Biology subjects

Doerr, T.

Publications and source records attributed to Doerr, T..

17 recordsLinked to original sources

bioRxiv2026

A digital droplet microarray for measuring tolerance to antibiotics

Antibiotic tolerance enables populations of microbes to survive normally lethal antibiotic concentrations, increasing the likelihood of reinfection and facilitating the evolution of resistance. Tolerance measurements typically involve quantifying viable cells after antibiotic exposure. Existing methods range from accessible but low-throughput approaches, such as plate counting, to higher-throughput but semi-quantitative techniques, such as the TDtest. Here, we develop a new system for rapid, precise and high-throughput tolerance measurements. We utilize Surface Patterned Omniphobic Tiles (SPOTs) to discretize cell suspensions into nano-to microliter droplets and estimate the viable cell concentrations following antibiotic exposure from the proportion of empty droplets using Poissonian statistics. We apply the platform to monitor Klebsiella pneumoniae tolerance to meropenem over time as a proof of concept. The resulting assay is accessible, compatible with multiple media, and boasts a large dynamic range, sufficient resolution, and rapid handling.

microbiology↗

bioRxiv2026

Shielded by the dead: how killed bacteria shape the dynamics and evolution of innate immunity

The outcome of an infection is determined by the dynamic interplay between microbial growth and host immunity. During a bacterial infection, bacteria killed by innate immune effectors can accumulate as corpses in the extracelluar space, where they can continue to bind (and thus sequester) immune effectors. The impacts on infection outcomes of continued biochemical activity ("sponginess") by the dead have been generally overlooked in theoretical and empirical studies of within-host disease dynamics. We develop a mechanism-based mathematical model of within-host dynamics that incorporates host microbial sensing, the production of immune effectors, the interaction of those effectors with microbes, and shutdown of the immune response after an infection has been controlled. Corpse sponginess impedes the hosts ability to control infection, but at the same time, the rapid mopping up of effectors by bacterial corpses also protects host tissue against autoimmune self-harm from immune effectors still circulating after the infection has been resolved. This dual impact of bacterial sponginess alters the trade-off between damage done by infecting bacteria versus autoimmune damage, consequently shifting the evolutionarily optimal immune activation and shutdown kinetics. Thus, the sponginess of bacterial corpses likely shapes both short-term infection dynamics and the long-term evolution of immune systems.

immunology↗

bioRxiv2025

Prevalence and mechanisms of high-level carbapenem antibiotic tolerance in clinical isolates of Klebsiella pneumoniae

Antibiotic tolerance is the ability of bacteria to survive normally lethal doses of antibiotics for extended time periods. Clinically significant Enterobacterales, for example, can exhibit high tolerance to the last-resort antibiotic meropenem. Meropenem tolerance is associated with formation of cell wall-deficient spheroplasts that readily recover to rod shape and normal growth upon removal of the antibiotic. Both the true prevalence of tolerance, and genetic mechanisms underlying it, remain poorly understood. Here, we find that meropenem tolerance is widespread among clinical Enterobacterales. Using forward genetics, we uncover novel tolerance factors in a hypertolerant isolate of the ESKAPE pathogen Klebsiella pneumoniae. We find that multiple mechanisms contribute to tolerance, and that cell envelope stress responses (PhoPQ, Cpx, Rcs and OmpR/EnvZ) collectively promote spheroplast stability and recovery, while the lytic transglycosylase MltB counteracts it. Our data indicate that tolerance is widespread among clinical isolates, and that outer membrane maintenance is a key factor promoting survival of tolerant K. pneumoniae.

