scholarly journals Screening for Inhibition of Vibrio cholerae VipA-VipB Interaction Identifies Small-Molecule Compounds Active against Type VI Secretion

2014 ◽  
Vol 58 (7) ◽  
pp. 4123-4130 ◽  
Author(s):  
Kun Sun ◽  
Jeanette Bröms ◽  
Moa Lavander ◽  
Bharat Kumar Gurram ◽  
Per-Anders Enquist ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Small Molecule ◽  
Secretion System ◽  
Eukaryotic Cells ◽  
Minimal Risk ◽  
Type Vi Secretion System ◽  
Content Type ◽  
Development Of Resistance ◽  
Type Vi Secretion ◽  
Bacterial Replication

ABSTRACTThe type VI secretion system (T6SS) is the most prevalent bacterial secretion system and an important virulence mechanism utilized by Gram-negative bacteria, either to target eukaryotic cells or to combat other microbes. The components show much variability, but some appear essential for the function, and two homologues, denoted VipA and VipB inVibrio cholerae, have been identified in all T6SSs described so far. Secretion is dependent on binding of an α-helical region of VipA to VipB, and in the absence of this binding, both components are degraded within minutes and secretion is ceased. The aim of the study was to investigate if this interaction could be blocked, and we hypothesized that such inhibition would lead to abrogation of T6S. A library of 9,600 small-molecule compounds was screened for their ability to block the binding of VipA-VipB in a bacterial two-hybrid system (B2H). After excluding compounds that showed cytotoxicity toward eukaryotic cells, that inhibited growth ofVibrio, or that inhibited an unrelated B2H interaction, 34 compounds were further investigated for effects on the T6SS-dependent secretion of hemolysin-coregulated protein (Hcp) or of phospholipase A1activity. Two compounds, KS100 and KS200, showed intermediate or strong effects in both assays. Analogues were obtained, and compounds with potent inhibitory effects in the assays and desirable physicochemical properties as predicted byin silicoanalysis were identified. Since the compounds specifically target a virulence mechanism without affecting bacterial replication, they have the potential to mitigate the virulence with minimal risk for development of resistance.

scholarly journals Type VI Secretion System Dynamics Reveals a Novel Secretion Mechanism in Pseudomonas aeruginosa

2018 ◽  
Vol 200 (11) ◽  
Author(s):  
Jacqueline Corbitt ◽  
Jun Seok Yeo ◽  
C. Ian Davis ◽  
Michele LeRoux ◽  
Paul A. Wiggins
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Vibrio Cholerae ◽  
Conformational Change ◽  
Secretion System ◽  
Force Generation ◽  
Type Vi Secretion System ◽  
Content Type ◽  
Evolutionary Diversification ◽  
Type Vi Secretion ◽  
Generation Mechanisms

ABSTRACT The type VI secretion system (T6SS) inhibits the growth of neighboring bacterial cells through a contact-mediated mechanism. Here, we describe a detailed characterization of the protein localization dynamics in the Pseudomonas aeruginosa T6SS. It has been proposed that the type VI secretion process is driven by a conformational-change-induced contraction of the T6SS sheath. However, although the contraction of an optically resolvable TssBC sheath and the subsequent localization of ClpV are observed in Vibrio cholerae , coordinated assembly and disassembly of TssB and ClpV are observed without TssB contraction in P. aeruginosa . These dynamics are inconsistent with the proposed contraction sheath model. Motivated by the phenomenon of dynamic instability, we propose a new model in which ATP hydrolysis, rather than conformational change, generates the force for secretion. IMPORTANCE The type VI secretion system (T6SS) is widely conserved among Gram-negative bacteria and is a central determinant of bacterial fitness in polymicrobial communities. The secretion system targets bacteria and secretes effectors that inhibit the growth of neighboring cells, using a contact-mediated-delivery system. Despite significant homology to the previously characterized Vibrio cholerae T6SS, our analysis reveals that effector secretion is driven by a distinct force generation mechanism in Pseudomonas aeruginosa . The presence of two distinct force generation mechanisms in T6SS represents an example of the evolutionary diversification of force generation mechanisms.

scholarly journals Vibrio cholerae Requires the Type VI Secretion System Virulence Factor VasX To Kill Dictyostelium discoideum

2011 ◽  
Vol 79 (7) ◽  
pp. 2941-2949 ◽  
Author(s):  
Sarah T. Miyata ◽  
Maya Kitaoka ◽  
Teresa M. Brooks ◽  
Steven B. McAuley ◽  
Stefan Pukatzki
Keyword(s):  
Vibrio Cholerae ◽  
Dictyostelium Discoideum ◽  
Virulence Factor ◽  
Membrane Lipids ◽  
Gene Clusters ◽  
Secretion System ◽  
Type Vi Secretion System ◽  
Content Type ◽  
Enzymatic Function ◽  
Type Vi Secretion

