Browsing by Subject "Aeromonas hydrophila"
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Item Important role of the type VI secretion system in the virulence of an emergent human pathogen Aeromonas hydrophila: Delineating the mechanism of action of its effectors(2010-07-02) Giovanni Suarez; Ashok Chopra; Vladimir Motin; Victor Reyes; Vernon Tesh; Johnny PetersonAeromonas hydrophila causes various human diseases, including gastroenteritis, wound infections and septicemia. In this study, we characterized the new type 6 secretion system (T6SS) from isolate SSU of A. hydrophila and demonstrated its role in bacterial virulence. We have provided evidence that the T6SS is independent of other secretion systems and that the vasH gene is essential for the expression of the genes encoding the T6SS gene cluster. We demonstrated T6SS’s ability to translocate effector proteins into the host cells and also showed that T6SS mutants were less toxic to human and murine cell lines, and more efficiently phagocytosed by macrophages. Importantly, bacterial-host cell interaction was needed for the T6SS to induce cytotoxicity in eukaryotic cells. Based on 2-dimensional gel electrophoresis and mass spectrometric analyses of bacterial supernatants, we identified a member of the VgrG protein family, (VgrG1), containing a vegetative insecticidal protein (VIP-2) domain at its carboxyl-terminal end, as well as the hemolysin co-regulated protein (Hcp). We provided evidence that VgrG1 showed ADP-ribosyltransferase activity associated with its VIP-2 domain. Our data indicated that episomal expression of the vgrG1 gene in HeLa Tet-Off cells disrupted the actin cytoskeleton, followed by a decrease in cell viability and an increase in apoptosis. We also provided evidence that the expression of the hcp gene in HeLa cells resulted in apoptosis of the host cells without changes in their morphology. In addition, we showed that the addition of exogenous recombinant Hcp (rHcp) reduced bacterial uptake by macrophages. These results were substantiated by the in vivo analysis of cytokine profiling and activation of host immune cells after infection with the ÄvasH mutant supplemented with rHcp indicating that this T6SS effector inhibited production of pro-inflammatory cytokines and induced production of immunosuppressive cytokines, such as IL-10 and TGF-â. In summary, we showed that the T6SS represent an important virulence determinant of A. hydrophila.Item Mechanism of action of AexU, a new type III secretion system effector from an emerging human pathogen Aeromonas hydrophila(2010-07-08) Johanna Carolina Sierra; Ashok Chopra; Vladimir Motin; Judith Johnson; Johnny Peterson; Eric SmithOur laboratory first reported the complete sequence of the type III secretion system (T3SS) from a diarrheal isolate SSU of A. hydrophila. We identified an effector protein (designated as AexU) of the T3SS, which exhibited ADP-ribosyltransferase (ADPRT) and GTPase-activating protein (GAP) activity. AexU was successfully expressed in the HeLa cell Tet-Off system and I provided evidence that cells expressing and producing the full length AexU showed actin reorganization followed by apoptosis. Earlier, we showed that the ÄaexU null mutant was attenuated in a mouse model, and I now demonstrated that while the parental A. hydrophila strain could be detected in the lung, liver, and spleen of infected mice, the ÄaexU mutant was rapidly cleared from these organs resulting in increased survivability of animals. The GAP activity of AexU was mainly responsible for host cell apoptosis and disruption of actin filaments. Further, AexU prevented phosphorylation of c-Jun, JNK and IêBá and inhibited IL-6 and IL-8 secretion from HeLa cells. Our data indicated that AexU operated by inhibiting NF-êB and inactivating Rho GTPases. Importantly, however, when the ÄaexU null mutant was complemented with the mutated aexU gene devoid of ADPRT and GAP activities, a higher mortality rate in mice with concomitant increase in the production of proinflammatory cytokines/chemokines was noted. These data indicated that either such a mutated AexU is a potent inducer of them or that AexU possesses yet another unknown activity that is modulated by ADPRT and GAP activities and results in this aberrant cytokine/chemokine production responsible for increased animal death.Item Studies on Waterborne Pathogen Reactivation after Disinfection(2013-12-09) Kaur, JasjeetReactivation of different Escherichia coli strains and Aeromonas hydrophila after ultraviolet (UV), ultrasound, and photocatalytic disinfection treatments was addressed in this study. Photocatalytic disinfection was carried out under low pressure ultraviolet (LP UV) irradiation at five titanium dioxide (TiO_(2)) concentrations (1 g/L, 0.5 g/L, 0.75 g/L, and 0.1 g/L) to achieve 5 log_(10) reduction of a laboratory E. coli K-12 strain (ATCC? 10798). Regrowth and reactivation of E. coli in dark and light was studied up to 4 h after disinfection period. During the repair period, flow cytometry had shown 4-5 log_(10) higher cell counts than culture based method. Photocatalysis at 0.1 g/L TiO_(2) had resulted in 50% cells with intact cell membrane during the repair period and has lowered the repair rate of the E. coli (ATCC? 10798) after disinfection. Then ultrasound (24 kHz) and UV-C irradiation were applied to inactivate four E. coli isolates (ATCC?10798, E. coli isolate from feces of feral hog and deer, and treated wastewater effluent) to nearly 8 log_(10) reduction. Photoreactivation and dark repair of E. coli isolates were studied over a 24 h period after disinfection. In general, ultrasound disinfection had resulted in higher inactivation rate of 0.52 log min^(-1) than UV-C (0.39 log min^(-1)) for E. coli isolates. The extent of percent log repair of ultrasound inactivated E. coli isolates after 24 h of dark repair and photoreactivation were 30% lower than after UV-C. The metabolic activity of E. coli cells was greatly reduced after ultrasound as shown by AlamarBlue? assay. Transmission electron micrographs of ultrasound disinfected E. coli revealed shearing and size reduction of bacterial cells. Aeromonas hydrophila (ATCC? 35654), an emerging pathogen, was inactivated using a 24 kHz continuous ultrasound and UV-C in combination with three TiO_(2) concentrations (1g/L, 0.1 g/L and 0 g/L). High inactivation rate of 1.52 log min^(-1) was observed for ultrasound disinfection in absence of TiO_(2). Aeromonas hydrophila had showed a net log reduction of 6 log_(10) after ultrasound exposure in comparison to a net 2 log_(10) reduction after UV-C over a 24 h repair period. Metabolic activity of Aeromonas hydrophila was adversely affected by ultrasonication as well. Ultrasound appears to be effective in inactivating environmental E. coli isolates and Aeromonas hydrophila in water through shearing and breaking effects, which decreased the metabolic activity as well as photoreactivation and dark repair.