The Listeria monocytogenes PASTA Kinase PrkA and Its Substrate YvcK Are Required for Cell Wall Homeostasis, Metabolism, and Virulence.

Obstacles to bacterial survival and replication in the cytosol of host cells, and the mechanisms used by bacterial pathogens to adapt to this niche are not well understood. Listeria monocytogenes is a well-studied Gram-positive foodborne pathogen that has evolved to invade and replicate within the host cell cytosol; yet the mechanisms by which it senses and responds to stress to survive in the cytosol are largely unknown. To assess the role of the L. monocytogenes penicillin-binding-protein and serine/threonine associated (PASTA) kinase PrkA in stress responses, cytosolic survival and virulence, we constructed a ΔprkA deletion mutant. PrkA was required for resistance to cell wall stress, growth on cytosolic carbon sources, intracellular replication, cytosolic survival, inflammasome avoidance and ultimately virulence in a murine model of Listeriosis. In Bacillus subtilis and Mycobacterium tuberculosis, homologues of PrkA phosphorylate a highly conserved protein of unknown function, YvcK. We found that, similar to PrkA, YvcK is also required for cell wall stress responses, metabolism of glycerol, cytosolic survival, inflammasome avoidance and virulence. We further demonstrate that similar to other organisms, YvcK is directly phosphorylated by PrkA, although the specific site(s) of phosphorylation are not highly conserved. Finally, analysis of phosphoablative and phosphomimetic mutants of YvcK in vitro and in vivo demonstrate that while phosphorylation of YvcK is irrelevant to metabolism and cell wall stress responses, surprisingly, a phosphomimetic, nonreversible negative charge of YvcK is detrimental to cytosolic survival and virulence in vivo. Taken together our data identify two novel virulence factors essential for cytosolic survival and virulence of L. monocytogenes. Furthermore, our data demonstrate that regulation of YvcK phosphorylation is tightly controlled and is critical for virulence. Finally, our data suggest that yet to be identified substrates of PrkA are essential for cytosolic survival and virulence of L. monocytogenes and illustrate the importance of studying protein phosphorylation in the context of infection.

Selective pharmacologic inhibition of a PASTA kinase increases Listeria monocytogenes susceptibility to ß-lactam antibiotics.

While ß-lactam antibiotics are a critical part of the antimicrobial arsenal, they are frequently compromised by various resistance mechanisms, including changes in penicillin binding proteins of the bacterial cell wall. Genetic deletion of the penicillin binding protein and serine/threonine kinase-associated protein (PASTA) kinase in methicillin-resistant Staphylococcus aureus (MRSA) has been shown to restore ß-lactam susceptibility. However, the mechanism remains unclear, and whether pharmacologic inhibition would have the same effect is unknown. In this study, we found that deletion or pharmacologic inhibition of the PASTA kinase in Listeria monocytogenes by the nonselective kinase inhibitor staurosporine results in enhanced susceptibility to both aminopenicillin and cephalosporin antibiotics. Resistance to vancomycin, another class of cell wall synthesis inhibitors, or antibiotics that inhibit protein synthesis was unaffected by staurosporine treatment. Phosphorylation assays with purified kinases revealed that staurosporine selectively inhibited the PASTA kinase of L. monocytogenes (PrkA). Importantly, staurosporine did not inhibit a L. monocytogenes kinase without a PASTA domain (Lmo0618) or the PASTA kinase from MRSA (Stk1). Finally, inhibition of PrkA with a more selective kinase inhibitor, AZD5438, similarly led to sensitization of L. monocytogenes to ß-lactam antibiotics. Overall, these results suggest that pharmacologic targeting of PASTA kinases can increase the efficacy of ß-lactam antibiotics.

A toxoplasma patatin-like protein changes localization and alters the cytokine response during toxoplasmic encephalitis.

Toxoplasma gondii is an obligate intracellular parasite that forms a lifelong infection within the central nervous system of its host. The T. gondii genome encodes six members of the patatin-like phospholipase family; related proteins are associated with host-microbe interactions in bacteria. T. gondii patatin-like protein 1 (TgPL1) was previously determined to be necessary for parasites to suppress nitric oxide and prevent degradation in activated macrophages. Here, we show that in the rapidly replicating tachyzoite stage, TgPL1 is localized within vesicles inside the parasite that are distinct from the dense granules; however, in the encysted bradyzoite stage, TgPL1 localizes to the parasitophorous vacuole (PV) and cyst wall. While we had not previously seen a defect of the TgPL1 deletion mutant (ΔTgPL1) during acute and early chronic infection, the localization change of TgPL1 in bradyzoites caused us to reevaluate the ΔTgPL1 mutant during late chronic infection and in a toxoplasmic encephalitis (TE) mouse model. Mice infected with ΔTgPL1 are more resistant to TE and have fewer inflammatory lesions than mice infected with the wild type and ΔTgPL1 genetically complemented with TgPL1. This increased resistance to TE could result from several contributing factors. First, we found that ΔTgPL1 bradyzoites did not convert back to tachyzoites readily in tissue culture. Second, a subset of cytokine levels were higher in ΔTgPL1-infected mice, including gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and monocyte chemotactic protein 1 (MCP-1). These studies suggest that TgPL1 plays a role in the maintenance of chronic T. gondii infection.