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A Disulfide Bond-forming Machine Is Linked to the Sortase-mediated Pilus Assembly Pathway in the Gram-positive Bacterium Actinomyces oris.

ABSTRACT: Export of cell surface pilins in Gram-positive bacteria likely occurs by the translocation of unfolded precursor polypeptides; however, how the unfolded pilins gain their native conformation is presently unknown. Here, we present physiological studies to demonstrate that the FimA pilin of Actinomyces oris contains two disulfide bonds. Alanine substitution of cysteine residues forming the C-terminal disulfide bridge abrogates pilus assembly, in turn eliminating biofilm formation and polymicrobial interaction. Transposon mutagenesis of A. oris yielded a mutant defective in adherence to Streptococcus oralis, and revealed the essential role of a vitamin K epoxide reductase (VKOR) gene in pilus assembly. Targeted deletion of vkor results in the same defects, which are rescued by ectopic expression of VKOR, but not a mutant containing an alanine substitution in its conserved CXXC motif. Depletion of mdbA, which encodes a membrane-bound thiol-disulfide oxidoreductase, abrogates pilus assembly and alters cell morphology. Remarkably, overexpression of MdbA or a counterpart from Corynebacterium diphtheriae, rescues the ?vkor mutant. By alkylation assays, we demonstrate that VKOR is required for MdbA reoxidation. Furthermore, crystallographic studies reveal that A. oris MdbA harbors a thioredoxin-like fold with the conserved CXXC active site. Consistently, each MdbA enzyme catalyzes proper disulfide bond formation within FimA in vitro that requires the catalytic CXXC motif. Because the majority of signal peptide-containing proteins encoded by A. oris possess multiple Cys residues, we propose that MdbA and VKOR constitute a major folding machine for the secretome of this organism. This oxidative protein folding pathway may be a common feature in Actinobacteria.

SUBMITTER: Reardon-Robinson ME

PROVIDER: S-EPMC4571867 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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A Disulfide Bond-forming Machine Is Linked to the Sortase-mediated Pilus Assembly Pathway in the Gram-positive Bacterium Actinomyces oris.

Reardon-Robinson Melissa E ME Osipiuk Jerzy J Chang Chungyu C Wu Chenggang C Jooya Neda N Joachimiak Andrzej A Das Asis A Ton-That Hung H

The Journal of biological chemistry 20150713 35

Export of cell surface pilins in Gram-positive bacteria likely occurs by the translocation of unfolded precursor polypeptides; however, how the unfolded pilins gain their native conformation is presently unknown. Here, we present physiological studies to demonstrate that the FimA pilin of Actinomyces oris contains two disulfide bonds. Alanine substitution of cysteine residues forming the C-terminal disulfide bridge abrogates pilus assembly, in turn eliminating biofilm formation and polymicrobial ...[more]

PMID: 26170452

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Project description:The Gram-positive bacterium Actinomyces oris, a key colonizer in the development of oral biofilms, contains 18 LPXTG motif-containing 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.Sec-mediated translocation of bacterial protein precursors across the cytoplasmic membrane involves cleavage of their signal peptide by a signal peptidase (SPase). Like many Gram-positive bacteria, A. oris expresses two SPases, LepB1 and LepB2. The latter is a genetic suppressor of lethal "glyco-stress" caused by membrane accumulation of glycosylated GspA when the housekeeping sortase srtA is genetically disrupted. We show here that LepB1 and LepB2 are capable of processing GspA, whereas only LepB2 is required for cleavage of fimbrial signal peptides. This is the first example of a type I SPase dedicated to LPXTG motif-containing fimbrial proteins. Thus, A. oris provides an experimental model with which to investigate the specificity mechanism of type I SPases.

