Biology subjects

Sanchez, J.

Publications and source records attributed to Sanchez, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Gut-derived ammonia modulates hypothalamic stress responses during viral infection

The gut-brain axis integrates microbial and host metabolism to regulate systemic physiology, yet its role during viral infection remains poorly defined. Viral infection induces behavioral changes and neuroendocrine stress responses accompanied by profound alterations in gut microbial metabolism. Here, we show that chronic viral infection in mice increases systemic levels of microbiota-derived ammonia in a CD8 T cell-dependent manner. Increased ammonia accumulates in the brain and selectively activates neurons within the paraventricular hypothalamus (PVH), driving corticosterone release into the circulation. Pharmacological inhibition of ammonia detoxification exacerbates these effects, leading to increased corticosterone levels, aggravated sickness behavior, and dampened antiviral responses. Together, these findings identify gut-derived ammonia as a previously unrecognized immunometabolic signal linking antiviral T cell responses to hypothalamic control of systemic stress during viral infection. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=186 SRC="FIGDIR/small/736796v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@1291dbcorg.highwire.dtl.DTLVardef@5f223corg.highwire.dtl.DTLVardef@1c0aafaorg.highwire.dtl.DTLVardef@190502_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

Boosting reversible photocontrol of a photoxenase by an engineered conformational shift

Our study successfully explores strategies to effectively improve the photocontrol efficiency of light-sensitive enzymes, dubbed photoxenases, with photoswitchable unnatural amino acids (UAAs). The engineering of photoxenases is a versatile method for the reversible photocontrol in various applications. To boost the photocontrol of an established allosteric and heterodimeric photoxenase based on imidazole glycerol phosphate synthase, we turned from an ineffective tuning of the UAA photochemistry to a semi-rational enzyme design. Remarkably, mutations at the catalytically important heterodimer interface increased the light-regulation factor (LRF) for the kcat up to ~100 with near-quantitative reversibility. Steady-state kinetic investigations combined with computationally determined correlation-based Shortest-Path-Map analysis and conformational landscapes revealed how photocontrol was altered in the two best hits. The LRF(kcat) correlated with a shift of a conformational equilibrium between an active and inactive population at the targeted active site and a tuned population productivity upon irradiation. While the overall reduced kcat values originated from a rewiring of the allosteric signal transmission, the increased LRF(kcat) resulted from a change in i) the size of the conformational shift, ii) the population productivity, and iii) the conformational heterogeneity. With this, our findings provide initial guidelines to boost photocontrol and underscore the power of photoxenase engineering.

biochemistry↗

bioRxiv2025

CD8+ T cells regulate the bioenergetic reprogramming of lymphoid organs and the heart during viral infection

The activation of the immune system is a bioenergetically-costly process1. Yet, essential bodily functions require a continuous energy supply, imposing energy constraints and trade-offs between competing processes2. Our understanding of the underlying bioenergetic adaptations reconciling rapid immune activation with other vital processes remains scarce. 3-6 Here, by using experimental models of viral infections, we identified an unexpected CD8+ T cell-driven redistribution of energy substrates between lymphoid organs and the heart. Viral infection promoted systemic hypoglycaemia and ketogenesis, together with systemic reallocation of energy substrates. Across organs analysed, secondary lymphoid organs and the heart showed the most dramatic changes. The former increased glucose uptake and oxidation while the heart showed the opposite, switching to preferential fatty acid utilization. These bioenergetic adaptations were absent in infected mice lacking CD8+ T cells or with T cells lacking the glucose transporter GLUT1. Pharmacological inhibition of fatty acid oxidation forced a systemic switch to glucose oxidation. This was associated with metabolic decompensation, reduced cardiac energetics, left ventricular stress, and mortality in otherwise nonlethal viral infections. Our results reveal how the energetic cost of immune cell activation imposes bioenergetic adaptations on non-lymphoid organs, posing a major challenge for the heart by completely relying on fatty acids.

