Biology subjects

Hanna, N.

Publications and source records attributed to Hanna, N..

17 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↗

bioRxiv2026

STAT transcription factors regulate host defences and vacuolar integrity during Mycobacterium marinum infection

Signal transducers and activators of transcription (STAT) are central regulators of cytokine-mediated immunity in metazoans, yet their ancestral functions prior to the emergence of interferon signalling remain poorly understood. Dictyostelium discoideum expresses four STAT-like factors that impact the intracellular fate of Mycobacterium marinum (Mm). Using dst knockout strains, we identify DstA and DstB as susceptibility factors, whereas DstC acts as a resistance factor, revealing a regulatory axis controlling the integrity of the Mm-containing vacuole (MCV). Despite the absence of canonical cytokine receptors and JAK kinases, DstA rapidly translocates to the nucleus in a damage-dependent manner, whereas DstB and DstC exhibit delayed cytosolic redistribution. DstA associates with VacA, a membrane microdomain component, regulates its transcription and accumulation at the MCV, while VacA acts as a cytoplasmic anchor that limits DstA nuclear translocation. Our findings establish STAT-like proteins as evolutionarily ancient regulators of vacuole integrity and host defence, providing new insights into the origins of STAT-mediated immune responses during mycobacterial infection.

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

Membrane microdomains are crucial for Mycobacterium marinum EsxA-dependent membrane damage, escape to the cytosol and infection

Infection by pathogenic mycobacteria such as Mycobacterium tuberculosis disrupts the membrane of the Mycobacterium-Containing Vacuole (MCV). The key effector EsxA, secreted via the ESX-1 type-VII system, is pivotal in this process, yet its membranolytic activity is not fully elucidated. Infecting the amoeba Dictyostelium discoideum with Mycobacterium marinum, we demonstrate that the composition of the MCV membrane, notably its sterol-rich microdomains, significantly influences damage and rupture. Disruption of these microdomains through the knockout of organizing proteins, termed vacuolins, or through sterol depletion, markedly diminishes M. marinum-induced membrane damage and cytosolic escape, thereby increasing cellular resistance to infection. Furthermore, we establish that vacuolins and sterols are essential for the in vitro partitioning of EsxA within membranes. Extending our findings to mammalian cells, we show that the role of microdomain organizers and sterols is evolutionarily conserved; specifically, flotillin knockdown and sterol depletion enhance the resistance of murine microglial cells to M. marinum infection. Our results underscore the critical role of host membrane microdomains in facilitating mycobacterial membranolytic activity and subsequent cytosolic access, pivotal for a successful infection.

cell biology↗

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↗

bioRxiv2024

ADRA2A promotes the classical/progenitor subtype and reduces disease aggressiveness of pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) manifests diverse molecular subtypes, including the classical/progenitor and basal-like/squamous subtypes, with the latter known for its aggressiveness. We employed integrative transcriptome and metabolome analyses to identify potential genes contributing to the molecular subtype differentiation and its metabolic features. Transcriptome analysis in PDAC patient cohorts revealed downregulation of adrenoceptor alpha 2A (ADRA2A) in the basal-like/squamous subtype, suggesting its potential role as a candidate suppressor of this subtype. Reduced ADRA2A expression was significantly associated with a high frequency of lymph node metastasis, higher pathological grade, advanced disease stage, and decreased survival among PDAC patients. In vitro experiments demonstrated that ADRA2A transgene expression and ADRA2A agonist inhibited PDAC cell invasion. Additionally, ADRA2A-high condition downregulated the basal-like/squamous gene expression signature, while upregulating the classical/progenitor gene expression signature in our PDAC patient cohort and PDAC cell lines. Metabolome analysis conducted on the PDAC cohort and cell lines revealed that elevated ADRA2A levels were associated with suppressed amino acid and carnitine/acylcarnitine metabolism, which are characteristic metabolic profiles of the classical/progenitor subtype. Collectively, our findings suggest that heightened ADRA2A expression induces transcriptome and metabolome characteristics indicative of classical/progenitor subtype with decreased disease aggressiveness in PDAC patients. These observations introduce ADRA2A as a candidate for diagnostic and therapeutic targeting in PDAC. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=186 SRC="FIGDIR/small/584316v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@edc5ceorg.highwire.dtl.DTLVardef@5579b6org.highwire.dtl.DTLVardef@1a9a000org.highwire.dtl.DTLVardef@147724a_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG HighlightsO_LIADRA2A is downregulated in the basal-like/squamous PDAC while its expression is maintained in the classical/progenitor PDAC subtype C_LIO_LIUpregulated ADRA2A expression correlates with improved PDAC survival and reduced invasion in PDAC cells C_LIO_LIUpregulated ADRA2A downregulates the MYC signaling pathway and promotes the classical/progenitor gene expression profile C_LIO_LIUpregulated ADRA2A induces a unique metabolic signature characterized by diminished amino acid and carnitine/acylcarnitine metabolism, resembling the classical/progenitor PDAC subtype C_LI

