Nitric oxide detoxification mechanisms of Neisseria meningitidis enhance CGMP activity during infection of human macrophages

Abstract

Rationale and hypothesis Increased bacterial load in meningococcal sepsis is positively correlated with severe disease and increased concentrations of circulating nitric oxide (NO) metabolites. As a vasorelaxant, NO binds to the soluble guanylyl cyclase (sGC), found in abundance in vascular smooth muscle and activates cGMP, a secondary messenger important for vasodilation, platelet inhibition etc. We previously showed that NO detoxification mechanisms of N. meningitidis reduce abundance of NO species in mammalian cells, so we tested the hypothesis that this activity alters cellular cGMP activity. Objectives To examine the effect of bacterial NO metabolism on cGMP produced by Human macrophages (MDM) and endothelial (HMEC-1) cells. Methodology Human MDMs and HMEC-1 cells were exposed to NO donor spermine NONOate, together with the phosphodiesterase inhibitor IBMX and a soluble guanyl cyclase sensitiser BAY412275. Cells were infected with wild type Neisseria meningitidis (serogroup B strain MC58), a mutant strain for putative Arginine Decarboxylase (ΔNMB0468) and an isogenic mutant unable to detoxify NO (ΔnorB). After two hours, cells were lysed and cGMP was measured by ELISA. Findings In the presence of spermine NONOate, IBMX and BAY 412275, the level of cGMP was detected at 5.69 pmol/mg protein in human MDMs; without these agonists no cGMP was detected (p