Biology subjects

Gutierrez, M.

Publications and source records attributed to Gutierrez, M..

At least 19 recordsLinked to original sources

bioRxiv2026

A multimodal precision oncology platform integrating genomics,single-cell transcriptomics, and functional drug screening expandstherapeutic options in pediatric brain tumors

Precision oncology for pediatric brain tumors mainly relies on clinically validated, targetable genomic alterations, which are often sparse, yield few matched therapies, and do not guide chemotherapy selection. We developed a tiered framework in which deep whole-genome sequencing and single-cell RNA sequencing extend targeted-therapy selection, with ex vivo functional drug screening supporting selection of both targeted therapies and chemotherapies. We applied it to 17 children with malignant CNS tumors. The approach expanded recommendations beyond genomics: functional screening provided every patient with at least one high-confidence targeted-therapy or chemotherapy nomination, while single-cell RNA sequencing identified an average of 37 druggable gene targets per patient, producing a fourfold increase in high-confidence targeted-therapy recommendations. In an exploratory analysis, functional classifications were directionally consistent with outcomes in 8 of 10 evaluable cases, although combination treatments and radiation precluded causal attribution. Single-cell profiling also identified subclone-specific vulnerabilities, supporting prospective evaluation of the platform's feasibility and predictive utility.

cancer biology↗

bioRxiv2026

A generalizable normalization framework to decouple protocol and instrument effects: Application to high-sensitivity proteomics multicentric study (PME13)

Multicenter studies are essential for benchmarking analytical workflows, yet their interpretation is often confounded by the combined effects of experimental protocols and instrumentation. To address this challenge, we introduce a simple normalization-based analytical framework, the recovery metric ({rho}), designed to decouple protocol driven effects from instrument dependent variability. We applied this framework to the 13th Proteomics Multicentric Experiment (PME13), a large multicentric proteomics dataset generated across 27 laboratories using high sensitivity workflows and varying sample preparation protocols. By leveraging a common digested reference sample, {rho} enables direct cross-comparison of all datasets on a unified scale, effectively minimizing instrument-related biases. Using this approach, we demonstrate that apparent instrument dependent trends are largely removed when evaluated through {rho}, revealing consistent protocol driven effects across laboratories. Statistical modeling identified key variables influencing {rho}, including sample input amount, reduction and alkylation, and the use of n-dodecyl-{beta}-D-maltoside (DDM). While DDM was associated with improved {rho}, reduction and alkylation and additional handling steps led to reduced performance, particularly at low input levels. We further highlight practical considerations for the application of ratio based normalization, including the occurrence of values exceeding theoretical bounds, which reflect deviations from underlying assumptions and require appropriate filtering. Overall, this work establishes a generalizable analytical strategy for disentangling confounding factors in multicentric datasets and provides practical guidelines for optimizing high sensitivity proteomics (HSP) workflows. The proposed framework is broadly applicable to other analytical fields where cross laboratory comparability is required.

bioinformatics↗

bioRxiv2026

Population analysis and host-disease associations of Shiga toxin-producing Escherichia coli from various sources across eleven European countries using whole genome sequencing