cancer biology↗

bioRxiv2024

Caspase-8 activity mediates TNFα production and restricts Coxiella burnetii replication during murine macrophage infection

Coxiella burnetii is an obligate intracellular bacteria which causes the global zoonotic disease Q Fever. Treatment options for infection are limited, and development of novel therapeutic strategies requires a greater understanding of how C. burnetii interacts with immune signaling. Cell death responses are known to be manipulated by C. burnetii, but the role of caspase-8, a central regulator of multiple cell death pathways, has not been investigated. In this research, we studied bacterial manipulation of caspase-8 signaling and the significance of caspase-8 to C. burnetii infection, examining bacterial replication, cell death induction, and cytokine signaling. We measured caspase, RIPK, and MLKL activation in C. burnetii-infected TNF/CHX-treated THP-1 macrophage-like cells and TNF/ZVAD-treated L929 cells to assess apoptosis and necroptosis signaling. Additionally, we measured C. burnetii replication, cell death, and TNF induction over 12 days in RIPK1-kinase-dead, RIPK3-kinase-dead, or RIPK3-kinase-dead-caspase-8-/- BMDMs to understand the significance of caspase-8 and RIPK1/3 during infection. We found that caspase-8 is inhibited by C. burnetii, coinciding with inhibition of apoptosis and increased susceptibility to necroptosis. Furthermore, C. burnetii replication was increased in BMDMs lacking caspase-8, but not in those lacking RIPK1/3 kinase activity, corresponding with decreased TNF production and reduced cell death. As TNF is associated with the control of C. burnetii, this lack of a TNF response may allow for the unchecked bacterial growth we saw in caspase-8-/- BMDMs. This research identifies and explores caspase-8 as a key regulator of C. burnetii infection, opening novel therapeutic doors.

microbiology↗

bioRxiv2023

LMO3 is a suppressor of the basal-like/squamous PDAC subtype and reduces disease aggressiveness of pancreatic cancer through glycerol 3-phosphate metabolism

