Medium alkalization due to carbon metabolism is largely responsible for inhibition of bacterial growth by Vibrio cholerae supernatants

Vibrio cholerae is the causative agent of the diarrheal disease cholera. Many Vibrio species secrete antimicrobial factors, though the identity of such a factor has not been determined for any V. cholerae strain. Such an antimicrobial factor could be relevant to pathogenesis of cholera, which disrupts the intestinal microbiome. In this study, we investigated the antimicrobial effects of supernatant from 72 hour old cultures of V. cholerae C6706 on Shigella flexneri CFS100. Inhibition of S. flexneri growth was found to be dependent on the alkaline pH of the supernatant. A 1:1 mixture of pH-adjusted supernatant and LB was found to inhibit S. flexneri growth at alkaline but not neutral pH, as was pH-adjusted LB alone. In minimal medium, elevation of supernatant pH by V. cholerae was dependent on nutritional factors, and this elevation of medium pH also correlated with increased S. flexneri growth inhibition. Though medium alkalization in LB is often attributed to amino acid catabolism and the consequent production of ammonia, supplementation of V. cholerae cultures in minimal medium with amino acids had a weaker effect on alkalization and inhibition than did supplementation with selected carbon sources. This suggests that some feature of carbon metabolism causes medium alkalization and the resultant antimicrobial activity. Several V. cholerae mutants in potentially relevant pathways were screened for alkalization and S. flexneri growth inhibition, but none had any effect.Complicating this picture is the finding that V. cholerae grown under microaerobic conditions produce a less alkaline supernatant with stronger S. flexneri growth inhibition. The significance of this is unknown.Molecular Bioscience

Identification of critical residues of the serotype modifying O-acetyltransferase of Shigella flexneri

BACKGROUND Thirteen serotypes of Shigella flexneri (S. flexneri) have been recognised, all of which are capable of causing bacillary dysentery or shigellosis. With the emergence of the newer S. flexneri serotypes, the development of an effective vaccine has only become more challenging. One of the factors responsible for the generation of serotype diversity is an LPS O-antigen modifying, integral membrane protein known as O-acetyltransferase or Oac. Oac functions by adding an acetyl group to a specific O-antigen sugar, thus changing the antigenic signature of the parent S. flexneri strain. Oac is a membrane protein, consisting of hydrophobic and hydrophilic components. Oac bears homology to several known and predicted acetyltransferases with most homology existing in the N-terminal transmembrane (TM) regions. RESULTS In this study, the conserved motifs in the TM regions and in hydrophilic loops of S. flexneri Oac were targeted for mutagenesis with the aim of identifying the amino acid residues essential for the function of Oac. We previously identified three critical arginines-R73, R75 and R76 in the cytoplasmic loop 3 of Oac. Re-establishing that these arginines are critical, in this study we suggest a catalytic role for R73 and a structural role for R75 and R76 in O-acetylation. Serine-glycine motifs (SG 52-53, GS 138-139 and SYG 274-276), phenylalanine-proline motifs (FP 78-79 and FPV 282-84) and a tryptophan-threonine motif (WT141-142) found in TM segments and residues RK 110-111, GR 269-270 and D333 found in hydrophilic loops were also found to be critical to Oac function. CONCLUSIONS By studying the effect of the mutations on Oac's function and assembly, an insight into the possible roles played by the chosen amino acids in Oac was gained. The transmembrane serine-glycine motifs and hydrophilic residues (RK 110-111, GR 269-270 and D333) were shown to have an affect on Oac assembly which suggests a structural role for these motifs. The phenylalanine-proline and the tryptophan-threonine motifs affect Oac function which could suggest a catalytic role for these amino acids.This work was supported by a grant from the National Health and Medical Research Council of Australia

Reaksi antigen-antibodi antara protein sub unit pili 18 KDa Shigella flexneri dengan sub unit outer membran protein (OMP) Shigella dysentriae: Strategi memperoleh vaksin shigellosis berbasis protein pili

Bloody diarrhea disease remains a major cause of the high morbidity and mortality of children globally. Diarrhea caused by S. flexneri and S. dysentriae commonly treated with antibiotics seen already started resistant to existing antibiotics varied while the prevention of shigellosis with Shigella vaccines were developed and used today are still of limited use. This study aims to explore the hemagglutinin protein sub units pili and Omp S. dysentriae suspected having similar characteristics. This study uses Dot Blot and Western Blot to determine the antigen-antibody reaction between the antibody protein sub unit pili S. flexneri with sub unit Omp S. dysentriae. The result showed that the protein sub unit 18 kDa S. flexneri is a protein that is able to agglutinate red blood cells of mice at the highest dilution and is able to recognize sub unit Omp S. dysentriae with a molecular weight of 18 kDa; 21 kDa; and 23 kDa. Protein sub units Omp S. dysentriae with a molecular weight of 18 kDa; 21 kDa; and 23 kDa possibility having epitopes that can be recognized by an antibody protein sub unit pili S. flexneri 18 kDa

Deletion of toxin–antitoxin systems in the evolution of Shigella sonnei as a host-adapted pathogen

