In vitro Trypanocidal Activity, Genomic Analysis of Isolates, and in vivo Transcription of Type VI Secretion System of Serratia marcescens Belonging to the Microbiota of Rhodnius prolixus Digestive Tract

Yersinia pestis cells were grown in vitro at 26 and 37 °C, the ambient temperatures of its flea vector and its mammalian hosts, respectively, and subjected to subcellular fractionation. Abundance changes at 26 vs 37 °C were observed for many outer-membrane (OM) proteins. The cell adhesion protein Ail (y1324) and three putative small β-barrel OM proteins (y1795, y2167 and y4083) were strongly increased at 37 °C. The Ail/Lom family protein y1682 (OmpX) was strongly increased at 26 °C. Several porins and TonB-dependent receptors, which control small molecule transport through the OM, were also altered in abundance in a temperature-dependent manner. These marked differences in the composition of the OM proteome are probably important for the adaptation of Y. pestis to its in vivo life stages. Thirteen proteins that appear to be part of an intact type VI secretion system (T6SS) were identified in membrane fractions of stationary-phase cells grown at 26 °C, but not at 37 °C. The corresponding genes are clustered in the Y. pestis KIM gene locus y3658–y3677. The proteins y3674 and y3675 were particularly abundant and co-fractionated in a M r range indicative of participation in a multi-subunit complex. The soluble haemolysin-coregulated protein y3673 was even more abundant. Its release into the extracellular medium was triggered by treatment of Y. pestis cells with trypsin. Proteases and other stress-response-inducing factors may constitute environmental cues resulting in the activation of the T6SS in Y. pestis.

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Bacterial symbionts use a type VI secretion system to eliminate competitors in their natural host

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1808302115 ◽

2018 ◽

Vol 115 (36) ◽

pp. E8528-E8537 ◽

Cited By ~ 56

Author(s):

Lauren Speare ◽

Andrew G. Cecere ◽

Kirsten R. Guckes ◽

Stephanie Smith ◽

Michael S. Wollenberg ◽

...

Keyword(s):

Intraspecific Competition ◽

Vibrio Fischeri ◽

Secretion System ◽

Dependent Manner ◽

Light Organ ◽

Type Vi Secretion System ◽

Type Vi Secretion ◽

Natural Isolates ◽

Physical Mixing

Intraspecific competition describes the negative interaction that occurs when different populations of the same species attempt to fill the same niche. Such competition is predicted to occur among host-associated bacteria but has been challenging to study in natural biological systems. Although many bioluminescentVibrio fischeristrains exist in seawater, only a few strains are found in the light-organ crypts of an individual wild-caughtEuprymna scolopessquid, suggesting a possible role for intraspecific competition during early colonization. Using a culture-based assay to investigate the interactions of differentV. fischeristrains, we found “lethal” and “nonlethal” isolates that could kill or not kill the well-studied light-organ isolate ES114, respectively. The killing phenotype of these lethal strains required a type VI secretion system (T6SS) encoded in a 50-kb genomic island. Multiple lethal and nonlethal strains could be cultured from the light organs of individual wild-caught adult squid. Although lethal strains eliminate nonlethal strains in vitro, two lethal strains could coexist in interspersed microcolonies that formed in a T6SS-dependent manner. This coexistence was destabilized upon physical mixing, resulting in one lethal strain consistently eliminating the other. When juvenile squid were coinoculated with lethal and nonlethal strains, they occupied different crypts, yet they were observed to coexist within crypts when T6SS function was disrupted. These findings, using a combination of natural isolates and experimental approaches in vitro and in the animal host, reveal the importance of T6SS in spatially separating strains during the establishment of host colonization in a natural symbiosis.

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Evolution of a cis-acting SNP that controls Type VI Secretion in Vibrio cholerae

10.1101/2022.01.11.475911 ◽

2022 ◽

Author(s):

Siu Lung Ng ◽

Sophia A. Kammann ◽

Gabi Steinbach ◽

Tobias Hoffmann ◽

Peter J. Yunker ◽

...