ABSTRACTThe type VI secretion system (T6SS) is recognized as an important virulence mechanism in several Gram-negative pathogens. InVibrio cholerae, the causative agent of the diarrheal disease cholera, a minimum of three gene clusters—one main cluster and two auxiliary clusters—are required to form a functional T6SS apparatus capable of conferring virulence toward eukaryotic and prokaryotic hosts. Despite an increasing understanding of the components that make up the T6SS apparatus, little is known about the regulation of these genes and the gene products delivered by this nanomachine. VasH is an important regulator of theV. choleraeT6SS. Here, we present evidence that VasH regulates the production of a newly identified protein, VasX, which in turn requires a functional T6SS for secretion. Deletion ofvasXdoes not affect export or enzymatic function of the structural T6SS proteins Hcp and VgrG-1, suggesting that VasX is dispensable for the assembly of the physical translocon complex. VasX localizes to the bacterial membrane and interacts with membrane lipids. We present VasX as a novel virulence factor of the T6SS, as aV. choleraemutant lackingvasXexhibits a phenotype of attenuated virulence towardDictyostelium discoideum.

scholarly journals The LonA Protease Regulates Biofilm Formation, Motility, Virulence, and the Type VI Secretion System in Vibrio cholerae

2016 ◽  
Vol 198 (6) ◽  
pp. 973-985 ◽  
Author(s):  
Andrew Rogers ◽  
Loni Townsley ◽  
Ana L. Gallego-Hernandez ◽  
Sinem Beyhan ◽  
Laura Kwuan ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Biofilm Formation ◽  
Secretion System ◽  
Lon Protease ◽  
Human Pathogen ◽  
Type Vi Secretion System ◽  
Cyclic Diguanylate ◽  
Content Type ◽  
Infant Mouse ◽  
Type Vi Secretion

ABSTRACTThe presence of the Lon protease in all three domains of life hints at its biological importance. The prokaryotic Lon protease is responsible not only for degrading abnormal proteins but also for carrying out the proteolytic regulation of specific protein targets. Posttranslational regulation by Lon is known to affect a variety of physiological traits in many bacteria, including biofilm formation, motility, and virulence. Here, we identify the regulatory roles of LonA in the human pathogenVibrio cholerae. We determined that the absence of LonA adversely affects biofilm formation, increases swimming motility, and influences intracellular levels of cyclic diguanylate. Whole-genome expression analysis revealed that the message abundance of genes involved in biofilm formation was decreased but that the message abundances of those involved in virulence and the type VI secretion system were increased in alonAmutant compared to the wild type. We further demonstrated that alonAmutant displays an increase in type VI secretion system activity and is markedly defective in colonization of the infant mouse. These findings suggest that LonA plays a critical role in the environmental survival and virulence ofV. cholerae.IMPORTANCEBacteria utilize intracellular proteases to degrade damaged proteins and adapt to changing environments. The Lon protease has been shown to be important for environmental adaptation and plays a crucial role in regulating the motility, biofilm formation, and virulence of numerous plant and animal pathogens. We find that LonA of the human pathogenV. choleraeis in line with this trend, as the deletion of LonA leads to hypermotility and defects in both biofilm formation and colonization of the infant mouse. In addition, we show that LonA regulates levels of cyclic diguanylate and the type VI secretion system. Our observations add to the known regulatory repertoire of the Lon protease and the current understanding ofV. choleraephysiology.

scholarly journals Pathoadaptive Conditional Regulation of the Type VI Secretion System in Vibrio cholerae O1 Strains

2011 ◽  
Vol 80 (2) ◽  
pp. 575-584 ◽  
Author(s):  
Takahiko Ishikawa ◽  
Dharmesh Sabharwal ◽  
Jeanette Bröms ◽  
Debra L. Milton ◽  
Anders Sjöstedt ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Secretion System ◽  
Wild Type ◽  
Type Vi Secretion System ◽  
Content Type ◽  
High Osmolarity ◽  
Secretion Pathway ◽  
Type Vi Secretion ◽  
Vibrio Cholerae O1 ◽  
K 12

