Search

Escherichia coliK-12 is a model organism for bacteriology and has served as a workhorse for molecular biology and biochemistry for over a century since its first isolation in 1922. However,Escherichia coliK-12 strains are phenotypically devoid of an O antigen (OAg) since early reports in the scientific literature. Recent studies reported the presence of independent mutations that abolish OAg biogenesis inE. coliK-12 strains from the same original source, suggesting unknown evolutionary forces have selected for loss of OAg during the early propagation of K-12. Here, we show for the first time that restoration of OAg inE. coliK-12 strain MG1655 synergistically sensitises bacteria to vancomycin with bile salts (VBS). Suppressor mutants surviving lethal doses of VBS mostly contained disruptions in OAg biogenesis. We present data supporting a model where the transient presence and accumulation of lipid-carried OAg intermediates in the bacterial periplasm interfere with peptidoglycan synthesis, causing growth defects that are synergistically enhanced by bile salts. Lastly, we demonstrate that continuous bile salt exposure of OAg-producing MG1655 in the laboratory, can recreate a scenario where OAg disruption is selected for. Hence our work provides a likely explanation for the long-held mystery of howE. coliK-12 lost its OAg production and opens new avenues for exploring long-standing questions on the intricate network coordinating the synthesis of different cell envelope components in Gram-negative bacteria.Significance statementEscherichia coliK-12 is the most studied microorganism, widely used in laboratories for studying bacteriology and as a tool for molecular biology. The reason why it is devoid of O antigen remains a long-standing question. Our work has uncovered a previously unknown selection pressure of bile salts on bacterial O antigen biogenesis, which provides a plausible scenario for how the early propagation ofE. coliK-12 strains in bile salt containing media could have led to loss of O antigen in K-12. Our results also suggest that the accumulation of O antigen intermediates in the bacterial periplasm may interfere with bacterial cell wall synthesis, which paves a new research direction into the interplay of different cell envelope component synthesis pathways.

Disulfide bond (Dsb) proteins catalyse oxidative protein folding governing bacterial survival and virulence. Dsb systems inEscherichia coliK-12 are well-studied, yet what determines dithiol oxidase or disulfide reductase activity remains unknown. Past studies suggest oligomerisation of periplasmic thiol oxidoreductases dictates the direction of thiol catalytic activity. Here, we studied three suppressor-of-copper-sensitivity C (ScsC) Dsb-like proteins known to exist in the reduced state and bind to copper. These proteins adopt different quaternary structures:Salmonella entericaScsC (StScsC) is monomeric, while ScsC fromProteus mirabilis(PmScsC) andCaulobacter crescentus(CcScsC) are trimeric. When expressed in the model organismE. coliK-12, we showed that all three ScsC proteins exhibit both dithiol oxidation and disulfide reduction activity, despite structural differences. Interestingly, while ScsC reductase function was supported by the canonicalE. coliDsbD reductase, oxidase activity depended on environmental oxidation. However, an engineered monomeric PmScsC synergises withE. coliDsbB to gain dithiol oxidase activity at the expense of reductase function. Thus, oligomerisation could be one mechanism by which ScsC proteins avoid interactions with the periplasmic thiol oxidase pathway. This tightly controls their re-oxidation and maintains ScsC proteins in the reduced state required for binding and sequestering toxic levels of cellular copper.

Aims: Thioredoxin (TRX)-fold proteins are ubiquitous in nature. This redox scaffold has evolved to enable a variety of functions, including redox regulation, protein folding, and oxidative stress defense. In bacteria, the TRX-like disulfide bond (Dsb) family mediates the oxidative folding of multiple proteins required for fitness and pathogenic potential. Conventionally, Dsb proteins have specific redox functions with monomeric and dimeric Dsbs exclusively catalyzing thiol oxidation and disulfide isomerization, respectively. This contrasts with the eukaryotic disulfide forming machinery where the modular TRX protein disulfide isomerase (PDI) mediates thiol oxidation and disulfide reshuffling. In this study, we identified and structurally and biochemically characterized a novel Dsb-like protein from Salmonella enterica termed bovine colonization factor protein H (BcfH) and defined its role in virulence. Results: In the conserved bovine colonization factor (bcf) fimbrial operon, the Dsb-like enzyme BcfH forms a trimeric structure, exceptionally uncommon among the large and evolutionary conserved TRX superfamily. This protein also displays very unusual catalytic redox centers, including an unwound α-helix holding the redox active site and a trans-proline instead of the conserved cis-proline active site loop. Remarkably, BcfH displays both thiol oxidase and disulfide isomerase activities contributing to Salmonella fimbrial biogenesis. Innovation and Conclusion: Typically, oligomerization of bacterial Dsb proteins modulates their redox function, with monomeric and dimeric Dsbs mediating thiol oxidation and disulfide isomerization, respectively. This study demonstrates a further structural and functional malleability in the TRX-fold protein family. BcfH trimeric architecture and unconventional catalytic sites permit multiple redox functions emulating in bacteria the eukaryotic PDI dual oxidoreductase activity. Antioxid. Redox Signal. 35, 21-39.

