Calcium promotes exopolysaccharide phase variation and biofilm formation of the resulting phase variants in the human pathogen Vibrio vulnificus

Vibrio vulnificus, an opportunistic human pathogen, is found as member of the normal microflora of shellfish and other seafoods, many of which are eaten raw. Though usually not harmful, V. vulnificus is responsible for causing fulminating septicemia in immunocompromised individuals. In previous light microscopic studies, we showed data suggesting that isogenic unencapsulated phase variants were more adherent to HeLa cells than were counterpart encapsulated phase variants. In this study, we extended our observations by comparing phase variant capsular morphology stained with Alcian blue (AB) and Ruthenium red (RR), and investigated the dynamics of biofilm formation by these organisms to glass coverslips (CS) using quantitative plate counts and scanning electron microscopy (SEM).To characterize the morphology of capsules expressed by these organisms, we stained cells grown on trypticase soy agar containing 1 % NaCl (TSA/NaCl) with AB and then prepared them for electron microscopy (EM) according to the method described by Hendley et al.

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Expression of Vibrio vulnificus Capsular Polysaccharide Inhibits Biofilm Formation

Journal of Bacteriology ◽

10.1128/jb.186.3.889-893.2004 ◽

2004 ◽

Vol 186 (3) ◽

pp. 889-893 ◽

Cited By ~ 85

Author(s):

Lavin A. Joseph ◽

Anita C. Wright

Keyword(s):

Virulence Factor ◽

Related Species ◽

Biofilm Formation ◽

Capsular Polysaccharide ◽

Vibrio Vulnificus ◽

Human Pathogen ◽

Multiple Strains ◽

Phase Variants

ABSTRACT Vibrio vulnificus is a human pathogen that produces lethal septicemia in susceptible persons, and the primary virulence factor for this organism is capsular polysaccharide (CPS). The role of the capsule in V. vulnificus biofilms was examined under a variety of conditions, by using either defined CPS mutants or spontaneous CPS expression phase variants derived from multiple strains. CPS expression was shown to inhibit attachment and biofilm formation, which contrasted with other studies describing polysaccharides as integral to biofilms in related species.

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Identification of a Group 1-Like Capsular Polysaccharide Operon for Vibrio vulnificus

Infection and Immunity ◽

10.1128/iai.69.11.6893-6901.2001 ◽

2001 ◽

Vol 69 (11) ◽

pp. 6893-6901 ◽

Cited By ~ 71

Author(s):

Anita C. Wright ◽

Jan L. Powell ◽

James B. Kaper ◽

J. Glenn Morris

Keyword(s):

Capsular Polysaccharide ◽

Vibrio Vulnificus ◽

Phase Variation ◽

Surface Expression ◽

Open Reading Frames ◽

Biosynthetic Genes ◽

Virulent Phenotype ◽

Phase Variants ◽

Group 1

ABSTRACT Virulence of Vibrio vulnificus correlates with changes in colony morphology that are indicative of a reversible phase variation for expression of capsular polysaccharide (CPS). Encapsulated variants are virulent with opaque colonies, whereas phase variants with reduced CPS expression are attenuated and are translucent. Using TnphoA mutagenesis, we identified a V.vulnificus CPS locus, which included an upstreamops element, a wza gene (wza Vv), and several open reading frames with homology to CPS biosynthetic genes. This genetic organization is characteristic of group 1 CPS operons. The wzagene product is required for transport of CPS to the cell surface inEscherichia coli. Polar transposon mutations inwza Vv eliminated expression of downstream biosynthetic genes, confirming operon structure. On the other hand, nonpolar inactivation of wza Vv was specific for CPS transport, did not alter CPS biosynthesis, and could be complemented in trans. Southern analysis of CPS phase variants revealed deletions or rearrangements at this locus. A survey of environmental isolates indicated a correlation between deletions inwza Vv and loss of virulent phenotype, suggesting a genetic mechanism for CPS phase variation. Full virulence in mice required surface expression of CPS and supported the essential role of capsule in the pathogenesis of V.vulnificus.