Keyword(s):

Vibrio Cholerae ◽

Phenotypic Diversity ◽

Constitutive Expression ◽

Regulatory Elements ◽

Regulatory Control ◽

Type Vi Secretion System ◽

Type Vi Secretion ◽

Intergenic Regions

Mutations in regulatory mechanisms that control gene expression contribute to phenotypic diversity and thus facilitate the adaptation of microbes to new niches. Regulatory architecture is often inferred from transcription factor identification and genome analysis using purely computational approaches. However, there are few examples of phenotypic divergence that arise from the rewiring of bacterial regulatory circuity by mutations in intergenic regions, because locating regulatory elements within regions of DNA that do not code for protein requires genomic and experimental data. We identify a single cis-acting single nucleotide polymorphism (SNP) dramatically alters control of the type VI secretion system (T6), a common weapon for inter-bacterial competition. Tight T6 regulatory control is necessary for adaptation of the waterborne pathogen Vibrio cholerae to in vivo conditions within the human gut, which we show can be altered by this single non-coding SNP that results in constitutive expression in vitro. Our results support a model of pathogen evolution through cis-regulatory mutation and preexisting, active transcription factors, thus conferring different fitness advantages to tightly regulated strains inside a human host and unfettered strains adapted to environmental niches.

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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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The Type VI Secretion System Encoded in Salmonella Pathogenicity Island 19 Is Required for Salmonella enterica Serotype Gallinarum Survival within Infected Macrophages

Infection and Immunity ◽

10.1128/iai.01165-12 ◽

2013 ◽

Vol 81 (4) ◽

pp. 1207-1220 ◽

Cited By ~ 36

Author(s):

Carlos J. Blondel ◽

Juan C. Jiménez ◽

Lorenzo E. Leiva ◽

Sergio A. Álvarez ◽

Bernardo I. Pinto ◽

...

Keyword(s):

Salmonella Enterica ◽

Pathogenicity Island ◽

Secretion System ◽

Host Cells ◽

Economic Losses ◽

Type Vi Secretion System ◽

Content Type ◽

Type Vi Secretion ◽

Core Components

ABSTRACTSalmonella entericaserotype Gallinarum is the causative agent of fowl typhoid, a disease characterized by high morbidity and mortality that causes major economic losses in poultry production. We have reported thatS. Gallinarum harbors a type VI secretion system (T6SS) encoded inSalmonellapathogenicity island 19 (SPI-19) that is required for efficient colonization of chicks. In the present study, we aimed to characterize the SPI-19 T6SS functionality and to investigate the mechanisms behind the phenotypes previously observedin vivo. Expression analyses revealed that SPI-19 T6SS core components are expressed and produced underin vitrobacterial growth conditions. However, secretion of the structural/secreted components Hcp1, Hcp2, and VgrG to the culture medium could not be determined, suggesting that additional signals are required for T6SS-dependent secretion of these proteins.In vitrobacterial competition assays failed to demonstrate a role for SPI-19 T6SS in interbacterial killing. In contrast, cell culture experiments with murine and avian macrophages (RAW264.7 and HD11, respectively) revealed production of a green fluorescent protein-tagged version of VgrG soon afterSalmonellauptake. Furthermore, infection of RAW264.7 and HD11 macrophages with deletion mutants of SPI-19 or strains with genes encoding specific T6SS core components (clpVandvgrG) revealed that SPI-19 T6SS contributes toS. Gallinarum survival within macrophages at 20 h postuptake. SPI-19 T6SS function was not linked toSalmonella-induced cytotoxicity or cell death of infected macrophages, as has been described for other T6SS. Our data indicate that SPI-19 T6SS corresponds to a novel tool used bySalmonellato survive within host cells.

