Biology subjects

Boraston, A.

Publications and source records attributed to Boraston, A..

3 recordsLinked to original sources

bioRxiv2025

Clinical stringent response activation promotes conjugal transfer of staphylococcal resistance plasmids

Conjugative transfer of plasmids represents a major route through which antibiotic resistance genes are spread. In the case of the prevalent and deadly pathogen Staphylococcus aureus, >90% of clinical isolates carry at least one plasmid. While plasmid-encoded mechanisms (e.g. plasmid copy number) can influence conjugation frequency, host factors and environmental stimuli can also affect transmission. In particular, stress responses like the stringent response have been associated with increased movement of mobile genetic elements. We have previously shown that clinical mutations in the stringent response controller, Rel, lead to elevated levels of the alarmones (p)ppGpp and antibiotic tolerance in S. aureus. Here, we report that stringent response activation in these strains promotes the conjugal transfer of diverse staphylococcal resistance plasmids. We observed that clinical Rel mutations promote donation, but not receipt, of plasmids from the three families of staphylococcal plasmid and a mobilisable plasmid. This increased conjugation frequency could also be induced by chemical induction of the stringent response by mupirocin. Intriguingly, detailed experimental analysis revealed that the effect of elevated (p)ppGpp on plasmid donation was not due to CodY derepression, SOS response induction, increased plasmid copy number or increased expression of conjugation machinery genes. Further, transcriptomic analysis failed to identify any other putative plasmid- or host-derived mechanisms to explain this observation. Further investigations are required to explore the mechanistic link between the stringent response and conjugation, given the pervasive transcriptional and post-translational effects of (p)ppGpp. Overall, the association between Rel mutation and increased plasmid donation is alarming, especially as Rel mutations are being increasingly identified among clinical isolates.

microbiology↗

bioRxiv2025

Substrate recognition and cleavage by mucin degrading O-glycopeptidases from the gut microbe Bacteroides caccae.

O-glycopeptidases are enzymes that hydrolyze the peptide bonds in glycoproteins by a mechanism that involves specific recognition of O-linked glycans on the substrate. Bacteroides caccae, an accomplished mucin degrader, is a member of the human gut microbiota with sixteen genes encoding putative O-glycopeptidases in the peptidase_M60 family. At present, the diversity of substrate selective in O- glycopeptidases is not well-understood nor is the rationale behind their expansion in bacteria such as B. caccae. Here we reveal the activity and diversity of the peptidase_M60 O-glycopeptidases encoded in the B. caccae genome. At least thirteen of the sixteen peptidase_M60 genes are active mucinases. Targeted functional studies by a high-throughput FRET screen combined with detailed kinetic analyses reveal that five examples in an uncharacterized clade of peptidase_M60 proteins have different substrate selectivities despite their relatively high degree of relatedness. Structural analyses of these enzymes, including bound complexes, reveal new insight into the molecular underpinnings of O-glycopeptidase diversity. This highlights the larger context of how varied the selectivity of peptidase_M60 O-glycopeptidases can be for the glycan moiety and/or the peptide portion of the substrates, and why mucin degraders like B. caccae diversify O-glycopeptidase substrate repertoires to potentially maximize breakdown of this extraordinarily complex polymer.

biochemistry↗

bioRxiv2020

The glycoprotease CpaA secreted by medically relevant Acinetobacter species targets multiple O-linked host glycoproteins

Glycans decorate proteins and affect their biological function, including protection against proteolytic degradation. However, pathogenic, and commensal bacteria have evolved specific glycoproteases that overcome the steric impediment posed by carbohydrates, cleaving glycoproteins precisely at their glycosylation site(s). Medically relevant Acinetobacter strains employ their type II secretion system (T2SS) to secrete the glycoprotease CpaA, which contributes to virulence. Previously, CpaA was shown to cleave two O-linked glycoproteins, factors V and XII, leading to reduced blood coagulation. In this work, we show that CpaA cleaves a broader range of O-linked human glycoproteins, including several glycoproteins involved in complement activation, such as CD55 and CD46. However, only CD55 was removed from the cell surface, while CD46 remained unaltered during the A. nosocomialis infection assay. We show that CpaA has a unique consensus target sequence that consists of a glycosylated serine or threonine residue after a proline residue (P-S/T), and its activity is not affected by sialic acids. Molecular modeling and mutagenesis analysis of CpaA suggest that the indole ring of Trp493 and the ring of the Pro residue in the substrate form a key interaction that contributes to CpaA sequence selectivity. Similar bacterial glycoproteases have recently gained attention as tools for proteomic analysis of human glycoproteins, and CpaA appears to be a robust and attractive new component of the glycoproteomics toolbox. Combined, our work provides insight into the function and possible application of CpaA, a member of a widespread class of broad-spectrum bacterial glycoproteases involved in host-pathogen interactions. IMPORTANCECpaA is a glycoprotease expressed by members of the Acinetobacter baumannii-calcoaceticus complex and it is the first bona fide secreted virulence factor identified in these species. Here, we show that CpaA cleaves multiple targets precisely at O-glycosylation sites preceded by a Pro residue. This feature, together with the observation that sialic acid does not impact CpaA activity, makes of this enzyme an attractive tool for the analysis of O-linked human protein for biotechnical and diagnostic purposes. Previous work identified proteins involved in blood coagulation as targets of CpaA. Our work broadens the set of targets of CpaA, pointing towards additional roles in bacteria-host interactions. We propose that CpaA belongs to an expanding class of functionally-defined glycoproteases that targets multiple O-linked host glycoproteins.

microbiology↗