microbiology↗

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

Determining the Youngs Modulus of the Bacterial Cell Envelope

Bacteria experience substantial physical forces in their natural environment including forces caused by osmotic pressure, growth in constrained spaces, and fluid shear. The cell envelope is the primary load-carrying structure of bacteria, but the mechanical properties of the cell envelope are poorly understood; reports of Youngs modulus of the cell envelope of E. coli are widely range from 2 MPa to 18 MPa. We have developed a microfluidic system to apply mechanical loads to hundreds of bacteria at once and demonstrated the utility of the approach for evaluating whole-cell stiffness. Here we extend this technique to determine Youngs modulus of the cell envelope of E. coli and of the pathogens V. cholerae and S. aureus. An optimization-based inverse finite element analysis was used to determine the cell envelope Youngs modulus from observed deformations. The Youngs modulus of the cell envelope was 2.06 {+/-} 0.04 MPa for E. coli, 0.84 {+/-} 0.02 MPa for E. coli treated with a chemical known to reduce cell stiffness, 0.12 {+/-} 0.03 MPa for V. cholerae, and 1.52 {+/-} 0.06 MPa for S. aureus (mean {+/-} SD). The microfluidic approach allows examining hundreds of cells at once and is readily applied to Gram-negative and Gram-positive organisms as well as rod-shaped and cocci cells, allowing further examination of the structural causes of differences in cell envelope Youngs modulus among bacteria species and strains.

bioengineering↗

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↗

bioRxiv2023

Novel role for peptidoglycan carboxypeptidases in maintaining the balance between bacterial cell wall synthesis and degradation

Peptidoglycan (PG) is the main component of the bacterial cell wall; it maintains cell shape while protecting the cell from internal osmotic pressure and external environmental challenges. PG synthesis is essential for bacterial growth and survival, and a series of PG modifications are required to allow expansion of the sacculus. Endopeptidases (EPs), for example, cleave the crosslinks between adjacent PG strands to allow the incorporation of newly synthesized PG. EPs are collectively essential for bacterial growth and must likely be carefully regulated to prevent sacculus degradation and cell death. However, EP regulation mechanisms are poorly understood. Here, we used TnSeq to uncover novel EP regulation factors in Vibrio cholerae. This screen revealed that the carboxypeptidase DacA1 (PBP5) alleviates EP toxicity. dacA1 is essential for viability on LB medium, and this essentiality was suppressed by EP overexpression, revealing that EP toxicity both mitigates, and is mitigated by, a defect in dacA1. A subsequent suppressor screen to restore viability of{Delta} dacA1 in LB medium was answered by hypomorphic mutants in the PG synthesis pathway, as well as mutations that promote PG degradation. Our data thus reveal a key role of DacA1 in maintaining the balance between PG synthesis and degradation.

microbiology↗

bioRxiv2022

Disrupting central carbon metabolism increases antibiotic susceptibility in Vibrio cholerae

Antibiotic tolerance, the ability of bacteria to sustain viability in the presence of typically bactericidal antibiotics for extended time periods, is an understudied contributor to treatment failure. The Gram-negative pathogen Vibrio cholerae, the causative agent of cholera disease, becomes highly tolerant to {beta}-lactam antibiotics (penicillin and related compounds) in a process requiring the two-component system VxrAB. VxrAB is induced by exposure to cell wall damage conditions, which results in the differential regulation of >100 genes. While the effectors of VxrAB are relatively well-known, VxrAB environment-sensing and activation mechanisms remain a mystery. Here, we used transposon mutagenesis to screen for mutants that spontaneously upregulate VxrAB signaling. This screen was answered by genes known to be required for proper cell envelope homeostasis, validating the approach. Unexpectedly, we also uncovered a new connection between central carbon metabolism and antibiotic tolerance. Inactivation of pgi (vc0374, coding for Glucose-6-phosphate isomerase) resulted in an intracellular accumulation of glucose-6-phosphate and fructose-6-phosphate, concomitant with a marked cell envelope defect, resulting in VxrAB induction. Deletion of pgi also increased sensitivity to {beta}-lactams and conferred a growth defect on salt-free LB; phenotypes that could be suppressed by deleting sugar uptake systems and by supplementing cell wall precursors in the growth medium. Our data suggest an important connection between central metabolism and cell envelope integrity and highlight a potential new target for developing novel antimicrobial agents. ImportanceAntibiotic tolerance (the ability to survive exposure to antibiotics) is a stepping-stone towards antibiotic resistance (the ability to grow in the presence of antibiotics), an increasingly common cause of antibiotic treatment failure. The mechanisms promoting tolerance are poorly understood. Herein, we discovered central carbon metabolism as a key contributor to antibiotic tolerance and resistance. A mutant in a sugar utilization pathway accumulates metabolites that likely shut down the synthesis of cell wall precursors, which weakens the cell wall and thus increases susceptibility to cell wall-active drugs. Our results illuminate the connection between central carbon metabolism and cell wall homeostasis in V. cholerae and suggest that interfering with metabolism may be a fruitful future strategy for development of antibiotic adjuvants.

