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Cell Host & Microbe Oct 2021The pore-forming protein gasdermin D (GSDMD) executes lytic cell death called pyroptosis to eliminate the replicative niche of intracellular pathogens. Evolution favors...
The pore-forming protein gasdermin D (GSDMD) executes lytic cell death called pyroptosis to eliminate the replicative niche of intracellular pathogens. Evolution favors pathogens that circumvent this host defense mechanism. Here, we show that the Shigella ubiquitin ligase IpaH7.8 functions as an inhibitor of GSDMD. Shigella is an enteroinvasive bacterium that causes hemorrhagic gastroenteritis in primates, but not rodents. IpaH7.8 contributes to species specificity by ubiquitinating human, but not mouse, GSDMD and targeting it for proteasomal degradation. Accordingly, infection of human epithelial cells with IpaH7.8-deficient Shigella flexneri results in increased GSDMD-dependent cell death compared with wild type. Consistent with pyroptosis contributing to murine disease resistance, eliminating GSDMD from NLRC4-deficient mice, which are already sensitized to oral infection with Shigella flexneri, leads to further enhanced bacterial replication and increased disease severity. This work highlights a species-specific pathogen arms race focused on maintenance of host cell viability.
Topics: Animals; Bacterial Proteins; Dysentery, Bacillary; Epithelial Cells; Female; Host-Pathogen Interactions; Humans; Mice; Mice, Knockout; Phosphate-Binding Proteins; Pore Forming Cytotoxic Proteins; Proteolysis; Shigella flexneri; Ubiquitin-Protein Ligases
PubMed: 34492225
DOI: 10.1016/j.chom.2021.08.010 -
Journal of Bacteriology Nov 2021Shigella flexneri is an intracellular human pathogen that invades colonic cells and causes bloody diarrhea. S. flexneri evolved from commensal Escherichia coli, and...
Shigella flexneri is an intracellular human pathogen that invades colonic cells and causes bloody diarrhea. S. flexneri evolved from commensal Escherichia coli, and genome comparisons reveal that S. flexneri has lost approximately 20% of its genes through the process of pathoadaptation, including a disproportionate number of genes associated with the turnover of the nucleotide-based second messenger cyclic di-GMP (c-di-GMP); however, the remaining c-di-GMP turnover enzymes are highly conserved. c-di-GMP regulates many behavioral changes in other bacteria in response to changing environmental conditions, including biofilm formation, but this signaling system has not been examined in S. flexneri. In this study, we expressed VCA0956, a constitutively active c-di-GMP synthesizing diguanylate cyclase (DGC) from Vibrio cholerae, in S. flexneri to determine if virulence phenotypes were regulated by c-di-GMP. We found that expressing VCA0956 in S. flexneri increased c-di-GMP levels, and this corresponds with increased biofilm formation and reduced acid resistance, host cell invasion, and plaque size. We examined the impact of VCA0956 expression on the S. flexneri transcriptome and found that genes related to acid resistance were repressed, and this corresponded with decreased survival to acid shock. We also found that individual S. flexneri DGC mutants exhibit reduced biofilm formation and reduced host cell invasion and plaque size, as well as increased resistance to acid shock. This study highlights the importance of c-di-GMP signaling in regulating S. flexneri virulence phenotypes. The intracellular human pathogen causes dysentery, resulting in as many as one million deaths per year. Currently, there is no approved vaccine for the prevention of shigellosis, and the incidence of antimicrobial resistance among species is on the rise. Here, we explored how the widely conserved c-di-GMP bacterial signaling system alters behaviors associated with pathogenesis. We found that expressing or removing enzymes associated with c-di-GMP synthesis results in changes in 's ability to form biofilms, invade host cells, form lesions in host cell monolayers, and resist acid stress.
Topics: Aquaculture; Cyclic GMP; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Genome, Bacterial; Mutation; Phosphorus-Oxygen Lyases; Shigella flexneri; Transcriptome; Virulence
PubMed: 34543105
DOI: 10.1128/JB.00242-21 -
International Journal of Infectious... Nov 2021Shigella flexneri (S. flexneri) is prevalent worldwide and the most common Shigella in many countries, causing highly contagious diarrhea, which seriously threatens...
OBJECTIVES
Shigella flexneri (S. flexneri) is prevalent worldwide and the most common Shigella in many countries, causing highly contagious diarrhea, which seriously threatens public health. This study aimed to develop a colorimetric loop-mediated isothermal amplification (LAMP) for the rapid, accurate, and visualization detection of S. flexneri.
