SummaryRickettsia conorii, an obligate intracellular tickborne pathogen and the causative agent of Mediterranean spotted fever, binds to and invades non-phagocytic mammalian cells. Previous work identified Ku70 as a mammalian receptor involved in the invasion process and identified the rickettsial autotransporter protein, rOmpB, as a ligand; however, little is known about the role of Ku70-rOmpB interactions in the bacterial invasion process. Using an Escherichia coli heterologous expression system, we show here that rOmpB mediates attachment to mammalian cells and entry in a Ku70-dependent process. A purified recombinant peptide corresponding to the rOmpB passenger domain interacts with Ku70 and serves as a competitive inhibitor of adherence. We observe that rOmpB-mediated infection culminates in actin recruitment at the bacterial foci, and that this entry process relies in part on actin polymerization likely imparted through protein tyrosine kinase and phosphoinositide 3-kinase-dependent activities and microtubule stability. Small-interfering RNA studies targeting components of the endocytic pathway reveal that entry by rOmpB is dependent on c-Cbl, clathrin and caveolin-2. Together, these results illustrate that rOmpB is sufficient to mediate Ku70-dependent invasion of mammalian cells and that clathrin-and caveolin-dependent endocytic events likely contribute to the internalization process.

Cytolethal distending toxins (CDTs) 6 are members of a group of bacterial toxins and effectors called "cyclomodulins" that interfere with the eukaryotic cell cycle rather than inducing overt cytotoxicity (1, 2). Inhibiting cell cycle disrupts many of the normal functions of rapidly dividing eukaryotic cells, including lymphocytes and epithelial cells, which provide immunity and physical barriers to microbial pathogens (3-5). Thus, it is not surprising that cdt genes are found in a diverse group of Gram-negative pathogens that colonize different niches within the host. Although a growing body of evidence supports the importance of CDTs in bacterial virulence and host-pathogen interactions (6), the manner in which individual CDTs interact with and intoxicate host cells remains poorly understood.CDTs are AB 2 toxins, consisting of a hetero-trimeric complex of three proteins (CdtA, CdtB, and CdtC) at a 1:1:1 molar ratio (5,7,8). The current model is that CdtA and CdtC are the binding "B" moieties that collaborate to facilitate binding and entry of the catalytic "A" subunit, CdtB, into mammalian cells. CdtB shares a common tertiary structure with DNase I and phosphatidylinositol 3,4,5-triphosphate phosphatase enzymes and displays both activities in cell-free systems (9 -13). It is not currently known which activity is of greater importance, and this may depend on the specific toxin and/or the host target cell type (12,14). CdtB enzymatic activity induces cell cycle arrest predominantly at the G 2 /M transition, resulting in cellular distension and ultimately cell death (5,15,16).Consistent with their proposed roles as binding subunits, CdtA and/or CdtC increase the ability of CdtB to associate with host cells and greatly enhance intoxication (7,(17)(18)(19)(20)(21)(22)(23)(24)(25). The identification of ricin-like lectin domains in CdtA and CdtC from structural and biochemical data first suggested that these subunits may interact with carbohydrates on the cell surface (13,26,27). Consistent with this hypothesis, CDT produced by Escherichia coli (Ec-CDT) was reported to require N-linked glycoproteins for binding and subsequent intoxication of HeLa cells (23). Moreover, Ec-CDT bound fucose in vitro, and fucose-specific lectins blocked Ec-CDT-mediated cell cycle arrest, presumably by preventing binding of toxin to its receptor. These findings suggested that fucose might serve as a binding determinant for Ec-CDT. Similarly, host glycans were reported to support Aggregatibacter actinomycetemcomitans (Aa-CDT) intoxication. Specifically, Aa-CDT bound three glycosphingolipids, GM1, GM2, and GM3, and intoxication of human monocytic U937 cells was blocked by preincubation of toxin with liposomes that contained G M3 (24). In addition, the CdtA subunit of Aa-CDT bound to the glycoprotein thyroglobulin (19). However, the functional significance of this binding is * This work was supported, in whole or in part, by National Institutes of Health Grants T32DE007296 (to A. E.), F31AI061837 (to F. J. M.-A.), and AI59095 (to S. R. B. ...

