DNA Adenine Methyltransferase Influences the Virulence of Aeromonas hydrophila

Abstract

Among the various virulence factors produced by Aeromonas hydrophila, a type II secretion system (T2SS)-secreted cytotoxic enterotoxin (Act) and the T3SS are crucial in the pathogenesis of Aeromonas-associated infections. Our laboratory molecularly characterized both Act and the T3SS from a diarrheal isolate, SSU of A. hydrophila, and defined the role of some regulatory genes in modulating the biological effects of Act. In this study, we cloned, sequenced, and expressed the DNA adenine methyltransferase gene of A. hydrophila SSU (dam AhSSU ) in a T7 promoter-based vector system using Escherichia coli ER2566 as a host strain, which could alter the virulence potential of A. hydrophila. Recombinant Dam, designated as M.AhySSUDam, was produced as a histidine-tagged fusion protein and purified from an E. coli cell lysate using nickel affinity chromatography. The purified Dam had methyltransferase activity, based on its ability to transfer a methyl group from S-adenosyl-L-methionine to N 6 -methyladenine-free lambda DNA and to protect methylated lambda DNA from digestion with DpnII but not against the DpnI restriction enzyme. The dam gene was essential for the viability of the bacterium, and overproduction of Dam in A. hydrophila SSU, using an arabinose-inducible, P BAD promoter-based system, reduced the virulence of this pathogen. Specifically, overproduction of M.AhySSUDam decreased the motility of the bacterium by 58%. Likewise, the T3SS-associated cytotoxicity, as measured by the release of lactate dehydrogenase enzyme in murine macrophages infected with the Dam-overproducing strain, was diminished by 55% compared to that of a control A. hydrophila SSU strain harboring the pBAD vector alone. On the contrary, cytotoxic and hemolytic activities associated with Act as well as the protease activity in the culture supernatant of a Dam-overproducing strain were increased by 10-, 3-, and 2.4-fold, respectively, compared to those of the control A. hydrophila SSU strain. The Dam-overproducing strain was not lethal to mice (100% survival) when given by the intraperitoneal route at a dose twice that of the 50% lethal dose, which within 2 to 3 days killed 100% of the animals inoculated with the A. hydrophila control strain. Taken together, our data indicated alteration of A. hydrophila virulence by overproduction of Dam.Aeromonas hydrophila is both a human and an animal pathogen, and the diseases associated with this bacterium in humans include gastroenteritis, wound infections, and septicemia (36, 88). In immuno-compromised individuals, some of the more serious complications associated with A. hydrophila infections are cellulitis, myonecrosis, and ecthyma gangrenosum, which often are fatal (2, 36). A variety of virulence factors/mechanisms, such as the surface layer, capsule, different forms of pili (e.g., type IV [Tap] and bundle-forming pili), toxins, quorum sensing, biofilm formation and, more recently, the type III secretion system (T3SS), were identified in various Aeromonas species that are involved in the pathogenesis...

Evaluation of the roles played by Hcp and VgrG type 6 secretion system effectors in Aeromonas hydrophila SSU pathogenesis

“…Protease activity in culture filtrates of various Aeromonas strains was measured using our previously described procedure (Erova et al, 2006). In short, protease activity was calculated per ml culture filtrate and was divided by the optical density of the culture to obtain the protease activity per unit of growth.…”

“…murine infection model (Erova et al, 2006;Khajanchi et al, 2009;Sierra et al, 2010Sierra et al, , 2011. Following a challenge dose of 4.0610 7 c.f.u.…”

“…In addition to motility, the production of proteases, in particular metalloproteases, also promotes A. hydrophila virulence (Erova et al, 2006). To determine contributions made by Hcp and/or VgrG proteins to protease activity, we measured the ability of Aeromonas mutant culture filtrates to catalyse the degradation of Hide powder azure substrate.…”

Altered Ca2+Regulation of Yop Secretion inYersinia enterocoliticaafter DNA Adenine Methyltransferase Overproduction Is Mediated by Clp-Dependent Degradation of LcrG

“…Furthermore, Dam has been shown to be essential for virulence in a number of human and animal pathogens, like Salmonella enterica, Yersinia pseudotuberculosis, Yersinia pestis, Haemophilus influenzae, Vibrio cholerae, Pasteurella multocida, and Aeromonas hydrophila (9,19,36,37,71). For example, Dam-deficient S. enterica serovar Typhimurium show reduced M-cell cytotoxicity and invasion of enterocytes.…”

“…In addition to having these functions, DNA methylation influences the binding of regulatory proteins to promoter regions, thereby imparting an epigenetic mechanism linked to replication on gene expression. The best-studied example is the expression of pyelonephritis-associated pili (pap) of uropathogenic E. coli, where Dam controls the binding of Lrp in the promoter region of the pap operon (29).Furthermore, Dam has been shown to be essential for virulence in a number of human and animal pathogens, like Salmonella enterica, Yersinia pseudotuberculosis, Yersinia pestis, Haemophilus influenzae, Vibrio cholerae, Pasteurella multocida, and Aeromonas hydrophila (9,19,36,37,71 (3,36,37). Attenuation of these strains was used effectively in the development of vaccine strains of Salmonella as well as Yersinia (17,18,26,36,57,66).…”