A type III secretion system is required for Aeromonas hydrophila AH-1 pathogenesis

Abstract

Aeromonas hydrophila is a gram-negative opportunistic pathogen in fish and humans. Many bacterial pathogens of animals and plants have been shown to inject anti-host virulence determinants into the hosts via a type III secretion system (TTSS). Degenerate primers based on lcrD family genes that are present in every known TTSS allowed us to locate the TTSS gene cluster in A. hydrophila AH-1. A series of genome walking steps helped in the identification of 25 open reading frames that encode proteins homologous to those in TTSSs in other bacteria. PCR-based analysis showed the presence of lcrD homologs (ascV) in all of the 33 strains of A. hydrophila isolated from various sources. Insertional inactivation of two of the TTSS genes (aopB and aopD) led to decreased cytotoxicity in carp epithelial cells, increased phagocytosis, and reduced virulence in blue gourami. These results show that a TTSS is required for A. hydrophila pathogenesis. This is the first report of sequencing and characterization of TTSS gene clusters from A. hydrophila. The TTSS identified here may help in developing suitable vaccines as well as in further understanding of the pathogenesis of A. hydrophila.

FIG. 1.

Genetic organizations of TTSSs in A. hydrophila and other bacteria. Arrows indicate the proposed directions of transcription.

FIG. 2.

Location of the ascV gene as determined by PFGE and Southern blot analysis. (A) PFGE of S1 nuclease-treated (lane 1) and intact (lane 2) genomic plugs. (B) Southern blot analysis with ascV as a probe against the gel in panel A. (C and D) Gel showing a PacI-digested genomic DNA plug from AH-1 (C) and Southern blot with ascV as a probe against the gel in panel C.

FIG. 3.

aopB and aopD mutants show a decrease in the pathogenesis of blue gourami infections. The number of fish alive after being injected with 10⁶ CFU of A. hydrophila is plotted against the number of days that it took for the fish to die. The fish were monitored for 7 days. WT, wild-type AH-1.

FIG. 4.

Micrographs of EPC cells (A and B) and blue gourami phagocytes (C D) infected with A. hydrophila AH-1 (A and C) and the aopB mutant (B and D). Phase-contrast micrographs of EPC monolayers were infected with A. hydrophila strains at 2.5 h postinfection (MOI of 1). Giemsa-stained bright-field micrographs of blue gourami phagocytes were infected with A. hydrophila strains at 3 h postinfection (MOI of 10). The aopB and aopD mutants had similar results in both EPC cell and phagocyte experiments. Bars, 50 μm (B) and 10 μm (D); panels A and C are at the same magnifications as panels B and D, respectively.

FIG. 5.

Phagocytosis assay. The percent phagocytosis of gourami phagocytes was calculated after infection with wild-type A. hydrophila (AH-1) (WT) or mutants (aopB and aopD). Results are expressed as the representative mean ± standard error of the mean from duplicate wells in triplicate experiments. Asterisks indicate a significant difference from results obtained with A. hydrophila AH-1 (P < 0.05).