Acute Hepatopancreatic Necrosis Disease-Causing Vibrio parahaemolyticus Strains Maintain an Antibacterial Type VI Secretion System with Versatile Effector Repertoires

Abstract

Acute hepatopancreatic necrosis disease (AHPND) is a newly emerging shrimp disease that has severely damaged the global shrimp industry. AHPND is caused by toxic strains of Vibrio parahaemolyticus that have acquired a "selfish plasmid" encoding the deadly binary toxins PirA^vp/PirB^vp To better understand the repertoire of virulence factors in AHPND-causing V. parahaemolyticus, we conducted a comparative analysis using the genome sequences of the clinical strain RIMD2210633 and of environmental non-AHPND and toxic AHPND isolates of V. parahaemolyticus Interestingly, we found that all of the AHPND strains, but none of the non-AHPND strains, harbor the antibacterial type VI secretion system 1 (T6SS1), which we previously identified and characterized in the clinical isolate RIMD2210633. This finding suggests that the acquisition of this T6SS might confer to AHPND-causing V. parahaemolyticus a fitness advantage over competing bacteria and facilitate shrimp infection. Additionally, we found highly dynamic effector loci in the T6SS1 of AHPND-causing strains, leading to diverse effector repertoires. Our discovery provides novel insights into AHPND-causing pathogens and reveals a potential target for disease control.IMPORTANCE Acute hepatopancreatic necrosis disease (AHPND) is a serious disease that has caused severe damage and significant financial losses to the global shrimp industry. To better understand and prevent this shrimp disease, it is essential to thoroughly characterize its causative agent, Vibrio parahaemolyticus Although the plasmid-encoded binary toxins PirA^vp/PirB^vp have been shown to be the primary cause of AHPND, it remains unknown whether other virulent factors are commonly present in V. parahaemolyticus and might play important roles during shrimp infection. Here, we analyzed the genome sequences of clinical, non-AHPND, and AHPND strains to characterize their repertoires of key virulence determinants. Our studies reveal that an antibacterial type VI secretion system is associated with the AHPND strains and differentiates them from non-AHPND strains, similar to what was seen with the PirA/PirB toxins. We propose that T6SS1 provides a selective advantage during shrimp infections.

Keywords: AHPND; MIX effectors; T3SS; T6SS; Vibrio; Vibrio parahaemolyticus; shrimp; type III secretion system; type VI secretion system.

FIG 1

V. parahaemolyticus clinical, non-AHPND, and AHPND strains contain a conserved T3SS1. (A) Comparison of T3SS1 clusters from clinical, non-AHPND, and AHPND strains of V. parahaemolyticus. All V. parahaemolyticus strains analyzed in this study contain a highly conserved T3SS1 that is further categorized into two subtypes, T3SS1a and T3SS1b. T3SS1a contains vp1676 to vp1679 (blue), and T3SS1b contains three newly identified genes (green). JP, Japan; VT, Vietnam; TH, Thailand; MX, Mexico; CN, China. (B) HeLa cells were infected with the indicated V. parahaemolyticus strains for 4 h at an MOI of 10. Lactate dehydrogenase (LDH) release was evaluated as the measure of cytotoxicity against host cells. Data are means ± standard deviations (SD); n = 3. Data are representative of three independent experiments.

FIG 2

V. parahaemolyticus clinical, non-AHPND, and AHPND strains contain a conserved but differentially regulated T6SS2. (A) Schematic representation of the V. parahaemolyticus T6SS2 gene cluster. RIMD2210633 strain locus numbers are shown above and gene names below. (B) Expression (Cell) and secretion (Media) of V. parahaemolyticus T6SS2 component Hcp2 by the indicated V. parahaemolyticus strains grown in LB at 23°C or 37°C for 5 h and analyzed by Western blotting using α-Hcp2 antibody. PAR, parental strain. LC, loading control.

FIG 3

All V. parahaemolyticus AHPND strains contain a T6SS1 cluster similar to that of the RIMD2210633 clinical strain. Schematic representation comparison of T6SS1 clusters from clinical and AHPND strains of V. parahaemolyticus. RIMD2210633 strain locus numbers are shown above and gene names below. Genes with variations between the strains analyzed are highlighted with different colors. RIMD2210633 genes are labeled in blue, and corresponding genes in AHPND strains are labeled in blue when they are the same or highly conserved but in other colors when otherwise. The same color indicates conserved genes (>90% amino acid identity). In RIMD2210633, the previously annotated vp1396 and vp1397 actually form one single gene, as in all AHPND strains (cyan). JP, Japan; VT, Vietnam; TH, Thailand; MX, Mexico; CN, China; MIX, T6SS MIX effector; Imm, immunity protein; AHH, AHH nuclease toxin domain.

FIG 4

AHPND-causing V. parahaemolyticus strains encode mobile T6SS MIX effectors. Mobile genetic elements containing T6SS MIX effectors from the clinical isolate RIMD2210633 and four AHPND strains, including TUMSAT_DE1_S1, NCKU_CV_CHN, FIM-S1708+, and M0605. Components within the mobile elements that are discussed in the text are highlighted with different colors: T6SS mobile MIX effector, blue and cyan; immunity, green; toxin/antitoxin module, orange; genes encoding proteins that mediate DNA rearrangement and mobility upstream of the E/I pair are in red and downstream of the E/I pair are in magenta. The number of omitted genes is shown in parentheses.

FIG 5

AHPND-causing V. parahaemolyticus strains contain a functional antibacterial T6SS1. (A) Expression (Cell) and secretion (Media) of T6SS1 component VgrG1 by the indicated V. parahaemolyticus strains grown in MLB at 30°C ± 20 μM phenamil were analyzed by Western blotting using α-VgrG1 antibody. PAR, parental strain. LC, loading control. (B and C) Viability counts of E. coli (B) or V. cholerae (C) prey before (0 h) and after (4 h) coculture with indicated V. parahaemolyticus attacker strains on MLB (3% NaCl) solid media at 30°C. Data are representative of three independent experiments.