Pathogenic Shigella spp. are the leading cause of bacterial dysentery, with Shigella flexneri and Shigella sonnei accounting for around 90% of cases worldwide. While S. flexneri causes most disease in low-income countries (following ingestion of contaminated food and/or water), S. sonnei predominates in wealthy countries and is mainly spread from person to person. Although both species contain a large virulence plasmid, pINV, that is essential for the organism to cause disease, little is known about its maintenance. Here, using a counterselectable marker within the virulence-encoding region of pINV, we show that the S. sonnei plasmid is less stable than that of S. flexneri, especially at environmental temperatures. GmvAT, a toxin–antitoxin system, is responsible for the difference in stability and is present in pINV from S. flexneri but absent in S. sonnei pINV; GmvT is an acetyltransferase toxin that inhibits protein translation. Loss of GmvAT and a second toxin–antitoxin system, CcdAB, from pINV reduces S. sonnei plasmid stability outside the host, reflecting the host-adapted lifestyle and person-to-person transmission of this species, and hence the striking differences in its epidemiology

Species-wide whole genome sequencing reveals historical global spread and recent local persistence in Shigella flexneri.

Shigella flexneri is the most common cause of bacterial dysentery in low-income countries. Despite this, S. flexneri remains largely unexplored from a genomic standpoint and is still described using a vocabulary based on serotyping reactions developed over half-a-century ago. Here we combine whole genome sequencing with geographical and temporal data to examine the natural history of the species. Our analysis subdivides S. flexneri into seven phylogenetic groups (PGs); each containing two-or-more serotypes and characterised by distinct virulence gene complement and geographic range. Within the S. flexneri PGs we identify geographically restricted sub-lineages that appear to have persistently colonised regions for many decades to over 100 years. Although we found abundant evidence of antimicrobial resistance (AMR) determinant acquisition, our dataset shows no evidence of subsequent intercontinental spread of antimicrobial resistant strains. The pattern of colonisation and AMR gene acquisition suggest that S. flexneri has a distinct life-cycle involving local persistence

Shigella flexneri utilize the spectrin cytoskeleton during invasion and comet tail generation

Abstract Background The spectrin cytoskeleton is emerging as an important host cell target of enteric bacterial pathogens. Recent studies have identified a crucial role for spectrin and its associated proteins during key pathogenic processes of Listeria monocytogenes and Salmonella Typhimurium infections. Here we investigate the involvement of spectrin cytoskeletal components during the pathogenesis of the invasive pathogen Shigella flexneri. Results Immunofluorescent microscopy reveals that protein 4.1 (p4.1), but not adducin or spectrin, is robustly recruited to sites of S. flexneri membrane ruffling during epithelial cell invasion. Through siRNA-mediated knockdowns, we identify an important role for spectrin and the associated proteins adducin and p4.1 during S. flexneri invasion. Following internalization, all three proteins are recruited to the internalized bacteria, however upon generation of actin-rich comet tails, we observed spectrin recruitment to those structures in the absence of adducin or p4.1. Conclusion These findings highlight the importance of the spectrin cytoskeletal network during S. flexneri pathogenesis and further demonstrate that pathogenic events that were once thought to exclusively recruit the actin cytoskeletal system require additional cytoskeletal networks.</p

Mitochondria mediate septin cage assembly to promote autophagy of Shigella

Septins, cytoskeletal proteins with well-characterised roles in cytokinesis, form cage-like structures around cytosolic Shigella flexneri and promote their targeting to autophagosomes. However, the processes underlying septin cage assembly, and whether they influence S. flexneri proliferation, remain to be established. Using single-cell analysis, we show that the septin cages inhibit S. flexneri proliferation. To study mechanisms of septin cage assembly, we used proteomics and found mitochondrial proteins associate with septins in S. flexneri-infected cells. Strikingly, mitochondria associated with S. flexneri promote septin assembly into cages that entrap bacteria for autophagy. We demonstrate that the cytosolic GTPase dynamin-related protein 1 (Drp1) interacts with septins to enhance mitochondrial fission. To avoid autophagy, actin-polymerising Shigella fragment mitochondria to escape from septin caging. Our results demonstrate a role for mitochondria in anti-Shigella autophagy and uncover a fundamental link between septin assembly and mitochondria

The emergence and fate of horizontally acquired genes in Escherichia coli

Bacterial species, and even strains within species, can vary greatly in their gene contents and metabolic capabilities. We examine the evolution of this diversity by assessing the distribution and ancestry of each gene in 13 sequenced isolates of Escherichia coli and Shigella. We focus on the emergence and demise of two specific classes of genes, ORFans (genes with no homologs in present databases) and HOPs (genes with distant homologs), since these genes, in contrast to most conserved ancestral sequences, are known to be a major source of the novel features in each strain. We find that the rates of gain and loss of these genes vary greatly among strains as well as through time, and that ORFans and HOPs show very different behavior with respect to their emergence and demise. Although HOPs, which mostly represent gene acquisitions from other bacteria, originate more frequently, ORFans are much more likely to persist. This difference suggests that many adaptive traits are conferred by completely novel genes that do not originate in other bacterial genomes. With respect to the demise of these acquired genes, we find that strains of Shigella lose genes, both by disruption events and by complete removal, at accelerated rates