OmpA-mediated rickettsial adherence to and invasion of human endothelial cells is dependent upon interaction with α2β1 integrin

OmpA-mediated rickettsial adherence to and invasionof human endothelial cells is dependent uponinteraction with a2b1 integrin

Robert D. Hillman, Jr,1,2 Yasmine M. Baktash1 andJuan J. Martinez1,2*†

1Department of Microbiology, The University of Chicago,920 East 58th Street, Cummings Life Sciences Center707A, Chicago, IL 60637, USA.2The Howard T. Ricketts Laboratory, 9700 S. CassAvenue, Building 204, Argonne, IL 60439, USA.

Summary

Rickettsia conorii, a member of the spotted fevergroup (SFG) of the genus Rickettsia and causativeagent of Mediterranean spotted fever, is an obli-gate intracellular pathogen capable of infectingvarious mammalian cell types. SFG rickettsiaeexpress two major immunodominant surface cellantigen (Sca) proteins, OmpB (Sca5) and OmpA(Sca0). While OmpB-mediated entry has been char-acterized, the contribution of OmpA has not beenwell defined. Here we show OmpA expression inEscherichia coli is sufficient to mediate adherenceto and invasion of non-phagocytic human endothe-lial cells. A recombinant soluble C-terminal OmpAprotein domain (954–1735) with predicted struc-tural homology to the Bordetella pertussis pertac-tin protein binds mammalian cells and perturbsR. conorii invasion by interacting with severalmammalian proteins including b1 integrin. Usingfunctional blocking antibodies, small interferingRNA transfection, and mouse embryonic fibroblastcell lines, we illustrate the contribution of a2b1integrin as a mammalian ligand involved inR. conorii invasion of primary endothelial cells. Wefurther demonstrate that OmpA-mediated attach-ment to mammalian cells is in part dependent on aconserved non-continuous RGD motif present in apredicted C-terminal ‘pertactin’ domain in OmpA.

Our results demonstrate that multiple adhesin–receptor pairs are sufficient in mediating efficientbacterial invasion of R. conorii.

Introduction

Members of the genus Rickettsia are Gram-negative, obli-gate intracellular bacteria that are transmitted to a humanhost via an arthropod vector. Rickettsial species are cat-egorized as either a member of the typhus group (TG) orthe spotted fever group (SFG), based on differences inantigenicity to lipopolysaccharide, the presence of certainouter membrane proteins, and in part on the diseasesthey cause (Anacker et al., 1987; Vishwanath, 1991;Walker et al., 1995; Feng and Walker, 2003). Members ofboth groups are the aetiological agents of severe emerg-ing infectious diseases throughout the world, with Rickett-sia prowazekii (TG) and Rickettsia rickettsii (SFG) beingclassified as select agents by the United States Centersfor Disease Control and Prevention (CDC) due to theseverity of the disease, the existence of antibiotic-resistant strains (Weiss and Dressler, 1962a,b), and thepotential for aerosol transmission (Oster et al., 1977).

Transmission of SFG rickettsiae, including Rickettsiaconorii, to the human host occurs primarily via tick-biteinoculation when the salivary contents of the infectedvector are transferred during a blood meal (Hackstadt,1996). Expansion of the bacterial population and horizontalcell–cell transmission near the inoculation site results in alocalized dermal and epidermal necrosis referred to as aneschar or tache noir (Walker et al., 1988). Once an infec-tion is established in the host, SFG rickettsiae primarilytarget the endothelial lining of the vasculature, causinginjury to the vascular endothelium and infiltration ofperivascular mononuclear cells, which leads to vasodila-tion, an increase in fluid in the interstitial space. Thesesymptoms are often accompanied by a characteristic‘spotted fever’ dermal rash in some infected patients (Handet al., 1970; Walker et al., 1988). Damage to targetendothelial cells, particularly in the lung and brain, canresult in severe pathology such as pulmonary oedema,interstitial pneumonia and neurological and other multi-

Received 12 April, 2012; revised 10 October, 2012; accepted 6November, 2012. *For correspondence. E-mail [email protected];Tel. (+1) 225 578 9297; Fax (+1) 225 578 9701.†Current address: Department of Pathobiological Sciences, LSUSchool of Veterinary Medicine, Baton Rouge, LA 70803, USA.

Cellular Microbiology (2013) 15(5), 727–741 doi:10.1111/cmi.12068First published online 6 December 2012

© 2012 Blackwell Publishing Ltd

cellular microbiology

organ manifestations (Raoult and Roux, 1997). Becauseinitial clinical manifestations mimic flulike symptoms,disease is often misdiagnosed or proper diagnosis isdelayed. Some broad-spectrum antibiotics, such as doxy-cycline, are capable of eradicating an infection; however, ifleft untreated, mortality rates are estimated to be as high as20% (Yagupsky and Wolach, 1993; Yagupsky, 2000).

Previous studies have identified a family of at least 19genes termed surface cell antigen (sca) proteins thatencode either predicted secreted proteins or outer mem-brane proteins (Blanc et al., 2005). While most of thesegenes are split, fragmented or absent in the majority ofrickettsial species, there are five sca genes that appear tohave evolved under positive selection and are present inthe genomes of nearly all rickettsial species: ompA (sca0),ompB (sca5), sca1, sca2 and sca4 (Blanc et al., 2005). Thepredicted proteins encoded by these genes, excludingsca4, share homology with a family of proteins in Gram-negative bacteria termed autotransporters, many of whichare known virulence factors (Henderson and Nataro,2001). In R. conorii, Sca1 has been shown to mediatebacterial adherence, while Sca2 has been proven suffi-cient to mediate both adherence to and invasion of mam-malian cells (Cardwell and Martinez, 2009; Riley et al.,2010). OmpB has also been shown to participate in boththe adherence and invasion processes (Uchiyama et al.,2006; Chan et al., 2009) and to play a role in humoral andcellular protective immune responses (Feng et al.,2004a,b; Chan et al., 2011). Previous studies have alsoidentified plasma membrane-associated Ku70 as a recep-tor for OmpB (Martinez et al., 2005), the only reportedmammalian host receptor to date. While OmpA has alsobeen shown to elicit humoral immune responses (Vishwa-nath et al., 1990; Diaz-Montero et al., 2001; Feng andWalker, 2003; Feng et al., 2004a) and to mediate adher-ence in a related rickettsial species, R. rickettsii (Li andWalker, 1998), its function in R. conorii remains largelyunknown.

The presence of ompA/sca0 in the R. conorii genomeand other SFG rickettsiae would suggest that, similar toother related Sca proteins, OmpA in R. conorii plays acritical role in mediating interactions with mammalian cells.To elucidate the role of OmpA in rickettsial–host cell inter-actions, we have adapted a heterologous expressionsystem in a surrogate Gram-negative species, Escherichiacoli, that has been previously utilized to successfully studySca1, Sca2 and OmpB protein function (Uchiyama et al.,2006; Cardwell and Martinez, 2009; Chan et al., 2009;Riley et al., 2010). Here, we show that R. conorii OmpA,when expressed at the outer membrane of E. coli cells, issufficient to mediate adherence to and invasion of mam-malian cells in vitro in the absence of additional virulencefactors. Our results also demonstrate that OmpA-mediatedcellular invasion is dependent on a2b1 integrin expression

and is independent from the OmpB-Ku70-mediated inva-sion pathway of R. conorii.

