Pandemic Vibrio cholerae shuts down site-specific recombination to retain an interbacterial defence mechanism

AbstractVibrio cholerae is an aquatic microbe that can be divided into three subtypes: harmless environmental strains, localised pathogenic strains, and pandemic strains causing global cholera outbreaks. Each type has a contact-dependent type VI secretion system (T6SS) that kills neighbouring competitors by translocating unique toxic effector proteins. Pandemic isolates possess identical effectors, indicating that T6SS effectors may affect pandemicity. Here, we show that one of the T6SS gene clusters (Aux3) exists in two states: a mobile, prophage-like element in a small subset of environmental strains, and a truncated Aux3 unique to and conserved in pandemic isolates. Environmental Aux3 can be readily excised from and integrated into the genome via site-specific recombination, whereas pandemic Aux3 recombination is reduced. Our data suggest that environmental Aux3 acquisition conferred increased competitive fitness to pre-pandemic V. cholerae, leading to grounding of the element in the chromosome and propagation throughout the pandemic clade.

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Analysis of Vibrio cholerae genomes using a novel bioinformatic tool identifies new, active Type VI Secretion System gene clusters

10.1101/526723 ◽

2019 ◽

Author(s):

Cristian V. Crisan ◽

Aroon T. Chande ◽

Kenneth Williams ◽

Vishnu Raghuram ◽

Lavanya Rishishwar ◽

...

Keyword(s):

Vibrio Cholerae ◽

Bacterial Species ◽

Gene Clusters ◽

Secretion System ◽

Effector Proteins ◽

Type Vi Secretion System ◽

Type Vi Secretion ◽

Bioinformatic Tool ◽

Immunity Factor ◽

Auxiliary Gene

AbstractBackgroundLike many bacteria, Vibrio cholerae, which causes fatal cholera, deploys a harpoon-like Type VI Secretion System (T6SS) to compete against other microbes in environmental and host settings. The T6SS punctures adjacent cells and delivers toxic effector proteins that are harmless to bacteria carrying cognate immunity factors. Only four effector/immunity pairs encoded on one large and three auxiliary gene clusters have been characterized from largely clonal, patient-derived strains of V. cholerae.ResultsWe sequenced two dozen V. cholerae strain genomes from diverse sources and developed a novel and adaptable bioinformatic tool based on Hidden Markov Models. We identified two new T6SS auxiliary gene clusters; one, Aux 5, is described here. Four Aux 5 loci are present in the host strain, each with an atypical effector/immunity gene organization. Structural prediction of the putative effector indicated it is a lipase, which we name TleV1 (Type VI lipase effector Vibrio, TleV1). Ectopic TleV1 expression induced toxicity in E. coli, which was rescued by co-expression of the TleV1 immunity factor. A clinical V. cholerae reference strain expressing the Aux 5 cluster used TleV1 to lyse its parental strain upon contact via its T6SS but was unable to kill parental cells expressing TleV1’s immunity factor.ConclusionWe developed a novel bioinformatic method and identified new T6SS gene clusters in V. cholerae. We also showed the TleV1 toxin is delivered in a T6SS-manner by V. cholerae and can lyse other bacterial cells. Our web-based tool may be modified to identify additional novel T6SS genomic loci in diverse bacterial species.

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

10.1101/868539 ◽

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.

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

Infection and Immunity ◽

10.1128/iai.01266-10 ◽

2011 ◽

Vol 79 (7) ◽

pp. 2941-2949 ◽

Cited By ~ 118

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.

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When the pandemic opts for the lockdown: Secretion system evolution in the cholera bacterium

Microbial Cell ◽

10.15698/mic2021.03.744 ◽

2021 ◽

Vol 8 (3) ◽

pp. 69-72

Author(s):

Francis J. Santoriello ◽

Stefan Pukatzki

Keyword(s):

Common Ancestor ◽

Diarrheal Disease ◽

Secretion System ◽

Effector Proteins ◽

Type Vi Secretion System ◽

Human Gastrointestinal Tract ◽

Type Vi Secretion ◽

T4 Bacteriophage ◽

The Common ◽

Pandemic Strains

Vibrio cholerae, the causative agent of the diarrheal disease cholera, is a microbe capable of inhabiting two different ecosystems: chitinous surfaces in brackish, estuarine waters and the epithelial lining of the human gastrointestinal tract. V. cholerae defends against competitive microorganisms with a contact-dependent, contractile killing machine called the type VI secretion system (T6SS) in each of these niches. The T6SS resembles an inverted T4 bacteriophage tail and is used to deliver toxic effector proteins into neighboring cells. Pandemic strains of V. cholerae encode a unique set of T6SS effector proteins, which may play a role in pathogenesis or pandemic spread. In our recent study (Santoriello et al. (2020), Nat Commun, doi: 10.1038/s41467-020-20012-7), using genomic and molecular biology tools, we demonstrated that the T6SS island Auxiliary Cluster 3 (Aux3) is unique to pandemic strains of V. cholerae. We went on to show that Aux3 is related to a phage-like element circulating in environmental V. cholerae strains and that two genetic domestication events formed the pandemic Aux3 cluster during the evolution of the pandemic clone. Our findings support two main conclusions: (1) Aux3 evolution from phage-like element to T6SS cluster offers a snapshot of phage domestication in early T6SS evolution and (2) chromosomal maintenance of Aux3 was advantageous to the common ancestor of V. cholerae pandemic strains.