ABSTRACTThe most recently discovered secretion pathway in Gram-negative bacteria, the type VI secretion system (T6SS), is present in many species and is considered important for the survival of non-O1 non-O139Vibrio choleraein aquatic environments. Until now, it was not known whether there is a functionally active T6SS in wild-typeV. choleraeO1 strains, the cause of cholera disease in humans. Here, we demonstrate the presence of a functionally active T6SS in wild-typeV. choleraeO1 strains, as evidenced by the secretion of the T6SS substrate Hcp, which required several gene products encoded within the putativevasgene cluster. Our analyses showed that the T6SS of wild-typeV. choleraeO1 strain A1552 was functionally activated when the bacteria were grown under high-osmolarity conditions. The T6SS was also active when the bacteria were grown under low temperature (23°C), suggesting that the system may be important for the survival of the bacterium in the environment. A test of the interbacterial virulence ofV. choleraestrain A1552 against anEscherichia coliK-12 strain showed that it was strongly enhanced under high osmolarity and that it depended on thehcpgenes. Interestingly, we found that the newly recognized osmoregulatory protein OscR plays a role in the regulation of T6SS gene expression and secretion of Hcp fromV. choleraeO1 strains.

scholarly journals Diversity of Clinical and Environmental Isolates of Vibrio cholerae in Natural Transformation and Contact-Dependent Bacterial Killing Indicative of Type VI Secretion System Activity

2016 ◽  
Vol 82 (9) ◽  
pp. 2833-2842 ◽  
Author(s):  
Eryn E. Bernardy ◽  
Maryann A. Turnsek ◽  
Sarah K. Wilson ◽  
Cheryl L. Tarr ◽  
Brian K. Hammer
Keyword(s):  
Vibrio Cholerae ◽  
Natural Transformation ◽  
Secretion System ◽  
Past Century ◽  
Dna Uptake ◽  
Type Vi Secretion System ◽  
Environmental Isolates ◽  
Bacterial Killing ◽  
Content Type ◽  
Type Vi Secretion

ABSTRACTThe bacterial pathogenVibrio choleraecan occupy both the human gut and aquatic reservoirs, where it may colonize chitinous surfaces that induce the expression of factors for three phenotypes: chitin utilization, DNA uptake by natural transformation, and contact-dependent bacterial killing via a type VI secretion system (T6SS). In this study, we surveyed a diverse set of 53 isolates from different geographic locales collected over the past century from human clinical and environmental specimens for each phenotype outlined above. The set included pandemic isolates of serogroup O1, as well as several serogroup O139 and non-O1/non-O139 strains. We found that while chitin utilization was common, only 22.6% of the isolates tested were proficient at chitin-induced natural transformation, suggesting that transformation is expendable. Constitutive contact-dependent killing ofEscherichia coliprey, which is indicative of a functional T6SS, was rare among clinical isolates (only 4 of 29) but common among environmental isolates (22 of 24). These results bolster the pathoadaptive model in which tight regulation of T6SS-mediated bacterial killing is beneficial in a human host, whereas constitutive killing by environmental isolates may give a competitive advantage in natural settings. Future sequence analysis of this set of diverse isolates may identify previously unknown regulators and structural components for both natural transformation and T6SS.

scholarly journals Transcriptional Silencing by TsrA in the Evolution of Pathogenic Vibrio cholerae Biotypes

mBio ◽  
2020 ◽  
Vol 11 (6) ◽  
Author(s):  
Florence Caro ◽  
José A. Caro ◽  
Nicole M. Place ◽  
John J. Mekalanos
Keyword(s):  
Gene Expression ◽  
Vibrio Cholerae ◽  
Virulence Factors ◽  
Virulence Genes ◽  
Secretion System ◽  
Type Vi Secretion System ◽  
Content Type ◽  
Type Vi Secretion ◽  
Genes Encoding ◽  
El Tor

