DksA coordinates bile-mediated regulation of virulence-associated phenotypes in type three secretion system-positive Vibrio cholerae

In order to cause disease, pathogenic strains of Vibrio cholerae rely on intricate regulatory networks to orchestrate the transition between their native aquatic environment and the human host. For example, bacteria in a nutrient-starved environment undergo a metabolic shift called the stringent response, which is mediated by the alarmone ppGpp and an RNA-polymerase binding transcriptional factor, DksA. In O1 serogroup strains of V. cholerae, which use the toxin co-regulated pilus (TCP) and cholera toxin (CT) as primary virulence factors, DksA was reported to have additional functions as a mediator of virulence gene expression. However, little is known about the regulatory networks coordinating virulence phenotypes in pathogenic strains that use TCP/CT-independent virulence mechanisms. We therefore investigated whether functions of DksA outside of the stringent response are conserved in type three secretion system (T3SS)-positive V. cholerae . In using the T3SS-positive clinically isolated O39 serogroup strain AM-19226, we observed an increase in dksA expression in the presence of bile at 37 °C. However, DksA was not required for wild-type levels of T3SS structural gene expression, or for colonization in vivo. Rather, data indicate that DksA positively regulates the expression of master regulators in the motility hierarchy. Interestingly, the ΔdksA strain forms a less robust biofilm than the WT parent strain at both 30 and 37 °C. We also found that DksA regulates the expression of hapR, encoding a major regulator of biofilm formation and protease expression. Athough DksA does not appear to modulate T3SS virulence factor expression, its activity is integrated into existing regulatory networks governing virulence-related phenotypes. Strain variations therefore may take advantage of conserved ancestral proteins to expand regulons responding to in vivo signals and thus coordinate multiple phenotypes important for infection.

vttRA and vttRB Encode ToxR Family Proteins That Mediate Bile-Induced Expression of Type Three Secretion System Genes in a Non-O1/Non-O139 Vibrio cholerae Strain

10.1128/iai.01073-09 ◽

2010 ◽

Vol 78 (6) ◽

pp. 2554-2570 ◽

Cited By ~ 21

Author(s):

Ashfaqul Alam ◽

Vincent Tam ◽

Elaine Hamilton ◽

Michelle Dziejman

Keyword(s):

Gene Expression ◽

Vibrio Cholerae ◽

Secretion System ◽

Regulatory Proteins ◽

Cholerae Strain ◽

Genes Encoding ◽

Intergenic Regions ◽

ABSTRACT Strain AM-19226 is a pathogenic non-O1/non-O139 serogroup Vibrio cholerae strain that does not encode the toxin-coregulated pilus or cholera toxin but instead causes disease using a type three secretion system (T3SS). Two genes within the T3SS pathogenicity island, herein named vttR A (locus tag A33_1664) and vttR B (locus tag A33_1675), are predicted to encode proteins that show similarity to the transcriptional regulator ToxR, which is found in all strains of V. cholerae. Strains with a deletion of vttR A or vttR B showed attenuated colonization in vivo, indicating that the T3SS-encoded regulatory proteins play a role in virulence. lacZ transcriptional reporter fusions to intergenic regions upstream of genes encoding the T3SS structural components identified growth in the presence of bile as a condition that modulates gene expression. Under this condition, VttRA and VttRB were necessary for maximal gene expression. In contrast, growth in bile did not substantially alter the expression of a reporter fusion to the vopF gene, which encodes an effector protein. Increased vttR B reporter fusion activity was observed in a ΔvttR B strain background, suggesting that VttRB may regulate its own expression. The collective results are consistent with the hypothesis that T3SS-encoded regulatory proteins are essential for pathogenesis and control the expression of selected T3SS genes.

Laboratory Adaptation of Bordetella pertussis Is Associated with the Loss of Type Three Secretion System Functionality

10.1128/iai.00136-11 ◽

2011 ◽

Vol 79 (9) ◽

pp. 3677-3682 ◽

Cited By ~ 23

Author(s):

M. E. Gaillard ◽

D. Bottero ◽

C. E. Castuma ◽

L. A. Basile ◽

D. Hozbor

Keyword(s):

