we have used fH coimmunopre-cipitation to confirm that the rOmpB �-peptide (�p), but not a�-peptide from a related Sca protein (Sca2 �p), serves as a rick-ettsial protein that interacts with fH (Fig. 3). In addition, expres-sion of rOmpB �p at the E. coli OM endows that bacterium withserum resistance (Fig. 4). Taken together, these data represent thefirst description as to how R. conorii possess inherent resistance tothe antibacterial affects of serum complement.

FIG 2 Association with factor H promotes R. conorii resistance to comple-ment-mediated killing. (A) R. conorii was incubated in 50% NHS or factorH-depleted human serum (fHDplHS), followed by quantitation of viable bac-teria remaining. Values are expressed as the percentage of bacteria recoveredcompared to the bacteria incubated in PBS. The data represent three reactionsfor each condition and a minimum of three repetitions. P values are derivedfrom unpaired Student t test. (B and C) Bacteria incubated with PBS (red),NHS (blue), or fHDplHS (green) were analyzed by flow cytometry for depo-sition of C3 (B) or the bactericidal MAC (C5b-9) (C) using fluorescently con-jugated antibodies. Incubation of the bacteria in fHDplHS (green) results inmore deposition of C3 and C5b-9 than NHS (blue) or PBS (red). Complementdeposition is also demonstrated by an increase in the percentage of bacteriathat have a fluorescence signal higher than the PBS-treated control. A total of100,000 bacteria were analyzed for each histogram. The bars in panels B and Cdemarcate the data used for the area-under-the-curve statistical analysis. Sizesare indicated in panels A and C in kilodaltons (kDa).

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The �p is the portion of the rOmpB autotransporter that re-mains imbedded within the rickettsial OM. Autotransporter�-peptides belong to a family of �-barrel membrane proteins withlimited conservation in both the number of membrane spanningsheets and makeup of adjoining loops but share a highly conservedtertiary barrel-like membrane-spanning structure (reviewed inreference 22). rOmpB �p contains 12 transmembrane strandswith six potentially exposed extracellular loops. Classically, afunction associated with the �p is translocation of the associatedautotransporter passenger domain across the OM, where it is sub-sequently liberated from the �p by an unknown mechanism(25, 27).

As stated above, rOmpB is processed in vivo from its nativeform to two independent polypeptides: the passenger domain and�p. The passenger domain remains loosely associated with theOM and is thought not to be covalently linked to the integral OM�p (25). We must therefore conclude that serum resistance is me-diated in the areas proximal to the bacterial OM and not in thepassenger domain-containing layer distal to the OM (12, 68). Thisis to be expected considering the propensity for complement de-position near a membrane and the fact that the MAC forms inconjunction with a targeted membrane (78).

A previous report aimed at identifying rickettsial/host interac-tions has identified the rOmpB �p as a protein that associates withan unknown ligand from endothelial cell extracts, suggesting that

FIG 4 Expression rOmpB �p is sufficient to mediate survival in human se-rum. (A) E. coli BL21(DE3) harboring empty vector (pEt22b) or pYC6(rOmpB �p) was incubated in PBS or NHS and analyzed for serum survival.Uninduced bacteria (�IPTG) do not survive in NHS. (B) Western immuno-blot analysis with anti-His6 antibody reveals that expression of rOmpB �p(arrow) at the OM correlates with the ability to survive in NHS.

FIG 3 rOmpB �-peptide binds factor H. (A) The indicated proteins were incubated, immunoprecipitated with goat anti-human fH antibody, separated onSDS-PAGE, and stained with Coomassie blue (upper panel). The arrow indicates the band that only coimmunoprecipitated when both fH and rickettsial lysatewere 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 �-peptide (�p) of the autotransporter proteinrOmpB. Amino acid sequence coverage of the rOmpB �p was 43.3%. No peptides were recovered from the remaining portion of rOmpB. Sizes are indicated inkilodaltons (kDa). (C) E. coli BL21 harboring the empty vector pEt22b, plasmid encoding for the rOmpB �p (pYC6), or control plasmid encoding for the Sca2�p (pMC014) was induced with IPTG. Total detergent soluble lysates from these bacteria were incubated with purified fH and subsequently subjected to anti-fHimmunoprecipitation. fH immunoprecipitated equally from the indicated samples (upper panel). A His6-reactive species only coimmunoprecipitated with fHupon expression of the rOmpB �p (middle panel). Total protein lysates from the indicated samples were analyzed by Western immunoblotting to confirmrecombinant protein expression (bottom panel, input).

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the �p may function as an adhesin (64). We describe here a addi-tional function for the rOmpB �p, namely, the interaction withfactor H and the promotion of serum resistance. These two bind-ing functions can be completely complementary because manyfactor H-binding proteins in Gram-negative bacteria were previ-ously identified as adhesins (16, 29, 60). However, in some species,factor H binding does not correlate with adherence (47). As such,

future analysis will need to be performed to clarify the relationshipbetween these functions of rOmpB �p.

