Abstract
The widely conserved LytR-CpsA-Psr (LCP) family of enzymes in Gram-positive bacteria is known to attach glycopolymers, including wall teichoic acid, to the cell envelope. However, it is undetermined if these enzymes are capable of catalyzing glycan attachment to surface proteins. In the actinobacterium Actinomyces oris, an LCP homolog here named LcpA is genetically linked to GspA, a glycoprotein that is covalently attached to the bacterial peptidoglycan by the housekeeping sortase SrtA. Here we show by X-ray crystallography that LcpA adopts an α-β-α structural fold, akin to the conserved LCP domain, which harbors characteristic catalytic arginine residues. Consistently, alanine substitution for these residues, R149 and R266, abrogates GspA glycosylation, leading to accumulation of an intermediate form termed GspALMM, which is also observed in the lcpA mutant. Unlike other LCP proteins characterized to date, LcpA contains a stabilizing disulfide bond, mutations of which severely affect LcpA stability. In line with the established role of disulfide bond formation in oxidative protein folding in A. oris, deletion of vkor, coding for the thiol-disulfide oxidoreductase VKOR, also significantly reduces LcpA stability. Biochemical studies demonstrated that the recombinant LcpA enzyme possesses pyrophosphatase activity, enabling hydrolysis of diphosphate bonds. Furthermore, this recombinant enzyme, which weakly interacts with GspA in solution, catalyzes phosphotransfer to GspALMM Altogether, the findings support that A. oris LcpA is an archetypal LCP enzyme that glycosylates a cell wall-anchored protein, a process that may be conserved in Actinobacteria, given the conservation of LcpA and GspA in these high-GC-content organisms.IMPORTANCE In Gram-positive bacteria, the conserved LCP family enzymes studied to date are known to attach glycopolymers, including wall teichoic acid, to the cell envelope. It is unknown if these enzymes catalyze glycosylation of surface proteins. We show here in the actinobacterium Actinomyces oris by X-ray crystallography and biochemical analyses that A. oris LcpA is an LCP homolog, possessing pyrophosphatase and phosphotransferase activities known to belong to LCP enzymes that require conserved catalytic Arg residues, while harboring a unique disulfide bond critical for protein stability. Importantly, LcpA mediates glycosylation of the surface protein GspA via phosphotransferase activity. Our studies provide the first experimental evidence of an archetypal LCP enzyme that promotes glycosylation of a cell wall-anchored protein in Gram-positive bacteria.
Highlights
The widely conserved LytR-CpsA-Psr (LCP) family of enzymes in Grampositive bacteria is known to attach glycopolymers, including wall teichoic acid, to the cell envelope
In addition to LcpA, A. oris MG1 encodes three additional proteins with LCP domains. ana_0299, designated lcpB, is adjacent to two conserved genes (Fig. 1A) coding for a UDP-N-acetyl-D-mannosaminuronic acid dehydrogenase and a homolog of glycosyl/glycerophosphate transferase TagF, which has previously been implicated in the wall teichoic acid (WTA) synthesis of Staphylococcus epidermidis [17]. ana_1577 and ana_1578 appear to reside in the same transcriptional unit (Fig. 1A)
We present here the first experimental evidence that A. oris LcpA— capable of catalyzing hydrolysis of diphosphate bonds and phosphotransfer— glycosylates the cell wall-anchored protein GspA prior to its attachment to peptidoglycan, a subsequent process that is facilitated by the housekeeping sortase SrtA [15]
Summary
The widely conserved LytR-CpsA-Psr (LCP) family of enzymes in Grampositive bacteria is known to attach glycopolymers, including wall teichoic acid, to the cell envelope. IMPORTANCE In Gram-positive bacteria, the conserved LCP family enzymes studied to date are known to attach glycopolymers, including wall teichoic acid, to the cell envelope It is unknown if these enzymes catalyze glycosylation of surface proteins. Our studies provide the first experimental evidence of an archetypal LCP enzyme that promotes glycosylation of a cell wall-anchored protein in Grampositive bacteria Glycopolymers, such as wall teichoic acids (WTAs), capsular polysaccharides, and lipoteichoic acids (LTAs), displayed on the cell envelope of Gram-positive bacteria play critical roles in cell physiology by modulating immunogenicity, host and bacterial surface interactions, protein stability, cell division, and affinity for charged molecules, including antimicrobial peptides and cations [1,2,3,4]. Further biochemical characterizations provide evidence that LcpA possesses pyrophosphatase activity and functions as a phosphotransferase, catalyzing glycosylation of the cell wall-anchored protein GspA
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