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

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

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

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

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

Oligonucleotide primers used in this study.

<p>Oligonucleotide primers used in this study.</p

The Δ<i>fbpA</i>Δ<i>sapM</i> double knockout (DKO) strain is more immunogenic in mouse macrophages and elicits stronger immune responses in mice:

<p><b>a</b>) BMs and DCs from C57Bl/6 mice were infected with mycobacteria (MOI 1∶1), washed and overlaid with Antigen 85B specific BB7 hybridoma T cells (1∶20 ratio). After 4 hrs, the supernatants collected were tested for IL-2 using sandwich ELISA. DKO induces BMs and DCs to prime T cells to secrete larger amounts of IL-2, indicating a better processing of DKO for Ag85B (4 experiments, SEM, * <0.009 vs. <b>Δ</b>fbpA <i>or </i><b>Δ</b>sapM; by t test). <b>b</b>). C57Bl/6 mice (3 per group) were vaccinated with mycobacterial strains at 10⁶ CFU per mouse given once subcutaneously. At time intervals, the spleen derived T cells were tested for Ag85B responsive T cells using IFN-γ coated plates and Elispot assay. DKO vaccination leads to a larger expansion of Ag85B specific T cells. All Elispot numbers represent Ag85B stimulated numbers subtracted from KLH protein stimulated T cells. T cells from naïve mice were stimulated with KLH alone (3 separate experiments, 3 mice per group per time point).</p

The Δ<i>fbpA</i>Δ<i>sapM</i> double knockout (DKO) strain shows enhanced lysosomal localization in mouse macrophages:

<p><i>gfpMtb</i> H37Rv or Oregon green stained mutant strains were phagocytosed into BMs, incubated, fixed 24 hrs later and stained with primary antibodies to lysosomal markers LAMP1 (IDB4), CD63 and rab7 followed by Texas red conjugated conjugates. Mycobacteria colocalizing with antibodies were scored using a Nikon fluorescence microscope and Metaview deconvolution software. <b>a</b>) Illustration that the DKO mutant colocalizes better with rab7 lysosomal marker. <b>b</b>) Percent colocalization was determined by counting 200 macrophages per well each with 1–3 mycobacteria and averaging counts from triplicate chambers (SD). One of three similar experiments is shown. Text below the bar diagram indicates the colocalization of each marker in relation to different strains (*p<0.01, t test).</p

The Δ<i>fbpA</i>Δ<i>sapM</i> double knockout (DKO) strain is attenuated in macrophages and induces stronger oxidant responses that reduce its viability:

<p>Macrophages from C57Bl/6 mouse bone marrow (BMs) and human THP1 macrophages (pre-activated with phorbol ester) were infected with mycobacteria (MOI 1∶1), washed, incubated, lysed and plated for viable colony counts (CFUs). <b>a)</b>. The DKO strain is more attenuated compared to wild type <i>Mtb</i> in BMs. <b>b-c)</b> Intracellular reactive oxygen species (ROS) and nitric oxide (NO) were measured respectively using dihydro-dichloro-fluorescein acetate (DCFDA) fluorescent probe and Greiss reagent. DKO induced elevated NO responses (p value by t test; panel <b>c</b>) but not ROS (panel <b>b</b>). <b>d-e)</b> DKO was attenuated in THP1 macrophages compared to Δ<i>fbpA</i>, Δ<i>sapM</i> or wild type H37Rv in BMs (p<0.01) that correlated with increased ROS responses (panel <b>e</b>). Nitric oxide responses of THP1 were not detectable (not shown). <b>f)</b>. Mycobacteria (10⁵ CFU/mL; baseline shown as dotted line) were exposed to the bactericidal action of the superoxide and NO donor 3-morpholinosydnonimine (10 mM; SIN-1) in 7H9 broth and viable counts determined at intervals (24 and 72 hr post treatment) by plating on 7H11 agar. DKO is markedly susceptible by 72 hrs <i>in vitro</i> to the oxidants released by SIN-1 (p value by t test).</p