The regulatory network of Vibrio parahaemolyticus type VI secretion system 1

Abstract

Summary Type VI secretion systems (T6SSs) are widespread, tightly regulated, protein delivery apparatuses used by Gram‐negative bacteria to outcompete their neighbours. The pathogen, Vibrio parahaemolyticus , encodes two T6SSs. These T6SSs are differentially regulated by external conditions. T6SS1, an antibacterial system predominantly found in pathogenic isolates, requires warm marine‐like conditions and surface sensing for activation. The regulatory network that governs this activation is not well understood. In this work, we devised a screening methodology that allows us to easily monitor the outcome of bacterial competitions and thus to identify mutants that are defective in T6SS1‐mediated bacterial killing. The methodology, termed Ba cterial Co mpetition F luorescence (BaCoF), relies on detection of a fluorescent signal as an indicator of the survival and growth of a T6SS‐sensitive, GFP‐expressing prey that has been co‐cultured with mutants derived from a T6SS + attacker of interest. Using BaCoF, we screened a random transposon insertion mutant library and identified genes required for V. parahaemolyticus T6SS1 activation, among them TfoY and Tmk. We used epistasis experiments to determine the relationships between the newly identified components and other regulators that were previously described. Thus, we present here a detailed biological understanding of the T6SS1 regulatory network.

A comparative genomics methodology reveals a widespread family of membrane-disrupting T6SS effectors

“…3). These plasmids were introduced into the surrogate platform strains, and their ability to confer T6SS1-mediated killing of a parental RIMD 2210633 prey was determined in a quantitative competition assay, as well as in a qualitative assay 43 by monitoring the survival of GFP-expressing prey. Notably, we confirmed the expression of all but one candidate E/I pair by immunoblot analysis ( Supplementary Fig.…”

“…Noteworthy, the development of a surrogate T6SS platform is pivotal for the future scale up of this methodology; it allows one to screen effector candidates from any isolate that harbors the T6SS in question, even from isolates that are not amenable for genetic manipulations. Moreover, the use of our previously developed methodology 43 , in which prey survival is qualitatively assessed by monitoring GFP levels expressed by the prey strain, obviates the need for laborious quantitative competition assays as an initial screen of candidates. To test a candidate effector in the surrogate platform, one simply needs to amplify the DNA sequence of the candidate E/I pair in question (or order the cassette from a variety of commercial vendors), introduce it into the surrogate platform strain (and into its T6SS − negative control derivative), and employ it in bacterial competition (either quantitative or qualitative) against the surrogate platform's parental strain that lacks immunity against the exogenous effectors.…”

“…Qualitative bacterial competition assays. Qualitative bacterial competition assays were performed as previously described 43 , with minor changes. In brief, surrogate platform strains carrying the indicated E/I candidates were grown overnight in 96well plates containing 100 µL of MLB supplemented with kanamycin (250 µg/mL).…”

Comparative Proteomics and Secretomics Revealed Virulence and Antibiotic Resistance-Associated Factors in Vibrio parahaemolyticus Recovered From Commonly Consumed Aquatic Products

“…T6SS1 is active under warm marine-like conditions (3% NaCl, 30°C), whereas T6SS2 is active under low salinity and low temperature (1% NaCl, 23°C) ( Salomon et al, 2013 ). Recent studies have indicated that T6SSs are tightly regulated in pathogenic bacteria, for example, Vibrio cholerae , Pseudomonas aeruginosa , and V. parahaemolyticus , and induced by external conditions and cues such as quorum sensing, salinity, temperature, mucin, chitin, surface sensing, and cell membrane damage ( Ben-Yaakov and Salomon, 2019 ).…”

The substrate‐dependent regulatory effects of the AfeI/R system in Acidithiobacillus ferrooxidans reveals the novel regulation strategy of quorum sensing in acidophiles

“…QS has been widely identified in Gramnegative and Gram-positive bacteria and is fundamental for cell-to-cell communication (Juhas et al, 2005;Kai and Bassler, 2016). Hundreds of traits can be regulated via QS in both pathogenic and environmental bacteria, such as EPS synthesis, biofilm formation, cell colonization, bioluminescence and the secretion of virulence factors (Goo et al, 2015;Ben-Yaakov and Salomon, 2019). QS can also regulate metabolic processes in some bacteria, such as sugar and phosphate metabolism, as well as secondary metabolites (Goo et al, 2015;Certner and Vollmer, 2018;Ha et al, 2018).…”