Role of Anaerobiosis in Capsule Production and Biofilm Formation in Vibrio vulnificus

Vibrio vulnificus, a pervasive human pathogen, can cause potentially fatal septicemia after consumption of undercooked seafood. Biotype 1 strains ofV. vulnificusare most commonly associated with human infection and are separated into two genotypes, clinical (C) and environmental (E), based on the virulence-correlated gene. For ingestion-based vibriosis to occur, this bacterium must be able to withstand multiple conditions as it traverses the gastrointestinal tract and ultimately gains entry into the bloodstream. One such condition, anoxia, has yet to be extensively researched inV. vulnificus. We investigated the effect of oxygen availability on capsular polysaccharide (CPS) production and biofilm formation in this bacterium, both of which are thought to be important for disease progression. We found that lack of oxygen elicits a reduction in both CPS and biofilm formation in both genotypes. This is further supported by the finding thatpilA,pilD, andmshAgenes, all of which encode type IV pilin proteins that aid in attachment to surfaces, were downregulated during anaerobiosis. Surprisingly, E-genotypes exhibited distinct differences in gene expression levels of capsule and attachment genes compared to C-genotypes, both aerobically and anaerobically. The importance of understanding these disparities may give insight into the observed differences in environmental occurrence and virulence potential between these two genotypes ofV. vulnificus.

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Implications of Chitin Attachment for the Environmental Persistence and Clinical Nature of the Human Pathogen Vibrio vulnificus

Applied and Environmental Microbiology ◽

10.1128/aem.03811-13 ◽

2013 ◽

Vol 80 (5) ◽

pp. 1580-1587 ◽

Cited By ~ 19

Author(s):

Tiffany C. Williams ◽

Mesrop Ayrapetyan ◽

James D. Oliver

Keyword(s):

Vibrio Vulnificus ◽

Protein A ◽

Aquatic Organisms ◽

The United States ◽

Major Constituent ◽

Marine Snow ◽

Pathogenic Potential ◽

Content Type ◽

Type Iv ◽

Type Iv Pilin

ABSTRACTVibrio vulnificusnaturally inhabits a variety of aquatic organisms, including oysters, and is the leading cause of seafood-related death in the United States. Strains of this bacterium are genetically classified into environmental (E) and clinical (C) genotypes, which correlate with source of isolation. E-genotype strains integrate into marine aggregates more efficiently than do C-genotype strains, leading to a greater uptake of strains of this genotype by oysters feeding on these aggregates. The causes of this increased integration of E-type strains into marine “snow” have not been demonstrated. Here, we further investigate the physiological and genetic causalities for this genotypic heterogeneity by examining the ability of strains of each genotype to attach to chitin, a major constituent of marine snow. We found that E-genotype strains attach to chitin with significantly greater efficiency than do C-genotype strains when incubated at 20°C. Type IV pili were implicated in chitin adherence, and even in the absence of chitin, the expression level of type IV pilin genes (pilA,pilD, andmshA) was found to be inherently higher by E genotypes than by C genotypes. In contrast, the level of expression ofN-acetylglucosamine binding protein A (gbpA) was significantly higher in C-genotype strains. Interestingly, incubation at a clinically relevant temperature (37°C) resulted in a significant increase in C-genotype attachment to chitin, which subsequently provided a protective effect against exposure to acid or bile, thus offering a clue into their increased incidence in human infections. This study suggests that C- and E-genotype strains have intrinsically divergent physiological programs, which may help explain the observed differences in the ecology and pathogenic potential between these two genotypes.

