Biology subjects

Hitch, T. C. A.

Publications and source records attributed to Hitch, T. C. A..

10 recordsLinked to original sources

bioRxiv2026

A microbiota-derived protease links phage susceptibility to host epithelial responses

Bacteriophages are major ecological drivers of gut microbial ecology, yet whether bacterial mechanisms that determine phage susceptibility have consequences for the mammalian host remains poorly understood. Here, we identify dipeptidyl peptidase 11 (Dpp11a), the predominant active serine protease of the prevalent gut commensal Phocaeicola vulgatus, as an unexpected bacterial defence factor. Dpp11a protects against environmental proteases and confers resistance to bacteriophage infection. Metatranscriptomic analyses further reveal increased expression of both dpp11a and P. vulgatus-associated phage transcripts in ulcerative colitis stool samples, indicating that both components of this interaction are transcriptionally active in disease-associated human microbiomes. Using the microfluidic gut-on-a-chip co-culture model HuMiX, we show that the absence of Dpp11 is accompanied by altered epithelial tight-junction remodelling during phage-bacterial infection. Together, our findings reveal that the consequences of bacterial phage defence can extend beyond phage-bacterium interactions to the mammalian epithelium.

microbiology↗

bioRxiv2026

Mixed acid fermentation products from Lachnospira eligens counteract myotube atrophy

IntroductionAcute myeloid leukemia (AML) is a hematological malignancy associated with muscle wasting. As the relative abundance of Lachnospira eligens was reduced in patients with AML compared to healthy individuals and correlated positively with muscle strength, we hypothesized that L. eligens positively impacts the muscle through the production of small metabolites reaching the systemic circulation. MethodsL. eligens levels were analyzed in two additional independent cohorts. Six L. eligens isolates were characterized through whole-genome sequencing to select clinically relevant strains. The composition of their culture supernatant was analyzed by metabolomics. The impact of L. eligens supernatant on dexamethasone- and interleukin-6-atrophied murine myotubes was assessed. Bioactive metabolites and their production mechanism were identified using among others bioactivity-guided fractionation. The underlying mechanism was also explored on the host side through myotubes transcriptome analysis and metabolic flux analysis. The relevance of bioactive metabolites and their production mechanism was evaluated through clinical data and samples analyses and in a mouse model of leukemia. ResultsThe levels of L. eligens are reduced in independent cohorts of patients with AML and its supernatant counteracts myotube atrophy. This anti-atrophic effect, conserved between strains of the same species, depends on the occurrence of mixed acid fermentation (MAF) in anoxic culture conditions and the presence of its acid end-products acetate, formate and D-lactate. Consistent with those results, blood levels of acetate are decreased and the relative abundance of fecal bacteria capable of performing aerobic respiration is increased in patients with AML. However, bacterial supernatant failed to prevent muscle atrophy and weakness in leukemic mice, likely due to insufficient sustained elevation of acid end-products in the blood. ConclusionThis work reveals the anti-atrophic effect of MAF end-products on myotubes and suggests the importance of considering gut electron acceptor levels (e.g. O2) in disorders affecting muscle health. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/729780v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@112529eorg.highwire.dtl.DTLVardef@1ee1929org.highwire.dtl.DTLVardef@b5f389org.highwire.dtl.DTLVardef@187bc40_HPS_FORMAT_FIGEXP M_FIG Mixed acid fermentation products from Lachnospira eligens counteract myotube atrophy. Our study suggests that gut anaerobiosis is disrupted in treatment-naive patients with acute myeloid leukemia (AML), leading to decreased circulating acetate levels and a reduced relative abundance of L. eligens, which significantly correlated with muscle strength. In line with this framework, in vitro experiments demonstrate that the culture supernatant of L. eligens, which contains mixed acid fermentation (MAF) end-products such as acetate, effectively counteracts C2C12 myotube atrophy in the presence of pro-atrophying stimuli. Further mechanistic experiments indicate a causal role for MAF end-products in this anti-atrophying effect. Created with BioRender.com. Legend: solid frames: experimental results; dashed frames: hypothetical conclusions derived from results; black solid arrow: established correlation; black dashed arrows: hypothetical causation. C_FIG

microbiology↗

bioRxiv2025

Function-Based Selection of Synthetic Communities Enables Mechanistic Microbiome Studies

