Abschlussbericht des Modellprojektes "Teil sein & Teil haben"

Mit dem demographischen Wandel steigt auch die Zahl der Menschen mit Komplexer Behinderung, die kaum über Verbalsprache verfügen und umfassender Unterstützung bei der täglichen Lebensgestaltung bedürfen. Zudem scheinen diese Menschen kaum von den gegenwärtigen behindertenpolitischen Trends zur Verwirklichung einer „vollen, wirksamen und gleichberechtigten Teilhabe an der Gesellschaft“ (u.a. Präambel und Art. 1 der UN-BRK) zu profitieren. Die Einrichtungen der Behindertenhilfe äußern sich zunehmend überfordert, diesem gesellschaftlichen Auftrag sowie den besonderen Bedürfnissen ihrer sich verändernden Klientel gerecht zu werden. Darüber hinaus fehlt es den Fachkräften häufig an pädagogischem Handlungswissen. Hier setzte das Forschungsprojekt „Teil ¬ sein & Tei l¬ haben®“ an, ein Modellprojekt zur Erfassung der Bedarfe von Menschen mit Komplexer Behinderung und zur Professionalisierung einer Teilhabeorientierten Pflege und Begleitung. Das Projekt wurde durch die Stiftung Wohlfahrtspflege NRW gefördert und wurde im Juni 2016 vom Verein KuBus e.V. ® auf den Weg gebracht und von Prof.'in Dr. Barbara Fornefeld geleitet. Ziel des Projektes war es, auf der Grundlage theoretischer wie empirischer Erkenntnisse, Handlungsempfehlungen für eine teilhabeorientierte Pflege und Begleitung des Personenkreises zu erarbeiten, indem „Teilhabe“ als Begriff und Konzept sowohl theoretisch als auch empirisch beleuchtet und auf die Implikationen für Menschen mit Komplexer Behinderung untersucht wird. In seiner über dreijährigen Laufzeit brachte „Teil ¬ sein & Tei l¬ haben®“ eine Vielzahl grundlagentheoretischer wie empirischer Erkenntnisse hervor, die im vorliegenden Abschlussbericht ein- und überblicksartig zusammengefasst werden

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

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

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 early autophagic response caused by <i>M</i>. <i>marinum</i> infection is dependent on ESX-1.

<p><b>A.</b> Percentage of cells containing GFP-Atg8⁺ <i>M</i>. <i>marinum</i> wt and ΔRD1 at 1.5, 7 and 24 hpi. Means and standard deviations from independent triplicates. A mean of 375 and 230 infected cells per time point was counted for <i>M</i>. <i>marinum</i> wt and ΔRD1 infection, respectively; <b>B.</b> Percentage of <i>M</i>. <i>marinum</i> wt and ΔRD1 bacteria positive for GFP-Ub. Mean and standard deviation from independent triplicates; <b>C.</b> GFP-Ub-expressing <i>atg1</i>- cells were infected with Vibrant DyeCycle Ruby-labelled <i>M</i>. <i>marinum</i> wt, ΔRD1 and ΔRD1::2F9. Means and standard deviations from independent duplicates of the percentage of bacteria positive for GFP-Ub at 7 hpi. A mean of 72–98 bacteria per infection was counted. Unpaired <i>t</i> test (**<i>p</i> ≤ 0.01); <b>D.</b> <i>D</i>. <i>discoideum</i> wt and <i>atg1</i>- cells were infected with <i>lux</i>-expressing <i>M</i>. <i>marinum</i> wt and ΔRD1. The intracellular bacteria growth (RLUs) from triplicates was monitored. Statistical differences at the end of the experiment were calculated with a Bonferroni post hoc test after two-way ANOVA (****<i>p</i> ≤ 0.0001); <b>E.</b> qPCR results of relative abundance of <i>atg8a</i>, <i>atg8b</i>, <i>atg1</i> and <i>p62</i> mRNAs at 2, 5 and 24 hpi with <i>M</i>. <i>marinum</i> wt (blue outline) and ΔRD1 (red outline). The mRNA level in non-infected cells is indicated as 1 in the figure (dashed line). The mRNA level of the housekeeping gene <i>gapdh</i> was used for normalization. Means and standard deviations from three independent experiments performed in triplicates are represented; <b>F.</b> GFP-Atg8-expressing cells were infected or mock-infected for 1.5 or 7 h with mCherry-expressing <i>M</i>. <i>marinum</i> wt or with DsRed-expressing <i>M</i>. <i>marinum</i> ΔRD1. Maximum projections were used to measure the Integrated Density (IntDen) of the cytosolic GFP-Atg8 fluorescence compared to the extracellular IntDen (background). Median with interquartile ranges of the cytosolic GFP-Atg8 IntDen per cell. Each dot represents one cell. 210–357 cells per condition from three independent experiments were counted. Mann-Whitney test (****<i>p</i> ≤ 0.0001; **<i>p</i> ≤ 0.01; ns, <i>p</i> > 0.05).</p

<i>M</i>. <i>marinum</i> induces an early autophagic response in <i>D</i>. <i>discoideum</i>.

<p><b>A.</b> Two representative maximum projections showing GFP-Atg8-expressing cells infected (bottom) or mock-infected (top) with mCherry-expressing wt <i>M</i>. <i>marinum</i>. Images were recorded live 1.5 h after infection. White arrowheads point to GFP-Atg8 structures. Scale bars, 10 μm; <b>B.</b> The median and interquartile ranges of the number of GFP-Atg8 structures per cell were calculated. For each condition, 300 cells from independent triplicates were counted. Mann-Whitney test (****<i>p</i> ≤ 0.0001); <b>C.</b> Single sections of live GFP-Atg8-expressing amoebae 1.5 h after infection with mCherry-expressing <i>M</i>. <i>marinum</i> wt. White, magenta and yellow arrowheads point to GFP-Atg8 dots, patches and GFP-Atg8-positive (GFP-Atg8⁺) MCV, respectively. Scale bars, 5 μm; <b>D.</b> At 1.5 hpi, 256 cells (100%) with GFP-Atg8⁺ <i>M</i>. <i>marinum</i> were classified as in <b>C</b>. Means and standard deviations from three independent infections are represented; <b>E.</b> EM of the different types of autophagosomes in the vicinity of the MCV at 7 hpi. White asterisks label bacteria, white arrowheads point to round phagophores and autophagosomes, magenta arrowheads point to extended autophagosomes, and the yellow arrowhead indicates a double membrane compartment containing mycobacteria. Nuclei . Scale bars, 1 μm; <b>F.</b> Sections (top) and maximum projections (bottom) showing co-localization (white arrowheads) of autophagy markers with bacteria at 1–1.5 hpi. Around 50% and 30% of the Atg8-positive bacteria were also positive for Ub and GFP-p62, respectively. Scale bars, 5 μm; <b>G.</b> qPCR results of relative abundance of <i>atg8a</i>, <i>atg8b</i>, <i>atg1</i> and <i>p62</i> mRNAs at 3 hpi. The mRNA level in non-infected cells is indicated as 1. The mRNA level of the housekeeping gene <i>gapdh</i> was used for normalization. Means and standard deviations from three independent experiments performed in triplicates are represented.</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

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