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Differential Expression of Vibrio vulnificus Capsular Polysaccharide

Infection and Immunity ◽

10.1128/iai.67.5.2250-2257.1999 ◽

1999 ◽

Vol 67 (5) ◽

pp. 2250-2257 ◽

Cited By ~ 55

Author(s):

Anita C. Wright ◽

Jan L. Powell ◽

Mike K. Tanner ◽

Lynne A. Ensor ◽

Arthur B. Karpas ◽

...

Keyword(s):

Capsular Polysaccharide ◽

Vibrio Vulnificus ◽

Phase Variation ◽

Surface Expression ◽

Ruthenium Red ◽

Cell Surface Expression ◽

Enzyme Linked Immunosorbent Assay ◽

Type I ◽

Transposon Mutants ◽

Phase Variants

ABSTRACT Vibrio vulnificus is a human pathogen whose virulence has been associated with the expression of capsular polysaccharide (CPS). Multiple CPS types have been described; however, virulence does not appear to correlate with a particular CPS composition. Reversible-phase variation for opaque and translucent colony morphologies is characterized by changes in CPS expression, as suggested by electron microscopy of cells stained nonspecifically with ruthenium red. Isolates with opaque colony morphologies are virulent and appear to be more thickly encapsulated than naturally occurring translucent-phase variants, which have reduced, patchy, or absent CPS. Previously, we have shown that the virulence of translucent-phase variants was intermediate between opaque-phase variants and acapsular transposon mutants, suggesting a correlation between virulence and the amount of CPS expressed. In the present study, CPS expression of phase variants and genetically defined mutants of V. vulnificusM06-24/O was examined by using a CPS-specific monoclonal antibody with an enzyme-linked immunosorbent assay, flow cytometry, and immunoelectron microscopy. Semiquantitative analyses of CPS expression correlated well among these assays, confirming that the translucent-phase variant was intermediate in CPS expression and retained type I CPS-specific epitopes. Cell surface expression of CPS varied with the growth phase, increasing during logarithmic growth and declining in stationary culture. Significantly greater CPS expression (P = 0.026) was observed for cells grown at 30°C than for those at 37°C. These studies confirm that phase variation and virulence in V. vulnificus correlate with the amount of CPS expressed and demonstrate the fluidity of bacterial polysaccharide expression in response to environmental conditions.

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Structure, Function, and Regulation of the Essential Virulence Factor Capsular Polysaccharide of Vibrio vulnificus

International Journal of Molecular Sciences ◽

10.3390/ijms21093259 ◽

2020 ◽

Vol 21 (9) ◽

pp. 3259 ◽

Cited By ~ 1

Author(s):

Gregg S. Pettis ◽

Aheli S. Mukerji

Keyword(s):

Coastal Waters ◽

Capsular Polysaccharide ◽

Vibrio Vulnificus ◽

Critical Role ◽

Phase Variation ◽

Innate Immune ◽

Repeat Sequences ◽

Life Threatening ◽

Inflammatory Cytokine Response ◽

Group 1

Vibrio vulnificus populates coastal waters around the world, where it exists freely or becomes concentrated in filter feeding mollusks. It also causes rapid and life-threatening sepsis and wound infections in humans. Of its many virulence factors, it is the V. vulnificus capsule, composed of capsular polysaccharide (CPS), that plays a critical role in evasion of the host innate immune system by conferring antiphagocytic ability and resistance to complement-mediated killing. CPS may also provoke a portion of the host inflammatory cytokine response to this bacterium. CPS production is biochemically and genetically diverse among strains of V. vulnificus, and the carbohydrate diversity of CPS is likely affected by horizontal gene transfer events that result in new combinations of biosynthetic genes. Phase variation between virulent encapsulated opaque colonial variants and attenuated translucent colonial variants, which have little or no CPS, is a common phenotype among strains of this species. One mechanism for generating acapsular variants likely involves homologous recombination between repeat sequences flanking the wzb phosphatase gene within the Group 1 CPS biosynthetic and transport operon. A considerable number of environmental, genetic, and regulatory factors have now been identified that affect CPS gene expression and CPS production in this pathogen.