Pancreatic ductal adenocarcinoma (PDAC) encompasses diverse molecular subtypes, including the classical/progenitor and basal-like/squamous subtypes, each exhibiting distinct characteristics, with the latter known for its aggressiveness. We employed an integrative approach combining transcriptomic and metabolomic analyses to pinpoint potential genes contributing to the basal-like/squamous subtype differentiation. Applying this approach to our NCI-UMD-German and a validation cohort, we identified LIM Domain Only 3 (LMO3), a transcription co-factor, as a candidate suppressor of the basal-like/squamous subtype. Reduced LMO3 expression was significantly associated with higher pathological grade, advanced disease stage, induction of the basal-like/squamous subtype, and decreased survival among PDAC patients. In vitro experiments demonstrated that LMO3 transgene expression inhibited PDAC cell proliferation and migration/invasion, concurrently downregulating the basal-like/squamous gene signature. Metabolomic analysis of patient tumors and PDAC cells revealed a metabolic program linked to elevated LMO3 expression and the classical/progenitor subtype, characterized by enhanced lipogenesis and suppressed amino acid metabolism. Notably, glycerol 3-phosphate (G3P) levels positively correlated with LMO3 expression and associated with improved patient survival. Furthermore, glycerol-3-phosphate dehydrogenase 1 (GPD1), a crucial enzyme in G3P synthesis, showed upregulation in LMO3-high and classical/progenitor PDAC, suggesting its potential role in mitigating disease aggressiveness. Collectively, our findings suggest that heightened LMO3 expression reduces transcriptomic and metabolomic characteristics indicative of basal-like/squamous tumors with decreased disease aggressiveness in PDAC patients. The observations describe LMO3 as a candidate for diagnostic and therapeutic targeting in PDAC. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/564448v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@15ab2f1org.highwire.dtl.DTLVardef@199f209org.highwire.dtl.DTLVardef@1e1661corg.highwire.dtl.DTLVardef@f6eb7e_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LILMO3 is downregulated in basal-like/squamous PDAC while its expression is maintained in the classical/progenitor PDAC subtype C_LIO_LIUpregulated LMO3 expression correlates with improved PDAC survival and reduced proliferation and migration/invasion in PDAC cells C_LIO_LIUpregulated LMO3 suppresses basal-like/squamous differentiation and induces a unique metabolic signature characterized by elevated lipogenesis and diminished amino acid metabolism, resembling the classical/progenitor PDAC subtype C_LIO_LIEnhanced LMO3 expression associates with elevated glycerol 3-phosphate levels in PDAC, correlating with improved patient survival in PDAC C_LI

cancer biology↗

bioRxiv2023

ELAPOR1 induces the classical/progenitor subtype and contributes to reduced disease aggressiveness through metabolic reprogramming in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is a heterogeneous disease with distinct molecular subtypes classified as classical/progenitor and basal-like/squamous. We hypothesized that integrative transcriptomic and metabolomic approaches can identify candidate genes whose inactivation contributes to the development of the aggressive basal-like/squamous subtype. Using our integrated approach, we identified endosome-lysosome associated apoptosis and autophagy regulator 1 (ELAPOR1/KIAA1324) as a candidate tumor suppressor in both our NCI-UMD-German cohort and validation cohorts. We found that decreased ELAPOR1 expression was significantly associated with high pathological grade, advanced disease stage, the basal-like/squamous subtype, and decreased survival in PDAC patients. In vitro experiments showed that ELAPOR1 transgene expression inhibited migration and invasion of PDAC cells. Metabolomic analysis of patient tumors and PDAC cells revealed a metabolic program associated with both upregulated ELAPOR1 and the classical/progenitor subtype, encompassing upregulated lipogenesis and downregulated amino acid metabolism. 1-methylnicotinamide, an oncometabolite derived from S-adenosylmethionine, was inversely associated with ELAPOR1 expression and promoted migration and invasion of PDAC cells in vitro. Taken together, our data suggest that enhanced ELAPOR1 expression promotes transcriptomic and metabolomic characteristics that are indicative of the classical/progenitor subtype, whereas its reduction associates with basal-like/squamous tumors with increased disease aggressiveness in PDAC patients. This positions ELAPOR1 as a promising candidate for diagnostic and therapeutic targeting in PDAC. Novelty and ImpactPancreatic ductal adenocarcinoma (PDAC) exhibits heterogeneous molecular subtypes: classical/progenitor and basal-like/squamous. Comprehensive transcriptome and metabolome analyses in the PDAC patient cohorts and PDAC cell lines revealed that elevated ELAPOR1 correlates with enhanced survival, reduced PDAC cell invasion, and a distinct metabolic signature resembling the classical/progenitor subtype. Additionally, 1-methylnicotinamide has been identified as an oncometabolite, showing an inverse correlation with ELAPOR1. These findings emphasize ELAPOR1s potential as a diagnostic and therapeutic target in PDAC. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/558894v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@f95c81org.highwire.dtl.DTLVardef@128334corg.highwire.dtl.DTLVardef@3fcb60org.highwire.dtl.DTLVardef@414011_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIELAPOR1 is downregulated in basal-like/squamous PDAC C_LIO_LIUpregulation of ELAPOR1 associates with improved PDAC survival and reduced migration and invasion in PDAC cells C_LIO_LIELAPOR1 expression induces a distinct metabolic signature as characterized by upregulation of lipogenesis and downregulation of amino acid metabolism, commonly observed in the classical/progenitor PDAC subtype C_LIO_LIThe oncometabolite, 1-methylnicotinamide (MNA) is decreased when ELAPOR1 is upregulated, and promotes the migration and invasion of PDAC cells C_LI