immunology↗

bioRxiv2025

Assessment of chemical methods in the extraction of spore surface layers in Clostridioides difficile spores

Clostridioides difficile spores are essential for initiation, recurrence, and transmission of C. difficile infections (CDI). These outermost layers of the spore, the exosporium and spore coat, are responsible for initial interactions with the host and spore resistance properties respectively. Several spore coat /exosporium extraction methods have been utilized to study the spore surface with differing procedures making comparison across studies difficult. Here, we tested how commonly used exosporium and spore coat extraction methods, termed EBB, USD, and Laemmli, remove the spore coat and exosporium layers of C. difficile spores. We assessed the impact of these extraction methods on the spore through transmission electron microscopy, phase contrast microscopy, western blotting, and lysozyme triggered cortex degradation. Transmission electron microscopy shows that treatment with EBB and USD, completely remove the spore coat and exosporium layer while leaving decoated spores intact. Western blots revealed differences in the ability to extract spore surface protein markers (CdeC, CdeM, CotA). In addition, lysozyme was able to degrade the cortex in decoated spores regardless of the treatment employed. Western blot analysis of lysozyme treated-decoated spores, reveals that EBB and USD treatment allows for detection and release of the spore core germination protease, GPR. Our results provide a comparison of commonly used extraction methods in C. difficile spore biology, standardizing their impact in spore coat and exosporium extraction for use in future studies. ImportanceThe outermost layers of C. difficile spores, the exosporium and spore coat, are essential for the spores resistance properties and initial interactions with the host. However, there is variability in extraction protocols making it difficult to compare across studies. This work evaluates the commonly used extraction methods EBB, USD, and Laemmli at removing the exosporium and spore coat and provides a foundation for improved reproducibility. Here, we identified the effectiveness of these different extraction methods allowing us to better understand these techniques to accurately analyze spore surface in C. difficile spore research.

microbiology↗

bioRxiv2025

Molecular mechanisms of 10-Butyl Ether Minocycline (BEM), a novel non-antibiotic tetracycline, as a potential treatment for inflammatory and neuroimmune-related disorders.

The pleiotropy of minocycline (MINO), including anti-inflammatory, antioxidant, anti-migratory, anti-MMP, and neuroprotective effects, has been extensively reported. A novel non-antibiotic minocycline derivative, 10-butyl ether minocycline (BEM), was synthesized to retain the pleiotropy of minocycline while minimizing side effects such as antibiotic resistance and gut dysbiosis. Previously, we showed that BEM reduced alcohol consumption in dependent murine and porcine models of Alcohol Use Disorder (AUD). In this study, we investigated the molecular mechanisms of BEM to determine its potential as a therapeutic agent for neuroimmune and inflammatory conditions such as AUD. Here, we report that BEM showed a nearly complete loss of antimicrobial activity against E. coli, S. typhi, and C. albicans. BEM showed a dose-dependent reduction in cell viability as measured by the MTT assay, similarly to MINO. BEM also suppressed LPS-induced microglial activation as shown by reduced Iba1 expression in immunohistochemistry and western blot analyses. Inhibition of MMP-9 by BEM (IC50 = 42.2 {micro}M) was improved compared to MINO (IC50 = 60.3 {micro}M) while MMP-8 inhibition was moderate (IC50: BEM = 69.4 {micro}M; MINO = 45.4 {micro}M). BEM was found to be effective in inhibiting VEGF-induced endothelial cell migration and L-glutamine-induced ROS levels. Limited inhibition of 15-LOX activity was observed (IC50: BEM = 92.6 {micro}M; MINO = 65.6 {micro}M). BEM was not toxic to mitochondria, even at high concentrations (200 {micro}M). By eliminating antimicrobial properties while preserving therapeutic pleiotropy, BEM presents an advancement in the development of a promising candidate with multimodal mechanisms to treat neuroimmune-inflammatory pathologies. ImpactOur current approach to correlate non-antibacterial actions of BEM to MINOs established mechanistic effects will enable the informed use of BEM for several medical indications including for inflammation and neuroimmune conditions. The focus on BEMs multimodal actions and long-term safety during drug discovery represents a paradigm shift toward complex therapeutic drug development and repositioning, improving upon traditional singular high-affinity target-based approaches. Such new drug discovery attempts could potentially enhance treatment relevance in complex disorders with multiple targets and theoretically guide the creation of second-generation analogs. Significance statementWe report mechanisms of action for BEM, a minocycline analog under evaluation for the treatment of Alcohol Use Disorder, which may also show efficacy for other complex disease processes that involve inflammatory or neuroimmune components. We show that BEM had a nearly complete loss of antimicrobial action, yet retained the pleiotropy of MINO, likely making it a better multimodal therapeutic for long-term treatment of complex diseases with neuroimmune-related components. Visual abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/661183v3_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@1ade11corg.highwire.dtl.DTLVardef@961d1borg.highwire.dtl.DTLVardef@15101f8org.highwire.dtl.DTLVardef@158810e_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2025