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Protein Nanowires: the Electrification of the Microbial World and Maybe Our Own

Journal of Bacteriology ◽

10.1128/jb.00331-20 ◽

2020 ◽

Vol 202 (20) ◽

Cited By ~ 2

Author(s):

Derek R. Lovley ◽

Dawn E. Holmes

Keyword(s):

Electron Transport ◽

Long Range ◽

Electronic Devices ◽

Electricity Production ◽

Microbial World ◽

Electrically Conductive ◽

Anaerobic Digesters ◽

Content Type ◽

Type Iv ◽

Type Iv Pilin

ABSTRACT Electrically conductive protein nanowires appear to be widespread in the microbial world and are a revolutionary “green” material for the fabrication of electronic devices. Electrically conductive pili (e-pili) assembled from type IV pilin monomers have independently evolved multiple times in microbial history as have electrically conductive archaella (e-archaella) assembled from homologous archaellin monomers. A role for e-pili in long-range (micrometer) extracellular electron transport has been demonstrated in some microbes. The surprising finding of e-pili in syntrophic bacteria and the role of e-pili as conduits for direct interspecies electron transfer have necessitated a reassessment of routes for electron flux in important methanogenic environments, such as anaerobic digesters and terrestrial wetlands. Pilin monomers similar to those found in e-pili may also be a major building block of the conductive “cables” that transport electrons over centimeter distances through continuous filaments of cable bacteria consisting of a thousand cells or more. Protein nanowires harvested from microbes have many functional and sustainability advantages over traditional nanowire materials and have already yielded novel electronic devices for sustainable electricity production, neuromorphic memory, and sensing. e-pili can be mass produced with an Escherichia coli chassis, providing a ready source of material for electronics as well as for studies on the basic mechanisms for long-range electron transport along protein nanowires. Continued exploration is required to better understand the electrification of microbial communities with microbial nanowires and to expand the “green toolbox” of sustainable materials for wiring and powering the emerging “Internet of things.”

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Key Role of Capsular Polysaccharide in the Induction of Systemic Infection and Abortion by Hypervirulent Campylobacter jejuni

Infection and Immunity ◽

10.1128/iai.00001-17 ◽

2017 ◽

Vol 85 (6) ◽

Cited By ~ 8

Author(s):

Orhan Sahin ◽

Samantha A. Terhorst ◽

Eric R. Burrough ◽

Zhangqi Shen ◽

Zuowei Wu ◽

...

Keyword(s):

Guinea Pig ◽

Campylobacter Jejuni ◽

Capsular Polysaccharide ◽

Systemic Infection ◽

The United States ◽

Content Type ◽

Model Following ◽

Capsule Production ◽

Guinea Pig Model

ABSTRACT Campylobacter jejuni is a zoonotic pathogen, and a hypervirulent clone, named clone SA, has recently emerged as the predominant cause of ovine abortion in the United States. To induce abortion, orally ingested Campylobacter must translocate across the intestinal epithelium, spread systemically in the circulation, and reach the fetoplacental tissue. Bacterial factors involved in these steps are not well understood. C. jejuni is known to produce capsular polysaccharide (CPS), but the specific role that CPS plays in systemic infection and particularly abortion in animals remains to be determined. In this study, we evaluated the role of CPS in bacteremia using a mouse model and in abortion using a pregnant guinea pig model following oral challenge. Compared with C. jejuni NCTC 11168 and 81-176, a clone SA isolate (IA3902) resulted in significantly higher bacterial counts and a significantly longer duration of bacteremia in mice. The loss of capsule production via gene-specific mutagenesis in IA3902 led to the complete abolishment of bacteremia in mice and abortion in pregnant guinea pigs, while complementation of capsule expression almost fully restored these phenotypes. The capsule mutant strain was also impaired for survival in guinea pig sera and sheep blood. Sequence-based analyses revealed that clone SA possesses a unique CPS locus with a mosaic structure, which has been stably maintained in all clone SA isolates derived from various hosts and times. These findings establish CPS as a key virulence factor for the induction of systemic infection and abortion in pregnant animals and provide a viable candidate for the development of vaccines against hypervirulent C. jejuni.

