scholarly journals Systematic Identification of Cyclic-di-GMP Binding Proteins in Vibrio cholerae Reveals a Novel Class of Cyclic-di-GMP-Binding ATPases Associated with Type II Secretion Systems

2015 ◽  
Vol 11 (10) ◽  
pp. e1005232 ◽  
Author(s):  
Kevin G. Roelofs ◽  
Christopher J. Jones ◽  
Sarah R. Helman ◽  
Xiaoran Shang ◽  
Mona W. Orr ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Binding Proteins ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Secretion Systems ◽  
Systematic Identification

scholarly journals Cyclic Di-GMP and VpsR Induce the Expression of Type II Secretion in Vibrio cholerae

2017 ◽  
Vol 199 (19) ◽  
Author(s):  
Rudolph E. Sloup ◽  
Ashley E. Konal ◽  
Geoffrey B. Severin ◽  
Michelle L. Korir ◽  
Mira M. Bagdasarian ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Protein Complexes ◽  
Second Messenger ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Secretion Systems ◽  
Content Type ◽  
Severe Diarrhea ◽  
Wide Range ◽  
Genes Encoding

ABSTRACT Vibrio cholerae is a human pathogen that alternates between growth in environmental reservoirs and infection of human hosts, causing severe diarrhea. The second messenger cyclic di-GMP (c-di-GMP) mediates this transition by controlling a wide range of functions, such as biofilms, virulence, and motility. Here, we report that c-di-GMP induces expression of the extracellular protein secretion (eps) gene cluster, which encodes the type II secretion system (T2SS) in V. cholerae. Analysis of the eps genes confirmed the presence of two promoters located upstream of epsC, the first gene in the operon, one of which is induced by c-di-GMP. This induction is directly mediated by the c-di-GMP-binding transcriptional activator VpsR. Increased expression of the eps operon did not impact secretion of extracellular toxin or biofilm formation but did increase expression of the pseudopilin protein EpsG on the cell surface. IMPORTANCE Type II secretion systems (T2SSs) are the primary molecular machines by which Gram-negative bacteria secrete proteins and protein complexes that are folded and assembled in the periplasm. The substrates of T2SSs include extracellular factors, such as proteases and toxins. Here, we show that the widely conserved second messenger cyclic di-GMP (c-di-GMP) upregulates expression of the eps genes encoding the T2SS in the pathogen V. cholerae via the c-di-GMP-dependent transcription factor VpsR.

scholarly journals Specificity of the Type II Secretion Systems of Enterotoxigenic Escherichia coli and Vibrio cholerae for Heat-Labile Enterotoxin and Cholera Toxin

2010 ◽  
Vol 192 (7) ◽  
pp. 1902-1911 ◽  
Author(s):  
Benjamin Mudrak ◽  
Meta J. Kuehn
Keyword(s):  
Escherichia Coli ◽  
Cholera Toxin ◽  
Vibrio Cholerae ◽  
Enterotoxigenic Escherichia Coli ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Wild Type ◽  
Secretion Systems ◽  
B Subunit ◽  
Heat Labile

ABSTRACT The Gram-negative type II secretion (T2S) system is a multiprotein complex mediating the release of virulence factors from a number of pathogens. While an understanding of the function of T2S components is emerging, little is known about what identifies substrates for export. To investigate T2S substrate recognition, we compared mutations affecting the secretion of two highly homologous substrates: heat-labile enterotoxin (LT) from enterotoxigenic Escherichia coli (ETEC) and cholera toxin (CT) from Vibrio cholerae. Each toxin consists of one enzymatic A subunit and a ring of five B subunits mediating the toxin's secretion. Here, we report two mutations in LT's B subunit (LTB) that reduce its secretion from ETEC without global effects on the toxin. The Q3K mutation reduced levels of secreted LT by half, and as with CT (T. D. Connell, D. J. Metzger, M. Wang, M. G. Jobling, and R. K. Holmes, Infect. Immun. 63:4091-4098, 1995), the E11K mutation impaired LT secretion. Results in vitro and in vivo show that these mutants are not degraded more readily than wild-type LT. The Q3K mutation did not significantly affect CT B subunit (CTB) secretion from V. cholerae, and the E11A mutation altered LT and CTB secretion to various extents, indicating that these toxins are identified as secretion substrates in different ways. The levels of mutant LTB expressed in V. cholerae were low or undetectable, but each CTB mutant expressed and secreted at wild-type levels in ETEC. Therefore, ETEC's T2S system seems to accommodate mutations in CTB that impair the secretion of LTB. Our results highlight the exquisitely fine-tuned relationship between T2S substrates and their coordinate secretion machineries in different bacterial species.

scholarly journals Hexamers of the Type II Secretion ATPase GspE from Vibrio cholerae with Increased ATPase Activity

Structure ◽  
2013 ◽  
Vol 21 (9) ◽  
pp. 1707-1717 ◽  
Author(s):  
Connie Lu ◽  
Stewart Turley ◽  
Samuel T. Marionni ◽  
Young-Jun Park ◽  
Kelly K. Lee ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Vibrio Cholerae ◽  
Type Ii Secretion ◽  
Type Ii

scholarly journals Role of fimV in type II secretion system-dependent protein secretion of Pseudomonas aeruginosa on solid medium

Microbiology ◽  
2011 ◽  
Vol 157 (7) ◽  
pp. 1945-1954 ◽  
Author(s):  
Gérard P. F. Michel ◽  
Anthony Aguzzi ◽  
Geneviève Ball ◽  
Chantal Soscia ◽  
Sophie Bleves ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Solid Medium ◽  
Early Stage ◽  
Growth Conditions ◽  
Classical Type ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Secretion Systems ◽  
Type Ii Secretion System ◽  
Dependent Protein

