The role of c-di-AMP in host-microbe interactions

Abstract

c-di-AMP is a second nucleotide messenger, which in gram-positive bacteria is known to be involved in stress tolerance, regulation of virulence, biofilm formation and growth. In S. mitis, two phosphodiesterase enzymes down-regulate the levels of c-di-AMP, and one di-adenylate cyclase synthesizes c-di-AMP. S. mitis is part of the normal microbiota of the oral cavity of humans and known to be opportunistic pathogenic in immunocompromised patients. From earlier studies, it has been observed that S. mitis Δpde2 knockout mutant was less pathogenic compared to the WT, ΔcdaA and Δpde1-mutants in a neutropenic mouse model and unable to survive in the mice.

The aim of this study was therefore to determine the basis for the c-di-AMP mediated effect on colonization and/or virulence of S. mitis. The WT strain, and three knock-out mutants (ΔcdaA, Δpde1 and Δpde2) were used to study the susceptibility of S. mitis against phagocytosis and antibacterial conditions encountered by bacteria during phagocytosis. This was done by challenging the bacterial strains in time-kill assays using different acidic pH adjusted by inorganic (HCl), and organic (Aa) acids as well as in time-kill assays using H2O2. The susceptibility of the different S. mitis strains to phagocytosis by different immune cells was tested by CFU enumeration of bacteria after exposure to neutrophils or macrophages. Flow cytometry was performed to analyze and estimate the interaction between macrophage cells and the bacterial strains, along with further analysis with fluorescence microscopy and confocal microscopy. Finally, the ability of the different S. mitis strains to induce cytokine expression from human epithelial cells was measured by qPCR. This was to analyze if the c-di-AMP signaling system has an effect on cytokine production from epithelial cells which can attract and regulate the activity of immune cells.

Our results showed that the c-di-AMP signaling system may be involved in acid stress survival since the Δpde1-mutant grew better than the other strains at low pH independent of the type of acid. C-di-AMP does not seem to be involved in regulation of H2O2 stress survival. Neutrophil killing assays suggested that c-di-AMP can reduce the susceptibility to phagocytosis and/or degradation. Killing assays with macrophages resulted in better survival of ΔcdaA than the other strains, which indicated that decreased c-di-AMP levels helps in survival regardless of if they are opsonized or not. Confocal microscopy showed that S. mitis is localized outside, but in close interaction with macrophages after incubation on ice. Flow cytometry to some extent confirmed that low c-di-AMP levels may protect S. mitis from phagocytosis. Morphological differences in S. mitis were detected with both decreased (longer chains of bacteria) and increased (shorter chain lengths) levels of c-di-AMP. Induction of cytokine production by epithelial cells and the survival of the bacteria inside the host may be affected by the c-di-AMP signaling system.