Comparative Genomics of Aeromonas hydrophila Secretion Systems and Mutational Analysis of hcp1 and vgrG1 Genes From T6SS

Abstract

Virulent Aeromonas hydrophila causes severe motile Aeromonas septicemia in warmwater fishes. In recent years, channel catfish farming in the U.S.A. and carp farming in China have been affected by virulent A. hydrophila, and genome comparisons revealed that these virulent A. hydrophila strains belong to the same clonal group. Bacterial secretion systems are often important virulence factors; in the current study, we investigated whether secretion systems contribute to the virulent phenotype of these strains. Thus, we conducted comparative secretion system analysis using 55 A. hydrophila genomes, including virulent A. hydrophila strains from U.S.A. and China. Interestingly, tight adherence (TaD) system is consistently encoded in all the vAh strains. The majority of U.S.A. isolates do not possess a complete type VI secretion system, but three core elements [tssD (hcp), tssH, and tssI (vgrG)] are encoded. On the other hand, Chinese isolates have a complete type VI secretion system operon. None of the virulent A. hydrophila isolates have a type III secretion system. Deletion of two genes encoding type VI secretion system proteins (hcp1 and vgrG1) from virulent A. hydrophila isolate ML09-119 reduced virulence 2.24-fold in catfish fingerlings compared to the parent strain ML09-119. By determining the distribution of genes encoding secretion systems in A. hydrophila strains, our study clarifies which systems may contribute to core A. hydrophila functions and which may contribute to more specialized adaptations such as virulence. Our study also clarifies the role of type VI secretion system in A. hydrophila virulence.

Keywords: Aeromonas hydrophila; Hcp; T6SS; VgrG; comparative genomics; secretion systems.

Figure 1

Average nucleotide identities (ANI) of A. hydrophila genomes and phylogenetic tree based on core genome. Note that branch lengths of the phylogenetic tree were reduced to fit the image.

Figure 2

Core and accessory proteins of secretion systems, T4P, Tad, and flagella encoded in A. hydrophila genomes. Numbers and color represent the number of copies of each listed gene. The strains are listed in the same order as Table 1 and Figure 1 (the first 27 strains are vAh strains). tssD is also known as hcp and tssI is also known as vgrG. A.G. indicates Accessory genes.

Figure 3

Virulence of vAh T6SS mutants in channel catfish fingerlings. (A) Percent mortalities in catfish fingerlings experimentally infected with vAh T6SS mutants and vAh wild type (WT) strain ML09-119. (B) Percent survival in catfish fingerlings surviving infection with T6SS mutants and re-challenged with vAh WT at 21 d post-infection. Data are the mean ± SE of three replicate tanks. Significant differences between challenged and non-vaccinated treatments are indicated with asterisks (p < 0.05).