Rickettsial outer-membrane protein B (rOmpB) mediates bacterial invasion through Ku70 in an actin, c-Cbl, clathrin and caveolin 2-dependent manner

Abstract

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.

Disruption of theRickettsia rickettsiiSca2 Autotransporter Inhibits Actin-Based Motility

“…Two members of the family, rickettsial OmpA (rOmpA) and rOmpB, are high-abundance antigenic proteins on the surfaces of rickettsiae and are targets of neutralizing antibody (3,39). The functions of most members of the Sca family of autotransporters are unknown, but the proposed roles of rOmpA and rOmpB as adhesins (15,39,61) have led to the suggestion that many members of the Sca family may also be involved in the attachment process (10). Indeed, R. conorii Sca2 has recently been associated with adherence (13).…”

Endothelial Cell Proteomic Response to Rickettsia conorii Infection Reveals Activation of the Janus Kinase (JAK)-Signal Transducer and Activator of Transcription (STAT)-Inferferon Stimulated Gene (ISG)15 Pathway and Reprogramming Plasma Membrane Integrin/Cadherin Signaling

“…Rickettsial organisms enter endothelial cells through a calcium-dependent zipper-like entry mechanism involving the actin cytoskeleton (2)(3)(4). Viable organisms subsequently exit the phagosomes via phospholipase D and hemolysin activities (5,6), replicate in the cytoplasm, and exhibit early intercellular spread as a consequence of directional actin polymerization without detectable cellular injury (7)(8)(9).…”

Multimethylation of Rickettsia OmpB Catalyzed by Lysine Methyltransferases

“…The precursor of OmpB consists of a signal peptide, a large N-terminal passenger domain and a C-terminal ␤-barrel domain (11,12). The passenger domain of rickettsial OmpB has been shown to participate in adhesion to mammalian cells in vitro, suggesting this may be the role of OmpB in the virulence of Rickettsia (5,6,13).…”