Shiga toxin-producing Escherichia coli (STEC) are important foodborne pathogens, able to cause severe disease in humans. In the DiSCoVeR project (https://onehealthejp.eu/jrp-discover/) a STEC inventory from human and non-human sources from 11 European countries was set up and [≥] 3500 strains were sequenced to perform comparative genomics analysis. We used this dataset to assess STEC population structure and to investigate potential associations between genomic features, host reservoirs and symptoms. Most STEC isolates analysed by Whole Genome Sequencing (WGS) in this study were collected between years 2010-2020. An ad hoc pipeline was deployed for a harmonised characterization of the STEC in the database, allowing the determination of serotyping, stx gene subtyping, 7-loci MLST, virulotyping and cgMLST. The results were analysed with Principal Component Analysis (PCoA) in relation with isolation source to assess clustering of STEC subpopulations. When human STEC data were analysed, the PCoA revealed three distinct human STEC subpopulations (STEC_1, STEC_2 and STEC_3), which were further analysed for associations between genomic features, symptoms and variance. The non-human STEC showed a more dispersed distribution, except for one subpopulation with genes linked to specific host species, and some virulence profiles overlapping with the STEC_1 population. In conclusion, our analysis identified distinct STEC subpopulations from human cases, each characterized by specific genetic features and associated with varying proportions of severe disease outcomes. These findings provide novel insights supporting the risk assessment of STEC. Impact statement[This lay summary of your article should be no more than 200 words, and should a) provide a perspective of how this article adds to the literature in the field; b) identify breadth of interest/utility; and c) state the significance of output (incremental or step), in terms of relevance.] This study is based on the establishment of a One Health STEC genomes database, including sequences from isolates of different sources. Most of the isolates had been isolated in the ten-years time span 2010-2020, in 11 different countries, for surveillance and monitoring activities or specific surveys and research purposes. The final dataset included the whole genome sequencing of 3,418 STEC isolates, mainly from human cases of infections. The metadata included the host symptoms, where available, for human STEC strains and the animal source the strains had been isolated from. We set up a pipeline for the harmonized analysis of STEC WGS, called Discover, made available though ARIES webserver or GitHub. The analysis allowed a deep characterization of STEC strains circulating in Europe. We used this resource to assess STEC population structure and to investigate potential associations between genomic features, host reservoirs, and various symptoms associated with STEC infection by PCoA. This analysis highlighted the presence of subpopulation of human STEC associated with specific features. We provide new information useful for risk characterization, as well as a large dataset genome database and associated metadata compiled from STEC strains, representing a valuable resource for the scientific community, enabling further investigations into STEC diversity, evolution, source attribution and public health relevance. Data summaryThe authors confirm all supporting data, including sequence data accession numbers, code and protocols have been provided within the article or through supplementary data files. One supplementary method and five supplementary tables are available with the online version of this article

genomics↗

bioRxiv2026

Loss of catalytic activity and impaired proteostasis in guanosine nucleotide-depleted LRRK2

Coding mutations in the Leucine-rich repeat kinase 2 (LRRK2) gene represent the most common cause of familial Parkinsons disease (PD), and are frequently observed in idiopathic PD. In addition, variation around the LRRK2 locus has been shown to alter PD risk by genome-wide association studies. Disease-causing mutations cluster within the catalytic core of LRRK2 - composed of GTPase (ROC) and serine-threonine kinase domains - and lead to an increase in kinase activity, resulting in hyperphosphorylation of a subset of RAB GTPases and consequent cellular toxicity. However, the interplay between LRRK2 GTPase and kinase domains, and with the surrounding scaffold regions has remained underexplored, with implications for the prediction of on- and off-target effects associated with kinase inhibition. To address this gap, here we dissected the contributions of kinase, GTPase and scaffold domains to LRRK2 function in murine macrophages and tissues expressing endogenous levels of GTP/GDP-binding deficient Lrrk2 T1348N. Guanosine nucleotide-free Lrrk2 is devoid of both GTPase and kinase activities but maintains the scaffold shell, leading to significant reshaping of Lrrk2 interactome and engagement in novel interactions. This altered functional state leads to impaired autophagy and accumulation of enlarged lysosomes and autophagic cargo in macrophages and kidneys. Since pharmacological inhibition of LRRK2 is under clinical evaluation, our results reveal retained scaffold functions upon loss of catalytic activity that warrant careful consideration.