Shigella IcsA is a versatile surface virulence factor required for both early and late pathogenesis stages, extracellularly to intracellularly. Despite IcsA serving as a model Type V secretion system (T5SS) autotransporter to study host pathogen interactions, its detailed molecular architecture is poorly understood. Recently, IcsA was found to switch to a different conformation for its adhesin activity upon sensing of the host stimuli by Shigella Type III secretion system (T3SS). Here, we report that the single cysteine residue (C130) near the N-terminus of IcsA passenger has a role in IcsA adhesin activity. We also show that the IcsA passenger (IcsAp) exists in multiple conformations, and the conformation populations are influenced by a central pair of cysteine residues (C375 and C379), which is not previously reported for any Type V autotransporter passengers. Disruption of either or both central cysteine residues alters the exposure of IcsA epitopes to polyclonal anti-IcsA antibodies previously shown to block Shigella adherence, yet without loss of IcsA intracellular functions in actin-based motility (ABM). Anti-IcsA antibody reactivity was restored when the IcsA paired cysteine substitution mutants were expressed in a ∆ipaD background with a constitutively active T3SS, highlighting an interplay between T3SS and T5SS. The work here uncovers a novel molecular switch empowered by a centrally localised, short-spaced cysteine pair in the Type V autotransporter IcsA that ensures conformational heterogeneity to aid IcsA evasion of host immunity.ImportanceShigella species are the leading cause of diarrheal related death globally by causing bacillary dysentery. The surface virulence factor IcsA which is essential for Shigella pathogenesis is a unique multi-functional autotransporter that is responsible for cell adhesion, and actin-based motility, yet detailed mechanistic understanding is lacking. Here, we show that the three cysteine residues in IcsA contribute to the protein’s distinct functions. The N terminus cysteine residue within the IcsA passenger domain plays a role in adhesin function, while a centrally localised cysteine pair provides conformational heterogeneity resulting in IcsA molecules with different reactivity to adhesion-blocking anti-IcsA antibodies. In synergy with the Type III secretion system, this molecular switch preserves biological function in distinct IcsA conformations for cell adhesion, actin-based motility and autophagy escape, providing a potential strategy by which Shigella evade host immunity targeting of this essential virulence factor.

Antibiotics are failing fast, and the development pipeline remains alarmingly dry. New drug research and development is being urged by world health officials, with new antibacterials against multidrug-resistant Gram-negative pathogens as the highest priority. Antivirulence drugs, which inhibit bacterial pathogenicity factors, are a class of promising antibacterials, however, their development is stifled by lack of standardised preclinical testing akin to what guides antibiotic development. The lack of established target-specific microbiological assays amenable to high-throughput, often means that cell-based testing of virulence inhibitors is absent from the discovery (hit-to-lead) phase, only to be employed at later-stages of lead optimization. Here, we address this by establishing a pipeline of bacterial cell-based assays developed for the identification and early preclinical evaluation of DsbA inhibitors, previously identified by biophysical and biochemical assays. Inhibitors of DsbA block oxidative protein folding required for virulence factor folding in pathogens. Here we use existing Escherichia coli DsbA inhibitors and uropathogenic E. coli (UPEC) as a model pathogen, to demonstrate that the combination of a cell-based sulfotransferase assay and a motility assay (both DsbA reporter assays), modified for a higher throughput format, can provide a robust and target-specific platform for the identification and evaluation of DsbA inhibitors.

