Biology subjects

Busch-Nentwich, E. M.

Publications and source records attributed to Busch-Nentwich, E. M..

10 recordsLinked to original sources

bioRxiv2026

Macrophage adaptation to hypoxia in the tuberculous granuloma potentiates mycobacterium-induced mitochondrial damage and granuloma necrosis

Granulomas, organized macrophage-rich aggregates, are pathological hallmarks of tuberculosis. Characteristically, as Mycobacterium tuberculosis replicates within macrophages, the granuloma enlarges, and then the center undergoes necrosis, promoting mycobacterial growth in the debris (caseum). This sequence results in large, mycobacterium-rich granulomas that can rupture into airways, facilitating disease transmission. Here, using the zebrafish-Mycobacterium marinum tuberculosis model, we find that both stages - granuloma enlargement and necrosis - depend on the mycobacterial virulence factor, EsxA, that induces mitochondrial damage and apoptosis of infected macrophages. Initially, dying macrophages are engulfed by newly recruited macrophages. Then, the enlarging granuloma core becomes hypoxic and induces Hypoxia Inducible Factor-1 (HIF-1). HIF-1 suppresses mitochondrial respiration, sensitizing macrophages to EsxA mitotoxicity. These sensitized macrophages undergo accelerated apoptosis which outstrips clearance, causing mycobacterium-rich necrotic debris to accumulate in the granuloma cores. Thus, tuberculosis transmission depends on the dynamic interplay between a mycobacterial virulence factor and an adaptive host metabolic program.

microbiology↗

bioRxiv2022

Gaucher Disease Protects Against Tuberculosis

Biallelic mutations in the glucocerebrosidase (GBA1) gene cause Gaucher disease, characterized by lysosomal accumulation of glucosylceramide and glucosylsphingosine in macrophages. This and other lysosomal diseases occur with high frequency in Ashkenazi Jews. It has been proposed that the underlying mutations confer a selective advantage, in particular conferring protection against tuberculosis. Here, using a zebrafish Gaucher disease model, we find that the mutation GBA1 N370S, predominant among Ashkenazi Jews, increases resistance to tuberculosis through the microbicidal activity of glucosylsphingosine in macrophage lysosomes. Consistent with lysosomal accumulation occurring only in homozygotes, heterozygotes remain susceptible to tuberculosis. Thus, our findings reveal a mechanistic basis for protection against tuberculosis by GBA1 N370S and provide biological plausibility for its selection if the relatively mild deleterious effects in homozygotes were offset by significant protection against tuberculosis, a rampant killer of the young in Europe through the Middle Ages into the 19th century. Significance StatementGaucher disease is a recessively inherited disorder in which the lipids glucosylceramide and glucosylsphingosine accumulate in lysosomes of macrophages. Macrophages are the first immune cells to engulf infecting bacteria and we find that glucosylsphingosine increases their ability to kill Mycobacterium tuberculosis that causes tuberculosis. Gaucher disease due to a particular mutation is frequent in Ashkenazi Jews. Since from the middle ages they were often confined to areas of high tuberculosis prevalence, it has been proposed that the mutation prevailed because heterozygotes, who do not accumulate lipids nor manifest Gaucher disease, were protected. Our findings raise the possibility that selection operated on homozygotes manifesting mild forms of Gaucher disease who were protected against tuberculosis which would often have been fatal.

microbiology↗

bioRxiv2022

mTOR-regulated Mitochondrial Metabolism Limits Mycobacterium-induced Cytotoxicity

Necrosis of macrophages in the tuberculous granuloma represents a major pathogenic event in tuberculosis. Through a zebrafish forward genetic screen, we identified the mTOR kinase, a master regulator of metabolism, as an early host resistance factor in tuberculosis. We found that mTOR complex 1 protects macrophages from mycobacterium-induced death by enabling infection-induced increases in mitochondrial energy metabolism fueled by glycolysis. These metabolic adaptations are required to prevent mitochondrial damage and death caused by the secreted mycobacterial virulence determinant ESAT-6. Thus, the host can effectively counter this early critical mycobacterial virulence mechanism simply by regulating energy metabolism, thereby allowing pathogen-specific immune mechanisms time to develop. Our findings may explain why Mycobacterium tuberculosis, albeit humanitys most lethal pathogen, is successful in only a minority of infected individuals.

immunology↗

bioRxiv2021

A red herring in zebrafish genetics: allele-specific gene expression can underlie altered transcript abundance in zebrafish mutants

In model organisms, RNA sequencing is frequently used to assess the effect of genetic mutations on cellular and developmental processes. Typically, animals heterozygous for a mutation are crossed to produce offspring with different genotypes. Resultant embryos are grouped by genotype to compare homozygous mutant embryos to heterozygous and wild-type siblings. Genes that are differentially expressed between the groups are assumed to reveal insights into the pathways affected by the mutation. Here we show that in zebrafish, differentially expressed genes are often overrepresented on the same chromosome as the mutation due to different levels of expression of alleles from different genetic backgrounds. Using an incross of haplotype-resolved wild-type fish, we found evidence of widespread allele-specific expression, which appears as differential expression when comparing embryos homozygous for a region of the genome to their siblings. When analysing mutant transcriptomes, this means that differentially expressed genes on the same chromosome as a mutation of interest may not be caused by that mutation. Typically, the genomic location of a differentially expressed gene is not considered when interpreting its importance with respect to the phenotype. This could lead to pathways being erroneously implicated or overlooked due to the noise of spurious differentially expressed genes on the same chromosome as the mutation. These observations have implications for the interpretation of RNA-seq experiments involving outbred animals and non-inbred model organisms.

