scholarly journals Inactivation of the sfgtr4 Gene of Shigella flexneri Induces Biofilm Formation and Affects Bacterial Pathogenicity

2020 ◽  
Vol 8 (6) ◽  
pp. 841
Author(s):  
Abdelmoughit Kaoukab-Raji ◽  
Latéfa Biskri ◽  
Abdelmounaaïm Allaoui
Keyword(s):  
Foodborne Pathogens ◽  
Biofilm Formation ◽  
Lipid A ◽  
Shigella Flexneri ◽  
Polymorphonuclear Neutrophils ◽  
Virulence Plasmid ◽  
Environmental Persistence ◽  
Bacterial Killing ◽  
Type Iii Effectors

Biofilm formation is a significant cause for the environmental persistence of foodborne pathogens. This phenomenon remains misunderstood in Shigella flexneri whose pathogenicity is mainly associated with the virulence plasmid pWR100. Sequence analysis of the latter predicts a putative lipopolysaccharides (LPS) glycosyltransferase (Gtr) encoded by Sfgtr4, which is the second gene of the SfpgdA-orf186-virK-msbB2 locus. We demonstrated here that purified SfGtr4 exhibited a Gtr activity in vitro by transferring glucose to lipid A. To establish the role of SfGtr4 in virulence, we generated a Sfgtr4 mutant and assessed its phenotype in vitro. Sfgtr4 mutant significantly reduced HeLa cells invasion without impairing type III effectors secretion, increased susceptibility to lysozyme degradation, and enhanced bacterial killing by polymorphonuclear neutrophils (PMNs). SfGtr4 is related to proteins required in biofilm formation. We established conditions whereby wild-type Shigella formed biofilm and revealed that its appearance was accelerated by the Sfgtr4 mutant. Additional phenotypical analysis revealed that single SfpdgA and double SfpgdA-Sfgtr4 mutants behaved similarly to Sfgtr4 mutant. Furthermore, a molecular interaction between SfGtr4 and SfPgdA was identified. In summary, the dual contribution of SfGtr4 and SfPgdA to the pathogenicity and the regulation biofilm formation by S. flexneri was demonstrated here.

scholarly journals Virulent Shigella flexneri subverts the host innate immune response through manipulation of antimicrobial peptide gene expression

2008 ◽  
Vol 205 (5) ◽  
pp. 1121-1132 ◽  
Author(s):  
Brice Sperandio ◽  
Béatrice Regnault ◽  
Jianhua Guo ◽  
Zhi Zhang ◽  
Samuel L. Stanley ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Shigella Flexneri ◽  
Host Cells ◽  
Virulence Plasmid ◽  
Cationic Peptides ◽  
Immunity Genes ◽  
Genes Encoding ◽  
Peptide Gene

Antimicrobial factors are efficient defense components of the innate immunity, playing a crucial role in the intestinal homeostasis and protection against pathogens. In this study, we report that upon infection of polarized human intestinal cells in vitro, virulent Shigella flexneri suppress transcription of several genes encoding antimicrobial cationic peptides, particularly the human β-defensin hBD-3, which we show to be especially active against S. flexneri. This is an example of targeted survival strategy. We also identify the MxiE bacterial regulator, which controls a regulon encompassing a set of virulence plasmid-encoded effectors injected into host cells and regulating innate signaling, as being responsible for this dedicated regulatory process. In vivo, in a model of human intestinal xenotransplant, we confirm at the transcriptional and translational level, the presence of a dedicated MxiE-dependent system allowing S. flexneri to suppress expression of antimicrobial cationic peptides and promoting its deeper progression toward intestinal crypts. We demonstrate that this system is also able to down-regulate additional innate immunity genes, such as the chemokine CCL20 gene, leading to compromised recruitment of dendritic cells to the lamina propria of infected tissues. Thus, S. flexneri has developed a dedicated strategy to weaken the innate immunity to manage its survival and colonization ability in the intestine.