Antibiotic resistance is one of the most prominent threats to modern medicine. In the latest World Health Organization list of bacterial pathogens that urgently require new antibiotics, nine out of 12 are Gram-negative, with four being of ‘Critical Priority’. One crucial barrier restricting antibiotic efficacy against Gram-negative bacteria is their unique cell envelope. While fatty acids are a shared constituent of all structural membrane lipids, their biosynthesis pathway in bacteria is distinct from eukaryotes making it an attractive target for new antibiotic development that remains less explored. Here, we interrogated the redundant components of the bacterial Type IIFattyAcidSynthesis (FAS II) pathway, showing that disrupting FAS II homeostasis inEscherichia colithrough deletion of thefabHgene damages the cell envelope of antibiotic susceptible and antibiotic resistant clinical isolates. ThefabHgene encodes the β-ketoacyl acyl carrier protein synthase III (KAS III), which catalyzes the initial condensation reactions during fatty acid biosynthesis. We show thatfabHnull mutation potentiated the killing of multi-drug resistantE. coliby a broad panel of previously ineffective antibiotics, despite the presence of relevant antibiotic resistance determinants, for example, carbapenemasekpc2. Enhanced antibiotic sensitivity was additionally demonstrated in the context of eradicating established biofilms and treating established human cell infectionin vitro. Our findings showcase the potential of FabH as a promising target that could be further explored in the development of therapies that may repurpose currently ineffective antibiotics or rescue failing last-resort antibiotics against Gram-negative pathogens.IMPORTANCEGram-negative pathogens are a major concern for global public health due to increasing rates of antibiotic resistance and the lack of new drugs. A major contributing factor towards antibiotic resistance in Gram-negative bacteria is their formidable outer membrane, which acts as a permeability barrier preventing many biologically active antimicrobials from reaching the intracellular targets and thus limiting their efficacy. Fatty acids are the fundamental building blocks of structural membrane lipids and their synthesis constitutes an attractive antimicrobial target as it follows distinct pathways in prokaryotes and eukaryotes. Herein, we identified a component of fatty acid synthesis, FabH, as a ‘gate-keeper’ of outer membrane barrier function. Without FabH, Gram-negative bacteria become susceptible to otherwise impermeable antibiotics and are re-sensitised to killing by last-resort antibiotics. This study supports FabH as a promising target for inhibition in future antimicrobial therapies.

Antibiotics are failing fast, and the development pipeline is alarmingly dry. New drug research and development is being urged by world health officials, with new antibacterials against multidrug-resistant Gram-negative pathogens as the highest priority. Antivirulence drugs, which are inhibitors of bacterial pathogenicity factors, are a class of promising antibacterials, however, their development is often stifled by lack of standardised preclinical testing akin to what guides antibiotic development. The lack of established target-specific microbiological assays amenable to high-throughput, often means that cell-based testing of virulence inhibitors is absent from the discovery (hit-to-lead) phase, only to be employed at later-stages of lead optimization. Here, we address this by establishing a pipeline of bacterial cell-based assays developed for the identification and early preclinical evaluation of DsbA inhibitors. Inhibitors of DsbA block bacterial oxidative protein folding and were previously identified by biophysical and biochemical assays. Here we use existing Escherichia coli DsbA inhibitors and uropathogenic E. coli (UPEC) as a model pathogen, to demonstrate that a combination of a cell-based AssT sulfotransferase assay and the UPEC motility assay, modified for a higher throughput format, can provide a robust and target-specific platform for the evaluation of DsbA inhibitors. Our pipeline could also be used in fragment and compound screening for the identification of new DsbA inhibitor classes or hits with a broad spectrum of activity. In conclusion, the establishment of accurate, high-throughput microbiological assays for antivirulence drug identification and early preclinical development, is a significant first step towards their translation into effective therapeutics.ImportanceThe safety net of last resort antibiotics is quickly vanishing as bacteria become increasingly resistant to most available drugs. If no action is taken, we will likely enter a post-antibiotic era, where common infections and minor injuries are once again lethal. The paucity in new antibiotic discovery of the past decades has compounded the problem of increasing antibiotic resistance, to the point that it now constitutes a global health crisis that demands global action. There is currently an urgent need for new antibacterial drugs with new targets and modes of action. To address this, research and development efforts into antivirulence drugs, such as DsbA inhibitors, have been ramping up globally. However, the development of microbiological assays as tools for effectively identifying and evaluating antivirulence drugs is lagging behind. Here, we present a high-throughput cell-based screening and evaluation pipeline, which could significantly advance development of DsbA inhibitor as antivirulence therapeutics.

