Biology subjects

Nitschke, J.

Publications and source records attributed to Nitschke, J..

5 recordsLinked to original sources

bioRxiv2026

Time-resolved transcriptomic profiling of Dictyostelium discoideum infection with Mycobacterium marinum reveals Atg9-dependent restriction via maintenance of vacuole integrity

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a major global health challenge, highlighting the need to better understand host mechanisms restricting mycobacteria infection. Mycobacterium marinum (Mm) shares key virulence mechanisms with Mtb and provides a suitable model for studying mycobacterial pathogenesis. Here, we used Dictyostelium discoideum (Dd), a genetically tractable phagocytic model with evolutionarily conserved pathways shared with mammalian macrophages, to investigate host responses to Mm infection. Time-resolved transcriptomic profiling across early, intermediate, and late stages of infection identified global infection-responsive genes, stage-specific pathways, and substantial conservation with transcriptional responses of human macrophages infected with Mtb. Autophagy and ESCRT pathways were dynamically regulated throughout infection, suggesting stage-specific roles in host defence. We focused on Atg9, an autophagy factor that was strongly induced during infection and recruited to damaged mycobacterium-containing vacuoles (MCVs). Atg9 promoted membrane damage control and maintained MCV integrity, thereby preventing premature bacteria escape to the cytosol of virulent Mm. Loss of Atg9 led to accumulation of MCV damage, accelerated escape to the cytosol, and enhanced intracellular bacteria growth, phenocopying atg1-deficient cells. Our findings further distinguish complementary host defence mechanisms acting at distinct stages of infection: Atg9-associated membrane repair, potentially involving ATG8ylation, limits vacuole damage and bacteria escape during early infection, whereas Atg9-dependent xenophagic restriction contributes to bacteria clearance at later stages. Together, these findings establish Dd as a powerful model for dissecting conserved host responses to mycobacteria and identify Atg9-dependent membrane protection as a key host resistance mechanism with potential relevance to TB pathogenesis and therapeutic intervention.

microbiology↗

bioRxiv2024

The Dictyostelium discoideum - Mycobacterium marinum infection model, a powerful high throughput screening platform for anti-infective compounds

Tuberculosis is among the worlds deadliest diseases, causing approximately 2 million deaths annually. The urgent need for new antitubercular drugs has been intensified by the rise of drug-resistant strains. Despite recent advancements, most hits identified through traditional target-based screening exhibit limited efficacy in vivo. Consequently, there is a growing demand for whole-cell-based approaches that directly utilize host-pathogen systems. The Dictyostelium discoideum-Mycobacterium marinum host-pathogen system is a well-established and powerful alternative model system to study mycobacterial infections. In this article, the phenotypic host-pathogen protocol assay is presented here which relies on monitoring M. marinum during its infection of the amoeba D. discoideum. This assay is characterized by its scalability for high-throughput screening, robustness, and ease of manipulation, making it an effective system for compound screening. This system provides not only bacterial load readout via a bioluminescent M. marinum strain, but now also host survival and growth via a fluorescent D. discoideum strain enabling further host characterization by quantifying growth inhibition and potential cytotoxicity. Finally, the system was benchmarked with selected antibiotics and anti-infectives and calculated IC50s and MICs where applicable, demonstrating its capability to differentiate between antibiotics and anti-infective compounds. ImportanceThis methods paper introduces a robust, scalable, and high-throughput phenotypic host-pathogen assay based on the well-established Dictyostelium discoideum-Mycobacterium marinum system. In contrast to conventional target-based drug screening approaches, which often struggle to translate effectively in vivo, this platform directly monitors pathogen-host interactions, providing comprehensive insights into bacterial load, host survival, and potential cytotoxicity. By employing bioluminescent M. marinum and fluorescent D. discoideum strains, we validated the system using established antibiotics and anti-infective compounds, effectively distinguishing their effects through IC50 and MIC calculations.

microbiology↗

bioRxiv2024

Discovery of anti-infective compounds against Mycobacterium marinum after biotransformation of simple natural stilbene scaffolds by a fungal secretome

