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
alpha-tubulin, and nuclear disintegration compared to untreated controls. DANA was a potent, non-toxic inhibitor of the
sialidase activity, equivalent to about 1mU 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.