cancer biology↗

bioRxiv2023

SERPINB3 induces the basal-like/squamous subtype and enhances disease progression in pancreatic cancer

Pancreatic cancer is a heterogeneous disease with distinct subtypes. Here, we investigated candidate driver genes of the highly aggressive basal-like/squamous molecular subtype of pancreatic ductal adenocarcinoma (PDAC). Integrative transcriptomic analyses identified the upregulated serine/cysteine protease inhibitor, SERPINB3 (squamous cell carcinoma antigen 1, SCCA1) in basal-like/squamous PDAC using discovery and validation approaches. Upregulation of SERPINB3 associated with decreased patient survival and a transcriptome profile indicative of the basal-like/squamous subtype. In human PDAC cell lines, SERPINB3 transgene expression enhanced their invasion capability. Moreover, upregulated expression of SERPINB3 in AsPC-1 cells resulted in enhanced lung metastasis in an orthotopic xenograft model. Molecular analysis of the primary tumor xenografts indicated activation of pathways related to metastasis, increased oxidative damage, and angiogenesis when SERPINB3 was upregulated. Furthermore, metabolomic analysis, using patient cohorts and PDAC cell lines showed a distinct metabolic pattern closely associated with both SERPINB3 and the basal-like/squamous subtype, which included upregulation of carnitine/acylcarnitine, amino acid, glutathione, and purine metabolic pathways, and glycolysis. Further RNA-seq and metabolomic analyses indicated that SERPINB3 may potentially induce the basal-like/squamous subtype and metabolic reprogramming through MYC activation. Taken together, our findings identified SERPINB3 as a candidate marker gene for the basal-like/squamous subtype, which may contribute to the disease aggressiveness in this subtype of PDAC. Abbreviations8-Hydroxy-2-deoxyguanosine (8-OHdG), Diaminobenzene (DAB), Gene Set Enrichment Analysis (GSEA), Human Metabolome Technologies, Inc. (HMT), Immunohistochemistry (IHC), Ingenuity pathway analysis (IPA), Pancreatic ductal adenocarcinoma (PDAC), Serine/Cysteine Proteinase Inhibitor Family B Member 3 (SERPINB3) Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/534766v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@8209f5org.highwire.dtl.DTLVardef@15be96dorg.highwire.dtl.DTLVardef@13a0c6corg.highwire.dtl.DTLVardef@58b2f_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LISERPINB3 is upregulated in basal-like/squamous PDAC and associates with decreased patient survival C_LIO_LISERPINB3 promotes differentiation into the basal-like/squamous subtype and enhances invasion and metastasis of PDAC C_LIO_LISERPINB3 induces metabolic reprogramming and MYC activation and a metabolic signature indicative of basal-like/squamous PDAC C_LI

cancer biology↗

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↗

bioRxiv2022

Recognition of non-self is necessary to activate Drosophila's immune response against an insect parasite