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Role of DegQ in differential stability of flagellin subunits in Vibrio vulnificus

npj Biofilms and Microbiomes ◽

10.1038/s41522-021-00206-7 ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

You-Chul Jung ◽

Mi-Ae Lee ◽

Han-Shin Kim ◽

Kyu-Ho Lee

Keyword(s):

Life Cycle ◽

Biofilm Formation ◽

Vibrio Vulnificus ◽

Differential Regulation ◽

Specific Manner ◽

Associated Proteins ◽

Differential Stability ◽

Methionine Residues

AbstractBiofilm formation of Vibrio vulnificus is initiated by adherence of flagellated cells to surfaces, and then flagellum-driven motility is not necessary during biofilm maturation. Once matured biofilms are constructed, cells become flagellated and swim to disperse from biofilms. As a consequence, timely regulations of the flagellar components’ expression are crucial to complete a biofilm life-cycle. In this study, we demonstrated that flagellins’ production is regulated in a biofilm stage-specific manner, via activities of a protease DegQ and a chaperone FlaJ. Among four flagellin subunits for V. vulnificus filament, FlaC had the highest affinities to hook-associated proteins, and is critical for maturating flagellum, showed the least susceptibility to DegQ due to the presence of methionine residues in its DegQ-sensitive domains, ND1 and CD0. Therefore, differential regulation by DegQ and FlaJ controls the cytoplasmic stability of flagellins, which further determines the motility-dependent, stage-specific development of biofilms.

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Presence of Nitric Oxide-Sensing Systems in the Human Pathogen Vibrio vulnificus

Biocontrol Science ◽

10.4265/bio.20.199 ◽

2015 ◽

Vol 20 (3) ◽

pp. 199-203 ◽

Cited By ~ 3

Author(s):

ABDELAZIZ ELGAML ◽

SHIN-ICHI MIYOSHI

Keyword(s):

Nitric Oxide ◽

Vibrio Vulnificus ◽

Human Pathogen ◽

Sensing Systems

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Influence of Glucose Concentrations on Biofilm Formation, Motility, Exoprotease Production, and Quorum Sensing in Aeromonas hydrophila

Journal of Food Protection ◽

10.4315/0362-028x.jfp-12-321 ◽

2013 ◽

Vol 76 (2) ◽

pp. 239-247 ◽

Cited By ~ 51

Author(s):

IQBAL KABIR JAHID ◽

NA-YOUNG LEE ◽

ANNA KIM ◽

SANG-DO HA

Keyword(s):

Quorum Sensing ◽

Aeromonas Hydrophila ◽

Biofilm Formation ◽

Opportunistic Pathogen ◽

Homoserine Lactone ◽

Protease Production ◽

Acyl Homoserine Lactone ◽

Human Pathogen ◽

Initial Biofilm ◽

Motility Inhibition

Aeromonas hydrophila recently has received increased attention because it is opportunistic and a primary human pathogen. A. hydrophila biofilm formation and its control are a major concern for food safety because biofilms are related to virulence. Therefore, we investigated biofilm formation, motility inhibition, quorum sensing, and exoprotease production of this opportunistic pathogen in response to various glucose concentrations from 0.05 to 2.5% (wt/vol). More than 0.05% glucose significantly impaired (P < 0.05) quorum sensing, biofilm formation, protease production, and swarming and swimming motility, whereas bacteria treated with 0.05% glucose had activity similar to that of the control (0% glucose). A stage shift biofilm assay revealed that the addition of glucose (2.5%) inhibited initial biofilm formation but not later stages. However, addition of quorum sensing molecules N-3-butanoyl-DL-homoserine lactone and N-3-hexanoyl homoserine lactone partially restored protease production, indicating that quorum sensing is controlled by glucose concentrations. Thus, glucose present in food or added as a preservative could regulate acyl-homoserine lactone quorum sensing molecules, which mediate biofilm formation and virulence in A. hydrophila.