Results

Expression of recombinant R. conorii OmpA in E. coli

Previously, an E. coli heterologous expression system wasused to study the function of other individual R. conorii Scaproteins (Uchiyama et al., 2006; Cardwell and Martinez,2009; Chan et al., 2009; Riley et al., 2010). We were ableto adapt this system to express OmpA at the E. coli outermembrane by cloning the full-length R. conorii ompA openreading frame into the IPTG-inducible vector, pET-22b,resulting in pMC022. Western immunoblot analysis ofE. coli outer membrane fractions confirmed that BL21(DE3)/pMC022 (ompA) expresses a high molecular weightHis6-reactive protein which corresponds to the predictedmobility of the full-length R. conorii OmpA protein (Fig. 1A,left panel). We confirmed that the expressed protein wasOmpA by using specific monoclonal antibodies (mAbs)against the protein (Fig. 1A, right panel). Furthermore,using flow cytometry analysis, we demonstrated the pres-ence of OmpA protein on the surface of inducedBL21(DE3)/pMC022 cells (Fig. S1). In addition, as hasbeen observed for other Sca proteins in this heterologousexpression system (Cardwell and Martinez, 2009; Chanet al., 2009; Riley et al., 2010), OmpA does not appear tobe proteolytically processed by E. coli.

OmpA is sufficient to mediate adherence to andinvasion of host cells

To assay for the putative contribution of R. conorii OmpAduring the initial host cell interaction, E. coli expressingOmpA at the outer membrane were initially assessed forthe capacity to associate with various cultured mamma-lian cells by a colony-forming unit (CFU) adherenceassay. As shown in Fig. 1B, expression of OmpA is suffi-cient to mediate cell association of E. coli to culturedHeLa and human microvascular lung endothelial(HMVEC-L) cells when compared to E. coli harbouring acontrol vector, pET-22b. Similar results were obtainedusing the human endothelial cell line, EA.hy 926 (data notshown). We confirmed the sufficiency of OmpA to mediatecell association to HMVEC-L cells using a fluorescence-based adherence assay (Fig. 1C). A standard gentamicinprotection assay was used to determine whether OmpAwas also sufficient to mediate host cell invasion, as hasbeen previously demonstrated for a cohort of Sca proteinsin R. conorii (Uchiyama et al., 2006; Cardwell and Mar-tinez, 2009; Chan et al., 2009). Confluent monolayers ofmammalian cells were infected with IPTG-induced E. coliharbouring either pET-22b, pMC022 or pYC9, a OmpB-expressing plasmid used as a positive control (Chan

728 R. D. Hillman, Jr, Y. M. Baktash and J. J. Martinez

© 2012 Blackwell Publishing Ltd, Cellular Microbiology, 15, 727–741

et al., 2009). Extracellular bacteria were killed by gen-tamicin, and internalized bacteria were quantified by CFUenumeration (Fig. 1B, right panels). These data illustratethat, in the absence of any additional R. conorii surfaceantigens, OmpA is sufficient to facilitate adherence to andinternalization into non-phagocytic human epithelial cellsand primary human endothelial cells.

Soluble OmpA protein binds mammalian cells andinhibits rickettsial invasion

We then sought to determine whether recombinant purifiedOmpA protein was able to associate with mammalian cellsand competitively inhibit R. conorii infection. Attempts toexpress and purify the full-length OmpApassenger domain(aa 35–1735) as a soluble protein were unsuccessful, likely

due to instability caused by several tandem repeats in theN-terminus of the protein. However, as shown in Fig. 2A,we were able to express and purify a portion of theR. conorii OmpA passenger domain comprised of twoN-terminal tandem repeats and the remainder of theC-terminus of the passenger domain (aa 954–1735) fusedto an N-terminal glutathione-S-transferase moiety (GST-OmpA954–1735). We incubated HeLa and HMVEC-L cells insuspension with GST-OmpA954–1735 or GST alone, andprocessed the samples for flow cytometric analysis. Asshown in Fig. 2B, GST-OmpA954–1735, but not GST alone, isable to bind to mammalian host cells.

For the evaluation of the specific contribution of OmpAto a rickettsial infection, we pre-incubated confluent mon-olayers of HMVEC-L cells with GST-OmpA954–1735 or GSTand then infected with R. conorii. In a concentration-

Fig. 1. R. conorii OmpA is sufficient to mediate adherence to and invasion of mammalian cells.A. E. coli BL21(DE3)/pET-22b (Lane 1), and E. coli BL21(DE3)/pMC022 (ompA) (Lane 2), were induced, biochemically fractionated to isolateouter membranes (OM), and OM protein fractions were probed with His6 polyclonal (left panel) or OmpA monoclonal antibodies (right panel).The asterisk denotes OmpA.B. CFU-based adherence and invasion assays confirm OmpA is sufficient to mediate adherence to and invasion of HeLa and HMVEC-L cells.E. coli BL21(DE3)/pMC022 are capable of binding (left panels) and invading (right panels) cultured cells when induced with 0.1 mM IPTG.E. coli BL21(DE3)/pET-22b and un-induced OmpA-expressing E. coli (pMC022-I) do not adhere and are non-invasive. E. coli BL21(DE3)(pYC9), which express R. conorii OmpB, were used as a positive control for invasion (Chan et al., 2009).C. An immunofluorescence microscopy adherence assay confirms that expression of OmpA in E. coli is sufficient to mediate adherence toHMVEC-L cells. Actin is depicted in red, bacteria in green, and nuclei are shown in blue. P-values were determined using a two-tailedStudent’s t-test, * < 0.005.

OmpA-a2b1 integrin mediated rickettsial invasion 729


dependent manner, incubation with GST-OmpA954–1735 isable to inhibit R. conorii association at levels up toapproximately 50% at the highest concentration used(Fig. 2C). To assess for the specificity of inhibition againstanother conserved Sca protein, we pre-incubated theEA.hy 926 human endothelial cell line with GST or GST-OmpA954–1735 and then infected these cells with E. coliBL21 (DE3) harbouring either pMC022 (ompA) or pYC9(ompB). As shown in Fig. 2D, GST-OmpA954–1735 competi-tively inhibits OmpA-mediated cellular association, butdoes not have any effect on the ability of OmpB-expressing bacteria to associate with endothelial cells.These data taken together demonstrate that recombinantOmpA protein is capable of binding mammalian cells, andis sufficient to competitively inhibit R. conorii and OmpA-mediated adherence to target mammalian cells.

