remodeling and consumption of host lipids during infection.

Bacterial pathogens have vastly distinct sites that they inhabit during infection. This requires adaptation due to changes in nutrient availability and antimicrobial stress. The bacterial surface is a primary barrier, and here, we show that the bacterial pathogen increases its surface decorations when it transitions to an intracellular lifestyle.

O antigen biogenesis sensitises Escherichia coli K-12 to bile salts, providing a plausible explanation for its evolutionary loss.

Escherichia coli K-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 coli K-12 strains are phenotypically devoid of an O antigen (OAg) since early reports in the scientific literature. Recent studies have reported the presence of independent mutations that abolish OAg repeating-unit (RU) biogenesis in E.

Adapts to Niche-Specific Stresses through Modifications in Cell Envelope Composition and Decoration.

is the primary causative agent of worldwide shigellosis. As the pathogen transverses the distinct niches of the gastrointestinal tract it necessitates dynamic adaptation strategies to mitigate host antimicrobials such as dietary fatty acids (FAs) and the bile salt, deoxycholate (DOC). This study investigates the dynamics of the cell envelope, by interrogating adaptations following FA or DOC exposure.

Cysteine-Dependent Conformational Heterogeneity of Shigella flexneri Autotransporter IcsA and Implications of Its Function.

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 Type III secretion system (T3SS).

Subcellular localization of the enterobacterial common antigen GT-E-like glycosyltransferase, WecG.

Enterobacterales have developed a specialized outer membrane polysaccharide (enterobacterial common antigen [ECA]). ECA biosynthesis begins on the cytoplasmic side of the inner membrane (IM) where glycosyltransferases sequentially add sugar moieties to form a complete repeat unit which is then translocated across the IM by WzxE before being polymerized into short linear chains by WzyE/WzzE. Research into WecG, the enzyme responsible for generating ECA lipid-II, has not progressed beyond Barr et al.

Identification of the Shigella flexneri Wzy Domain Modulating Wzz Interaction and Detection of the Wzy/Wzz/Oag Complex.

Shigella flexneri implements the Wzy-dependent pathway to biosynthesize the O antigen (Oag) component of its surface lipopolysaccharide. The inner membrane polymerase Wzy catalyzes the repeat addition of undecaprenol-diphosphate-linked Oag (Und-PP-RUs) to produce a polysaccharide, the length of which is tightly regulated by two competing copolymerase proteins, Wzz (short-type Oag; 10 to 17 RUs) and Wzz (very-long-type Oag; >90 RUs). The nature of the interaction between Wzy and Wzz/Wzz in Oag polymerization remains poorly characterized, with the majority of the literature characterizing the individual protein constituents of the Wzy-dependent pathway.

A method for increasing electroporation competence of Gram-negative clinical isolates by polymyxin B nonapeptide.

The study of clinically relevant bacterial pathogens relies on molecular and genetic approaches. However, the generally low transformation frequency among natural isolates poses technical hurdles to widely applying common methods in molecular biology, including transformation of large constructs, chromosomal genetic manipulation, and dense mutant library construction. Here we demonstrate that culturing clinical isolates in the presence of polymyxin B nonapeptide (PMBN) improves their transformation frequency via electroporation by up to 100-fold in a dose-dependent and reversible manner.

Topology of the Enterobacterial Common Antigen polymerase WzyE.

Enterobacteriales have evolved a specialized outer membrane polysaccharide [Enterobacterial Common Antigen (ECA)] which allows them to persist in various environmental niches. Biosynthesis of ECA initiates on the cytoplasmic leaflet of the inner membrane (IM) where glycosyltransferases assemble ECA repeat units (RUs). Complete RUs are then translocated across the IM and assembled into polymers by ECA-specific homologues of the Wzy-dependent pathway.

Interdependence of Shigella flexneri O Antigen and Enterobacterial Common Antigen Biosynthetic Pathways.

Outer membrane (OM) polysaccharides allow bacteria to resist harsh environmental conditions and antimicrobial agents, traffic to and persist in pathogenic niches, and evade immune responses. Shigella flexneri has two OM polysaccharide populations, being enterobacterial common antigen (ECA) and lipopolysaccharide (LPS) O antigen (Oag); both are polymerized into chains by separate homologs of the Wzy-dependent pathway. The two polysaccharide pathways, along with peptidoglycan (PG) biosynthesis, compete for the universal biosynthetic membrane anchor, undecaprenyl phosphate (Und-P), as the finite pool of available Und-P is critical in all three cell wall biosynthetic pathways.