microbiology↗

bioRxiv2022

Mechanical stimuli activate gene expression via a cell envelope stress sensing pathway

In tissues with mechanical function, the regulation of remodeling and repair processes is often controlled by mechanosensitive mechanisms; damage to the tissue structure is detected by changes in mechanical stress and strain, stimulating matrix synthesis and repair. While this mechanoregulatory feedback process is well recognized in animals and plants, it is not known whether such a process occurs in bacteria. In Vibrio cholerae, antibiotic-induced damage to the load-bearing cell wall promotes increased signaling by the two-component system VxrAB, which stimulates cell wall synthesis. Here we show that changes in mechanical stress and strain within the cell envelope are sufficient to stimulate VxrAB signaling in the absence of antibiotics. We applied mechanical forces to individual bacteria using three distinct loading modalities: extrusion loading within a microfluidic device, compression, and hydrostatic pressure. In all three cases, VxrAB signaling, as indicated by a fluorescent protein reporter, was increased in cells submitted to greater magnitudes of mechanical loading, hence diverse forms of mechanical stimuli activate VxrAB signaling. Mechanosensitivity of VxrAB signaling was lost following removal of the VxrAB stimulating endopeptidase ShyA, suggesting that VxrAB may not be directly sensing mechanical forces, but instead relies on other factors including lytic enzymes in the periplasmic space. Our findings suggest that mechanical signals play an important role in regulating cell wall homeostasis in bacteria. Significance StatementBiological materials with mechanical function (bones, muscle, etc.) are often maintained through mechanosensitive mechanisms, in which damage-induced reductions in stiffness stimulate remodeling and repair processes that restore mechanical function. Here we show that a similar process can occur in bacteria. We find that mechanical stresses in the bacterial cell envelope (the primary load-bearing structure in bacteria) regulate signaling of a two-component system involved in cell wall synthesis. These findings suggest that the mechanical stress state within the cell envelope can contribute to cell wall homeostasis. Furthermore, these findings demonstrate the potential to use mechanical stimuli to regulate gene expression in bacteria.

microbiology↗

bioRxiv2021

Peptidoglycan recycling contributes to outer membrane integrity and carbapenem tolerance in Acinetobacter baumannii

