Genetic interaction mapping reveals functional relationships between peptidoglycan endopeptidases and carboxypeptidases.

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 regulators 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 ΔdacA1 in LB medium identified hypomorphic mutants in the PG synthesis pathway, as well as mutations that promote EP activation. Our data thus reveal a more complex role of DacA1 in maintaining PG homeostasis than previously assumed.

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. cloacae's 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, our data suggest that multiple, independent mechanisms contribute to AMP resistance in E. cloacae.

High-level carbapenem tolerance requires antibiotic-induced outer membrane modifications.

Antibiotic tolerance is an understudied potential contributor to antibiotic treatment failure and the emergence of multidrug-resistant bacteria. The molecular mechanisms governing tolerance remain poorly understood. A prominent type of ß-lactam tolerance relies on the formation of cell wall-deficient spheroplasts, which maintain structural integrity via their outer membrane (OM), an asymmetric lipid bilayer consisting of phospholipids on the inner leaflet and a lipid-linked polysaccharide (lipopolysaccharide, LPS) enriched in the outer monolayer on the cell surface. How a membrane structure like LPS, with its reliance on mere electrostatic interactions to maintain stability, is capable of countering internal turgor pressure is unknown. Here, we have uncovered a novel role for the PhoPQ two-component system in tolerance to the ß-lactam antibiotic meropenem in Enterobacterales. We found that PhoPQ is induced by meropenem treatment and promotes an increase in 4-amino-4-deoxy-L-aminoarabinose [L-Ara4N] modification of lipid A, the membrane anchor of LPS. L-Ara4N modifications likely enhance structural integrity, and consequently tolerance to meropenem, in several Enterobacterales species. Importantly, mutational inactivation of the negative PhoPQ regulator mgrB (commonly selected for during clinical therapy with the last-resort antibiotic colistin, an antimicrobial peptide [AMP]) results in dramatically enhanced tolerance, suggesting that AMPs can collaterally select for meropenem tolerance via stable overactivation of PhoPQ. Lastly, we identify histidine kinase inhibitors (including an FDA-approved drug) that inhibit PhoPQ-dependent LPS modifications and consequently potentiate meropenem to enhance lysis of tolerant cells. In summary, our results suggest that PhoPQ-mediated LPS modifications play a significant role in stabilizing the OM, promoting survival when the primary integrity maintenance structure, the cell wall, is removed.

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

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 presence strongly correlates with pandemicity; however, the contribution of these islands to V. cholerae's 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. cholerae's zinc-starvation response in vitro, we noticed that a mutant constitutively expressing zinc starvation genes (Δzur) congregates at the bottom of a culture tube when grown in a nutrient-poor medium. Using transposon mutagenesis, we found that flagellar motility, chemotaxis, and VSP-II encoded genes were required for congregation. 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 an activator of the nearby AerB chemoreceptor. AerB interfaces with the chemotaxis system to drive oxygen-dependent congregation and energy taxis. Importantly, this work suggests a functional link between VSP-II, zinc-starved environments, and energy taxis, yielding insights into the role of VSP-II in a metal-limited host or aquatic reservoir.

Cleave a Septum, Leave a Cell: Bdellovibrio bacteriovorus Secretes a Specialized Lytic Transglycosylase to Clear Prey Cell Septum Obstruction.

Predatory microbes like Bdellovibrio feed on other bacteria by invading their periplasm, replicating within the bacterial shell that is now a feeding trough, and ultimately lysing the prey and disseminating. A new study by E. J. Banks, C. Lambert, S. Mason, J. Tyson, et al. (J Bacteriol 205:e00475-22, 2023, https://doi.org/10.1128/jb.00475-22) highlights the great lengths to which Bdellovibrio must go to affect host cell remodeling: A secreted cell wall lytic enzyme with specificity for the host septal cell wall maximizes the size of the attacker's meal and the size of the restaurant in which it can spread out. This study provides novel insights into bacterial predator-prey dynamics and showcases elegant co-option of an endogenous cell wall turnover enzyme refurbished as a warhead to enhance prey consumption.

