Biology subjects

Motooka, D.

Publications and source records attributed to Motooka, D..

At least 19 recordsLinked to original sources

bioRxiv2026

Purification-free cryo-EM reveals endogenous assemblies from minute tardigrade samples

Tardigrades are minute animals renowned for environmental stress tolerance, and their remarkable biology has motivated extensive molecular studies using biochemical and biophysical approaches. Yet the native structures of endogenous macromolecular assemblies in tardigrades remain largely inaccessible, in part because only limited biological material is available. Here we show that purification-free single-particle cryo-EM, guided by long-read cDNA sequence assignment, can recover multiple endogenous assemblies directly from crude extract of a wild-derived Macrobiotus cf. shonaicus culture. We reconstructed a dimeric vitellogenin (Vg) particle at 3.10 [A] resolution, a previously uncharacterized tardigrade ferritin at 3.09 [A] resolution, and a low-resolution ribosome map. The Vg particle is a glycosylated and metal-containing assembly with structural evidence of proteolytic processing and contains lipid-like densities occupying a broad chamber whose walls include N-linked glycans. The ferritin structure revealed a divergent C-terminal helix that lines a mineral-like density observed at a position corresponding to previously described mineralization sites in ferritin-family cages. These results establish purification-free cryo-EM as a route to structural analysis of endogenous macromolecular assemblies from minute animal samples. In tardigrades, this approach provides a foundation for future state-dependent structural studies, including desiccation, rehydration and reproduction.

biochemistry↗

bioRxiv2026

Core genome MLST reveals genetic and BafA-associated phenotypic diversities in Bartonella henselae strains

Bartonella henselae is a zoonotic pathogen associated with cat-scratch disease. Although multilocus sequence typing (MLST) has been used for strain classification, its resolution for distinguishing between B. henselae isolates remains limited. We herein developed a B. henselae-specific core genome MLST (cgMLST) scheme based on whole-genome sequencing data and examined the genetic and phenotypic diversities of 80 strains derived from cats, humans, mongooses, and masked palm civets. Using the conventional MLST scheme, the 80 strains were classified into nine sequence types (STs), while cgMLST subdivided them into 72 cgSTs, demonstrating a marked improvement in discriminatory power. The cgMLST scheme comprised 1,183 core genes and showed high applicability across the 80 strains. A phylogenetic analysis revealed that ST1, which has been associated with cat-scratch disease, was further subdivided into three major clusters and two singletons, indicating high genetic heterogeneity within this ST. We also found that the bafA subtypes clustered in a manner that was largely consistent with the cgMLST-based phylogenetic structure, suggesting a close relationship between bafA variations and the genomic background of B. henselae strains. In a human umbilical vein endothelial cell proliferation assay, strains belonging to distinct cgSTs exhibited strain-dependent differences in proliferative capacity, which were associated with the bafA subtype classification. Some strains induced focal cell fragmentation and a reduced cell density at a high multiplicity of infection, indicating strain-dependent differences in endothelial cell injury. Collectively, the present results establish a high-resolution cgMLST framework for B. henselae and demonstrate that genetically distinct strains have diverse endothelial cell phenotypes.

microbiology↗

bioRxiv2026

Real-time near-infrared imaging distinguishes nasopharyngeal colonization from aspiration of Streptococcus pneumoniae and identifies aspiration as a trigger of severe disease

Streptococcus pneumoniae asymptomatically colonizes the nasopharynx but can invade the lower respiratory tract to cause life-threatening disease, particularly in older adults. However, whether the initial site of bacterial deposition following intranasal inoculation determines disease progression has not been directly examined. Here, we developed a near-infrared (NIR) fluorescence imaging approach using indocyanine green (ICG)-labeled S. pneumoniae TIGR4 to visualize early bacterial distribution in real time. ICG labeling by simple mixing, without genetic or chemical modification, neither impaired bacterial growth at 33 or 37{degrees}C, nor altered acid tolerance. Continuous video imaging during the first 10 min of infection resolved two distinct patterns: bacteria confined to the nasopharynx (colonization) and those aspirated into the lower respiratory tract (aspiration). Kaplan-Meier analysis revealed markedly higher mortality in the aspiration group in both young (hazard ratio = 7.9) and aged (hazard ratio = 8.4) mice, despite a 10-fold lower inoculum used for aged animals, with deaths beginning on day 3. Systemic profiling of blood at 24 h by RNA sequencing and plasma proteomics revealed that early aspiration in aged mice was associated with the activation of inflammatory and hematopoietic programs, enrichment of complement and coagulation cascades, and phagocytic pathways. Together, these findings establish aspiration into the lower respiratory tract as a trigger of severe pneumococcal disease and introduce real-time NIR imaging as a technique for linking early infection dynamics to systemic host responses.

