Identification of Two Putative Rickettsial Adhesins by Proteomic Analysis
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Research in Microbiology 157 (2006) 605–612 www.elsevier.com/locate/resmic Identification of two putative rickettsial adhesins by proteomic analysis Patricia Renesto a,∗, Laurent Samson a, Hiroyuki Ogata b, Saïd Azza a, Patrick Fourquet c, Jean-Pierre Gorvel c, Robert A. Heinzen d, Didier Raoult a a Unité des Rickettsies, CNRS UMR 6020, IFR-48, Faculté de Médecine, 27 Boulevard Jean Moulin, 13385 Marseille, France b Information Génomique et Structurale, CNRS UPR 2589, IBSM, Parc Scientifique de Luminy, 13288 Marseille, France c CIML, Parc scientifique et technologique de Luminy, 13288 Marseille, France d Laboratory of Intracellular Parasites, NIAID, NIH, Hamilton, MO, USA Received 6 September 2005; accepted 7 February 2006 Available online 28 February 2006 Abstract The rickettsial membrane proteins that promote their uptake by eukaryotic host cells are unknown. To identify rickettsial ligand(s) that bind host cell surface proteins, biotinylated epithelial cells were used to probe a nitrocellulose membrane containing rickettsial extracts separated by SDS– PAGE. This overlay assay revealed that two close rickettsial ligands of approximately 32–30 kDa were recognized by host cells. Both proteins were identified using high resolution 2D-PAGE coupled with mass spectrometry analysis. One protein was identified as the C-terminal extremity of rOmpB called the β-peptide. The second interacting protein was identified as a protein of unknown function encoded by RC1281 and RP828 in Rickettsia conorii and in Rickettsia prowazekii, respectively, that shares strong similarities with other bacterial adhesins. Both proteins are highly conserved within the Rickettsia genus and might play a critical role in their pathogenicity. These data may have important implications for the development of future vaccines against rickettsial infections. © 2006 Elsevier SAS. All rights reserved. Keywords: Rickettsiae; Adhesin; rOmpB 1. Introduction The attachment of microorganisms to a biological surface is a complex process involving both non-specific and specific events. Non-specific attachment can be mediated by electro- Rickettsia species are small, Gram-negative bacilli that are static forces, lipophilic and hydrophobic interactions. Specific obligate intracellular parasites of eukaryotic cells. This genus “lock and key”-type interactions between lectin-like bacterial consists of two antigenically defined groups: the spotted fever binding proteins (adhesins) and complex glycoprotein recep- group (SFG) and the typhus group (TG), which are respec- tors on host tissues have also been described [15,48]. Adhesin tively responsible for spotted fevers and typhus [36]. Electron proteins can be located at the distal tip of non-flagellar surface microscopy studies show that initial contact between these bac- appendages known as pili or fibrillae [21]. Afimbrial adhesins, teria and endothelial cells results in a dramatic alteration of which include most other adherence molecules, have also been the cell surface, with the formation of numerous microvillar reported. Characterized afimbrial adhesins include BabA ad- extensions associated with membrane ruffling [42]. However, hesin from Helicobacter pylori [18], outer-membrane proteins the molecular mechanisms through which rickettsiae attach to (Vomp) from Bartonella quintana [51], filamentous hemag- and induce their internalization by host cells are still largely un- glutinin and pertactin from Bordetella pertussis [7], opacity known [47]. proteins (Opas) from Neisseria and invasin from enteropatho- genic Yersinia [19]. Another major constituent of the outer membrane of Gram-negative bacteria that plays a critical role * Corresponding author. in the interface between microorganisms and the environment E-mail address: [email protected] (P. Renesto). is lipopolysaccharide (LPS). Its role as an adhesin has been 0923-2508/$ – see front matter © 2006 Elsevier SAS. All rights reserved. doi:10.1016/j.resmic.2006.02.002 606 P. Renesto et al. / Research in Microbiology 157 (2006) 605–612 suggested for several bacteria including Actinobacillus pleu- to identification of 2 putative rickettsial adhesins recognized by ropneumoniae, Campylobacter jejuni, Helicobacter pylori and host cells. Neisseria gonorrhoeae [20]. Rickettsiae express two major surface proteins, termed rick- 2. Materials and methods ettsial outer membrane proteins A (rOmpA) and B (rOmpB) [2,24]. Both proteins are present in SFG rickettsiae, but only 2.1. Rickettsiae and cells rOmpB is present in TG [26,50]. As a consequence of a gene degradation of rompA [5,37], rOmpA is exclusive for SFG. It is R. conorii Seven (Malish), ATCC VR-610T and R. prowaze- well established that rOmpA and rOmpB