Antimycobacterial drug discovery using Mycobacteria-infected amoebae identifies anti-infectives and new molecular targets

Tuberculosis remains a serious threat to human health world-wide, and improved efficiency of medical treatment requires a better understanding of the pathogenesis and the discovery of new drugs. In the present study, we performed a whole-cell based screen in order to complete the characterization of 168 compounds from the GlaxoSmithKline TB-set. We have established and utilized novel previously unexplored host-model systems to characterize the GSK compounds, i.e. the amoeboid organisms D. discoideum and A. castellanii, as well as a microglial phagocytic cell line, BV2. We infected these host cells with Mycobacterium marinum to monitor and characterize the anti-infective activity of the compounds with quantitative fluorescence measurements and high-content microscopy. In summary, 88.1% of the compounds were confirmed as antibiotics against M. marinum, 11.3% and 4.8% displayed strong anti-infective activity in, respectively, the mammalian and protozoan infection models. Additionally, in the two systems, 13-14% of the compounds displayed pro-infective activity. Our studies underline the relevance of using evolutionarily distant pathogen and host models in order to reveal conserved mechanisms of virulence and defence, respectively, which are potential "universal" targets for intervention. Subsequent mechanism of action studies based on generation of over-expresser M. bovis BCG strains, generation of spontaneous resistant mutants and whole genome sequencing revealed four new molecular targets, including FbpA, MurC, MmpL3 and GlpK

Mycobacterium marinum antagonistically induces an autophagic response while repressing the autophagic flux in a TORC1- and ESX-1-dependent manner.

Autophagy is a eukaryotic catabolic process also participating in cell-autonomous defence. Infected host cells generate double-membrane autophagosomes that mature in autolysosomes to engulf, kill and digest cytoplasmic pathogens. However, several bacteria subvert autophagy and benefit from its machinery and functions. Monitoring infection stages by genetics, pharmacology and microscopy, we demonstrate that the ESX-1 secretion system of Mycobacterium marinum, a close relative to M. tuberculosis, upregulates the transcription of autophagy genes, and stimulates autophagosome formation and recruitment to the mycobacteria-containing vacuole (MCV) in the host model organism Dictyostelium. Antagonistically, ESX-1 is also essential to block the autophagic flux and deplete the MCV of proteolytic activity. Activators of the TORC1 complex localize to the MCV in an ESX-1-dependent manner, suggesting an important role in the manipulation of autophagy by mycobacteria. Our findings suggest that the infection by M. marinum activates an autophagic response that is simultaneously repressed and exploited by the bacterium to support its survival inside the MCV

A mitotically inheritable unit containing a MAP kinase module

Prions are novel kinds of hereditary units, relying solely on proteins, that are infectious and inherited in a non-Mendelian fashion. To date, they are either based on autocatalytic modification of a 3D conformation or on autocatalytic cleavage. Here, we provide further evidence that in the filamentous fungus Podospora anserina, a MAP kinase cascade is probably able to self-activate and generate C, a hereditary unit that bears many similarities to prions and triggers cell degeneration. We show that in addition to the MAPKKK gene, both the MAPKK and MAPK genes are necessary for the propagation of C, and that overexpression of MAPK as that of MAPKKK facilitates the appearance of C. We also show that a correlation exists between the presence of C and localization of the MAPK inside nuclei. These data emphasize the resemblance between prions and a self-positively regulated cascade in terms of their transmission. This thus further expands the concept of protein-base inheritance to regulatory networks that have the ability to self-activate

The autophagic flux is blocked during wt <i>M</i>. <i>marinum</i> infection.

