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Bacterial protein glycosylation is commonly mediated by oligosaccharyltransferases (OTases) that transfer oligosaccharides en bloc from preassembled lipid-linked precursors to acceptor proteins. Natively, O-linking OTases usually transfer a single repeat unit of the O-antigen or capsular polysaccharide to the side chains of serine or threonine on acceptor proteins. Three major families of bacterial O-linking OTases have been described: PglL, PglS, and TfpO. TfpO is limited to transferring short oligosaccharides both in its native context and when heterologously expressed in glycoengineered Escherichia coli. On the other hand, PglL and PglS can transfer long-chain polysaccharides when expressed in glycoengineered E. coli. Herein, we describe the discovery and functional characterization of a novel family of bacterial O-linking OTases termed TfpM from Moraxellaceae bacteria. TfpM proteins are similar in size and sequence to TfpO enzymes but can transfer long-chain polysaccharides to acceptor proteins. Phylogenetic analyses demonstrate that TfpM proteins cluster in distinct clades from known bacterial OTases. Using a representative TfpM enzyme from Moraxella osloensis, we determined that TfpM glycosylates a C-terminal threonine of its cognate pilin-like protein and identified the minimal sequon required for glycosylation. We further demonstrated that TfpM has broad substrate tolerance and can transfer diverse glycans including those with glucose, galactose, or 2-N-acetyl sugars at the reducing end. Last, we find that a TfpM-derived bioconjugate is immunogenic and elicits serotype-specific polysaccharide IgG responses in mice. The glycan substrate promiscuity of TfpM and identification of the minimal TfpM sequon renders this enzyme a valuable additional tool for expanding the glycoengineering toolbox.

Industrial hog operation (IHO) workers are at increased risk of carryingStaphylococcus aureusin their nares, particularly strains that are livestock-associated (LA) and multidrug-resistant. The pathogenicity of LA-S. aureusstrains remains unclear, with some prior studies suggesting reduced transmission and virulence in humans compared to community-associated methicillin-resistant (CA-MRSA)S. aureus. The objective of this study was to determine the degree to which LA-S. aureusstrains contracted by IHO workers cause disease relative to a representative CA-MRSA strain in a mouse model of skin and soft tissue infection (SSTI). Mice infected with CC398 LA-S. aureusstrains (IHW398-1 and IHW398-2) developed larger lesion sizes with higher bacterial burden than mice infected with CA-MRSA (SF8300) (p < 0.05). The greatest lesion size and bacterial burden was seen with a CC398 strain that produced a recurrent SSTI in an IHO worker. The LA-S. aureusinfected mice had decreased IL-1β protein levels compared with CA-MRSA-infected mice (p < 0.05), suggesting a suboptimal host response to LA-S. aureusSSTIs. WGSA revealed heterogeneity in virulence factor and antimicrobial resistance genes carried by LA-S. aureusand CA-MRSA strains. The observed pathogenicity suggest that more attention should be placed on preventing the spread of LA-S. aureusinto human populations.