Project description:Posttranslocational protein folding in the Gram-positive biofilm-forming actinobacterium Actinomyces oris is mediated by a membrane-bound thiol-disulfide oxidoreductase named MdbA, which catalyzes oxidative folding of nascent polypeptides transported by the Sec translocon. Reoxidation of MdbA involves a bacterial vitamin K epoxide reductase (VKOR)-like protein that contains four cysteine residues, C93/C101 and C175/C178, with the latter forming a canonical CXXC thioredoxin-like motif; however, the mechanism of VKOR-mediated reoxidation of MdbA is not known. We present here a topological view of the A. oris membrane-spanning protein VKOR with these four exoplasmic cysteine residues that participate in MdbA reoxidation. Like deletion of the VKOR gene, alanine replacement of individual cysteine residues abrogated polymicrobial interactions and biofilm formation, concomitant with the failure to form adhesive pili on the bacterial surface. Intriguingly, the mutation of the cysteine at position 101 to alanine (C101A mutation) resulted in a high-molecular-weight complex that was positive for MdbA and VKOR by immunoblotting and was absent in other alanine substitution mutants and the C93A C101A double mutation and after treatment with the reducing agent ?-mercaptoethanol. Consistent with this observation, affinity purification followed by immunoblotting confirmed this MdbA-VKOR complex in the C101A mutant. Furthermore, ectopic expression of the Mycobacterium tuberculosis VKOR analog in the A. oris VKOR deletion (?VKOR) mutant rescued its defects, in contrast to the expression of M. tuberculosis VKOR variants known to be nonfunctional in the disulfide relay that mediates reoxidation of the disulfide bond-forming catalyst DsbA in Escherichia coli Altogether, the results support a model of a disulfide relay, from its start with the pair C93/C101 to the C175-X-X-C178 motif, that is required for MdbA reoxidation and appears to be conserved in members of the class ActinobacteriaIMPORTANCE It has recently been shown in the high-GC Gram-positive bacteria (or Actinobacteria) Actinomyces oris and Corynebacterium diphtheriae that oxidative folding of nascent polypeptides transported by the Sec machinery is catalyzed by a membrane-anchored oxidoreductase named MdbA. In A. oris, reoxidation of MdbA requires a bacterial VKOR-like protein, and yet, how VKOR mediates MdbA reoxidation is unknown. We show here that the A. oris membrane-spanning protein VKOR employs two pairs of exoplasmic cysteine residues, including the canonical CXXC thioredoxinlike motif, to oxidize MdbA via a disulfide relay mechanism. This mechanism of disulfide relay is essential for pilus assembly, polymicrobial interactions, and biofilm formation and appears to be conserved in members of the class Actinobacteria, including Mycobacterium tuberculosis.

Project description:BACKGROUND:Actinomyces oris is a Gram-positive bacterium that has been associated with healthy and diseased sites in the human oral cavity. Most pathogenic bacteria require iron to survive, and in order to acquire iron in the relatively iron-scarce oral cavity A. oris has been shown to produce iron-binding molecules known as siderophores. The genes encoding these siderophores and transporters are thought to be regulated by the amount of iron in the growth medium and by the metal-dependent repressor, AmdR, which we showed previously binds to the promoter of proposed iron-regulated genes. OBJECTIVE:The purpose of this study was to characterize siderophore and associated iron transport systems in A. oris. DESIGN:We examined gene expression of the putative iron transport genes fetA and sidD in response to low- and high-iron environments. One of these genes, sidD, encoding a putative Fe ABC transporter protein, was insertionally inactivated and was examined for causing growth defects. To gain a further understanding of the role of iron metabolism in oral diseases, clinical isolates of Actinomyces spp. were examined for the presence of the gene encoding AmdR, a proposed global regulator of iron-dependent gene expression in A. oris. RESULTS:When A. oris was grown under iron-limiting conditions, the genes encoding iron/siderophore transporters fetA and sidD showed increased expression. One of these genes (sidD) was mutated, and the sidD::Km strain exhibited a 50% reduction in growth in late log and stationary phase cells in media that contained iron. This growth defect was restored when the sidD gene was provided in a complemented strain. We were able to isolate the AmdR-encoding gene in seven clinical isolates of Actinomyces. When these protein sequences were aligned to the laboratory strain, there was a high degree of sequence similarity. CONCLUSIONS:The growth of the sidD::Km mutant in iron-replete medium mirrored the growth of the wild-type strain grown in iron-limiting medium, suggesting that the sidD::Km mutant was compromised in iron uptake. The known iron regulator AmdR is well conserved in clinical isolates of A. oris. This work provides additional insight into iron metabolism in this important oral microbe.

Dataset Information

A Disulfide Bond-forming Machine Is Linked to the Sortase-mediated Pilus Assembly Pathway in the Gram-positive Bacterium Actinomyces oris.

Publications

A Disulfide Bond-forming Machine Is Linked to the Sortase-mediated Pilus Assembly Pathway in the Gram-positive Bacterium Actinomyces oris.

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