Calpain-2 facilitates infection of the intracellular bacteria Listeria monocytogenes and invasion intestinal immune barrier by impairing nitric oxide homeostasis

The ubiquitously expressed enzymes, calpain-1 and -2 (CAPN1 and 2) play important roles in a wide variety of physiological and pathological processes including infection and immune responses. CAPN2 is particular interest due to its role in regulating intracellular bacterial infection and invasion, but the mechanisms by which this occurs have not been elucidated. In this study, we focused on intestinal CAPN2 involved in infection by the intracellular bacteria Listeria monocytogenes (L. monocytogenes) which invade the intestinal immune barrier Peyer's patches by regulating nitric oxide (NO) homeostasis. L. monocytogenes infection by intragastric inoculation and intestinal ligated loops was performed in mice with deletion of the endogenous calpain inhibitory protein, calpastatin (CAST), or the use of a CAPN2 specific inhibitor (CAPN2-Inh). L. monocytogenes invasion, CAPN2 activation, and their dependence on NO was investigated using iNOS knockout mice. The effects of CAPN mediated decrease in NO production and the subsequent modulation of L. monocytogenes infection and cell-to-cell transmission was confirmed in macrophages using flow cytometry and fluorescence tracing. Database analysis indicated dynamic CAPN2 activation and expression during L. monocytogenes infection, which was confirmed in our infection models as well as macrophage experiments. CAPN2 was found to regulate iNOS mediated NO production in a manner that impaired NO homeostasis, which subsequently affected L. monocytogenes invasion of the intestinal immune barrier through Peyer's patches. Furthermore, proliferation and cell-to-cell spread of L. monocytogenes were decreased significantly in macrophages treated with a CAPN2-Inh or through the abatement of NO signaling. Collectively, these data reveal a NO-dependent role for CAPN2 activation in promoting L. monocytogenes invasion of the host intestinal immune barrier, which highlights a potential strategy for targeting CAPN2 and thereby preventing intracellular bacterial infection and invasion of L. monocytogenes.