scholarly journals Genetic Variation in the Vibrio vulnificus Group 1 Capsular Polysaccharide Operon

2006 ◽  
Vol 188 (5) ◽  
pp. 1987-1998 ◽  
Author(s):  
Maria Chatzidaki-Livanis ◽  
Melissa K. Jones ◽  
Anita C. Wright
Keyword(s):  
Capsular Polysaccharide ◽  
Vibrio Vulnificus ◽  
Allelic Variation ◽  
Phase Variation ◽  
Phase Variable ◽  
Deletion Mutations ◽  
Underlying Illness ◽  
Dna Elements ◽  
Repetitive Dna Elements ◽  
Group 1

ABSTRACT Vibrio vulnificus produces human disease associated with raw-oyster consumption or wound infections, but fatalities are limited to persons with chronic underlying illness. Capsular polysaccharide (CPS) is required for virulence, and CPS expression correlates with opaque (Op) colonies that show “phase variation” to avirulent translucent (Tr) phenotypes with reduced CPS. The results discussed here confirmed homology of a V. vulnificus CPS locus to the group 1 CPS operon in Escherichia coli. However, two distinct V. vulnificus genotypes or alleles were associated with the operon, and they diverged at sequences encoding hypothetical proteins and also at unique, intergenic repetitive DNA elements. Phase variation was examined under conditions that promoted high-frequency transition of Op to Tr forms. Recovery of Tr isolates in these experiments showed multiple genotypes, which were designated TR1, TR2, and TR3: CPS operons of TR1 isolates were identical to the Op parent, and cells remained phase variable but expressed reduced CPS. TR2 and TR3 showed deletion mutations in one (wzb) or multiple genes, respectively, and deletion mutants were acapsular and locked in the Tr phase. Complementation in trans restored the Op phenotype in strains with the wzb deletion mutation. Allelic variation in repetitive elements determined the locations, rates, and extents of deletion mutations. Thus, different mechanisms are responsible for reversible phase variation in CPS expression versus genetic deletions in the CPS operon of V. vulnificus. Repetitive-element-mediated deletion mutations were highly conserved within the species and are likely to promote survival in estuarine environments.

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

2020 ◽  
Vol 21 (9) ◽  
pp. 3259 ◽  
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.

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

2001 ◽  
Vol 69 (11) ◽  
pp. 6893-6901 ◽  
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.

scholarly journals Mutational analysis of genes implicated in LPS and capsular polysaccharide biosynthesis in the opportunistic pathogen Bacteroides fragilis

Microbiology ◽  
2009 ◽  
Vol 155 (4) ◽  
pp. 1039-1049 ◽  
Author(s):  
Sheila Patrick ◽  
Simon Houston ◽  
Zubin Thacker ◽  
Garry W. Blakely
Keyword(s):  
Capsular Polysaccharide ◽  
Mutational Analysis ◽  
Bacteroides Fragilis ◽  
Gene Clusters ◽  
Opportunistic Pathogen ◽  
Extracellular Polysaccharides ◽  
Phase Variable ◽  
Multiple Gene ◽  
Polysaccharide Biosynthesis ◽  
Group 1

The obligate anaerobe Bacteroides fragilis is a normal resident of the human gastrointestinal tract. The clinically derived B. fragilis strain NCTC 9343 produces an extensive array of extracellular polysaccharides (EPS), including antigenically distinct large, small and micro- capsules. The genome of NCTC 9343 encodes multiple gene clusters potentially involved in the biosynthesis of EPS, eight of which are implicated in production of the antigenically variable micro-capsule. We have developed a rapid and robust method for generating marked and markerless deletions, together with efficient electroporation using unmodified plasmid DNA to enable complementation of mutations. We show that deletion of a putative wzz homologue prevents production of high-molecular-mass polysaccharides (HMMPS), which form the micro-capsule. This observation suggests that micro-capsule HMMPS constitute the distal component of LPS in B. fragilis. The long chain length of this polysaccharide is strikingly different from classical enteric O-antigen, which consists of short-chain polysaccharides. We also demonstrate that deletion of a putative wbaP homologue prevents expression of the phase-variable large capsule and that expression can be restored by complementation. This suggests that synthesis of the large capsule is mechanistically equivalent to production of Escherichia coli group 1 and 4 capsules.

