Further characterization of a type III secretion system (T3SS) and of a new effector protein from a clinical isolate of Aeromonas hydrophila-Part I

A type III secretion system (T3SS)-associated cytotoxin, AexT, with ADP-ribosyltransferase activity and homology to Pseudomonas aeruginosa bifuncational toxins ExoT/S, was recently identified from a fish pathogen Aeromonas salmonicida. In this study, we reported the molecular characterization of an aexT-like toxin gene (designated as aexU) from a diarrheal isolate SSU of A. hydrophila. The aexU gene was 1539 bp in length and encoded a protein of 512 amino acid (aa) residues. The NH2-terminus of AexU (aa residues 1-231) exhibited a 67% homology with the NH2-terminus of AexT from A. salmonicida. Importantly, its COOH-terminus (aa residues 232-512) had no homology with any known functional proteins in the database; however, the full-length AexU retained ADP-ribosyltransferase activity. The expression and subsequent secretion of AexU was T3SS dependent, as inactivation of the ascV gene that codes for an inner-membrane component of the T3SS channel from the wild-type (WT) bacterium, blocked translocation of AexU in HT-29 human colonic epithelial cells. We provided evidence that inactivation of acrV and axsE genes (homologs of lcrV and exsE in Yersinia species and P. aeruginosa, respectively) from A. hydrophila SSU, altered expression and/or secretion of AexU. We deleted an aexU gene from the WT, as well as from the ΔaopB mutant, of A. hydrophila, generating a single knockout (ΔaexU) and a double knockout mutant, ΔaopB/ΔaexU. Increased phagocytosis was observed in RAW264.7 murine macrophages infected with the ΔaopB/ΔaexU mutant, as compared to macrophages when infected with the parental ΔaopB strain. Further, mice infected with the ΔaexU mutant had a 60% survival rate, compared to animals infected with the WT or the ΔaexU-complemented strain that caused 90-100% of the animals to die at a 2-3 LD50s dose. Immunization of mice with the recombinant AexU protected them from subsequent lethal challenge dose by the WT bacterium. Finally, we detected specific anti-AexU antibodies in the sera of mice that survived challenge by the WT bacterium, which may indicate that AexU plays an important role in the pathogenesis of Aeromonas infections.