Uncovering the hidden diversity and functional roles of root endophytic Streptomyces under drought stress

The genus Streptomyces has consistently been found to be enriched in drought-stressed plant root microbiomes, yet the ecological basis and functional variation underlying this enrichment at the strain and isolate level remain unclear. Using two 16S rRNA sequencing methods with different levels of taxonomic resolution, we confirmed drought-associated enrichment of Streptomyces in field-grown sorghum roots and identified five closely related but distinct ASVs belonging to the genus with variable drought enrichment patterns. From a culture collection of sorghum root endophytes, we selected 12 Streptomyces isolates representing these ASVs for phenotypic and genomic characterization. Whole-genome sequencing revealed substantial variation in gene content, even among closely related isolates, and exometabolomic profiling showed distinct metabolic responses to media supplemented with drought-versus well-watered root tissue. Traits linked to drought survival, including osmotic stress tolerance, siderophore production, and carbon utilization, varied widely among isolates and were not phylogenetically conserved. Using a broader panel of 48 Streptomyces, we demonstrate that drought enrichment (DE) scores, determined through mono-association experiments in gnotobiotic sorghum systems, showed high variability and lacked correlation with plant growth promotion. Pangenome-wide association identified gene clusters involved in osmolyte transport (e.g., proP) and membrane biosynthesis (e.g., fabG) as positively associated with DE, though most associations lacked phylogenetic signal. Collectively, these results demonstrate that Streptomyces enrichment under drought is not a conserved genus-level trait but is instead strain-specific and functionally heterogeneous. Furthermore, enrichment in the root microbiome does not predict beneficial effects on plant growth. This work underscores the need to resolve functional traits at the strain level and highlights the complexity of microbe-host-environment interactions under abiotic stress. ImportanceUnderstanding how beneficial microbes respond to drought is critical for developing microbiome-based strategies to enhance crop resilience. The Streptomyces genus is consistently enriched in drought-stressed root microbiomes, yet our study reveals that this enrichment is not a uniform isolate-level response. Instead, we show that drought enrichment is highly strain-specific and functionally diverse, with key associated traits like osmotic stress tolerance and siderophore production varying independently of phylogeny. Importantly, enrichment does not predict a microbes ability to promote plant growth under stress. By integrating high-resolution taxonomic, genomic, and metabolomic approaches, we uncover the ecological and functional complexity underlying Streptomyces responses to drought. These findings emphasize the importance of resolving microbial function at the strain level and caution against assuming functional traits based on taxonomic identity itself.

microbiology↗

bioRxiv2025

Microbiota comparison of individual and pooled cow fecal samples from PEI dairy farms