ABSTRACT Vibrio cholerae is a globally important pathogen responsible for the severe epidemic diarrheal disease called cholera. The current and ongoing seventh pandemic of cholera is caused by El Tor strains, which have completely replaced the sixth-pandemic classical strains of V. cholerae. To successfully establish infection and disseminate to new victims, V. cholerae relies on key virulence factors encoded on horizontally acquired genetic elements. The expression of these factors relies on the regulatory architecture that coordinates the timely expression of virulence determinants during host infection. Here, we apply transcriptomics and structural modeling to understand how type VI secretion system regulator A (TsrA) affects gene expression in both the classical and El Tor biotypes of V. cholerae. We find that TsrA acts as a negative regulator of V. cholerae virulence genes encoded on horizontally acquired genetic elements. The TsrA regulon comprises genes encoding cholera toxin (CT), the toxin-coregulated pilus (TCP), and the type VI secretion system (T6SS), as well as genes involved in biofilm formation. The majority of the TsrA regulon is carried on horizontally acquired AT-rich genetic islands whose loss or acquisition could be directly ascribed to the differences between the classical and El Tor strains studied. Our modeling predicts that the TsrA protein is a structural homolog of the histone-like nucleoid structuring protein (H-NS) oligomerization domain and is likely capable of forming higher-order superhelical structures, potentially with DNA. These findings describe how TsrA can integrate into the intricate V. cholerae virulence gene expression program, controlling gene expression through transcriptional silencing. IMPORTANCE Pathogenic Vibrio cholerae strains express multiple virulence factors that are encoded by bacteriophage and chromosomal islands. These include cholera toxin and the intestinal colonization pilus called the toxin-coregulated pilus, which are essential for causing severe disease in humans. However, it is presently unclear how the expression of these horizontally acquired accessory virulence genes can be efficiently integrated with preexisting transcriptional programs that are presumably fine-tuned for optimal expression in V. cholerae before its conversion to a human pathogen. Here, we report the role of a transcriptional regulator (TsrA) in silencing horizontally acquired genes encoding important virulence factors. We propose that this factor could be critical to the efficient acquisition of accessory virulence genes by silencing their expression until other signals trigger their transcriptional activation within the host.

scholarly journals Secretome Analysis of Vibrio cholerae Type VI Secretion System Reveals a New Effector-Immunity Pair

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Emrah Altindis ◽  
Tao Dong ◽  
Christy Catalano ◽  
John Mekalanos
Keyword(s):  
Vibrio Cholerae ◽  
Bioinformatics Analysis ◽  
Secretion System ◽  
Comparative Proteomics ◽  
Gram Negative Bacteria ◽  
Type Vi Secretion System ◽  
Immunity Protein ◽  
Gram Negative ◽  
Content Type ◽  
Type Vi Secretion

ABSTRACT The type VI secretion system (T6SS) is a dynamic macromolecular organelle that many Gram-negative bacteria use to inhibit or kill other prokaryotic or eukaryotic cells. The toxic effectors of T6SS are delivered to the prey cells in a contact-dependent manner. In Vibrio cholerae, the etiologic agent of cholera, T6SS is active during intestinal infection. Here, we describe the use of comparative proteomics coupled with bioinformatics to identify a new T6SS effector-immunity pair. This analysis was able to identify all previously identified secreted substrates of T6SS except PAAR (proline, alanine, alanine, arginine) motif-containing proteins. Additionally, this approach led to the identification of a new secreted protein encoded by VCA0285 (TseH) that carries a predicted hydrolase domain. We confirmed that TseH is toxic when expressed in the periplasm of Escherichia coli and V. cholerae cells. The toxicity observed in V. cholerae was suppressed by coexpression of the protein encoded by VCA0286 (TsiH), indicating that this protein is the cognate immunity protein of TseH. Furthermore, exogenous addition of purified recombinant TseH to permeabilized E. coli cells caused cell lysis. Bioinformatics analysis of the TseH protein sequence suggest that it is a member of a new family of cell wall-degrading enzymes that include proteins belonging to the YD repeat and Rhs superfamilies and that orthologs of TseH are likely expressed by species belonging to phyla as diverse as Bacteroidetes and Proteobacteria. IMPORTANCE The Gram-negative bacterium Vibrio cholerae causes cholera, a severe and often lethal diarrheal disease. The 2010-2012 epidemic in Haiti and new explosive epidemics in Africa show that cholera remains a significant global public health problem. The type VI secretion system (T6SS) is a dynamic organelle expressed by many Gram-negative bacteria, which use it to inject toxic effector proteins into eukaryotic and bacterial prey cells. In this study, we applied a comparative proteomics approach to the V. cholerae T6SS secretome to identify new substrates of this secretion apparatus. We show that the product of the gene VCA0285 is likely a new peptidoglycan hydrolase that is secreted by T6SS and that its cognate immunity protein is encoded by the gene that is immediately downstream (VCA0286). Bioinformatics analysis shows that VCA0285 carries four conserved motifs that likely define a large family of hydrolases with antibacterial activity. The identification of new antibacterial T6SS effectors provides useful information for the development of novel antibiotics and therapeutic agents.

scholarly journals Vibrio cholerae Type VI Secretion System Auxiliary Cluster 3 is a Pandemic-associated Mobile Genetic Element