Although classical type II secretion systems (T2SSs) are widely present in Gram-negative bacteria, atypical T2SSs can be found in some species. In Pseudomonas aeruginosa, in addition to the classical T2SS Xcp, it was reported that two genes, xphA and xqhA, located outside the xcp locus were organized in an operon (PaQa) which encodes the orphan PaQa subunit. This subunit is able to associate with other components of the classical Xcp machinery to form a functional hybrid T2SS. In the present study, using a transcriptional lacZ fusion, we found that the PaQa operon was more efficiently expressed (i) on solid LB agar than in liquid LB medium, (ii) at 25 °C than at 37 °C and (iii) at an early stage of growth. These results suggested an adaptation of the hybrid system to particular environmental conditions. Transposon mutagenesis led to the finding that vfr and fimV genes are required for optimal expression of the orphan PaQa operon in the defined growth conditions used. Using an original culturing device designed to monitor secretion on solid medium, the ring-plate system, we found that T2SS-dependent secretion of exoproteins, namely the elastase LasB, was affected in a fimV deletion mutant. Our findings led to the discovery of an interplay between FimV and the global regulator Vfr triggering the modulation of the level of Vfr and consequently the modulation of T2SS-dependent secretion on solid medium.

scholarly journals Docking and Assembly of the Type II Secretion Complex of Vibrio cholerae

2009 ◽  
Vol 191 (9) ◽  
pp. 3149-3161 ◽  
Author(s):  
Suzanne R. Lybarger ◽  
Tanya L. Johnson ◽  
Miranda D. Gray ◽  
Aleksandra E. Sikora ◽  
Maria Sandkvist
Keyword(s):  
Vibrio Cholerae ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
Outer Membrane Proteins ◽  
Subcellular Location ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Protein Fusion ◽  
Green Fluorescent

ABSTRACT Secretion of cholera toxin and other virulence factors from Vibrio cholerae is mediated by the type II secretion (T2S) apparatus, a multiprotein complex composed of both inner and outer membrane proteins. To better understand the mechanism by which the T2S complex coordinates translocation of its substrates, we are examining the protein-protein interactions of its components, encoded by the extracellular protein secretion (eps) genes. In this study, we took a cell biological approach, observing the dynamics of fluorescently tagged EpsC and EpsM proteins in vivo. We report that the level and context of fluorescent protein fusion expression can have a bold effect on subcellular location and that chromosomal, intraoperon expression conditions are optimal for determining the intracellular locations of fusion proteins. Fluorescently tagged, chromosomally expressed EpsC and EpsM form discrete foci along the lengths of the cells, different from the polar localization for green fluorescent protein (GFP)-EpsM previously described, as the fusions are balanced with all their interacting partner proteins within the T2S complex. Additionally, we observed that fluorescent foci in both chromosomal GFP-EpsC- and GFP-EpsM-expressing strains disperse upon deletion of epsD, suggesting that EpsD is critical to the localization of EpsC and EpsM and perhaps their assembly into the T2S complex.

scholarly journals Inner-membrane GspF of the bacterial type II secretion system is a dimeric adaptor mediating pseudopilus biogenesis

2018 ◽  
Author(s):  
Wouter Van Putte ◽  
Tatjana De Vos ◽  
Wim Van Den Broeck ◽  
Henning Stahlberg ◽  
Misha Kudryashev ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Virulence Factors ◽  
Protein Complex ◽  
Secretion System ◽  
Structural Basis ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Secretion Systems ◽  
Inner Membrane ◽  
Type Ii Secretion System

AbstractThe type II secretion system (T2SS), a protein complex spanning the bacterial envelope, is pivotal to bacterial pathogenicity. Central to T2SS function is the extrusion of protein cargos from the periplasm into the extracellular environment mediated by a pseudopilus and motorized by a cytosolic ATPase. GspF, an inner-membrane component of T2SS has long been considered to be a key player in this process, yet the structural basis of its role had remained elusive. Here, we employed single-particle electron microscopy based on XcpS (GspF) from the T2SS of pathogenicP. aeruginosastabilized by a nanobody, to show that XcpS adopts a dimeric structure mediated by its transmembrane helices. This assembly matches in terms of overall organization and dimensions the basal inner-membrane cassette of a T2SS machinery. Thus, GspF is poised to serve as an adaptor involved in the mediation of propeller-like torque generated by the motor ATPase to the secretion pseudopilus.Non-technical author summaryAntibiotic resistance by bacteria imposes a worldwide threat that can only be overcome through a multi-front approach: preventive actions and the parallel development of novel molecular strategies to combat antibiotic resistance mechanisms. One such strategy might focus on antivirulence drugs that prevent host invasion and spreading by pathogenic bacteria, without shutting down essential functions related to bacterial survival. The rationale behind such an approach is that it might limit selective pressure leading to slower evolutionary rates of resistant bacterial strains. Bacterial secretion systems are an appropriate target for such therapeutic approaches as their impairment will inhibit the secretion of a multitude of virulence factors. This study focuses on the structural characterization of one of the proteins residing in the inner-membrane cassette of the type II secretion system (T2SS), a multi-protein complex in multiple opportunistic pathogens that secretes virulence factors. The targeted protein is essential for the assembly of the pseudopilus, a rod-like supramolecular structure that propels the secretion of virulence factors by pathogenic Gram-negative bacteria. Our study crucially complements growing evidence supporting a rotational assembly model of the pseudopilus and contributes to a better understanding of the functioning of the T2SS and the related secretion systems. We envisage that such knowledge will facilitate targeting of these systems for therapeutic purposes.

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