Biology subjects

Sychantha, D.

Publications and source records attributed to Sychantha, D..

5 recordsLinked to original sources

bioRxiv2026

Denuded peptidoglycan oligosaccharides enable the biochemical investigation of bacterial cell wall recognition, modification, and degradation

Peptidoglycan is an essential component of the bacterial cell wall, providing mechanical strength and maintaining cell shape. It consists of glycan chains crosslinked by short peptide stems, resulting in a chemically heterogeneous macromolecule that remains challenging to study in a well-defined form. Access to discrete peptidoglycan fragments has therefore been critical for advancing biochemical and structural studies of cell wall-active enzymes. However, current synthetic, semi-synthetic, and cell wall extraction approaches remain limited by the complexity of carbohydrate chemistry and the difficulty of isolating pure, well-defined material. Here, we report a facile enzymatic approach for generating defined, denuded peptidoglycan oligosaccharides from the cell walls of two Staphylococcus species. These oligosaccharides, which terminate in N-acetylglucosamine and range from two to five disaccharide units in length, serve as substrates for a diverse panel of peptidoglycan-active enzymes that cleave or chemically modify the glycan backbone. We further show that these denuded oligosaccharides can be used in lysozyme-catalyzed transglycosylation reactions to generate p-nitrophenyl derivatives, enabling continuous colorimetric monitoring of peptidoglycan-cleaving enzymes. This method provides a practical route to defined peptidoglycan glycans and establishes a platform for further structural diversification, including stem peptide reattachment, quantitative enzyme assays, and structural characterization of peptidoglycan-binding proteins.

biochemistry↗

bioRxiv2025

A ubiquitous Streptomyces biosynthetic megacluster encodes an arsenal of synergistic biotin-targeting antibiotics

The rise of multidrug-resistant pathogens underscores the urgent need for antibiotics that act through new targets and mechanisms. Biotin metabolism, essential in most bacteria, remains underexploited therapeutically. Here, we uncover a highly conserved, co-located biosynthetic megacluster in Streptomyces, a striking "cluster of clusters", that encodes four distinct natural product families: acidomycin, stravidins, dapamycins, and -methyl-KAPA, and is flanked by genes that encode streptavidin, a high-affinity biotin-binding protein. Remarkably, all molecules target different steps in bacterial biotin metabolism, revealing a multi-pronged natural strategy for biotin starvation. This arrangement of four functionally convergent biosynthetic gene clusters at a single genomic locus is without precedent. Even more surprisingly, we find that this anti-biotin megacluster is widespread across Streptomyces bacteria, suggesting a deeply conserved evolutionary solution to microbial competition. Mechanistically, the compounds inhibit biotin biosynthesis through enzyme blockade, prodrug activation, covalent cofactor mimicry, and biotin sequestration via co-expressed streptavidin. Stravidin S2 and -methyl-KAPA are effective in a murine model of multidrug-resistant E. coli infection. These findings expose a coordinated biosynthetic logic in microbial secondary metabolites and point to higher-order biosynthetic architectures as promising reservoirs of antibiotic innovation.

microbiology↗

bioRxiv2025

Rapidly evolving orphan immunity genes protect human gut bacteria from intoxication by the type VI secretion system

Bacteria encode diverse mechanisms for mediating interbacterial antagonism through the exchange of toxic effector proteins. Although the structure, function, and regulation of these pathways has been well established for many organisms, an understanding of their ecological and evolutionary dynamics lags behind. Type VI secretion systems (T6SS) deliver effectors between competing Gram-negative bacteria, including among mammalian gut Bacteroidales, resulting in the evolution of elaborate defense mechanisms that protect against T6SS attack. One such mechanism is the recombinase-associated acquired interbacterial defence (rAID) system, which harbors arrays of orphan immunity genes that diverge in sequence from T6SS-associated cognate immunity genes. It is not known if such sequence divergence impacts rAID orphan immunity function, or how rAID distribution across microbiomes relates to the T6SS. Here, we show that divergent rAID orphan immunity factors that possess SUKH domains allow bacteria to survive intoxication by cognate effectors. Such protection is due to high affinity protein-protein interactions between orphan immunity and effector that are comparable to that of cognate effector-immunity. Unlike other examples of T6SS effector-immunity interactions, we find that the binding interface is comprised of electrostatic interactions with a high degree of redundancy underlying its protective capacity. Finally, we quantify orphan immunity and effector gene abundance and dynamics across human gut metagenomes, revealing patterns of co-occurrence indicative of positive selection. Population genetic analyses of longitudinal data suggests that orphan immunity genes accumulate non-synonymous mutations that lie at the predicted effector-immunity interface. Together, our findings establish rAID orphan immunity genes as important bacterial fitness determinants in the human gut.