scholarly journals Capsular Polysaccharide Phase Variation in Vibrio vulnificus

2006 ◽  
Vol 72 (11) ◽  
pp. 6986-6993 ◽  
Author(s):  
Tamara Hilton ◽  
Tom Rosche ◽  
Brett Froelich ◽  
Benjamin Smith ◽  
James Oliver
Keyword(s):  
Oxidative Stress ◽  
Quorum Sensing ◽  
Cell Surface ◽  
Serum Iron ◽  
Capsular Polysaccharide ◽  
Vibrio Vulnificus ◽  
Phase Variation ◽  
Fatality Rate ◽  
Single Colony ◽  
And Oxidative Stress

ABSTRACT Commonly found in raw oysters, Vibrio vulnificus poses a serious health threat to immunocompromised individuals and those with serum iron overload, with a fatality rate of approximately 50%. An essential virulence factor is its capsular polysaccharide (CPS), which is responsible for a significant increase in virulence compared to nonencapsulated strains. However, this bacterium is known to vary the amount of CPS expressed on the cell surface, converting from an opaque (Op) colony phenotype to a translucent (Tr) colony phenotype. In this study, the consistency of CPS conversion was determined for four strains of V. vulnificus. Environmental conditions including variations in aeration, temperature, incubation time, oxidative stress, and media (heart infusion or modified maintenance medium agar) were investigated to determine their influence on CPS conversion. All conditions, with the exception of variations in media and oxidative stress, significantly affected the conversion of the population, with high ranges of CPS expression found even within cells from a single colony. The global quorum-sensing regulators RpoS and AI-2 were also examined. While RpoS was found to significantly mediate phenotypic conversion, quorum sensing was not. Finally, 12 strains that comprise the recently found clinical (C) and environmental (E) genotypes of V. vulnificus were examined to determine their rates of population conversion. C-genotype strains, which are most often associated with infection, had a significantly lower rate of population conversion from Op to Tr phenotypes than did E-genotype strains (ca. 38% versus ca. 14%, respectively). Biofilm capabilities of these strains, however, were not correlated with increased population conversion.

Role of GacA in virulence of Vibrio vulnificus

Microbiology ◽  
2010 ◽  
Vol 156 (12) ◽  
pp. 3722-3733 ◽  
Author(s):  
Julie D. Gauthier ◽  
Melissa K. Jones ◽  
Patrick Thiaville ◽  
Jennifer L. Joseph ◽  
Rick A. Swain ◽  
...  
Keyword(s):  
Capsular Polysaccharide ◽  
Vibrio Vulnificus ◽  
Phase Variation ◽  
Dependent Manner ◽  
Wild Type ◽  
Significant Impairment ◽  
Life Threatening ◽  
Two Component Signal Transduction ◽  
Systemic Infections