R. conorii OmpA interacts with specific mammaliansurface protein complexes

We next sought to identify mammalian interacting pro-teins that could potentially serve as receptors for OmpA.We performed an in vitro ‘GST pull-down’ assay usingsoluble HeLa cell proteins incubated with either GST-OmpA954–1735 or GST alone. The resulting complexeswere captured with glutathione-sepharose and elutedwith excess glutathione. Unique putative interacting pro-teins that were exclusive to GST-OmpA954–1735, but notGST, were analysed by tandem mass spectrometry. Thisanalysis revealed that GST-OmpA954–1735 appears tointeract with several high molecular weight mammalianproteins including filamin-A, epiplakin and dynein(Fig. 3B). Each of these cytoskeletal linker proteins are

Fig. 2. OmpA binds mammalian cells and competitively inhibits rickettsial invasion.A. Coomassie-stained SDS-polyacrylamide gel depicting the soluble protein GST-OmpA954–1735, denoted by the arrow.B. HeLa (left) and HMVEC-L (right) cells in suspension were fixed and incubated with 10 mM GST-OmpA954–1735 or GST in serum-free DMEM,washed, and processed for flow cytometry analysis. The two traces represent GST (red) and GST-OmpA954–1735 (blue) binding.C. Soluble OmpA protein inhibits R. conorii association. HMVEC-L cells were incubated with GST (100 mg ml-1) or GST-OmpA954–1735

(10 mg ml-1, 50 mg ml-1 and 100 mg ml-1) for 20 min, washed, and infected with R. conorii at an MOI of 10 for 60 min. Samples were fixed,processed for immunofluorescence staining, and total R. conorii were counted.D. GST-OmpA954–1735 is sufficient to inhibit association of OmpA-expressing E. coli (pMC022), but not OmpB-expressing E. coli (pYC9) toEA.hy 926 cells. P-values were determined using a two-tailed Student’s t-test, * < 0.005, ** < 0.05.



involved in sub-membranous complexes, but none aredirectly associated with the plasma membrane (Mar-gadant et al., 2011) suggesting that they are not viablecandidates for R. conorii receptors. Interestingly, themost abundant species found in the analysis, filamin-A,has been shown to form an adhesion complex with b1integrin, and integrins have been demonstrated to beinvolved both as receptors and as downstream signallingeffectors linked specifically to bacterial and viral invasionprocesses (Isberg and Barnes, 2001; Eto et al., 2007;Stewart and Nemerow, 2007; Cantor et al., 2008).We therefore hypothesized that b1 integrin may functionas a mammalian ligand for OmpA. To test this, weanalysed the samples sent for mass spectrometry byWestern immunoblot analysis with antibodies specific toseveral b and a integrin subunits. We revealed the pres-ence of both b1 and a2 integrin, but not b4 and a1integrin in the GST-OmpA954–1735 eluates (Fig. 3C). Takentogether, these results suggest that a2b1 integrin maybe a mammalian interacting protein with R. conoriiOmpA.

a2b1 integrin is involved in R. conorii invasion ofmammalian cells

We then investigated whether b1 integrin colocalized withinvading R. conorii in infected endothelial cells. HMVEC-Lcells were infected with R. conorii and then processed forimmunofluorescence microscopy. Analysis of single crosssections from confocal Z-stacks reveals that b1 integrincolocalizes with several invading R. conorii (Fig. 4A andB, arrows). These results suggest that infection of themammalian host cell with R. conorii leads to the recruit-ment of b1 integrin to entry foci and that some b1 integrin-containing receptors at the plasma membrane areinvolved in the uptake process.

We sought to further investigate whether b1 integrin orother integrin subunits could serve as a mammalian ligandfor R. conorii. We utilized a panel of functional blockingmAbs specific to b integrin subunits (Eto et al., 2007) toconfirm the involvement of b1 integrin in the R. conoriiinvasion pathway. Pre-incubation of HMVEC-L cells withfunctional blocking b integrin mAbs had no significant effect

Fig. 3. OmpA interacts with mammalianprotein complexes including a2 and b1integrin.A. Identification of interacting proteins byaffinity chromatography. Fifty micrograms ofGST or GST-OmpA954–1735 was incubated withdetergent soluble HeLa cell lysate andinteracting proteins were captured onimmobilized glutathione sepharose. Boundproteins were washed and eluted with excessglutathione. Host proteins that associate withGST-OmpA954–1735 but not GST (denoted byasterisks and enlarged in the boxed region)were excised and analysed by microcapillaryLC/MS/MS.B. GST-OmpA954–1735 interacts with distinctmammalian protein. Analysis of the doubletband in A, denoted by the larger asterisk,revealed the presence of peptidescorresponding to filamin-A, epiplakin 1 anddynein 1 heavy chain.C. Eluted samples of HeLa cell proteinsincubated with either GST-OmpA954–1735

(Lane 1) or GST (Lane 2) were analysed byWestern immunoblot analysis against variousb and a integrin subunits. HeLa whole celllysate (Lane 3) was used as positive control.b1 and a2 integrin both co-elute withGST-OmpA954–1735 using an affinitychromatography approach.



on total R. conorii cell association (data not shown);however, pre-incubation with a mAb against b1 integrin(MAB1951) significantly perturbed invasion compared toother b integrin mAbs and an isotype-matched control mAb(9E10) (Fig. 5A). These results are consistent with previ-ous findings using functional blocking antibody againstKu70, a previously identified R. conorii receptor (Martinezet al., 2005). Small interfering RNA (siRNA) transfectionwas then used to validate the phenotype illustrated by theblocking mAbs. We transfected HMVEC-L cells withscrambled siRNA or siRNAs against b1 integrin andinfected these cells with R. conorii. As shown in Fig. 5B,reduction of endogenous b1 integrin expression leads to acorresponding significant inhibition of R. conorii invasion,similar to levels observed in the mAb functional blockingassay, but does not affect the R. conorii adherence (datanot shown). To further confirm the role of b1 integrin as aligand involved in rickettsial host cell internalization, weperformed similar cell association and internalizationassays using a mouse embryonic fibroblast cell line, GD25,in which the gene encoding for b1 integrin has been

inactivated and in GD25b1A cells, a stably transfectedderivative of GD25 that encodes the widely expressedwild-type b1 integrin splice variant, b1A (Fèassler andMeyer, 1995). As shown in Fig. 5C, rickettsial invasion ofthe b1 integrin-null cell line was significantly decreasedrelative to cells in which b1 integrin expression has beenrestored.

Integrins are heterodimeric proteins consisting of a andb subunits (Margadant et al., 2011). Prior analysis of affin-ity chromatography eluates by Western immunoblot analy-sis showed the presence of a2 integrin; therefore, wesought to then elucidate whether this was the a subunit thatpairs with b1 integrin to form the functional OmpA ligand.Using a panel of functional blocking mAbs against aintegrin subunits, we demonstrated that inhibition of a2integrin function, but not other a integrins, significantlydiminished R. conorii invasion of HMVEC-L cells (Fig. 5D).As shown in Fig. 5E, we observed a similar inhibition ofR. conorii invasion using siRNA transfection against a2integrin, suggesting that a2b1 integrin is a specificR. conorii mammalian internalization factor.

