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 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.

Fig. 1

Receptor and ligand expression by primary alligator pulmonary fibroblasts. Nuclei were stained with 4′,6′-diamidino-2-phenylindole hydrochloride (blue) and cytoskeletal proteins were immunolabeled with complementary fluorophores. (a) Hyaluronan receptor CD44 (yellow double-tagged with fluorescein and tetramethylrhodamine) immunolabeled with mAb IM7; (b) Fas receptor CD95 (green) immunolabeled with polyclonal IgG ab13550, and a-tubulin (red) immunolabeled with mAb DM1A (Sigma-Aldrich); (c) Fas ligand CD178 (red) immunolabeled with mAb H11, and β-actin (green) immunolabeled with mAb 13E5 (Cell Signaling Technology); (d) CD95 (green) upregulation and redistribution following incubation with Mycoplasma alligatoris.

Fig. 2

Mycoplasma alligatoris infection promotes surface CD95 expression and apoptosis of primary pulmonary fibroblasts. Subconfluent fibroblasts were infected 24 hr with 10⁹ CFU M. alligatoris, then CD95 expression was measured with primary antibody ab13550, and 5-bromo-2′deoxyuridine (BrdU) DNA nick end-labeling was measured with primary antibody PRB-1 (Molecular Probes), by flow cytometry. Control fibroblasts were incubated with medium only. Gate = 0.5% of secondary antibody controls. n = number of replicate wells tested. Bars = 1 standard error.

Fig. 3

Caspase expression in primary alligator pulmonary fibroblasts. Nuclei were stained with 4′,6′-diamidino-2-phenylindole hydrochloride (blue) and a-tubulin (green) was immunolabeled with mAb DM1A. (a) Caspase 3 (red) immunolabeled with polyclonal antibody 9662; (b) Caspase 7 (red) immunolabeled with polyclonal antibody 9492; (c) Caspase 9 (red) immunolabeled with polyclonal antibody 9502; (d) Western blots of M. alligatoris-infected (I) and control (U) fibroblast lysates probed with primary antibodies against uncleaved poly(ADP-ribose) polymerase (PARP; 9542) and caspases 3, 7, and 9. Alkaline phosphatase-conjugated secondary antibodies were detected with 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt plus nitroblue tetrazolium. Blots were digitized by using an AlphaImager (Alpha Innotech, San Leandro, CA). Contrast was normalized for better clarity by using the Auto Contrast feature after cropping and alignment with Adobe Photoshop CS 8.0 (Adobe Systems, San Jose, CA). Filled arrowheads indicate bands consistent with the predicted sizes of uncleaved and cleaved mammalian caspases and PARP. Unfilled arrowheads indicate absence of predicted bands.

Fig. 4

DANA protects pulmonary fibroblasts from Mycoplasma alligatoris-induced increases in surface CD95 expression and apoptosis in a dose-dependent fashion. Subconfluent fibroblasts were infected 24 hr with 10⁹ CFU M. alligatoris, then CD95 expression (open bars, n = 2 replicates) was measured with primary antibody ab13550, and 5-bromo-2′deoxyuridine (BrdU) DNA nick end-labeling (filled bars, n = 3 replicates) was measured with primary antibody PRB-1, by flow cytometry. Error bars = 1 standard error, † = P< 0.10, ** = P< 0.01 by Fisher’s Protected Least Significant Difference test.