Role of sialidase in Mycoplasma alligatoris-induced pulmonary fibroblast apoptosis

Abstract

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 a-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.

Small molecule inhibitors of mammalian glycosylation

“…The sialidase inhibitor 2-deoxy-2,3-didehydro- N -acetylneuraminic acid (DANA) inhibits mammalian sialidases in cultured human leukaemia cells, primary pulmonary fibroblasts, and primary rat hippocampal neurons at a concentration of 1 mM, in pancreatic beta cells at 100 µM, and at 10 mg/kg i.p. in mice [210] , [211] , [212] , [213] . However, DANA is still much more efficient in the inhibition of viral and bacterial sialidases [214] .…”

Genetic variation in sialidase and linkage to N-acetylneuraminate catabolism in Mycoplasma synoviae

“…Most bacterial sialidases preferentially cleave α-(2-3)-linked sialic acids, and are found in species that live in close contact with vertebrate host cells as commensals or facultative pathogens. Sialidase activity is involved in bacterial colonization and dissemination, ECM degradation, and induced host-cell death [12,20,21,22,23]. It has also been proposed that bacterial desialylation of host glycoconjugates could expose or form new host antigens to play a role in autoimmune complications of infection [24,25].…”

Sialidase Activity in Mycoplasma Synoviae

“…Sialidase (EC 3.2.1.18) activity is common in other pathogens, and has been associated with bacterial colonization, extracellular matrix degradation, nutrition, dissemination, and induction of apoptosis (7,15,19,25,40). The enzyme catalyzes hydrolysis of α-(2-3)-, α-(2-6)-, and α-(2-8)-glycosidic linkages of terminal sialic residues in oligosaccharides, glycoproteins, glycolipids, colominic acid and synthetic substrates.…”