Identification of a Transcription Factor That Regulates Host Cell Exit and Virulence of <i>Mycobacterium tuberculosis</i>

<div><p>The interaction of <i>Mycobacterium tuberculosis</i> (Mtb) with host cell death signaling pathways is characterized by an initial anti-apoptotic phase followed by a pro-necrotic phase to allow for host cell exit of the bacteria. The bacterial modulators regulating necrosis induction are poorly understood. Here we describe the identification of a transcriptional repressor, Rv3167c responsible for regulating the escape of Mtb from the phagosome. Increased cytosolic localization of MtbΔ<i>Rv3167c</i> was accompanied by elevated levels of mitochondrial reactive oxygen species and reduced activation of the protein kinase Akt, and these events were critical for the induction of host cell necrosis and macroautophagy. The increase in necrosis led to an increase in bacterial virulence as reflected in higher bacterial burden and reduced survival of mice infected with MtbΔ<i>Rv3167c</i>. The regulon of Rv3167c thus contains the bacterial mediators involved in escape from the phagosome and host cell necrosis induction, both of which are crucial steps in the intracellular lifecycle and virulence of Mtb.</p></div

Mitochondrial ROS is required for cell death and autophagy induction by MtbΔ<i>Rv3167c</i>.

<p>Uninfected (UI), wild-type Mtb (Mtb), <i>Rv3167c</i> deletion mutant Mtb (MtbΔ) or complemented mutant Mtb (MtbΔC) bacteria infected cells were analyzed as described. (A) <i>Left</i>: Representative histogram of mitochondrial ROS measurement using MitoSOX Red staining of BMDMs at 24h. <i>Right</i>: Quantification of mitochondrial ROS by estimating increase in MitoSOX Red fluorescence intensity (mean ± S.E.M, n = 4) (B) <i>Left</i>: Representative histogram of cellular ROS measurement using DCFDA staining of BMDMs at 24h. <i>Right</i>: Quantification of cellular ROS by estimating increase in DCFDA fluorescence intensity (means ± S.E.M, n = 4). For (A) and (B), percentage increase in mean fluorescent intensity of infected cells compared to UI cells was calculated after subtracting background fluorescence. (C) Necrosis induction in WT and <i>Nox2</i>^<i>-/-</i> BMDMs was determined by PI staining and flow cytometry at 48h (mean ± S.E.M, n = 6). (D) Necrosis induction in BMDMs from WT and mitochondrial targeted catalase (mCAT) knock-in mice was determined by PI staining and flow cytometry at 24h (mean ± S.E.M, n = 3). (E) Effect of the Akt activator sc-79 on mitochondrial ROS was measured by staining with MitoSOX Red and flowcytometry (mean ± S.E.M, n = 3).</p

Mechanisms of Stage-Transcending Protection Following Immunization of Mice with Late Liver Stage-Arresting Genetically Attenuated Malaria Parasites

<div><p>Malaria, caused by <i>Plasmodium</i> parasite infection, continues to be one of the leading causes of worldwide morbidity and mortality. Development of an effective vaccine has been encumbered by the complex life cycle of the parasite that has distinct pre-erythrocytic and erythrocytic stages of infection in the mammalian host. Historically, malaria vaccine development efforts have targeted each stage in isolation. An ideal vaccine, however, would target multiple life cycle stages with multiple arms of the immune system and be capable of eliminating initial infection in the liver, the subsequent blood stage infection, and would prevent further parasite transmission. We have previously shown that immunization of mice with <i>Plasmodium yoelii</i> genetically attenuated parasites (GAP) that arrest late in liver stage development elicits stage-transcending protection against both a sporozoite challenge and a direct blood stage challenge. Here, we show that this immunization strategy engenders both T- and B-cell responses that are essential for stage-transcending protection, but the relative importance of each is determined by the host genetic background. Furthermore, potent anti-blood stage antibodies elicited after GAP immunization rely heavily on F_C-mediated functions including complement fixation and F_C receptor binding. These protective antibodies recognize the merozoite surface but do not appear to recognize the immunodominant merozoite surface protein-1. The antigen(s) targeted by stage-transcending immunity are present in both the late liver stages and blood stage parasites. The data clearly show that GAP-engendered protective immune responses can target shared antigens of pre-erythrocytic and erythrocytic parasite life cycle stages. As such, this model constitutes a powerful tool to identify novel, protective and stage-transcending T and B cell targets for incorporation into a multi-stage subunit vaccine.</p></div

Mtb Rv3167c is important for inhibition of host cell death.