Fig. 4. R. conorii colocalizes with b1 integrin during infection of cultured primary endothelial cells.A. Confluent monolayers of HMVEC-L cells were infected with R. conorii for 30 min, fixed, and processed for immunofluorescence staining.Arrows are used to denote sites that have been magnified in the inset, showing b1 integrin interacting with invading R. conorii.B. Areas in the inset in A marked by a white line were analysed by the ‘Measure RGB’ function in Image J and are presented as a colourhistogram measuring an area in the image from left to right. Colocalization is indicated by an overlap of the red and green channelhistograms. Scale bar denotes 10 mm.



OmpA is the R. conorii surface protein that interactswith a2b1 integrin

OmpB is a related rickettsial autotransporter protein andwas previously demonstrated to be sufficient to interactwith plasma membrane-associated Ku70 and lead to bac-terial internalization (Martinez et al., 2005; Chan et al.,2009). We wanted to confirm that R. conorii OmpA, and

not another rickettsial outer membrane autotransporterprotein, was the corresponding rickettsial ligand that inter-acted with b1 integrin. We initially infected GD25 andGD25b1A cells with E. coli harbouring pET-22b, pMC022(ompA) and pYC9 (ompB) and then determined the abilityof bacteria to associate with these cells. As shown inFig. 6A, OmpA-mediated adherence is diminished tolevels similar to the non-adherent control (pET-22b) in

Fig. 5. a2b1 integrin is involved in R. conorii invasion of mammalian cell.A. Primary human endothelial cells (HMVEC-L) were incubated with a panel of b integrin functional blocking antibodies, in addition to a 9E10IgG1 isotype-matched negative control, prior to infection with R. conorii. The antibody against b1 integrin significantly reduced levels ofinvasion. Data are presented normalized against the negative control, which is set to 100%. The % invasion of R. conorii into cells treated withthe 9E10 isotype control was 57.28 � 10.25%, while invasion into cells treated with the b1 integrin functional blocking antibody was34.01 � 9.04%.B. Reduction in endogenous b1 integrin expression inhibits R. conorii invasion. Transfection of cells with b1 integrin siRNA, when comparedagainst a scrambled negative control, significantly limits the invasive capacity of R. conorii. Transfection efficiency is illustrated by immunoblotand was quantified at 57% knockdown by densitometry analysis.C. Cells devoid of b1 integrin expression limit the efficacy of R. conorii invasion. Mouse embryonic fibroblast cells either devoid of (GD25) orcomplemented for (GD25b1A) b1 integrin expression were infected with R. conorii for 20 min and analysed for invasion. The invasivecapability of R. conorii is significantly reduced in the GD25 cell line.D. In HMVEC-L cells, functional blocking antibodies against a integrin subunits show a statistically significant perturbation of R. conoriiinvasion only when a2 integrin is functionally blocked. 9E10 was used as a negative control, against which all values are normalized. Invasionof R. conorii into 9E10 treated cells was 54.56 � 8.73%, while invasion into a2 integrin antibody treated cells was 39.61 � 7.85%.E. Reduction in endogenous a2 integrin expression inhibits R. conorii invasion. Transfection of cells with a2 integrin siRNA significantly limitsthe invasive capacity of R. conorii into HMVEC-L cells. Transfection efficiency is illustrated by immunoblot and was quantified at 68%knockdown by densitometry analysis. P-values were determined using a two-tailed Student’s t-test, * < 0.005, ** < 0.05.



cells that are genetically depleted for b1 integrin (GD25cells). In contrast, OmpB-mediated adherence is notaffected. To further support these findings, we thenreduced endogenous a2 and b1 integrin expression inEA.hy 926 cells using siRNA transfection, and infectedthese cells with E. coli BL21 (DE3) strains harbouring thecontrol plasmid pET-22b, pMC022 (ompA) or pYC9(ompB). Reduction of endogenous b1 and a2 integrinexpression by siRNA transfection, both individually and intandem, perturbs OmpA-mediated association with humanendothelial cells, but it has no effect on OmpB-mediatedbacterial adherence. Taken together, these results demon-strate that OmpA and a2b1 integrin represent a bona fideadhesin–mammalian ligand pair involved in cellular attach-ment to and ultimately entry into non-phagocytic mamma-lian cells in a mechanism that is independent from OmpB-Ku70-mediated rickettsial entry pathway.

The OmpA ‘pertactin domain’ contains a non-continuousRGD motif involved in cellular adherence

We next sought to characterize the molecular details gov-erning OmpA–integrin interactions. A Blastp-based bioin-formatic analysis of the functional blocking R. conorii

OmpA domain (aa 954–1735) did not reveal any signifi-cant amino acid sequence homology to known adhesinsfrom other pathogenic Gram-negative bacteria. However,using a web-based structural homology prediction soft-ware suite, Phyre (Kelley and Sternberg, 2009), we deter-mined that R. conorii OmpA954–1735 is predicted to adopt aright-handed b-helix fold (Fig. 7A) similar to a fold found inthe passenger domain of the Bordetella pertussis pertac-tin autotransporter protein (Junker et al., 2006). Pertactinbinds host mammalian cells in an integrin-dependentmanner through a canonical RGD integrin-binding motif(Everest et al., 1996). Proteins containing linear RGDmotifs are sufficient to mediate b integrin-dependent inter-actions, but are not absolutely required for this process ashas been demonstrated for the FimH protein of type 1piliated uropathogenic E. coli (UPEC) (Eto et al., 2007)and the invasin protein from Yersinia pseudotuberculosis(Leong et al., 1990; 1991). A canonical linear RGDsequence is not present within the OmpA passengerdomain (aa 35–1735); however, OmpA does contain aRNIGD sequence (aa 1106–1110, Fig. 7B), which isinvariant in OmpA proteins from other pathogenic SFGrickettsiae (Fig. 7C). This motif is predicted to be surface-exposed and positioned so as to interact with mammalian

Fig. 6. a2b1 integrin is a mammalian ligandfor R. conorii OmpA.A. Absence of b1 integrin ablatesOmpA-mediated adherence. Mouseembryonic fibroblast cells devoid of (GD25) orcomplemented for (GD25b1A) b1 integrinexpression were infected with E. colicontaining pMC022 (ompA), pYC9 (ompB) orthe empty vector pET-22b, and adherencewas enumerated by CFU assay.OmpA-mediated adherence is significantlyreduced, while OmpB-mediated adherenceremains unaffected.B. E. coli BL21(DE3) (pMC022) (ompA) showa reduced ability to bind EA.hy 926 cells thathave been transfected with siRNAs againsteither b1 integrin or a2 integrin or incombination. E. coli BL21(DE3)/pYC9,encoding the R. conorii OmpB protein, are notaffected in their ability to associate with thesecells. P-values were determined using atwo-tailed Student’s t-test: *P < 0.005,**P < 0.05.