METHODS
According to the screened specific genes of S. flexneri, three groups of LAMP primers were designed and evaluated, and the colorimetric LAMP reaction volume was optimized. The specificity of the colorimetric LAMP was validated by 20 S. flexneri and 96 non-S. flexneri clinical isolates. In addition, the sensitivity of the developed assay was evaluated by the serial 10-fold dilutions of plasmid DNA.
RESULTS
A colorimetric LAMP assay was developed based on the specific S. flexneri hypothetical protein gene (Accession: AE014073 Region: 4170556.4171068). The colorimetric LAMP method had good specificity for detecting S. flexneri and enabled detection of S. flexneri within 30 minutes, with a plasmid detection limit of 7*10° copies/μL. The results of amplification could be easily identified by color.
CONCLUSIONS
This colorimetric LAMP assay could be used for rapid and accurate diagnosis of S. flexneri infection, especially in remote hospitals and laboratories with under-equipped medical facilities, and in situations where an urgent diagnosis is needed.
Topics: Colorimetry; Humans; Molecular Diagnostic Techniques; Nucleic Acid Amplification Techniques; Sensitivity and Specificity; Shigella flexneri
PubMed: 34517048
DOI: 10.1016/j.ijid.2021.09.015 -
Journal of Bacteriology Dec 2023Bacterial pathogens have vastly distinct sites that they inhabit during infection. This requires adaptation due to changes in nutrient availability and antimicrobial...
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. We also observed changes in bacterial and host cell fatty acid homeostasis. Specifically, intracellular increased the expression of their fatty acid degradation pathway, while the host cell lipid pool was significantly depleted. Importantly, bacterial proliferation could be inhibited by fatty acid supplementation of host cells, thereby providing novel insights into the possible link between human malnutrition and susceptibility to .
Topics: Humans; Bacterial Proteins; Shigella flexneri; Fatty Acids; Lipids
PubMed: 37991380
DOI: 10.1128/jb.00320-23 -
BMC Microbiology Jul 2021The natural hosts of Shigella are typically humans and other primates, but it has been shown that the host range of Shigella has expanded to many animals. Although...
BACKGROUND
The natural hosts of Shigella are typically humans and other primates, but it has been shown that the host range of Shigella has expanded to many animals. Although Shigella is becoming a major threat to animals, there is limited information on the genetic background of local strains. The purpose of this study was to assess the presence of virulence factors and the molecular characteristics of S. flexneri isolated from calves with diarrhea.
RESULTS
Fifty-four S. flexneri isolates from Gansun, Shanxi, Qinghai, Xinjiang and Tibet obtained during 2014 to 2016 possessed four typical biochemical characteristics of Shigella. The prevalences of ipaH, virA, ipaBCD, ial, sen, set1A, set1B and stx were 100 %, 100 %, 77.78 %, 79.63 %, 48.15 %, 48.15 and 0 %, respectively. Multilocus variable number tandem repeat analysis (MLVA) based on 8 variable number of tandem repeat (VNTR) loci discriminated the isolates into 39 different MLVA types (MTs), pulsed field gel electrophoresis (PFGE) based on NotI digestion divided the 54 isolates into 31 PFGE types (PTs), and multilocus sequence typing (MLST) based on 15 housekeeping genes differentiated the isolates into 7 MLST sequence types (STs).
CONCLUSIONS
The findings from this study enrich our knowledge of the molecular characteristics of S. flexneri collected from calves with diarrhea, which will be important for addressing clinical and epidemiological issues regarding shigellosis.
Topics: Animals; Cattle; Diarrhea; Dysentery, Bacillary; Electrophoresis, Gel, Pulsed-Field; Minisatellite Repeats; Shigella flexneri; Virulence Factors
PubMed: 34271864
DOI: 10.1186/s12866-021-02277-0 -
Infection and Immunity Oct 2021infection remains a public health problem in much of the world. Classic models of pathogenesis suggest that microfold epithelial cells in the small intestine are the...