Bordetella Pertussis ◽

Growth Conditions ◽

Secretion System ◽

Clinical Isolates ◽

Content Type ◽

Associated Proteins ◽

ABSTRACTAlthoughBordetella pertussiscontains and transcribes loci encoding type III secretion system (TTSS) homologues, expression of TTSS-associated proteins has been reported only for non-laboratory-adapted Irish clinical isolates. Here we confirm such a result for clinical isolates obtained from patients treated in Argentinean hospitals. Moreover, we demonstrate that the expression of TTSS-associated proteins is independent both of the year in which the isolate was obtained and of the types of polymorphic alleles for other virulence factors but is dependent on environmental growth conditions. Interestingly, we observed that TTSS-associated protein expression is lost after successivein vitropassages but becomes operative again when bacteria come into contact with the host. Thisin vivoactivation of TTSS expression was observed not only for clinical isolates previously adapted to the laboratory after successivein vitropassages but also for vaccine strains that did not express the systemin vitro. The reversibility of TTSS expression, demonstrated by its switching off-on when the bacterium comes into contact with the host, appears to be an adaptive response of this pathogen.

HilD, HilC, and RtsA Form Homodimers and Heterodimers To Regulate Expression of the Salmonella Pathogenicity Island I Type III Secretion System

Journal of Bacteriology ◽

10.1128/jb.00012-20 ◽

2020 ◽

Vol 202 (9) ◽

Cited By ~ 1

Author(s):

Koh-Eun Narm ◽

Marinos Kalafatis ◽

James M. Slauch

Keyword(s):

Gene Expression ◽

Dna Sequences ◽

Systemic Disease ◽

Pathogenicity Island ◽

Secretion System ◽

Dimerization Domain ◽

Content Type ◽

Network Function ◽

ABSTRACT Salmonella enterica serovar Typhimurium colonizes and invades host intestinal epithelial cells using the type three secretion system (T3SS) encoded on Salmonella pathogenicity island 1 (SPI1). The level of SPI1 T3SS gene expression is controlled by the transcriptional activator HilA, encoded on SPI1. Expression of hilA is positively regulated by three homologous transcriptional regulators, HilD, HilC, and RtsA, belonging to the AraC/XylS family. These regulators also activate the hilD, hilC, and rtsA genes by binding to the same DNA sequences upstream of these promoters, forming a complex feed-forward loop to control SPI1 expression. Despite the apparent redundancy in function, HilD has a unique role in SPI1 regulation because the majority of external regulatory inputs act exclusively through HilD. To better understand SPI1 regulation, the nature of interaction between HilD, HilC, and RtsA has been characterized using biochemical and genetic techniques. Our results showed that HilD, HilC, and RtsA can form heterodimers as well as homodimers in solution. Comparison with other AraC family members identified a putative α-helix in the N-terminal domain, which acts as the dimerization domain. Alanine substitution in this region results in reduced dimerization of HilD and HilC and also affects their ability to activate hilA expression. The dimer interactions of HilD, HilC, and RtsA add another layer of complexity to the SPI1 regulatory circuit, providing a more comprehensive understanding of SPI1 T3SS regulation and Salmonella pathogenesis. IMPORTANCE The SPI1 type three secretion system is a key virulence factor required for Salmonella to both cause gastroenteritis and initiate serious systemic disease. The system responds to numerous environmental signals in the intestine, integrating this information via a complex regulatory network. Here, we show that the primary regulatory proteins in the network function as both homodimers and heterodimers, providing information regarding both regulation of virulence in this important pathogen and general signal integration to control gene expression.

In-vitro and in-vivo analysis of the production of the Bordetella type three secretion system effector A in Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica

Microbes and Infection ◽

10.1016/j.micinf.2013.02.006 ◽

2013 ◽

Vol 15 (5) ◽

pp. 399-408 ◽

Cited By ~ 14

Author(s):

Nicolas Hegerle ◽

Lamya Rayat ◽

Grégory Dore ◽

Nora Zidane ◽

Hugues Bedouelle ◽

...

Keyword(s):

Bordetella Pertussis ◽

Secretion System ◽

Bordetella Bronchiseptica ◽

Bordetella Parapertussis ◽

In Vivo Analysis ◽

Regulation by ToxR-Like Proteins Converges onvttRBExpression To Control Type 3 Secretion System-Dependent Caco2-BBE Cytotoxicity in Vibrio cholerae

Journal of Bacteriology ◽

10.1128/jb.00130-16 ◽

2016 ◽

Vol 198 (11) ◽

pp. 1675-1682 ◽

Cited By ~ 6

Author(s):

Kelly A. Miller ◽

Madeline K. Sofia ◽

Jacob W. A. Weaver ◽

Christopher H. Seward ◽

Michelle Dziejman

Keyword(s):