cell biology↗

bioRxiv2025

IGF2BP3 remodels the microRNA targeting landscape in MLL-AF4 leukemia

Insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3/I3) is a multi-domain RNA-binding protein required for MLL-AF4-driven leukemogenesis, but its mechanism of action remains enigmatic. We hypothesized from our previous work that I3 amplifies oncogenic gene expression by modulating RNA induced silencing complex (RISC) mRNA interactions. To test this, we performed miR-eCLIP of AGO2, the catalytic RISC subunit, in I3 knock-out (I3KO) as well as control B-cell acute lymphoblastic cell lines (B-ALL) and identified I3-dependent AGO2 binding sites on 111 3'UTRs. Analyzing chimeric miRNA-mRNA reads, we observed differential miRNA occupancy in the I3KO compared to control, including increased targeting by miR-181a, a regulator of leukocyte differentiation. Notably, miR-181a overexpression phenocopied I3 loss, implicating I3 in restricting miR-181a-mediated repression. Biochemical assays confirmed direct competition between I3 and AGO2-miRNA complexes for 3'-UTR binding. Taken together, our results provide a model for how I3 promotes leukemogenesis by antagonizing RISC-mediated repression of oncogenic mRNAs.

cancer biology↗

bioRxiv2025

Supervised and unsupervised machine learning methods for modelling current and future habitat of Peruvian anchovy

Understanding the drivers and potential impacts of environmental variability on the distribution of Peruvian anchovies, the largest single-species fishery on the planet, is essential for their proper management in a changing world. However, the intricate interactions of these organisms and environmental variability require the use of complex models such as machine learning methods. In this study, we compared three methods for producing habitat maps of anchovies: the traditional Generalised Additive Models, the XGBoost which is a form of supervised machine learning and a new method based on clustering water types as a form of unsupervised machine learning. We optimised the three methods with a parameter grid search algorithm and compared their capability to replicate the mean state of anchovies by comparing them with presence-absence observations along the Peruvian coastline between 1990 and 2010. We used the output of a physical-biogeochemical model as input for the habitat models to produce distribution maps of anchovy. All models successfully simulated the distribution of anchovies along the Peruvian coastline in normal years and a reduced area of distribution with most of the anchovies in the southern part of the domain during the canonical El Nino 97/98. We then applied the models to predict potential changes in the distribution of anchovies under projected temperature and wind conditions by the end of the century. We observed a reduction in the probability of anchovy occurrence under conditions of higher temperature and weaker winds. Two of the three habitat models predicted a severe maximum decline by 90% (GAM) and 75% (XGBoost) whereby the clustering model predicted a moderate maximum decline in anchovy occurrence by 20%.

ecology↗

bioRxiv2024

Metabolic regulation of RNA methylation by the m6A-reader IGF2BP3

The interplay of RNA modifications - deposited by "writers", removed by "erasers" and identified by RNA binding proteins known as "readers" - forms the basis of the epitranscriptomic gene regulation hypothesis. Recent studies have identified the oncofetal RNA-binding protein IGF2BP3 as a "reader" of the N6-methyladenosine (m6A) modification and crucial for regulating gene expression. Yet, how its function as a reader overlaps with its critical oncogenic function in leukemia remains an open question. Here, we report the novel finding that the reader IGF2BP3 reprograms cellular metabolism, resulting in an altered ability of the "writers" to modify the epitranscriptome. In leukemia cells, IGF2BP3 supports increased glycolytic flux and one-carbon metabolism, leading to increased production of S-adenosyl methionine (SAM), a key substrate for methylation reactions within the cell. IGF2BP3 directly regulates the translation of MAT2B, the regulatory subunit of the methionine-adenosyltransferase complex, which is the final enzyme in a pathway leading to SAM production. This, in turn, results in increased m6A modifications on RNA, resulting in positive feedback regulation. This novel mechanism illustrates how metabolism mutually acts with epitranscriptomic modifications, underscoring the pervasive impact of IGF2BP3 in gene regulatory mechanisms governing a broad range of cancer-specific processes.