infection remains a public health problem in much of the world. Classic models of pathogenesis suggest that microfold epithelial cells in the small intestine are the preferred initial site of invasion. However, recent evidence supports an alternative model in which primarily infects a much wider range of epithelial cells that reside primarily in the colon. Here, we investigated whether the luminal pH difference between the small intestine and the colon could provide evidence in support of either model of Shigella flexneri pathogenesis. Because virulence factors culminating in cellular invasion are linked to biofilms in S. flexneri, we examined the effect of pH on the ability of S. flexneri to form and maintain adherent biofilms induced by deoxycholate. We showed that a basic pH (as expected in the small intestine) inhibited formation of biofilms and dispersed preassembled mature biofilms, while an acidic pH (similar to the colonic environment) did not permit either of these effects. To further elucidate this phenomenon at the molecular level, we probed the transcriptomes of biofilms and S. flexneri grown under different pH conditions. We identified specific amino acid (cysteine and arginine) metabolic pathways that were enriched in the bacteria that formed the biofilms but decreased when the pH increased. We then utilized a type III secretion system reporter strain to show that increasing pH reduced deoxycholate-induced virulence of S. flexneri in a dose-dependent manner. Taken together, these experiments support a model in which infection is favored in the colon because of the local pH differences in these organs.
Topics: Base Sequence; Biofilms; Deoxycholic Acid; Gastrointestinal Tract; Hydrogen-Ion Concentration; Shigella flexneri; Transcriptome; Virulence
PubMed: 34424745
DOI: 10.1128/IAI.00387-21 -
Journal of Bacteriology Sep 2022Shigella flexneri implements the Wzy-dependent pathway to biosynthesize the O antigen (Oag) component of its surface lipopolysaccharide. The inner membrane polymerase...
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. Here, we report instead a major investigation into the specific binding interactions of Wzy with its copolymerase counterparts. For the first time, a region of Wzy that forms a unique binding site for Wzz has been identified. Specifically, this work has elucidated key Wzy moieties at the N- and C-terminal domains (NTD and CTD) that form an intramolecular pocket modulating the Wzz interaction. Novel copurification data highlight that disruption of residues within this NTD-CTD pocket impairs the interaction with Wzz without affecting Wzz binding, thereby specifically disrupting polymerization of longer polysaccharide chains. This study provides a novel understanding of the molecular interaction of Wzy with Wzz/Wzz in the Wzy-dependent pathway and, furthermore, detects the Wzy/Wzz/Und-PP-Oag complex for the first time. Beyond S. flexneri, this work may be extended to provide insight into the interactions between protein homologues expressed by related species, especially members of , that produce dual Oag chain length determinants. Shigella flexneri is a pathogen causing significant morbidity and mortality, predominantly devastating the pediatric age group in developing countries. A major virulence factor contributing to S. flexneri pathogenesis is its surface lipopolysaccharide, which is comprised of three domains: lipid A, core oligosaccharide, and O antigen (Oag). The Wzy-dependent pathway is the most common biosynthetic mechanism implemented for Oag biosynthesis by Gram-negative bacteria, including S. flexneri. The nature of the interaction between the polymerase, Wzy, and the polysaccharide copolymerases, Wzz and Wzz, in Oag polymerization is poorly characterized. This study investigates the molecular interplay between Wzy and its copolymerases, deciphering key interactions in the Wzy-dependent pathway that may be extended beyond S. flexneri, providing insight into Oag biosynthesis in Gram-negative bacteria.
Topics: Bacterial Proteins; Child; Diphosphates; Humans; Lipid A; Lipopolysaccharides; O Antigens; Shigella flexneri; Virulence Factors
PubMed: 35980183
DOI: 10.1128/jb.00224-22 -
FEMS Microbiology Reviews Feb 2004Shigella flexneri is a gram-negative bacterium which causes the most communicable of bacterial dysenteries, shigellosis. Shigellosis causes 1.1 million deaths and over... (Review)
Review
Shigella flexneri is a gram-negative bacterium which causes the most communicable of bacterial dysenteries, shigellosis. Shigellosis causes 1.1 million deaths and over 164 million cases each year, with the majority of cases occurring in the children of developing nations. The pathogenesis of S. flexneri is based on the bacteria's ability to invade and replicate within the colonic epithelium, which results in severe inflammation and epithelial destruction. The molecular mechanisms used by S. flexneri to cross the epithelial barrier, evade the host's immune response and enter epithelial cells have been studied extensively in both in vitro and in vivo models. Consequently, numerous virulence factors essential to bacterial invasion, intercellular spread and the induction of inflammation have been identified in S. flexneri. The inflammation produced by the host has been implicated in both the destruction of the colonic epithelium and in controlling and containing the Shigella infection. The host's humoral response to S. flexneri also appears to be important in protecting the host, whilst the role of the cellular immune response remains unclear. The host's immune response to shigellosis is serotype-specific and protective against reinfection by the same serotype, making vaccination a possibility. Since the 1940s vaccines for S. flexneri have been developed with little success, however, the growing understanding of S. flexneri's pathogenesis and the host's immune response is assisting in the generation of more refined vaccine strategies. Current research encompasses a variety of vaccine types, which despite disparity in their efficacy and safety in humans represent promising progress in S. flexneri vaccine development.
