The regulatory network of Vibrio parahaemolyticus type VI secretion system 1

Abstract

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 Bacterial Competition Fluorescence (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.

Figure 1

BaCoF screen reveals V. parahaemolyticus mutants defective in T6SS1‐mediated bacterial killing. (A) Schematic representation of the BaCoF methodology. Erm, erythromycin; ON, overnight. (B and C) Genomic map of BaCoF hits' transposon insertion sites (red triangles) within known T6SS1 gene clusters and modules (B) and previously non‐T6SS1 associated operons (C). Arrows indicate the direction of gene transcription. Locus numbers are listed above and gene names or annotations are listed below. In C, serial numbers of transposon (Tn5) mutant BaCoF hits are shown below in red.

Figure 2

BaCoF hits present T6SS1‐mediated bacterial killing, secretion and growth defects. (A and B) Viability counts of V. parahaemolyticus POR1/Δvp1415‐6 (A) and E. coli DH5α (B) prey before (0 h) and after (4 h) co‐culture with the indicated V. parahaemolyticus attackers on MLB media (containing 3% NaCl) at 30°C. Asterisks mark statistical significance between samples and POR1 parental attacker at 4 h timepoint by unpaired, two‐tailed Student's t‐test (*P < 0.05). DL, Detection limit. (C) Expression (cells) and secretion (media) of VgrG1 were detected by immunoblotting using specific antibodies against VgrG1. Loading control (LC), visualized as trihalo compounds' fluorescence of the immunoblot membrane, is shown for total protein lysates. (D) Growth of V. parahaemolyticus strains in MLB at 30°C is shown as OD₆₀₀ measurements. Data are mean ± SD, n = 3.

Figure 3

TfoY is an essential positive regulator of V. parahaemolyticus T6SS1. (A and B) Viability counts of V. parahaemolyticus POR1/Δvp1415‐6 (A) and E. coli DH5α (B) prey before (0 h) and after (4 h) co‐culture with the indicated V. parahaemolyticus attackers on MLB media (containing 3% NaCl) containing 0.1% L‐arabinose at 30°C. Asterisks mark statistical significance between samples and POR1 + pEmpty parental attacker at 4 h timepoint by unpaired, two‐tailed Student's t‐test (*P < 0.05). DL, Detection limit. (C and D) Expression (cells) and secretion (media) of VgrG1 were detected by immunoblotting using specific antibodies against VgrG1. Loading control (LC), visualized as trihalo compounds' fluorescence of the immunoblot membrane, is shown for total protein lysates. As indicated, the strains contained arabinose‐inducible expression vectors of TfoY (pTfoY), TfoX (pTfoX), or an empty vector (pEmpty).

Figure 4

VP2049 (Tmk) is required for T6SS1 activity. (A and B) Viability counts of V. parahaemolyticus POR1/Δvp1415‐6 (A) and E. coli DH5α (B) prey before (0 h) and after (4 h) co‐culture with the indicated V. parahaemolyticus attackers on MLB media (containing 3% NaCl) with 0.1% L‐arabinose at 30°C. Asterisks mark statistical significance between samples and POR1 + pEmpty parental attacker at 4 h timepoint by unpaired, two‐tailed Student's t‐test (*P < 0.05). (C) Expression (cells) and secretion (media) of VgrG1 were detected by immunoblotting using specific antibodies against VgrG1. Loading control (LC), visualized as trihalo compounds' fluorescence of the immunoblot membrane, is shown for total protein lysates. (D) Growth of V. parahaemolyticus strains in MLB containing 0.1% L‐arabinose and 250 μg ml⁻¹ kanamycin at 30°C is shown as OD₆₀₀ measurements. Data are mean ± SD, n = 3. As indicated, the strains contained arabinose‐inducible expression vectors of VP2050 (pVP2050), VP2049 (pVP2049), or an empty vector (pEmpty).

Figure 5

T6SS1 is regulated by a complex network of positive and negative regulators. (A) Expression (cells) and secretion (media) of VgrG1 were detected by immunoblotting using specific antibodies against VgrG1. Loading control (LC), visualized as trihalo compounds' fluorescence of the immunoblot membrane, is shown for total protein lysates. (B and C) Viability counts of V. parahaemolyticus POR1/Δvp1415‐6 prey before (0 h) and after (4 h) co‐culture with the indicated V. parahaemolyticus attackers on MLB media (containing 3% NaCl) at 30°C. DL, Detection limit. (D) Expression (cells) and secretion (media) of VgrG1 were detected by immunoblotting using specific antibodies against VgrG1. Loading control (LC), visualized as trihalo compounds' fluorescence of the immunoblot membrane, is shown for total protein lysates. (E) Viability counts of V. parahaemolyticus POR1/Δvp1415‐6 prey before (0 h) and after (4 h) co‐culture with the indicated V. parahaemolyticus attackers on MLB media containing 0.1% L‐arabinose at 30°C. As indicated, the strains contained the arabinose‐inducible expression vector of TfoY (pTfoY) or an empty vector (pEmpty). Asterisks mark statistical significance between samples and POR1 or POR1 + pEmpty parental attacker at 4 h timepoint by unpaired, two‐tailed Student's t‐test (*P < 0.05).

Figure 6

Model of the V. parahaemolyticus T6SS1 regulatory network. T6SS1 is induced under warm marine‐like conditions and upon surface sensing (via inhibition of the polar flagella). This leads to activation of TfoY, which in turn, activates VP1391 and VP1407 (positive regulators encoded within the T6SS1 cluster) that turn on the antibacterial T6SS1. The quorum‐sensing master regulator, OpaR, represses T6SS1 downstream of TfoY, possibly by direct binding to promoters of T6SS1 cluster operons, whereas H‐NS represses the system upstream of TfoY. Dashed arrows denote links that may be indirect.