Bacteriophage Sf6 host range mutant that infects Shigella flexneri serotype 2a2 strains.

Shigella flexneri serotype 2a2 (II:9;10) is the most prevalent strain in causing bacillary dysentery in developing countries. Chemical modifications such as glucosylation, O-acetylation, and phosphoethanolamine modifications of lipopolysaccharide (LPS) O antigen (Oag) contribute to the emergence of various serotypes. Sf6 is a Shigella-specific bacteriophage that infects only a limited range of S.

Detection of a disulphide bond and conformational changes in Shigella flexneri Wzy, and the role of cysteine residues in polymerase activity.

Shigella flexneri utilises the Wzy-dependent pathway for the production of a plethora of complex polysaccharides, including the lipopolysaccharide O-antigen (Oag) component. The inner membrane protein Wzy polymerises Oag repeat units, whilst two co-polymerase proteins, Wzz and Wzz, together interact with Wzy to regulate production of short- (S-Oag) and very long- (VL-Oag) Oag modal lengths, respectively. The 2D arrangement of Wzy transmembrane and soluble regions has been previously deciphered, however, attaining information on the 3D structural and conformational arrangement of Wzy or any homologue, has proven difficult.

Identification of a Region in Shigella flexneri WzyB Disrupting the Interaction with Wzz.

Shigella flexneri can synthesize polysaccharide chains having complex sugars and a regulated number of repeating units. S. flexneri lipopolysaccharide O antigen (Oag) is synthesized by the Wzy-dependent pathway, which is the most common pathway used in bacteria for polysaccharide synthesis.

Large metabolic rewiring from small genomic changes between strains of .

The instability of genomes has been described, but how this instability causes phenotypic differences within the species is largely unknown and likely variable. We describe herein the genome of strain PE577, originally a clinical isolate, which exhibits several phenotypic differences compared to the model strain 2457T. Like many previously described strains of , PE577 lacks discernible, functional CRISPR and restriction-modification systems.

Polysaccharide co-polymerase WzzB/WzzE chimeras reveal transmembrane 2 region of WzzB is important for interaction with WzyB.

The ability of bacteria to synthesise complex polysaccharide chains at a controlled number of repeating units has wide implications for a range of biological activities that include: symbiosis, biofilm formation and immune system avoidance. Complex polysaccharide chains such as the O antigen (Oag) component of lipopolysaccharide and the enterobacterial common antigen (ECA) are synthesised by the most common polysaccharide synthesis pathway used in bacteria, known as the Wzy-dependent pathway. The Oag and ECA are polymerized into chains via the inner membrane proteins WzyB and WzyE, respectively, while the respective co-polymerases WzzB and WzzE modulate the number of repeat units in the chains or "the modal length" of the polysaccharide via a hypothesised interaction.

Influence of Shigella flexneri 2a O Antigen Acetylation on Its Bacteriophage Sf6 Receptor Activity and Bacterial Interaction with Human Cells.

is a major causative agent of bacillary dysentery in developing countries, where serotype 2a is the prevalent strain. To date, approximately 30 serotypes have been identified for , and the major contribution to the emergence of new serotypes is chemical modifications of the lipopolysaccharide (LPS) component O antigen (Oag). Glucosylation, O-acetylation, and phosphoethanolamine (PEtN) modifications increase the Oag diversity, providing benefits to LPS Oag acts as a primary receptor for bacteriophage Sf6, which infects only a limited range of serotypes (Y and X).

Targets Human Colonic Goblet Cells by O Antigen Binding to Sialyl-Tn and Tn Antigens via Glycan-Glycan Interactions.

targets colonic cells in humans to initiate invasive infection processes that lead to dysentery, and direct interactions between their lipopolysaccharide O antigens and blood group A related glycans are involved in the cell adherence interactions. Here, we show that treatment with Tn and sialyl-Tn glycans, monoclonal antibodies and lectins reactive to Tn/sialyl-Tn, and luteolin (a Tn antigen synthesis inhibitor) all significantly inhibited adherence and invasion of cells . Surface plasmon resonance analysis showed that lipopolysaccharide O antigen had a high affinity interaction with Tn/sialyl-Tn.

The virulence domain of Shigella IcsA contains a subregion with specific host cell adhesion function.

