Conjugative Transfer of Acute Hepatopancreatic Necrosis Disease-Causing pVA1-Type Plasmid Is Mediated by a Novel Self-Encoded Type IV Secretion System

Abstract

The pathogenic pVA1-type plasmids that carry pirAB toxin genes are the genetic basis for Vibrio to cause acute hepatopancreatic necrosis disease (AHPND), a lethal shrimp disease posing an urgent threat to shrimp aquaculture. Emerging evidence also demonstrate the rapid spread of pVA1-type plasmids across Vibrio species. The pVA1-type plasmids have been predicted to encode a self-encoded type IV secretion system (T4SS). Here, phylogenetic analysis indicated that the T4SS is a novel member of Trb-type. We further confirmed that the T4SS was able to mediate the conjugation of pVA1-type plasmids. A trbE gene encoding an ATPase and a traG gene annotated as a type IV coupling protein (T4CP) were characterized as key components of the T4SS. Deleting either of these 2 genes abolished the conjugative transfer of a pVA1-type plasmid from AHPND-causing Vibrio parahaemolyticus to Vibrio campbellii, which was restored by complementation of the corresponding gene. Moreover, we found that bacterial density, temperature, and nutrient levels are factors that can regulate conjugation efficiency. In conclusion, we proved that the conjugation of pVA1-type plasmids across Vibrio spp. is mediated by a novel T4SS and regulated by environmental factors. IMPORTANCE AHPND is a global shrimp bacteriosis and was listed as a notifiable disease by the World Organization for Animal Health (WOAH) in 2016, causing losses of more than USD 7 billion each year. Several Vibrio species such as V. parahaemolyticus, V. harveyi, V. campbellii, and V. owensii harboring the virulence plasmid (designated as the pVA1-type plasmid) can cause AHPND. The increasing number of Vibrio species makes prevention and control more difficult, threatening the sustainable development of the aquaculture industry. In this study, we found that the horizontal transfer of pVA1-type plasmid is mediated by a novel type IV secretion system (T4SS). Our study explained the formation mechanism of pathogen diversity in AHPND. Moreover, bacterial density, temperature, and nutrient levels can regulate horizontal efficiency. We explore new ideas for controlling the spread of virulence plasmid and form the basis of management strategies leading to the prevention and control of AHPND.

Keywords: acute hepatopancreatic necrosis disease (AHPND); conjugative transfer; pVA1-type plasmid; type IV secretion system (T4SS).

FIG 1

Analysis and annotation of the novel Trb-type T4SS found in pVA1-type plasmids. (A) The annotation diagrams of the type IV secretion system in the pVPGX1 of Vibrio parahaemolyticus 20130629002S01 compared to those in chromosome 2 of Photobacterium profundum SS9. (B) Amino acid identities of the T4SS components across pVA1-type plasmids against the reference pVPGX1. (C) Predicted pattern of the pVA1-type plasmid-borne T4SS based on that of the VirB-type T4SS. “?” means those components have no counterparts in the VirB-type T4SS.

FIG 2

Phylogenetic analyses of T4SS amino acid sequences. We used the amino acid sequences of TraG + TrbL+TrbE of the three components of Trb-type T4SS, and searched the corresponding components of the other types of T4SS. Percentage bootstrap values (1000 replicates) 85% are shown. GenBank accession numbers of the reference sequences are shown in Table S1. Scale bar represents the number of amino acid substitutions per site.

FIG 3

Growth curves of the mutants. R²: the square value of correlation coefficient of Logistic fitting curve. X0: the time when growth curve reaches maximum growth rate, and the result was presented as average value ± standard error (SE). (A) Vp2S01::cat, (B) Vp2S01ΔtrbE, (C) Vp2S01ΔtraG, (D) Vp2S01ΔtrbE::pRK415-trbE, (E) Vp2S01ΔtraG::pRK415-traG.

FIG 4

The conjugation efficiency of the deletion strain, wild strain, and complementation strain. (A) The conjugation efficiency of the trbE gene deletion strain, wild strain, and complementation strains. (B) The conjugation efficiency of the traG gene deletion strain, wild strain, and complementation strain. *, P < 0.05, **, P < 0.01. <d.l.: below detection limit.

FIG 5

Effects of different factors on conjugation efficiency. (A) Derivation graph of the influence of different factors on conjugative transfer. (B) The effect of different bacterial densities on conjugation efficiency. (C) The effect of different temperatures on conjugation efficiency. (D) The effect of different nutrient levels on conjugation efficiency. HP.F: the filtrate of shrimp hepatopancreas, S.S.W: sterile seawater, S.F.F: shrimp feed filtrate. ***, P < 0.001. ns: no significant difference. <d.l.: below detection limit.