The specific role of γδ T cells in non-tuberculous mycobacteria (NTM) infections remains incompletely understood. Here we characterized the immune landscape of pulmonary tissues from a Mycobacterium abscessus-infected NTM mice model. Interleukin-17 (IL-17)A+ γδ T cells were essential for controlling M. abscessus infection by directly eliminating extracellular bacteria. These cells conferred protective immunity in two mouse models of infection combined with either pulmonary fibrosis or a lack of functional interferon-gamma (IFNγ)-mediated immunity. IL-17A+ γδ T cells exerted bactericidal activity via granzyme B-dependent cytotoxic pathways. Absence of IL-17A substantially impaired bacterial recognition and cytotoxic function of IL-17A+ γδ T cells, highlighting the cell-intrinsic requirement for IL-17A signalling in γδ T cell activation. M. abscessus recognition through Toll-like receptor 2 induced macrophage production of IL-1β and IL-23, promoting the expansion and activation of IL-17A+ γδ T cells. Single-cell RNA sequencing on human peripheral blood mononuclear cells suggests that anti-IFNγ autoantibodies compromise γδ T cell function. Our findings establish IL-17A+ γδ T cells as a promising therapeutic target for NTM infections, particularly when IFNγ-dependent immunity is compromised.
The anti-apoptotic molecule BCL-2 favours the maintenance of the CD4+ T-cell reservoir during HIV infection. Whether inhibition of BCL-2 can lead to long-term reduction of the HIV reservoir is unclear. Here we initiated antiretroviral therapy (ART) in 24 simian immunodeficiency virus (SIV)-infected rhesus macaques at 14 days post infection (p.i.), alone or combined with a 10-day treatment of venetoclax or venetoclax and CD8α depletion, with a follow-up to day 294 p.i. We report a rapid and sustained reduction of the intact SIV reservoir in venetoclax-treated rhesus macaques in blood and lymph nodes. CD4+ T cells that persisted after venetoclax treatment showed partial reduction in apoptotic sensitivity in ex vivo assays. These exhibited elevated expression of anti-apoptotic BCL-2 and BCL-xL, and showed reduced expression of pro-apoptotic molecules such as PUMA. These findings support the rationale for extended venetoclax dosing and suggest that combining BCL-2 inhibition with agents targeting additional anti-apoptotic molecules could enhance clearance of the viral reservoir in HIV cure strategies.
Integrons capture functional genes in mobile genetic elements called integron cassettes, which represent an untapped source of genes of biotechnological interest. Here we present two tools, cassette gatherer and cassette hunter, that enable high-throughput establishment of gene libraries either from genetically tractable strains or directly from DNA. We re-engineered a class 1 integron into counterselection markers on a plasmid or on the chromosome of a naturally competent Vibrio cholerae, which enabled capture of single cassettes in a sequence- and function-independent manner. When applied to Vibrio strains and genomic libraries, our tools recovered hundreds of single cassettes per assay with more than 99% specificity. We further subjected the library of cassettes generated by the hunter and gatherer tools to screens against phages ICP2 and T4, and identified nine phage-defence systems, including five previously undescribed. These tools enable rapid and large-scale recovery of integron cassettes that could be leveraged for functional gene discovery.
Platforms for community-led but globally connected knowledge and resource sharing can move us towards more equitable and more effective research and solutions for infectious diseases.
Recovering high-quality microbial genomes from metagenomic sequencing data is essential for accurate profiling and understanding microbial variation. However, existing clustering methods often suffer from limited accuracy and scalability. Here we present MetaCAT (Metagenome Clustering and Association Tool), a framework that combines recovery of microbial genomes from metagenomic data and analysis of their associations with host traits. MetaCAT incorporates a Sparse Weighted Dirichlet Process Gaussian Mixture Model (SWDPGMM) to accurately and efficiently decompose complex datasets and combines k-mer frequency with read coverage to improve genome reconstruction. It also provides a dedicated workflow for microbial single-nucleotide polymorphism identification and metagenome-wide association studies with the host. MetaCAT outperforms existing methods in both clustering accuracy and computational efficiency across diverse datasets. Using metagenomic data from colorectal cancer cohorts, it revealed previously unrecognized marker species and microbial single-nucleotide polymorphisms associated with colorectal cancer. MetaCAT provides a scalable framework for microbial community profiling and advances our understanding of host-microbe interactions.
The journey of Mycobacterium tuberculosis between hosts in aerosol droplets has often been viewed as a passive phase in its life cycle. Recent evidence shows that the inevitable desiccation associated with aerosol transmission triggers a program that imperfectly repairs DNA damage, generating genetic diversity and increased recovery of antibiotic-resistant mutants.