cells (Fig. 7B). We therefore sought to determine whetherOmpA-mediated cellular association is dependent on anRGD sequence. We pre-incubated EA.hy 926 cells witha control peptide and a peptide containing the RGDsequence and then infected these cells with E. coli BL21(DE3) harbouring pMC022 (ompA), pYC9 (ompB) andpRI203, a plasmid encoding for the Y. pseudotuberculosisinvasin protein (Isberg et al., 1987). As shown in Fig. 7D,the RGD containing peptide is sufficient to inhibit OmpA-and invasin-mediated cellular association, but had noeffect on OmpB-mediated adherence. We then deter-mined whether the OmpA ‘pertactin domain’ is sufficient toinhibit cellular adherence mediated by an unrelatedantigen whose function is also blocked by RGD containing

proteins. As shown in Fig. 7E, GST-OmpA954–1735, but notGST alone, is capable to competitively inhibiting Y. pseu-dotuberculosis invasin-mediated adherence to mamma-lian cells. Taken together, these results suggest that theR. conorii OmpA protein contains a non-linear, but func-tional RGD-like motif that is likely involved in mediatingassociation with plasma membrane b1 integrin-containingreceptors.

Discussion

Invasion of target host cells is essential for the subse-quent intracellular survival and proliferation of obligateintracellular pathogens like R. conorii. Previous work has

Fig. 7. OmpA-mediated adherence isdependent on a non-continuous RGD motif inthe R. conorii OmpA ‘pertactin domain’.A. OmpA is predicted to adopt fold similar toother known integrin-binding autotransporterproteins. The web-based structural homologyprediction software suite, Phyre, predicts thatOmpA954–1735 adopts a right-handed b-helixfold, similar to ‘passenger domains’ of otherautotransporter proteins in pathogenicGram-negative bacterial species. Amino acids1044–1606 are modelled onto the pertactinprotein of Bordetella pertussis (pdb1DAB).Predicted loops and turns are coloured ingreen, while b-strands are coloured in yellow.B. A putative integrin interacting motif(RNIGD) appears to be surface-exposed andpositioned to interact with mammalian ligands(highlighted in red in A and enlarged fordetail).C. A alignment of OmpA sequences frompathogenic SFG rickettsial species harbouredin different tick vectors and located ondifferent continents reveals that a RNIGDmotif (in bold) is widely conserved.Superscripts refer for the amino acid positionsin each individual OmpA protein in theindicated rickettsial species.D. Pretreatment of EA.hy 926 cells withan RGD containing peptide, but not acontrol peptide, is sufficient to inhibit OmpA-(pMC022) and invasin- (pRI203) mediatedcellular association, but does not havean effect on OmpB (pYC9) adherence.E. The purified recombinant R. conorii OmpApertactin domain (GST-OmpA954–1735), but notGST, can competitively inhibit adherence ofan unrelated antigen, invasin (pRI203), to theEA.hy 926 endothelial cell line. P-values weredetermined using a two-tailed Student’s t-test,* < 0.005, ** < 0.05.



demonstrated that R. conorii specifically binds to plasmamembrane-associated Ku70 in a OmpB-dependentmanner (Martinez et al., 2005; Chan et al., 2009), and thatinvasion is dependent on Arp2/3, an actin-nucleatingprotein complex, as well as a multitude of host signallingevents mediated by c-Cbl, clathrin, caveolin 2, Cdc-42,phosphoinositide 3-kinase, c-Src and other kinases (Mar-tinez and Cossart, 2004; Chan et al., 2009). Here wedemonstrate that R. conorii invasion is also mediated byan interaction between OmpA and the mammalian inter-acting factor, a2b1 integrin.

The a2b1 integrin heterodimer has been previouslyshown to be a receptor for a variety of matrix and non-matrix ligands including collagen type I, laminins,E-cadherin and several viruses (Languino et al., 1989;Mizuno et al., 2000; Graham et al., 2003). This receptor isexpressed on various cell types, including epithelial andendothelial cells, and interactions between extracellularligands and a2b1 integrin have been implicated in severalbiological processes such as caveolin- and clathrin-dependent endocytosis (Shi and Sottile, 2008; Ezrattyet al., 2009), inflammation and immunity (Gahmberg et al.,1998). Engagement of extracellular ligands at the cellsurface is known to induce integrin clustering, and this canhave positive feedback both on the binding avidity of theintegrin receptor itself as well as the activation of an arrayof downstream signalling events (Huveneers and Danen,2009). Our immunofluorescence results presented hereshow that R. conorii recruits b1 integrin to sites of bacterialentry in primary endothelial cells, suggesting that engage-ment of a rickettsial ligand (OmpA) can induce integrinreceptor clustering and activation. Of note, several bonafide integrin ligands initiate signalling cascades, leadingto the phosphorylation of conserved tyrosine resides onthe b integrin cytoplasmic domain and activation of c-Srcand focal adhesion kinase (FAK) (Cantor et al., 2008).R. conorii invasion into non-phagocytic mammalian cellsinvolves the activation of src-family tyrosine kinases andtyrosine phosphorylation of FAK (Martinez and Cossart,2004), two signalling events strongly associated with b1integrin activation (Parsons, 2003). In addition, F-actindynamics are altered downstream of integrin clustering byrecruitment and activation of several proteins, GTPasesand lipid kinases, including Cdc42, and PI 3-kinase(Huveneers and Danen, 2009). It is possible that, byengaging integrin receptors, R. conorii may stimulatethese signalling pathways ultimately leading to theobserved F-actin polymerization required for efficientbacterial internalization (Martinez and Cossart, 2004).Whether OmpA-expressing bacteria can directly triggersignalling cascades known to be associated with b1integrin activation is currently under investigation.

An intact ompA gene is present in other pathogenicSFG rickettsiae such as R. rickettsii, Rickettsia japonica

and Rickettsia africae; however, ompA is split, fragmentedor otherwise absent in species belonging to the TG includ-ing R. prowazekii and Rickettsia typhi (Blanc et al., 2005),suggesting that different autotransporter–mammalianligand pairs govern the invasion of this class of obligateintracellular pathogens. Interestingly, a mutation in ompAin the SFG rickettsial strain R. rickettsii Iowa results in thelack of observable OmpA protein and is correlated with adefect in virulence in a guinea pig model of infectivity(Ellison et al., 2008), further highlighting the importance ofOmpA for infections mediated by SFG rickettsiae. Whilethe ompA gene products expressed in SFG rickettsiaevary in predicted molecular weight, each OmpA proteincontains a conserved putative integrin interaction motif,RNIGD. Proteins containing non-continuous RGD motifsof varying lengths including R . . . D, KQAGDV, LDV/IDSand RLD/KRLDGS sequences have been shown to beinvolved in mediating interactions with various integrinreceptors (Ruoslahti, 1996). Our results demonstrate thatR. conorii OmpA contains a functional non-continuousRGD domain that is involved in mediating interactionswith b1 integrins. Whether the RNIGD motif present inthe OmpA C-terminal ‘pertactin’ domain is required forinteractions with a2b1 integrin is currently under activeinvestigation.