Understanding the complex interactions between microbes and their environment requires robust model systems such as synthetic communities (SynComs). We developed a functionally directed approach to generate SynComs by selecting strains that encode key functions identified in metagenomes. This approach enables the rapid construction of SynComs tailored to any ecosystem. To optimize community design, we implemented genome-scale metabolic models, providing in silico evidence for cooperative strain coexistence prior to experimental validation. Using this strategy, we designed multiple host-specific SynComs, including those for the rumen, mouse, and human microbiomes. By weighting functions differentially enriched in diseased versus healthy individuals, we constructed SynComs that capture complex host-microbe interactions. Notably, we designed an inflammatory bowel disease SynCom of 10 members that successfully induced colitis in gnotobiotic IL10-/- mice, demonstrating the potential of this method to model disease-associated microbiomes. Our study establishes a targeted framework for designing SynComs to advance mechanistic insights into host-microbe interactions. HighlightsO_LIAutomated functional selection of SynComs based on metagenomic data C_LIO_LIEcosystem-specific SynComs capture the functional landscape of microbiota C_LIO_LIColitis-inducing SynCom developed as a model for inflammatory bowel diseases C_LI

microbiology↗

bioRxiv2025

Benchmarking of shotgun sequencing depth highlights strain-level limitations of metagenomic analysis

Shallow metagenomics promises taxonomic and functional insights into samples at an affordable price. To determine the depth of sequencing required for specific analysis, benchmarking is required using defined microbial communities. We used complex mixtures of DNA from cultured gut bacteria and analysed taxonomic composition, strain-level resolution, and functional profiles at up to eleven sequencing depths (0.1-50.0 Gb). Reference-based analysis provided accurate taxonomic, and strain-level insights at 0.5-1.0 Gb. In contrast, de-novo metagenome-assembled genome (MAG) reconstruction required deep sequencing (>10 Gb), and even high-quality MAGs were chimeric, with 54.5 to 81.8 % accurately representing the original strains, depending on the bioinformatic approach used. However, the issue of chimeric MAGs can be reduced by using strain-aware assembly methods or long-read sequencing. Functionally, 2 Gb provided reliable insights at the pathway level, but sufficient proteome coverage was only achieved at or above 10 Gb. Library preparation and host DNA contamination were identified as confounders in shallow metagenomic analysis. This comprehensive analysis using complex mock communities provides guidance to an increasing community of scientists interested in using shallow metagenomics, and highlights the limitations of MAGs in accurately capturing strain-level diversity.

microbiology↗

bioRxiv2025

Robust genome-based delineation of bacterial genera

BackgroundGenomic analysis has become essential in bacterial taxonomy, enabling fast classification of bacteria. However, quantifiable measures to make informed decision on the taxonomic placement of bacterial taxa above the level of species are rare, which hampers the stability of knowledge in databases and articles. In this work, we focused on bacterial classification at the genus level and revisited the concept of Percentage Of Conserved Proteins (POCP). Whilst POCP is broadly used as an overall genome relatedness index, the underlying tool and method of calculation differ, resulting in disparate implementations and misnomers unfit for the increasing wealth of public genomes available. ResultsWe evaluated 10 protein alignments methods and found evidence for a scalable yet robust alternative to BLASTP for POCP based on 2,358,466 pairwise comparisons of 4,767 genomes across 35 families. However, we showed that certain combinations of tools and parameters are not suited for POCP calculations owing to drastic over-or underestimation. Therefore, we bring forward a clearer definition of POCP using only unique matches, termed POCPu, that showed better genus delineation than with POCP. We suggested tentative family-specific thresholds when the standard of 50% was not resolutive enough. ConclusionsWe propose that faster bacterial genus delineation should be calculated with DIAMOND using very-sensitive settings and only unique matches (POCPu). We advise microbiologists to carefully assess that the tools used for genus assignment match their biological assumptions to avoid misguided inferences.

microbiology↗

bioRxiv2024

Broad diversity of human gut bacteria accessible via a traceable strain deposition system

Numerous bacteria in the human gut microbiome remain unknown and/or have yet to be cultured. While collections of human gut bacteria have been published, few strains have been made publicly available. A major hurdle in making strains publicly available is their deposition to public culture collections. We propose a framework for the bulk-deposition of strains to culture collections, which removes many of the barriers previously identified (www.dsmz.de/bulk-deposit). Using this bulk-deposition system we have created a publicly available collection of human gut isolates. The Human intestinal Bacteria Collection (HiBC) (www.hibc.rwth-aachen.de) contains 340 strains representing 198 species within 29 families and 7 phyla, of which 29 previously unknown species are taxonomically described and named. These included two butyrate-producing species of Faecalibacterium and new dominant species associated with health and inflammatory bowel disease, Ruminococcoides intestinale and Blautia intestinihominis, respectively. Plasmids were prolific within the HiBC isolates, with almost half (46%) of strains containing plasmids, with a maximum of six within a strain. This included a broadly occurring plasmid (pBAC) that exists in three diverse forms across Bacteroidales species. Megaplasmids were identified within two strains, the pMMCAT megaplasmid is globally present within multiple Bacteroidales species. This collection of easily searchable and publicly available gut bacterial isolates will facilitate functional studies of the gut microbiome.

microbiology↗

bioRxiv2024

Lineage-specific microbial protein prediction enables large-scale exploration of protein ecology within the human gut