microbiology↗

bioRxiv2025

The histidine kinase VraS orchestrates the cell-wall stress response in Staphylococcus aureus via its direct interaction with glycopeptides and β-lactams

Multidrug-resistant Staphylococcus aureus is a major global health threat, with the VraTSR three-component system playing a key role in sensing and conferring resistance to cell-wall active antibiotics, particularly vancomycin. VraTSR comprises the membrane histidine kinase VraS, the cytoplasmic response regulator VraR, and the uncharacterized membrane protein VraT, which regulate the cell wall stress stimulon. However, the molecular signals sensed by VraTSR remain unknown. To elucidate the activation mechanism of this regulatory system, we investigated interactions with {beta}-lactams and glycopeptides. Using a transcriptional reporter strain, we confirmed VraTSR activation by {beta}-lactams, glycopeptides, a vancomycin-derived photoprobe (VPP), and the previously unreported activators A47934 and moenomycin A. Photo-crosslinking assays with VPP and full-length VraS expressed in membranes revealed a direct interaction with vancomycin, which was further confirmed in purified VraS reconstituted in liposomes. VPP binding was concentration-dependent, saturable, and displaced by vancomycin. Saturation transfer difference (STD) Nuclear Magnetic Resonance (NMR) experiments confirmed vancomycin binding to VraS and demonstrated ampicillin interaction, highlighting the involvement of aryl protons from both antibiotics. These findings establish VraS as a receptor for vancomycin and ampicillin. In contrast, assays with membrane vesicles expressing only VraT or co-expressing VraS/VraT did not show covalent adduct formation between VraT and VPP. While VraTs exact role remains unclear, its participation in antibiotic sensing or signal transduction cannot yet be excluded. These results demonstrate that vancomycin and ampicillin directly activate VraS, providing critical insights into the activation of the cell wall stress stimulon and the mechanisms underlying antibiotic resistance. Disrupting VraTSR signaling is a promising strategy to combat multidrug resistance in S. aureus, and we provide invaluable in vitro platforms for identifying potential VraS inhibitors. Author SummaryMultidrug-resistant Staphylococcus aureus poses a major global health threat due to its resistance to cell-wall active antibiotics. Our study focuses on the VraTSR three-component system, a key regulator of the cell wall stress response in S. aureus, whose activation signals have remained unknown. We demonstrate that VraS, the membrane histidine kinase of the system, acts as a direct receptor for vancomycin and ampicillin--two structurally distinct antibiotics. These findings uncover the activation mechanism of VraTSR and position VraS as a central player in antibiotic sensing and resistance. By identifying VraS as a direct antibiotic receptor, we provide a promising target for developing inhibitors to disrupt VraTSR signaling and restore antibiotic efficacy. Additionally, the in vitro platforms we established enable the identification and testing of potential VraS inhibitors. This study highlights the importance of understanding bacterial stress-response pathways to combat antibiotic resistance, offering critical insights for developing new therapeutic strategies against multidrug-resistant S. aureus, a growing global health challenge.

biochemistry↗

bioRxiv2023

A feedback control mechanism governs the synthesis of lipid-linked precursors of the bacterial cell wall

Many bacterial surface glycans such as the peptidoglycan (PG) cell wall, O-antigens, and capsules are built from monomeric units linked to a polyprenyl lipid carrier. How this limiting lipid carrier is effectively distributed among competing pathways has remained unclear for some time. Here, we describe the isolation and characterization of hyperactive variants of Pseudomonas aeruginosa MraY, the essential and conserved enzyme catalyzing the formation of the first lipid-linked PG precursor called lipid I. These variants result in the elevated production of the final PG precursor lipid II in cells and are hyperactive in a purified system. Amino acid substitutions within the activated MraY variants unexpectedly map to a cavity on the extracellular side of the dimer interface, far from the active site. Our structural evidence and molecular dynamics simulations suggest that the cavity is a binding site for lipid II molecules that have been transported to the outer leaflet of the membrane. Overall, our results support a model in which excess externalized lipid II allosterically inhibits MraY, providing a feedback mechanism to prevent the sequestration of lipid carrier in the PG biogenesis pathway. MraY belongs to the broadly distributed polyprenyl-phosphate N-acetylhexosamine 1-phosphate transferase (PNPT) superfamily of enzymes. We therefore propose that similar feedback mechanisms may be widely employed to coordinate precursor supply with demand by polymerases, thereby optimizing the partitioning of lipid carriers between competing glycan biogenesis pathways.

microbiology↗