c-di-AMP secreted by intracellular Listeria monocytogenes activates a host type I interferon response

Abstract

Intracellular bacterial pathogens, such as Listeria monocytogenes, are detected in the cytosol of host immune cells. Induction of this host response is often dependent on microbial secretion systems and, in L. monocytogenes, is dependent on multidrug efflux pumps (MDRs). Using L. monocytogenes mutants that overexpressed MDRs, we identified cyclic diadenosine monophosphate (c-di-AMP) as a secreted molecule able to trigger the cytosolic host response. Overexpression of the di-adenylate cyclase, dacA (lmo2120), resulted in elevated levels of the host response during infection. c-di-AMP thus represents a putative bacterial secondary signaling molecule that triggers a cytosolic pathway of innate immunity and is predicted to be present in a wide variety of bacteria and archea.

Figure 1. Characterization and isolation of L. monocytogenes secreted IFN-β stimulatory ligand

(A) IFN-β production by BMMs in response to solid phase extracts (SPE) of marR- L. monocytogenes supernatants in the presence (black bars) and absence (grey bars) of digitonin. IFN-β activity was measured using ISRE L929 cells that generate luciferase in response to type-I IFN stimulation. Data are mean of biological replicates (N=2). (B) IFN-β activity by BMMs in response to solid phase extracts of sterile filtered culture supernatants from mdrM-, wild-type (WT), marR-, and tetR::Tn917 strains of L. monocytogenes. Negative control consists of digitonin permeabilizing solution alone (dig). Data are mean ± SD (N=2). Data representative of two independent experiments. (C) IFN-β stimulatory activity of culture supernatants fractionated using reversed-phase HPLC. Activity measured as in (A). Data are the mean activity of biological replicates (N=2).

Figure 2. Cyclic di-AMP is an IFN-β activating ligand

(A) Tandem mass spectrum resulting from collisionally activated dissociation of the singly charged positive ion at m/z = 659.11 formed from an active fraction of Listeria monocytogenes. (B) Tandem mass spectrum of commercially obtained sample of c-di-AMP (BioLog Life Sciences Institute, Denmark). Fragmentation pathways of c-di-AMP are shown in (C). The fragment ions at m/z = 641.10 and 312.05 correspond to neutral losses of 18 Da from the precursor ion (m/z = 659.11) and from the fragment ion at m/z = 330.06, respectively, and are consistent with neutral loss of water molecules from these respective ions. (D) Commercial c-di-AMP standard was added to BMMs in increasing amount. IFN-β production by BMMs was detected using the type I IFN reporter cell line (ISRE L929). Commercial c-di-AMP standard and the active L. monocytogenes fraction were treated with snake venom phosphodiesterase (SVPD). Data are mean of biological replicates ± SD (N=2).

Figure 3. The di-adenylate cyclase gene dacA (lmo2120) alters CSP activation during infection

(A) Predicted operon of genes lmo2120, renamed here dacA, and lmo2119. The gene product of lmo2119 contains three ybbR domains of unknown function. The gene product of lmo2120 contains a single di-adenylate cyclase (DAC) domain. Trans-membrane spanning segments (TM) predicted using Topcons (http://topcons.cbr.su.se/index.php). (B) Intracellular growth curves of WT L. monocytogenes (closed circles) and L. monocytogenes with an integration vector (pLIV2) containing IPTG inducible dacA in the absence (open circles) and presence (open squares) of IPTG (1 mM) in BMMs. Data are mean ± SD (N=3). (C) qRT-PCR analysis of IFN-β induction by each strain in BMMs. Data are mean ± SD (N=2). Data representative of two independent experiments.