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.

FIG. 1.

Alignment of deduced amino acid sequences of the lspA genes from R. typhi (Rt) (GenBank accession no. NC_006142), R. prowazekii (Rp) (GenBank accession no. AJ235271), R. bellii (Rb) (GenBank accession no. NZ_AARC01000001), R. canadensis (Rcan) (GenBank accession no. NZ_AAFF01000001), R. akari (Ra) (GenBank accession no. NZ_AAFE01000001), R. conorii (Rc) (GenBank accession no. NC_003103), R. sibirica (Rs) (GenBank accession no. AABW01000001), R. rickettsii (Rr) (GenBank accession no. NZ_AADJ01000001), R. felis (Rf) (GenBank accession no. NC_007109), and E. coli (Ec) (GenBank accession no. X00776). Molecular masses and isoelectric points were computed with MacVector 7.1.1 software. The conserved amino acid domains (boxes A through E) are shown in bold. The catalytic residues are marked by asterisks.

FIG. 2.

Kinetics of lepB, lgt, and lspA transcription during the R. typhi life cycle in in vitro host (NCTC clone 929) cells determined by two-step real-time qRT-PCR. The MNEs (± standard errors) of the target genes (lepB, lgt, and lspA) were calculated relative to the expression of the reference (16S rRNA) gene by Q-Gene software with amplification efficiency correction. At the top, the change in the level of MNE at different times of growth for each target gene is given with respect to that of the preinfection time point (0 h), set at 1.00. The expression levels of lgt and lspA at 24 and 48 h postinfection were found to be significantly different (P < 0.05 [single-factor ANOVA]) from those at 1 to 8 h postinfection. However, there is a lack of statistical significance to support the same for lepB, which may be due to variations in expression levels.

FIG. 3.

Globomycin resistance assay for R. typhi lspA. E. coli cells carrying the designated plasmids were incubated in the presence of various concentrations of globomycin, and growth of the cultures was measured as the optical density at 600 nm (OD₆₀₀). The mean OD₆₀₀ ± standard error is plotted against the globomycin concentration.

FIG. 4.

Western blot analysis of the expression of R. typhi SPase II in E. coli Top10 cells. Total proteins from E. coli cells carrying the appropriate plasmid were probed with anti-HisG monoclonal antibody. Lane 1, total proteins from E. coli Top10/pTrcHisA; lane2, total proteins from E. coli Top10/pTrcHisRTlspA127; lane3, total proteins from E. coli Top10/pTrcHisEClspA7. Arrows indicate the sizes of Bio-Rad Kaleidoscope prestained marker bands (32.1 and 17.5 kDa). The minor bands below the 17.5-kDa bands in lanes 2 and 3 may have resulted from the degradation of recombinant SPase II or nonspecific binding in the total proteins.