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A genomic island in Vibrio cholerae with VPI-1 site-specific recombination characteristics contains CRISPR-Cas and type VI secretion modules

Scientific Reports ◽

10.1038/srep36891 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 18

Author(s):

Maurizio Labbate ◽

Fabini D. Orata ◽

Nicola K. Petty ◽

Nathasha D. Jayatilleke ◽

William L. King ◽

...

Keyword(s):

Vibrio Cholerae ◽

Genomic Island ◽

Site Specific ◽

Site Specific Recombination ◽

Type Vi Secretion

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Interbacterial competition and anti-predatory behavior of environmental Vibrio cholerae strains

10.1101/2020.07.24.220178 ◽

2020 ◽

Author(s):

Natália C. Drebes Dörr ◽

Melanie Blokesch

Keyword(s):

Whole Genome Sequencing ◽

Vibrio Cholerae ◽

Genome Sequencing ◽

Secretion System ◽

Whole Genome ◽

Type Vi Secretion System ◽

Environmental Isolates ◽

Type Vi Secretion ◽

Pandemic Strains ◽

Constitutively Active

SummaryVibrio cholerae isolates responsible for cholera pandemics represent only a small portion of the diverse strains belonging to this species. Indeed, most V. cholerae are encountered in aquatic environments. To better understand the emergence of pandemic lineages, it is crucial to discern what differentiates pandemic strains from their environmental relatives. Here, we studied the interaction of environmental V. cholerae with eukaryotic predators or competing bacteria and tested the contributions of the hemolysin and the type VI secretion system (T6SS) to those interactions. Both of these molecular weapons are constitutively active in environmental isolates but subject to tight regulation in the pandemic clade. We showed that several environmental isolates resist amoebal grazing and that this anti-grazing defense relies on the strains’ T6SS and its actin-cross-linking domain (ACD)-containing tip protein. Strains lacking the ACD were unable to defend themselves against grazing amoebae but maintained high levels of T6SS-dependent interbacterial killing. We explored the latter phenotype through whole-genome sequencing of fourteen isolates, which unveiled a wide array of novel T6SS effector and (orphan) immunity proteins. By combining these in silico predictions with experimental validations, we showed that highly similar but nonidentical immunity proteins were insufficient to provide cross-immunity among those wild strains.Originality-Significance StatementThis work contributes to the understanding of phenotypic consequences that differentiate diverse Vibrio cholerae strains. We focused on the type VI secretion system (T6SS) and the pore forming toxin hemolysin, which are tightly regulated in pandemic strains but remain constitutively active in non-pandemic isolates. We unveiled diverse arrays of T6SS effector/immunity modules in a set of environmental strains by long-read whole genome sequencing and de novo assembly. These modules determine whether the strains are able to evade amoebal predation and dictate their level of compatibility or competitiveness with one another.

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Analysis of Vibrio cholerae genomes identifies new type VI secretion system gene clusters

Genome Biology ◽

10.1186/s13059-019-1765-5 ◽

2019 ◽

Vol 20 (1) ◽

Cited By ~ 9

Author(s):

Cristian V. Crisan ◽

Aroon T. Chande ◽

Kenneth Williams ◽

Vishnu Raghuram ◽

Lavanya Rishishwar ◽

...

Keyword(s):

Vibrio Cholerae ◽

Gene Clusters ◽

Secretion System ◽

Type Vi Secretion System ◽

Type Vi Secretion ◽

New Type

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Type VI secretion system mutations reduced competitive fitness of classical Vibrio cholerae biotype

Nature Communications ◽

10.1038/s41467-021-26847-y ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Benjamin Kostiuk ◽

Francis J. Santoriello ◽

Laura Diaz-Satizabal ◽

Fabiana Bisaro ◽

Kyung-Jo Lee ◽

...

Keyword(s):

Vibrio Cholerae ◽

Diarrhoeal Disease ◽

Secretion System ◽

Type Vi Secretion System ◽

Genetic Determinants ◽

Microbial Competition ◽

Type Vi Secretion ◽

Pandemic Strains ◽

El Tor ◽

Microbial Attack

AbstractThe gram-negative bacterium Vibrio cholerae is the causative agent of the diarrhoeal disease cholera and is responsible for seven recorded pandemics. Several factors are postulated to have led to the decline of 6th pandemic classical strains and the rise of El Tor biotype V. cholerae, establishing the current 7th pandemic. We investigated the ability of classical V. cholerae of the 2nd and 6th pandemics to engage their type six secretion system (T6SS) in microbial competition against non-pandemic and 7th pandemic strains. We report that classical V. cholerae underwent sequential mutations in T6SS genetic determinants that initially exposed 2nd pandemic strains to microbial attack by non-pandemic strains and subsequently caused 6th pandemic strains to become vulnerable to El Tor biotype V. cholerae intraspecific competition. The chronology of these T6SS-debilitating mutations agrees with the decline of 6th pandemic classical strains and the emergence of 7th pandemic El Tor V. cholerae.

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