Disrupting Central Carbon Metabolism Increases ß-Lactam 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, becomes highly tolerant to ß-lactam antibiotics (penicillin and related compounds) in a process requiring the two-component system VxrAB. VxrAB is induced by exposure to cell wall damaging 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 in Vibrio cholerae. 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 ß-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. IMPORTANCE Antibiotic tolerance (the ability to survive exposure to antibiotics) is a stepping stone toward 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. Here, we identified central carbon metabolism as a key contributor to antibiotic tolerance and resistance. A strain with a mutation 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 the development of antibiotic adjuvants.

Masters of Misdirection: Peptidoglycan Glycosidases in Bacterial Growth.

The dynamic composition of the peptidoglycan cell wall has been the subject of intense research for decades, yet how bacteria coordinate the synthesis of new peptidoglycan with the turnover and remodeling of existing peptidoglycan remains elusive. Diversity and redundancy within peptidoglycan synthases and peptidoglycan autolysins, enzymes that degrade peptidoglycan, have often made it challenging to assign physiological roles to individual enzymes and determine how those activities are regulated. For these reasons, peptidoglycan glycosidases, which cleave within the glycan strands of peptidoglycan, have proven veritable masters of misdirection over the years. Unlike many of the broadly conserved peptidoglycan synthetic complexes, diverse bacteria can employ unrelated glycosidases to achieve the same physiological outcome. Additionally, although the mechanisms of action for many individual enzymes have been characterized, apparent conserved homologs in other organisms can exhibit an entirely different biochemistry. This flexibility has been recently demonstrated in the context of three functions critical to vegetative growth: (i) release of newly synthesized peptidoglycan strands from their membrane anchors, (ii) processing of peptidoglycan turned over during cell wall expansion, and (iii) removal of peptidoglycan fragments that interfere with daughter cell separation during cell division. Finally, the regulation of glycosidase activity during these cell processes may be a cumulation of many factors, including protein-protein interactions, intrinsic substrate preferences, substrate availability, and subcellular localization. Understanding the true scope of peptidoglycan glycosidase activity will require the exploration of enzymes from diverse organisms with equally diverse growth and division strategies.

A multifaceted cellular damage repair and prevention pathway promotes high-level tolerance to ß-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 ß-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 ß-lactam antibiotics cause an increase in intracellular free iron levels and collateral oxidative damage, which is exacerbated in the ∆vxrAB mutant. Mutating major iron uptake systems dramatically increases ∆vxrAB tolerance to ß-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 ß-lactam therapy in a mouse model of cholera infection. Our results suggest that a microorganism's ability to counteract diverse antibiotic-induced stresses promotes high-level antibiotic tolerance and highlights the complex secondary responses elicited by antibiotics.

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., ß-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 Domain 1 from the M23 active site, likely through conformational rearrangement in vivo.

Host-pathogen immune feedbacks can explain widely divergent outcomes from similar infections.

A long-standing 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.

Lytic transglycosylases RlpA and MltC assist in Vibrio cholerae daughter cell separation.

The cell wall is a crucial structural feature in the vast majority of bacteria and comprises a covalently closed network of peptidoglycan (PG) strands. While PG synthesis is important for survival under many conditions, the cell wall is also a dynamic structure, undergoing degradation and remodeling by 'autolysins', enzymes that break down PG. Cell division, for example, requires extensive PG remodeling, especially during separation of daughter cells, which depends heavily upon the activity of amidases. However, in Vibrio cholerae, we demonstrate that amidase activity alone is insufficient for daughter cell separation and that lytic transglycosylases RlpA and MltC both contribute to this process. MltC and RlpA both localize to the septum and are functionally redundant under normal laboratory conditions; however, only RlpA can support normal cell separation in low-salt media. The division-specific activity of lytic transglycosylases has implications for the local structure of septal PG, suggesting that there may be glycan bridges between daughter cells that cannot be resolved by amidases. We propose that lytic transglycosylases at the septum cleave PG strands that are crosslinked beyond the reach of the highly regulated activity of the amidase and clear PG debris that may block the completion of outer membrane invagination.

Characterization of an anti-FLAG antibody binding protein in V. cholerae.

