Lethality of sortase depletion in <scp><i>A</i></scp><i>ctinomyces oris</i> caused by excessive membrane accumulation of a surface glycoprotein

Wu, Chenggang; Huang, I-Hsiu; Chang, Chia‐Wei; Reardon-Robinson, Melissa E.; Das, Asis; Ton‐That, Hung

doi:10.1111/mmi.12780

Cited by 52 publications

(141 citation statements)

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“…Consistent with these observations, the deletion of srtA in the gspA deletion strain did not generate any detrimental defects, nor did overexpression of GspA lacking the CWSS in the srtA gspA double mutant. Additionally, mutagenesis screening also revealed 12 other suppressors, one of which was mapped to ANA_1190, which encodes a type I SPase (8). Farther upstream of ANA_1190 is another type I SPase-encoding gene (ANA_1188).…”

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“…Folded proteins, embedded into the membrane via the hydrophobic domain of the CWSS, are cleaved at the LPXTG motif between threonine and glycine and then anchored to the cell wall by the housekeeping sortase SrtA. Unlike that in other Gram-positive bacteria studied to date, deletion of srtA in A. oris is lethal to cells (8). The basis for srtA essentiality was identified by transposon mutagenesis screening for srtA genetic suppressors.…”

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“…The CWSS is composed of the signature LPXTG motif, a hydrophobic domain, and a tail of positively charged residues (10). In all Gram-positive bacteria, a conserved transpeptidase enzyme called the housekeeping sortase carries out the cell wall-anchoring function of surface proteins via the LPXTG motif (11-13).According to the current models proposed for A. oris (1,8,14), membrane translocation of surface protein precursors is mediated by the Sec translocase via their signal peptides, which are removed by a SPase after translocation. Posttranslocational folding of the Citation Siegel SD, Wu C, Ton-That H. 2016.…”

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“…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).…”

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confidence: 99%

“…According to the current models proposed for A. oris (1,8,14), membrane translocation of surface protein precursors is mediated by the Sec translocase via their signal peptides, which are removed by a SPase after translocation. Posttranslocational folding of the surface proteins with multiple cysteine residues is catalyzed by the thiol-disulfide oxidoreductase MdbA (14,15).…”

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

Siegel

Ton‐That

2016

J Bacteriol

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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...

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confidence: 99%

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confidence: 99%

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confidence: 99%

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confidence: 99%

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confidence: 99%

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

Siegel

Ton‐That

2016

J Bacteriol

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Anchoring of LPXTG-Like Proteins to the Gram-Positive Cell Wall Envelope

Siegel¹,

Reardon²,

Ton‐That³

2016

Current Topics in Microbiology and Immunology

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In Gram-positive bacteria, protein precursors with a signal peptide and a cell wall sorting signal (CWSS)-which begins with an LPXTG motif, followed by a hydrophobic domain and a tail of positively charged residues-are targeted to the cell envelope by a transpeptidase enzyme call sortase. Evolution and selective pressure gave rise to six classes of sortase, i.e., SrtA-F. Only class C sortases are capable of polymerizing substrates harboring the pilin motif and CWSS into protein polymers known as pili or fimbriae, whereas the others perform cell wall anchoring functions. Regardless of the products generated from these sortases, the basic principle of sortase-catalyzed transpeptidation is the same. It begins with the cleavage of the LPXTG motif, followed by the cross-linking of this cleaved product at the threonine residue to a nucleophile, i.e., an active amino group of the peptidoglycan stem peptide or the lysine residue of the pilin motif. This chapter will summarize the efforts to identify and characterize sortases and their associated pathways with emphasis on the cell wall anchoring function.

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