cancer biology↗

bioRxiv2024

ATG9A and ARFIP2 cooperate to regulate PI4P levels for lysosomal repair

Lysosome damage activates multiple pathways to prevent lysosome-dependent cell death, including a repair mechanism involving ER-lysosome membrane contact sites, phosphatidylinositol 4-kinase- 2a (PI4K2A), phosphatidylinositol-4 phosphate (PI4P) and oxysterol-binding protein-related proteins (ORPs), lipid transfer proteins. PI4K2A localizes to trans-Golgi network and endosomes yet how it is delivered to damaged lysosomes remains unknown. During acute sterile damage, and damage caused by intracellular bacteria, we show that ATG9A-containing vesicles perform a critical role in delivering PI4K2A to damaged lysosomes. ADP ribosylation factor interacting protein 2 (ARFIP2), a component of ATG9A vesicles, binds and sequesters PI4P on lysosomes, balancing ORP- dependent lipid transfer and promoting retrieval of ATG9A vesicles through recruitment of the adaptor protein complex-3 (AP-3). Our results reveal a role for mobilized ATG9A vesicles and ARFIP2 in lysosome homeostasis after damage and bacterial infection.

cell biology↗

bioRxiv2024

Mycobacterium tuberculosis ESX-T7SS impacts the 3D architecture of tuberculous lesion in susceptible mice

Tuberculosis (TB) is characterized by the formation of heterogenous, immune-rich granulomas present in various forms in the lungs. Both host and pathogen contribute to this heterogeneity however the molecular and cellular drivers of within-host granuloma heterogeneity remain to be fully elucidated. This knowledge gap is due to a lack of experimental approaches that can fully capture the complex dynamics of the lung architecture, dynamics of host-pathogen interplay and pathogenesis. Here, we developed an approach that combines PACT-based clearing with light sheet fluorescent microscopy for visualizing lesion architecture development and lung involvement in M. tuberculosis-infected C3HeB/FeJ susceptible mice. This 3D modelling of whole lung lobes approach revealed critical architectural features in lesion development and lung involvement that were not apparent using traditional thin section imaging. Wild type M. tuberculosis infection triggered a clear and well-organized granulomatous-like organization with substantial growth throughout the infection period that invaded a high percentage of the total lung volume. In contrast, infection with the avirulent ESX-1 deletion mutant strain Mtb {Delta}RD1 showed an altered growth pattern with diffuse and sparsely organized CD11b recruitment to sites of infection. Moreover, most of the Mtb {Delta}RD1-triggered lesions were present in the periphery of the lungs and did not display any formal organization. We further provide a novel way of interrogating PACT-cleared tissue for high ultrastructural content using volumetric correlative light and electron microscopy, allowing individual immune cell populations to be tracked and their fate within the granuloma captured. Ultimately, the combination of both modalities allowed an unprecedented view of the architectural distribution of M. tuberculosis in the lungs and the progression of lesion development over time. Our data highlight that ESX-1 from M. tuberculosis is required for lesion architecture progression in a susceptible mouse model of TB.

microbiology↗

bioRxiv2023

Congenital heart defects in Down syndrome are caused by increased dosage of DYRK1A

Down syndrome (DS), trisomy 21, is a gene dosage disorder which results in multiple phenotypes including congenital heart defects (CHD). This clinically important pathology is caused by a third copy of one or more of the [~]230 genes on human chromosome 21 (Hsa21), but the identity of the causative dosage-sensitive genes is unknown and hence pathological mechanisms remain obscure. We show that embryonic hearts from human fetuses with DS and mouse models of DS have reduced expression of mitochondrial respiration and cell proliferation genes correlating with CHD. Using systematic genetic mapping, we determine that three copies of the Dyrk1a gene, encoding a serine/threonine protein kinase, are required to cause CHD. Reducing Dyrk1a copy number from three to two reverses defects in proliferation and mitochondrial respiration in embryonic cardiomyocytes and rescues septation defects in DS hearts. Furthermore, treatment of pregnant mice with a DYRK1A inhibitor developed for clinical use partially reduces the incidence of CHD among Dp1Tyb embryos. Thus, increased dosage of DYRK1A is required to impair mitochondrial function and cause CHD in DS, revealing a therapeutic target for this common human condition. One Sentence SummaryIncreased dosage of DYRK1A causes mitochondrial dysfunction and congenital heart defects in Down syndrome and is ameliorated in utero by a drug.

