<i>Toxoplasma gondii</i> GRA7-Targeted ASC and PLD1 Promote Antibacterial Host Defense via PKCα

<div><p>Tuberculosis is a global health problem and at least one-third of the world’s population is infected with <i>Mycobacterium tuberculosis</i> (MTB). MTB is a successful pathogen that enhances its own intracellular survival by inhibiting inflammation and arresting phago-lysosomal fusion. We previously demonstrated that <i>Toxoplasma gondii</i> (<i>T</i>. <i>gondii</i>) dense granule antigen (GRA) 7 interacts with TNF receptor-associated factor 6 via Myeloid differentiation primary response gene 88, enabling innate immune responses in macrophages. To extend these studies, we found that GRA7 interacts with host proteins involved in antimicrobial host defense mechanisms as a therapeutic strategy for tuberculosis. Here, we show that protein kinase C (PKC)α-mediated phosphorylation of <i>T</i>. <i>gondii</i> GRA7-I (Ser52) regulates the interaction of GRA7 with PYD domain of apoptosis-associated speck-like protein containing a carboxy-terminal CARD, which is capable of oligomerization and inflammasome activation can lead to antimicrobial defense against MTB. Furthermore, GRA7-III interacted with the PX domain of phospholipase D1, facilitating its enzyme activity, phago-lysosomal maturation, and subsequent antimicrobial activity in a GRA7-III (Ser135) phosphorylation-dependent manner via PKCα. Taken together, these results underscore a previously unrecognized role of GRA7 in modulating antimicrobial host defense mechanism during mycobacterial infection.</p></div

PKCα-dependent phosphorylation of GRA7 was essential for interaction with ASC and PLD1.

<p>(<b>A</b>) THP-1 cells were stimulated with rGRA7 (5 μg/ml) for the indicated times (left) or 30 min. (right), followed by IP with αHis-agarose bead (left) or αPKCα (right) and IB with αPKCα, αPKCβI, αPKCγ, αHis, and αActin. (<b>B</b>) Phos-tag and SDS-PAGE analyses of GST-GRA7, GRA7-I^WT, GRA7-I^S52A, GRA7-III^WT, GRA7-III^T121A, or GRA7-III^S135A expressed together with Flag-tagged PKCα in 293T cells left untreated (CIP-) or treated with calf intestinal alkaline phosphatase (CIP+), and subjected to GST pulldown, followed by IB with αGST. WCLs were used for IB with αFlag or αActin. (<b>C</b>) Mapping of PKCα phosphorylation sites on GRA7 by tiled peptide array analysis using purified recombinant PKCα. Phosphorylation intensity of 15-amino acid peptides that span full-length GRA7 and are each shifted by 3 amino acids was detected using MultiGauge version 3.0. The serines in the two peptides that showed a phosphorylation signal stronger than 100 PSL/mm² are indicated above the corresponding peaks. (<b>D</b>) 293T cells were co-transfected with GST or GST-GRA7-I and truncated mutant constructs together with V5-ASC (left), or GST or GST-GRA7-III and truncated mutant constructs together with AU1-PLD1 (right), and subjected to GST pulldown, followed by IB with αV5 or αAU1. WCLs were used for IB with αGST, αV5, αAU1, αPKCα, or αActin. (<b>E</b>) BMDMs from PKCα^+/+ and PKCα^-/- were stimulated with rGRA7 for 30 min., followed by IP with αHis-agarose bead and IB with αASC, αPLD1, αPKCα, and αActin. The data are representative of four independent experiments with similar results (<b>A</b> to <b>E</b>).</p

Parameters influencing BCR free survival after early salvage radiotherapy.