FLAG-tags are commonly used for protein abundance measurements and for identification of protein-protein interactions in living cells. We have observed that the cholera pathogen Vibrio cholerae encodes a FLAG-antibody-reactive protein and identified this protein as an outer membrane porin, Porin4, which contains a sequence very similar to the 3xFLAG epitope tag. We have demonstrated the binding affinity of the conserved peptide sequence (called Porin 4 tag) in Porin4 against monoclonal anti-FLAG M2 antibody. In addition, we created a porin4 deletion mutant, which can be used for background-less FLAG antibody detection experiments.

Spheroplast-Mediated Carbapenem Tolerance in Gram-Negative Pathogens.

Antibiotic tolerance, the ability to temporarily sustain viability in the presence of bactericidal antibiotics, constitutes an understudied and yet potentially widespread cause of antibiotic treatment failure. We have previously shown that the Gram-negative pathogen Vibrio cholerae can tolerate exposure to the typically bactericidal ß-lactam antibiotics by assuming a spherical morphotype devoid of detectable cell wall material. However, it is unclear how widespread ß-lactam tolerance is. Here, we tested a panel of clinically significant Gram-negative pathogens for their response to the potent, broad-spectrum carbapenem antibiotic meropenem. We show that clinical isolates of Enterobacter cloacae, Klebsiella aerogenes, and Klebsiella pneumoniae, but not Escherichia coli, exhibited moderate to high levels of tolerance of meropenem, both in laboratory growth medium and in human serum. Importantly, tolerance was mediated by cell wall-deficient spheroplasts, which readily recovered wild-type morphology and growth upon removal of antibiotic. Our results suggest that carbapenem tolerance is prevalent in clinically significant bacterial species, and we suggest that this could contribute to treatment failure associated with these organisms.

A cell wall damage response mediated by a sensor kinase/response regulator pair enables beta-lactam tolerance.

The bacterial cell wall is critical for maintenance of cell shape and survival. Following exposure to antibiotics that target enzymes required for cell wall synthesis, bacteria typically lyse. Although several cell envelope stress response systems have been well described, there is little knowledge of systems that modulate cell wall synthesis in response to cell wall damage, particularly in Gram-negative bacteria. Here we describe WigK/WigR, a histidine kinase/response regulator pair that enables Vibrio cholerae, the cholera pathogen, to survive exposure to antibiotics targeting cell wall synthesis in vitro and during infection. Unlike wild-type V. cholerae, mutants lacking wigR fail to recover following exposure to cell-wall-acting antibiotics, and they exhibit a drastically increased cell diameter in the absence of such antibiotics. Conversely, overexpression of wigR leads to cell slimming. Overexpression of activated WigR also results in increased expression of the full set of cell wall synthesis genes and to elevated cell wall content. WigKR-dependent expression of cell wall synthesis genes is induced by various cell-wall-acting antibiotics as well as by overexpression of an endogenous cell wall hydrolase. Thus, WigKR appears to monitor cell wall integrity and to enhance the capacity for increased cell wall production in response to damage. Taken together, these findings implicate WigKR as a regulator of cell wall synthesis that controls cell wall homeostasis in response to antibiotics and likely during normal growth as well.

Don't let sleeping dogmas lie: new views of peptidoglycan synthesis and its regulation.

Bacterial cell wall synthesis is the target for some of our most powerful antibiotics and has thus been the subject of intense research focus for more than 50 years. Surprisingly, we still lack a fundamental understanding of how bacteria build, maintain and expand their cell wall. Due to technical limitations, directly testing hypotheses about the coordination and biochemistry of cell wall synthesis enzymes or architecture has been challenging, and interpretation of data has therefore often relied on circumstantial evidence and implicit assumptions. A number of recent papers have exploited new technologies, like single molecule tracking and real-time, high resolution temporal mapping of cell wall synthesis processes, to address fundamental questions of bacterial cell wall biogenesis. The results have challenged established dogmas and it is therefore timely to integrate new data and old observations into a new model of cell wall biogenesis in rod-shaped bacteria.

Genetic Determinants of Penicillin Tolerance in Vibrio cholerae.