scholarly journals Development, Interlaboratory Evaluations, and Application of a Simple, High-ThroughputShigellaSerum Bactericidal Assay

mSphere ◽  
2018 ◽  
Vol 3 (3) ◽  
Author(s):  
Moon H. Nahm ◽  
Jigui Yu ◽  
Hailey P. Weerts ◽  
Heather Wenzel ◽  
Chitradevi S. Tamilselvi ◽  
...  
Keyword(s):  
Vaccine Development ◽  
Shigella Flexneri ◽  
Binding Activity ◽  
Serum Samples ◽  
Bacterial Killing ◽  
Content Type ◽  
Bactericidal Assay ◽  
Serum Bactericidal Assay ◽  
Reference Serum

ABSTRACTShigellais an important cause of diarrhea worldwide, with serotypesShigella flexneri2a,S. flexneri3a, andShigella sonneidemonstrating epidemiological prevalence. Many development efforts are focused onShigellalipopolysaccharide (LPS)-based vaccines, as O antigen-specific conjugate vaccines are immunogenic and efficacious. Immunization withShigellavaccines containing LPS can elicit antibodies capable of killingShigellain a serotype-specific manner. Thus, to facilitateShigellavaccine development, we have developed a serum bactericidal assay (SBA) specific for threeShigellaserotypes that measures killing of target bacteria at multiple serum dilutions and in the presence of exogenous complement. The SBA has a high analytical throughput and uses simple technologies and readily available reagents. The SBA was characterized with human sera with bactericidal antibodies againstS. flexneri2a,S. flexneri3a, andS. sonnei. Purified LPS of a homologous serotype, but not a heterologous serotype, inhibited bacterial killing. Assessment of precision found median intra-assay precision to be 13.3% and median interassay precision to be 19 to 30% for the three serotypes. The SBA is linear, with slight deviations for samples with low (~40) killing indices. The SBA was sensitive enough to allow about 100-fold predilution of serum samples. Repeat assays yielded results with less than 2-fold deviations, indicating the robustness of the assay. Assay results from four different laboratories were highly comparable when normalized with a reference serum. TheShigellaSBA, combined with a reference serum, should facilitate the development ofShigellavaccines across the field.IMPORTANCEShigellais an important cause of diarrhea worldwide, and efforts are ongoing to produce a safe and effectiveShigellavaccine. Although a clear immune correlate of protection has not been established, antibodies with bactericidal capacity may provide one means of protecting against shigellosis. Thus, it is important to measure the functional capacity of antibodies, as opposed to only binding activity. This article describes a simple, robust, and high-throughput serum bactericidal assay capable of measuringShigella-specific functional antibodiesin vitro. We show for the first time that this assay was successfully performed by multiple laboratories and generated highly comparable results, particularly when SBA titers were normalized using a reference standard. The serum bactericidal assay, along with a reference serum, should greatly facilitateShigellavaccine development.

scholarly journals Differential Regulation by Magnesium of the Two MsbB Paralogs of Shigella flexneri

2008 ◽  
Vol 190 (10) ◽  
pp. 3526-3537 ◽  
Author(s):  
Seth R. Goldman ◽  
Yupeng Tu ◽  
Marcia B. Goldberg
Keyword(s):  
Lipid A ◽  
Shigella Flexneri ◽  
Differential Regulation ◽  
Virulence Plasmid ◽  
Transferase Activity ◽  
Dependent Manner ◽  
Transcriptional Fusion ◽  
Environmental Signals ◽  
Stressful Environments ◽  
Insight Into

ABSTRACT Shigella flexneri, a gram-negative enteric pathogen, is unusual in that it contains two nonredundant paralogous genes that encode the myristoyl transferase MsbB (LpxM) that catalyzes the final step in the synthesis of the lipid A moiety of lipopolysaccharide. MsbB1 is encoded on the chromosome, and MsbB2 is encoded on the large virulence plasmid present in all pathogenic shigellae. We demonstrate that myristoyl transferase activity due to MsbB2 is detected in limited magnesium medium, but not in replete magnesium medium, whereas that due to MsbB1 is detected under both conditions. MsbB2 increases overall hexa-acylation of lipid A under limited magnesium conditions. Regulation of MsbB2 by magnesium occurs at the level of transcription and is dependent on the conserved magnesium-inducible PhoPQ two-component regulatory pathway. Direct hexanucleotide repeats within the promoter upstream of msbB2 were identified as a putative PhoP binding site, and mutations within the repeats led to diminished PhoP-dependent expression of a transcriptional fusion of lacZ to this promoter. Thus, the virulence plasmid-encoded paralog of msbB is induced under limited magnesium in a PhoPQ-dependent manner. PhoPQ regulates the response of many Enterobacteriaceae to environmental signals, which include modifications of lipid A that confer increased resistance of the organism to stressful environments and antimicrobial peptides. The findings reported here are the first example of gene duplication in which one paralog has selectively acquired the mechanism for differential regulation by PhoPQ. Our findings provide molecular insight into the mechanisms by which each of the two MsbB proteins of S. flexneri likely contributes to pathogenesis.