This work was designed to investigate the phenolic composition, antioxidant and pancreatic lipase inhibitory activities of Rhus chinensis Mill. fruits extracted by different solvents, and to illustrate which major phenolic compounds were responsible for lipase inhibition and to evaluate their interactions. Results showed that R. chinensis Mill. fruits were rich in phenolics, which included 13 types identified and quantified by UHPLC-ESI-HRMS/MS. Among the identified phenolics, myricetin-3-O-rhamnoside and quercetin-3-O-rhamnoside were the most dominant detected in all extracts. Extracts with 80% methanol, 80% ethanol and 80% acetone exhibited strong antioxidant and pancreatic lipase inhibitory activities in vitro, and these activities were positively correlated with phenolic contents. Myricetin-3-O-rhamnoside and quercetin-3-O- rhamnoside demonstrated good lipase inhibitory activities in a dose-dependent manner and synergistically inhibited lipase. This work may provide insights into the potential uses of R. chinensis Mill. fruits in food and nutraceutical industries.

Capsules are a critical virulence factor in many pathogenic Escherichia coli, of which groups 2 and 3 capsules are synthesised by the ABC transporter pathway. The well-studied forms are in group 2 and much of our knowledge of group 3 is inferred from our understanding of group 2. We analyse six group 3 gene clusters including representatives of K10, K11 and K96, and find unexpected diversity. Groups 2 and 3 both have gene clusters with terminal regions 1 and 3 containing mostly genes shared by all members of both groups, plus a central region 2, that in group 2 has the genes for synthesising the serotype-specific repeat unit. We find that in all but one case group 3 gene clusters include, in addition to serotype-specific genes, a previously unrecognised set of shared genes in region 2 that probably codes for an additional structural element. Also, the six shared genes in regions 1 and 3 of group 3 exist in two very different sequence forms. It appears that the E. coli ABC transporter capsules have a very long history, with more fundamental diversity present in group 3, but greater diversity in the exposed strongly antigenic serotype-specific component encoded by region 2.

The Wzx/Wzy-dependent pathway is the predominant pathway for O-antigen production in Gram-negative bacteria. The O-antigen repeat unit (O unit) is an oligosaccharide that is assembled at the cytoplasmic face of the membrane on undecaprenyl pyrophosphate. Wzx then flips it to the periplasmic face for polymerization by Wzy, which adds an O unit to the reducing end of a growing O-unit polymer in each round of polymerization. Wzx and Wzy both exhibit enormous sequence diversity. It has recently been shown that, contrary to earlier reports, the efficiency of diverse Wzx forms can be significantly reduced by minor structural variations to their native O-unit substrate. However, details of Wzy substrate specificity remain unexplored. The closely related galactose-initiated Salmonella O antigens present a rare opportunity to address these matters. The D1 and D2 O units differ only in an internal mannose-rhamnose linkage, and D3 expresses both in the same chain. D1 and D2 polymerases were shown to be specific for O units with their respective α or β configuration for the internal mannose-rhamnose linkage. The Wzy encoded by D3 gene cluster polymerizes only D1 O units, and deleting the gene does not eliminate polymeric O antigen, both observations indicating the presence of an additional wzy gene. The levels of Wzx and Wzy substrate specificity will affect the ease with which new O units can evolve, and also our ability to modify O antigens, capsules or secreted polysaccharides by glyco-engineering, to generate novel polysaccharides, as the Wzx/Wzy-dependent pathway is responsible for much of the diversity.

Lipoic acid is synthesized by a remarkably atypical pathway in which the cofactor is assembled on its cognate proteins. An octanoyl moiety diverted from fatty acid synthesis is covalently attached to the acceptor protein, and sulfur insertion at carbons 6 and 8 of the octanoyl moiety form the lipoyl cofactor. Covalent attachment of this cofactor is required for function of several central metabolism enzymes, including the glycine cleavage H protein (GcvH). In Bacillus subtilis, GcvH is the sole substrate for lipoate assembly. Hence lipoic acid-requiring 2-oxoacid dehydrogenase (OADH) proteins acquire the cofactor only by transfer from lipoylated GcvH. Lipoyl transfer has been argued to be the primordial pathway of OADH lipoylation. The Escherichia coli pathway where lipoate is directly assembled on both its GcvH and OADH proteins, is proposed to have arisen later. Because roughly 3 billion years separate the divergence of these bacteria, it is surprising that E. coli GcvH functionally substitutes for the B. subtilis protein in lipoyl transfer. Known and putative GcvHs from other bacteria and eukaryotes also substitute for B. subtilis GcvH in OADH modification. Because glycine cleavage is the primary GcvH role in ancestral bacteria that lack OADH enzymes, lipoyl transfer is a “moonlighting” function: that is, development of a new function while retaining the original function. This moonlighting has been conserved in the absence of selection by some, but not all, GcvH proteins. Moreover, Aquifex aeolicus encodes five putative GcvHs, two of which have the moonlighting function, whereas others function only in glycine cleavage.