microbiology↗

bioRxiv2026

Leptin receptor deficiency suppresses gastric tumorigenesis by limiting stromal activation and tumor microenvironment development.

Leptin receptor (LEPR) signaling has been implicated in multiple malignancies; however, its role in gastric tumors remains poorly defined. We previously demonstrated that mice with gastrointestinal epithelial cell-specific deletion of suppressor of cytokine signaling 3 (SOCS3 cKO), a negative feedback regulator of LEPR signaling, develop gastric tumors due to aberrant leptin production and LEPR activation. Here, we demonstrate that concurrent deletion of both Socs3 and Lepr (double knockout; DKO) under the same promoter substantially suppresses gastric tumorigenesis and markedly prolonged survival. Whereas SOCS3 cKO mice exhibited early stromal activation, increased TGF-{beta}1 production, accumulation of cancer-associated fibroblasts (CAFs) and collagen deposition, these tumor-promoting alterations were substantially attenuated in DKO mice. Additionally, DKO mice showed reduced inflammatory cytokine and chemokine signaling, decreased the accumulation of Gr-1+CD11b+ myeloid-derived suppressor cells, and reduced LEPR and TGF-{beta} signaling. Analysis of The Cancer Genome Atlas stomach adenocarcinoma cohort revealed high LEPR expression in the chromosomal instability and genomically stable subtypes, correlating with poor prognosis. Moreover, LEPR expression was mutually exclusive with CLDN18 and ERBB2, two major therapeutic biomarkers, and positively correlated with a CAF-related transcriptional signature. Our findings identify LEPR signaling in epithelial cells as a key driver of gastric tumorigenesis through promotion of stromal activation and tumor microenvironment development. They further highlight LEPR as a promising therapeutic target for patients with gastric cancer who are unlikely to benefit from current ERBB2/HER2- or CLDN18-directed therapies.

cancer biology↗

bioRxiv2026

Acquisition of Stickland-metabolizing bacteria during infancy prevents Clostridium botulinum infection

Infant botulism is caused by intestinal colonization with Clostridium botulinum, whereas healthy adults are resistant. Although the gut microbiota has long been implicated in protection against C. botulinum, its bacterial basis has remained unknown. Here, we show that specific amino acid-metabolizing bacteria confer resistance to C. botulinum colonization through nutrient competition. Longitudinally collected human infant microbiotas exhibited a transition from susceptibility to resistance after transplantation into germ-free mice. 5-Aminovalerate marked the resistant microbiota, implicating Stickland metabolism. Resistant microbiotas were enriched in Stickland-metabolizing Clostridia, including Clostridioides difficile. Metabolomics revealed overlapping amino acid utilization, and C. difficile suppressed C. botulinum expansion through amino acid competition. These findings demonstrate that acquisition of Stickland-metabolizing Clostridia during infancy prevents C. botulinum infection through competition for shared amino-acid-dependent nutritional niches.

microbiology↗

bioRxiv2026

Retinoid signaling promotes frontonasal identity while repressing maxillary identity during craniofacial development

