A screen for kinase inhibitors identifies antimicrobial imidazopyridine aminofurazans as specific inhibitors of the Listeria monocytogenes PASTA kinase PrkA

Bacterial signaling systems such as protein kinases and quorum sensing have become increasingly attractive targets for the development of novel antimicrobial agents in a time of rising antibiotic resistance. The family of bacterial Penicillin-binding-protein And Serine/Threonine kinase-Associated (PASTA) kinases is of particular interest due to the role of these kinases in regulating resistance to β-lactam antibiotics. As such, small-molecule kinase inhibitors that target PASTA kinases may prove beneficial as treatments adjunctive to β-lactam therapy. Despite this interest, only limited progress has been made in identifying functional inhibitors of the PASTA kinases that have both activity against the intact microbe and high kinase specificity. Here, we report the results of a small-molecule screen that identified GSK690693, an imidazopyridine aminofurazan-type kinase inhibitor that increases the sensitivity of the intracellular pathogen Listeria monocytogenes to various β-lactams by inhibiting the PASTA kinase PrkA. GSK690693 potently inhibited PrkA kinase activity biochemically and exhibited significant selectivity for PrkA relative to the Staphylococcus aureus PASTA kinase Stk1. Furthermore, other imidazopyridine aminofurazans could effectively inhibit PrkA and potentiate β-lactam antibiotic activity to varying degrees. The presence of the 2-methyl-3-butyn-2-ol (alkynol) moiety was important for both biochemical and antimicrobial activity. Finally, mutagenesis studies demonstrated residues in the back pocket of the active site are important for GSK690693 selectivity. These data suggest that targeted screens can successfully identify PASTA kinase inhibitors with both biochemical and antimicrobial specificity. Moreover, the imidazopyridine aminofurazans represent a family of PASTA kinase inhibitors that have the potential to be optimized for selective PASTA kinase inhibition

Phagocytes produce prostaglandin E2 in response to cytosolic Listeria monocytogenes

Listeria monocytogenes is an intracellular bacterium that elicits robust CD8+ T-cell responses. Despite the ongoing development of L. monocytogenes-based platforms as cancer vaccines, our understanding of how L. monocytogenes drives robust CD8+ T-cell responses remains incomplete. One overarching hypothesis is that activation of cytosolic innate pathways is critical for immunity, as strains of L. monocytogenes that are unable to access the cytosol fail to elicit robust CD8+ T-cell responses and in fact inhibit optimal T-cell priming. Counterintuitively, however, activation of known cytosolic pathways, such as the inflammasome and type I IFN, lead to impaired immunity. Conversely, production of prostaglandin E2 (PGE2) downstream of cyclooxygenase-2 (COX-2) is essential for optimal L. monocytogenes T-cell priming. Here, we demonstrate that vacuole-constrained L. monocytogenes elicit reduced PGE2 production compared to wild-type strains in macrophages and dendritic cells ex vivo. In vivo, infection with wild-type L. monocytogenes leads to 10-fold increases in PGE2 production early during infection whereas vacuole-constrained strains fail to induce PGE2 over mock-immunized controls. Mice deficient in COX-2 specifically in Lyz2+ or CD11c+ cells produce less PGE2, suggesting these cell subsets contribute to PGE2 levels in vivo, while depletion of phagocytes with clodronate abolishes PGE2 production completely. Taken together, this work demonstrates that optimal PGE2 production by phagocytes depends on L. monocytogenes access to the cytosol, suggesting that one reason cytosolic access is required to prime CD8+ T-cell responses may be to facilitate production of PGE2

Contribution of pht transporters to nutrient acquisition and differentiation of intracellular <italic>Legionella pneumophila</italic>.

