Contribution of nuclease to the pathogenesis of Aeromonas hydrophila

Abstract

Aeromonas hydrophila is a gram-negative bacterium that is widely distributed in aquatic environments and can cause septicemia in both fish and humans. However, the underlying mechanisms leading to severe infection are not well understood. In this study, an A. hydrophila nuclease (ahn) deletion mutant was constructed to investigate its contribution to pathogenesis. This mutant did not differ from the wild-type strain in terms of its growth or hemolytic phenotype. However, the ahn-deficient mutant was more susceptible to being killed by fish macrophages and mouse blood in vitro. Furthermore, evidence obtained using both fish and murine infection models strongly indicated that the inactivation of Ahn impaired the ability of A. hydrophila to evade innate immune clearance in vivo. More importantly, the virulence of the mutant was attenuated in both fish and mice, with reductions in dissemination capacities and mortality rates. These findings implicate Ahn in A. hydrophila virulence, with important functions in evading innate immune defenses.

Keywords: Aeromonas hydrophila; nuclease; pathogenesis.

Figure 1.

Construction and phenotypic characterization of the Δahn mutant and the complement strain, CΔahn. (A) Strategy for the deletion of ahn by homologous recombination. (B) Growth curves of the WT, Δahn and CΔahn strains. (C) β-Hemolytic phenotype of the strains grown on sheep's blood agar. (D) Scanning electron microscopic pictures of different strains (scale bar = 1 μm).

Figure 2.

Effect of Ahn deletion as determined by bactericidal assays. (A) Percentage of CFUs following 1 h of incubation with head-kidney macrophages. (B) Percentage of CFUs following 1 h of incubation at 37°C with heparinized mouse blood. The experiments were performed 3 times independently. The data shown were obtained from a representative experiment. *P < 0.05.

Figure 3.

Role of Ahn in resistance against clearance in vivo. (A) Severe accumulation of ascites in the abdomen of a blunt snout bream (Megalobrama amblycephala) infected with A. hydrophila. Scale bar = 1 cm. (B) Bacterial loads recovered from fish ascites after 24 h post-i.p. inoculation. (C) In vivo competitive indices of Δahn versus the WT strain in fish abdominal cavity and mouse air sac infection models.

Figure 4.

Inactivation of ahn impairs A. hydrophila systemic dissemination. (A) Numbers of viable A. hydrophila bacteria in mouse blood at 24 h after i.p.inoculation. Numbers of viable A. hydrophila bacteria in fish kidneies (B) and livers (C) at 24 h after i.p. inoculation. (D) Pathological examination of kidneies of fish infected with the indicated A. hydrophila strains. The arrow indicates renal tubular cell swelling. Scale bar = 200 μm.

Figure 5.

Ahn contributes to the virulence of A. hydrophila. (A) Survival curves for fish challenged with A. hydrophila wild-type and deletion strains. (B) Survival curves for mice challenged with A. hydrophila.