developmental biology↗

bioRxiv2023

ER-dependent membrane repair of mycobacteria-induced vacuole damage

Several intracellular pathogens, such as Mycobacterium tuberculosis, damage endomembranes to access the cytosol and subvert innate immune responses. The host counteracts endomembrane damage by recruiting repair machineries that retain the pathogen inside the vacuole. Here, we show that the endoplasmic reticulum (ER)-Golgi protein oxysterol binding protein (OSBP) and its Dictyostelium discoideum homologue OSBP8 are recruited to the Mycobacterium-containing vacuole (MCV) after ESX-1-dependent membrane damage. Lack of OSBP8 causes a hyperaccumulation of phosphatidylinositol-4-phosphate (PI4P) on the MCV and decreased cell viability. OSBP8-depleted cells had reduced lysosomal and degradative capabilities of their vacuoles that favoured mycobacterial growth. In agreement with a function of OSBP8 in membrane repair, human macrophages infected with M. tuberculosis recruited OSBP in an ESX-1 dependent manner. These findings identified an ER-dependent repair mechanism for restoring MCVs in which OSBP8 functions to equilibrate PI4P levels on damaged membranes. ImportanceTuberculosis still remains a global burden and is one of the top infectious diseases from a single pathogen. Mycobacterium tuberculosis, the causative agent, has perfected many ways to replicate and persist within its host. While mycobacteria induce vacuole damage to evade the toxic environment and eventually escape into the cytosol, the host recruits repair machineries to restore the MCV membrane. However, how lipids are delivered for membrane repair is poorly understood. Using advanced fluorescence imaging and volumetric correlative approaches, we demonstrate that this involves the recruitment of the ER-Golgi lipid transfer protein OSBP8 in the D. discoideum/ M. marinum system. Strikingly, depletion of OSBP8 affects lysosomal function accelerating mycobacterial growth. This indicates that an ER-dependent repair pathway constitutes a host defence mechanism against intracellular pathogens such as M. tuberculosis.

cell biology↗

bioRxiv2023

Rescue of blood coagulation Factor VIII exon-16 mis-splicing by antisense oligonucleotides

The human Factor VIII (F8) protein is essential for the blood coagulation cascade and specific F8 mutations cause the rare bleeding disorder Hemophilia A (HA). Here, we investigated the impact of HA-causing single-nucleotide mutations on F8 pre-mRNA splicing. We found that 14/97 ([~]14.4%) coding sequence mutations tested in our study induced exon skipping. Splicing patterns of 4/11 ([~]36.4%) F8 exons tested were especially sensitive to the presence of common disease-causing mutations. RNA-chemical probing analyses revealed a three-way junction structure at the 3' end of intron 15 (TWJ-3-15). TWJ-3-15 sequesters the polypyrimidine tract, a key determinant of 3' splice site strength. Using exon-16 of the F8 gene as a model, we designed specific antisense oligonucleotides (ASOs) that target TWJ-3-15 and identified three that promote the splicing of F8 exon-16. Interaction of TWJ-3-15 with ASOs increases accessibility of the polypyrimidine tract and inhibits the binding of hnRNPA1-dependent splicing silencing factors. Moreover, ASOs targeting TWJ-3-15 rescue diverse splicing-sensitive HA-causing mutations, most of which are distal to the 3 splice site being impacted. The TWJ-3-15 structure and its effect on mRNA splicing provide a model for HA etiology in patients harboring specific F8 mutations and provide a framework for precision RNA-based HA therapies.

molecular biology↗

bioRxiv2022

First reported detection of the mobile colistin resistance genes, mcr-8 and mcr-9, in the Irish environment