Translocation of lipid-linked oligosaccharides is a common theme across prokaryotes and eukaryotes. For bacteria, such activity is used in cell wall construction, polysaccharide synthesis, and the relatively recently discovered protein glycosylation. To the best of our knowledge, the Gram-negative inner membrane flippase Wzx was the first protein identified as being involved in oligosaccharide translocation, and yet we still have only a limited understanding of this protein after 3 decades of research. At present, Wzx is known to be a multitransmembrane protein with enormous sequence diversity that flips oligosaccharide substrates with varied degrees of preference. In this review, we provide an overview of the major findings for this protein, with a particular focus on substrate preference.

The most common system for synthesis of cell surface polysaccharides is the Wzx/Wzy-dependent pathway, which involves synthesis, on the cytoplasmic face of the cell membrane, of repeat units, which are then translocated to the periplasmic face by a Wzx translocase and then polymerized by Wzy to generate the polysaccharide. One such polysaccharide is O antigen, which is incorporated into lipopolysaccharide (LPS). The O antigen is extremely variable, with over 186 forms in Escherichia coli . Wzx proteins are also very diverse, but they have been thought to be specific only for the first sugar of the repeat units. However, recent studies demonstrated examples in which Wzx translocases have considerable preference for their native repeat unit, showing that specificity can extend well beyond the first sugar. These results appear to be in conflict with the early conclusions, but they involved specificity for side branch residues and could be a special case. Here we take six Wzx translocases that were critical in the earlier studies on the importance of the first sugar and assess their ability to translocate the Escherichia coli O16 and O111 repeat units. We use gene replacements to optimize maintenance of expression level and show that under these conditions the native translocases are the most effective for their native repeat unit, being, respectively, 64-fold and 4-fold more effective than the next best. We conclude that Wzx translocases are commonly adapted to their native repeat unit, which provides an explanation for the great diversity of wzx genes.

Most Gram-negative bacteria have an O antigen, a polysaccharide with many repeats of a short oligosaccharide that is a part of the lipopolysaccharide, the major lipid in the outer leaflet of the outer membrane. Lipopolysaccharide is variable with 46 forms in Salmonella enterica that underpin the serotyping scheme. Repeat units are assembled on a lipid carrier that is embedded in the cell membrane, and are then translocated by the Wzx translocase from the cytoplasmic face to the outer face of the cell membrane, followed by polymerization. The O antigen is then incorporated into lipopolysaccharide and exported to the outer membrane. The Wzx translocase is widely thought to be specific only for the first sugar of the repeat unit, despite extensive variation in both O antigens and Wzx translocases. However, we found for S. enterica groups B, D2 and E that Wzx translocation exhibits significant specificity for the repeat-unit structure, as variants with single sugar differences are translocated with lower efficiency and little long-chain O antigen is produced. It appears that Wzx translocases are specific for their O antigen for normal levels of translocation.

The Wzx/Wzy O-antigen pathway involves synthesis of a repeat unit (O unit) consisting of 3 to 8 sugars on an inner-membrane-embedded lipid carrier. These O units are translocated across the membrane to its periplasmic face by Wzx, while retaining linkage to the carrier, and then polymerized by Wzy to O-antigen polymer, which WaaL ligase transfers to a lipopolysaccharide precursor to complete lipopolysaccharide synthesis, concomitantly releasing the lipid carrier. This lipid carrier is also used for peptidoglycan assembly, and sequestration is known to be toxic. Thus, O-unit synthesis must involve precise regulation to meet demand but avoid overproduction. Here we show that loss of WaaL reverses a known growth defect in a Salmonella mutant that otherwise accumulates O-unit intermediates and propose that WaaL is also involved in a novel feedback mechanism to regulate O-unit synthesis, based on the availability of O units on the periplasmic face of the membrane.