Gene Expression ◽

Cell Death ◽

Vibrio Cholerae ◽

Secretion System ◽

Content Type ◽

Transcriptional Regulatory ◽

Type 3 Secretion System ◽

Type 3

ABSTRACTGenes carried on the type 3 secretion system (T3SS) pathogenicity island ofVibrio choleraenon-O1/non-O139 serogroup strain AM-19226 must be precisely regulated in order for bacteria to cause disease. Previously reported results showed that both T3SS function and the presence of bile are required to cause Caco2-BBE cell cytotoxicity during coculture with strain AM-19226. We therefore investigated additional parameters affectingin vitrocell death, including bacterial load and the role of three transmembrane transcriptional regulatory proteins, VttRA, VttRB, and ToxR. VttRAand VttRBare encoded on the horizontally acquired T3SS genomic island, whereas ToxR is encoded on the ancestral chromosome. While strains carrying deletions in any one of the three transcriptional regulatory genes are unable to cause eukaryotic cell death, the results of complementation studies point to a hierarchy of regulatory control that converges onvttRBexpression. The data suggest both that ToxR and VttRAact upstream of VttRBand that modifying the level of eithervttRAorvttRBexpression can strongly influence T3SS gene expression. We therefore propose a model whereby T3SS activity and, hence,in vitrocytotoxicity are ultimately regulated byvttRBexpression.IMPORTANCEIn contrast to O1 and O139 serogroupV. choleraestrains that cause cholera using two main virulence factors (toxin-coregulated pilus [TCP] and cholera toxin [CT]), O39 serogroup strain AM-19226 uses a type 3 secretion system as its principal virulence mechanism. Although the regulatory network governing TCP and CT expression is well understood, the factors influencing T3SS-associated virulence are not. Using anin vitromammalian cell model to investigate the role of three ToxR-like transmembrane transcriptional activators in causing T3SS-dependent cytotoxicity, we found that expression levels and a hierarchical organization were important for promoting T3SS gene expression. Furthermore, our results suggest that horizontally acquired, ToxR-like proteins act in concert with the ancestral ToxR protein to orchestrate T3SS-mediated pathogenicity.

Regulatory Effects of CsrA in Vibrio cholerae

mBio ◽

10.1128/mbio.03380-20 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Heidi A. Butz ◽

Alexandra R. Mey ◽

Ashley L. Ciosek ◽

Alexander A. Crofts ◽

Bryan W. Davies ◽

...

Keyword(s):

Gene Expression ◽

Vibrio Cholerae ◽

Virulence Gene ◽

Growth Cycle ◽

Mammalian Host ◽

Aqueous Environment ◽

Content Type ◽

Human Host

ABSTRACT CsrA is a posttranscriptional global regulator in Vibrio cholerae. Although CsrA is critical for V. cholerae survival within the mammalian host, the regulatory targets of CsrA remain mostly unknown. To identify pathways controlled by CsrA, RNA-seq transcriptome analysis was carried out by comparing the wild type and the csrA mutant grown to early exponential, mid-exponential, and stationary phases of growth. This enabled us to identify the global effects of CsrA-mediated regulation throughout the V. cholerae growth cycle. We found that CsrA regulates 22% of the V. cholerae transcriptome, with significant regulation within the gene ontology (GO) processes that involve amino acid transport and metabolism, central carbon metabolism, lipid metabolism, iron uptake, and flagellum-dependent motility. Through CsrA-RNA coimmunoprecipitation experiments, we found that CsrA binds to multiple mRNAs that encode regulatory proteins. These include transcripts encoding the major sigma factors RpoS and RpoE, which may explain how CsrA regulation affects such a large proportion of the V. cholerae transcriptome. Other direct targets include flrC, encoding a central regulator in flagellar gene expression, and aphA, encoding the virulence gene transcription factor AphA. We found that CsrA binds to the aphA mRNA both in vivo and in vitro, and CsrA significantly increases AphA protein synthesis. The increase in AphA was due to increased translation, not transcription, in the presence of CsrA, consistent with CsrA binding to the aphA transcript and enhancing its translation. CsrA is required for the virulence of V. cholerae and this study illustrates the central role of CsrA in virulence gene regulation. IMPORTANCE Vibrio cholerae, a Gram-negative bacterium, is a natural inhabitant of the aqueous environment. However, once ingested, this bacterium can colonize the human host and cause the disease cholera. In order to successfully transition between its aqueous habitat and the human host, the bacterium must sense changes in its environment and rapidly alter gene expression. Global regulators, including CsrA, play an integral role in altering the expression of a large number of genes to promote adaptation and survival, which is required for intestinal colonization. We used transcriptomics and a directed CsrA-RNA coimmunoprecipitation to characterize the CsrA regulon and found that CsrA alters the expression of more than 800 transcripts in V. cholerae. Processes regulated by CsrA include motility, the rugose phenotype, and virulence pathways. CsrA directly binds to the aphA transcript and positively regulates the production of the virulence regulator AphA. Thus, CsrA regulates multiple processes that have been linked to pathogenesis.