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Pathogenesis of Gram-Negative Bacteremia

Clinical Microbiology Reviews ◽

10.1128/cmr.00234-20 ◽

2021 ◽

Vol 34 (2) ◽

Author(s):

Caitlyn L. Holmes ◽

Mark T. Anderson ◽

Harry L. T. Mobley ◽

Michael A. Bachman

Keyword(s):

Immune Responses ◽

Global Economy ◽

Human Infection ◽

Model Systems ◽

Small Subset ◽

Gram Negative ◽

Content Type ◽

Capsule Production ◽

Gram Negative Bacteremia ◽

Species Specific

SUMMARY Gram-negative bacteremia is a devastating public health threat, with high mortality in vulnerable populations and significant costs to the global economy. Concerningly, rates of both Gram-negative bacteremia and antimicrobial resistance in the causative species are increasing. Gram-negative bacteremia develops in three phases. First, bacteria invade or colonize initial sites of infection. Second, bacteria overcome host barriers, such as immune responses, and disseminate from initial body sites to the bloodstream. Third, bacteria adapt to survive in the blood and blood-filtering organs. To develop new therapies, it is critical to define species-specific and multispecies fitness factors required for bacteremia in model systems that are relevant to human infection. A small subset of species is responsible for the majority of Gram-negative bacteremia cases, including Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. The few bacteremia fitness factors identified in these prominent Gram-negative species demonstrate shared and unique pathogenic mechanisms at each phase of bacteremia progression. Capsule production, adhesins, and metabolic flexibility are common mediators, whereas only some species utilize toxins. This review provides an overview of Gram-negative bacteremia, compares animal models for bacteremia, and discusses prevalent Gram-negative bacteremia species.

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Molecular and Physical Factors That Influence Attachment of Vibrio vulnificus to Chitin

Applied and Environmental Microbiology ◽

10.1128/aem.00753-15 ◽

2015 ◽

Vol 81 (18) ◽

pp. 6158-6165 ◽

Cited By ~ 13

Author(s):

Tiffany C. Williams ◽

Mesrop Ayrapetyan ◽

James D. Oliver

Keyword(s):

Quorum Sensing ◽

Vibrio Vulnificus ◽

The United States ◽

Type Iv Pili ◽

Negative Regulator ◽

Physical Factors ◽

Wound Infections ◽

Environmental Persistence ◽

Content Type ◽

Type Iv

ABSTRACTThe human pathogenVibrio vulnificusis the leading cause of seafood-related deaths in the United States. Strains are genotyped on the basis of alleles that correlate with isolation source, with clinical (C)-genotype strains being more often implicated in disease and environmental (E)-genotype strains being more frequently isolated from oysters and estuarine waters. Previously, we have shown that the ecologically distinct C- and E-genotype strains ofV. vulnificusdisplay different degrees of chitin attachment, with C-genotype strains exhibiting reduced attachment relative to their E-genotype strain counterparts. We identified type IV pili to be part of the molecular basis for this observed genotypic variance, as E-genotype strains exhibit higher levels of expression of these genes than C-genotype strains. Here, we used a C-genotype quorum-sensing (QS) mutant to demonstrate that quorum sensing is a negative regulator of type IV pilus expression, which results in decreased chitin attachment. Furthermore, calcium depletion reduced E-genotype strain attachment to chitin, which suggests that calcium is necessary for proper functioning of the type IV pili in E-genotype strains. We also found that starvation or dormancy can alter the efficiency of chitin attachment, which has significant implications for the environmental persistence ofV. vulnificus. With the increasing incidence of wound infections caused byV. vulnificus, we investigated a subset of E-genotype strains isolated from human wound infections and discovered that they attached to chitin in a manner more similar to that of C-genotype strains. This study enhances our understanding of the molecular and physical factors that mediate chitin attachment inV. vulnificus, providing insight into the mechanisms that facilitate the persistence of this pathogen in its native environment.

