Biology subjects

Rosch, K. M.

Publications and source records attributed to Rosch, K. M..

4 recordsLinked to original sources

bioRxiv2025

Differential endopeptidase requirements during adaptation to changing growth conditions in Vibrio cholerae

The bacterial cell wall is a covalently linked meshwork of peptidoglycan (PG) that establishes cell shape and prevents osmotic lysis. This structure must be flexible enough to accommodate transenvelope protein complexes, but strong enough to withstand high intracellular pressure. In order to elongate and divide, cells must remodel the cell wall through the concerted action of PG synthesis and degradation. Endopeptidases, a class of PG degrading enzymes, facilitate cell growth by hydrolyzing PG crosslinks. Vibrio cholerae encodes several functionally redundant endopeptidases, two of which are nearly identical: ShyA and ShyC. To investigate differential roles of these enzymes, we assessed growth and morphology of ShyA and ShyC mutants. We found that ShyA, but not ShyC, is required for normal adaptation to low osmolarity medium. Cells lacking ShyA exhibited longer lag phase and aberrant morphology during adaptation, and reduced survival in the presence of a beta-lactam antibiotic. Lastly, our experiments revealed that cells lacking ShyAs LysM domain exhibited more severe defects than cells lacking ShyA altogether, implicating the LysM domain in proper regulation of ShyA activity.

microbiology↗

bioRxiv2024

Sugar phosphate-mediated inhibition of peptidoglycan precursor synthesis

Antibiotic tolerance, the widespread ability of diverse pathogenic bacteria to sustain viability in the presence of typically bactericidal antibiotics for extended time periods, is an understudied steppingstone towards antibiotic resistance. The Gram-negative pathogen Vibrio cholerae, the causative agent of cholera, is highly tolerant to {beta}-lactam antibiotics. We previously found that the disruption of glycolysis, via deletion of pgi (vc0374, glucose-6-phosphate isomerase), resulted in significant cell wall damage and increased sensitivity towards {beta}-lactam antibiotics. Here, we uncover the mechanism of this resulting damage. We find that glucose causes growth inhibition, partial lysis, and a damaged cell envelope in {Delta}pgi. Supplementation with N-acetylglucosamine, but not other carbon sources (either from upper glycolysis, TCA cycle intermediates, or cell wall precursors) restored growth, re-established antibiotic resistance towards {beta}-lactams, and recovered cellular morphology of a pgi mutant exposed to glucose. Targeted metabolomics revealed the cell wall precursor synthetase enzyme GlmU (vc2762, coding for the bifunctional enzyme that converts glucosamine-1P to UDP-GlcNAc) as a critical bottleneck and mediator of glucose toxicity in {Delta}pgi. In vitro assays of GlmU revealed that sugar phosphates (primarily glucose-1-phosphate) inhibit the acetyltransferase activity of GlmU (likely competitively), resulting in compromised PG and LPS biosynthesis. These findings identify GlmU as a critical branchpoint enzyme between central metabolism and cell envelope integrity and reveal the molecular mechanism of {Delta}pgi glucose toxicity in Vibrio cholerae. ImportanceSugar-phosphate toxicity is a well characterized phenomenon that is seen within diverse bacterial species, and yet the molecular underpinnings remain elusive. We previously discovered that disrupting Vibrio choleraes ability to eat glucose (by disrupting the pgi gene), also resulted in a damaged cell envelope. Upon deletion of pgi, glucose-phosphate levels rapidly build and inhibit the enzymatic activity of GlmU, a key step of bacterial peptidoglycan precursor synthesis. GlmU inhibition causes enhanced killing by antibiotics and a pronounced cell envelope defect. Thus, GlmU serves as a prime target for novel drug development. This research opens new routes through which central metabolism and sugar-phosphate toxicity modulate antibiotic susceptibility.

microbiology↗

bioRxiv2024

Multiple resistance factors collectively promote inoculum-dependent dynamic survival during antimicrobial peptide exposure in Enterobacter cloacae

Antimicrobial peptides (AMPs) are a promising tool with which to fight rising antibiotic resistance. However, pathogenic bacteria are equipped with several AMP defense mechanisms, whose contributions to AMP resistance are often poorly defined. Here, we evaluate the genetic determinants of resistance to an insect AMP, cecropin B, in the opportunistic pathogen Enterobacter cloacae. Single-cell analysis of E. cloacaes response to cecropin revealed marked heterogeneity in cell survival, phenotypically reminiscent of heteroresistance (the ability of a subpopulation to grow in the presence of supra-MIC concentration of antimicrobial). The magnitude of this response was highly dependent on initial E. cloacae inoculum. We identified 3 genetic factors which collectively contribute to E. cloacae resistance in response to the AMP cecropin: The PhoPQ-two-component system, OmpT-mediated proteolytic cleavage of cecropin, and Rcs-mediated membrane stress response. Altogether, this evidence suggests that multiple, independent mechanisms contribute to AMP resistance in E. cloacae.

microbiology↗

bioRxiv2021

Lytic transglycosylases mitigate periplasmic crowding by degrading soluble cell wall turnover products

The peptidoglycan cell wall is a predominant structure of bacteria, determining cell shape and supporting survival in diverse conditions. Peptidoglycan is dynamic and requires regulated synthesis of new material, remodeling, and turnover - or autolysis - of old material. Despite exploitation of peptidoglycan synthesis as an antibiotic target, we lack a fundamental understanding of how peptidoglycan synthesis and autolysis intersect to maintain the cell wall. Here, we uncover a critical physiological role for a widely misunderstood class of autolytic enzymes, lytic transglycosylases (LTGs). We demonstrate that LTG activity is essential to survival by contributing to periplasmic processes upstream and independent of peptidoglycan recycling. Defects accumulate in Vibrio cholerae LTG mutants due to generally inadequate LTG activity, rather than absence of specific enzymes, and essential LTG activities are likely independent of protein-protein interactions, as heterologous expression of a non-native LTG rescues growth of a conditionally LTG-null mutant. Lastly, we demonstrate that soluble, uncrosslinked, endopeptidase-dependent peptidoglycan chains, also detected in the wild-type, are enriched in LTG mutants, and that LTG mutants are hypersusceptible to the production of diverse periplasmic polymers. Collectively, our results suggest that LTGs prevent toxic crowding of the periplasm with synthesis-derived peptidoglycan polymers and contrary to prevailing models, that this autolytic function can be temporally separate from peptidoglycan synthesis.

microbiology↗