Obligate intracellular bacteria of the genus Rickettsia must adhere to and invade the host endothelium in order to establish an infection. These processes require the interaction of rickettsial surface proteins with mammalian host cell receptors. A previous bioinformatic analysis of sequenced rickettsial species identified a family of at least 17 predicted "surface cell antigen" (sca) genes whose products resemble autotransporter proteins. Two members of this family, rOmpA and rOmpB of spotted fever group (SFG) rickettsiae have been identified as adhesion and invasion factors, respectively; however, little is known about the putative functions of the other sca gene products. An intact sca2 gene is found in the majority of pathogenic SFG rickettsiae and, due to its sequence conservation among these species, we predict that Sca2 may play an important function at the rickettsial surface. Here we have shown that sca2 is transcribed and expressed in Rickettsia conorii and have used a heterologous gain-of-function assay in E. coli to determine the putative role of Sca2. Using this system, we have demonstrated that expression of Sca2 at the outer membrane of nonadherent, noninvasive E. coli is sufficient to mediate adherence to and invasion of a panel of mammalian cells, including endothelial cells. Furthermore, soluble Sca2 protein is capable of diminishing R. conorii invasion of cultured mammalian cells. This is the first evidence that Sca2 participates in the interaction between SFG rickettsiae and host cells and suggests that in addition to other surface proteins, Sca2 may play a critical role in rickettsial pathogenesis.

The pathogenesis of spotted fever group (SFG) Rickettsia species, including R. conorii and R. rickettsii, is acutely dependent on adherence to and invasion of host cells, including cells of the mammalian endothelial system. Bioinformatic analyses of several rickettsia genomes revealed the presence of a cohort of genes designated sca genes that are predicted to encode proteins with homology to autotransporter proteins of Gram-negative bacteria. Previous work demonstrated that three members of this family, rOmpA (Sca0), Sca2, and rOmpB (Sca5) are involved in the interaction with mammalian cells; however, very little was known about the function of other conserved rickettsial Sca proteins. Here we demonstrate that sca1, a gene present in nearly all SFG rickettsia genomes, is actively transcribed and expressed in R. conorii cells. Alignment of Sca1 sequences from geographically diverse SFG Rickettsia species showed that there are high degrees of sequence identity and conservation of these sequences, suggesting that Sca1 may have a conserved function. Using a heterologous expression system, we demonstrated that production of R. conorii Sca1 in the Escherichia coli outer membrane is sufficient to mediate attachment to but not invasion of a panel of cultured mammalian epithelial and endothelial cells. Furthermore, preincubation of a recombinant Sca1 peptide with host cells blocked R. conorii cell association. Together, these results demonstrate that attachment to mammalian cells can be uncoupled from the entry process and that Sca1 is involved in the adherence of R. conorii to host cells.

Spotted fever group (SFG) rickettsial species are obligate intracellular tick-borne pathogens that are responsible for important human diseases. Previous reports have demonstrated the feasibility of using recombinant surface cell antigen Sca5/OmpB to elicit protective immunity against homologous challenges using murine models of Mediterranean spotted fever and Rocky Mountain spotted fever. In addition, the feasibility of generating cross-protective immunity against related rickettsial species has also been established, but the molecular basis for these phenomena was not explored. Here, we demonstrate that vaccination of C3H/HeN mice with a recombinant OmpB domain derived from Rickettsia conorii induced high titer humoral immune responses that are capable of recognizing the native OmpB protein at the R. rickettsii outer membrane, but this immunization was not sufficient to induce effective protective immunity. In contrast, animals vaccinated with a corresponding OmpB domain derived from R. rickettsii protected animals from fatal outcomes. These results demonstrate that vaccination with nearly identical antigens may not be an effective strategy to induce wide-ranging protective immunity against related SFG Rickettsia species.