Role of sialidase in Mycoplasma alligatoris-induced pulmonary fibroblast apoptosis

Mycoplasma alligatoris causes acute lethal cardiopulmonary disease of susceptible hosts. A survey of its genome implicated sialidase and hyaluromdase, synergistic regulators of hyaluronan receptor CD44-mediated signal transcluction 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 (X-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, coincubation 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-deoxyuri dine incorporation measured in a terminal dUTP nick end-labeling apoptosis assay. In contrast, incubation with 200-fold excess purified C perfringens sialiclase alone did not affect CD95 expression or chromatin integrity, or induce fibroblast apoptosis. From those observations we conclude that interaction of its sialiclase with hyaluronidase or another virulence factor(s) is necessary to elicit the pro-apoptotic effects of M. alligatoris infection. (c) 2006 Elsevier B.X. All rights reserved.