Biochemical and physiological characterization of the GTP-binding protein Obg of Mycobacterium tuberculosis

<p>Abstract</p> <p>Background</p> <p>Obg is a highly conserved GTP-binding protein that has homologues in bacteria, archaea and eukaryotes. In bacteria, Obg proteins are essential for growth, and they participate in spore formation, stress adaptation, ribosome assembly and chromosomal partitioning. This study was undertaken to investigate the biochemical and physiological characteristics of Obg in <it>Mycobacterium tuberculosis</it>, which causes tuberculosis in humans.</p> <p>Results</p> <p>We overexpressed <it>M. tuberculosis </it>Obg in <it>Escherichia coli </it>and then purified the protein. This protein binds to, hydrolyzes and is phosphorylated with GTP. An anti-Obg antiserum, raised against the purified Obg, detects a 55 kDa protein in immunoblots of <it>M. tuberculosis </it>extracts. Immunoblotting also discloses that cultured <it>M. tuberculosis </it>cells contain increased amounts of Obg in the late log phase and in the stationary phase. Obg is also associated with ribosomes in <it>M</it>. <it>tuberculosis</it>, and it is distributed to all three ribosomal fractions (30 S, 50 S and 70 S). Finally, yeast two-hybrid analysis reveals that Obg interacts with the stress protein UsfX, indicating that <it>M. tuberculosis </it>Obg, like other bacterial Obgs, is a stress related protein.</p> <p>Conclusions</p> <p>Although its GTP-hydrolyzing and phosphorylating activities resemble those of other bacterial Obg homologues, <it>M. tuberculosis </it>Obg differs from them in these respects: (a) preferential association with the bacterial membrane; (b) association with all three ribosomal subunits, and (c) binding to the stress protein UsfX, rather than to RelA. Generation of mutant alleles of Obg of <it>M. tuberculosis</it>, and their characterization in vivo, may provide additional insights regarding its role in this important human pathogen.</p

The fbpA/sapM Double Knock Out Strain of Mycobacterium tuberculosis Is Highly Attenuated and Immunogenic in Macrophages

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the leading cause of death due to bacterial infections in mankind, and BCG, an attenuated strain of Mycobacterium bovis, is an approved vaccine. BCG sequesters in immature phagosomes of antigen presenting cells (APCs), which do not fuse with lysosomes, leading to decreased antigen processing and reduced Th1 responses. However, an Mtb derived ΔfbpA attenuated mutant underwent limited phagosome maturation, enhanced immunogenicity and was as effective as BCG in protecting mice against TB. To facilitate phagosome maturation of ΔfbpA, we disrupted an additional gene sapM, which encodes for an acid phosphatase. Compared to the wild type Mtb, the ΔfbpAΔsapM (double knock out; DKO) strain was attenuated for growth in mouse macrophages and PMA activated human THP1 macrophages. Attenuation correlated with increased oxidants in macrophages in response to DKO infection and enhanced labeling of lysosomal markers (CD63 and rab7) on DKO phagosomes. An in vitro Antigen 85B peptide presentation assay was used to determine antigen presentation to T cells by APCs infected with DKO or other mycobacterial strains. This revealed that DKO infected APCs showed the strongest ability to present Ag85B to T cells (>2500 pgs/mL in 4 hrs) as compared to APCs infected with wild type Mtb or ΔfbpA or ΔsapM strain (<1000 pgs/mL in 4 hrs), indicating that DKO strain has enhanced immunogenicity than other strains. The ability of DKO to undergo lysosomal fusion and vacuolar acidification correlated with antigen presentation since bafilomycin, that inhibits acidification in APCs, reduced antigen presentation. Finally, the DKO vaccine elicited a better Th1 response in mice after subcutaneous vaccination than either ΔfbpA or ΔsapM. Since ΔfbpA has been used in mice as a candidate vaccine and the DKO (ΔfbpAΔsapM) mutant is more immunogenic than ΔfbpA, we propose the DKO is a potential anti-tuberculosis vaccine

Methionine Sulfoxide Reductase A (MsrA) Deficient Mycoplasma genitalium Shows Decreased Interactions with Host Cells