Type three secretion system in non-toxigenic Vibrio cholerae O1, Mexico

Journal of Medical Microbiology ◽

10.1099/jmm.0.078295-0 ◽

2014 ◽

Vol 63 (12) ◽

pp. 1760-1762 ◽

Cited By ~ 4

Author(s):

Jamil Mahmud ◽

Shah M. Rashed ◽

Tarequl Islam ◽

Saiful Islam ◽

Haruo Watanabe ◽

...

Keyword(s):

Vibrio Cholerae ◽

Secretion System ◽

Vibrio Cholerae O1 ◽

Role for the Burkholderia pseudomallei Type Three Secretion System Cluster 1bpscNGene in Virulence

10.1128/iai.01351-10 ◽

2011 ◽

Vol 79 (9) ◽

pp. 3659-3664 ◽

Cited By ~ 23

Author(s):

Tanya D'Cruze ◽

Lan Gong ◽

Puthayalai Treerat ◽

Georg Ramm ◽

John D. Boyce ◽

...

Keyword(s):

Mammalian Cells ◽

Burkholderia Pseudomallei ◽

Gene Clusters ◽

Secretion System ◽

Host Cells ◽

Infection Model ◽

Content Type ◽

ABSTRACTBurkholderia pseudomallei, the causal agent of melioidosis, employs a number of virulence factors during its infection of mammalian cells. One such factor is the type three secretion system (TTSS), which is proposed to mediate the transport and secretion of bacterial effector molecules directly into host cells. TheB. pseudomalleigenome contains three TTSS gene clusters (designated TTSS1, TTSS2, and TTSS3). Previous research has indicated that neither TTSS1 nor TTSS2 is involved inB. pseudomalleivirulence in a hamster infection model. We have characterized aB. pseudomalleimutant lacking expression of the predicted TTSS1 ATPase encoded bybpscN. This mutant was significantly attenuated for virulence in a respiratory melioidosis mouse model of infection. In addition, analysesin vitroshowed diminished survival and replication in RAW264.7 cells and an increased level of colocalization with the autophagy marker protein LC3 but an unhindered ability to escape from phagosomes. Taken together, these data provide evidence that the TTSS1bpscNgene product plays an important role in the intracellular survival ofB. pseudomalleiand the pathogenesis of murine infection.

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

mBio ◽

10.1128/mbio.02901-20 ◽

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.

Type Three Secretion System Island-Encoded Proteins Required for Colonization by Non-O1/Non-O139 Serogroup Vibrio cholerae

10.1128/iai.03020-14 ◽

2015 ◽

Vol 83 (7) ◽

pp. 2862-2869 ◽

Cited By ~ 15

Author(s):

Mudit Chaand ◽

Kelly A. Miller ◽

Madeline K. Sofia ◽

Cory Schlesener ◽

Jacob W. A. Weaver ◽

...

Keyword(s):

Vibrio Cholerae ◽

Protein Transport ◽

Secretion System ◽

Secretory Diarrhea ◽

Content Type ◽

Infant Mouse ◽

Colonization Factor ◽

Translocated Proteins ◽

Type 3

Vibrio choleraeis a genetically diverse species, and pathogenic strains can encode different virulence factors that mediate colonization and secretory diarrhea. Although the toxin-coregulated pilus (TCP) is the primary colonization factor in epidemic-causingV. choleraestrains, other strains do not encode the TCP and instead promote colonization via the activity of a type 3 secretion system (T3SS). Using the infant mouse model and T3SS-positive O39 serogroup strain AM-19226, we sought to determine which of 12 previously identified, T3SS-translocated proteins (Vops) are important for host colonization. We constructed in-frame deletions in each of the 12 loci in strain AM-19226 and identified five Vop deletion strains, including ΔVopM, which were severely attenuated for colonization. Interestingly, a subset of deletion strains was also incompetent for effector protein transport. Our collective data therefore suggest that several translocated proteins may also function as components of the structural apparatus or translocation machinery and indicate that while VopM is critical for establishing an infection, the combined activities of other effectors may also contribute to the ability of T3SS-positive strains to colonize host epithelial cell surfaces.