The GacS/GacA two-component signal transduction system regulates virulence, biofilm formation and symbiosis in Vibrio species. The present study investigated this regulatory pathway in Vibrio vulnificus, a human pathogen that causes life-threatening disease associated with the consumption of raw oysters and wound infections. Small non-coding RNAs (csrB1, csrB2, csrB3 and csrC) commonly regulated by the GacS/GacA pathway were decreased (P<0.0003) in a V. vulnificus CMCP6 ΔgacA : : aph mutant compared with the wild-type parent, and expression was restored by complementation of the gacA deletion mutation in trans. Of the 20 genes examined by RT-PCR, significant reductions in the transcript levels of the mutant in comparison with the wild-type strain were observed only for genes related to motility (flaA), stationary phase (rpoS) and protease (vvpE) (P=0.04, 0.01 and 0.002, respectively). Swimming motility, flagellation and opaque colony morphology indicative of capsular polysaccharide (CPS) were unchanged in the mutant, while cytotoxicity, protease activity, CPS phase variation and the ability to acquire iron were decreased compared with the wild-type (P<0.01). The role of gacA in virulence of V. vulnificus was also demonstrated by significant impairment in the ability of the mutant strain to cause either skin (P<0.0005) or systemic infections (P<0.02) in subcutaneously inoculated, non-iron-treated mice. However, the virulence of the mutant was equivalent to that of the wild-type in iron-treated mice, demonstrating that the GacA pathway in V. vulnificus regulates the virulence of this organism in an iron-dependent manner.

scholarly journals A novel paralogous gene family involved in phase-variable flagella-mediated motility in Campylobacter jejuni

Microbiology ◽  
2002 ◽  
Vol 148 (2) ◽  
pp. 473-480 ◽  
Author(s):  
Andrey V Karlyshev ◽  
Dennis Linton ◽  
Norman A Gregson ◽  
Brendan W Wren
Keyword(s):  
Campylobacter Jejuni ◽  
Insertional Mutagenesis ◽  
Capsular Polysaccharide ◽  
Ex Vivo ◽  
Phase Variation ◽  
Phase Variable ◽  
Accessory Factor ◽  
Flagellar Biosynthesis ◽  
Major Factors

Flagella-mediated motility is recognized to be one of the major factors contributing to virulence in Campylobacter jejuni. Motility of this bacterium is known to be phase variable, although the mechanism of such variation remains unknown. C. jejuni genome sequencing revealed a number of genes prone to phase variation via a slipped-strand mispairing mechanism. Many of these genes are hypothetical and are clustered in the regions involved in formation of three major cell surface structures: capsular polysaccharide, lipooligosaccharide and flagella. Among the genes of unknown function, the flagellar biosynthesis and modification region contains seven hypothetical paralogous genes designated as the motility accessory factor (maf) family. Remarkably, two of these genes (maf1 and maf4) were found to be identical and both contain homopolymeric G tracts. Using insertional mutagenesis it was demonstrated that one of the genes, maf5, is involved in formation of flagella. Phase variation of the maf1 gene via slipped-strand mispairing partially restored motility of the maf5 mutant. The maffamily represents a new class of bacterial genes related to flagellar biosynthesis and phase variation. Reversible expression of flagella may be advantageous for the adaptation of C. jejunito the varied in vivo and ex vivo environments encountered during its life cycle, as well in evasion of the host immune response.

scholarly journals Differential Expression of Vibrio vulnificus Capsular Polysaccharide

1999 ◽  
Vol 67 (5) ◽  
pp. 2250-2257 ◽  
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.

Comparative Cytogenetics in Four Leptodactylus Species (Amphibia, Anura, Leptodactylidae): Evidence of Inner Chromosomal Diversification in Highly Conserved Karyotypes

2021 ◽  
pp. 1-11
Author(s):  
David S. da Silva ◽  
Heriberto F. da Silva Filho ◽  
Marcelo B. Cioffi ◽  
Edivaldo H.C. de Oliveira ◽  
Anderson J.B. Gomes
Keyword(s):  
18S Rdna ◽  
Repetitive Sequences ◽  
Molecular Cytogenetics ◽  
Comparative Cytogenetics ◽  
The Family ◽  
Agnor Staining ◽  
Dna Elements ◽  
Conventional Cytogenetics ◽  
Species Specific ◽  
Repetitive Dna Elements