<p>Cell death induction in uninfected (UI), wild-type Mtb (Mtb), <i>Rv3167c</i> deletion mutant Mtb (MtbΔ) or complemented mutant Mtb (MtbΔC) bacteria infected cells was determined. (A) TUNEL staining and flow cytometry of THP1 cells at 24h (mean ± S.E.M, n = 6). (B) Cell death induction in human monocyte-derived macrophages (hMDMs) at 48h was measured by hypodiploid staining and flow cytometry. Cells were obtained from two independent donors (mean ± S.E.M, n = 6). (C) Release of adenylate kinase into supernatants from uninfected and infected THP1 cells was measured by Toxilight assay at the indicated time points (mean ± S.E.M, n = 6). Cell death induction in (D) wild type (WT) and (E) <i>Casp3</i>^<i>-/-</i> BMDMs was determined by TUNEL staining and flow cytometry at 24h. Cells were treated with the pan caspase inhibitor zVAD-fmk (40μM) for one hour prior to and throughout the infection (mean ± S.E.M, n = 3). (F) PARP cleavage in THP1 cells at 24h detected by western blotting of whole cell lysates obtained from infected cells or after treatment with apoptosis inducer staurosporine (Sts). Image is representative of three independent experiments.</p

MtbΔ<i>Rv3167c</i> mediates cell death via programmed necrosis.

<p>(A) Necrosis induction in WT and <i>Ripk3</i>^<i>-/</i>- BMDMs was measured by PI staining and flow cytometry at 24h (mean ± S.E.M, n = 3) in uninfected (UI), wild-type Mtb (Mtb), <i>Rv3167c</i> deletion mutant Mtb (MtbΔ) or complemented mutant Mtb (MtbΔC) bacteria infected cells. (B) Necrosis induction in WT and <i>Parp1</i>^<i>-/-</i> BMDMs at 72h was determined by PI staining (mean ± S.E.M, n = 3). (C) Necrosis induction in WT and <i>Casp1/11</i>^<i>-/-</i> BMDMs was determined by PI staining at 48h (mean ± S.E.M, n = 6). (D) Increases in lysosomal permeabilization were measured by acridine orange staining at indicated time points (mean ± S.E.M, n = 6).</p

Host kinase Akt1/2 regulates MtbΔ<i>Rv3167c</i>-mediated autophagy and necrosis signaling.

<p>Uninfected (UI), wild-type Mtb (Mtb), <i>Rv3167c</i> deletion mutant Mtb (MtbΔ) or complemented mutant Mtb (MtbΔC) bacteria infected cells were analyzed as described. (A) JNK and p38 MAPK phosphorylation in THP1 LC3GFP cells at 18h detected by western blotting of whole cell lysates. Image is representative of three independent experiments. (B) JNK and p38 MAPK phosphorylation in human monocyte derived macrophages (hMDMs) at 24h detected by western blotting of whole cell lysates. Image is representative of three independent experiments. Cells were obtained from two donors. (C) Autophagy induction in presence of JNK (JNK-I—SP600125) and p38 MAPK (p38-I-SB203580) inhibitors detected by flow cytometry (mean ± S.E.M, n = 6). (D) Effect of JNK inhibitor (JNK-I-SP600125, 25μM) and (E) p38MAPK inhibitor (p38-I-SB203580, 25μM) on necrosis measured by PI staining and flow cytometry at 24h (mean ± S.E.M, n = 6). (F) Akt phosphorylation in THP1 LC3GFP cells detected at 16h by western blotting. Image is representative of three independent experiments. (G) Autophagy induction in presence of Akt activator (Akt—A—sc-79) examined by flow cytometry at 16h (mean ± S.E.M, n = 8). (H) Necrosis induction in presence of Akt activator (Akt—A—sc-79) in THP1 macrophages measured by flow cytometry at 24h (means ± S.E.M, n = 6). For immunoblots, numbers below indicate fold change in band intensity compared to Mtb sample after normalization to loading control.</p

MtbΔ<i>Rv3167c</i> causes increased macroautophagy but not autophagic cell death.