scholarly journals Cross-Sectional Analysis of Clinical and Environmental Isolates of Pseudomonas aeruginosa: Biofilm Formation, Virulence, and Genome Diversity

2004 ◽  
Vol 72 (1) ◽  
pp. 133-144 ◽  
Author(s):  
Nathan E. Head ◽  
Hongwei Yu
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Capsular Polysaccharide ◽  
Subtractive Hybridization ◽  
Genomic Diversity ◽  
Virulence Plasmid ◽  
Reactive Oxygen Intermediate ◽  
Cross Sectional ◽  
Growth Modes

ABSTRACT Chronic lung infections with Pseudomonas aeruginosa biofilms are associated with refractory and fatal pneumonia in cystic fibrosis (CF). In this study, a group of genomically diverse P. aeruginosa isolates were compared with the reference strain PAO1 to assess the roles of motility, twitching, growth rate, and overproduction of a capsular polysaccharide (alginate) in biofilm formation. In an in vitro biofilm assay system, P. aeruginosa displayed strain-specific biofilm formation that was not solely dependent on these parameters. Compared with non-CF isolates, CF isolates expressed two opposing growth modes: reduced planktonic growth versus efficient biofilm formation. Planktonic cells of CF isolates showed elevated sensitivity to hydrogen peroxide, a reactive oxygen intermediate, and decreased lung colonization in an aerosol infection mouse model. Despite having identical genomic profiles, CF sequential isolates produced different amounts of biofilm. While P. aeruginosa isolates exhibited genomic diversity, the genome size of these isolates was estimated to be 0.4 to 19% (27 to 1,184 kb) larger than that of PAO1. To identify these extra genetic materials, random amplification of polymorphic DNA was coupled with PAO1-subtractive hybridization. Three loci were found within the genomes of two CF isolates encoding one novel homolog involved in retaining a Shigella virulence plasmid (mvpTA) and two divergent genes that function in removing negative supercoiling (topA) and biosynthesis of pyoverdine (PA2402). Together, P. aeruginosa biodiversity could provide one cause for the variation of morbidity and mortality in CF. P. aeruginosa may possess undefined biofilm adhesins that are important to the development of an antibiofilm therapeutic target.

scholarly journals Clinically Relevant Concentrations of Polymyxin B and Meropenem Synergistically Kill Multidrug-Resistant Pseudomonas aeruginosa and Minimize Biofilm Formation

Antibiotics ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 405
Author(s):  
Hasini Wickremasinghe ◽  
Heidi H. Yu ◽  
Mohammad A. K. Azad ◽  
Jinxin Zhao ◽  
Phillip J. Bergen ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Respiratory Infections ◽  
Population Analysis ◽  
Membrane Damage ◽  
Membrane Integrity ◽  
Polymyxin B ◽  
Multidrug Resistant ◽  
Bacterial Killing