Our results also demonstrate that similar to otherinvasive ‘zipper’ mechanism pathogens such as Listeriamonocytogenes and UPEC, R. conorii expresses severalantigens conserved in other SFG rickettsiae that are suf-ficient to mediate bacterial adherence to and internaliza-tion of mammalian cells independent of one another. Wehave also demonstrated that purified, recombinant pro-teins containing portions of Sca1, Sca2, OmpB (Cardwelland Martinez, 2009; Chan et al., 2009; Riley et al., 2010)and now OmpA are capable of competitively inhibitingR. conorii adherence and invasion of mammalian cells,suggesting that these Sca proteins are interacting withputative mammalian receptors. Interestingly, Ku70 anda2b1 integrin are expressed at the plasma membrane ofendothelial cells (Senger et al., 1997; Muller et al., 2005)suggesting R. conorii may utilize these receptors to ini-tially gain access to an intracellular niche after beingintroduced into the vasculature of a host by a feeding tick.It is possible that other antigens, such as Sca1 and Sca2,may also contribute to this initial attachment and invasionof endothelial cells respectively. Alternatively, these pro-teins may be involved in allowing R. conorii and otherSFG rickettsiae to target organs such as the lungs, liverand spleen where pathology is prevalent in models ofdisseminated disease (Walker et al., 1994; Chan et al.,2011). While several Sca proteins have now been dem-onstrated to be sufficient for invasion of mammalian cells,we propose that effective entry of SFG rickettsiae intotarget cells in vivo likely involves OmpA and other Sca



proteins working in concert to generate the signal trans-duction cascades necessary to trigger the entry process.

The results presented here highlight the involvement ofintegrins and integrin-dependent signalling events in theinitial rickettsia–host cell interaction. However, we do notexclude the possibility that b integrins may play additionalroles during an infection. R. conorii infections of endothe-lial cells have been correlated with the disruption of cel-lular adherens and tight junctions leading to an increase incellular permeability (Valbuena and Walker, 2005). Whilethe molecular details of pathology associated with rickett-sial disease are not well understood, studies have shownthat neutrophil-mediated vascular permeability is integrin-dependent (DiStasi and Ley, 2009). Understanding themechanisms that lead to alterations in endothelial junc-tions may prove useful in efforts to identify host and bac-terial targets that may be exploited for the development ofanti-rickettsial therapies.

Experimental procedures

Cell lines and bacterial strains

The human cervical adenocarcinoma cell line HeLa and thehuman endothelial cell line EA.hy 926 were maintained in Dul-becco’s modified Eagle’s medium (DMEM) supplemented with10% heat-inactivated fetal bovine serum and 1¥ non-essentialamino acids (cDMEM). Human lung microvascular endothelial(HMVEC-L) cells were grown in endothelial growth medium sup-plemented with a -2-MV bullet kit (Lonza). The b1 integrin-null cellline GD25 and its stably transfected b1 integrin-expressingderivative GD25b1A (kindly provided by Dr D.F. Mosher, theUniversity of Wisconsin-Madison) were grown in cDMEM. Puro-mycin was supplemented at 10 mg ml-1 to maintain the stabletransfection. All mammalian cells were grown at 37°C/5% CO2.Bacterial genetic manipulations were performed in E. coli OneShot® TOP10 chemically competent cells (Invitrogen) grown at37°C in LB broth supplemented with carbenicillin (50 mg ml-1)where appropriate. E. coli BL21 (DE3) cells were transformedwith the indicated plasmids and plated on selective LB agarcontaining carbenicillin (50 mg ml-1). R. conorii Malish 7 waspropagated in Vero cells and isolated and stored as previouslydescribed (Martinez and Cossart, 2004). Titres were determinedby a limiting-dilution infectivity assay and calculated using theReed and Muench formula.

Antibodies and other reagents

Anti-GST polyclonal rabbit antibody Z-5, polyclonal antibodiesagainst b1 integrin (sc-6622, sc-8978) and b4 integrin (sc-9090)were purchased from Santa Cruz Biotechnology. The RGD con-taining peptide (GRGDNP) and the control peptide (GRGESP)were also purchased from Santa Cruz Biotechnology. Alexa Fluor488/546-conjugated goat anti-rabbit IgG and Alexa Fluor 488/546-conjugated goat anti-mouse IgG, Texas Red-conjugatedphalloidin and DAPI (4′,6′-diamidino-2-phenylindole) were pur-chased from Molecular Probes. Rabbit polyclonal antiseraagainst the R. conorii OmpA passenger domain were generated

using recombinant protein purified from BL21 (DE3)/pBOB001(described below) essentially as described in Chan et al. (2011).Rabbit polyclonal anti-R. conorii antiserum (RCPFA) generatedagainst paraformaldehyde (PFA)-fixed bacteria has beendescribed (Chan et al., 2011). Rabbit polyclonal anti-E. coliantiserum was generated as described (Chan et al., 2009).IRDye 800CW-conjugated donkey anti-rabbit IgG and IRDye680LT-conjugated donkey anti-mouse IgG were obtained fromLI-COR. mAb against OmpA (clone 13-3) (Anacker et al., 1985;1987) was kindly provided by R. Heinzen, PhD (NIAID, RockyMountain Laboratories). mAbs against b1 integrin (MAB1951F,FCMAB375A4, 217648) and polyclonal antibody against a2integrin (AB1936) were purchased from Millipore. b (ECM440)and a (ECM430) integrin antibody screening kits were also pur-chased from Millipore. Complete protease inhibitor tablets werepurchased from Roche. Annealed siRNAs against b1 integrinsense strand, a2 integrin sense strand and Silencer negativecontrol siRNA were purchased from Ambion. RNAiMAX was pur-chased from Invitrogen.