Innate immune responses can be activated by pathogen-associated molecular patterns (PAMPs) or danger signals released by damaged tissues. As PAMPs are typically conserved across broad groups of pathogens but absent from the host, it is unclear whether they allow hosts to recognize parasites that are phylogenetically related to themselves, such as parasitoid wasps infecting insects. Parasitoids must penetrate the cuticle of Drosophila larvae to inject their eggs. In line with previous results, we find that the danger signal of wounding triggers the differentiation of specialized immune cells called lamellocytes. However, using oil droplets to mimic infection by a parasitoid wasp egg, we find that the activation of melanization response that kills parasitoids also requires exposure to a parasitoid wasp molecule that acts as a PAMP. The unidentified factor enhances the transcriptional response in hemocytes and induces a specific response in the fat body that includes Tep1, which is essential for efficient melanization. We conclude that a combination of danger signals and PAMPs are required activate Drosophilas immune response against parasitic insects.

immunology↗

bioRxiv2021

Disruption of vacuolin microdomains in the host Dictyostelium discoideum increases resistance to Mycobacterium marinum-induced membrane damage and infection

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, manipulates the host phagosome maturation pathway to replicate intracellularly. Mycobacterium marinum, a closely-related species, and Dictyostelium discoideum, a social amoeba and alternative phagocytic host, have been used as models to study host-pathogen interactions occurring during mycobacterial infections. Vacuolins, functional homologues of the mammalian flotillins, organize membrane microdomains and play a role in vesicular trafficking. Various pathogens have been reported to manipulate their membrane association and function. During infection of D. discoideum with M. marinum, Vacuolin C was specifically and highly induced and all three vacuolin isoforms were enriched at the mycobacteria-containing-vacuole (MCV). In addition, absence of vacuolins reduced escape from the MCV and conferred resistance to M. marinum infection. Moreover, ESAT-6, the membrane-disrupting virulence factor of M. marinum, was less associated with membranes when vacuolins were absent. Together, these results suggest that vacuolins are important host factors that are manipulated by mycobacteria to inflict membrane damage and escape from their compartment.

microbiology↗

bioRxiv2019

Time-resolved RNA-seq profiling of the infection of Dictyostelium discoideum by Mycobacterium marinum reveals an integrated host response to damage and stress

Tuberculosis remains the most pervasive infectious disease and the recent emergence of multiple drug-resistant strains emphasizes the need for more efficient drug treatments. The experimentally versatile Dictyostelium discoideum - Mycobacterium marinum infection model provides a powerful system to study mycobacteria pathogenicity and host response. In this study, a time-resolved transcriptomic analysis of the amoeba D. discoideum was performed to decipher the different host pathways impacted during infection. We investigated how D. discoideum fine-tunes its gene expression in response to M. marinum infection by assessing the transcriptomic profile covering the critical stages of entry, establishment of a permissive niche, proliferation and dissemination (1, 3, 6, 12, 24 and 48 hours post infection). Differential gene expression provided a fingerprint of the transcriptome of the host cell in the presence of mycobacteria, and helped identify specific markers and molecular signatures of infection. Enrichment pathway analysis showed that most of the Biological Processes (BP) of upregulated genes at early time point of infection hinted towards damage response and cellular defence, especially in specific pathways involved in membrane repair (ESCRT) and bacteria elimination (autophagy). Whereas at late time points of infection, BP related to starvation were upregulated. Some other signatures were more unexpected, such as cell cycle (downregulation of cytosolic large & small ribosomal subunits) and upregulation of metabolic adaptations (lipids transport).

systems biology↗

bioRxiv2019

Dictyostelium discoideum flotillin homologues are essential for phagocytosis and participate in plasma membrane recycling and lysosome biogenesis

The metazoan flotillins are lipid rafts residents involved in membrane trafficking and recycling of plasma membrane proteins. Dictyostelium discoideum, a social soil amoeba, uses phagocytosis to digest, kill and feed on bacteria. D. discoideum possesses three flotillin-like proteins, termed VacA, VacB and the recently identified VacC. All three vacuolins gradually accumulate on postlysosomes and, like flotillins, are strongly associated with membranes and partly with lipid rafts. Vacuolins are absolutely required for uptake of various particles. Their absence impairs particle recognition possibly because of defective recycling of plasma membrane or cortex-associated proteins. In addition, vacuolins are involved in phagolysosome biogenesis, although this does not impact digestion and killing of a wide range of bacteria. Furthermore, vacuolin knockout affects early recruitment of the WASH complex on phagosomes, suggesting that vacuolins may be involved in the WASH-dependent plasma membrane recycling. Altogether, these results indicate that vacuolins act as the functional homologues of flotillins in D. discoideum.

