A Type I Signal Peptidase Is Required for Pilus Assembly in the Gram-Positive, Biofilm-Forming Bacterium Actinomyces oris

Abstract

The Gram-positive bacterium Actinomyces oris, a key colonizer in the development of oral biofilms, contains 18 LPXTG motifcontaining proteins, including fimbrillins that constitute two fimbrial types critical for adherence, biofilm formation, and polymicrobial interactions. Export of these protein precursors, which harbor a signal peptide, is thought to be mediated by the Sec machine and require cleavage of the signal peptide by type I signal peptidases (SPases). Like many Gram-positive bacteria, A. oris expresses two SPases, named LepB1 and LepB2. The latter has been linked to suppression of lethal "glyco-stress," caused by membrane accumulation of the LPXTG motif-containing glycoprotein GspA when the housekeeping sortase srtA is genetically disrupted. Consistent with this finding, we show here that a mutant lacking lepB2 and srtA was unable to produce high levels of glycosylated GspA and hence was viable. However, deletion of neither lepB1 nor lepB2 abrogated the signal peptide cleavage and glycosylation of GspA, indicating redundancy of SPases for GspA. In contrast, the lepB2 deletion mutant failed to assemble the wild-type levels of type 1 and 2 fimbriae, which are built by the shaft fimbrillins FimP and FimA, respectively; this phenotype was attributed to aberrant cleavage of the fimbrillin signal peptides. Furthermore, the lepB2 mutants, including the catalytically inactive S101A and K169A variants, exhibited significant defects in polymicrobial interactions and biofilm formation. Conversely, lepB1 was dispensable for the aforementioned processes. These results support the idea that LepB2 is specifically utilized for processing of fimbrial proteins, thus providing an experimental model with which to study the basis of type I SPase specificity. A ctinomyces oris is a key colonizer in the development of dental plaque or oral biofilms because of its ability to adhere to many oral colonizers and host surfaces. This multiadhesive property is attributed to the presence of two types of fimbriae or pili called type 1 and 2 fimbriae (1, 2). Type 1 fimbriae-made of the shaft fimbrillin FimP and the tip fimbrillin FimQ-mediate bacterial adherence to proline-rich proteins that coat tooth enamel (2-4). Type 2 fimbriae-composed of the shaft fimbrillin FimA and the tip fimbrillin FimB-promote polymicrobial interactions or bacterial coaggregation, biofilm formation, and adhesion to host cells (2,5,6). A. oris also encodes 14 cell wall-anchored proteins (AcaA to AcaN), one of which, CafA (previously named AcaF), has been shown to be the major coaggregation factor that forms a distinct fimbrial tip of type 2 fimbriae (7). It was recently shown that the cell wall-anchored protein AcaC (named GspA) is glycosylated, and its glycosylation appears to be coupled to protein secretion and sortase SrtA-catalyzed cell wall anchoring (8). Shared features found in these cell wall-anchored proteins and pilins (or fimbrillins) are an N-terminal signal peptide and a C-terminal cell wall sorting signal (CWSS). The N-terminal signal...

Cell-to-cell interaction requires optimal positioning of a pilus tip adhesin modulated by gram-positive transpeptidase enzymes

“…S1A). To explore if overexpression of CafA in the ΔgspA/ΔsrtA double mutant rescues its coaggregation defect, we introduced into this strain a multicopy plasmid-expressing CafA under the control of a constitutive promoter (pCafA) (29), and coaggregation was quantitatively determined as described before (37). Compared to the MG1 strain, which coaggregation efficiency was normalized to 1, overexpression of CafA could not rescue the coaggregation defect of the ΔgspA/ΔsrtA mutant (SI Appendix, Fig.…”

Reoxidation of the Thiol-Disulfide Oxidoreductase MdbA by a Bacterial Vitamin K Epoxide Reductase in the Biofilm-Forming Actinobacterium Actinomyces oris

“…Both PCR products were used as templates for overlapping PCR with primers VKOR-HindIII-F and HA-NdeI-R. The PCR product generated was gel purified and digested with HindIII and NdeI (New England BioLabs [NEB]) and subsequently ligated into pCWU10 (36). The resulting plasmid was introduced into DH5␣, and plasmid DNA was purified for sequencing using primer pVKORseq (see Table S1) to confirm the insertion sequence.…”

“…Cell fractionation was performed as previously described (36). Briefly, log-phase cell cultures of various strains were obtained and normalized to an OD 600 of 1.0 (36). Cells were fractionated into culture medium (S), cell wall (W), membrane (M), and cytoplasmic (C) fractions; proteins were precipitated using trichloroacetic acid (TCA) and washed with acetone.…”

“…Protoplasts of log-phase A. oris cells were prepared as previously described (13,36) and used in a protease protection assay (39). The protoplasts were treated with proteinase K (Sigma) in PBS at final concentrations of 0, 10, 50, 100, and 200 ng/l at room temperature for 15 min.…”

“…Cell fractionation was performed as previously described (36). Briefly, log-phase cell cultures of various strains were obtained and normalized to an OD 600 of 1.0 (36).…”