The <i>Listeria monocytogenes</i> PASTA Kinase PrkA and Its Substrate YvcK Are Required for Cell Wall Homeostasis, Metabolism, and Virulence

<div><p>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. <i>Listeria monocytogenes</i> 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 <i>L</i>. <i>monocytogenes</i> <u>p</u>enicillin-binding-protein <u>a</u>nd <u>s</u>erine/<u>t</u>hreonine <u>a</u>ssociated (PASTA) kinase PrkA in stress responses, cytosolic survival and virulence, we constructed a Δ<i>prkA</i> 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 <i>Bacillus subtilis</i> and <i>Mycobacterium tuberculosis</i>, 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 <i>in vitro</i> and <i>in vivo</i> 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 <i>in vivo</i>. Taken together our data identify two novel virulence factors essential for cytosolic survival and virulence of <i>L</i>. <i>monocytogenes</i>. 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 <i>L</i>. <i>monocytogenes</i> and illustrate the importance of studying protein phosphorylation in the context of infection.</p></div

PrkA is required for intracellular replication, cytosolic survival, evasion of inflammasome activation and virulence.

<p><b>(A)</b> Intracellular growth of wild-type (black circles) and Δ<i>prkA</i> mutants (blue triangles) was determined in bone marrow-derived macrophages (BMDMs) following infection at an MOI of 0.2. <b>(B)</b> Intracellular lysis of <i>L</i>. <i>monocytogenes</i> was measured in immortalized interferon α/β receptor (IFNAR)^-/- macrophages. Macrophages were infected at an MOI of 5 by <i>L</i>. <i>monocytogenes</i> strains carrying pBHE573 and macrophage luciferase expression was measured 6 hours post infection. <b>(A, B)</b> Mean values are reported. Experiments are representative of three or more biological replicates and error bars represent the standard deviation of technical triplicates. * indicates statistical significance by Student’s T-test (P<0.05). <b>(C)</b> Host Cell Death induced in wild-type C57/Bl6 (solid) or Gbp^Chr3-/- (hashed) BMDMs was analyzed by lactate dehydrogenase release 6 hours post infection at an MOI of 5. Data is an average of three biological replicates and error bars represent standard deviation of the mean. * indicates statistical significance by Student’s T-test (P<0.05). <b>(D)</b> C57Bl6 mice were infected intravenously with 1x10⁵ wild-type (black circles) or Δ<i>prkA</i> mutants (blue triangles) <i>in vivo</i>. Spleens (left) and Livers (right) were harvested 48 hours post infection homogenized and plated for CFU. The median (solid bar) and limit of detection (line) for each experiment is indicated. Data are representative of two independent experiments with 5 mice each. * indicates statistical significance by Mann-Whitney Test (P<0.05).</p

The Δ<i>prkA</i> mutant has altered growth and morphology in minimal media with glycerol as the primary carbon source.

<p><b>(A)</b> Growth of the Δ<i>prkA</i> mutant in BHI, <b>(B)</b> or in Improved Minimal Media (IMM) with glucose-6-phosphate (filled symbols and solid lines) or glycerol (open symbols and dashed lines) as the sole carbon source. Data are averages of three biological replicates and error bars represent standard deviation of the mean. <b>(C)</b> Morphology of wild-type and the Δ<i>prkA</i> mutant in BHI, IMM Glucose-6-phosphate, and IMM Glycerol at OD 0.5 (BHI) or 6 hours post inoculation.</p

PrkA phosphorylates YvcK on 2 independent threonines.

<p><b>(A)</b> PrkA was incubated with [γ-³²P]ATP separately (lane 1), with YvcK (lane 2) or with YvcK point mutants (lanes 3–5) overnight, proteins were separated by SDS-PAGE and analyzed either by coomasie to demonstrate equivalent amounts of YvcK in each reaction (Top) or autoradiography (Bottom). <b>(B)</b> MS/MS of phosphorylated YvcK identified the doubly charged ion 693.34 m/z (monoisotopic mass 1384.67 Da) matched to a doubly phosphorylated YvcK 247–261 tryptic peptide with one missed cleavage site. Phosphorylated threonines (red) 252 and 256 can be identified definitively by either b or y ions.</p

Phosphomimetic YvcK mutants phenocopy Δ<i>yvcK</i> mutant <i>ex vivo</i> and <i>in vivo</i>, but not <i>in vitro</i>.

