Biological characterization of a new type III secretion system effector from a clinical isolate of Aeromonas hydrophila-Part II

Johanna C. Sierra, Giovanni Suarez, Jian Sha, Sheri M. Foltz, Vsevolod Popov, Cristi L. Galindo, Harold R. Garner, Ashok Chopra

Research output: Contribution to journalArticle

32 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)147-160
Number of pages14
JournalMicrobial Pathogenesis
Volume43
Issue number4
DOIs
StatePublished - Oct 2007

Fingerprint

Aeromonas hydrophila
HeLa Cells
ADP Ribose Transferases
Apoptosis
Staining and Labeling
Phalloidine
Amino Acids
Caspase 9
Bromides
Histidine
Confocal Microscopy
Sodium Dodecyl Sulfate
Caspase 3
Histones
Pseudomonas aeruginosa
Chromatin
Actins
Polyacrylamide Gel Electrophoresis
Electron Microscopy
Fishes

Keywords

  • ADP-ribosyltransferase activity
  • Aeromonas hydrophila
  • Cell death
  • Effector proteins
  • Expression in prokaryotic and eukaryotic systems
  • T3SS

ASJC Scopus subject areas

  • Microbiology
  • Infectious Diseases

Cite this

Biological characterization of a new type III secretion system effector from a clinical isolate of Aeromonas hydrophila-Part II. / Sierra, Johanna C.; Suarez, Giovanni; Sha, Jian; Foltz, Sheri M.; Popov, Vsevolod; Galindo, Cristi L.; Garner, Harold R.; Chopra, Ashok.

In: Microbial Pathogenesis, Vol. 43, No. 4, 10.2007, p. 147-160.

Research output: Contribution to journalArticle

Sierra, Johanna C. ; Suarez, Giovanni ; Sha, Jian ; Foltz, Sheri M. ; Popov, Vsevolod ; Galindo, Cristi L. ; Garner, Harold R. ; Chopra, Ashok. / Biological characterization of a new type III secretion system effector from a clinical isolate of Aeromonas hydrophila-Part II. In: Microbial Pathogenesis. 2007 ; Vol. 43, No. 4. pp. 147-160.
@article{74293c6382734a4d876c2a7bd0596d89,
title = "Biological characterization of a new type III secretion system effector from a clinical isolate of Aeromonas hydrophila-Part II",
abstract = "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.",
keywords = "ADP-ribosyltransferase activity, Aeromonas hydrophila, Cell death, Effector proteins, Expression in prokaryotic and eukaryotic systems, T3SS",
author = "Sierra, {Johanna C.} and Giovanni Suarez and Jian Sha and Foltz, {Sheri M.} and Vsevolod Popov and Galindo, {Cristi L.} and Garner, {Harold R.} and Ashok Chopra",
year = "2007",
month = "10",
doi = "10.1016/j.micpath.2007.05.003",
language = "English (US)",
volume = "43",
pages = "147--160",
journal = "Microbial Pathogenesis",
issn = "0882-4010",
publisher = "Academic Press Inc.",
number = "4",

}

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

AU - Galindo, Cristi L.

AU - Garner, Harold R.

AU - Chopra, Ashok

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

VL - 43

SP - 147

EP - 160

JO - Microbial Pathogenesis

JF - Microbial Pathogenesis

SN - 0882-4010

IS - 4

ER -