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A new family of Type VI secretion system-delivered effector proteins displays ion-selective pore-forming activity

10.1101/676247 ◽

2019 ◽

Author(s):

Giuseppina Mariano ◽

Katharina Trunk ◽

David J. Williams ◽

Laura Monlezun ◽

Henrik Strahl ◽

...

Keyword(s):

Effector Proteins ◽

Type Vi Secretion System ◽

Secretion Systems ◽

New Family ◽

Type Vi Secretion ◽

Outer Membrane Permeability ◽

Polymicrobial Communities ◽

Bacterial Effectors

AbstractType VI secretion systems (T6SSs) are nanomachines widely used by bacteria to compete with rivals. T6SSs deliver multiple toxic effector proteins directly into neighbouring cells and play key roles in shaping diverse polymicrobial communities. A number of families of T6SS-dependent anti-bacterial effectors have been characterised, however the mode of action of others remains unknown. Here we report that Ssp6, an anti-bacterial effector delivered by theSerratia marcescensT6SS, is an ion-selective pore-forming toxin.In vivo, Ssp6 inhibits growth by causing depolarisation of the inner membrane of intoxicated cells and also leads to increased outer membrane permeability, whilst reconstruction of Ssp6 activityin vitrodemonstrated that it forms cation-selective pores. A survey of bacterial genomes revealed that Ssp6-like effectors are widespread in Enterobacteriaceae and often linked with T6SS genes. We conclude that Ssp6 represents a new family of T6SS-delivered anti-bacterial effectors, further diversifying the portfolio of weapons available for deployment during inter-bacterial conflict.

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Differential Gene Expression Patterns ofYersinia pestisandYersinia pseudotuberculosisduring Infection and Biofilm Formation in the Flea Digestive Tract

mSystems ◽

10.1128/msystems.00217-18 ◽

2019 ◽

Vol 4 (1) ◽

Cited By ~ 8

Author(s):

Iman Chouikha ◽

Daniel E. Sturdevant ◽

Clayton Jarrett ◽

Yi-Cheng Sun ◽

B. Joseph Hinnebusch

Keyword(s):

Digestive Tract ◽

Biofilm Formation ◽

Yersinia Pseudotuberculosis ◽

Expression Patterns ◽

Type Vi Secretion System ◽

Genetic Changes ◽

Content Type ◽

Biofilm Development

ABSTRACTYersinia pestis, the etiologic agent of plague, emerged as a fleaborne pathogen only within the last 6,000 years. Just five simple genetic changes in theYersinia pseudotuberculosisprogenitor, which served to eliminate toxicity to fleas and to enhance survival and biofilm formation in the flea digestive tract, were key to the transition to the arthropodborne transmission route. To gain a deeper understanding of the genetic basis for the development of a transmissible biofilm infection in the flea foregut, we evaluated additional gene differences and performedin vivotranscriptional profiling ofY. pestis, aY. pseudotuberculosiswild-type strain (unable to form biofilm in the flea foregut), and aY. pseudotuberculosismutant strain (able to produce foregut-blocking biofilm in fleas) recovered from fleas 1 day and 14 days after an infectious blood meal. Surprisingly, theY. pseudotuberculosismutations that increased c-di-GMP levels and enabled biofilm development in the flea did not change the expression levels of thehmsgenes responsible for the synthesis and export of the extracellular polysaccharide matrix required for mature biofilm formation. TheY. pseudotuberculosismutant uniquely expressed much higher levels ofYersiniatype VI secretion system 4 (T6SS-4) in the flea, and this locus was required for flea blockage byY. pseudotuberculosisbut not for blockage byY. pestis. Significant differences between the two species in expression of several metabolism genes, the Psa fimbrial genes, quorum sensing-related genes, transcription regulation genes, and stress response genes were evident during flea infection.IMPORTANCEY. pestisemerged as a highly virulent, arthropod-transmitted pathogen on the basis of relatively few and discrete genetic changes fromY. pseudotuberculosis. Parallel comparisons of thein vitroandin vivotranscriptomes ofY. pestisand twoY. pseudotuberculosisvariants that produce a nontransmissible infection and a transmissible infection of the flea vector, respectively, provided insights into howY. pestishas adapted to life in its flea vector and point to evolutionary changes in the regulation of metabolic and biofilm development pathways in these two closely related species.

