scholarly journals Type VI Secretion System-Associated Gene Clusters Contribute to Pathogenesis of Salmonella enterica Serovar Typhimurium

2012 ◽  
Vol 80 (6) ◽  
pp. 1996-2007 ◽  
Author(s):  
David T. Mulder ◽  
Colin A. Cooper ◽  
Brian K. Coombes
Keyword(s):  
Salmonella Enterica ◽  
Gene Clusters ◽  
Secretion System ◽  
Host Cells ◽  
Type Vi Secretion System ◽  
Macrophage Infection ◽  
Content Type ◽  
Type Vi Secretion ◽  
Systemic Dissemination ◽  
Serovar Typhimurium

ABSTRACTThe enteropathogenSalmonella entericaserovar Typhimurium employs a suite of tightly regulated virulence factors within the intracellular compartment of phagocytic host cells resulting in systemic dissemination in mice. A type VI secretion system (T6SS) withinSalmonellapathogenicity island 6 (SPI-6) has been implicated in this process; however, the regulatory inputs and the roles of noncore genes in this system are not well understood. Here we describe four clusters of noncore T6SS genes in SPI-6 based on a comparative relationship with the T6SS-3 ofBurkholderia malleiand report that the disruption of these genes results in defects in intracellular replication and systemic dissemination in mice. In addition, we show that the expression of the SPI-6-encoded Hcp and VgrG orthologs is enhanced during late stages of macrophage infection. We identify six regions that are transcriptionally active during cell infections and that have regulatory contributions from the regulators of virulence SsrB, PhoP, and SlyA. We show that levels of protein expression are very weak underin vitroconditions and that expression is not enhanced upon the deletion ofssrB,phoP,slyA,qseC,ompR, orhfq, suggesting an unknown activating factor. These data suggest that the SPI-6 T6SS has been integrated into theSalmonellaTyphimurium virulence network and customized for host-pathogen interactions through the action of noncore genes.

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

2013 ◽  
Vol 81 (4) ◽  
pp. 1207-1220 ◽  
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.

scholarly journals H-NS Silencing of the Salmonella Pathogenicity Island 6-Encoded Type VI Secretion System Limits Salmonella enterica Serovar Typhimurium Interbacterial Killing

2015 ◽  
Vol 83 (7) ◽  
pp. 2738-2750 ◽  
Author(s):  
Yannick R. Brunet ◽  
Ahmad Khodr ◽  
Laureen Logger ◽  
Laurent Aussel ◽  
Tâm Mignot ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Secretion System ◽  
Bacterial Toxin ◽  
Type Vi Secretion System ◽  
Secretion Systems ◽  
Mobility Shift ◽  
Content Type ◽  
Type Vi Secretion ◽  
Serovar Typhimurium ◽  
Electrophoretic Mobility Shift Assays

The secretion of bacterial toxin proteins is achieved by dedicated machineries called secretion systems. The type VI secretion system (T6SS) is a widespread versatile machine used for the delivery of protein toxins to both prokaryotic and eukaryotic cells. InSalmonella entericaserovar Typhimurium, the expression of the T6SS genes is activated during macrophage or mouse infection. Here, we show that the T6SS gene cluster is silenced by the histone-like nucleoid structuring H-NS protein using a combination of reporter fusions, electrophoretic mobility shift assays, DNase footprinting, and fluorescence microscopy. We further demonstrate that derepression of theS. Typhimurium T6SS genes induces T6SS-dependent intoxication of competing bacteria. Our results suggest that relieving T6SS H-NS silencing may be used as a sense-and-kill mechanism that will helpS. Typhimurium to homogenize and synchronize the microbial population to gain efficiency during infection.

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.

scholarly journals Fur-Dam Regulatory Interplay at an Internal Promoter of the Enteroaggregative Escherichia coli Type VI Secretion sci1 Gene Cluster

2020 ◽  
Vol 202 (10) ◽  
Author(s):  
Yannick R. Brunet ◽  
Christophe S. Bernard ◽  
Eric Cascales
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Gene Clusters ◽  
Secretion System ◽  
Target Cells ◽  
Type Vi Secretion System ◽  
Content Type ◽  
Internal Promoter ◽  
E Coli ◽  
Type Vi Secretion

ABSTRACT The type VI secretion system (T6SS) is a weapon for delivering effectors into target cells that is widespread in Gram-negative bacteria. The T6SS is a highly versatile machine, as it can target both eukaryotic and prokaryotic cells, and it has been proposed that T6SSs are adapted to the specific needs of each bacterium. The expression of T6SS gene clusters and the activation of the secretion apparatus are therefore tightly controlled. In enteroaggregative Escherichia coli (EAEC), the sci1 T6SS gene cluster is subject to a complex regulation involving both the ferric uptake regulator (Fur) and DNA adenine methylase (Dam)-dependent DNA methylation. In this study, an additional, internal, promoter was identified within the sci1 gene cluster using +1 transcriptional mapping. Further analyses demonstrated that this internal promoter is controlled by a mechanism strictly identical to that of the main promoter. The Fur binding box overlaps the −10 transcriptional element and a Dam methylation site, GATC-32. Hence, the expression of the distal sci1 genes is repressed and the GATC-32 site is protected from methylation in iron-rich conditions. The Fur-dependent protection of GATC-32 was confirmed by an in vitro methylation assay. In addition, the methylation of GATC-32 negatively impacted Fur binding. The expression of the sci1 internal promoter is therefore controlled by iron availability through Fur regulation, whereas Dam-dependent methylation maintains a stable ON expression in iron-limited conditions. IMPORTANCE Bacteria use weapons to deliver effectors into target cells. One of these weapons, the type VI secretion system (T6SS), assembles a contractile tail acting as a spring to propel a toxin-loaded needle. Its expression and activation therefore need to be tightly regulated. Here, we identified an internal promoter within the sci1 T6SS gene cluster in enteroaggregative E. coli. We show that this internal promoter is controlled by Fur and Dam-dependent methylation. We further demonstrate that Fur and Dam compete at the −10 transcriptional element to finely tune the expression of T6SS genes. We propose that this elegant regulatory mechanism allows the optimum production of the T6SS in conditions where enteroaggregative E. coli encounters competing species.