Mycoplasma genitalium is an important sexually transmitted pathogen that affects both men and women. In genital-mucosal tissues, it initiates colonization of epithelial cells by attaching itself to host cells via several identified bacterial ligands and host cell surface receptors. We have previously shown that a mutant form of M. genitalium lacking methionine sulfoxide reductase A (MsrA), an antioxidant enzyme which converts oxidized methionine (Met(O)) into methionine (Met), shows decreased viability in infected animals. To gain more insights into the mechanisms by which MsrA controls M. genitalium virulence, we compared the wild-type M. genitalium strain (G37) with an msrA mutant (MS5) strain for their ability to interact with target cervical epithelial cell lines (HeLa and C33A) and THP-1 monocytic cells. Infection of epithelial cell lines with both strains revealed that MS5 was less cytotoxic to HeLa and C33A cell lines than the G37 strain. Also, the MS5 strain was more susceptible to phagocytosis by THP-1 cells than wild type strain (G37). Further, MS5 was less able to induce aggregation and differentiation in THP-1 cells than the wild type strain, as determined by carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling of the cells, followed by counting of cells attached to the culture dish using image analysis. Finally, MS5 was observed to induce less proinflammatory cytokine TNF-α by THP-1 cells than wild type G37 strain. These results indicate that MsrA affects the virulence properties of M. genitalium by modulating its interaction with host cells

The Mycoplasma genitalium MG_454 Gene Product Resists Killing by Organic Hydroperoxides▿ †

Mycoplasma genitalium is the smallest self-replicating organism and a successful human pathogen associated with a range of genitourinary maladies. As a consequence of its restricted genome size, genes that are highly conserved in other bacteria are absent in M. genitalium. Significantly, genes that encode antioxidants like superoxide dismutase and catalase-peroxidase are lacking. Nevertheless, comparative genomics has revealed that MG_454 of M. genitalium encodes a protein with putative function as an organic hydroperoxide reductase (Ohr). In this study, we found that an M. genitalium transposon mutant that lacks expression of MG_454 was sensitive to killing by t-butyl hydroperoxide and cumene hydroperoxide. To understand whether this sensitivity to hydroperoxides was linked to MG_454, we cloned this gene behind an arabinose-inducible PBAD promoter in plasmid pHERD20T and transformed this construct (pHERDMG454) into a Pseudomonas aeruginosa strain having deletion in its ohr gene (ohr mutant) and showing sensitivity to organic hydroperoxides. The P. aeruginosa ohr mutant harboring pHERDMG454, when induced with arabinose, was able to reverse its sensitivity to organic hydroperoxides, thus supporting the notion that the product of MG_454 resists organic hydroperoxides in M. genitalium. Surprisingly, real-time reverse transcription-PCR showed that expression of MG_454 in M. genitalium was not elevated in response to oxidative stress but was elevated in response to physical stresses, like salt (NaCl) and heat. Although failure of MG_454 to respond to oxidative stress in M. genitalium implies the absence of a known oxidative stress response regulator in the genome of M. genitalium, elevated expression of MG_454 due to physical stress suggests its control by an unidentified regulator

Phagocytosis of <i>M. genitalium</i> strains by THP-1 cells.

<p><b>A. Determination of phagocytosis by color change method.</b> Phagocytosis of <i>M. genitalium</i> strains by THP-1 cells were determined by a change in color after adding MTS solution (Promega) as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036247#s2" target="_blank">Materials and Methods</a> section. The solid bars indicate absorbance (<i>A</i>₄₉₀) of the control wells (Mycoplasmas without THP-1 cells) and striped bars represent absorbance (<i>A</i>₄₉₀) of the experimental wells (THP-1 cells infected with mycoplasmas). Results represent Mean ± SD from three independent experiments. G37, MGRE and MS5 indicate infection of cells with <i>M. genitalium</i> wild type G37 strain, control strain MGRE and <i>msrA</i> mutant strain MS5, respectively. * = p≤0.05 vs wild type G37. <b>B. Visualization of phagocytosed </b><b><i>M. genitalium</i></b><b> G37, MGRE and MS5 strains.</b> G37, MGRE and MS5 bacteria were labeled with FITC as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036247#s2" target="_blank">Material and Methods</a>. FITC and DIC represent fluorescence and differential interference contrast of the same field. Merge represents overlay of FITC and DIC. PBS indicate uninfected control cells; G37, MGRE and MS5 indicate infection of cells with <i>M. genitalium</i> wild type G37 strain, control strain MGRE and <i>msrA</i> mutant strain MS5, respectively.</p

Differentiation of THP-1 cells by <i>M. genitalium</i> strains.