Plasmid DNA constructs

The full-length ompA open reading frame was amplified by PCRfrom a chromosomal preparation of R. conorii Malish 7 usingforward and reverse primers 5′-AGGATCCAGCGAATATTTCTCCAAAATTATTT-3′ and 5′-AACTCGAGAAATTAACACGAACTTTCACACT-3′ respectively. The resulting PCR product con-tained the restriction sites BamHI and XhoI incorporated 5′ and3′ of the ompA gene. The PCR product was initially TOPATA-cloned into pCR2.1 (Invitrogen), resulting in pMC003, thendigested with BamHI and XhoI for insertion into the expressionvector pET-22b (Novagen), resulting in plasmid pMC022. Thesequence coding for the entire OmpA passenger domain (aa35–1735) was PCR amplified from pMC022 as a template usingprimers 5′-AAGGATCCATTGCTGTTTCAGGTGTTATTG-3′ and5′-AACTCGAGTTACATATCTTCATCACCAGAAGAA-3′. ThePCR product was digested with BamHI and XhoI and directionallycloned into pYC55 (Chan et al., 2011) resulting in pBOB001. TheC-terminal half of the OmpA passenger domain encompassingamino acids 954–1735 was PCR amplified from pMC022 usingthe forward and reverse primers 5′-AAGGATCCACATTACAAGCTGGAGGAAG-3′ and 5′-AACTCGAGTTACATATCTTCATCACCAGAAGAA-3′ respectively. The resulting PCR productwas digested with BamHI and XhoI and ligated into pGEX-2TKP(a generous gift from T. Kouzarides, Gurdon Institute, UK), result-ing in the plasmid pBOB002. Construction of pYC9 is describedelsewhere (Chan et al., 2009). The pRI203 plasmid containingthe Y. pseudotuberculosis inv gene has been described (Isberget al., 1987) and was kindly provided by M. Mulvey, PhD (TheUniversity of Utah).

E. coli outer membrane protein fractionation

Outer membrane protein fractions from E. coli were preparedessentially as described in Nikaido (1994). Briefly, 10 ml ofinduced E. coli BL21(DE3) culture was pelleted and resuspendedin 1 ml of lysis buffer (20 mM Tris, pH 8.0, plus protease inhibitor).Cells were lysed by sonication on ice at amplitude 4, 15 s on and15 s off, repeated four times or until lysates became translucent.Unbroken cells were spun down by centrifugation at 8000 g for



2 min at 4°C. The supernatant containing the whole cell lysatewas added to a clean tube, and 50 ml of 10% sarkosyl was added(final concentration 0.5%) at room temperature to extract innermembrane proteins. Outer membrane proteins were pelleted byhigh-speed centrifugation (100 000 g) at 4°C for 30 min. Mem-branes were resuspended in 0.5 ml of 20 mM Tris, pH 8.0, 0.3 MNaCl, resolved by SDS-PAGE, and transferred to nitrocellulosemembrane. Membranes were analysed by immunoblot with anti-His6 rabbit polyclonal serum and anti-OmpA mouse mAbs. Immu-noreactive species were revealed with chemiluminescence andfilm exposure or on a LI-COR Odyssey CLx imaging system. Thespecificity of the OmpA serum was verified by Western immuno-blotting using purified OmpA35–1735-His, purified GST-OmpB36–1334

(Chan et al., 2009), the isolated OmpA serum and a mAb (mAb6B.6) against OmpB (Chan et al., 2011).

Protein expression and purification

Overnight cultures of E. coli BL21 (DE3) harbouring pBOB001were diluted 1:10 into fresh media containing carbenicillin andgrown at 37°C to mid-exponential phase (OD600 = 0.6). Proteinexpression was induced by the addition of 0.1 mM isopropylb-D-1-thiogalactopyranoside (IPTG) for 4 h at 30°C. Bacteriawere harvested by centrifugation and resuspended in denatur-ing lysis buffer (100 mM NaH2PO4, 10 mM Tris-Cl, 8 M urea pH8.0 containing protease inhibitor cocktail) and lysed by sonica-tion (40% amplitude, 5 s on/10 s off, 20 min – Fisher Scientificsonic dismembrator model 500). Lysates were cleared by cen-trifugation at 13 000 g for 30 min at 4°C and applied to aNi-NTA FF column. The column was washed extensively withwash buffer (100 mM NaH2PO4, 10 mM Tris-Cl, 8 M urea pH6.3) and then proteins were eluted (100 mM NaH2PO4, 10 mMTris-Cl, 8 M urea, pH 5.9). Fractions containing protein(OmpA35–1735-His) were analysed by SDS-PAGE and then dia-lysed into phosphate-buffered saline (PBS) before being storedat -80°C until use.

Overnight bacterial cultures of E. coli BL21 (DE3) transformedwith pBOB002 were diluted 1:10 into 1 l of fresh LB medium withcarbenicillin and grown at 37°C to mid-exponential phase(OD600 = 0.6). The expression of GST-OmpA954–1735 was inducedby the addition of 0.1 mM isopropyl b-D-1-thiogalactopyranoside(IPTG) for 3.5 h at 30°C. Bacteria were harvested by centrifuga-tion, resuspended in PBS, pH 7.4, containing protease inhibitor,and lysed by sonication as described above. Lysates werecleared by centrifugation at 13 000 g for 30 min at 4°C andapplied to a GST-TRAP FF column on an Åkta fast protein liquidchromatography (FPLC). Protein was eluted with 30 mM glutath-ione and fractions were dialysed into PBS before being stored at-80°C until use.

In vitro pull-down assay

Fifty micrograms of soluble GST or GST-OmpA954–1735 proteinwas incubated with cleared HeLa whole cell lysate in NP40 lysisbuffer [20 mM Tris (pH 8.0), 150 mM NaCl, 1% NP40, 10% glyc-erol] for 2 h at room temperature. To this suspension, 50 ml of a50% slurry of immobilized glutathione-agarose in PBS was addedand incubated overnight at 4°C. The agarose beads were thenspun down 5 min at 600 g and washed five times in 1 ml of a 1:10NP40 buffer dilution in PBS, rocking each time for 4 min. To elute

bound protein, 30 ml of 30 mM glutathione in PBS was added andsamples were incubated with gentle rocking for 30 min at roomtemperature. The beads were again spun down at 600 g and thesupernatant was transferred to a clean tube. 6¥ SDS-PAGEsample buffer was added to each sample; proteins were resolvedby SDS-PAGE and then analysed by silver stain or Westernimmunoblot analysis. Protein identification by micro-capillaryLC/MS/MS analysis was performed at the Taplin Mass Spectrom-etry Facility (https://taplin.med.harvard.edu/)

Cell association and invasion assays

Cell association and invasion assays for R. conorii and E. coliexpressing R. conorii antigens in HeLa cells, EA.hy 926 cells andHMVEC-L cells were performed as previously described (Chanet al., 2009). BL21 (DE3)/pMC022 (ompA) was grown overnightat 37°C, diluted 1:10 into fresh media, grown to mid-logarithmicphase and induced with 0.1 mM IPTG for 3 h at 30°C, while BL21(DE3)/pYC9 (ompB) was induced with 0.1 mM IPTG for 3 h at37°C. The BL21 (DE3)/pRI203 (inv) strain was grown overnight at37°C, diluted 1:10 into fresh media and then grown to mid-logarithmic phase prior to use. To further demonstrate adherenceof the indicated E. coli strains to mammalian cells, HMVEC-Lcells were seeded onto sterile coverslips in 24-well plates andthen infected with 50 ml of an OD600 = 1.0 dilution of BL21 (DE3)containing pET-22b (empty vector) or pMC022 (ompA). Cover-slips were processed for immunofluorescence analysis asdescribed in Chan et al. (2009) using Texas Red phalloidin(1:200), DAPI (1:10 000), rabbit anti-E. coli antiserum (1:500)and Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:1000).

