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ABSTRACT Bacterial capsules provide protection against environmental challenges and host immunity. Historically, Escherichia coli K serotyping scheme, which relies on the hypervariable capsules, has identified around 80 K forms that fall into four distinct groups. Based on recent work by us and others, we predicted that E. coli capsular diversity is grossly underestimated. We exploited group 3 capsule gene clusters, the best genetically defined capsule group in E. coli, to analyze publicly available E. coli sequences for overlooked capsular diversity within the species. We report the discovery of seven novel group 3 clusters that fall into two distinct subgroups (3A and 3B). The majority of the 3B capsule clusters were found on plasmids, contrary to the defining feature of group 3 capsule genes localizing at the serA locus on the E. coli chromosome. Other new group 3 capsule clusters were derived from ancestral sequences through recombination events between shared genes found within the serotype variable central region 2. Intriguingly, flanking regions 1 and 3, known to be conserved areas among capsule clusters, showed considerable intra-subgroup variation in clusters from the 3B subgroup, containing genes of shared ancestry with other Enterobacteriaceae species. Variation of group 3 kps clusters within dominant E. coli lineages, including multidrug-resistant pathogenic lineages, further supports that E. coli capsules are undergoing rigorous change. Given the pivotal role of capsular polysaccharides in phage predation, our findings raise attention to the need of monitoring kps evolutionary dynamics in pathogenic E. coli in supporting phage therapy. IMPORTANCE Capsular polysaccharides protect pathogenic bacteria against environmental challenges, host immunity, and phage predations. The historical Escherichia coli K typing scheme, which relies on the hypervariable capsular polysaccharide, has identified around 80 different K forms that fall into four distinct groups. Taking advantage of the supposedly compact and genetically well-defined group 3 gene clusters, we analyzed published E. coli sequences to identify seven new gene clusters and revealed an unexpected capsular diversity. Genetic analysis revealed that group 3 gene clusters shared closely related serotype-specific region 2 and were diversified through recombination events and plasmid transfer between multiple Enterobacteriaceae species. Overall, capsular polysaccharides in E. coli are undergoing rigorous change. Given the pivotal role capsules play in phage interactions, this work highlighted the need to monitor the evolutionary dynamics of capsules in pathogenic E. coli for effective phage therapy.

ABSTRACT Shigella IcsA is a versatile surface virulence factor required for early and late pathogenesis stages extracellularly and 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 the host stimuli by Shigella Type III secretion system (T3SS). Here, we reported that the single cysteine residue (C130) near the N terminus of the IcsA passenger had a role in IcsA adhesin activity. We also showed that the IcsA passenger (IcsAp) existed in multiple conformations, and the conformation populations were influenced by a central pair of cysteine residues (C375 and C379), which was not previously reported for any Type V autotransporter passengers. Disruption of either or both central cysteine residues altered 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 an ΔipaD background with a constitutively active T3SS, highlighting an interplay between T3SS and T5SS. The work here uncovered a novel molecular switch empowered by a centrally localized, short-spaced cysteine pair in the Type V autotransporter IcsA that ensured conformational heterogeneity to aid IcsA evasion of host immunity. IMPORTANCE Shigella 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 multifunctional autotransporter that is responsible for cell adhesion, and actin-based motility, yet detailed mechanistic understanding is lacking. Here, we showed that the three cysteine residues in IcsA contributed to the protein’s distinct functions. The N-terminal cysteine residue within the IcsA passenger domain played a role in adhesin function, while a centrally localized cysteine pair provided conformational heterogeneity that resulted in IcsA molecules with different reactivity to adhesion-blocking anti-IcsA antibodies. In synergy with the Type III secretion system, this molecular switch preserved biological function in distinct IcsA conformations for cell adhesion, actin-based motility, and autophagy escape, providing a potential strategy by which Shigella evades host immunity and targets this essential virulence factor.

ABSTRACT 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, 9 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 II fatty acid synthesis (FAS II) pathway, showing that disrupting FAS II homeostasis in Escherichia coli through deletion of the fabH gene damages the cell envelope of antibiotic-susceptible and antibiotic-resistant clinical isolates. The fabH gene encodes the β-ketoacyl acyl carrier protein synthase III (KAS III), which catalyzes the initial condensation reactions during fatty acid biosynthesis. We show that fabH null mutation potentiated the killing of multidrug-resistant E. coli by a broad panel of previously ineffective antibiotics, despite the presence of relevant antibiotic resistance determinants, for example, carbapenemase kpc2. Enhanced antibiotic sensitivity was additionally demonstrated in the context of eradicating established biofilms and treating established human cell infection in 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. IMPORTANCE Gram-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 toward 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. Here, 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 resensitized to killing by last-resort antibiotics. This study supports FabH as a promising target for inhibition in future antimicrobial therapies.

ABSTRACT 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.

The O antigen (OAg) polysaccharide is one of the most diverse surface molecules of Gram-negative bacterial pathogens. The structural classification of OAg, based on serological typing and sequence analysis, is important in epidemiology and the surveillance of outbreaks of bacterial infections. Despite the diverse chemical structures of OAg repeating units (RUs), the genetic basis of RU assembly remains poorly understood and represents a major limitation in assigning gene functions in polysaccharide biosynthesis. Here, we describe a genetic approach to interrogate the functional order of glycosyltransferases (GTs). Using Shigella flexneri as a model, we established an initial glycosyltransferase (IT)-controlled system, which allows functional order allocation of the subsequent GT in a 2-fold manner as follows: (i) first, by reporting the growth defects caused by the sequestration of UndP through disruption of late GTs and (ii) second, by comparing the molecular sizes of stalled OAg intermediates when each putative GT is disrupted. Using this approach, we demonstrate that for RfbF and RfbG, the GT involved in the assembly of S. flexneri backbone OAg RU, RfbG, is responsible for both the committed step of OAg synthesis and the third transferase for the second L-Rha. We also show that RfbF functions as the last GT to complete the S. flexneri OAg RU backbone. We propose that this simple and effective genetic approach can be also extended to define the functional order of enzymatic synthesis of other diverse polysaccharides produced both by Gram-negative and Gram-positive bacteria. [ABSTRACT FROM AUTHOR]

The most common system for synthesis of cell surface polysaccharides is the Wzx/Wzy-dependent pathway, which involves syn-thesis, 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, re-cent studies demonstrated examples in which Wzx translocases have considerable preference for their native repeat unit, show-ing 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 016 and Olli 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 explana-tion for the great diversity of wzx genes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]