TY - JOUR
T1 - Biological characterization of a new type III secretion system effector from a clinical isolate of Aeromonas hydrophila-Part II
AU - Sierra, Johanna C.
AU - Suarez, Giovanni
AU - Sha, Jian
AU - Foltz, Sheri M.
AU - Popov, Vsevolod L.
AU - Galindo, Cristi L.
AU - Garner, Harold R.
AU - Chopra, Ashok K.
N1 - Funding Information:
This research was supported by grants from the NIH/NIAID (AI041611), the Environmental Protection Agency, and NIH/NIAID Western Regional Center of Excellence 1U54 AI057156-01. Cristi L. Galindo received support from an NIH cardiology fellowship, Cardiology Department, University of Texas Southwestern Medical Center. We thank Dr. Tom Albrecht and Eugene P. Knutson, Department of Microbiology and Immunology, UTMB, for providing their expertise in planning studies related to confocal microscopy and for the facilities of the Infectious Diseases and Toxicology Optical Imaging Core. We thank Ms. Mardelle Susman for editorial assistance.
PY - 2007/10
Y1 - 2007/10
N2 - We recently identified a novel type III secretion system (T3SS) effector, AexU, from a diarrheal isolate SSU of Aeromonas hydrophila, and demonstrated that mice infected with the ΔaexU mutant were significantly protected from mortality. Although the NH2-terminal domain of this toxin exhibits homology to AexT of A. salmonicida, a fish pathogen, and ExoT/S of Pseudomonas aeruginosa, the COOH-terminal domain of AexU is unique, with no homology to any known proteins in the NCBI database. In this study, we purified the full-length AexU and its NH2-terminal (amino acid residues 1-231) and COOH-terminal (amino acid residues 232-512) domains after expression of their corresponding genes in Escherichia coli as histidine-tag fusion proteins using the bacteriophage T7 RNA polymerase/promoter-based pET-30a vector system. The full-length and NH2- and COOH-terminal domains of AexU exhibited ADP-ribosyltransferase activity, with the former two exhibiting much higher activity than the latter. These different forms of AexU were also successfully expressed and produced in the HeLa Tet-Off cell system using a pBI-EGFP vector, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot analysis, and intracellular staining of the toxin using flow cytometric analysis. Production of AexU in HeLa cells resulted in possible actin reorganization and cell rounding, as determined by phalloidin staining and confocal microscopy. Based on electron microscopy, the toxin also caused chromatin condensation, which is indicative of apoptosis. Apoptosis of HeLa cells expressing and producing AexU was confirmed by 7-amino actinomycin D (7-AAD) and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide] assays, by detection of cytoplasmic histone-associated DNA fragments, and by activation of caspases 3 and 9. These effects were much more pronounced in host cells that expressed and produced the full-length or NH2-terminal domain of AexU, compared to those that expressed and produced the COOH-terminal domain or the vector alone. This study represents the first characterization of this novel T3SS effector.
AB - We recently identified a novel type III secretion system (T3SS) effector, AexU, from a diarrheal isolate SSU of Aeromonas hydrophila, and demonstrated that mice infected with the ΔaexU mutant were significantly protected from mortality. Although the NH2-terminal domain of this toxin exhibits homology to AexT of A. salmonicida, a fish pathogen, and ExoT/S of Pseudomonas aeruginosa, the COOH-terminal domain of AexU is unique, with no homology to any known proteins in the NCBI database. In this study, we purified the full-length AexU and its NH2-terminal (amino acid residues 1-231) and COOH-terminal (amino acid residues 232-512) domains after expression of their corresponding genes in Escherichia coli as histidine-tag fusion proteins using the bacteriophage T7 RNA polymerase/promoter-based pET-30a vector system. The full-length and NH2- and COOH-terminal domains of AexU exhibited ADP-ribosyltransferase activity, with the former two exhibiting much higher activity than the latter. These different forms of AexU were also successfully expressed and produced in the HeLa Tet-Off cell system using a pBI-EGFP vector, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot analysis, and intracellular staining of the toxin using flow cytometric analysis. Production of AexU in HeLa cells resulted in possible actin reorganization and cell rounding, as determined by phalloidin staining and confocal microscopy. Based on electron microscopy, the toxin also caused chromatin condensation, which is indicative of apoptosis. Apoptosis of HeLa cells expressing and producing AexU was confirmed by 7-amino actinomycin D (7-AAD) and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide] assays, by detection of cytoplasmic histone-associated DNA fragments, and by activation of caspases 3 and 9. These effects were much more pronounced in host cells that expressed and produced the full-length or NH2-terminal domain of AexU, compared to those that expressed and produced the COOH-terminal domain or the vector alone. This study represents the first characterization of this novel T3SS effector.
KW - ADP-ribosyltransferase activity
KW - Aeromonas hydrophila
KW - Cell death
KW - Effector proteins
KW - Expression in prokaryotic and eukaryotic systems
KW - T3SS
UR - http://www.scopus.com/inward/record.url?scp=34547600796&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=34547600796&partnerID=8YFLogxK
U2 - 10.1016/j.micpath.2007.05.003
DO - 10.1016/j.micpath.2007.05.003
M3 - Article
C2 - 17582731
AN - SCOPUS:34547600796
SN - 0882-4010
VL - 43
SP - 147
EP - 160
JO - Microbial Pathogenesis
JF - Microbial Pathogenesis
IS - 4
ER -