Fadl Amin A, Galindo Cristi L, Sha Jian, Erova Tatiana E, Houston Clifford W, Olano Juan P, Chopra Ashok K
Department of Microbiology and Immunology, The University of Texas Medical Branch, Medical Research Building, 301 University Boulevard, Galveston, TX 77555-1070, USA.
Microb Pathog. 2006 May;40(5):198-210. doi: 10.1016/j.micpath.2006.01.003. Epub 2006 Apr 19.
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.
在我们之前的研究中，我们从嗜水气单胞菌的细胞毒性肠毒素基因（act）缺失的腹泻分离株SSU中删除了编码气单胞菌外膜蛋白B（AopB）的基因，AopB是III型分泌系统（T3SS）的一个结构成分。我们实验室还对细胞毒性肠毒素（Act）进行了分子特征分析，该毒素由细菌利用II型分泌系统（T2SS）分泌。act/aopB突变体对培养细胞（如RAW 264.7小鼠巨噬细胞和HT-29人结肠上皮细胞）的细胞毒性显著降低，并且在小鼠中无致病性。在本研究中，我们构建了另外的嗜水气单胞菌突变体，其中T3SS相关的ascV和acrV基因被单独或与act基因组合删除，以研究宿主-病原体相互作用。与感染野生型（WT）嗜水气单胞菌的动物相比，感染这些突变体的小鼠血清中炎症细胞因子和趋化因子的诱导显著降低。在用WT和act/aopB突变体感染后，我们对上述小鼠巨噬细胞和人结肠上皮细胞的RNA进行了微阵列分析，以检查整体细胞转录反应。基于三个独立实验，WT嗜水气单胞菌改变了RAW 264.7细胞中434个基因和HT-29细胞中80个基因的表达。与用WT细菌感染细胞相比，当使用act/aopB突变体时，209个巨噬细胞基因和32个上皮细胞基因的表达改变减少，表明Act和/或AopB参与了这些基因的转录调控。我们通过实时逆转录-聚合酶链反应验证了15个基因的上调，并证实了嗜水气单胞菌WT与突变体诱导RAW 264.7和HT-29细胞上清液中细胞因子/趋化因子的产生。这是对宿主细胞对嗜水气单胞菌感染的转录反应的首次描述。
