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.

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Envelope viruses maturate by macromolecule assembly and budding. To investigate these steps, we generated virus-like particles (VLPs) by co-expression of structural proteins of Sendai virus (SeV), a prototype of the family Paramyxoviridae. Simultaneous expression of matrix (M), nucleo- (N), fusion (F), and hemagglutinin-neuraminidase (HN) proteins resulted in the generation of VLPs that had morphology and density similar to those of authentic virus particles, although the efficiency of release from cells was significantly lower than that of the virus. By using this VLP formation as a model of virus budding, roles of individual proteins in budding were investigated. The M protein was a driving force of budding, and the F protein facilitated and the HN protein suppressed VLP release. Either of the glycoproteins, F or HN, as well as the N protein, significantly shifted density of VLPs to that of virus particles, suggesting that viral proteins bring about integrity of VLPs by protein-protein interactions. We further found that co-expression of a nonstructural protein, C, but not V, enhanced VLP release to a level comparable to that of virus particles, demonstrating that the C protein plays a role in virus budding.

We propose the name Rickettsia japonica sp. nov. (with type strain YH [= ATCC VR-13631) for a serologically specific species of spotted fever group rickettsiae that are pathogenic for humans (J. Infect. Dis. 159:1122-1126, 1989; J. Clin. Microbiol. 28:1177-1180, 1990). The biologic and genomic characteristics of the organism (G+C content, 31.2 +-0.7 mol%) are essentially the same as those of other pathogenic spotted fever group rickettsiae, although the R. japonica isolates cause a persistent infection in Vero cells for many subcultures.In this paper we formally describe Rickettsia japonica (23,25), which has been identified as the causative agent of a human disease. The first isolate, strain YHT (T = type strain), was isolated in 1985 from the blood of a patient with febrile exanthematous illness in Japan by using a tissue culture technique (20). Five strains of the causative agent, including strain YHT, have been isolated from patients that were serodiagnosed as having a spotted fever group (SFG) rickettsiosis (22).Justification for a new species. The justification for establishing a new species for this organism is based on the current standard method for comparing rickettsial taxonomic types by serologic analysis (23). Experiments involving reciprocal cross-reactions of mouse polyclonal antibodies to strains of the new species and other species of SFG rickettsiae produced by the standard method were carried out to calculate the specificity differences. By using this approach we demonstrated that all five strains belong to a single species that is distinct from all of the previously described SFG rickettsiae that are known to be pathogenic for humans (23). In addition, none of the isolates reacted with mouse monoclonal antibodies that are species specific for other pathogenic SFG rickettsiae (23). Furthermore, the results of Western immunoblotting revealed different electrophoretic mobilities and antigenic reactivities for the major immunodominant high-molecular-weight surface polypeptides of the Japanese isolates and standard pathogenic SFG rickettsial strains (23). Species-specific monoclonal antibodies to R. japonica reacted only with strains of R. japonica, supporting the conclusion that R. japonica is a new species of SFG rickettsiae (25).

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