are immunodominant kii BreinL, ATCC VR-142T were propagated on a conflu- antigens containing protective epitopes. Protection afforded by ent monolayer of African green monkey kidney cells (Vero the recombinant truncated rOmpA protein against rickettsial in- cells, ATCC C1587) [36] and purified on renographin gra- fection has been demonstrated in a R. rickettsii–mouse model dient as previously reported [13]. Bacterial pellets were sol- [28,41], and in a R. conorii–guinea pig model [46]. Vaccine ubilized in Laemmli buffer and separated by sodium dode- efficacy of plasmid DNA encoding recombinant rOmpA has cyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in also been reported [9,41]. Protection against lethal infection a 12% acrylamide gel [22]. of C3H/HeN SCID mice by R. conorii was induced by mAbs against rOmpA and rOmpB [45]. Similarly, protection of mice 2.2. Biotinylation of epithelial cell proteins against lethal challenge with R. typhi was induced by im- munization with native R. typhi rOmpB protein [6,10,45]. In Vero cells grown to confluence in 125 cm2 flasks were contrast, passive immunization with antibodies against LPS washed with phosphate buffered saline (PBS), detached with does not protect mice from infection with viable rickettsiae beads and centrifuged. The pellet was resuspended in PBS con- [1,14,45]. taining 10 mg/ml of EZ-link sulfo-NHS-LC-biotin (Pierce) and With the exception of R. akari (the agent of rickettsialpox) ◦ incubated 1 h at 37 C. After washing, pellets were submitted which infects macrophages, rickettsiae target primarily en- to sonication and insoluble components removed by centrifuga- dothelial cells [36]. Several lines of evidence suggest that ◦ tion (10 000 g for 15 min at 4 C). Biotinylated proteins were rOmpA and rOmpB are involved in adherence of these microor- ◦ stored at −80 C. ganisms to eukaryotic cells [26]. Two additional outer mem- brane proteins of 31 and 29.5 kDa are also implicated in adher- 2.3. Overlay assays ence of R. prowazekii [11]. The 31 kDa protein is thought to correspond to the cleavable C-terminal domain of rOmpB [17], whereas the 29.5 kDa protein is the preprotein translocase SecA Rickettsial extracts separated in one dimension by SDS– subunit [12]. PAGE (20 µg) or by two-dimensional (2D)-PAGE (200 µg) were transferred to nitrocellulose membranes (Amersham). Despite numerous efforts [35,39,43], genetic manipulation ◦ of Rickettsia remained impossible for years [49]. While recent Membranes were incubated for 1.5 h at 4 C with biotiny- progress in the field of the genetic transformation of Rickettsia lated epithelial cells diluted 1:100 in phosphate buffer saline is hopeful [33], it is still difficult to specifically knock out a rick- (PBS) supplemented with 0.02% Tween-20 and 0.5% milk ettsial gene and to firmly demonstrate the biological role of (PBS–Tween–milk). After washing three times, the blots were the corresponding encoded protein. Recognition of and bind- incubated with peroxidase-labeled streptavidin (1:1000, Amer- ing to the host cell is a key step in the pathogenesis of many sham) and biotinylated proteins detected by chemilumines- virulent bacterial strains, and identification of the molecular ba- cence (ECL, Amersham). sis of rickettsial attachment to host cells remains an important objective. This is particularly true when considering the fact 2.4. Rickettsial membrane preparation for 2D-electrophoresis that these strictly intracellular bacteria, which are considered as a threat for use as bioterrorism agents [4], must enter host cells The R. conorii and R. prowazekii pellets purified as de- to replicate and survive. scribed above were resuspended in 5 ml of 5 mM Tris–HCl The last few years have seen a dramatic increase in the num- buffer (pH 7.6) and lysed by two passes through a constant ber of sequenced bacterial genomes, accompanied by a rapid cell disruption system (2 kbar). Cell debris and unbroken cells acceleration in the development of new bioinformatics tools. were removed by centrifugation at 5600 g for 20 min. Bac- Large-scale DNA sequencing has also stimulated the devel- terial membranes present in the supernatant were pelleted by opment of technological changes in proteomics, namely two- ultracentrifugation at 100 000 g for 2 h and washed in 5 mM ◦ dimensional polyacrylamide gel electrophoresis (2D-PAGE) Tris–HCl, pH 7.6 before storage at −80 C. coupled with mass spectrometry which allows identification of proteins in a complex mixture. In this work, we used a global 2.5. 2-D electrophoresis and silver staining proteomic approach to identify rickettsial ligands involved in bacteria-host cell interactions. This study, which was carried ImmobilineTM DryStrips (18 cm, pH 3–11 or 6–11 Amer- out on both R. conorii (the agent of Mediterranean spotted sham) were rehydrated overnight with 350