<p><b>A.</b> GFP-Atg8-expressing cells were infected or mock-infected for 0.5 or 6 h with mCherry-expressing <i>M</i>. <i>marinum</i> wt or with DsRed-expressing <i>M</i>. <i>marinum</i> ΔRD1 and treated or not with a PI cocktail at 2.5× for one additional hour. Representative maximum projections of live imaging at 1.5 hpi are shown. Scale bars, 10 μm; <b>B.</b> Medians with interquartile ranges of the number of GFP-Atg8 structures per cell from the infections carried out in <b>A</b>. Each dot represents one cell. 178–338 cells per condition from three independent experiments were counted. The λ that define the Poisson distribution of each data set and differences between them were calculated as described in Materials and Methods (*<i>p</i> ≤ 0.05; ****<i>p</i> ≤ 0.0001; ns, <i>p</i> > 0.05); <b>C.</b> Mean and standard deviation of the log₂ (<b>λ</b>_PI/<b>λ</b>_mock) from the three independent replicates represented in <b>B</b>. A log₂ (<b>λ</b>_PI/<b>λ</b>_mock) of zero implies that there was a total autophagic block. <i>p</i>-values calculated as described in Materials and Methods (****<i>p</i> ≤ 0.0001; ns, <i>p</i> > 0.05). <b>D.</b> Samples from infections (<b>A</b>) were immunoblotted against GFP. Longer exposure of the free GFP bands is shown as "GFP (high)". Ponceau-S staining as loading control. Representative result from four independent experiments <b>E.</b> Means and standard deviations of the ratio GFP/GFP-Atg8 from the immunoblots represented in <b>D</b>. Unpaired <i>t</i> test (*<i>p</i> ≤ 0.05; **<i>p</i> ≤ 0.01; ns, <i>p</i> > 0.05). <b>F.</b> EM of <i>D</i>. <i>discoideum</i> infected with <i>M</i>. <i>marinum</i> wt for 7 h and incubated or not with PI at 2.5× for an additional hour. White and green asterisks label bacteria and autophagosomes, respectively. White arrowheads point to membrane extensions originating at the MCV. The black arrowhead marks the zoomIn. Scale bars, 2 μm.</p

Artificial induction of autophagy restricts <i>M</i>. <i>marinum</i> proliferation.

<p><b>A.</b> GFP-Atg8-expressing cells were infected for 5 h with mCherry-expressing <i>M</i>. <i>marinum</i> wt and treated or not with AR-12 at 2.5 μM for 2 additional hours. Representative maximum projections of live imaging are shown. White arrowheads point to GFP-Atg8 recruitment to MCV. Scale bars, 10 μm; <b>B.</b> Quantification of the percentage of infected cells with GFP-Atg8⁺ bacteria at 7 hpi. Means and standard deviations from six (mock) and three (AR-12) independent replicates are represented and an unpaired <i>t</i> test showed no statistical significance (ns, <i>p</i> > 0.05). 688 and 297 infected cells were counted for the mock and the AR-12 treatments, respectively; <b>C.</b> Classification of types of GFP-recruitment for infected mock (258 cells) and AR-12 (141 cells) treated. Means and standard deviations from six (mock) and three (AR-12) independent replicates are represented. Unpaired <i>t</i> test (**<i>p</i> ≤ 0.01; ***<i>p</i> ≤ 0.001); <b>D.</b> Cells infected with <i>lux</i>-expressing <i>M</i>. <i>marinum</i> wt bacteria were treated or not with AR-12 at 2.5 μM. Intracellular bacteria growth [relative luminescence units (RLU)] is represented as the mean and standard deviation from triplicates. Statistical differences were calculated with a Bonferroni post hoc test after two-way ANOVA (*<i>p</i> ≤ 0.05).</p

Autophagy is necessary for the maintenance of the MCV.

<p><b>A.</b> The intracellular growth of <i>lux</i>-expressing <i>M</i>. <i>marinum</i> wt was monitored inside wt and <i>atg1</i>- cells. RLUs are represented as the mean and standard deviation from quadruplicates. Statistical differences were calculated with a Bonferroni post hoc test after two-way ANOVA (**<i>p</i> ≤ 0.01; (****<i>p</i> ≤ 0.0001); <b>B.</b> EM of the locations of <i>M</i>. <i>marinum</i> wt inside wt and <i>atg1</i>- cells at 0.25, 1 and 6 hpi. Black and orange asterisks label bacteria inside a compartment or in the cytosol, respectively. Black arrowheads mark the zoomIn. Nuclei . Scale bars, 2 μm; <b>C.</b> <i>D</i>. <i>discoideum</i> wt, <i>atg8</i>- and <i>atg1</i>- cells were infected with mCherry-expressing <i>M</i>. <i>marinum</i>, fixed and stained against Ub (green) and mCherry (red). Representative maximum projections at 0.25 and 6 hpi are shown. White arrowheads label the ubiquitinated bacteria. Scale bars, 10 μm; <b>D.</b> Quantification of the percentage of intracellular bacteria/MCVs (red) positive for Ub (green) in wt, <i>atg8</i>-, <i>atg1</i>- and <i>atg1</i>-Atg1-GFP cells at 0.25 and 6 hpi. Means and standard deviations from 2–3 independent experiments. 128–299 infected cells were counted per time point and cell line. Unpaired <i>t</i> test (*<i>p</i> ≤ 0.05; ***<i>p</i> ≤ 0.001; **** <i>p</i> ≤ 0.0001).</p

Model of the <i>D</i>. <i>discoideum</i> autophagic response during <i>M</i>. <i>marinum</i> infection.