This study evaluated the efficacy of a high-throughput Dictyostelium discoideum - Mycobacterium marinum Dd-Mm infection system by first benchmarking it against a set of antibiotics and second in screening a library of natural product (NP) derivatives for anti-infective activity against intracellular Mycobacterium marinum (Mm). The study observed no activity of pyrazinamide against Mm, consistent with known resistance patterns, and confirmed other antibiotics, such as rifampicin and bedaquiline, with activity below defined antibacterial susceptibility breakpoints. From screening a small library of NP derivatives, trans-{delta}-viniferins emerged as promising anti-infective scaffolds, particularly two compounds which exhibited an anti-infective activity on Mm during infection but not on Mm in broth, 17 with an IC50 of 18.1 {micro}M, and 19 with an IC50 of 9 {micro}M). Subsequent exploration via halogenation and structure-activity relationship (SAR) studies led to the identification of derivatives with improved selectivity and potency. The observed anti-infective phenotype may involve mechanisms such as blocking mycobacterial virulence factors or boosting host defense. Furthermore, the study highlights the potential of natural product-inspired derivatization approaches for drug discovery and underscores the utility of the Dd-Mm infection system in identifying novel anti-infective compounds. IMPORTANCEThis study underscores the significance of leveraging natural product-inspired approaches and innovative infection models in search for novel anti-infective compounds. By benchmarking and employing high-throughput Dictyostelium discoideum-Mycobacterium marinum infection system on a small, focused library of natural product derivatives, the study identified trans-{delta}-viniferins as promising anti-infective scaffolds against Mycobacterium marinum, opening potential therapeutic avenues for combating tuberculosis. The findings highlight the value of exploring nature-inspired chemistry for drug discovery and addressing global health challenges.

microbiology↗

bioRxiv2023

Temporal genome-wide fitness analysis of Mycobacterium marinum during infection reveals genetic requirement for virulence and survival in amoebae and microglial cells

Tuberculosis remains the most pervasive infectious disease and the recent emergence of multiple or even fully drug-resistant strains increases the risk and emphasizes the need for more efficient and better drug treatments. A key feature of mycobacteria pathogenesis is the metabolic switch during infection and expression of virulence genes is often adapted to specific infection conditions. This study aims to identify genes that are involved in the establishment and maintenance of the infection. To answer these questions, we have applied Transposon Sequencing (Tn-Seq) in M. marinum, an unbiased genome-wide strategy that combines saturation insertional mutagenesis and high throughput sequencing. This approach allowed us to precisely identify the localization and relative abundance of insertions in pools of Tn mutants. The essentiality and fitness cost, in terms of growth advantage and disadvantage of over 105 mutants were quantitatively compared between in vitro and different stages of infection in two evolutionary distinct hosts, D. discoideum and BV2 microglial cells. We found that 57% of TA sites in the M. marinum genome were disrupted and that 568 genes (10.2%) are essential for M. marinum, which is comparable to previous Tn-Seq studies on M. tuberculosis. The major pathways involved in the survival of M. marinum during infection of D. discoideum were related to vitamin metabolism, the esx-1 operon, as well as the mce1 operon.

microbiology↗

bioRxiv2021

The Dictyostelium discoideum E3 ubiquitin ligase TrafE coordinates endolysosomal damage response and cell-autonomous immunity to Mycobacterium marinum.

Cells are perpetually challenged by pathogens, protein aggregates or chemicals, that induce plasma membrane or endolysosomal compartments damage, recognised as severe stress and controlled downstream by the endosomal sorting complex required for transport (ESCRT) and the autophagy machineries that are recruited to damaged membranes to either repair or to remove membrane remnants. Yet little is known about the upstream endolysosomal damage response (ELDR) factors that sense damage and lead to extensive tagging of the damaged organelles with signals, such as K63-polyubiquitin, required for the recruitment of ELDR components. To explore ELDR key factors responsible for detection and marking of damaged compartments we use the professional phagocyte Dictyostelium discoideum. We found an evolutionary conserved E3-ligase, TrafE, that is robustly recruited to intracellular compartments disrupted after infection with Mycobacterium marinum or after sterile damage caused by chemical compounds. TrafE acts at the intersection of ESCRT and autophagy pathways and plays a key role in functional recruitment of the ESCRT subunits ALIX, Vps32 and Vps4 to damage sites or maturing autophagosomes. Importantly, we show that the absence of TrafE severely compromises the xenophagy restriction of bacteria as well as ESCRT-mediated and autophagy-mediated ELDR, resulting in early cell death.

microbiology↗