Vertebrate facial development depends on the correct specification of embryonic facial prominences, a process known to be governed by region-specific reciprocal signaling pathways between the craniofacial ectoderm, endoderm and mesenchyme. This process is further modulated by transcriptional and epigenetic mechanisms that regulate the expression of essential genes in mesenchymal progenitors derived from cranial neural crest cells. Retinoid signaling plays a critical role in facial development, and both gain-and loss-of-function results in a wide spectrum of facial defects, including orofacial clefts. In this study, we identified retinoid signaling as a critical regulator of cranial neural crest cell specification toward a frontonasal process identity in mice. Rdh10 oxidizes vitamin A (all-trans retinol) to retinal, which is a rate limiting step in the synthesis of retinoic acid. Rdh10 loss-of-function resulted in ectopic formation of whisker pads - a derivative of the maxillary process - within the frontonasal process region. This transformation was evidenced by the mis-expression of maxillary specific transcription factors including Meis2 and Lhx6 in the frontonasal mesenchyme. Furthermore, these transcription factors exhibited increased chromatin accessibility at their consensus binding sites following the loss of retinoid signaling in frontonasal cranial neural crest cells. These results indicate that retinoid signaling acts as a critical regulator specifying frontonasal identity and fate of cranial neural crest cells as they migrate into the frontonasal process, while concomitantly repressing maxillary process fate. These results not only advance our understanding of frontonasal prominence specification and the evolutionary development of craniofacial structures but also offers valuable insights into the etiology and pathogenesis of craniofacial malformations such as orofacial clefts.

developmental biology↗

bioRxiv2026

Inhibition of the gut ceramidase Asah2 decelerates the vertebrate ageing rate

The pace of ageing varies markedly among vertebrate species and individuals. However, the mechanisms underlying these differences in ageing rates remain unclear. Here, we show that the activity of the gut ceramidase Asah2 determines species- and strain-specific rates of vertebrate ageing. Comparative genomic analyses of vertebrate species and strains with different ageing rates reveal an association between low Asah2 activity and increased lifespan. Using the ultra-short-lived killifish Nothobranchius furzeri as a model, we demonstrate that knockout of Asah2 (asah2 KO) extends lifespan and attenuates systemic ageing phenotypes, including declines in locomotor activity, abnormal protein accumulation in the brain, and accumulation of senescent cells in the liver. asah2 KO elevates levels of ceramide species with long-chain fatty acids in the intestine, and supplementation with these ceramide species suppresses ageing phenotypes and extends lifespan in wild-type fish. asah2 KO and ceramide supplementation alter gut microbiota composition, and asah2 KO-derived microbiota transplantation attenuates ageing phenotypes, suggesting that reduced Asah2 activity prevents ageing through intestinal ceramide-mediated modulation of the microbiota. Given the evolutionary conservation of the Asah2 gene and its age-dependent upregulation in fish and humans, Asah2 and ceramides may act as ageing accelerators and decelerators, respectively, across animal species.

developmental biology↗

bioRxiv2025

Nutrient Acquisition Drives Edwardsiella tarda Pathogenesis in Necrotizing Soft Tissue Infection

Necrotizing soft tissue infections (NSTIs) are rapidly progressive and life-threatening diseases caused by diverse bacterial pathogens. While classical virulence factors such as toxins and secretion systems have been extensively characterized, the role of metabolic fitness in supporting bacterial survival within the nutrient-restricted host environment remains underexplored. Edwardsiella tarda, a human-pathogenic bacterium implicated in NSTIs, represents an emerging model for studying non-canonical pathogenic strategies. Here, we employed transposon-directed insertion site sequencing (TraDIS) to identify genes critical for E. tarda survival in a murine soft tissue infection model. A genome-wide screen revealed 41 genes significantly depleted during the infection, including those involved in iron and zinc acquisition (fetB, zupT), vitamin biosynthesis (pdxK, cobA), and polyamine metabolism (speB). Functional assays using defined minimal media demonstrated that supplementation with vitamin B6 or putrescine enhanced bacterial growth, validating their contribution to fitness under nutrient-limited conditions. Our findings indicate that E. tarda pathogenesis is driven not solely by classical virulence factors but also by its ability to acquire essential nutrients and adapt metabolically to host-imposed nutritional stress. This study provides the first genome-wide fitness map for E. tarda during soft tissue infection and reveals new targets for therapeutic intervention that disrupt nutrient acquisition systems. These results also emphasize the broader relevance of metabolic adaptation as a determinant of virulence in invasive bacterial infections. IMPORTANCENecrotizing soft tissue infections (NSTIs) are severe, rapidly progressing bacterial infections with high morbidity and mortality. Although classical virulence factors such as toxins have been widely studied, much less is known about how pathogens adapt metabolically to survive within the nutrient-restricted environment in host tissues. This study uses Edwardsiella tarda, an emerging NSTI pathogen, as a model to identify genes required for in vivo fitness using transposon insertion sequencing (TraDIS). By revealing the critical roles of nutrient acquisition and metabolic adaptation, rather than toxin production alone, this work challenges conventional paradigms of bacterial virulence. Our findings suggest that targeting bacterial nutrient acquisition pathways may offer a novel therapeutic approach to control invasive infections. Furthermore, this study provides the first genome-wide fitness map of E. tarda during soft tissue infection, offering a valuable resource for future research into polymicrobial wound infections and host-pathogen nutrient competition.