Bacterial Wzx flippases translocate (flip) short oligosaccharide repeat units (O units) across the inner membrane into the periplasm, which is a critical step in the assembly of many O antigens, capsules and other surface polysaccharides. There is enormous diversity in O antigens and capsules in particular, even within species. Wzx proteins are similarly diverse, but it has been widely accepted that they have significant specificity only for the first sugar of an O unit. In this study, we analysed the Wzx from the Salmonella enterica group C2 O antigen gene cluster, which is a unique and divergent member of a set of gene clusters that produce galactose-initiated O antigens. We demonstrate that this Wzx has a strong preference for the presence of an abequose side-branch, which manifests in a reduction of long-chain O antigen and a major growth defect. This contributes to a growing body of evidence that, contrary to earlier proposals, Wzx flippases commonly exhibit a strong preference for the structure of their native O unit.

O antigens are polysaccharides consisting of repeat units of three to eight sugars, generally assembled by genes in a discrete O antigen gene cluster. Salmonella enterica produces 46 forms of O antigen, and most of the variation is determined by genes in the gene cluster. However in some cases the structures are modified by enzymes encoded outside of the gene cluster, and several such modifications have been reported for Salmonella enterica group E, some with the genes on bacteriophages and one gene at a distant chromosomal site. We identified the enzyme, WbaK, that is responsible for O-acetylating the subgroup E1 O antigen, and found that the gene is located just downstream of the gene cluster as currently known. The wbaK gene appears to have been imported by a recombination event that also replaced the last 37 bp of the wbaP gene, indicating that homologous recombination was involved. Some of the group E strains we studied must have the original gene cluster, as they lack wbaK and the sequence downstream of wbaP is very similar to that in several other S. enterica O antigen gene clusters. In effect the gene cluster was extended by one gene in subgroup E1. It appears that a function that is usually encoded by a gene outside of the gene cluster has been added to the gene cluster, in this case giving an example of how such gene clusters can evolve.

Most Gram-negative bacteria have an O antigen, a polysaccharide with many repeats of a short oligosaccharide that is a part of the lipopolysaccharide, the major lipid in the outer leaflet of the outer membrane. Lipopolysaccharide is variable with 46 forms in Salmonella enterica that underpin the serotyping scheme. Repeat units are assembled on a lipid carrier that is embedded in the cell membrane, and are then translocated by the Wzx translocase from the cytoplasmic face to the outer face of the cell membrane, followed by polymerization. The O antigen is then incorporated into lipopolysaccharide and exported to the outer membrane. The Wzx translocase is widely thought to be specific only for the first sugar of the repeat unit, despite extensive variation in both O antigens and Wzx translocases. However, we found for S. enterica groups B, D2 and E that Wzx translocation exhibits significant specificity for the repeat-unit structure, as variants with single sugar differences are translocated with lower efficiency and little long-chain O antigen is produced. It appears that Wzx translocases are specific for their O antigen for normal levels of translocation.

Inhibition of the DiSulfide Bond (DSB) oxidative protein folding machinery, a major facilitator of virulence in Gram-negative bacteria, represents a promising antivirulence strategy. We previously developed small molecule inhibitors of DsbA from Escherichia coli K-12 (EcDsbA) and showed that they attenuate virulence of Gram-negative pathogens by directly inhibiting multiple diverse DsbA homologues. Here we tested the evolutionary robustness of DsbA inhibitors as antivirulence antimicrobials against Salmonella enterica serovar Typhimurium under pathophysiological conditions in vitro. We show that phenylthiophene DsbA inhibitors slow S. Typhimurium growth in minimal media, phenocopying S. Typhimurium isogenic dsbA null mutants. Through passaging experiments, we found that DsbA inhibitor resistance was not induced under conditions that rapidly induced resistance to ciprofloxacin, an antibiotic commonly used to treat Salmonella infections. Furthermore, no mutations were identified in the dsbA gene of inhibitor-treated S. Typhimurium, and S. Typhimurium virulence remained susceptible to DsbA inhibitors. Our work demonstrates that under in vitro pathophysiological conditions, DsbA inhibitors can have both antivirulence and antibiotic action. Importantly, our finding that DsbA inhibitors appear to be evolutionarily robust offers promise for their further development as next-generation antimicrobials against Gram-negative pathogens.