<p><b>(A</b>) Expression of YvcK in wild-type, Δ<i>yvcK</i>, <i>yvcK</i> T252A T256A, <i>yvcK</i> T252E T256E, and Δ<i>prkA</i> at mid-log in BHI (1–5) or with 250 μg/mL (6–10). <b>(B)</b> Growth of wild-type (black circles), <i>yvcK</i> T252A T256A (orange squares filled on the left side), <i>yvcK</i> T252E T256E (purple squares filled on the right side) in IMM with glycerol as the sole carbon source. Data are averages of three biological replicates and error bars represent standard deviation of the mean. <b>(C)</b> Intracellular lysis of <i>L</i>. <i>monocytogenes</i> was measured in immortalized IFNAR^-/- macrophages. Macrophages were infected at an MOI of 5 by <i>L</i>. <i>monocytogenes</i> strains carrying pBHE573 and macrophage luciferase expression was measured 6 hours post infection. Mean values are reported. Experiments are representative of three or more biological replicates and error bars represent the standard deviation of technical triplicates. * indicates statistical significance by Student’s T-test (P<0.05). <b>(D)</b> Host Cell Death induced in wild-type C57/Bl6 BMDMs was analyzed by lactate dehydrogenase release 6 hours post infection at an MOI of 5. Data is an average of three biological replicates and error bars represent standard deviation of the mean. * indicates statistical significance by Student’s T-test (P<0.05). <b>(E)</b> C57Bl6 mice were infected intravenously with 1x10⁵ wild-type (black circles), <i>yvcK</i> T252A T256A (red squares filled on the left side), <i>yvcK</i> T252E T256E (red squares filled on the right side) <i>in vivo</i>. Spleens (left) and Livers (right) were harvested 48 hours post infection homogenized and plated for CFU. The median (solid bar) and limit of detection (line) for each experiment is indicated. Data are representative of two independent experiments with 5 mice each. * indicates statistical significance by Mann-Whitney Test (P<0.05).</p

Minimum inhibitory concentrations of cell wall targeting agents in BHI.

<p>Values are mean minimum inhibitory concentrations and standard deviations in μg/mL. Values were determined by five or more biological replicates of serial 2-fold dilutions up or down from 1 μg/mL. Shaded boxes are statistically significant compared to wild-type (Student’s T-Test P< 0.05).</p

Δ<i>yvcK</i> mutants phenocopy Δ<i>prkA</i> mutants <i>in vitro</i>, <i>ex vivo</i> and <i>in vivo</i>.

<p><b>(A</b>) Growth of the Δ<i>yvcK</i> mutant in BHI, <b>(B)</b> or in IMM with glucose-6-phosphate (filled symbols and solid lines) or glycerol (open symbols and dashed lines) as the sole carbon source. Data are averages of three biological replicates and error bars represent standard deviation of the mean. <b>(C)</b> Morphology of wild-type and the Δ<i>yvcK</i> mutant in BHI, IMM Glucose-6-phosphate, and IMM Glycerol at OD 0.5 (BHI) or 6 hours post inoculation. <b>(D)</b> Intracellular growth of wild-type (black circles) and Δ<i>yvcK</i> mutants (red squares) was determined in bone marrow-derived macrophages (BMDMs) following infection at an MOI of 0.2. <b>(E)</b> Intracellular lysis of <i>L</i>. <i>monocytogenes</i> was measured in immortalized IFNAR^-/- macrophages. Macrophages were infected at an MOI of 5 by <i>L</i>. <i>monocytogenes</i> strains carrying pBHE573 and macrophage luciferase expression was measured 6 hours post infection. <b>(D, E)</b> Mean values are reported. Experiments are representative of three or more biological replicates and error bars represent the standard deviation of technical triplicates. * indicates statistical significance by Student’s T-test (P<0.05). <b>(F)</b> Host Cell Death induced in wild-type C57/Bl6 BMDMs was analyzed by lactate dehydrogenase release 6 hours post infection at an MOI of 5. Data is an average of three biological replicates and error bars represent standard deviation of the mean. * indicates statistical significance by Student’s T-test (P<0.05). <b>(G)</b> C57Bl6 mice were infected intravenously with 1x10⁵ wild-type (black circles) or Δ<i>yvcK</i> mutants (red squares) <i>in vivo</i>. Spleens (left) and Livers (right) were harvested 48 hours post infection homogenized and plated for CFU. The median (solid bar) and limit of detection (line) for each experiment is indicated. Data are representative of two independent experiments with 5 mice each. * indicates statistical significance by Mann-Whitney Test (P<0.05).</p

Minimum inhibitory concentrations of cell wall targeting agents in BHI.

<p>Values are mean minimum inhibitory concentrations and standard deviations in μg/mL. Values were determined by three or more biological replicates of serial 2-fold dilutions up or down from 1 μg/mL. Shaded boxes are statistically significant compared to wild-type (Student’s T-Test P< 0.05).</p