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 Cytosporone B, an Inhibitor of the Type III Secretion System of Salmonella enterica Serovar Typhimurium

2013 ◽  
Vol 57 (5) ◽  
pp. 2191-2198 ◽  
Author(s):  
Jianfang Li ◽  
Chao Lv ◽  
Weiyang Sun ◽  
Zhenyu Li ◽  
Xiaowei Han ◽  
...  
Keyword(s):  
Type Iii Secretion ◽  
Salmonella Enterica ◽  
Bacterial Resistance ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Effector Proteins ◽  
Host Cells ◽  
Content Type ◽  
Type Iii ◽  
Serovar Typhimurium

ABSTRACTBacterial virulence factors have been increasingly regarded as attractive targets for development of novel antibacterial agents. Virulence inhibitors are less likely to generate bacterial resistance, which makes them superior to traditional antibiotics that target bacterial viability.Salmonella entericaserovar Typhimurium, an important food-borne human pathogen, has type III secretion system (T3SS) as its major virulence factor. T3SS secretes effector proteins to facilitate invasion into host cells. In this study, we identified several analogs of cytosporone B (Csn-B) that strongly block the secretion ofSalmonellapathogenicity island 1 (SPI-1)-associated effector proteins, without affecting the secretion of flagellar protein FliCin vitro. Csn-B and two other derivatives exhibited a strong inhibitory effect on SPI-1-mediated invasion to HeLa cells, while no significant toxicity to bacteria was observed. Nucleoid proteins Hha and H-NS bind to the promoters of SPI-1 regulator geneshilD,hilC, andrtsAto repress their expression and consequently regulate the expression of SPI-1 apparatus and effector genes. We found that Csn-B upregulated the transcription ofhhaandhns, implying that Csn-B probably affected the secretion of effectors through the Hha–H-NS regulatory pathway. In summary, this study presented an effective SPI-1 inhibitor, Csn-B, which may have potential in drug development against antibiotic-resistantSalmonella.

scholarly journals Barcoded Consortium Infections Resolve Cell Type-Dependent Salmonella enterica Serovar Typhimurium Entry Mechanisms

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Maria Letizia Di Martino ◽  
Viktor Ek ◽  
Wolf-Dietrich Hardt ◽  
Jens Eriksson ◽  
Mikael E. Sellin
Keyword(s):  
Host Cell ◽  
Salmonella Enterica ◽  
Cell Types ◽  
Secretion System ◽  
Host Cells ◽  
Host Cell Invasion ◽  
Cell Type ◽  
Content Type ◽  
Invasion Mechanisms ◽  
Serovar Typhimurium

ABSTRACT Bacterial host cell invasion mechanisms depend on the bacterium’s virulence factors and the properties of the target cell. The enteropathogen Salmonella enterica serovar Typhimurium (S.Tm) invades epithelial cell types in the gut mucosa and a variety of immune cell types at later infection stages. The molecular mechanism(s) of host cell entry has, however, been studied predominantly in epithelial cell lines. S.Tm uses a type three secretion system (TTSS-1) to translocate effectors into the host cell cytosol, thereby sparking actin ruffle-dependent entry. The ruffles also fuel cooperative invasion by bystander bacteria. In addition, several TTSS-1-independent entry mechanisms exist, involving alternative S.Tm virulence factors, or the passive uptake of bacteria by phagocytosis. However, it remains ill-defined how S.Tm invasion mechanisms vary between host cells. Here, we developed an internally controlled and scalable method to map S.Tm invasion mechanisms across host cell types and conditions. The method relies on host cell infections with consortia of chromosomally tagged wild-type and mutant S.Tm strains, where the abundance of each strain can be quantified by qPCR or amplicon sequencing. Using this methodology, we quantified cooccurring TTSS-1-dependent, cooperative, and TTSS-1-independent invasion events in epithelial, monocyte, and macrophage cells. We found S.Tm invasion of epithelial cells and monocytes to proceed by a similar MOI-dependent mix of TTSS-1-dependent and cooperative mechanisms. TTSS-1-independent entry was more frequent in macrophages. Still, TTSS-1-dependent invasion dominated during the first minutes of interaction also with this cell type. Finally, the combined action of the SopB/SopE/SopE2 effectors was sufficient to explain TTSS-1-dependent invasion across both epithelial and phagocytic cells. IMPORTANCE Salmonella enterica serovar Typhimurium (S.Tm) is a widespread and broad-host-spectrum enteropathogen with the capacity to invade diverse cell types. Still, the molecular basis for the host cell invasion process has largely been inferred from studies of a few selected cell lines. Our work resolves the mechanisms that Salmonellae employ to invade prototypical host cell types, i.e., human epithelial, monocyte, and macrophage cells, at a previously unattainable level of temporal and quantitative precision. This highlights efficient bacterium-driven entry into innate immune cells and uncovers a type III secretion system effector module that dominates active bacterial invasion of not only epithelial cells but also monocytes and macrophages. The results are derived from a generalizable method, where we combine barcoding of the bacterial chromosome with mixed consortium infections of cultured host cells. The application of this methodology across bacterial species and infection models will provide a scalable means to address host-pathogen interactions in diverse contexts.

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

2012 ◽  
Vol 79 (1) ◽  
pp. 32-38 ◽  
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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