<p><b>A. Adherent THP-1 cells showing fluorescence.</b> CFSE labeled THP-1 cells were infected with <i>M. genitalium</i> strains (MOI 1∶5). Images of adherent cells were acquired using confocal laser scanning microscope with 10× objective and 488 nm laser. G37, MS5 and MGRE are wild type, <i>msrA</i> mutant and control <i>M. genitalium</i> strains respectively. HKG37 represents heat killed bacteria of wild type <i>M. genitalium</i>. <b>B. Graph showing the amount of adherent cells for each infection.</b> The number of labeled cells in each image were counted using the particle plugin of Image J software. Average cell numbers from five different optical fields and from three independent experiments were used for determining the number of adherent mononuclear cells in each infection. Labels are as described in “<b>A</b>”. * = p≤0.05 vs wild type G37 strain.</p

Cytotoxic effect of <i>M. genitalium</i> strains on cervical epithelial cells based on cell survival assay.

<p>HeLa (<b>A</b>) and C33A (<b>B</b>) epithelial cells infected with <i>M. genitalium</i> G37 (wild type) and MS5 (<i>msrA</i> mutant) strains at different multiplicity of infections and cells survived was determined by SRB (sulforhodamine B) assays. Solid bars in figures <b>A</b> and <b>B</b> represent <i>M. genitalium</i> wild type strain G37. Bars with downward stripes in figures <b>A</b> and <b>B</b> represent <i>M. genitalium msrA</i> mutant strain MS5. Both strains were tested at various MOI (1∶0 −1∶100). * = p≤0.05 percent cell survival of MS5 strain is higher vs G37 strain. Results represent Mean ± SD of three independent experiments.</p

Microscopic observation of cytotoxic effect of <i>M. genitalium</i> strains on cervical epithelial cells.

<p>HeLa and C33A epithelial cells were infected with <i>M. genitalium</i> G37 and MS5 strains and analyzed using differential interference contrast at 488 nm in a confocal laser scanning microscope with 20× objective. PBS indicates uninfected control cells; G37, MGRE, MS5 and HKG37 indicate infection of cells with <i>M. genitalium</i> wild type G37 strain, control strain MGRE, <i>msrA</i> mutant strain MS5 and heat killed G37 bacteria, respectively.</p

Cytokines released by THP-1 cells after infection with mycoplasma strains.

<p>Supernatants from THP-1 cells infected with mycoplasma strains (MOI 1∶10) were collected and IL-1β and TNF-α concentrations were determined using an ELISA kit from eBioscience. <b>A.</b> Release of IL-1β by THP-1 cells and <b>B.</b> Release of TNF-α by THP-1 cells. PBS, phosphate buffered saline control; G37 and MS5 are wild type <i>msrA</i> mutant <i>M. genitalium</i> strains respectively; HKG37 represents heat killed wild type <i>M. genitalium</i>. * = p≤0.05 vs wild type G37.</p

Generation of Reactive Oxygen Species (ROS) by phagocytic RAW264.7 cells upon infection with <i>M. genitalium</i> strains.

<p><b>A. Confocal images of RAW264.7 cells showing ROS generation.</b> RAW264.7 cells were infected with G37, MS5, MGRE or treated with heat killed G37 (MOI 1∶10) and generation of ROS was detected by addition of DCF-DA. Images were captured using an Olympus confocal laser scanning microscope with 488 nm laser. DCF-DA and DIC indicate fluorescence of DCF-DA and differential interference contrast of the same field; G37, MS5 and MGRE represent cells infected with <i>M. genitalium</i> wild type, <i>msrA</i> mutant and control strains. HKG37 represents cells treated with heat killed wild type <i>M. genitalium</i>. <i>t</i>-BHP represents ROS induced with 1 µM <i>t</i>-butyl hydroperoxide for 30 min. <b>B. Graphical representation of ROS generated by RAW264.7 cells.</b> Each bar represents ROS generated by RAW264.7 cells in response to infection/induction. Images were captured using a confocal laser scanning microscope. Total fluorescence counts were determined from images using NIH image J software from ten different fields and three independent experiments. Arbitrary fluorescence units for each infection are given as Mean ± SD. * = p≤0.05 vs wild type G37. Labels are as described in “<b>A</b>”.</p