<p>Uninfected (UI), wild-type Mtb (Mtb), <i>Rv3167c</i> deletion mutant Mtb (MtbΔ) or complemented mutant Mtb (MtbΔC) bacteria infected cells were analyzed as described. (A) Autophagy induction by AF647-NHS ester stained bacteria in THP1 LC3GFP cells at 8h examined by confocal microscopy. <i>Left</i>: Representative fluorescent image. The white box indicates bacteria colocalizing with LC3GFP. <i>Right</i>: Quantitative analysis of autophagic cells and cells with LC3+ bacteria. At least 450 cells were counted per condition per experiment. Statistical analysis was performed using unpaired Student’s t test (mean ± S.E.M, n = 3). (B) <i>Left</i>: Representative TEM image showing absence of colocalization between bacteria and autophagosomes at 24h. *—bacteria; AP—autophagosome; M—mitochondria. <i>Right</i>: Quantitative analysis of bacteria within autophagosomes as observed by TEM. Atleast 10 cells were counted per condition per experiment (n = 2). (C) Autophagy induction in THP1 LC3GFP cells measured at 16h by flowcytometry (mean ± S.E.M, n = 6). (D) Conversion of GFP tagged and endogenous LC3I to LC3II detected by western blotting in whole cell lysates. Image is representative of three independent experiments. (E) Inhibition of MtbΔ<i>Rv3167c</i>-induced autophagy in presence of 3-MA (5mM) measured by flowcytometry at 16h (mean ± S.E.M, n = 4). (F) Necrosis induction in autophagy competent (Atg5^+/+) and autophagy deficient (Atg5^-/-) BMDMs measured using Toxilight assay (mean ± S.E.M, n = 6).</p

Quantification of staining patterns of sera from LAGAP-immunized mice.

<p>Quantification of staining patterns of sera from LAGAP-immunized mice.</p

Antibodies elicited by LAGAP in C57BL/6 but not BALB/cJ mice are of broad isotypes and recognize the merozoite surface.

<p>Serum was collected from LAGAP immunized C57BL/6 (top 3 rows) and BALB/cJ (bottom 3 rows) mice as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004855#ppat.1004855.g004" target="_blank">Fig 4</a> and used in a blood stage IFA. Secondary antibodies against total IgG, IgG1 and IgG2<u>b</u> were used to visualize isotype-specific anti-blood stage antibodies. Parasites were also visualized with antibodies recognizing MSP1 and DNA visualized with 4',6-diamidino-2-phenylindole (DAPI). Antibodies in LAGAP-immunized C57BL/6 and BALB/cJ mice do not differ in isotype but rather in antigen specificity with only C57BL/6 serum recognizing the merozoite surface.</p

<i>Rv3167c</i> regulates virulence of Mtb.

<p>(A) Survival of C57Bl6 mice infected via the aerosol route with 100 CFU of wild-type Mtb (Mtb), <i>Rv3167c</i> deletion mutant Mtb (MtbΔ) or complemented mutant Mtb (MtbΔC) bacteria (n = 7 per experimental group) (B) Lung, (C) spleen and (D) liver bacterial burdens were determined at indicated times (mean ± S.E.M, n = 6) (E) Cytokines and (F) chemokines in lung tissue homogenates at 56d were quantified using a multiplex ELISA (mean ± S.E.M, n = 3). (G) <i>Left</i>: Representative image of H and E stained section of lung tissue. <i>Right</i>: Quantitative analysis of lung area exhibiting cellular infiltration at 56d performed using ImageJ (mean ± S.E.M, n = 3).</p