The emergence and dissemination of mobile colistin resistance (mcr) genes across the globe poses a significant threat to public health, as colistin remains one of the last line treatment options for multi-drug resistant infections. Environmental samples (157 water and 157 wastewater) were collected in Ireland between 2018 and 2020. Samples collected were assessed for the presence of antimicrobial resistant bacteria using Brilliance ESBL, Brilliance CRE, mSuperCARBA and McConkey agar containing a ciprofloxacin disc. All water and integrated constructed wetland influent and effluent samples were filtered and enriched in buffered peptone water prior to culture, while wastewater samples were cultured directly. Isolates collected were identified via MALDI-TOF, were tested for susceptibility to 16 antimicrobials, including colistin, and subsequently underwent whole genome sequencing. Overall, eight mcr positive Enterobacterales (one mcr-8 and seven mcr-9) were recovered from six samples (freshwater (n=2), healthcare facility wastewater (n=2), wastewater treatment plant influent (n=1) and integrated constructed wetland influent (piggery farm waste) (n=1)). While the mcr-8 positive K. pneumoniae displayed resistance to colistin, all seven mcr-9 harbouring Enterobacterales remained susceptible. All isolates demonstrated multi-drug resistance and through whole genome sequencing analysis, were found to harbour a wide variety of antimicrobial resistance genes i.e., 30 {+/-} 4.1 (10-61), including the carbapenemases, blaOXA-48 (n=2) and blaNDM-1 (n=1), which were harboured by three of the isolates. The mcr genes were located on IncHI2, IncFIIK and IncI1-like plasmids. The findings of this study highlight potential sources and reservoirs of mcr genes in the environment and illustrate the need for further research to gain a better understanding of the role the environment plays in the persistence and dissemination of antimicrobial resistance.

microbiology↗

bioRxiv2022

Emergence of a proton exchange-based isomerization and lactonization mechanism in the plant coumarin synthase COSY

Plants contain rapidly evolving specialized metabolic enzymes to support the synthesis of a myriad of functionally diverse natural products. In the case of coumarin biosynthesis, a BAHD acyltransferase-family enzyme COSY was recently discovered in Arabidopsis that catalyzes coumarin formation from o-hydroxylated trans-hydroxycinnamoyl-CoA substrates. COSY is the first and only BAHD enzyme known to date that catalyzes an intramolecular acyl transfer reaction. Here we combine structural, biochemical, and computational approaches to investigate the mechanistic basis for the unique coumarin synthase activity of COSY. Comparative analyses of crystal structures of Arabidopsis thaliana COSY relative to other BAHD proteins reveal that COSY possesses an unconventional active-site configuration adapted to its specialized activity. Through deuterium exchange experiments, we discover a unique proton exchange mechanism at the -carbon of the o-hydroxylated trans-hydroxycinnamoyl-CoA substrates during the catalytic cycle of COSY. Mutagenesis studies and quantum mechanical cluster modeling further support that this mechanism is key to COSYs ability to lower the activation energy of the trans-to-cis isomerization of the hydroxycinnamoyl-CoA substrates, a critical rate-limiting step leading to coumarin production. This study unveils the emergence of an unconventional catalytic mechanism mediated by a BAHD-family enzyme, and sheds light on the potential evolutionary origin of COSY and its recruitment to the evolutionarily new coumarin biosynthetic pathway in eudicots.

biochemistry↗

bioRxiv2022

Electroencephalographic Signatures of Canine Cognitive Dysfunction

Canine cognitive dysfunction (CCD) is a highly prevalent neurodegenerative disease considered the canine analog of Alzheimers disease (AD). Unfortunately, CCD cannot be cured. However, early therapeutic interventions can slow the progression of cognitive decline and improve quality of life of the patients; therefore, early diagnosis is ideal. In humans, electroencephalogram (EEG) findings specific to AD have been described, and some of them have successfully detect early stages of the disease. In this study we characterized the EEG correlates of CCD, and we compared them with the EEGs of healthy aging dogs and dogs at risk of developing CCD. EEG recordings were performed in 25 senior dogs during wakefulness. Dogs were categorized in normal, at risk of CCD or with CCD according to their score in the Rofina questionnaire. We demonstrated that, quantitative EEG can detect differences between normal dogs and dogs with CCD. Dogs with CCD experience a reduction in beta and gamma interhemispheric coherence, and higher Joint Lempel Ziv complexity. Dogs at risk of developing CCD, had higher alpha power and interhemispheric coherence, making these features potential markers of early stages of the disease. These results demonstrate that EEG could be an additional biomarker that can contribute to the diagnosis of CCD, and reinforce the CCD as a translational model of AD.