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In vivo actin cross-linking induced by Vibrio cholerae type VI secretion system is associated with intestinal inflammation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0915156107 ◽

2010 ◽

Vol 107 (9) ◽

pp. 4365-4370 ◽

Cited By ~ 120

Author(s):

A. T. Ma ◽

J. J. Mekalanos

Keyword(s):

Vibrio Cholerae ◽

Intestinal Inflammation ◽

Secretion System ◽

Cross Linking ◽

Type Vi Secretion System ◽

Type Vi Secretion

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Promoter Swapping Unveils the Role of the Citrobacter rodentium CTS1 Type VI Secretion System in Interbacterial Competition

Applied and Environmental Microbiology ◽

10.1128/aem.02504-12 ◽

2012 ◽

Vol 79 (1) ◽

pp. 32-38 ◽

Cited By ~ 34

Author(s):

Erwan Gueguen ◽

Eric Cascales

Keyword(s):

Gene Clusters ◽

Secretion System ◽

Citrobacter Rodentium ◽

Type Vi Secretion System ◽

Content Type ◽

Type Vi Secretion ◽

Artificial Induction ◽

Release Assay ◽

Promoter Swapping

ABSTRACT The type VI secretion system (T6SS) is a versatile secretion machine dedicated to various functions in Gram-negative bacteria, including virulence toward eukaryotic cells and antibacterial activity. Activity of T6SS might be followed in vitro by the release of two proteins, Hcp and VgrG, in the culture supernatant. Citrobacter rodentium , a rodent pathogen, harbors two T6SS gene clusters, cts1 and cts2 . Reporter fusion and Hcp release assays suggested that the CTS1 T6SS was not produced or not active. The cts1 locus is composed of two divergent operons. We therefore developed a new vector allowing us to swap the two divergent endogenous promoters by P tac and P BAD using the λ red recombination technology. Artificial induction of both promoters demonstrated that the CTS1 T6SS is functional as shown by the Hcp release assay and confers on C. rodentium a growth advantage in antibacterial competition experiments with Escherichia coli .

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TheVibrio choleraeType VI Secretion System Can Modulate Host Intestinal Mechanics to Displace Commensal Gut Bacteria

10.1101/226472 ◽

2017 ◽

Cited By ~ 2

Author(s):

Savannah L. Logan ◽

Jacob Thomas ◽

Jinyuan Yan ◽

Ryan P. Baker ◽

Drew S. Shields ◽

...

Keyword(s):

Secretion System ◽

Effector Proteins ◽

Target Cells ◽

Type Vi Secretion System ◽

Zebrafish Model ◽

Microbial Competition ◽

Aeromonas Veronii ◽

Type Vi Secretion ◽

Commensal Strain

AbstractHost-associated microbiota help defend against bacterial pathogens; the mechanisms that pathogens possess to overcome this defense, however, remain largely unknown. We developed a zebrafish model and used live imaging to directly study how the human pathogenVibrio choleraeinvades the intestine. The gut microbiota of fish mono-colonized by commensal strainAeromonas veroniiwas displaced byV. choleraeexpressing its Type VI Secretion System (T6SS), a syringe-like apparatus that deploys effector proteins into target cells. Surprisingly, displacement was independent of T6SS-mediated killing ofAeromonas, driven instead by T6SS-induced enhancement of zebrafish intestinal movements that led to expulsion of the resident commensal by the host. Deleting an actin crosslinking domain from the T6SS apparatus returned intestinal motility to normal and thwarted expulsion, without weakeningV. cholerae′sability to killAeromonas in vitro. Our finding that bacteria can manipulate host physiology to influence inter-microbial competition has implications for both pathogenesis and microbiome engineering.

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