Many bacteria are resistant to killing (tolerant) by typically bactericidal antibiotics due to their ability to counteract drug-induced cell damage. Vibrio cholerae, the cholera agent, displays an unusually high tolerance to diverse inhibitors of cell wall synthesis. Exposure to these agents, which in other bacteria leads to lysis and death, results in a breakdown of the cell wall and subsequent sphere formation in V. cholerae Spheres readily recover to rod-shaped cells upon antibiotic removal, but the mechanisms mediating the recovery process are not well characterized. Here, we found that the mechanisms of recovery are dependent on environmental conditions. Interestingly, on agarose pads, spheres undergo characteristic stages during the restoration of rod shape. Drug inhibition and microscopy experiments suggest that class A penicillin binding proteins (aPBPs) play a more active role than the Rod system, especially early in sphere recovery. Transposon insertion sequencing (TnSeq) analyses revealed that lipopolysaccharide (LPS) and cell wall biogenesis genes, as well as the sigma E cell envelope stress response, were particularly critical for recovery. LPS core and O-antigen appear to be more critical for sphere formation/integrity and viability than lipid A modifications. Overall, our findings demonstrate that the outer membrane is a key contributor to beta lactam tolerance and suggest a role for aPBPs in cell wall biogenesis in the absence of rod-shape cues. Factors required for postantibiotic recovery could serve as targets for antibiotic adjuvants that enhance the efficacy of antibiotics that inhibit cell wall biogenesis.

An Ambient Temperature Electrolyte with Superior Lithium Ion Conductivity based on a Self-Assembled Block Copolymer.

In searching for polymer-based electrolytes with improved performance for lithium ion and lithium metal batteries, we studied block copolymer electrolytes with high amounts of bis(trifluoromethane)sulfonimide lithium obtained by macromolecular co-assembly of a poly(isoprene)-block-poly(styrene)-block-poly(ethylene oxide) and the salt from tetrahydrofuran. Particularly, an ultra-short poly(ethylene oxide) block of 2100 g mol-1 was applied, giving rise to 2D continuous lamellar microstructures. The macroscopic stability was ensured with major blocks from poly(isoprene) and poly(styrene), which separated the ionic conductive PEO/salt lamellae. Thermal annealing led to high ionic conductivities of 1.4 mS cm-1 at 20 °C with low activation energy and a superior lithium ion transference number of 0.7, accompanied by an improved mechanical stability (storage modulus of up to 107  Pa). With high Li:O ratios >1, we show a viable concept to achieve fast Li+ transport in block copolymers (BCP), decoupled from slow polymer relaxation.

Ordered Mesoporous Titania/Carbon Hybrid Monoliths for Lithium-ion Battery Anodes with High Areal and Volumetric Capacity.

Free-standing, binder-free, and conductive additive-free mesoporous titanium dioxide/carbon hybrid electrodes were prepared from co-assembly of a poly(isoprene)-block-poly(styrene)-block-poly(ethylene oxide) block copolymer and a titanium alkoxide. By tailoring an optimized morphology, we prepared macroscopic mechanically stable 300 µm thick monoliths that were directly employed as lithium-ion battery electrodes. High areal mass loading of up to 26.4 mg cm-2 and a high bulk density of 0.88 g cm-3 were obtained. This resulted in a highly increased volumetric capacity of 155 mAh cm-3 , compared to cast thin film electrodes. Further, the areal capacity of 4.5 mAh cm-2 represented a 9-fold increase compared to conventionally cast electrodes. These attractive performance metrics are related to the superior electrolyte transport and shortened diffusion lengths provided by the interconnected mesoporous nature of the monolith material, assuring superior rate handling, even at high cycling rates.

Endopeptidase-mediated beta lactam tolerance.

In many bacteria, inhibition of cell wall synthesis leads to cell death and lysis. The pathways and enzymes that mediate cell lysis after exposure to cell wall-acting antibiotics (e.g. beta lactams) are incompletely understood, but the activities of enzymes that degrade the cell wall ('autolysins') are thought to be critical. Here, we report that Vibrio cholerae, the cholera pathogen, is tolerant to antibiotics targeting cell wall synthesis. In response to a wide variety of cell wall--acting antibiotics, this pathogen loses its rod shape, indicative of cell wall degradation, and becomes spherical. Genetic analyses revealed that paradoxically, V. cholerae survival via sphere formation required the activity of D,D endopeptidases, enzymes that cleave the cell wall. Other autolysins proved dispensable for this process. Our findings suggest the enzymes that mediate cell wall degradation are critical for determining bacterial cell fate--sphere formation vs. lysis--after treatment with antibiotics that target cell wall synthesis.