With 82 species currently described, the genus <i>Leptodactylus</i> is the most diverse and representative one in the family Leptodactylidae. Concerning chromosomal organization, this genus represents an interesting and underexplored group since data from molecular cytogenetics are incipient, and little is known about the organization and distribution of repetitive DNA elements in the karyotypes. In this sense, this study aimed at providing a comparative analysis in 4 <i>Leptodactylus</i> species (<i>L. macrosternum, L. pentadactylus, L. fuscus,</i> and <i>Leptodactylus</i> cf<i>. podicipinus</i>), combining conventional cytogenetics (Giemsa staining, C-banding, and AgNOR staining) and mapping of molecular markers (18S rDNA, telomeric and microsatellite probes), to investigate mechanisms underlying their karyotype differentiation process. The results showed that all species had karyotypes with 2n = 22 and FN = 44, except for <i>Leptodactylus</i> cf. <i>podicipinus</i> which presented FN = 36. The 18S rDNA was observed in pair 8 of all analyzed species (corresponding to pair 4 in <i>L. pentadactylus</i>), coinciding with the secondary constrictions and AgNOR staining. FISH with microsatellite DNA probes demonstrated species-specific patterns, as well as an association of these repetitive sequences with constitutive heterochromatin blocks and ribosomal DNA clusters, revealing the dynamics of microsatellites in the genome of the analyzed species. In summary, our data demonstrate an ongoing process of genomic divergence inside species with almost similar karyotype, driven most likely by a series of pericentric inversions, followed by differential accumulation of repetitive sequences.

scholarly journals Identification and Functional Mapping of the Mycoplasma fermentans P29 Adhesin

2002 ◽  
Vol 70 (9) ◽  
pp. 4925-4935 ◽  
Author(s):  
Spencer A. Leigh ◽  
Kim S. Wise
Keyword(s):  
Disulfide Bond ◽  
Hela Cells ◽  
Mammalian Cells ◽  
Flow Cytometric Analysis ◽  
Phase Variation ◽  
Surface Proteins ◽  
Mycoplasma Species ◽  
Phase Variable ◽  
Binding Region ◽  
Mycoplasma Fermentans

ABSTRACT Initial adherence interactions between mycoplasmas and mammalian cells are important for host colonization and may contribute to subsequent pathogenic processes. Despite significant progress toward understanding the role of specialized, complex tip structures in the adherence of some mycoplasmas, particularly those that infect humans, less is known about adhesins through which other mycoplasmas of this host bind to diverse cell types, even though simpler surface components are likely to be involved. We show by flow cytometric analysis that a soluble recombinant fusion protein (FP29), representing the abundant P29 surface lipoprotein of Mycoplasma fermentans, binds human HeLa cells and inhibits M. fermentans binding to these cells, in both a quantitative and a saturable manner, whereas analogous fusion proteins representing other mycoplasma surface proteins did not. Constructs representing nested N- or C-terminal truncations of FP29 allowed initial mapping of this specific adherence function to a central region of the P29 sequence containing a 36-amino-acid disulfide loop. A derivative of FP29 containing a mutation converting one participating Cys to Ser, precluding intrachain disulfide bond formation, retained full activity. Together these results suggest that the direct interaction of M. fermentans with a ligand on the HeLa cell surface involves a limited segment of the P29 surface lipoprotein and requires neither the disulfide bond nor the contribution of adjacent portions of the protein. Earlier results indicating phase-variable display of monoclonal antibody surface epitopes on P29, now recognized to be outside this ligand binding region, raise the possibility that variation of mycoplasma surface architecture might alter the presentation of the binding region and the adherence phenotype. Preliminary results further indicated that FP29 could inhibit binding to HeLa cells by Mycoplasma hominis, a distinct human mycoplasma species displaying the phase-variable adhesin Vaa, but not that by Mycoplasma capricolum, an organism infecting caprine species. This result raises the additional, testable possibility that a common host cell ligand for two human mycoplasma species may be recognized through structurally dissimilar adhesins that undergo phase variation by two distinct mechanisms, governing protein expression (Vaa) or surface masking (P29).

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