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A Vibrio vulnificus Type IV Pilin Contributes to Biofilm Formation, Adherence to Epithelial Cells, and Virulence

Infection and Immunity ◽

10.1128/iai.73.3.1411-1422.2005 ◽

2005 ◽

Vol 73 (3) ◽

pp. 1411-1422 ◽

Cited By ~ 109

Author(s):

Rohinee N. Paranjpye ◽

Mark S. Strom

Keyword(s):

Vibrio Vulnificus ◽

Type Ii Secretion ◽

Secretion Pathway ◽

Type Iv ◽

Secreted Factors ◽

Pilus Biogenesis ◽

Type Iv Pilin ◽

Group A ◽

Prepilin Peptidase ◽

Reduced Adherence

ABSTRACT Vibrio vulnificus expresses a multitude of cell-associated and secreted factors that potentially contribute to pathogenicity, although the specific roles of most of these factors have been difficult to define. Previously we have shown that a mutation in pilD (originally designated vvpD), which encodes a type IV prepilin peptidase/N-methyltransferase, abolishes expression of surface pili, suggesting that they belong to the type IV class. In addition, a pilD mutant exhibits reduced adherence to HEp-2 cells, a block in secretion of several exoenzymes that follow the type II secretion pathway, and decreased virulence. In this study, we have cloned and characterized a V. vulnificus type IV pilin (PilA) that shares extensive homology to group A type IV pilins expressed by many pathogens, including Vibrio cholerae (PilA), Pseudomonas aeruginosa (PilA), and Aeromonas hydrophila (TapA). The V. vulnificus pilA gene is part of an operon and is clustered with three other pilus biogenesis genes, pilBCD. Inactivation of pilA reduces the ability of V. vulnificus to form biofilms and significantly decreases adherence to HEp-2 cells and virulence in iron dextran-treated mice. Southern blot analysis demonstrates the widespread presence of both pilA and pilD in clinical as well as environmental strains of V. vulnificus.

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USA300 and USA500 Clonal Lineages of Staphylococcus aureus Do Not Produce a Capsular Polysaccharide Due to Conserved Mutations in the cap5 Locus

mBio ◽

10.1128/mbio.02585-14 ◽

2015 ◽

Vol 6 (2) ◽

Cited By ~ 42

Author(s):

Susan Boyle-Vavra ◽

Xue Li ◽

Md Tauqeer Alam ◽

Timothy D. Read ◽

Julia Sieth ◽

...

Keyword(s):

United States ◽

Staphylococcus Aureus ◽

Capsular Polysaccharide ◽

The United States ◽

Whole Genome Sequence ◽

Content Type ◽

Capsule Production ◽

Usa300 Strain

ABSTRACTThe surface capsular polysaccharide (CP) is a virulence factor that has been used as an antigen in several successful vaccines against bacterial pathogens. A vaccine has not yet been licensed againstStaphylococcus aureus, although two multicomponent vaccines that contain CP antigens are in clinical trials. In this study, we evaluated CP production in USA300 methicillin-resistantS. aureus(MRSA) isolates that have become the predominant community-associated MRSA clones in the United States. We found that all 167 USA300 MRSA and 50 USA300 methicillin-susceptibleS. aureus(MSSA) isolates were CP negative (CP−). Moreover, all 16 USA500 isolates, which have been postulated to be the progenitor lineage of USA300, were also CP−. Whole-genome sequence analysis of 146 CP−USA300 MRSA isolates revealed they all carry acap5locus with 4 conserved mutations compared with strain Newman. Genetic complementation experiments revealed that three of these mutations (in thecap5promoter,cap5Dnucleotide 994, andcap5Enucleotide 223) ablated CP production in USA300 and that Cap5E75 Asp, located in the coenzyme-binding domain, is essential for capsule production. All but three USA300 MSSA isolates had the same fourcap5mutations found in USA300 MRSA isolates. Most isolates with a USA500 pulsotype carried three of these four USA300-specific mutations, suggesting the fourth mutation occurred in the USA300 lineage. Phylogenetic analysis of thecaploci of our USA300 isolates as well as publicly available genomes from 41 other sequence types revealed that the USA300-specificcap5mutations arose sequentially inS. aureusin a common ancestor of USA300 and USA500 isolates.IMPORTANCEThe USA300 MRSA clone emerged as a community-associated pathogen in the United States nearly 20 years ago. Since then, it has rapidly disseminated and now causes health care-associated infections. This study shows that the CP-negative (CP−) phenotype has persisted among USA300 isolates and is a universal and characteristic trait of this highly successful MRSA lineage. It is important to note that a vaccine consisting solely of CP antigens would not likely demonstrate high efficacy in the U.S. population, where about half of MRSA isolates comprise USA300. Moreover, conversion of a USA300 strain to a CP-positive (CP+) phenotype is unlikelyin vivoorin vitrosince it would require the reversion of 3 mutations. We have also established that USA300 MSSA isolates and USA500 isolates are CP−and provide new insight into the evolution of the USA300 and USA500 lineages.