The emergence of antibiotic resistance has severely impaired the treatment of chronic respiratory infections caused by multidrug-resistant (MDR) Pseudomonas aeruginosa. Since the reintroduction of polymyxins as a last-line therapy against MDR Gram-negative bacteria, resistance to its monotherapy and recurrent infections continue to be reported and synergistic antibiotic combinations have been investigated. In this study, comprehensive in vitro microbiological evaluations including synergy panel screening, population analysis profiling, time-kill kinetics, anti-biofilm formation and membrane damage analysis studies were conducted to evaluate the combination of polymyxin B and meropenem against biofilm-producing, polymyxin-resistant MDR P. aeruginosa. Two phylogenetically unrelated MDR P. aeruginosa strains, FADDI-PA060 (MIC of polymyxin B [MICpolymyxin B], 64 mg/L; MICmeropenem, 64 mg/L) and FADDI-PA107 (MICpolymyxin B, 32 mg/L; MICmeropenem, 4 mg/L) were investigated. Genome sequencing identified 57 (FADDI-PA060) and 50 (FADDI-PA107) genes predicted to confer resistance to a variety of antimicrobials, as well as multiple virulence factors in each strain. The presence of resistance genes to a particular antibiotic class generally aligned with MIC results. For both strains, all monotherapies of polymyxin B failed with substantial regrowth and biofilm formation. The combination of polymyxin B (16 mg/L)/meropenem (16 mg/L) was most effective, enhancing initial bacterial killing of FADDI-PA060 by ~3 log10 CFU/mL, followed by a prolonged inhibition of regrowth for up to 24 h with a significant reduction in biofilm formation (* p < 0.05). Membrane integrity studies revealed a substantial increase in membrane depolarization and membrane permeability in the surviving cells. Against FADDI-PA107, planktonic and biofilm bacteria were completely eradicated. In summary, the combination of polymyxin B and meropenem demonstrated synergistic bacterial killing while reinstating the efficacy of two previously ineffective antibiotics against difficult-to-treat polymyxin-resistant MDR P. aeruginosa.

scholarly journals Requirement of the Shigella flexneri Virulence Plasmid in the Ability To Induce Trafficking of Neutrophils across Polarized Monolayers of the Intestinal Epithelium

1998 ◽  
Vol 66 (9) ◽  
pp. 4237-4243 ◽  
Author(s):  
Beth A. McCormick ◽  
Andrew M. Siber ◽  
Anthony T. Maurelli
Keyword(s):  
Intestinal Epithelium ◽  
Shigella Flexneri ◽  
Basolateral Membrane ◽  
Epithelial Cell Line ◽  
Virulence Plasmid ◽  
Intestinal Epithelial ◽  
Epithelial Monolayers ◽  
Human Pmns

ABSTRACT Attachment of an array of enteric pathogens to epithelial surfaces is accompanied by recruitment of polymorphonuclear leukocytes (PMN) across the intestinal epithelium. In this report, we examine howShigella-intestinal epithelium interactions evoke the mucosal inflammatory response. We modeled these interactions in vitro by using polarized monolayers of the human intestinal epithelial cell line, T84, isolated human PMNs, and Shigella flexneri. We show that Shigella attachment to T84-cell basolateral membranes was a necessary component in the signaling cascade for induction of basolateral-to-apical directed transepithelial PMN migration, the direction of PMN transepithelial migration in vivo. In contrast, attachment of Shigella to the T84-cell apical membrane failed to stimulate a directed PMN transepithelial migration response. Importantly, the ability of Shigella to induce PMN migration across epithelial monolayers was dependent on the presence of the 220-kb virulence plasmid. Moreover, examination ofShigella genes necessary to signal subepithelial neutrophils established the requirement of a functional type III secretion system. Our results indicate that the ability ofShigella to elicit transepithelial signaling to neutrophils from the basolateral membrane of epithelial cells represents a mechanism involved in Shigella-elicited enteritis in humans.

scholarly journals Evaluation of the Antioxidative, Antibacterial, and Anti-Inflammatory Effects of theAloeFermentation Supernatant ContainingLactobacillus plantarumHM218749.1

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Meixiu Jiang ◽  
Kan Deng ◽  
Chunling Jiang ◽  
Mingui Fu ◽  
Chunlan Guo ◽  
...  
Keyword(s):  
Foodborne Pathogens ◽  
Salmonella Enteritidis ◽  
Shigella Flexneri ◽  
Aloe Vera ◽  
Protein Levels ◽  
Cosmetic Ingredients ◽  
Potential Benefits ◽  
Inhibition Zones ◽  
Antioxidant Effects

