The type III secretion system and cytotoxic enterotoxin alter the virulence of Aeromonas hydrophila

Abstract

Many gram-negative bacteria use a type III secretion system (TTSS) to deliver effector proteins into host cells. Here we report the characterization of a TTSS chromosomal operon from the diarrheal isolate SSU of Aeromonas hydrophila. We deleted the gene encoding Aeromonas outer membrane protein B (AopB), which is predicted to be involved in the formation of the TTSS translocon, from wild-type (WT) A. hydrophila as well as from a previously characterized cytotoxic enterotoxin gene (act)-minus strain of A. hydrophila, thus generating aopB and act/aopB isogenic mutants. The act gene encodes a type II-secreted cytotoxic enterotoxin (Act) that has hemolytic, cytotoxic, and enterotoxic activities and induces lethality in a mouse model. These isogenic mutants (aopB, act, and act/aopB) were highly attenuated in their ability to induce cytotoxicity in RAW 264.7 murine macrophages and HT-29 human colonic epithelial cells. The act/aopB mutant demonstrated the greatest reduction in cytotoxicity to cultured cells after 4 h of infection, as measured by the release of lactate dehydrogenase enzyme, and was avirulent in mice, with a 90% survival rate compared to that of animals infected with Act and AopB mutants, which caused 50 to 60% of the animals to die at a dose of three 50% lethal doses. In contrast, WT A. hydrophila killed 100% of the mice within 48 h. The effects of these mutations on cytotoxicity could be complemented with the native genes. Our studies further revealed that the production of lactones, which are involved in quorum sensing (QS), was decreased in the act (32%) and aopB (64%) mutants and was minimal (only 8%) in the act/aopB mutant, compared to that of WT A. hydrophila SSU. The effects of act and aopB gene deletions on lactone production could also be complemented with the native genes, indicating specific effects of Act and the TTSS on lactone production. Although recent studies with other bacteria have indicated TTSS regulation by QS, this is the first report describing a correlation between the TTSS and Act of A. hydrophila and the production of lactones.

FIG. 1.

(A) Cytotoxicity induced by WT A. hydrophila, its various mutants, and complemented strains in RAW 264.7 cells. Macrophages in triplicate wells were infected with live Aeromonas strains at an MOI of 10 for 2 h and 4 h. After incubation, host cell culture supernatants were used to measure LDH release. (B and C) Cytotoxicity induced by overnight-grown bacterial culture supernatants from WT A. hydrophila, its various mutants, and complemented strains in HT-29 (B) and RAW 264.7 (C) cells. Arithmetic means ± standard deviations from three independent experiments were used for plotting the data. Single asterisks denote statistically significant differences (by Student's t test) between various mutant strains and the WT bacterium. A double asterisk denotes a statistically significant difference between 2- and 4-h treatment of host cells with the aopB mutant. A triple asterisk indicates a statistically significant difference between the act mutant and the WT bacterium at 4 h. The actual P values are presented in the text. aopB⁺ and act⁺, complemented strains of aopB and act mutants.

FIG. 2.

Alteration of cell morphology induced by WT A. hydrophila and its various mutants in HT-29 (A) and RAW 264.7 (B) cells. Host cells were infected at an MOI of 10 and incubated for 2 h. Cells were then washed in PBS, fixed with 4% paraformaldehyde, and examined by Nomarski differential interference microscopy. Noninfected cells (control) and those infected with the aopB or act/aopB mutants exhibited normal cell morphology, while cell morphology was significantly altered (highly vacuolated and flattened [HT-29] or rounded [macrophages]) in cells infected with the WT or the act mutant. The experiment was performed in triplicate, and representative pictures are shown. For the cell detachment assay (C), HT-29 or RAW 264.7 cells were infected with the above-mentioned bacteria at an MOI of 10 for 2 and 4 h. The percentage of cells detached from the monolayers was calculated by Giemsa staining and solubilization of macrophages/HT-29 cells to release the blue color, the intensity of which was measured at 590 nm as described in Materials and Methods. Single asterisks denote statistically significant differences (by Student's t test) between various mutant strains and the WT bacterium. A double asterisk denotes a statistically significant difference between 2- and 4-h treatment of host cells with the aopB mutant. A triple asterisk indicates a statistically significant difference between the act mutant and the WT bacterium at 4 h.

FIG. 3.

Mutations in both the act and aopB genes rendered A. hydrophila SSU avirulent in a mouse model. Swiss Webster mice (n = 10 per group) were injected intraperitoneally with three 50% lethal doses of indicated mutants and the WT A. hydrophila SSU and monitored for death over a period of 16 days. The data were statistically analyzed using Fisher's exact test. Three independent experiments were performed, and data from a typical experiment are shown. Asterisks denote statistically significant differences between act/aopB mutant and WT bacteria. The actual P values are presented in the text.

FIG. 4.

Effect on lactone production by the TTSS and Act of A. hydrophila SSU. WT A. hydrophila, its various mutants, and complemented strains were grown to various densities (OD₆₀₀ of 0.3 to 1.2). The cultures were centrifuged, and the supernatants were measured for β-galactosidase activity (presented in Miller units), indicative of AHL production, using an A. tumefaciens β-galactosidase reporter strain. Three to five independent experiments were performed, and the arithmetic mean ± deviation was plotted. Single asterisks denote statistically significant differences (by Student's t test) in lactone production between WT and its various mutant derivatives at an OD of 1.2. A double asterisk denotes a statistically significant difference in lactone production in WT and its act mutant at ODs of 0.9 and 1.2. The increase in lactone production by the complemented strains was significantly higher than that in the corresponding mutants but was statistically insignificant compared to lactone production in the WT bacterium. Strains designated act⁺ and aopB⁺ indicate complementation of the corresponding mutants. The actual P values are presented in the text. Compl, complementation.