animal behavior and cognition↗

bioRxiv2021

Sorting droplets into many outlets

Droplet microfluidics is a commercially successful technology, widely used in single cell sequencing and droplet PCR. Combining droplet making with droplet sorting has also been demonstrated, but so far found limited use, partly due to difficulties in scaling manufacture with injection molded plastics. We introduce a droplet sorting system with several new elements, including: 1) an electrode design combining metallic and ionic liquid parts, 2) a modular, multi-sorting fluidic design with features for keeping inter-droplet distances constant, 3) using timing parameters calculated from fluorescence or scatter signal triggers to precisely actuate dozens of sorting electrodes, 4) droplet collection techniques, including ability to collect a single droplet, and 5) a new emulsion breaking method to collect aqueous samples for downstream analysis. We use these technologies to build a fluorescence based cell sorter that sorts with high purity. We also show that these microfluidic designs can be translated into injection molded thermoplastic, suitable for industrial production. Finally, we tally the advantages and limitations of these devices.

bioengineering↗

bioRxiv2020

Correlative Light Electron Ion Microscopy reveal in vivo localisation of bedaquiline in Mycobacterium tuberculosis infected lungs

Correlative light, electron and ion microscopy (CLEIM) offers huge potential to track the intracellular fate of antibiotics, with organelle-level resolution. However, a correlative approach that enables subcellular antibiotic visualisation in pathogen-infected tissue is lacking. Here, we developed CLEIM in tissue (CLEIMiT), and used it to identify the cell-type specific accumulation of an antibiotic in lung lesions of mice infected with Mycobacterium tuberculosis. Using CLEIMiT, we found that the anti-TB drug bedaquiline is localised not only in foamy macrophages in the lungs during infection but also accumulate in polymorphonuclear (PMN) cells.

microbiology↗

bioRxiv2020

Macrophage-specific responses to human- and animal-adapted tubercle bacilli reveal pathogen and host factors driving multinucleated cell formation

The Mycobacterium tuberculosis complex (MTBC) is a group of related pathogens that cause tuberculosis (TB) in mammals. MTBC species are distinguished by their ability to sustain in distinct host populations. While Mycobacterium bovis (Mbv) sustains transmission cycles in cattle and wild animals and causes zoonotic TB, M. tuberculosis (Mtb) affects human populations and seldom causes disease in cattle. However, the host and pathogen determinants driving host tropism between MTBC species are still unknown. Macrophages are the main host cell that encounters mycobacteria upon initial infection and we hypothesised that early interactions between the macrophage and mycobacteria influence species-specific disease outcome. To identify factors that contribute to host tropism, we analysed both blood-derived primary human and bovine macrophages (hM{phi} or bM{phi}, respectively) infected with Mbv and Mtb. We show that Mbv and Mtb reside in different cellular compartments and differentially replicate in hM{phi} whereas both Mbv and Mtb efficiently replicate in bM{phi}. Specifically, we show that out of the four infection combinations, only the infection of bM{phi} with Mbv promoted the formation of multinucleated cells (MNCs), a hallmark of tuberculous granulomas. Mechanistically, we demonstrate that both MPB70 from Mbv and extracellular vesicles released by Mbv-infected bM{phi} promote macrophage multi-nucleation. Importantly, we extend our in vitro studies to show that granulomas from Mbv-infected but not Mtb-infected cattle contained higher numbers of MNCs. Our findings implicate MNC formation in the contrasting pathology between Mtb and Mbv for the bovine host, and identify MPB70 from Mbv and extracellular vesicles from bM{phi} as mediators of this process.

microbiology↗