Strains of Avian Pathogenic Escherichia coli (APEC) Induce Caspase 3 Activation in HD11 Cell Line of Chicken Macrophages
F. Horn1,2; N.L. Barbieri1; M. Nordhoff2; C. Ewers2, L.H. Wieler2
1Departamento de Biofísica, Universidade Federal do Rio Grande do Sul, P.O. Box 15005, 91501-970, Porto Alegre, RS, Brasil; 2Institut für Mikrobiologie und Tierseuchen, Freie Universität Berlin, Germany
Avian pathogenic Escherichia coli (APEC) causes extra-intestinal infections in poultry, primarily respiratory infections that develop into pericarditis, perihepatitis and septicaemia. After adhering to tracheal epithelium, APEC must resist the bactericidal effect of serum and evade the host's phagocytic defense systems in order to infect internal organs and cause disease. Previous work showed that one strain of APEC (APEC17, ONT:H5) induced apoptosis in murine macrophage J744 cells. In this work, we investigate the ability of 5 APEC strains, distinguished by virulence-associated gene with a distinct patterns of virulence genes, to induce caspase 3/7 activation, the central caspases in apoptosis, in chicken macrophage HD11 cells. We used a non-pathogenic avian E. coli strain (IMT5104) as a negative control.
Monolayers of HD11 cells were infected with bacteria for 1 h at a multiplicity of infection of 20 or 150 bacteria/cell; cells were then washed and reincubated in medium containing 100 mg/ml gentamycin. At different time points s, cell extracts were prepared and assayed for caspase 3/7 activity using Ac-DEVD-AMC substrate. Our results show that 3 strains (APEC17, MT78 and TK3) induced caspase 3/7 activation; APEC strains IMT5155 and IMT9300 induced caspase activation at levels only marginally higher compared with IMT5104 (non-pathogenic). Activity was detected at 4 h and 6 h post-infection; it was neither detected as early as 2 h nor as late as 20 h post-infection. Analysis by confocal microscopy of infected macrophages showed that APEC are intracellularly located.
Our results suggest that other APEC strains, in addition to APEC17, cause apoptosis in infected macrophages. Further in vivo investigations are under way We wonder if apoptosis of defence cells is to verify the relevance of these findings in the establishment of disease.
Financial support: CAPES; BIC-Propesq UFRGS
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