PrfA is a key regulator of Listeria monocytogenes pathogenesis and induces the expression of multiple virulence factors within the infected host. PrfA is post-translationally regulated such that the protein becomes activated upon bacterial entry into the cell cytosol. The signal that triggers PrfA activation remains unknown, however mutations have been identified (prfA* mutations) that lock the protein into a high activity state. In this report we examine the consequences of constitutive PrfA activation on L. monocytogenes fitness both in vitro and in vivo. Whereas prfA* mutants were hyper-virulent during animal infection, the mutants were compromised for fitness in broth culture and under conditions of stress. Broth culture prfA*-associated fitness defects were alleviated when glycerol was provided as the principal carbon source; under these conditions prfA* mutants exhibited a competitive advantage over wild type strains. Glycerol and other three carbon sugars have been reported to serve as primary carbon sources for L. monocytogenes during cytosolic growth, thus prfA* mutants are metabolically-primed for replication within eukaryotic cells. These results indicate the critical need for environment-appropriate regulation of PrfA activity to enable L. monocytogenes to optimize bacterial fitness inside and outside of host cells.

Listeria monocytogenes secretes two chitinases and one chitin binding protein. Mutants lacking chiA, chiB, or lmo2467 exhibited normal growth in cultured cells but were defective for growth in the livers and spleens of mice. Mammals lack chitin; thus, L. monocytogenes may have adapted chitinases to recognize alternative substrates to enhance pathogenesis.

Earlier, we had demonstrated that treatment with low dose of GM-CSF can prevent the development of experimental autoimmune thyroiditis (EAT), myasthenia gravis (EAMG) and type-1 diabetes; and could also reverse ongoing EAT and EAMG. The protective effect was mediated through the induction of tolerogenic CD11C+CD8α− DCs and consequent expansion of Foxp3+ T-regulatory cells (Tregs). Subsequently, we showed that GM-CSF acted specifically on bone marrow precursors and facilitated their differentiation into tolerogenic DCs (GM-BMDCs), which directed Treg expansion in a contact dependent manner. This novel mechanism of Treg expansion was independent of TCR mediated signalling but required exogenous IL-2 and co-signalling from DC bound OX40L. In the present study, we observed that OX40L mediated signalling by GM-BMDCs although necessary was not sufficient for Treg expansion and required signalling by Jagged1. Concurrent signalling induced by OX40L and Jagged1 via OX40 and Notch3 receptors expressed on Tregs was essential for the Treg expansion with sustained FoxP3 expression. Adoptive transfer of only OX40L+Jagged1+ BMDCs led to Treg expansion, increased production of IL-4 and IL-10, and suppression of EAT in the recipient mice. These results showed a critical role for OX40L and Jagged1 induced co-signalling in GM-BMDC-induced Treg expansion.

Bacteria withstand starvation during long-term stationary phase through the acquisition of mutations that increase bacterial fitness. The evolution of the Growth Advantage in Stationary Phase (GASP) phenotype results in the ability of bacteria from an aged culture to outcompete bacteria from a younger culture when the two are mixed together. The GASP phenotype was first described for Escherichia coli but has not been examined for an environmental bacterial pathogen which must balance long-term survival strategies that promote fitness in the outside environment with those that promote fitness within the host. Listeria monocytogenes is an environmental bacterium that lives as a saprophyte in soil but is capable of replicating within the cytosol of mammalian cells. Here we demonstrate the ability of L. monocytogenes to express GASP via the acquisition of mutations during long-term stationary growth. L. monocytogenes GASP occurred through mechanisms that were both dependent and independent of the stress responsive alternative sigma factor SigB. Constitutive activation of the central virulence transcriptional regulator PrfA interfered with the development of GASP, however L. monocytogenes GASP cultures retained full virulence in mice. These results indicate that L. monocytogenes can accrue mutations that optimize fitness during long-term stationary growth without negatively impacting virulence.