<p>Early after engulfment, <i>M</i>. <i>marinum</i> damages the membrane of its MCV in an ESX-1-dependent manner. Membrane perforations might block lysosome fusion and, consequently, autophagic flux. In addition, ESX-1 inhibits TORC1 activity early during infection (1.5 hpi), presumably through a nutrient-sensing pathway as described for other bacteria [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006344#ppat.1006344.ref075" target="_blank">75</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006344#ppat.1006344.ref077" target="_blank">77</a>]. Downregulation of TORC1, which is always bound to the wt MCV membrane, induces the formation of autophagosomes that somehow repair the membrane damages and provide the MCV with cytoplasmic material. TORC1 re-activation at 7 hpi leads to the decrease in autophagosome formation and recruitment to the MCV, and enhances the blockade of the autophagic flux, which generates proto-lysosomal tubules (ALR). The block in autophagic flux prevents bacteria killing in autolysosomes and degradation of membranes and cytoplasmic material delivered to the MCV via the recruited autophagosomes.</p

The autophagic flux is blocked during wt <i>M</i>. <i>marinum</i> infection.

<p><b>A.</b> GFP-Atg8-expressing cells were infected or mock-infected for 0.5 or 6 h with mCherry-expressing <i>M</i>. <i>marinum</i> wt or with DsRed-expressing <i>M</i>. <i>marinum</i> ΔRD1 and treated or not with a PI cocktail at 2.5× for one additional hour. Representative maximum projections of live imaging at 1.5 hpi are shown. Scale bars, 10 μm; <b>B.</b> Medians with interquartile ranges of the number of GFP-Atg8 structures per cell from the infections carried out in <b>A</b>. Each dot represents one cell. 178–338 cells per condition from three independent experiments were counted. The λ that define the Poisson distribution of each data set and differences between them were calculated as described in Materials and Methods (*<i>p</i> ≤ 0.05; ****<i>p</i> ≤ 0.0001; ns, <i>p</i> > 0.05); <b>C.</b> Mean and standard deviation of the log₂ (<b>λ</b>_PI/<b>λ</b>_mock) from the three independent replicates represented in <b>B</b>. A log₂ (<b>λ</b>_PI/<b>λ</b>_mock) of zero implies that there was a total autophagic block. <i>p</i>-values calculated as described in Materials and Methods (****<i>p</i> ≤ 0.0001; ns, <i>p</i> > 0.05). <b>D.</b> Samples from infections (<b>A</b>) were immunoblotted against GFP. Longer exposure of the free GFP bands is shown as "GFP (high)". Ponceau-S staining as loading control. Representative result from four independent experiments <b>E.</b> Means and standard deviations of the ratio GFP/GFP-Atg8 from the immunoblots represented in <b>D</b>. Unpaired <i>t</i> test (*<i>p</i> ≤ 0.05; **<i>p</i> ≤ 0.01; ns, <i>p</i> > 0.05). <b>F.</b> EM of <i>D</i>. <i>discoideum</i> infected with <i>M</i>. <i>marinum</i> wt for 7 h and incubated or not with PI at 2.5× for an additional hour. White and green asterisks label bacteria and autophagosomes, respectively. White arrowheads point to membrane extensions originating at the MCV. The black arrowhead marks the zoomIn. Scale bars, 2 μm.</p

Expression of ESX-1 is essential to devoid the MCV of proteolytic activity.

<p>Cells expressing GFP-Rab11c, GFP-Rab7a or VatB-RFP, or incubated with LysoSensor Green or DQ Green BSA were infected for 1.5 and 7h with <i>M</i>. <i>marinum</i> wt or ΔRD1 labelled with Vibrant DyeCycle Ruby or expressing mCherry, DsRed or GFP. Representative sections of live imaging at 7 hpi are shown in <b>A</b>, <b>C</b>, <b>E</b>, <b>G</b> and <b>I</b>. White arrowheads point to the sites of recruitment/co-localization to the MCV. Scale bars, 10 μm; The percentage of bacteria/MCVs positive for GFP-Rab11c (<b>B</b>), GFP-Rab7a (<b>D</b>), VatB-RFP (<b>F</b>), LysoSensor Green (<b>H</b>) or DQ Green BSA (<b>J</b>) at 1.5 and 7 hpi was quantified. Mean and standard deviation from 2–3 independent experiments. A minimum of 100 infected cells were counted for each cell line. Unpaired <i>t</i> test compared wt and ΔRD1 bacteria (*<i>p</i> ≤ 0.05; **<i>p</i> ≤ 0.01).</p