Cellular respiration depends on transferring electrons to hydrophobic quinones in membrane bilayers, meaning bioenergetic capacity is constrained by available membrane surface area. While Gram-negative bacteria expand this capacity through internal membrane invaginations and eukaryotes use membrane-bound organelles, whether Gram-positive bacteria have alternative capacity-generating mechanisms is unknown. Here we show that Bacillus subtilis forms a quinone-transporting pseudomembrane composed of filaments of the NADH dehydrogenase Ndh and the quinone-transporting protein Ncp. Cryo-EM, lipidomics and molecular dynamics reveal that Ndh and Ncp co-assemble with lipids into a 4:4 stoichiometric complex with a solvent-excluded hydrophobic lumen containing phospholipids, which sequesters quinones. These complexes further assemble into filaments, linking chambers into a continuous conduit that amplifies quinone reduction. Phylogenetic analysis suggests that this capacity is widespread in Bacillota. Quinone-transporting filaments thus reveal a strategy to expand the quinone pool and bioenergetic capacity while occupying minimal membrane space.
Microbial genomes continuously adapt to environmental conditions, but identifying universal signatures of adaptation remains challenging. Here we show that environmental temperature can be accurately predicted across ecosystems from DNA composition alone (R2 = 0.75), using tetranucleotide frequencies from 1,235 marine and soil metagenomes and a machine learning approach. This predictive signal was also apparent within individual taxa, consistent with a fundamental temperature-associated signature. By contrast, GC content exhibited opposite correlations with temperature in soil (positive) and marine (negative) environments. This phenomenon was probably driven by differences in nutrient availability, as GC content increases with nutrients while nutrients decrease with temperature in marine samples. By integrating these observations, we identified specific tetranucleotides, with 50% GC, that displayed consistent and robust temperature correlations across environments and may have contributed to the stability of predictions. This work highlights metagenome-wide DNA-temperature associations, relevant for understanding microbial community responses to global changes.
Bacteriophages are the most diverse biological entities on Earth, but the processes influencing the evolution of genomic diversity in phages are poorly understood. Here, we show that phage genomes contain contingency loci, hypermutable DNA regions that promote reversible frameshift mutations through DNA polymerase slippage on simple sequence repeats. Contingency loci have been extensively described in bacteria, archaea and eukaryotes, yet are understudied in phages. We use experimental evolution and genome sequencing to demonstrate that contingency loci in E. coli phages T2 and T4 reversibly generate genomic and phenotypic heterogeneity in progeny that allow them to hedge their bets against host defences. We find that simple sequence repeats are widespread in diverse E. coli phages and vary in abundance across genes with different functions. Collectively, our study describes a previously unappreciated facet of phage replication in which mutagenic simple sequence repeats drive genetic diversification and population heterogeneity, allowing phages to exploit hosts despite varying defence mechanisms.
Nucleocytoplasmic large DNA and Mirusviricota viruses exhibit taxonomic richness which continually expands due to metagenomic sequencing. Here we curate a database of giant virus metagenome-assembled genomes (GVMAGs V2), comprising 8,508 species-level representatives from 18,727 GVMAGs, a sixfold increase from the previous giant virus phylogenetic frameworks. Phylogenomics revealed 712 previously undescribed genera, 13 previously unknown viral families and an order we propose named Mycodnavirales. By accounting for alternative and custom genetic codes, we improved gene calling in over 1,300 GVMAGs, enabling more accurate identification of protein-coding genes. Database mining uncovered putative endogenous viral elements in hosts spanning algae, fungi and parasitic protists, highlighting that giant virus integration is widespread and evolutionarily persistent. Protein-level analysis revealed enriched genes for pollutant degradation in Algavirales and widespread biosynthetic gene clusters linked to antimicrobial-like and antibiotic resistance gene-like activity. This public resource will serve as a foundation for expanding giant virus diversity, uncovering virus-host interactions and exploring viral evolution.
The gut microbiome is a dynamic ecosystem in which microorganisms constantly adjust their transcriptional programmes. Here we developed metastrand, a framework that integrates strand-aware metatranscriptomics and metagenomics to quantify mRNAs and antisense RNAs (asRNAs) in complex microbial communities at gene-level resolution. In inflammatory bowel disease (IBD), microbial asRNA programmes converged across patients during active disease, correlated with faecal metabolites and calprotectin levels and remained stable during persistent inflammation, highlighting their potential as biomarkers of inflammatory activity in the gut. These programmes involved antisense-to-sense transcriptional shifts at insertion sequence elements with functionally diverse passenger genes and preceded their detection at new genomic locations, linking asRNA dynamics to structural genome rearrangements and redistribution of adaptive functions under selective pressure. Similar dynamics were observed in a mouse model of colitis, oxidative stress in vitro and in patients with pathogen-confirmed gastroenteritis, establishing asRNAs as an important dimension of microbial adaptation in health and disease.