developmental biology↗

bioRxiv2020

Loss of slc39a14 causes simultaneous manganese deficiency and hypersensitivity in zebrafish

Mutations in SLC39A14, a manganese uptake transporter, lead to a neurodegenerative disorder characterised by accumulation of manganese in the brain and rapidly progressive dystonia-parkinsonism (Hypermanganesemia with Dystonia 2, HMNDYT2). Similar to the human phenotype, zebrafish slc39a14U801-/- mutants show prominent brain manganese accumulation and abnormal locomotor behaviour. In order to identify novel potential targets of manganese neurotoxicity, we performed transcriptome analysis of individual homozygous mutant and sibling slc39a14U801 zebrafish at five days post fertilisation unexposed and exposed to MnCl2. Anatomical gene enrichment analysis confirmed that differentially expressed genes map to the central nervous system and eye. Biological interpretation of differentially expressed genes suggests that calcium dyshomeostasis, activation of the unfolded protein response, oxidative stress, mitochondrial dysfunction, lysosomal disruption, apoptosis and autophagy, and interference with proteostasis are key events in manganese neurotoxicity. Differential expression of visual phototransduction genes also predicted visual dysfunction in mutant larvae which was confirmed by the absence of visual background adaptation and a diminished optokinetic reflex. Surprisingly, we found a group of differentially expressed genes in mutant larvae that normalised upon MnCl2 treatment suggesting that, in addition to neurotoxicity, manganese deficiency is present either subcellularly or in specific cells or tissues. This may have important implications for treatment as manganese chelation may aggravate neurological symptoms. Our analyses show that slc39a14U801-/- mutant zebrafish present a powerful model to study the cellular and molecular mechanisms underlying disrupted manganese homeostasis. Significance statementManganese neurotoxicity leading to progressive dystonia-parkinsonism is a characteristic feature of Hypermanganesemia with dystonia 2 (HMNDYT2) caused by mutations in SLC39A14, a manganese uptake transporter. Transcriptional profiling in slc39a14U801 loss-of-function zebrafish suggests that, in addition to manganese neurotoxicity, subcellular or cell type specific manganese deficiency contributes to the disease phenotype. Both manganese overload and deficiency appear to be associated with Ca2+ dyshomeostasis. We further demonstrate that activation of the unfolded protein response, oxidative stress, mitochondrial dysfunction, apoptosis and autophagy, and disrupted proteostasis are likely downstream events in manganese neurotoxicity. Our study shows that the zebrafish slc39a14U801 loss-of-function mutant is a powerful model to elucidate the mechanistic basis of diseases affected by manganese dyshomeostasis.

neuroscience↗

bioRxiv2019

A missense mutation dissociates triglyceride and phospholipid transfer activities in zebrafish and human microsomal triglyceride transfer protein

Microsomal triglyceride transfer protein (MTP) transfers triglycerides and phospholipids and is essential for the assembly of Apolipoprotein B (ApoB)-containing lipoproteins in the endoplasmic reticulum. We have discovered a zebrafish mutant (mttpc655) expressing a C-terminal missense mutation (G863V) in Mttp, one of the two subunits of MTP, that is defective at transferring triglycerides, but retains phospholipid transfer activity. Mutagenesis of the conserved glycine in the human MTTP protein (G865V) also eliminates triglyceride but not phospholipid transfer activity. The G863V mutation reduces the production and size of ApoB-containing lipoproteins in zebrafish embryos and results in the accumulation of cytoplasmic lipid droplets in the yolk syncytial layer. However, mttpc655 mutants exhibit only mild intestinal lipid malabsorption and normal growth as adults. In contrast, zebrafish mutants bearing the previously identified mttpstl mutation (L475P) are deficient in transferring both triglycerides and phospholipids and exhibit gross intestinal lipid accumulation and defective growth. Thus, the G863V point mutation provides the first evidence that the triglyceride and phospholipid transfer functions of a vertebrate MTP protein can be separated, arguing that selective inhibition of the triglyceride transfer activity of MTP may be a feasible therapeutic approach for dyslipidemia.

physiology↗

bioRxiv2018

Population based forward genetic screen of mutagenized zebrafish identifies loci associated with nicotine preference and human smoking behavior