The Gram-negative cell envelope is an essential structure that not only protects the cell against lysis from the internal turgor, but also forms a barrier to limit entry of antibiotics. Some of our most potent bactericidal antibiotics, the {beta}-lactams, exploit the essentiality of the cell envelope by inhibiting its biosynthesis, typically inducing lysis and rapid death. However, many Gram-negative bacteria exhibit "antibiotic tolerance", the ability to sustain viability in the presence of {beta}-lactams for extended time periods. Despite several studies showing that antibiotic tolerance contributes directly to treatment failure, and is a steppingstone in acquisition of true resistance, the molecular factors that promote intrinsic tolerance are not well-understood. Acinetobacter baumannii is a critical-threat nosocomial pathogen notorious for its ability to rapidly develop multidrug resistance. While typically reserved to combat multidrug resistant infections, carbapenem {beta}-lactam antibiotics (i.e., meropenem) are first-line prescriptions to treat A. baumannii infections. Meropenem tolerance in Gram-negative pathogens is characterized by morphologically distinct populations of spheroplasts, but the impact of spheroplast formation is not fully understood. Here, we show that susceptible A. baumannii clinical isolates demonstrate high intrinsic tolerance to meropenem, form spheroplasts with the antibiotic and revert to normal growth after antibiotic removal. Using transcriptomics and genetics screens, we characterized novel tolerance factors and found that outer membrane integrity maintenance, drug efflux and peptidoglycan homeostasis collectively contribute to meropenem tolerance in A. baumannii. Furthermore, outer membrane integrity and peptidoglycan recycling are tightly linked in their contribution to meropenem tolerance in A. baumannii. ImportanceCarbapenem treatment failure associated with "superbug" infections has rapidly increased in prevalence, highlighting an urgent need to develop new therapeutic strategies. Antibiotic tolerance can directly lead to treatment failure but has also been shown to promote acquisition of true resistance within a population. While some studies have addressed mechanisms that promote tolerance, factors that underlie Gram-negative bacterial survival during carbapenem treatment are not well-understood. Here, we characterized a role for peptidoglycan recycling in outer membrane integrity maintenance and carbapenem tolerance in A. baumannii. These studies suggest that the pathogen limits antibiotic concentrations in the periplasm and highlights physiological processes that could be targeted to improve antimicrobial treatment.

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↗

bioRxiv2021

Vibrio cholerae's mysterious Seventh Pandemic island (VSP-II) encodes novel Zur-regulated zinc starvation genes involved in chemotaxis and autoaggregation

Vibrio cholerae is the causative agent of cholera, a notorious diarrheal disease that is typically transmitted via contaminated drinking water. The current pandemic agent, the El Tor biotype, has undergone several genetic changes that include horizontal acquisition of two genomic islands (VSP-I and VSP-II). VSP-I and -2 presence strongly correlates with pandemicity; however, the contribution of these islands to V. choleraes life cycle, particularly the 26-kb VSP-II, remains poorly understood. VSP-II-encoded genes are not expressed under standard laboratory conditions, suggesting that their induction requires an unknown signal from the host or environment. One signal that bacteria encounter under both host and environmental conditions is metal limitation. While studying V. choleraes zinc-starvation response in vitro, we noticed that a mutant constitutively expressing zinc-starvation genes ({Delta}zur) aggregates in nutrient-poor media. Using transposon mutagenesis, we found that flagellar motility, chemotaxis, and VSP-II encoded genes are required for aggregation. The VSP-II genes encode an AraC-like transcriptional activator (VerA) and a methyl-accepting chemotaxis protein (AerB). Using RNA-seq and lacZ transcriptional reporters, we show that VerA is a novel Zur target and activator of the nearby AerB chemoreceptor. AerB interfaces with the chemotaxis system to drive oxygen-dependent autoaggregation and energy taxis. Importantly, this work suggests a functional link between VSP-II, zinc-starved environments, and aerotaxis, yielding insights into the role of VSP-II in a metal-limited host or aquatic reservoir. Author SummaryThe Vibrio Seventh Pandemic island was horizontally acquired by El Tor pandemic strain, but its role in pathogenicity or environmental persistence is unknown. A major barrier to VSP-II study was the lack of stimuli favoring its expression. We show that zinc starvation induces expression of this island and describe a transcriptional network that activates a VSP-II encoded aerotaxis receptor. Importantly, aerotaxis may enable V. cholerae to locate more favorable microenvironments, possibly to colonize anoxic portions of the gut or environmental sediments.

microbiology↗

bioRxiv2021

Host-pathogen Immune Feedbacks Can Explain Widely Divergent Outcomes from Similar Infections