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Pseudomonas aeruginosa transition from environmental generalist to human pathogen

Avances en Ciencias e Ingeniería ◽

10.18272/aci.v13i1.2225 ◽

2021 ◽

Vol 13 (1) ◽

pp. 11

Author(s):

Gabriela Vasco ◽

Gabriel Trueba

Keyword(s):

Pseudomonas Aeruginosa ◽

Human Body ◽

Biofilm Formation ◽

Reduction Reaction ◽

Human Pathogen ◽

Opportunistic Bacteria ◽

Oxidation Reduction ◽

Mammalian Tissues ◽

Oxidation Reduction Reaction ◽

Source Sink

Opportunistic bacteria Pseudomonas aeruginosa is one of the major concerns as an etiological agent of nosocomial infections in humans. Many virulence factors used to colonize the human body are the same as those used by P. aeruginosa to thrive in the environment such as membrane transport, biofilm formation, oxidation/reduction reaction, among others. P. aeruginosa origin is mainly from the environment, the adaptation to mammalian tissues may follow a source-sink evolution model; the environment is the source of many lineages, some of them capable of adaptation to the human body. Some lineages may adapt to humans and go through reductive evolution in which some genes are lost. The understanding of this process may be critical to implement better methods to control outbreaks in hospitals.

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Finding of Agr Phase Variants in Staphylococcus aureus

mBio ◽

10.1128/mbio.00796-19 ◽

2019 ◽

Vol 10 (4) ◽

Cited By ~ 3

Author(s):

Vishal Gor ◽

Aya J. Takemura ◽

Masami Nishitani ◽

Masato Higashide ◽

Veronica Medrano Romero ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Virulence Factors ◽

Phase Variation ◽

Accessory Gene ◽

Content Type ◽

Accessory Gene Regulator ◽

Immune Mediated ◽

A Minor ◽

First Time ◽

Phase Variants

ABSTRACT Staphylococcus aureus is an important human pathogen whose success is largely attributed to its vast arsenal of virulence factors that facilitate its invasion into, and survival within, the human host. The expression of these virulence factors is controlled by the quorum sensing accessory gene regulator (Agr) system. However, a large proportion of clinical S. aureus isolates are consistently found to have a mutationally inactivated Agr system. These mutants have a survival advantage in the host but are considered irreversible mutants. Here we show, for the first time, that a fraction of Agr-negative mutants can revert their Agr activity. By serially passaging Agr-negative strains and screening for phenotypic reversion of hemolysis and subsequent sequencing, we identified two mutational events responsible for reversion: a genetic duplication plus inversion event and a poly(A) tract alteration. Additionally, we demonstrate that one clinical Agr-negative methicillin-resistant S. aureus (MRSA) isolate could reproducibly generate Agr-revertant colonies with a poly(A) tract genetic mechanism. We also show that these revertants activate their Agr system upon phagocytosis. We propose a model in which a minor fraction of Agr-negative S. aureus strains are phase variants that can revert their Agr activity and may act as a cryptic insurance strategy against host-mediated stress. IMPORTANCE Staphylococcus aureus is responsible for a broad range of infections. This pathogen has a vast arsenal of virulence factors at its disposal, but avirulent strains are frequently isolated as the cause of clinical infections. These isolates have a mutated agr locus and have been believed to have no evolutionary future. Here we show that a fraction of Agr-negative strains can repair their mutated agr locus with mechanisms resembling phase variation. The agr revertants sustain an Agr OFF state as long as they exist as a minority but can activate their Agr system upon phagocytosis. These revertant cells might function as a cryptic insurance strategy to survive immune-mediated host stress that arises during infection.

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