For protein blocking experiments, HMVEC-L and EA.hy 926cells were pre-incubated with the indicated concentrations ofGST [100 mg ml-1 (3.84 mM)] and GST-OmpA954–1735 [100 mg ml-1

(1.3 mM)] for 30 min prior to the addition of bacteria. Inhibitionassays using the RGD peptide (GRGDNP, 100 mM) and thecontrol peptide (GRGESP, 100 mM) in EA.hy 926 cells were per-formed essentially as described in Leininger et al. (1991). Forantibody inhibition experiments, HMVEC-L cells were incubatedwith 10 mg ml-1 mAbs against b integrins and a integrins for30 min in serum-free DMEM (SF DMEM) prior to the addition ofR. conorii at an MOI of 10. For experiments using BL21 (DE3)derivatives, bacteria were induced as described above withdiluted to an OD600 of 1.0 and then 50 ml of each strain was usedto infect the indicated mammalian cell line. Data are representa-tive of at least three independent experiments. P-values weredetermined using a standard Student’s t-test.

RNA interference (RNAi)

HMVEC-L and EA.hy 926 cells were plated onto six-well plates at2.0 ¥ 105 cells per well and transfected using 30 pmol of thedescribed siRNAs and 6 ml of RNAiMAX. Forty-eight hours posttransfection, cells were harvested by trypsinization and replatedfor an additional 24 h onto sterile glass coverslips in 24-wellplates for association and invasion assays as described above,or six-well tissue culture plates for subsequent Western immuno-blot analysis of protein expression. Actin was used as a loadingcontrol to demonstrate equal loading. The efficacy of transfectionwas determined by densitometry analysis of immunoblot imagescaptured on the LI-COR Odyssey CLx Imager.



Immunofluorescence

To illustrate colocalization of b1 integrin with R. conorii, HMVEC-Lcells were seeded onto sterile glass coverslips in 24-well plates,infected for 30 min with R. conorii, and then processed for immun-ofluorescence as described (Martinez and Cossart, 2004) usinganti-RCPFA, and Alexa Fluor 488 goat anti-rabbit IgG antibodies tolabel R. conorii. b1 integrin was visualized in infected non-permeabilized cells with 2 mg ml-1 anti-b1 integrin mAb (clone4B7R) and Alexa Fluor 546-conjugated anti-mouse IgG. Imageswere captured on a Leica TCS SP2 AOBS Laser Scanning Con-focal microscope. Analysis of colocalization was performed onindividual confocal images using the ‘Measure RGB’ function inthe public domain NIH ImageJ 1.46r analysis software package(http://imagej.nih.giv/ij) with colocalization depicted as overlap-ping histograms of the green and red channels.

Flow cytometry

HMVEC-L and HeLa cells in a 10 cm culture dish were washedthoroughly with PBS and dislodged using 5 ml of PBS + 1 mMEDTA with incubation at 37°C/5% CO2. Cells were collected andpelleted by centrifugation, 200 g for 7 min. Cells were resus-pended in serum-free DMEM (SF-DMEM) and aliquoted insamples of 1.0 ¥ 106 cells in 1 ml volume. Purified recombinantGST (5 mM) or GST-OmpA954–1735 (5 mM) was added to cells andincubated for 30 min at room temperature. Cells were pelleted asdescribed above, washed with 500 ml of PBS, and fixed in 2%PFA for 20 min. Samples were subsequently washed, blocked in2% bovine serum albumin (BSA)/PBS and processed for analysisusing rabbit anti-GST (Z-5) and Alexa Fluor 488 goat anti-rabbitIgG antibodies. Samples were analysed on a BD LSR-II flowcytometer using fluorescein isothiocyanate (FITC) parametersand the FloJo software package. A minimum of 50 000 eventswere counted for each sample.

For localization of OmpA at the surface of E. coli, BL21 (DE3)containing either pET22-b (empty vector) or pMC022 (ompA)were grown under non-inducing and inducing conditions asdescribed above. Bacteria were washed three times in PBS andthen diluted to an OD600 = 0.5. Five hundred microlitres of thisbacterial suspension was incubated using PBS/2% BSA con-taining anti-OmpA antisera (1:500), Alexa Fluor 488 goat anti-rabbit IgG antibodies and DAPI (5 mg ml-1). Samples wereanalysed on a BD LSR-II flow cytometer using fluorescein iso-thiocyanate (FITC) and DAPI parameters and the FloJo soft-ware package. A minimum of 100 000 events were counted foreach sample.

Bioinformatic analysis and protein modelling

OmpA sequences from SFG rickettsiae were aligned using theClustalW protein analysis function in the MacVector softwarepackage. Accession numbers for OmpA sequences in the analy-sis are as follows: R. conorii Malish 7 (AAA17405.1), R. rickettsii‘Sheila Smith’ (ABV76839.1), R. japonica YH (BAK97125.1) andR. africae (AAC35172.2). Amino acids 954–1735 from R. conoriiMalish 7 OmpA were submitted online to the Phyre2 server foranalysis (Kelley and Sternberg, 2009). The highest scoring modelwas based on the B. pertussis p62 pertactin protein structure(pdb1DAB) (Emsley et al., 1996).

Acknowledgements

We would like to thank M.M. Cardwell for constructing thepMC022 plasmid. We also thank members of the Martinez lab forcritical review of the manuscript. An award to J. J. M. from theNational Institute of Allergy and Infectious Diseases (NIAID),Infectious Diseases Branch (AI 072606) supported this work. Wealso wish to acknowledge membership within the Region V GreatLakes Regional Center of Excellence (GLRCE) in Biodefenseand Emerging Infectious Diseases Consortium (NIAID AwardU54-AI-057153).

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Supporting information

Additional Supporting Information may be found in the onlineversion of this article:

Fig. S1. Localization of OmpA on the surface of induced E. coliBL21 (DE3)/pMC022 (ompA).A. 2 mg of the indicated purified proteins were separated onSDS-PAGE, transferred to nitrocellulose and immunoblotted witheither anti-OmpA serum or anti-OmpB mAb 6B.6. The OmpAserum is reactive against purified OmpA, but not purified OmpB,while the OmpB mAb does not react against OmpA.B. Flow cytometry analysis of E. coli BL21 (DE3) harbouringpMC022 (ompA). Bacteria were left un-induced or induced withIPTG and then processed for flow cytometry using the anti-OmpAserum and appropriate Alexa 488-conjugated secondary anti-body. The red trace represents bacteria harbouring the emptyvector (pET22-b), the blue trace is un-induced bacteria harbour-ing pMC022 and the green trace is IPTG-induced bacteria har-bouring pMC022. A minimum of 100 000 E. coli bacteria wereanalysed per spectrum.



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OmpA-mediated rickettsial adherence to and invasion of human endothelial cells is dependent upon interaction with α2β1 integrin