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Nontypeable Pneumococci Can Be Divided into Multiple cps Types, Including One Type Expressing the Novel Gene pspK

mBio ◽

10.1128/mbio.00035-12 ◽

2012 ◽

Vol 3 (3) ◽

Cited By ~ 59

Author(s):

In Ho Park ◽

Kyung-Hyo Kim ◽

Ana Lucia Andrade ◽

David E. Briles ◽

Larry S. McDaniel ◽

...

Keyword(s):

Pathogenic Bacteria ◽

Capsular Polysaccharide ◽

Genetic Locus ◽

The Novel ◽

Content Type ◽

Capsule Production ◽

Polysaccharide Synthesis ◽

Definition Of ◽

Survival Mechanisms ◽

Helical Region

ABSTRACTAlthough virulence ofStreptococcus pneumoniaeis associated with its capsule, some pathogenicS. pneumoniaeisolates lack capsules and are serologically nontypeable (NT). We obtained 64 isolates that were identified as NT “pneumococci” (i.e., bacteria satisfying the conventional definition but without the multilocus sequence typing [MLST]-based definition ofS. pneumoniae) by the traditional criteria. All 64 were optochin sensitive and hadlytA, and 63 hadply. Twelve isolates hadcpsA, suggesting the presence of a conventional but defective capsular polysaccharide synthesis (cps) locus. The 52cpsA-negative isolates could be divided into three null capsule clades (NCC) based onaliC(aliB-like ORF1),aliD(aliB-like ORF2), and our newly discovered gene,pspK, in theircpsloci.pspKencodes a protein with a long alpha-helical region containing an LPxTG motif and a YPT motif known to bind human pIgR. There were nine isolates in NCC1 (pspK+but negative foraliCandaliD), 32 isolates in NCC2 (aliC+aliD+but negative forpspK), and 11 in NCC3 (aliD+but negative foraliCandpspK). Among 52cpsA-negative isolates, 41 were identified asS. pneumoniaeby MLST analysis. All NCC1 and most NCC2 isolates wereS. pneumoniae, whereas all nine NCC3 and two NCC2 isolates were notS. pneumoniae. Several NCC1 and NCC2 isolates from multiple individuals had identical MLST andcpsregions, showing that unencapsulatedS. pneumoniaecan be infectious among humans. Furthermore, NCC1 and NCC2S. pneumoniaeisolates could colonize mice as well as encapsulatedS. pneumoniae, althoughS. pneumoniaewith an artificially disruptedcpslocus did not. Moreover, an NCC1 isolate withpspKdeletion did not colonize mice, suggesting thatpspKis critical for colonization. Thus, PspK may provide pneumococci a means of surviving in the nasopharynx without capsule.IMPORTANCEThe presence of a capsule is critical for many pathogenic bacteria, including pneumococci. Reflecting the pathogenic importance of the pneumococcal capsule, pneumococcal vaccines are designed to elicit anticapsule antibodies. Additional evidence for the pathogenic importance of the pneumococcal capsule is the fact that in pneumococci all the genes necessary for capsule production are together in one genetic locus, which is called thecpslocus. However, there are occasional pathogenic pneumococci without capsules, and how they survive in the host without the capsule is unknown. Here, we show that in these acapsular pneumococci, thecpsloci have been replaced with various novel genes and they can colonize mouse nasopharynges as well as capsulated pneumococci. Since the genes that replace thecpsloci are likely to be important in host survival, they may show new and/or alternative capsule-independent survival mechanisms used by pneumococci.

