Mycoplasma alligatoris infection promotes CD95 (FasR) expression and apoptosis of primary cardiac fibroblasts

Abstract

Mycoplasma alligatoris causes acute lethal primary infection of susceptible hosts. A genome survey implicated sialidase and hyaluronidase, potential promoters of CD95-mediated eukaryotic cell death, as virulence factors of M. alligatoris. We used immunofluorescence imaging and flow cytometry to examine the effects of M. alligatoris infection in vitro on CD95 expression and apoptosis by alligator cardiac fibroblasts, a major cell type of a target organ of M. alligatoris infection in vivo. A uniform distribution of CD95 in primary cultured cardiac, skeletal muscle, and embryonic fibroblasts was demonstrated by using polyclonal antibodies against the N or C terminus of mouse or human CD95. Anti-CD95 antibodies reacted on Western blots of fibroblast lysates with a band with the predicted apparent molecular weight of CD95, but soluble CD95 was not detected in plasma from control or M. alligatoris-infected alligators. The proportion of CD95-gated cardiac fibroblasts increased threefold (P<0.01) 48 h after inoculation with M. alligatoris. Infection induced morphological changes in cardiac fibroblasts, including translocation of CD95 characteristic of apoptosis and an eightfold increase (P<0.16) in 5-bromo-2'-deoxyuridine (BrdU) incorporation measured in a terminal deoxynucleotide transferase dUTP nick end-labeling apoptosis assay. The proportion of BrdU-gated controls activated with agonistic immunoglobulin M against human CD95 also increased threefold (P<0.03 for muscle). Heat-inactivated M. alligatoris and sterile M. alligatoris-conditioned culture supernatant had no effect. This is the first report of a CD95 homolog in the class Reptilia and establishes a new model that can be used to test the direct bacterial interaction with upstream components of the CD95 signal transduction pathway.

FIG. 1.

Alligator cardiac fibroblasts express CD95. The CD95 of control fibroblasts (upper panels) was labeled with antibody ab13550 against a synthetic peptide mapping at N-terminal residues 29 to 44 of mouse CD95 and visualized by Alexa Fluor 488-conjugated secondary antibody (green). For imaging, the fibroblast nuclei were labeled with 4′,6′-diamidino-2-phenylindole hydrochloride (DAPI; blue). Cytoskeletal α-tubulin was labeled with primary antibody against chick brain α-tubulin and visualized by tetramethylrhodamine-conjugated secondary antibody (red). The apoptotic changes induced by 48 h of activation with the CD95-agonistic monoclonal IgM antibody CH11 (center panels) were less severe than those following 48 h of incubation with 10⁹ CFU M. alligatoris (lower panels).

FIG. 2.

Fibroblast CD95 is detectable by Western blot assay. Antibody ab13550 reacted with a band (solid arrows) in positive control HeLa (lane A) and alligator fibroblast (lane B) cell lysates, consistent with the predicted apparent molecular mass of glycosylated CD95 of approximately 44 to 48 kDa (lane M, molecular mass standards). Monomeric (dashed arrow) or polymeric soluble CD95 could not be detected in plasma from control or M. alligatoris-infected alligators (lane C) by probing with ab13550 (shown) or any of the other antibodies tested in this study. 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).

FIG. 3.

Mycoplasma alligatoris infection promotes CD95 expression of cardiac fibroblasts. Subconfluent fibroblasts were incubated for 48 h with 10⁹ CFU of M. alligatoris, and then CD95 expression was measured with primary antibody ab13550 by flow cytometry (n = 3 replicates for embryo fibroblasts; n = 4 replicates for cardiac and muscle cells) (upper panel). Control fibroblasts were incubated with either IgM CH11 or medium only. The gate was set to exclude approximately 99.5% of negative control cells. Bars, 1 standard error. Statistical significance of the increase from untreated controls is indicated (**, P < 0.01; †, P < 0.10) (lower panel).

FIG. 4.

Mycoplasma alligatoris promotes apoptotic death of cardiac fibroblasts. Subconfluent fibroblasts were incubated for 48 h with 10⁹ CFU M. alligatoris, and then the TUNEL assay was performed with primary antibody PRB-1 by flow cytometry (n = 4 replicates for cardiac cells, and n = 2 replicates for muscle cells) (upper panel). Control fibroblasts were incubated with either IgM CH11 or medium only. The gate was set to exclude approximately 99.5% of negative control cells. Bars, 1 standard error. Statistical significance of the increase from untreated controls is indicated (*, P < 0.05) (lower panel).