A lymphostatin homologue from <i>Chlamydia pecorum</i> inhibits mitogen-activated bovine T cell proliferation and IFNγ production

Pathogens frequently produce proteins to evade or inhibit host immune responses. One such protein is lymphostatin from attaching and effacing <i>Escherichia coli</i> (also known as lymphocyte inhibitory factor A; LifA), which influences intestinal colonization and inhibits mitogen- and antigen-activated proliferation of T lymphocytes and pro-inflammatory cytokine synthesis. Here, we report the cloning, purification and characterization of a LifA homologue from <i>Chlamydia pecorum</i>. The predicted 382 KDa protein (CPE2_0552) exhibited 36% identity and 55% similarity over 3171 amino acids to lymphostatin from enteropathogenic <i>E. coli</i> strain E2348/69. CPE2_0552 shares glycosyltransferase and cysteine protease motifs required for lymphostatin activity, including similarity in the tertiary structure of these domains predicted by AlphaFold 3. Purified CPE2_0552 did not share the L-shaped globular structure of lymphostatin when analyzed by transmission electron microscopy. CPE2_0552 inhibited concanavalin A-stimulated proliferation of bovine T cells in a concentration-dependent manner, with an inhibitory dose 50 (ID₅₀) of 812 pg/mL. This was 38-fold higher than the ID₅₀ of <i>E. coli</i> E2348/69 lymphostatin tested in parallel on T cells from the same donors (21 pg/mL), but was similar to another LifA homologue from <i>E. coli</i> O157:H7 (ToxB). Moreover, CPE2_0552 inhibited the secretion of interferon gamma (IFNγ), a key cytokine that influences the outcome of <i>Chlamydia</i> infections. At the concentrations at which CPE2_0552 inhibited T lymphocyte proliferation and IFNγ secretion, negligible cytotoxicity was observed after 72 h of stimulation. Our study indicates that <i>E. coli</i> lymphostatin belongs to a wider family of lymphocyte-inhibitory molecules that exist in distantly related bacterial pathogens.