Deletion of the genes encoding the type III secretion system and cytotoxic enterotoxin alters host responses to Aeromonas hydrophila infection

Abstract: In our previous study, we deleted the gene encoding Aeromonas outer membrane protein B (AopB), a structural component of the type III secretion system (T3SS) from a cytotoxic enterotoxin gene (act)-minus diarrheal isolate SSU of Aeromonas hydrophila. Our laboratory also molecularly characterized the cytotoxic enterotoxin (Act), which is secreted by the bacterium utilizing the type II secretion system (T2SS). The act/aopB mutant exhibited significantly reduced cytotoxicity to cultured cells (e.g. RAW 264.7 murine macrophages and HT-29 human colonic epithelial cells) and was avirulent in mice. In this study, we developed additional A. hydrophila mutants in which T3SS-associated ascV and acrV genes were deleted, either individually or in combination with that of the act gene, to examine host–pathogen interactions. A significant reduction in the induction of inflammatory cytokines and chemokines was noted in the sera of mice infected with these mutants when compared to animals infected with wild-type (WT) A. hydrophila. After infection with the WT and act/aopB mutant, we performed microarray analyses on RNA from the above-mentioned murine macrophages and human colonic epithelial cells to examine global cellular transcriptional responses. Based on three independent experiments, WT A. hydrophila altered the expression of 434 genes in RAW 264.7 cells and 80 genes in HT-29 cells. Alteration in the expression of 209 macrophage and 32 epithelial cell genes was reduced when the act/aopB mutant was used, compared to when cells were infected with the WT bacterium, indicating the involvement of Act and/or AopB in transcriptional regulation of these genes. We verified up-regulation of 15 genes by real-time reverse transcriptase-polymerase chain reaction and confirmed A. hydrophila WT–versus mutant-induced production of cytokines/chemokines in supernatants from RAW 264.7 and HT-29 cells. This is the first description of host cell transcriptional responses to A. hydrophila infection. [Copyright &y& Elsevier]