A longstanding question in infection biology is why two very similar individuals, with very similar pathogen exposures, may have very different outcomes. Recent experiments have found that even isogenic Drosophila melanogaster hosts, given identical inoculations of some bacterial pathogens at suitable doses, can experience very similar initial bacteria proliferation but then diverge to either a lethal infection or a sustained chronic infection with much lower pathogen load. We hypothesized that divergent infection outcomes are a natural result of mutual negative feedbacks between pathogens and the host immune response. Here we test this hypothesis in silico by constructing process-based dynamic models for bacterial population growth, host immune induction, and the feedbacks between them, based on common mechanisms of immune system response. Mathematical analysis of a minimal conceptual model confirms our qualitative hypothesis that mutual negative feedbacks can magnify small differences among hosts into life-or-death differences in outcome. However, explaining observed features of chronic infections requires an extension of the model to include induced pathogen modifications that shield themselves from host immune responses at the cost of reduced proliferation rate. Our analysis thus generates new, testable predictions about the mechanisms underlying bimodal infection outcomes.

systems biology↗

bioRxiv2020

Class A Penicillin-Binding Protein-mediated cell wall synthesis promotes structural integrity during peptidoglycan endopeptidase insufficiency

The bacterial cell wall is composed primarily of peptidoglycan (PG), a poly-aminosugar that is essential to sustain cell shape, growth and structural integrity. PG is synthesized by two different types of synthase complexes (class A Penicillin-binding Proteins [PBP]s/Lpos and Shape, Elongation, Division, Sporulation [SEDS]/class B PBP pairs) and degraded by autolytic enzymes to accommodate growth processes. It is thought that autolsyin activity (and particulary the activity of endopeptidases, EPs) is required for PG synthesis and incorporation by creating gaps that are patched and paved by PG synthases, but the exact relationship between autolysins and the separate synthesis machineries remains incompletely understood. Here, we have probed the consequences of EP depletion for PG synthesis in the diarrheal pathogen Vibrio cholerae. We found that EP depletion resulted in severe morphological defects, increased cell mass, a decline in viability, and continuing (yet aberrant) incorporation of cell wall material. Mass increase and cell wall incorporation proceeded in the presence of Rod system inhibitors, but was abolished upon inhibition of aPBPs. However, the Rod system remained functional (i.e., exhibited sustained directed motion) even after prolonged EP depletion, without effectively promoting cell elongation. Lastly, heterologous expression of an EP from Neisseria gonorrhoeae could fully complement growth and morphology of an EP-insufficient V. cholerae. Overall, our findings suggest that in V. cholerae, the Rod system requires endopeptidase activity (but not necessarily direct interaction with EPs) to promote cell expansion and substantial PG incorporation, whereas aPBPs are able to engage in sacculus construction even during severe EP insufficiency. ImportanceSynthesis and turnover of the bacterial cell wall must be tightly co-ordinated to avoid structural integrity failure and cell death. Details of this coordination are poorly understood, particularly if and how cell wall turnover enzymes are required for the activity of the different cell wall synthesis machines. Our results suggest that in Vibrio cholerae, one class of turnover enzymes, the endopeptidases, are required only for substantial PG incorporation mediated by the Rod system, while the aPBPs maintain structural integrity during endopeptidase insufficiency. Our results suggest that aPBPs are more versatile than the Rod system in their ability to recognize cell wall gaps formed by autolysins other than the major endopeptidases, adding to our understanding of the co-ordination between autolysins and cell wall synthases. A detailed understanding of autolysin biology may promote the development of antibiotics that target these essential turnover processes.