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EDTA Inhibits Biofilm Formation, Extracellular Vesicular Secretion, and Shedding of the Capsular Polysaccharide Glucuronoxylomannan by Cryptococcus neoformans

Applied and Environmental Microbiology ◽

10.1128/aem.01953-12 ◽

2012 ◽

Vol 78 (22) ◽

pp. 7977-7984 ◽

Cited By ~ 44

Author(s):

Emma J. Robertson ◽

Julie M. Wolf ◽

Arturo Casadevall

Keyword(s):

Cryptococcus Neoformans ◽

Biofilm Formation ◽

Capsular Polysaccharide ◽

Inhibitory Effect ◽

Antifungal Drugs ◽

Biofilm Growth ◽

Content Type ◽

Sublethal Concentrations ◽

Vesicular Secretion

ABSTRACTThe fungal pathogenCryptococcus neoformanscan grow as a biofilm on a range of synthetic and prosthetic materials. Cryptococcal biofilm formation can complicate the placement of shunts used to relieve increased intracranial pressure in cryptococcal meningitis and can serve as a nidus for chronic infection. Biofilms are generally advantageous to pathogensin vivo, as they can confer resistance to antimicrobial compounds, including fluconazole and voriconazole in the case ofC. neoformans. EDTA can inhibit biofilm formation by several microbes and enhances the susceptibility of biofilms to antifungal drugs. In this study, we evaluated the effect of sublethal concentrations of EDTA on the growth of cryptococcal biofilms. EDTA inhibited biofilm growth byC. neoformans, and the inhibition could be reversed by the addition of magnesium or calcium, implying that the inhibitory effect was by divalent cation starvation. EDTA also reduced the amount of the capsular polysaccharide glucuronoxylomannan shed into the biofilm matrix and decreased vesicular secretion from the cell, thus providing a potential mechanism for the inhibitory effect of this cation-chelating compound. Our data imply that the growth ofC. neoformansbiofilms requires the presence of divalent metals in the growth medium and suggest that cations are required for the export of materials needed for biofilm formation, possibly including extracellular vesicles.

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Molecular Dissection of Bacterial Nanowires

mBio ◽

10.1128/mbio.00270-13 ◽

2013 ◽

Vol 4 (3) ◽

Cited By ~ 43

Author(s):

Thomas Boesen ◽

Lars Peter Nielsen

Keyword(s):

Conclusive Evidence ◽

Geobacter Sulfurreducens ◽

Electron Conduction ◽

Content Type ◽

Type Iv ◽

Pilin Subunit ◽

Type Iv Pilin ◽

Final Electron ◽

Conductive Properties ◽

Final Electron Acceptor

ABSTRACTThe discovery of bacterial conductive structures, termed nanowires, has intrigued scientists for almost a decade. Nanowires enable bacteria to transfer electrons over micrometer distances to extracellular electron acceptors such as insoluble metal oxides or electrodes. Nanowires are pilus based and inGeobacter sulfurreducensare composed of the type IV pilin subunit PilA. Multihemec-type cytochromes have been shown to attach to nanowire pili. Two hypotheses have been proposed for electron conduction in nanowires. The first (termed the metal-like conductivity or MLC hypothesis) claims that the pilus itself has the electron-conductive properties and the attached cytochromes mediate transfer to the final electron acceptor, whereas the second hypothesis (termed the superexchange conductivity or SEC hypothesis) suggests that electrons are “hopping” between heme groups in cytochromes closely aligned with the pilus as a scaffold. In their recent article inmBio, Vargas et al. [M. Vargas, N. S. Malvankar, P.-L. Tremblay, C. Leang, J. A. Smith, P. Patel, O. Snoeyenbos-West, K. P. Nevin, and D. R. Lovley, mBio 4(2):e00210-13, 2013] address this ambiguity through an analysis of strain Aro-5, aG. sulfurreducensPilA mutant lacking aromatic residues in the nonconserved portion of PilA. These residues were suspected of involvement in electron transport according to the MLC hypothesis. TheG. sulfurreducensmutant had reduced conductive properties, lending important support to the MLC hypothesis. The data also highlight the need for further and more conclusive evidence for one or the other hypothesis.

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