Silvia Bulgheresi muses on how studying bacterial symbionts of worms and mammals made her question whether the absence of a nucleus enables cellular freedom, potentiating architectural innovation and functional adaptation.
Candida albicans is a major opportunistic pathogen in humans that is capable of breaching mucosal barriers and causing severe systemic infections with high mortality. How the host controls mucosal infection and prevents dissemination remains unclear but is essential for improving disease outcomes. Here we demonstrate that C. albicans induces specific IL-1 family members, which are critical for initiating mucosal protection by controlling antimicrobial peptides, IL-17 and neutrophil responses. The loss of combined IL-1 family signalling led to severe oropharyngeal C. albicans infection, which was eventually resolved by a potent neutrophil response. However, in neutropenic conditions, a key patient risk factor, abolishing IL-1 family signalling resulted in C. albicans dissemination, predominantly to the liver, mirroring clinical disease and leading to mortality. This study highlights the IL-1 family as a key initiator of mucosal immunity, restricting mucosal invasion and cooperating with neutrophils to prevent life-threatening systemic infections.
Non-typhoidal Salmonella use molybdenum cofactor-containing MopB- or DMSO reductase-family members to respire chemically diverse substrates, including formate, nitrate and methionine sulfoxide, during infection. The DmsABC enzymatic complex encodes one such DMSO reductase to promote oxidative stress resistance. The Salmonella genome encodes several gene paralogues but their role in virulence is unclear. Here we characterize three Salmonella MopB-family extracytoplasmic sulfate reductases, which we call Xsr1A, Xsr2A and Xsr3A. Infection experiments in mice and macrophages show that these sulfate reductases support Salmonella growth and virulence in the gut and during systemic infection, countering the oxidative effects of host respiratory burst activity. Further experiments show that they are molybdenum cofactor-independent enzymes, and instead depend on the nearby redox-active [4Fe-4S] prosthetic group for catalytic activity. Orthologues of these sulfate reductases were found across distant evolutionary branches, suggesting that [4Fe-4S]-dependent catalysis may occur across the ubiquitous MopB superfamily. Our findings offer insights into the modular evolution of redox centres in the widespread MopB superfamily.
The emergence of antimalarial drug resistance threatens malaria control and elimination efforts in Africa. Ethiopia, once a success story in case reduction, is now experiencing a resurgence. Here we examine key drug resistance genes (Pfmdr1, Pfcrt, Pfk13, Pfdhfr and Pfdhps) and mitochondrial genomes from 605 Plasmodium falciparum isolates collected across 15 districts in Ethiopia with varying transmission intensity and Plasmodium vivax co-endemicity. Although chloroquine was withdrawn for P. falciparum long ago, it remains the first-line treatment for P. vivax; this overlapping use may shape selection pressure in co-endemic settings that influences resistance markers to artemether-lumefantrine, the current first-line therapy for P. falciparum. A dominant PfMDR1 NFSND haplotype, associated with reduced lumefantrine susceptibility, was identified alongside near fixation of the chloroquine-resistant PfCRT CVIET haplotype in specific areas. Concerningly, PfK13 variants associated with partial artemisinin resistance, R622I (10%), A675V (1.7%) and P441L (1.1%), were expanding. Multilevel models demonstrated robust, independent associations of R622I with PfCRT CVIET and PfDHFR AICNI, while ecological predictors were weaker and less consistent. These findings highlight genetic co-occurrence of Pfcrt and Pfk13 mutations in P. vivax-P. falciparum co-endemic settings and can inform antimalarial policy in Ethiopia.
Excessive foraging of colonic mucin glycans by gut bacteria is associated with diseases such as inflammatory bowel disease. Although Akkermansia muciniphila is an important mucin degrader, the role of carbohydrate sulfatases that facilitate digestion of these heavily sulfated glycans remains unclear. Combining in vitro digestion assays, proteomics and structural biology, we show that A. muciniphila sulfatases, such as Amuc1755 and Amuc0953, have rare adaptations targeted towards known sulfated mucin structures. They show larger degrees of modularity, including a previously unknown mucin-binding domain. When grown on colonic mucin substrates, glycoproteins of reduced size were important for the growth of A. muciniphila. Further mutational analysis and localization studies revealed that desulfation of N-acetyl-D-glucosamine was periplasmic, while desulfation of D-galactose occurred extracellularly and in the periplasm. These data improve our understanding of contexts for the positive health correlations of A. muciniphila while metabolizing colonic mucin as its sole carbon source.