microbiology↗

bioRxiv2025

A cattle-derived human H5N1 isolate suppresses innate immunity despite efficient replication in human respiratory organoids

The H5N1 high pathogenicity avian influenza virus (HPAIV) of clade 2.3.4.4b, which spreads globally via wild birds, has become a major public health concern because it can infect a variety of mammals, including humans. In 2024, infection of dairy cattle with H5N1 HPAIV clade 2.3.4.4b was confirmed in the United States, and subsequent human cases were reported. Although these viruses are highly pathogenic in animal models, human infections have generally been mild, revealing a striking discrepancy. Here, we characterized the cattle-derived human H5N1 isolate A/Texas/37/2024 (TX37-H5N1) using three-dimensional human respiratory organoids derived from induced pluripotent stem (iPS) cells. Despite efficient replication, TX37-H5N1 induced minimal interferon and inflammatory cytokine responses. Bulk and single-cell RNA sequencing revealed reduced STAT1-mediated transcriptional activity in TX37-H5N1-infected organoids compared to the historic H5N1 human isolate A/Vietnam/1203/2004. These findings suggest that TX37-H5N1 fails to trigger the strong innate responses, including robust cytokine production, that are typically associated with severe H5N1 disease and are thought to contribute to cytokine storm-medicated pathogenesis. This attenuated response may help explain the discrepancy between the high pathogenicity of TX37-H5N1 in animal models and its mild clinical presentation in humans. While zoonotic influenza risk is often assessed using cell lines or animal models, our study highlights the value of using human respiratory organoids to evaluate human-specific virus-host interactions. This platform provides a complementary tool for assessing the risk of emerging avian influenza viruses. HighlightsO_LIHuman respiratory organoids were used to model zoonotic B3.13 H5N1 infections. C_LIO_LIA cattle-derived human isolate, TX37-H5N1, replicated more efficiently than historical VN1203-H5N1. C_LIO_LITX37-H5N1 suppressed STAT-IRF-mediated innate immune responses. C_LIO_LITX37-H5N1 was sensitive to baloxavir and oseltamivir but less sensitive to favipiravir. C_LIO_LIHuman respiratory organoids offer a complementary platform for zoonotic influenza risk assessment. C_LI In briefOur study used human iPSC-derived respiratory organoids to investigate the mild clinical presentation of zoonotic B3.13 H5N1 viruses. We found that TX37-H5N1 replicates efficiently but suppresses innate immune responses, providing mechanistic insight into species-specific pathogenesis and highlighting the utility of human organoids for zoonotic risk assessment.

microbiology↗

bioRxiv2025

Oral antigen exposure under co-stimulation blockade generates Treg cells to establish immune tolerance despite prior sensitization