Legionella pneumophila is a ubiquitous aquatic bacterium that parasitizes protozoa and as a result has become an opportunistic pathogen, replicating within human amoeba-like alveolar macrophages. A central paradigm of Legionella pathogenesis is the dependence on bacterial differentiation: the bacterium survives ingestion by phagocytic cells in one phase, the transmissive phase, and efficiently replicates within these cells in a second phase, the replicative phase. We analyzed the biogenesis of the unique Legionella replication vacuole and subsequently analyzed the bacterial mechanisms of nutrient acquisition within this distinct environment. First we compared the biogenesis of the vacuoles containing Legionella pneumophila and the related intracellular pathogen Coxiella burnetii. We determined that although structural components of their type IV secretion systems required for vacuole biogenesis are functionally homologous, the vacuoles in which these bacteria reside are distinct. Furthermore, we discovered that the host cell plays an essential role in the establishment of the Legionella pneumophila replication vacuole: the vacuole is acidic and lysosome-like in murine macrophages and neutral and endoplasmic reticulum-derived in human cells. This work accentuates the versatility of L. pneumophila as an intracellular pathogen. To extend our knowledge of the L. pneumophila intracellular life cycle, we began to study the mechanisms of survival and replication within the unique replication vacuole. We identified and then analyzed a member of a novel transporter family, the phagosomal t&barbelow;ransporters (pht), PhtA, for its role in intracellular growth, differentiation and nutrient acquisition. We determined that PhtA is required for threonine acquisition and intracellular replication. In addition, we extended the paradigm of nutrient-regulated differentiation to the in vivo life cycle as we determined that a defect in threonine acquisition leads to a lack of intracellular differentiation. Following the identification and examination of phtA, we extended our analysis to determine the role of the 10 remaining L. pneumophila pht genes. We determined that PhtJ, like PhtA, is required for intracellular growth in macrophages and that this is due to its role in valine acquisition. In addition to PhtA and PhtJ, four other Pht proteins are required for growth within macrophages and five are dispensable for replication. We next assessed the role of the Pht proteins in replication in a natural host, Acanthamoeba castellanii. Genes required for growth in amoeba were also required for growth in macrophages, emphasizing the importance of the amoeba to the emergence of L. pneumophila as an opportunistic pathogen. In conclusion, by analyzing the biogenesis of the L. pneumophila replication vacuole and the machinery bacteria use to survive and replicate within host cells, we put forth a new model for how intracellular pathogens sense their unique vacuolar environments and determine whether or not to differentiate and subsequently replicate. The Pht system paradigm extends beyond L. pneumophila, and likely governs the intracellular life cycle of other important pathogens including Coxiella burnetii and Francisella tularensis.Ph.D.Biological SciencesMicrobiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/125873/2/3224736.pd

Listeria monocytogenes: The Impact of Cell Death on Infection and Immunity

Listeria monocytogenes has evolved exquisite mechanisms for invading host cells and spreading from cell-to-cell to ensure maintenance of its intracellular lifecycle. As such, it is not surprising that loss of the intracellular replication niche through induction of host cell death has significant implications on the development of disease and the subsequent immune response. Although L. monocytogenes can activate multiple pathways of host cell death, including necrosis, apoptosis, and pyroptosis, like most intracellular pathogens L. monocytogenes has evolved a series of adaptations that minimize host cell death to promote its virulence. Understanding how L. monocytogenes modulates cell death during infection could lead to novel therapeutic approaches. In addition, as L. monocytogenes is currently being developed as a tumor immunotherapy platform, understanding how cell death pathways influence the priming and quality of cell-mediated immunity is critical. This review will focus on the mechanisms by which L. monocytogenes modulates cell death, as well as the implications of cell death on acute infection and the generation of adaptive immunity

Inflammasome-Mediated Inhibition of <i>Listeria monocytogenes</i>-Stimulated Immunity Is Independent of Myelomonocytic Function

