The lspA Gene, Encoding the Type II Signal Peptidase of Rickettsia typhi : Transcriptional and Functional Analysis

Abstract

Lipoprotein processing by the type II signal peptidase (SPase II) is known to be critical for intracellular growth and virulence for many bacteria, but its role in rickettsiae is unknown. Here, we describe the analysis of lspA, encoding a putative SPase II, an essential component of lipoprotein processing in gram-negative bacteria, from Rickettsia typhi. Alignment of deduced amino acid sequences shows the presence of highly conserved residues and domains that are essential for SPase II activity in lipoprotein processing. The transcription of lspA, lgt (encoding prolipoprotein transferase), and lepB (encoding type I signal peptidase), monitored by real-time quantitative reverse transcription-PCR, reveals a differential expression pattern during various stages of rickettsial intracellular growth. The higher transcriptional level of all three genes at the preinfection time point indicates that only live and metabolically active rickettsiae are capable of infection and inducing host cell phagocytosis. lspA and lgt, which are involved in lipoprotein processing, show similar levels of expression. However, lepB, which is involved in nonlipoprotein secretion, shows a higher level of expression, suggesting that LepB is the major signal peptidase for protein secretion and supporting our in silico prediction that out of 89 secretory proteins, only 14 are lipoproteins. Overexpression of R. typhi lspA in Escherichia coli confers increased globomycin resistance, indicating its function as SPase II. In genetic complementation, recombinant lspA from R. typhi significantly restores the growth of temperature-sensitive E. coli Y815 at the nonpermissive temperature, supporting its biological activity as SPase II in prolipoprotein processing.

Functional Characterization of a Phospholipase A 2 Homolog from Rickettsia typhi

“…However, the secretion pathway of RT0522 protein into the host cytoplasm, its posttranslocational modification (if any), and the mechanism of interaction with host organelles remain unknown. In silico analysis revealed that the protein RT0522 does not contain a signal peptide sequence, suggesting that this protein is not secreted to the host cell cytoplasm through the Sec-dependent protein secretion pathway of rickettsiae (1,(29)(30)(31). However, other secretory pathways, e.g., the type IV protein secretion pathway, have been identified in rickettsiae (9).…”

“…The titers of partially purified R. typhi from Vero76 cells were determined using the Live/ Dead BacLight bacterial viability assay (Molecular Probes, Eugene, OR) as previously described (29). A monolayer of Vero76 cells (approximately 1 ϫ 10 6 cells) was infected with R. typhi at a multiplicity of infection of approximately 100 rickettsiae.…”

“…We checked the expression of RT0522 from pYES-522 transformed into INVSc1 cells under inducing conditions and found that there was no detectable expression of RT0522 in yeast cells (data not shown). This result prompted us to consider codon optimization of RT0522 (as Rickettsia genomes are AT rich) for expression in mammalian cells (10,19,29). The yeast strain INVSc1 transformed with plasmid pYES-522HS carrying the codon-optimized RT0522 gene expresses the expected protein (Fig.…”

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

“…Our previous work on R. typhi protein secretion has focused mainly on the characterization of the Sec pathway (49)(50)(51) and the rvh T4SS (26,27). Little is known about the function of the rickettsial T1SS, despite the presence of genes encoding the T1SS components within all sequenced Rickettsia genomes.…”

Proteomic Analysis of and Immune Responses toEhrlichia chaffeensisLipoproteins

“…Bacterial SPase II cleaves the signal peptide from glyceride-modified prolipoproteins, while they are translocated across the cytoplasmic membrane (37). It was shown recently that overexpression of R. typhi LspA in E. coli confers resistance to globomycin, a specific inhibitor of SPase II (18), and genetically complements the growth defect of a SPase II-deficient E. coli mutant (38). Although the percentage of amino acid identity between R. typhi LspA and E. chaffeensis LspA is only 36%, five domains and catalytic residues that are considered to be essential for the enzymatic activity of bacterial SPase II (37) are conserved in the LspA proteins of E. chaffeensis and other members of the family Anaplasmataceae (Fig.…”