Little work is done to developAloe vera(AV) using probiotics. To explore the potential benefits, the antioxidant effects and the antibacterial effects on foodborne pathogens ofAloefermentation supernatant were evaluated in vitro. Our results indicated that theAloefermentation supernatant fermented byLactobacillus plantarumHM218749.1 had very strong scavenging capacities of the DPPH (86%),O2•-(85%),OH•(76%), andFe2+chelation (82%) and reducing powers (242.5 mg/L), and the inhibition zones forSalmonella typhimurium,Salmonella enteritidis,Shigella flexneri,Escherichia coli,Listeria monocytogenes,S. dysenteriae301,Staphylococcus aureusCowan1, andPropionibacterium acneswere 16, 15, 19, 20, 21, 20, and 27 mm. Moreover, the low concentration ofAloefermentation supernatant had significantly reduced the production of IL-1β, TNF-α, and IL-6 in both mRNA and protein levels (P<0.01). Therefore, theAloefermentation supernatant can be used as functional beverage or cosmetic ingredients to guard human intestinal health, delaying senescence, and prevent chronic diseases.

scholarly journals Anti-Pseudomonas aeruginosa IgY Antibodies Induce Specific Bacterial Aggregation and Internalization in Human Polymorphonuclear Neutrophils

2015 ◽  
Vol 83 (7) ◽  
pp. 2686-2693 ◽  
Author(s):  
K. Thomsen ◽  
L. Christophersen ◽  
T. Bjarnsholt ◽  
P. Ø. Jensen ◽  
C. Moser ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Egg Yolk ◽  
Polymorphonuclear Neutrophils ◽  
Bacterial Killing ◽  
Content Type ◽  
Bacterial Fitness ◽  
Bacterial Hydrophobicity ◽  
Biofilm Infection

Polymorphonuclear neutrophils (PMNs) are essential cellular constituents in the innate host response, and their recruitment to the lungs and subsequent ubiquitous phagocytosis controls primary respiratory infection. Cystic fibrosis pulmonary disease is characterized by progressive pulmonary decline governed by a persistent, exaggerated inflammatory response dominated by PMNs. The principal contributor is chronicPseudomonas aeruginosabiofilm infection, which attracts and activates PMNs and thereby is responsible for the continuing inflammation. Strategies to prevent initial airway colonization withP. aeruginosaby augmenting the phagocytic competence of PMNs may postpone the deteriorating chronic biofilm infection. Anti-P. aeruginosaIgY antibodies significantly increase the PMN-mediated respiratory burst and subsequent bacterial killing ofP. aeruginosain vitro. The mode of action is attributed to IgY-facilitated formation of immobilized bacteria in aggregates, as visualized by fluorescence microscopy and the induction of increased bacterial hydrophobicity. Thus, the present study demonstrates that avian egg yolk immunoglobulins (IgY) targetingP. aeruginosamodify bacterial fitness, which enhances bacterial killing by PMN-mediated phagocytosis and thereby may facilitate a rapid bacterial clearance in airways of people with cystic fibrosis.

scholarly journals LECT2 protects mice against bacterial sepsis by activating macrophages via the CD209a receptor

2012 ◽  
Vol 210 (1) ◽  
pp. 5-13 ◽  
Author(s):  
Xin-Jiang Lu ◽  
Jiong Chen ◽  
Chao-Hui Yu ◽  
Yu-Hong Shi ◽  
Yu-Qing He ◽  
...  
Keyword(s):  
Immune Responses ◽  
Protective Immunity ◽  
Polymorphonuclear Neutrophils ◽  
Bacterial Sepsis ◽  
Bacterial Killing ◽  
Improved Outcome ◽  
Multifunctional Cytokine

Leukocyte cell–derived chemotaxin 2 (LECT2) is a multifunctional cytokine and reduced plasma levels were found in patients with sepsis. However, precise functions and mechanisms of LECT2 remain unclear. The aim of the present study was to determine the role of LECT2 in modulating immune responses using mouse sepsis models. We found that LECT2 treatment improved outcome in mice with bacterial sepsis. Macrophages (MΦ), but not polymorphonuclear neutrophils, mediated the beneficial effect of LECT2 on bacterial sepsis. LECT2 treatment could alter gene expression and enhance phagocytosis and bacterial killing of MΦ in vitro. CD209a was identified to specifically interact with LECT2 and mediate LECT2-induced MΦ activation. CD209a-expressing MΦ was further confirmed to mediate the effect of LECT2 on sepsis in vivo. Our data demonstrate that LECT2 improves protective immunity in bacterial sepsis, possibly as a result of enhanced MΦ functions via the CD209a receptor. The modulation of MΦ functions by LECT2 may serve as a novel potential treatment for sepsis.

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