<div><p>Activation of the Nlrc4 inflammasome results in the secretion of IL-1β and IL-18 through caspase-1 and induction of pyroptosis. <i>L. monocytogenes</i> engineered to activate Nlrc4 by expression of <i>Legionella pneumophilia</i> flagellin (<i>L. monocytogenes L</i>.p.<i>FlaA</i>) are less immunogenic for CD8⁺ T cell responses than wt <i>L. monocytogenes</i>. It is also known that IL-1β orchestrates recruitment of myelomonocytic cells (MMC), which have been shown to interfere with T cell-dendritic cells (DC) interactions in splenic white pulp (WP), limiting T cell priming and protective immunity. We have further analyzed the role of MMCs in the immunogenicity of <i>L. monocytogenes L</i>.p.<i>FlaA</i>. We confirmed that MMCs infiltrate the WP between 24–48 hours in response to wt <i>L. monocytogenes</i> infection and that depletion of MMCs enhances CD8⁺ T cell priming and protective memory. <i>L. monocytogenes</i> L.p.FlaA elicited accelerated recruitment of MMCs into the WP. While MMCs contribute to control of <i>L. monocytogenes L</i>.p.<i>FlaA</i>, MMC depletion did not increase immunogenicity of L.p.FlaA expressing strains. There was a significant decrease in <i>L. monocytogenes</i> L.p.FlaA in CD8α⁺ DCs independent of MMCs. These findings suggest that limiting inflammasome activation is important for bacterial accumulation in CD8α⁺ DCs, which are known to be critical for T cell response to <i>L. monocytogenes</i>. </p> </div

Increased CD8⁺ T cell generation and protective immunity after MMC depletion.

<p>(A) 8-10 week old LysM^+/eGFP mice were infected intravenously with 2.5x10⁴ wt <i>L. monocytogenes</i> for 12, 24, and 48 hours. At the indicated time spleens were excised, sectioned, and image using multi-photon microscopy. Multi-photon images of LysM^+/eGFP mice at 12, 24, and 48 hours after infection. Top panel (non-infected) –grayscale images. Dextran-TMR-red, LysM^+/eGFP- green, yellow dotted line-marginal zone, WP-white pulp, RP-red pulp (B) Relative GFP intensity inside individual WP nodule (A.U.). Data representative of 3 independent experiments. (C) Isolated naïve OT-I cells were transferred into 8-10 week old B6.SJL mice 24 hours prior to 250µg RB6-8C5 mAb. 5 hours after mAb treatment mice were infected i.v. with 1x10⁴<i>ΔactA/InlB </i><i>L. monocytogenes</i> expressing ova peptide. OT-I percentages as analyzed by FACs from blood day 7 post-infection. Bar graph (median plus range). Data representative of 3 independent experiments. (D) 8-10 week old C57BL/6 mice were treated with 250µg RB6-8C5 mAb and then immunized 5 hours later with 1x10³ ΔactA/InlB. 30 days post-immunization, mice were infected with 2x10⁵ wt <i>L. monocytogenes</i> for 3 days. Bacterial CFUs in liver and spleen day 3 post-infection. Dotted line – limit of detection. Data are representative of four independent experiments. *P < 0.05 by Mann-Whitney test.</p

CD8α⁺ DCs harbor significantly less after <i>L. monocytogenes</i> inflammasome activation.

<p>(A) 8-10 week old C57Bl/6 mice were infected intravenously with PBS, 2.5x10⁴ wt or 1x10⁵ L. <i>monocytogenes</i> L.p.FlaA for 12 and 18 hours. After infection, spleens were harvested, dissociated, and isolated DCs were stained with CD8α⁺, CD11b⁺, CD11c+, CD86⁺ antibodies and analyzed using FACS. Bar graph of percentage of CD11c^hiCD8α^hi DCs. The average ± SD of three replicates from 3 independent experiments. (B) Histogram of CD86 expression in CD11c^hiCD8α^hi DCs. (C) 8-10 week old C57Bl/6 mice were infected intravenously with PBS, 2.5x10⁴ wt or 1x10⁵ L. <i>monocytogenes</i> L.p.FlaA for 24 hours. After infection, spleens were harvested, dissociated, and CD8α⁺ DCs were purified using bead purification. Total isolated spleens were plated on BHI with bacterial counts taken after 24 hours. The average ± SD of four replicates from 4 independent experiments. (D) C57Bl/6 mice were treated for 5 hours with 250µg RB6-8C5 mAb and then infected intravenously with PBS, 2.5x10⁴ wt or 1x10⁵ L. <i>monocytogenes</i> L.p.FlaA for 24 hours. After infection, spleens were harvested, dissociated, and CD8α⁺ DCs were purified using bead purification. Total isolated spleens were plated on BHI with bacterial counts taken after 24 hours. The average ± SD of four replicates from 4 independent experiments.</p