Microbes use a range of genetic codes and gene structures, yet these are ignored during metagenomic analysis. This causes spurious protein predictions, preventing functional assignment which limits our understanding of ecosystems. To resolve this, we developed a lineage-specific gene prediction approach that uses the correct genetic code based on the taxonomic assignment of genetic fragments, removes partial predictions, and optimises prediction of small proteins. Applied to 9,634 metagenomes and 3,594 genomes from the human gut, this approach increased the landscape of captured expressed microbial proteins by 78.9%, including previously hidden functional groups. Optimised small protein prediction captured 3,772,658 small protein clusters, many with antimicrobial activity. Integration of the protein sequences and sample metadata into a tool, InvestiGUT, enables association of protein prevalence with host parameters. Accurate prediction of proteins is critical for understanding the functionality of microbiomes, hence this work will enhance understanding mechanistic interactions between microbes and hosts.

bioinformatics↗

bioRxiv2022

Enhanced cultured diversity of the mouse gut microbiota enables custom-made synthetic communities

Microbiome research is hampered by the fact that many bacteria are still unknown and by the lack of publicly available isolates. Fundamental and clinical research is in need of comprehensive and well-curated repositories of cultured bacteria from the intestine of mammalian hosts. In this work, we expanded the mouse intestinal bacterial collection (www.dsmz.de/miBC) to 212 strains, all publicly available and taxonomically described. This includes the study of strain-level diversity, small-sized bacteria, and the isolation and characterization of the first cultured members of one novel family, 10 novel genera, and 39 novel species. We demonstrate the value of this collection by performing two studies. First, metagenome-educated design allowed establishing custom synthetic communities (SYNs) that reflect different susceptibilities to DSS-induced colitis. Second, nine phylogenetically and functionally diverse species were used to amend the Oligo-Mouse Microbiota (OMM)12 model [Brugiroux et al. 2016 Nat Microbiol]. These strains compensated for differences observed between gnotobiotic OMM12 and specific pathogen-free (SPF) mice at multiple levels, including body composition and immune cell populations (e.g., T-cell subtypes) in the intestine and associated lymphoid tissues. Ready-to-use OMM stocks are available to the community for use in future studies. In conclusion, this work improves our knowledge of gut microbiota diversity in mice and enables functional studies via the modular use of isolates.

microbiology↗

bioRxiv2020

Diversity and function of microbial lipases within the mammalian gut

Triglycerides are a major dietary component and excessive intake has been linked to many health conditions. While the impact of host lipases on fat degradation and absorption have been well documented, bacterial lipases have been largely ignored. Here, we identified the landscape of microbial lipases within the gut of mice, pigs, and humans. In total, 373 microbial lipases were identified and formed two major clusters based on protein sequences, with the sequences containing highly conserved motifs. Metagenomic and cultivation approaches identified lipase-positive bacteria as diverse, occurring within seven phyla, and abundant within the human gut (9.1% {+/-} 4.6%). Pathway analysis in lipase-positive species identified a low prevalence of fatty acid utilisation and a higher frequency of glycerol metabolism. In vitro testing with stable isotopes confirmed that human gut bacteria can utilise the released glycerol. Overall, this study highlights the role of bacterial lipolytic activity in triglycerides metabolism within the gut.

microbiology↗

bioRxiv2019

Sporaefaciens musculi gen. nov., sp. nov., a novel bacterium isolated from the caecum of an obese mouse

A bacterial strain, designated WCA-9-b2, was isolated from the caecal content of an 18-week-old obese C57BL/6NTac male mouse. According to phenotypic analyses, the isolate is rod-shaped, Gram-positive, strictly anaerobic, spore-forming and non-motile under the conditions tested. Bacterial colonies were irregular and non-pigmented. Analysis of the 16S rRNA gene indicated that the isolate belonged to the family Lachnospiraceae with Clostridium scindens ATTC 35704 (94.9% sequence identity) and Dorea formicigenerans ATCC 27755 (94.8%) being the closest relatives. Whole genome sequencing showed average nucleotide identity (ANI) ranging from 69.80-74.23% and percentage of conserved proteins (POCP) values < 50% against the nine closest relatives. The genome-based G+C content of genomic DNA was 44.4%. The predominant metabolic end products of glucose fermentation were acetate and succinate. Based on these data, we propose that strain WCA-9-b2 represents a novel species within a novel genus, for which the name Sporaefaciens musculi gen. nov., sp. nov. is proposed. The type strain is WCA-9-b2T (=DSM 106039T = CCUG pending IDT). RepositoriesThe GenBank accession number for the 16S rRNA gene sequence of strain WCA-9-b2T is MN756014, and the accession number for the genome assembly is PRJNA592877. Raw sequencing Illumina NextSeq (PRJEB35655) and ONT MinION (PRJEB35656) data can be accessed at EMBL-EBI.

microbiology↗