The aim of this study was to compare the microbiota diversity captured in individual versus pooled fecal samples from dairy cattle and evaluate the feasibility of using pooled sampling to assess the microbiota of dairy cattle at the herd level. A cross-sectional study used animals from 28 Prince Edward Island (PEI) dairy farms in Canada. The farms were visited between July and December 2020. Both free-stall and tie-stall housing systems were eligible. Manure samples from dry and lactating cows were obtained. Then, approximately 20g of fecal samples from each group were pooled. DNA extractions on all subsamples were performed using the Qiagen PowerMax Soil Kit and submitted for 16S rRNA gene amplification and sequencing. Operational taxonomic units were determined, and four alpha diversity indices were computed. A total of 128 and 132 manure samples from pos and prepartum cows, respectively, were analyzed. Mixed-effects random slope models were employed, incorporating herd-level random effects to estimate the correlation among individual samples and between individual and pooled samples. The estimated Shannon observed features, Pielou evenness and Faith PD were 5.9, 580, 0.93 and 44.1 from the individual and 5.93, 587, 0.93 and 45.5 from the pooled samples, respectively. All alpha diversity metrics were not significantly different between individual and pooled samples. Overall, pooled sampling does not significantly affect diversity and provides comparable results with individual samples, though it tends to show slightly higher diversity in some indices. This sampling strategy could be used in microbiota studies of dairy herds.

microbiology↗

bioRxiv2025

fMRIPrep Lifespan: Extending A Robust Pipeline for Functional MRI Preprocessing to Developmental Neuroimaging

The adoption of a standardized preprocessing workflow is vital for fostering community, sharing, and reproducibility. fMRIPrep has been a critical advancement towards this end, however, it is limited in its capacity to be applied to data across the lifespan, starting from infancy. Here, we introduce fMRIPrep Lifespan, an extension of fMRIPrep that extends the standardized processing from childhood to senescence to include neonatal, infant, and toddler structural and functional MRI data preprocessing. This effort involves a NiPreps integration of 1) a workflow akin to fMRIPrep optimized for MRI data in the first years of life (previously NiBabies) and 2) upstream enhancements to the entire NiPreps suite, including multi-echo data processing, modularization of workflow components, and convergence of processing with other popular workflows (ABCD-BIDS, Human Connectome Project Pipelines). Using data from the Baby Connectome Project (participants 1-43 months of age), we demonstrate that fMRIPrep Lifespan produces high-quality outputs across a wide age range. Moving forward, the scalable, modular infrastructure of fMRIPrep Lifespan will ensure adaptability to data from birth to old age while maintaining robust and reproducible frameworks for functional MRI research across the lifespan.

bioinformatics↗

bioRxiv2025

Microbes regulate glomerular filtration rate in health and chronic kidney disease in mice

Microbes are implicated in a variety of host physiological and pathophysiological processes. In this study, we tested the hypothesis that microbes modulate glomerular filtration rate (GFR). Microbiota were depleted in mice using oral antibiotics (ABX; a mixture of ampicillin, neomycin, and vancomycin). GFR was significantly increased in ABX-treated mice. To confirm that the increase in GFR was due to decreased microbes, we also measured GFR in germ-free (GF) mice. GFR was increased in GF mice as compared to both conventional and conventionalized GF (CGF) mice. We next used the murine adenine diet model to ask if suppressing gut microbes with ABX also increases GFR in a setting of chronic kidney disease (CKD), where GFR is impaired. In females on an adenine diet, ABX increased GFR versus adenine alone on weeks 4 and 6. In males, ABX elevated GFR on week 2. Adenine diet significantly increased plasma creatinine and kidney fibrosis; this was suppressed by ABX in both sexes. To explore the mechanism of this increase, we tested the hypothesis that altered tubuloglomerular feedback (TGF) contributes to elevated GFR using the sodium-glucose cotransporter 2 (SGLT2) inhibitor empagliflozin (EMPA); EMPA impairs Na+ reabsorption in the proximal tubule, altering TGF. We found that EMPA impaired ABX-induced GFR increases on week 3 but not week 5, suggesting that altered TGF contributes to the initial increase in GFR. In conclusion, the microbiome plays a key role in setting baseline GFR by a mechanism which partially involves TGF, and, suppressing gut microbes can elevate GFR even in CKD. Translational StatementThis study reports that GFR is elevated when gut microbes are absent or suppressed in mice, indicating a role for commensal microbes to help establish baseline GFR in health. Likewise, suppressing gut microbes also elevates GFR in a chronic kidney disease model. These data suggest a future possibility of modulating the commensal microbes to elevate GFR in a clinical setting.

