| As obligate, intracellular, vector-borne bacteria, rickettsiae must adapt to both mammalian and arthropod host cell environments, and this adaptation must encompass factors involved in bacterial attachment, invasion, intracellular trafficking, and immune evasion. Deciphering the molecular mechanisms of the interactions between rickettsiae and their host cells has largely been hindered by the genetic intractability of these organisms; however, research in other Gram-negative pathogens has demonstrated that many bacterial determinants of attachment, entry, and infection-induced cellular injury comprise extracytoplasmic proteins. The annotations of several rickettsial genomes indicate the presence of a Sec translocon, the major route for bacterial protein secretion from the cytoplasm. For Rickettsia typhi, the etiologic agent of murine typhus, homologs of the Sec translocon-associated proteins LepB, LspA, and SecA have been functionally characterized; therefore, we hypothesize that the R. typhi Sec apparatus represents a mechanism for the secretion of rickettsial proteins, including virulence factors, into the extracytoplasmic environment. Our objective was to characterize such Sec-dependent R. typhi proteins in the context of a mammalian host cell infection. Beginning with a genome-wide computational screen, we identified 191 R. typhi proteins predicted to contain an N-terminal signal peptide to target them for secretion via the Sec system. We then tested the ability of 102 of these putative signal peptides to direct the Sec-dependent secretion of the alkaline phosphatase enzyme in E. coli, and we found that 84 (82%) of these candidates could function as signal peptides within this heterologous system. Furthermore, we demonstrated that at least 54 of the genes encoding these Sec-dependent proteins undergo active gene transcription during R. typhi infections in HeLa cells, indicating that these proteins may be involved in rickettsial growth and virulence in mammalian host cells. We then used this work as a platform from which to launch more in-depth studies of two specific R. typhi proteins: RT0216, a TolC homolog, and RT0218, a conserved rickettsial hypothetical protein. This contribution to rickettsial molecular biology could eventually reveal proteins which could be exploited for diagnostic, therapeutic, or preventive measures against rickettsial infections. |
