Characterization of Sec-translocon-dependent extracytoplasmic proteins of Rickettsia typhi

Abstract

As obligate intracellular, vector-borne bacteria, rickettsiae must adapt to both mammalian and arthropod host cell environments. 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 pathogenesis are extracytoplasmic proteins. The annotations of several rickettsial genomes indicate the presence of homologs of the 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, SecA, and LspA have been functionally characterized; therefore, 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. By using the web-based programs LipoP, SignalP, and Phobius, a total of 191 R. typhi proteins were predicted to contain signal peptides targeting them to the Sec translocon. Of these putative signal peptides, 102 were tested in an Escherichia coli-based alkaline phosphatase (PhoA) gene fusion system. Eighty-four of these candidates exhibited signal peptide activity in E. coli, and transcriptional analysis indicated that at least 54 of the R. typhi extracytoplasmic proteins undergo active gene expression during infections of HeLa cells. This work highlights a number of interesting proteins possibly involved in rickettsial growth and virulence in mammalian cells.

FIG. 1.

The alkaline phosphatase (′phoA) gene fusion vectors. (A) The composition of the pJDT and pJDT-SDM ′phoA gene fusion vectors. Restriction sites are underlined, and nucleotides highlighted in gray boxes represent the extra nucleotides added to generate three different reading frames. The first six amino acids of the ′PhoA protein are listed below the DNA sequence, along with the two amino acids introduced by the BamHI or MfeI sites and the frame-altering nucleotides. (B) The primers for site-directed mutagenesis of the pJDT vectors to convert the BamHI restriction sites into MfeI sites. Altered nucleotides are in bold and underlined, and the MfeI sites are in green.

FIG. 2.

Gene expression of R. typhi extracytoplasmic proteins in HeLa cells. HeLa cells were infected with R. typhi at an MOI of ∼10, and the bacteria were isolated for RNA extraction at 4 days postinfection. Two-step, real-time RT-qPCR was used to assess gene expression of the putative R. typhi extracytoplasmic proteins relative to the rpsL gene. The mean relative expression and the standard errors are presented for each gene; results are from three independent infections. Individual data points are listed in Table S5 in the supplemental material.