TY - JOUR
T1 - Deletion of the genes encoding the type III secretion system and cytotoxic enterotoxin alters host responses to Aeromonas hydrophila infection
AU - Fadl, Amin A.
AU - Galindo, Cristi L.
AU - Sha, Jian
AU - Erova, Tatiana E.
AU - Houston, Clifford
AU - Olano, Juan P.
AU - Chopra, Ashok K.
N1 - Funding Information:
This work was supported by a grant from the NIH/NIAID (AI41611) and the Gastrointestinal Research Interdisciplinary Program (GRIP), University of Texas Medical Branch (UTMB), Galveston, TX. Funds provided by the American Waters Works Association research Foundation were also crucial for the completion of these studies. Cristi L. Galindo, a predoctoral fellow, obtained funding from the National Science Foundation. A.A. Fadl was supported by the McLaughlin Postdoctoral Fellowship. Dr T. Wood from the department of HBC&G at UTMB, provided facility of his core laboratory for microarray studies. Dr V. Reyes, from the Department of Pediatrics at UTMB, provided facility of his core laboratory for cytokine profile assays.
PY - 2006/5
Y1 - 2006/5
N2 - 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.
AB - 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.
KW - Aeromonas hydrophila
KW - Affymetrix microarrays
KW - Cytokine/chemokine production
KW - Isogenic mutants
KW - Mouse model of infection
KW - Murine macrophages and human colonic epithelial cell lines
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U2 - 10.1016/j.micpath.2006.01.003
DO - 10.1016/j.micpath.2006.01.003
M3 - Article
C2 - 16626931
AN - SCOPUS:33747792784
SN - 0882-4010
VL - 40
SP - 198
EP - 210
JO - Microbial Pathogenesis
JF - Microbial Pathogenesis
IS - 5
ER -