physiology↗

bioRxiv2025

Functional profiling reveals a non-enzymatic role of NUDT5 in repressing purine de novo synthesis

Folate metabolism is intricately linked to purine de novo synthesis through the incorporation of folate-derived one-carbon units into the purine scaffold. Here, we investigate the chemical and genetic dependencies caused by mutations in the folate enzyme MTHFD1 and discover a key role for Nudix hydrolase 5 (NUDT5) in regulating purine de novo synthesis. Through genetic knockout and development of a selective chemical NUDT5 degrader, we uncover an unprecedented scaffolding role rather than NUDT5 enzymatic activity is responsible for this phenotype. We find that NUDT5 interacts with the rate-limiting enzyme of purine de novo synthesis, PPAT, to repress the pathway in response to elevated purine levels. Our findings establish NUDT5 as an important regulator of purine de novo synthesis and elucidate its role in mediating sensitivities to 6-thioguanine in cancer treatment and to adenosine in MTHFD1 deficiency.

molecular biology↗

bioRxiv2025

Distal mutations enhance catalysis in designed enzymes by facilitating substrate binding and product release

The role of amino-acid residues distant from an enzymes active site in facilitating the complete catalytic cycle--including substrate binding, chemical transformation, and product release-- remains poorly understood. Here, we investigate how distal mutations promote the catalytic cycle by engineering mutants of three de novo Kemp eliminases containing either active-site or distal mutations identified through directed evolution. Kinetic analyses, X-ray crystallography, and molecular dynamics simulations reveal that while active-site mutations create preorganized catalytic sites for efficient chemical transformation, distal mutations enhance catalysis by facilitating substrate binding and product release through tuning structural dynamics to widen the active-site entrance and reorganize surface loops. These distinct contributions work synergistically to improve overall activity, demonstrating that a well-organized active site, though necessary, is not sufficient for optimal catalysis. Our findings reveal critical roles that distal residues play in shaping the catalytic cycle to enhance efficiency, yielding valuable insights for enzyme design.

biochemistry↗

bioRxiv2024

Ubiquitination mediates protein localization in RNA virus-infected cells

Viruses trigger monocytes proinflammatory and antiviral responses. Ubiquitination, a post-translational modification primarily marking proteins for degradation, regulates cellular responses to virus infection. However, a comprehensive analysis of virus-induced ubiquitination in monocytes is lacking. Here we identified a widespread increase of ubiquitination under viral RNA challenge or influenza infection in monocytes. Systematic proteome studies revealed that influenza infection elicits dynamic ubiquitinome alterations, with a notable transition from early to late stage. Most of this increased ubiquitination is not proteolytic and targets proteins involved in subcellular localization, such as the mitochondrial protein COA7 which, when ubiquitinated during infection, translocates to the nucleus and inhibits stress granules formation and TNF- expression. Blocking ubiquitination halts viral ribonucleoproteins nuclear export, highlighting ubiquitinations importance for protein localization during virus infection.

molecular biology↗

bioRxiv2024

Nutrient competition predicts gut microbiome restructuring under drug perturbations