Of the factor H-binding proteins from Gram-negative bacte-ria—Salmonella enterica Rck and Yersinia enterocolitica Ail—share a tertiary �-barrel structure similar to the predicted struc-ture of rOmpB �p (7, 8, 16). These two other proteins have8-stranded �-barrels compared to the 12 strands of rOmpB �p. A

FIG 5 Analysis of rOmpB �p conservation and structure. (A) Alignment of rOmpB �p from diverse pathogenic Rickettsia spp. Indicated below the proteinsequences are the predicted transmembrane �-sheets (yellow arrows) and six surface-exposed peptide loops (bracketed). (B) The model of rOmpB �p in thebacterial OM clearly demonstrates the �-barrel structure with 12 transmembrane �-sheets (yellow), a central -helix that is the precleavage link to the passengerdomain (red), and an approximation of the membrane localization (blue). The six extracellular loops protrude away from the OM and are readily exposed to theextracellular environment.

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common structure of these three fH-binding proteins is the sur-face-exposed loops, which contain the fH attachment domains ofboth Rck and Ail (8, 16, 51). Using the Phyre protein modelingserver (38), we were able to build an approximation of the orien-tation and location of the rOmpB �p in the rickettsial OM (Fig. 5).The central �-barrel consists of 12 �-strands that form a lipophilicouter surface with a central hydrophilic core containing the alpha-helical link to the passenger domain (passenger domain notshown). The extracellular loops are largely polar and appear toprotrude some distance away from the outer leaflet of the OM.The model demonstrates that extracellular loops 3, 4, and 5 (fromthe N terminus) extend to a maximum of �35 Å from the bacterialsurface. Due to the polarity and surface exposure of these peptideloops, we hypothesize that these are the peptides of rOmpB �pthat interact with fH, as has been demonstrated in other Gram-negative pathogens.

It is of particular interest that the depletion of factor H fromNHS resulted in an increase of only �73% killing of R. conorii(Fig. 2A). Similarly, fH depletion only increased complementcomponent deposition on a subset of bacteria (Fig. 2B and C).When combined with the fact that nonpathogenic E. coli experi-ence about six times the rate of killing under similar serum con-ditions (Fig. 4A), these results suggest that R. conorii must possessother attributes responsible for serum resistance. Various meth-ods of acquired serum resistance have been demonstrated in in-vasive pathogens, including the acquisition of other soluble com-plement regulators, direct C3 inhibition, and the expression ofcomplement-specific bacterial proteases (reviewed in reference11). It is conceivable that Rickettsia spp. possess one or more ofthese serum resistance factors that function synergistically to pro-mote resistance to complement-mediated killing. The identifica-tion of these factors is an ongoing task in this laboratory.

Factor H is a large protein that at the macromolecular levelresembles “beads on a string” (5, 19, 41, 54, 56, 58). The 20 SCRs(short consensus repeats) of fH have been analyzed for associationwith its various natural substrates, including C3b, heparin, C-re-active protein, zinc, and sialic acid, as well as association withmany proteins of bacterial origin (reviewed in references 11 and65). Many bacterial fH-binding proteins associate with SCR5 toSCR7 or SCR19 to SCR20 of the polypeptide. Bacterial associationwith these sites promotes association with the cell surface whilepermitting the complement regulatory functions associated withC3b and factor I binding through SCR1 to SCR4. Whether R.conorii acquisition of fH functions in a similar manner is an areaongoing investigation.

The rOmpB �p is found in all pathogenic Rickettsia spp. se-quenced do date, and these species infect an extremely wide rangeof mammalian hosts and blood-feeding arthropods (10, 81). The�p is impressively well conserved in rickettsiae that are separatedby significant phylogeny and territory. It is conceivable that thestrong positive selective pressure to maintain the integrity ofrOmpB �p in all sequenced rickettsial species to date is in part dueto the ability of this protein to promote resistance to serum killingin zoonotic and human hosts. Interestingly, among the spottedfever group, host diversity is quite extraordinary. It will thereforebe intriguing to query the species specificity of fH binding (69).Since amino acid sequences and glycosylation patterns of fH differfrom animal to animal (2, 33, 41), rOmpB �p may only bind fHfrom a certain segment of mammals. Although many variableshave been associated with rickettsial host diversity (e.g., host avail-

ability, presence of arthropod vector, and immune state of thehost), fH binding and subsequent complement resistance mayinfluence host range of these severely pathogenic bacteria.

In conclusion, we have demonstrated here the first interactionbetween R. conorii and a soluble serum regulator of complement.This interaction with complement factor H yields strong resis-tance to serum, and the absence of this host effector results in asignificant loss of bacterial viability in serum. These molecularinteractions give us insight into the ability of R. conorii and poten-tially other rickettsial species to successfully infect humans whileexposed to the complement system through tick-bite inoculationand dissemination throughout the infected individual. Selectiveinhibition of the rOmpB �p-factor H interaction could make R.conorii more sensitive to the antibacterial effects of serum and mayhave a positive effect on patient outcome.

ACKNOWLEDGMENTS

We thank Marissa Cardwell, Robert Hillman, Yvonne Chan, and JessLeber for their assistance in experimental design and analysis and V.Thammavongosa and O. Schneewind for reagents.

This study is supported in part by a grant from the National Instituteof Allergy and Infectious Diseases (AI-72606) to J.J.M. We acknowledgemembership within the Region V Great Lakes Regional Center of Excel-lence in Biodefense and the Emerging Infectious Diseases Consortium(U54-AI-057153).

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