To facilitate smoking genetics research we determined whether a screen of mutagenized zebrafish for nicotine preference could predict loci affecting smoking behaviour. Of 30 ENU mutagenized families screened, two showed increased or decreased nicotine preference. Out of 25 inactivating mutations in the families, one in the slit3 gene segregated with increased nicotine preference in heterozygous individuals. Focussed SNP analysis of the human SLIT3 locus in cohorts from UK (n=863) and Finland (n=1715) identified two variants that predict cigarette consumption and likelihood of cessation. Characterisation of slit3 mutant larvae and adult fish revealed decreased sensitivity to the dopaminergic and serotonergic antagonist amisulpride, known to affect startle reflex that is correlated with addiction in humans, and increased htr1aa mRNA expression in mutant larvae. No effect on neuronal pathfinding was detected. These findings reveal a role for SLIT3 in development of pathways affecting responses to nicotine in zebrafish and smoking in humans.

genetics↗

bioRxiv2018

The gene regulatory basis of genetic compensation during neural crest induction

BackgroundThe neural crest (NC) is a vertebrate-specific cell type that contributes to a wide range of different tissues across all three germ layers. The gene regulatory network (GRN) responsible for the formation of neural crest is conserved across vertebrates. Central to the induction of the NC GRN are AP-2 and SoxE transcription factors but detailed interactions within the network remain to be resolved.\n\nResultsWe have used gene knockout and RNA sequencing strategies to dissect NC differentiation in zebrafish. We establish that initiation of the NC GRN takes place just after genome activation. We genetically ablate the NC using double mutants of tfap2a;tfap2c or remove specific subsets of the NC with sox10 and mitfa knockouts and characterise genome-wide gene expression levels across multiple time points. We find that although a single allele of tfap2c is capable of maintaining early NC induction and differentiation in the absence of tfap2a function, expression of many target genes remains abnormal and sensitive to tfap2 dosage. This separation of morphological and molecular phenotypes identifies a core set of genes required for early NC development. Using gene knockouts, we associate previously uncharacterised genes with pigment cell development and establish a role for maternal Hippo signalling in melanocyte differentiation.\n\nConclusionsStepwise genetic ablation of the NC identifies the core gene module required for neural crest induction. This work extends and refines the NC GRN while also uncovering the complex transcriptional basis of genetic compensation via paralogues.

developmental biology↗

bioRxiv2017

mRNA processing in mutant zebrafish lines generated by chemical and CRISPR-mediated mutagenesis produces potentially functional transcripts

As model organism-based research shifts from forward to reverse genetics approaches, largely due to the ease of genome editing technology, allow frequency of abnormal phenotypes is being observed in lines with mutations predicted to lead to deleterious effects on the encoded protein. In zebrafish, this low frequency is in part explained by compensation by genes of redundant or similar function, often resulting from the additional round of teleost-specific whole genome duplication within vertebrates. Here we offer additional explanations for the low frequency of mutant phenotypes. We analyzed mRNA processing in seven zebrafish lines with mutations expected to disrupt gene function, generated by CRISPR/Cas9 or ENU mutagenesis methods. Five of the seven lines showed evidence of genomic compensation by means of altered mRNA processing: one through a skipped exon that did not lead to a frame shift, one through nonsense-associated splicing that did not lead to a frame shift, and three through the use of cryptic splice sites. These results highlight the need for a methodical analysis of the mRNA produced in mutant lines before making conclusions or embarking on studies that assume loss of function as a result of a given genomic change. Furthermore, recognition of the types of genomic adaptations that can occur may inform the strategies of mutant generation.\n\nAuthor summaryThe recent rise of reverse genetic, gene targeting methods has allowed researchers to readily generate mutations in any gene of interest with relative ease. Should these mutations have the predicted effect on the mRNA and encoded protein, we would expect many more abnormal phenotypes than are typically being seen in reverse genetic screens. Here we set out to explore some of the reasons for this discrepancy by studying seven separate mutations in zebrafish. We present evidence that thorough cDNA sequence analysis is a key step in assessing the likelihood that a given mutation will produce hypomorphic or null alleles. This study reveals that alternative mRNA processing in the mutant background often produces transcripts that escape nonsense-mediated decay, thereby potentially preserving gene function. By understanding the ways that cells avoid the deleterious consequences of mutations, researchers can better design reverse genetic strategies to increase the likelihood of gene disruption.

molecular biology↗

bioRxiv2017

A high-resolution mRNA expression time course of embryonic development in zebrafish

We have produced an mRNA expression time course of zebrafish development across 18 time points from 1-cell to 5 days post-fertilisation sampling individual and pools of embryos. Using poly(A) pulldown stranded RNA-seq and a 3' end transcript counting method we characterise the temporal expression profiles of 23,642 genes. We identify temporal and functional transcript co-variance that associates 5,024 unnamed genes with distinct developmental time points. Specifically, a class of over 100 previously uncharacterised zinc finger domain containing genes, located on the long arm of chromosome 4, is expressed in a sharp peak during zygotic genome activation. The data reveal complex and widespread differential use of exons and previously unidentified 3' ends across development, new primary microRNA transcripts and temporal divergence of gene paralogues generated in the teleost genome duplication. To make this dataset a useful baseline reference, the data are accessible to browse and download at Expression Atlas and Ensembl.

developmental biology↗