Antigen-specific oral tolerance is effective in preventing harmful immune responses in antigen-non-sensitized animals but difficult to be induced in those already antigen-primed. Here, we show in mice that feeding of antigen-containing diet generates peripherally derived regulatory T (pTreg) cells that exhibit a tissue-adapted effector phenotype. The antigen feeding also enables the generated pTreg cells to acquire Treg-specific epigenomic changes in Treg signature genes including Foxp3, hence the stability of Treg-specific function. Cessation of antigen feeding, however, results in decline in oral tolerance with diminution of pTreg cells. Transcriptomic analysis has revealed that the induced pTreg cells predominantly express CD101. CD101+ Treg cells with a similar phenotype and epigenetic alterations can also be generated in vitro from antigen-primed naive CD4+ T cells by blockade of CD28-mediated co-stimulation during TGF-{beta}-dependent Treg induction. Furthermore, in vivo CD28 signal blockade by CTLA-4-immunoglobulin fusion protein (CTLA4-Ig) before antigen feeding can establish oral tolerance in the mice precedingly antigen-sensitized by a non-oral route. The blockade facilitates differentiation of antigen-specific conventional T cells into CD101+ pTreg cells. Thus, continuous oral antigen administration combined with CD28 co-stimulation blockade generates CD101+ antigen-specific functionally stable pTreg cells, which can establish long-term systemic antigen-specific immune tolerance even in antigen-pre-sensitized animals.

immunology↗

bioRxiv2024

Identifying Genetic Variations in emm89 Streptococcus pyogenes Linked to Severe Invasive Infections

Streptococcus pyogenes causes mild human infections as well as life-threatening invasive diseases. Since the mutations known to enhance virulence to date account for only half of the severe invasive infections, additional mechanisms/mutations need to be identified. Here, we conducted a genome-wide association study of emm89 S. pyogenes strains to comprehensively identify pathology-related bacterial genetic factors (SNPs, indels, genes, or k-mers). Japanese (n=311) and global (n=666) cohort studies of strains isolated from invasive or non-invasive infections revealed 17 and 1,075 SNPs/indels and 2 and 169 genes, respectively, that displayed associations with invasiveness. We validated one of them, a non-invasiveness-related point mutation, fhuB T218C, by structure predictions and introducing it into a severe invasive strain and confirmed that the mutant showed slower growth in human blood. Thus, we report novel mechanisms that convert emm89 S. pyogenes to an invasive phenotype and a platform for establishing novel treatments and prevention strategies.

microbiology↗

bioRxiv2024

Shaving black fur uncovers hidden issues in p16-3MR mice

The p16-3MR mouse model has previously been reported to effectively visualize and eliminate senescent cells in vivo. However, we now report significant issues with this model. Noninvasive bioluminescence imaging without shaving the black fur detected almost no luminescence signals, but they became visible after shaving. However, these signals were very faint and attributed to the auto-luminescence of coelenterazine-h, rather than reporter gene expression. Further experiments showed no significant bioluminescent changes with aging, doxorubicin treatment, or during wound healing, contradicting earlier reports. Comparative analysis between p16-3MR mice from different sources revealed no differences, suggesting functional issues with Renilla luciferase (Rluc) and herpes simplex virus 1 thymidine kinase (HSV-TK) expression in this mouse model. Our findings underscore the necessity of using white-furred mice or ensuring proper fur shaving for accurate in vivo bioluminescence imaging. We recommend the reevaluation of previously published studies that used p16-3MR mice without proper controls to ensure data accuracy. These results provide critical insights for researchers using p16-3MR mice in senescence studies.

molecular biology↗

bioRxiv2024

Spatial transcriptomics elucidates medulla niche supporting germinal center response in myasthenia gravis thymoma

Myasthenia gravis (MG) is known to be epidemiologically associated with abnormalities of the thymus, an organ that maintains central tolerance. However, due to the complexity of the thymus, specific characteristics related to the pathogenesis of MG remain elusive. In our study, we attempted to narrow down the features associated with MG using spatial transcriptome analysis of thymoma and thymic hyperplasia samples. We found that the majority of thymomas were constituted by the cortical region, whereas the medullary region was localized in comparatively restricted areas. Moreover, the medullary region contained polygenic enrichment, MG-specific germinal center structures, and a supporting immune microenvironment. Additionally, neuromuscular medullary thymic epithelial cells (nmTECs), previously identified as MG-specific autoantigen-producing cells, were situated at the cortico-medullary junction. The immune microenvironment in the medulla was characterized by a specific chemokine pattern and specific immune cells, such as CCR7+ migratory dendritic cells (migDCs) and effector regulatory T (Treg) cells. Furthermore, similar germinal center structures and immune microenvironments were observed in the medulla during thymic hyperplasia. This study indicates that the medulla and junction areas are related to the pathology of MG, suggesting that these areas should be the focus of future studies on MG pathogenesis and drug targeting.