<p>Parameters influencing BCR free survival after early salvage radiotherapy.</p

Additional file 2: of MASTL inhibition promotes mitotic catastrophe through PP2A activation to inhibit cancer growth and radioresistance in breast cancer cells

Figure S2. MASTL depletion increases G2 arrest and the accumulation of pH 3. a The quantification of the relative percentage of cells expressing red fluorescence (pH 3). b Representative images of a normal mitotic cells (left panel) and MASTL-depleted mitotic defect cells stained with anti-acetyl-tubulin antibody (green), anti-phospho-Histone H3 antibody (red), and DAPI (blue). Scale bar = 10 μm. (TIF 763 kb

Dental Hygiene Laboratory, Coleman Bldg., Westbrook College, 1970s

Four Westbrook College dental hygiene students work in the dental hygiene laboratory in Coleman in this 1970s photograph by Ellis Herwig Photography of Cambridge, Mass.https://dune.une.edu/wchc_photos_labs/1028/thumbnail.jp

Characteristics of patients.

<p>BCR, biochemical recurrence; PSA, prostate specific antigen; RT, radiotherapy; PSADT, PSA doubling time; ADT, androgen deprivation therapy; RP, radical prostatectomy.</p

Kaplan-Meier estimates of overall biochemical recurrence free survival.

<p>Kaplan-Meier estimates of overall biochemical recurrence free survival.</p

GRA7-III-induced activation of PLD1 was dependent on interaction with PLD1.

<p>(<b>A</b>) Bacterially purified 6xHis-GRA7-III and its mutants were analyzed by Coomassie blue staining (left) or IB with αHis (right). (<b>B</b>) BMDMs from PKCα^+/+ and PKCα^-/- mice were stimulated with rGRA7-III (5 μg/ml) and its mutants for the indicated times, followed by IB to detect phosphorylated and total forms of PLD1 and PLD2. Actin was used as a loading control. (<b>C</b>) BMDMs were stimulated with rGRA7-III and its mutants for 30 min. and analyzed for PLD activity assay <i>in vitro</i>. Data shown are the means ± SD of five experiments. (<b>D</b>) Fluorescence confocal images in BMDMs were stimulated with rGRA7 and its mutants for 30 min., fixed, immunostained with antibodies for His (Alexa 488) and PLD1 (Alexa 568). Scale bar, 20 μm. The data are representative of three independent experiments with similar results (<b>A</b>, <b>B</b>, and <b>D</b>).</p

Parameters influencing BCR free survival after salvage radiotherapy.

<p>BCR, biochemical recurrence; PSA, prostate specific antigen; RT, radiotherapy; PSADT, PSA doubling time; ADT, androgen deprivation therapy; RP, radical prostatectomy.</p

GRA7-III-induced activation of PLD1 and phagosomal maturation were required for antimicrobial activity in MTB-infected macrophages.

<p>(<b>A</b> and <b>B</b>) BMDMs were infected with MTB (MOI = 1) for 4 h and then stimulated with rGRA7 (5 μg/ml) and its mutants for 18 h. Mycobacteria-containing phagosome fractions were subsequently purified by sucrose-step-gradient-ultra-centrifugations, followed by IB to detect αRab5, αRab7, αLAMP1, αLAMP2, αPLD1, αPKCα, and αActin (<b>A</b>) or IP with αPLD1 and IB with αHis, αPLD1, and αActin (<b>B</b>). Quantitative analysis of the PLD1 interacts with His-rGRA7 in MTB-containing phagosomes band normalized to Actin is shown (lower). (<b>C</b> and <b>D</b>) Intracellular survival of MTB was assessed by CFU assay. BMDMs were infected with MTB for 4 h, followed by treatment with rGRA7, and then lysed to determine intracellular bacterial loads. (<b>D</b>, right) IB with αPLD1, αΑSC, and αActin in BMDMs. The data are representative of five independent experiments with similar results (<b>A</b> and <b>B</b>). Data shown are the mean ± SD of five experiments (<b>C</b> and <b>D</b>). Significant differences (*<i>P</i> < 0.05; **<i>P</i> < 0.01; ***<i>P</i> < 0.001) compared with PKCα+/+ and PKCα-/- (<b>B</b>) or rVector (<b>C</b> and <b>D</b>). CFU, colony-forming units. ns, not significant.</p