Reduced MMC accumulation after Inflammasome activation.

<p>(A) 8-10 week old C57BL/6 mice were infected intravenously with 2.5x10⁴ wt or 1x10⁵ L. <i>monocytogenes</i> L.p.FlaA for 12, 24, and 48 hours. Bacterial CFU from spleen and liver were collected at the indicated time points. Dotted line – limit of detection. The median of four replicates from 4 independent experiments. *P < 0.05 by Mann-Whitney test. (B) LysM^+/eGFP mice were infected intravenously with 2.5x10⁴ wt or 1x10⁵ L. <i>monocytogenes</i> L.p.FlaA for 12, 24, and 48 hours. At the indicated time spleens were excised, sectioned, and image using multi-photon microscopy. Multi-photon images of LysM^+/eGFP mice at 12, 24, and 48 hours after infection. LysM^+/eGFP- green, yellow dotted line-marginal zone, WP-white pulp, RP-red pulp (C) Relative GFP intensity inside individual WP nodule (A.U.). Data representative of 3 independent experiments.</p

Partial restoration of <i>L</i>. <i>monocytogenes</i> L.p.FlaA virulence after MMC depletion.

<p>8-10 week old CD11c-YFP transgenic x LysM^+/eGFP mice were treated with 250µg RB6-8C5 mAb and then infected i.v. with 2.5x10⁴ wt or 1x10⁵ L. <i>monocytogenes</i> L.p.FlaA both expressing RFP for up to 48 hours. After infection spleens were excised, sectioned, and image using multi-photon microscopy. (A) Multi-photon images of CD11c-YFP transgenic x LysM^+/eGFP mice at 48 hours after infection. <i>L. monocytogenes</i>-RFP, MZ-705 quantum dots-yellow. (B) Relative RFP intensity inside individual WP nodule (A.U.). Data representative of 3 independent experiments. (C) Bacterial CFU from liver and spleen at the indicated time points after RB6-8C5 antibody treatment. The median of at least three replicates from 3 independent experiments. *P < 0.05 by Mann-Whitney test.</p

Inhibition of cell-mediated immunity despite MMC depletion.

<p>(A) 5x10⁴ naïve OT-I cells from C57BL/6 mice were transferred into 8-10 week old B6.SJL mice 24 hours prior to RB6-8C5 mAb (250µg) treatment. 5 hours after antibody treatment mice were infected intravenously with 1x10⁴<i>ΔactA/InlB</i> or <i>ΔactA/InlB</i> L.p.FlaA <i>L. monocytogenes</i> both expressing ova peptide. OT-I percentages as analyzed by FACS from blood day 7 post-infection. Data representative of one experiment. (B) Bar graph (median plus range) of OT-I percentages as analyzed by FACS from blood day 7 post-infection. Data combined from three independent experiments. (C) 8-10 week old C57Bl/6 mice were treated with 250µg RB6-8C5 mAb and then immunized 5 hours later with 1x10³<i>ΔactA/InlB</i> or <i>ΔactA/InlB</i> L.p.FlaA <i>L. monocytogenes</i>. 30 days post immunization mice were infected with 2x10⁵ wt <i>L. monocytogenes</i> for 3 days. Bacterial CFUs in liver and spleen day 3 post-infection. Dotted line – limit of detection. Data are representative of four independent experiments. *P < 0.05 by Mann-Whitney test.</p