microbiology↗

bioRxiv2019

Structural basis of peptidoglycan endopeptidase regulation

Most bacteria surround themselves with a cell wall, a strong meshwork consisting primarily of the polymerized aminosugar peptidoglycan (PG). PG is essential for structural maintenance of bacterial cells, and thus for viability. PG is also constantly synthesized and turned over, the latter process is mediated by PG cleavage enzymes, for example the endopeptidases (EPs). EPs themselves are essential for growth, but also promote lethal cell wall degradation after exposure to antibiotics that inhibit PG synthases (e.g., {beta}-lactams). Thus, EPs are attractive targets for novel antibiotics and their adjuvants. However, we have a poor understanding of how these enzymes are regulated in vivo, depriving us of novel pathways for the development of such antibiotics. Here, we have solved crystal structures of the LysM/M23 family peptidase ShyA, the primary EP of the cholera pathogen Vibrio cholerae. Our data suggest that ShyA assumes two drastically different conformations; a more open form that allows for substrate binding, and a closed form, which we predicted to be catalytically inactive. Mutations expected to promote the open conformation caused enhanced activity in vitro and in vivo, and these results were recapitulated in EPs from the divergent pathogens Neisseria gonorrheae and Escherichia coli. Our results suggest that LysM/M23 EPs are regulated via release of the inhibitory Domain1 from the M23 active site, likely through conformational re-arrangement in vivo. SignificanceBacteria digest their cell wall following exposure to antibiotics like penicillin. The endopeptidases (EPs) are among the proteins that catalyze cell wall digestion processes after antibiotic exposure, but we do not understand how these enzymes are regulated during normal growth. Herein, we present the structure of the major EP from the diarrheal pathogen Vibrio cholerae. Surprisingly, we find that EPs from this and other pathogens appear to be produced as a largely inactive precursor that undergoes a conformational shift exposing the active site to engage in cell wall digestion. These results enhance our understanding of how EPs are regulated and could open the door for the development of novel antibiotics that overactivate cell wall digestion processes.

microbiology↗

bioRxiv2019

A multifaceted cellular damage repair and prevention pathway promotes high level tolerance to β-lactam antibiotics

Bactericidal antibiotics are powerful agents due to their ability to convert essential bacterial functions into lethal processes. However, many important bacterial pathogens are remarkably tolerant against bactericidal antibiotics due to inducible damage repair responses. The cell wall damage response two-component system VxrAB of the gastrointestinal pathogen Vibrio cholerae promotes high-level {beta}-lactam tolerance and controls a gene network encoding highly diverse functions, including negative control over multiple iron uptake systems. How this system contributes to tolerance is poorly understood. Here, we show that {beta}-lactam antibiotics cause an increase in intracellular free iron levels and collateral oxidative damage, which is exacerbated in the {Delta}vxrAB mutant. Mutating major iron uptake systems drastically increased {Delta}vxrAB tolerance to {beta}-lactams. We propose that VxrAB reduces antibiotic-induced toxic iron and concomitant metabolic perturbations by downregulating iron uptake transporters and show that iron sequestration enhances tolerance against {beta}-lactam therapy in a mouse model of cholera infection. Our results suggest that a microorganisms ability to counteract diverse antibiotic-induced stresses promotes high-level antibiotic tolerance, and highlights the complex secondary responses elicited by antibiotics.

microbiology↗

bioRxiv2019

Modulation of peptidoglycan synthesis by recycled cell wall tetrapeptides

The bacterial cell wall is made of peptidoglycan (PG), a polymer that is essential for maintenance of cell shape and survival. During growth, bacteria remodel their PG, releasing fragments that are predominantly re-internalized by the cell, where they are recycled for synthesis of new PG. Although the PG recycling pathway is widely conserved, its components are not essential and its roles in cell wall homeostasis are not well-understood. Here, we identified LdcV, a Vibrio cholerae L,D-carboxypeptidase that cleaves the terminal D-Alanine from recycled murotetrapeptides. In the absence of ldcV, recycled tetrapeptides accumulated in the cytosol, leading to two toxic consequences for the cell wall. First, incorporation of tetrapeptide-containing PG precursors into the cell wall led to reduction in D,D-cross-linkage between stem peptides, diminishing PG integrity. Second, tetrapeptide accumulation led to a decrease in canonical UDP-pentapeptide precursors, reducing PG synthesis. Thus, LdcV and the recycling pathway promote optimal cell wall assembly and composition. Furthermore, Ldc substrate preference for murotetrapeptides containing canonical (D-Alanine) vs. non-canonical (D-Methionine) D-amino acids is conserved, suggesting that accumulation of tetrapeptide recycling intermediates may modulate PG homeostasis in environments enriched in non-canonical-muropeptides via substrate competition.

microbiology↗