Human gut commensal bacteria are routinely exposed to various stresses, including therapeutic drugs, and collateral effects are difficult to predict. To systematically interrogate community-level effects of drug perturbations, we screened stool-derived in vitro communities with 707 clinically relevant small molecules. Across [~]5,000 community-drug interaction conditions, compositional and metabolomic responses were predictably impacted by nutrient competition, with certain species exhibiting improved growth due to adverse impacts on competitors. Changes to community composition were generally reversed by reseeding with the original community, although occasionally species promotion was long-lasting, due to higher-order interactions, even when the competitor was reseeded. Despite strong selection pressures, emergence of resistance within communities was infrequent. Finally, while qualitative species responses to drug perturbations were conserved across community contexts, nutrient competition quantitatively affected their abundances, consistent with predictions of consumer-resource models. Our study reveals that quantitative understanding of the interaction landscape, particularly nutrient competition, can be used to anticipate and potentially mitigate side effects of drug treatment on the gut microbiota.

microbiology↗

bioRxiv2024

How does the ion concentration affect the functions of kinesin BimC

BimC family proteins are bipolar motor proteins belonging to the kinesin superfamily which promote mitosis by crosslinking and sliding apart antiparallel microtubules. Understanding the binding mechanism between the kinesin and the microtubule is crucial for researchers to make advances in the treatment of cancer and other malignancies. Experimental research has shown that the ion concentration affects the function of BimC significantly. But the insights of the ion-dependent function of BimC remain unclear. By combining molecular dynamics (MD) simulations with a series of computational approaches, we studied the electrostatic interactions at the binding interfaces of BimC and the microtubule under different KCl concentrations. We found the electrostatic interaction between BimC and microtubule is stronger at 0 mM KCl compared to 150 mM KCl, which is consistent with experimental conclusions. Furthermore, important salt bridges and residues at the binding interfaces of the complex were identified, which illustrates the details of the BimC-microtubule interactions. Molecular dynamics analyses of salt bridges identified that the important residues on the binding interface of BimC are positively charged, while those residues on the binding interface of the tubulin heterodimer are negatively charged. The finding in this work reveals some important mechanisms of kinesin-microtubule binding, which helps the future drug design for cancer therapy.

biophysics↗

bioRxiv2024

Role of Glycogen Metabolism in Clostridioides difficile Virulence

Glycogen plays a vital role as an energy reserve in various bacterial and fungal species. Clostridioides difficile possesses a glycogen metabolism operon that contains genes for both glycogen synthesis and utilization. In our investigation, we focused on understanding the significance of glycogen metabolism in the physiology and pathogenesis of C. difficile. To explore this, we engineered a C. difficile JIR8094 strain lacking glycogen synthesis capability by introducing a group II intron into the glgC gene, the operons first component. Quantification of intracellular glycogen levels validated the impact of this modification. Interestingly, the mutant strain exhibited a 1.5-fold increase in toxin production compared to the parental strain, without significant changes in sporulation rate. Our analysis also revealed that wild-type C. difficile spores contained glycogen, whereas spores from the mutant strain lacking stored glycogen showed increased sensitivity to physical and chemical treatments and had a shorter storage life. By suppressing glgP expression, the gene coding for glycogen-phosphorylase, via CRISPRi, we demonstrated that glycogen accumulation but not the utilization is needed for spore resilience in C. difficile. Transmission Electron Microscopy (TEM) analysis revealed a significantly lower core/cortex ratio in glgC mutant strain spores. In hamster challenge experiments, both the parental and glgC mutant strains colonized hosts similarly; however, the mutant strain failed to induce infection relapse after antibiotic treatment cessation. These findings highlight the importance of glycogen metabolism in C. difficile spore resilience and suggest its role in disease relapse. ImportanceThis study on the role of glycogen metabolism in C. difficile highlights its critical involvement in the pathogens energy management, its pathogenicity and resilience. Our results also revealed that glycogen presence in spores is pivotal for their structural integrity and resistance to adverse conditions, which is essential for their longevity and infectivity. Importantly, the inability of the mutant strain to cause infection relapse in hamsters post-antibiotic treatment pinpoints a potential target for therapeutic interventions, highlighting the importance of glycogen in disease dynamics. This research thus significantly advances our understanding of C. difficile physiology and pathogenesis, offering new avenues for combating its persistence and recurrence.