2019 ◽  
Author(s):  
Francis J. Santoriello ◽  
Lina Michel ◽  
Daniel Unterweger ◽  
Stefan Pukatzki
Keyword(s):  
Vibrio Cholerae ◽  
Horizontal Transfer ◽  
Mobile Genetic Element ◽  
Genomic Analysis ◽  
Secretion System ◽  
Specific Factor ◽  
Genetic Element ◽  
Type Vi Secretion System ◽  
Site Specific ◽  
Type Vi Secretion

AbstractAll sequenced Vibrio cholerae isolates encode a contact-dependent type VI secretion system (T6SS) in three loci that terminate in a toxic effector and cognate immunity protein (E/I) pair, allowing for competitor killing and clonal expansion in aquatic environments and the host gut. Recent studies have demonstrated variability in the toxic effectors produced by different V. cholerae strains and the propensity for effector genes to undergo horizontal gene transfer. Here we demonstrate that a fourth cluster, auxiliary cluster 3 (Aux3), encoding the E/I pair tseH/tsiH, is located directly downstream from two putative recombinases and is flanked by repeat elements resembling att sites. Genomic analysis of 749 V. cholerae isolates, including both pandemic and environmental strains, revealed that Aux3 exists in two states: a ∼40 kb prophage-like element in nine environmental isolates and a ∼6 kb element in pandemic isolates. These findings indicate that Aux3 in pandemic V. cholerae is evolutionarily related to an environmental prophage-like element. In both states, Aux3 excises from the chromosome via site-specific recombination to form a circular product, likely priming the module for horizontal transfer. Finally, we show that Aux3 can integrate into the Aux3-naïve chromosome in an integrase-dependent, site-specific manner. This highlights the potential of Aux3 to undergo horizontal transfer by a phage-like mechanism, which based on pandemic coincidence may confer currently unknown fitness advantages to the recipient V. cholerae cell.Significance StatementV. cholerae is a human pathogen that causes pandemics affecting 2.8 million people annually (1). The O1 El Tor lineage is responsible for the current pandemic. A subset of non-O1 strains cause cholera-like disease by producing the major virulence factors cholera toxin and toxin co-regulated pilus but fail to cause pandemics. The full set of V. cholerae pandemic factors is unknown. Here we describe the type VI secretion system (T6SS) Aux3 element as a largely pandemic-specific factor that is evolutionarily related to an environmental prophage-like element circulating in non-pathogenic strains. These findings shed light on V. cholerae T6SS evolution and indicate the Aux3 element as a pandemic-enriched mobile genetic element.

scholarly journals The Type VI Secretion System Modulates Flagellar Gene Expression and Secretion in Citrobacter freundii and Contributes to Adhesion and Cytotoxicity to Host Cells

2015 ◽  
Vol 83 (7) ◽  
pp. 2596-2604 ◽  
Author(s):  
Liyun Liu ◽  
Shuai Hao ◽  
Ruiting Lan ◽  
Guangxia Wang ◽  
Di Xiao ◽  
...  
Keyword(s):  
Secretion System ◽  
Host Cells ◽  
Target Cells ◽  
Deletion Mutants ◽  
Citrobacter Freundii ◽  
Wild Type ◽  
Type Vi Secretion System ◽  
Content Type ◽  
Type Vi Secretion ◽  
Mutant Strains

The type VI secretion system (T6SS) as a virulence factor-releasing system contributes to virulence development of various pathogens and is often activated upon contact with target cells.Citrobacter freundiistrain CF74 has a complete T6SS genomic island (GI) that containsclpV,hcp-2, andvgrT6SS genes. We constructedclpV,hcp-2,vgr, and T6SS GI deletion mutants in CF74 and analyzed their effects on the transcriptome overall and, specifically, on the flagellar system at the levels of transcription and translation. Deletion of the T6SS GI affected the transcription of 84 genes, with 15 and 69 genes exhibiting higher and lower levels of transcription, respectively. Members of the cell motility class of downregulated genes of the CF74ΔT6SS mutant were mainly flagellar genes, including effector proteins, chaperones, and regulators. Moreover, the production and secretion of FliC were also decreased inclpV,hcp-2,vgr, or T6SS GI deletion mutants in CF74 and were restored upon complementation. In swimming motility assays, the mutant strains were found to be less motile than the wild type, and motility was restored by complementation. The mutant strains were defective in adhesion to HEp-2 cells and were restored partially upon complementation. Further, the CF74ΔT6SS, CF74ΔclpV, and CF74Δhcp-2mutants induced lower cytotoxicity to HEp-2 cells than the wild type. These results suggested that the T6SS GI in CF74 regulates the flagellar system, enhances motility, is involved in adherence to host cells, and induces cytotoxicity to host cells. Thus, the T6SS plays a wide-ranging role inC. freundii.

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