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Role of sialidase in Mycoplasma alligatoris-induced pulmonary fibroblast apoptosis

Hunt, M.E., Brown, D.R.
Veterinary microbiology 2007 v.121 no.1-2 pp. 73-82
sialidase, Mycoplasma alligatoris, mycoplasmosis, lungs, fibroblasts, animal diseases, enzyme activity, apoptosis, virulence, respiratory tract diseases, alligators, cell culture, protein synthesis, signal transduction, in vitro studies
Mycoplasma alligatoris causes acute lethal cardiopulmonary disease of susceptible hosts. A survey of its genome implicated sialidase and hyaluronidase, synergistic regulators of hyaluronan receptor CD44-mediated signal transduction leading to apoptotic cell death, as virulence factors of M. alligatoris. In this study, after the existence of a CD44 homolog in alligators was established by immunolabeling primary pulmonary fibroblasts with monoclonal antibody IM7 against murine CD44, the sialidase inhibitor 2,3-didehydro-2-deoxy-N-acetylneuraminic acid (DANA) was used to examine the effects of sialidase on fibroblast apoptosis following in vitro infection with M. alligatoris. While their CD44 expression remained constant, infected cells exhibited morphologic changes characteristic of apoptosis including decreased size, rounding, disordered α-tubulin, and nuclear disintegration compared to untreated controls. DANA was a potent, non-toxic inhibitor of the sialidase activity, equivalent to about 1 mU of Clostridium perfringens Type VI sialidase, expressed by M. alligatoris in the inoculum. Although DANA did not measurably reduce the proportion of infected fibroblasts labeled by a specific ligand of activated caspases, co-incubation with DANA protected (P < 0.01) fibroblasts in a concentration-dependent fashion from the M. alligatoris-induced trends toward increased apoptosis receptor CD95 expression, and increased 5-bromo-2'-deoxyuridine incorporation measured in a terminal dUTP nick end-labeling apoptosis assay. In contrast, incubation with 200-fold excess purified C. perfringens sialidase alone did not affect CD95 expression or chromatin integrity, or induce fibroblast apoptosis. From those observations we conclude that interaction of its sialidase with hyaluronidase or another virulence factor(s) is necessary to elicit the pro-apoptotic effects of M. alligatoris infection.