microbiology↗

bioRxiv2023

The Horizontal Ladder Test (HLT) protocol: A novel, optimized, and reliable means of assessing motor coordination in Sus scrofa domesticus

Pigs can be an important model for preclinical biological research, including neurological diseases such as Alcohol Use Disorder. Such research often involves longitudinal assessment of changes in motor coordination as the disease or disorder progresses. Current motor coordination tests in pigs are derived from behavioral assessments in rodents and lack critical aspects of face and construct validity. While such tests may permit for the comparison of experimental results to rodents, a lack of validation studies of such tests in the pig itself may preclude the drawing of meaningful conclusions. Here, we present a novel, optimized, and reliable horizontal ladder test (HLT) test protocol for evaluating motor coordination in pigs and an initial validation of its construct validity using voluntary alcohol consumption as an experimental manipulation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=157 SRC="FIGDIR/small/571517v2_ufig1.gif" ALT="Figure 1"> View larger version (101K): org.highwire.dtl.DTLVardef@167924eorg.highwire.dtl.DTLVardef@b674f2org.highwire.dtl.DTLVardef@b7c369org.highwire.dtl.DTLVardef@7e5a42_HPS_FORMAT_FIGEXP M_FIG C_FIG

animal behavior and cognition↗

bioRxiv2023

A mutant fitness compendium in Bifidobacteria reveals molecular determinants of colonization and host-microbe interactions

Bifidobacteria commonly represent a dominant constituent of human gut microbiomes during infancy, influencing nutrition, immune development, and resistance to infection. Despite interest as a probiotic therapy, predicting the nutritional requirements and health-promoting effects of Bifidobacteria is challenging due to major knowledge gaps. To overcome these deficiencies, we used large-scale genetics to create a compendium of mutant fitness in Bifidobacterium breve (Bb). We generated a high density, randomly barcoded transposon insertion pool in Bb, and used this pool to determine Bb fitness requirements during colonization of germ-free mice and chickens with multiple diets and in response to hundreds of in vitro perturbations. To enable mechanistic investigation, we constructed an ordered collection of insertion strains covering 1462 genes. We leveraged these tools to improve models of metabolic pathways, reveal unexpected host- and diet-specific requirements for colonization, and connect the production of immunomodulatory molecules to growth benefits. These resources will greatly reduce the barrier to future investigations of this important beneficial microbe.

microbiology↗

bioRxiv2023

Coxsackievirus infection induces direct pancreatic β-cell killing but poor anti-viral CD8+ T-cell responses

Coxsackievirus B (CVB) infection of pancreatic {beta} cells is associated with {beta}-cell autoimmunity. We investigated how CVB impacts human {beta} cells and anti-CVB T-cell responses. {beta} cells were efficiently infected by CVB in vitro, downregulated HLA Class I and presented few, selected HLA-bound viral peptides. Circulating CD8+ T cells from CVB-seropositive individuals recognized only a fraction of these peptides, and only another sub-fraction was targeted by effector/memory T cells that expressed the exhaustion marker PD-1. T cells recognizing a CVB epitope cross-reacted with the {beta}-cell antigen GAD. Infected {beta} cells, which formed filopodia to propagate infection, were more efficiently killed by CVB than by CVB-reactive T cells. Thus, our in-vitro and ex-vivo data highlight limited T-cell responses to CVB, supporting the rationale for CVB vaccination trials for type 1 diabetes prevention. CD8+ T cells recognizing structural and non-structural CVB epitopes provide biomarkers to differentially follow response to infection and vaccination.

immunology↗