immunology↗

bioRxiv2023

Age-associated aberrations of cumulus-oocyte interaction and microfilamentous structure in the zona pellucida decline female fertility

One of the major age-related declines in female reproductive function is the reduced quantity and quality of oocytes. Here we demonstrate that structural changes in the zona pellucida (ZP) decrease fertilization rates from 34- to 38-week-old female mice, equivalent to the mid-reproductive of human females. In middle-age ovaries, the decline in the number of transzonal projections was accompanied by a decrease in cumulus cell-oocyte interactions, resulting in a deterioration of the oocyte quality. Scanning electron microscopy showed the ZP surface microfilament structure transitioning from rugged to smooth with age, leading to decreased fertilization rates due to impaired sperm binding to the ZP. Moreover, the fertilization rate of middle-age mice was restored to a comparable level to that of young mice by destabilizing the ZP in the presence of glutathione. These results suggest that the age-related structural changes in the ZP is a key for successful fertilization at reproductive age.

developmental biology↗

bioRxiv2023

Fibroblast Nrf2 inhibits profibrotic transcription with Ddx54 and mitigates pathological fibrosis in the mouse heart and kidney

BackgroundTissue fibrosis is a common feature of many organ dysfunctions, such as heart failure and chronic kidney disease. However, no fundamental treatment has been developed. This study aims to identify novel molecular mechanisms for antifibrotic intervention, focusing on fibroblast activation. MethodsWe performed a forward genetic screen using a genome-wide CRISPR library in the context of transforming growth factor {beta} (TGF-{beta})-mediated connective tissue growth factor (CTGF) expression, and used unbiased techniques such as Cleavage Under Targets and Tagmentation (CUT&Tag) and proximity-dependent biotin labeling by TurboID to reveal the detailed molecular mechanisms. ResultsCRISPR library screening identified a number of players in both the canonical Smad pathway and the non-canonical pathway. In addition to the known factors, the Keap1-Nrf2 pathway was identified as a predominant regulator of TGF-{beta}-mediated CTGF expression. Keap1 deletion and consequent Nrf2 activation broadly suppressed profibrotic gene expression, independently of conventional antioxidant effects. CUT&Tag revealed that Nrf2 bound to the proximity of fibrosis-related genes including Ctgf and Fn1. Subsequent individual analysis revealed Smad3 and RNA polymerase II binding to the Nrf2 peak site, which was attenuated by Keap1 deletion. TurboID experiments further discovered that Nrf2 interacts with Ddx54, which acts as a corepressor. Consistently, Keap1 deletion-mediated repression of profibrotic gene expression was reversed by additional Ddx54 deletion. The impact of the Keap1-Nrf2 pathway on pathological fibrosis was examined using tamoxifen-inducible fibroblast-specific Keap1 knockout mice. Pressure overload for 4 weeks robustly induced cardiac hypertrophy, fibrosis and contractile dysfunction. However, deletion of Keap1 in the Postn lineage attenuated these cardiac pathologies. The anti-fibrotic effects of Keap1 deletion were also confirmed in renal fibrosis in the unilateral ureteral obstruction (UUO) model. ConclusionsFibroblast Nrf2 transcriptionally represses fibrosis-related genes in cooperation with the corepressor Ddx54. Fibroblast-specific deletion of Keap1 attenuated pathological fibrosis in pressure overload heart failure and renal fibrosis.

cell biology↗

bioRxiv2023

Deep mutational scanning of whole SARS-CoV-2 spike in an inverted infection system

