TolC-Dependent Secretion of an Ankyrin Repeat-Containing Protein of Rickettsia typhi

Abstract

bRickettsia typhi, the causative agent of murine (endemic) typhus, is an obligate intracellular pathogen with a life cycle involving both vertebrate and invertebrate hosts. In this study, we characterized a gene (RT0218) encoding a C-terminal ankyrin repeat domain-containing protein, named Rickettsia ankyrin repeat protein 1 (RARP-1), and identified it as a secreted effector protein of R. typhi. RT0218 showed differential transcript abundance at various phases of R. typhi intracellular growth. RARP-1 was secreted by R. typhi into the host cytoplasm during in vitro infection of mammalian cells. Transcriptional analysis revealed that RT0218 was cotranscribed with adjacent genes RT0217 (hypothetical protein) and RT0216 (TolC) as a single polycistronic mRNA. Given one of its functions as a facilitator of extracellular protein secretion in some Gram-negative bacterial pathogens, we tested the possible role of TolC in the secretion of RARP-1. Using Escherichia coli C600 and an isogenic tolC insertion mutant as surrogate hosts, our data demonstrate that RARP-1 is secreted in a TolC-dependent manner. Deletion of either the N-terminal signal peptide or the C-terminal ankyrin repeats abolished RARP-1 secretion by wild-type E. coli. Importantly, expression of R. typhi tolC in the E. coli tolC mutant restored the secretion of RARP-1, suggesting that TolC has a role in RARP-1 translocation across the outer membrane. This work implies that the TolC component of the putative type 1 secretion system of R. typhi is involved in the secretion process of RARP-1.

Secretome of obligate intracellularRickettsia

“…typhi requires both the Sec translocon and TolC (Kaur et al ., 2012) . Secretion of Rickettsia ankyrin repeat protein 1 (RARP-1) into the host cytoplasm was shown during R .…”

“…Since RARP-1 is already in the PP and processed at its required NT, the CTD may be required not only for docking the protein to TolC, but also for facilitating its extracellular secretion. Interestingly, the ANK domains are highly conserved across all Rickettsia RARP-1 homologs, with other regions of the protein exhibiting extreme levels of divergence and additional repeats (Kaur et al ., 2012) . Thus the RARP-1 ANK repeats may have a conserved role in secretion, with more variable regions of the protein interacting with host cell molecules.…”

“…typhi TolC cannot interact with the AcrA/AcrB efflux complex of E . coli (Kaur et al ., 2012) . In vivo studies, as well as more direct experiments designed to complement an apposite T1SS mutant strain, are needed to determine the energy requirements (if any) for RARP-1 secretion.…”

“…The OEP component, annotated as TolC, has a predicted structure similar to E . coli TolC, with strict conservation of essential residues involved in channel formation and regulation (Kaur et al ., 2012) . As no other OEP proteins are encoded within Rickettsia genomes, TolC is likely bifunctional, participating in protein secretion (Kaur et al ., 2012) and drug efflux pumps.…”

“…coli TolC, with strict conservation of essential residues involved in channel formation and regulation (Kaur et al ., 2012) . As no other OEP proteins are encoded within Rickettsia genomes, TolC is likely bifunctional, participating in protein secretion (Kaur et al ., 2012) and drug efflux pumps. This is supported by the presence of dozens of genes encoding components of non-T1SS transporters (Gillespie et al ., 2008) .…”