Topics: Bacterial Vaccines; Dysentery, Bacillary; Humans; Shigella flexneri; Virulence
PubMed: 14975529
DOI: 10.1016/j.femsre.2003.07.002 -
Frontiers in Cellular and Infection... 2020Regulation of the environmental stress response and virulence of may involve multiple signaling pathways; however, these mechanisms are not well-defined. In bacteria,...
Regulation of the environmental stress response and virulence of may involve multiple signaling pathways; however, these mechanisms are not well-defined. In bacteria, small regulatory RNAs (sRNAs) regulate bacterial growth, metabolism, virulence, and environmental stress response. Therefore, identifying novel functional sRNAs in could help elucidate pathogenic adaptations to host micro-environmental stresses and associated virulence. The aim of this study was to confirm the presence of an sRNA, , in and to determine its functions and possible mechanism of action. was found to regulate tolerance and virulence under hyperosmotic pressure. Its expression was verified by qRT-PCR and Northern blotting, and its genomic position was confirmed by 5'-rapid amplification of cDNA ends. expression was significantly decreased (~ 80%) under hyperosmotic conditions (680 mM NaCl), and the survival rate of the deletion strain increased by 20% under these conditions. This suggested that has been selected to promote host survival under hyperosmotic conditions. Additionally, virulence assessment, including guinea pig Sereny test and competitive invasion assays in mouse lungs, revealed that deletion significantly decreased virulence. Two-dimensional gel analyses suggest that may modulate the expression of , and genes, which may affect the virulence and survival of under osmotic pressures. Furthermore, expression has been shown to improve the survival of under osmotic pressures. These results suggest that has a broad range of action in response to hyperosmotic environmental stresses and in controlling its virulence to adapt to environmental stresses encountered during host infection.
Topics: Animals; Bacterial Proteins; Gene Expression Regulation, Bacterial; Guinea Pigs; Mice; Shigella flexneri; Stress, Physiological; Virulence
PubMed: 33042862
DOI: 10.3389/fcimb.2020.00483 -
Genes Sep 2020Bacillary dysentery caused by is a major cause of under-five mortality in developing countries, where a novel serotype 1c has become very common since the 1980s....
Bacillary dysentery caused by is a major cause of under-five mortality in developing countries, where a novel serotype 1c has become very common since the 1980s. However, the origin and diversification of serotype 1c remain poorly understood. To understand the evolution of serotype 1c and their antimicrobial resistance, we sequenced and analyzed the whole-genome of 85 clinical isolates from the United Kingdom, Egypt, Bangladesh, Vietnam, and Japan belonging to serotype 1c and related serotypes of 1a, 1b and Y/Yv. We identified up to three distinct O-antigen modifying genes in 1c strains, which were acquired from three different bacteriophages. Our analysis shows that 1c strains have originated from serotype 1a and serotype 1b strains after the acquisition of bacteriophage-encoding operon. The maximum-likelihood phylogenetic analysis using core genes suggests two distinct 1c lineages, one specific to Bangladesh, which originated from ancestral serotype 1a strains and the other from the United Kingdom, Egypt, and Vietnam originated from ancestral serotype 1b strains. We also identified 63 isolates containing multiple drug-resistant genes in them conferring resistance against streptomycin, sulfonamide, quinolone, trimethoprim, tetracycline, chloramphenicol, and beta-lactamase. Furthermore, antibiotic susceptibility assays showed 83 (97.6%) isolates as either complete or intermediate resistance to the WHO-recommended first- and second-line drugs. This changing drug resistance pattern demonstrates the urgent need for drug resistance surveillance and renewed treatment guidelines.
Topics: Anti-Bacterial Agents; Australia; Bacteriophages; Drug Resistance, Microbial; Dysentery, Bacillary; Humans; O Antigens; Phylogeny; Serogroup; Shigella flexneri; Viral Proteins; Virus Integration
PubMed: 32899396
DOI: 10.3390/genes11091042