In order to investigate SARS-CoV-2 mutations and their impact on immune evasion and infectivity, we developed a Deep Mutational Scanning (DMS) platform utilizing an inverted infection assay to measure spike expression, ACE2 affinity, and viral infectivity in human cells. Surprisingly, our analysis reveals that spike protein expression, rather than ACE2 affinity, is the primary factor affecting viral infectivity and correlated with SARS-CoV-2 evolution. Notably, within the N-terminal domain (NTD), spike expression and infectivity-enhancing mutations are concentrated in flexible loops. We also observed that Omicron variants BA.1 and BA.2 exhibit immune evasion through receptor binding domain (RBD) mutations, although these mutations reduce structural stability. Interestingly, the NTD has evolved to increase stability, compensating for the RBD instability and resulting in heightened overall infectivity. Our findings, available in SpikeScanDB, emphasize the importance of spike expression levels and compensatory mutations in both the NTD and RBD domains for shaping Omicron variant infectivity.

microbiology↗

bioRxiv2023

Single-cell transcriptome landscape of circulating CD4+ T cell populations in human autoimmune diseases

CD4+ T cells are a key mediator of various autoimmune diseases; however, how they contribute to disease development remains obscure primarily because of their cellular heterogeneity. Here, we evaluated CD4+ T cell subpopulations by decomposition-based transcriptome characterization together with canonical clustering strategies. This approach identified 12 independent transcriptional gene programs governing whole CD4+ T cell heterogeneity, which can explain the ambiguity of canonical clustering. In addition, we performed a meta-analysis using public single-cell data sets of over 1.8M peripheral CD4+ T cells from 953 individuals by projecting cells onto the reference and cataloged cell frequency and qualitative alterations of the populations in 20 diseases. The analyses revealed that the 12 transcriptional programs were useful in characterizing each autoimmune disease and predicting its clinical status. Moreover, genetic variants associated with autoimmune diseases showed disease-specific enrichment within the 12 gene programs. The results collectively provide a landscape of single-cell transcriptomes of CD4+ T cell subpopulations involved in autoimmune disease.

immunology↗

bioRxiv2022

Genetic and lipidomic identification of tuberculostearic acid as a controller of mycobacterial membrane compartmentalization

Mycobacteria diverge in a basic way from other bacterial and eukaryotic cells based on their distinct membrane structures. Here we report genome-wide transposon sequencing to discover the controllers of membrane compartmentalization in Mycobacterium smegmatis. cfa, a gene that encodes a putative cyclopropane-fatty-acyl-phospholipid synthase, shows the most significant effect on recovery from a membrane destabilizer, dibucaine. Lipidomic analysis of cfa deletion mutants demonstrates an essential role of Cfa in the synthesis of specific membrane lipids containing a C19:0 monomethyl-branched stearic acid. This molecule, also known as tuberculostearic acid (TBSA), has been intensively studied for decades due to its high level and genus-specific expression in mycobacteria. The proposed Cfa-mediated conversion of an unsaturation to a methylation matched well with its proposed role in lateral membrane organization, so we used new tools to determine the non-redundant effects of Cfa and TBSA in mycobacterial cells. cfa expression regulated major classes of membrane lipids including phosphatidylinositols, phosphatidylethanolamines and phosphatidylinositol mannosides. Cfa localized within the intracellular membrane domain (IMD), where it controls both cellular growth and recovery from membrane fluidization by facilitating subpolar localization of the IMD. Overall, cfa controls lateral membrane partitioning but does not detectably alter orthogonal transmembrane permeability. More generally, these results support the proposed role of the subpolar IMD as a subcellular site of mycobacterial control of membrane function. SignificanceMycobacteria remain major causes of disease worldwide based in part on their unusual membrane structures, which interface with the host. Here we discover the long sought biosynthetic origin of tuberculostearic acid (TBSA), a major fatty acid found selectively in mycobacteria, as well as its role in mycobacterial cells. The lipid is produced by an enzyme called Cfa, whose loss causes a growth defect and slow reformation of a membrane domain near the pole of the rod-shaped cell. Thus, our study offers mechanistic insights to the intrinsic molecular factors critical for mycobacterial plasma membrane partitioning.

microbiology↗