Shigella species cause bacillary dysentery, especially among young individuals. Shigellae target the human colon for invasion; however, the initial adhesion mechanism is poorly understood. The Shigella surface protein IcsA, in addition to its role in actin-based motility, acts as a host cell adhesin through unknown mechanism(s).

Specific blood group antibodies inhibit Shigella flexneri interaction with human cells in the absence of spinoculation.

The classical models of investigating Shigella flexneri adherence and invasion of tissue culture cells involve either bacterial centrifugation (spinoculation) or the use of AfaE adhesin to overcome the low infection rate observed in vitro. However clinically, S. flexneri clearly adheres and invades the human colon in the absence of 'spinoculation'.

In vitro characterization and identification of potential substrates of a low molecular weight protein tyrosine phosphatase in Streptococcus pneumoniae.

Streptococcus pneumoniae is a major human pathogen responsible for significant mortality and morbidity worldwide. Within the annotated genome of the pneumococcus lies a previously uncharacterized protein tyrosine phosphatase which shows homology to low molecular weight protein tyrosine phosphatases (LMWPTPs). LMWPTPs modulate many processes critical for the pathogenicity of a number of bacteria including capsular polysaccharide biosynthesis, stress response and persistence in host macrophages.

Role of Streptococcus pneumoniae OM001 operon in capsular polysaccharide production, virulence and survival in human saliva.

Streptococcus pneumoniae is the leading cause of community-acquired pneumonia in all ages worldwide, and with ever-increasing antibiotic resistance, the understanding of its pathogenesis and spread is as important as ever. Recently, we reported the presence of a Low Molecular Weight Tyrosine Phosphatase (LMWPTP) Spd1837 in the pneumococcus. This protein is encoded in an operon, OM001 with two other genes, with previous work implicating this operon as important for pneumococcal virulence.

Unprecedented Abundance of Protein Tyrosine Phosphorylation Modulates Shigella flexneri Virulence.

Evidence is accumulating that protein tyrosine phosphorylation plays a crucial role in the ability of important human bacterial pathogens to cause disease. While most works have concentrated on its role in the regulation of a major bacterial virulence factor, the polysaccharide capsule, recent studies have suggested a much broader role for this post-translational modification. This prompted us to investigate protein tyrosine phosphorylation in the human pathogen Shigella flexneri.

Conserved transmembrane glycine residues in the Shigella flexneri polysaccharide co-polymerase protein WzzB influence protein-protein interactions.

The O antigen (Oag) component of lipopolysaccharides (LPS) is crucial for virulence and Oag chain-length regulation is controlled by the polysaccharide co-polymerase class 1 (PCP1) proteins. Crystal structure analyses indicate that structural conservation among PCP1 proteins is highly maintained, however the mechanism of Oag modal-chain-length control remains to be fully elucidated. Shigella flexneri PCP1 protein WzzBSF confers a modal-chain length of 10-17 Oag repeat units (RUs), whereas the Salmonella enterica Typhimurium PCP1 protein WzzBST confers a modal-chain length of ~16-28 Oag RUs.

Glycan:glycan interactions: High affinity biomolecular interactions that can mediate binding of pathogenic bacteria to host cells.

Cells from all domains of life express glycan structures attached to lipids and proteins on their surface, called glycoconjugates. Cell-to-cell contact mediated by glycan:glycan interactions have been considered to be low-affinity interactions that precede high-affinity protein-glycan or protein-protein interactions. In several pathogenic bacteria, truncation of surface glycans, lipooligosaccharide (LOS), or lipopolysaccharide (LPS) have been reported to significantly reduce bacterial adherence to host cells.

Structural and Biochemical Analysis of a Single Amino-Acid Mutant of WzzBSF That Alters Lipopolysaccharide O-Antigen Chain Length in Shigella flexneri.

Lipopolysaccharide (LPS), a surface polymer of Gram-negative bacteria, helps bacteria survive in different environments and acts as a virulence determinant of host infection. The O-antigen (Oag) component of LPS exhibits a modal chain-length distribution that is controlled by polysaccharide co-polymerases (PCPs). The molecular basis of the regulation of Oag chain-lengths remains unclear, despite extensive mutagenesis and structural studies of PCPs from Escherichia coli and Shigella.

A small conserved motif supports polarity augmentation of Shigella flexneri IcsA.

The rod-shaped enteric intracellular pathogen Shigella flexneri and other Shigella species are the causative agents of bacillary dysentery. S. flexneri are able to spread within the epithelial lining of the gut, resulting in lesion formation, cramps and bloody stools.