cell biology↗

bioRxiv2019

Zinc intoxication of Mycobacterium marinum during Dictyostelium discoideum infection is counteracted by induction of the pathogen Zn2+ exporter CtpC

Macrophages use diverse strategies to kill or restrict intracellular pathogens. Some of these strategies involve the deprivation of bacteria from (micro)nutrients such as transition metals, and the bacteria intoxication through metal accumulation. Little is known about the chemical warfare between Mycobacterium marinum, a close relative of the human pathogen M. tuberculosis, and its hosts. Here we use the professional phagocyte Dictyostelium discoideum to investigate the role of Zn2+ during M. marinum infection. We show that M. marinum infection induces the accumulation of Zn2+ inside the Mycobacterium-containing vacuole (MCV), achieved by the induction and recruitment of the D. discoideum Zn2+ efflux pumps ZntA and ZntB. In cells lacking the ZntA detoxifying transporter there is further attenuation of M. marinum growth, possibly due to a compensatory efflux of Zn2+ into the MCV. This efflux is presumably carried out by ZntB, the main Zn2+ transporter in endosomes and phagosomes. Counterintuitively, M. marinum growth is also impaired in zntB KO cells, where MCVs accumulate less Zn2+. We also demonstrate that M. marinum senses toxic levels of Zn2+ and responds by upregulating its Zn2+ exporter CtpC, which supports bacteria survival under these restrictive conditions. Attenuation of M. marinum intracellular proliferation in zntA and zntB KO cells is accentuated in the absence of CtpC, confirming that mycobacteria face noxious levels of Zn2+. Altogether, we show for the first time that M. marinum infection induces a deleterious Zn2+ elevation in D. discoideum, which is counteracted by the bacteria with the induction of its Zn2+ exporter CtpC.

cell biology↗

bioRxiv2018

Identification of novel anti-bacterial compounds from a chemically highly diverse pathways-based library using phenotypic screens in amoebae host models

Legionella pneumophila and tubercular Mycobacteria are the causative agents of potentially fatal diseases due to their pathogenesis but also to the emergence of antibiotic resistance that limits treatment strategies. The aim of our study is to explore the antimicrobial activity of a small ligand-based chemical library of 1,255 structurally diverse compounds. These compounds were screened in a combination of three assays, two monitoring the intracellular growth of the pathogenic bacteria, Mycobacterium marinum and L. pneumophila, and an additional anti-virulence \"plaque\" assay for M. marinum. We set up these assays using two amoeba strains, the genetically tractable Dictyostelium discoideum and the free-living amoeba Acanthamoeba castellanii. In summary, sixty-four compounds showed anti-infective/anti-virulence activity in at least one of the 3 assays. The intracellular assays hit rate varied between 1.7% (n=22) for M. marinum and 2.8% (n=35) for L pneumophila with 7 compounds in common between both pathogens. In parallel, 1.2 % (n= 15) of the tested compounds were able to restore D. discoideum growth in presence of M. marinum spiked in a lawn of Klebsiella pneumoniae. We also validated the generality of the hit compounds identified using the A. castellanii-M. marinum anti-infective screen in the powerful D. discoideum-M. marinum host-pathogen model. The characterization of anti-infective and antibacterial hits in the latter infection model revealed compounds able to reduce intracellular growth more than 50% at 30 M. Our studies underline the relevance of using a combination of low-cost and low-complexity assays with full 3R compliance associated with a rationalized focused library of compounds to help identifying new chemical scaffolds and dissect some of their properties prior to run further compounds development steps.

microbiology↗