mSystems最新文献

In response to host-generated stresses, Mycobacterium tuberculosis (Mtb) reprograms its physiology in myriad ways to establish and maintain an infection, yet the signals that underlie this transformation are not well defined. The abundant toxin-antitoxin (TA) systems harbored in the Mtb genome, including 11 in the mazEF family, are thought to act as stress sensors, yet their roles are largely unknown. Although TA systems from other bacteria are generally thought to impart reversible growth arrest in response to stress, the exquisite specificity of Mtb tRNase toxins instead portends a more nuanced role. Here, we used a proteomics approach to track de novo protein synthesis to uncover molecular events initiated by the Mtb MazF-mt9 toxin (MazF7, Rv2063A). First, we documented striking enrichment of enzymes and transporters derived from the contiguous 36-gene region for phthiocerol dimycocerosate (PDIM) synthesis without an accompanying increase in PDIM lipid production. This paradox was reconciled by concomitant downregulation of proteins comprising the Mce1 transporter (imports host fatty acids), cholesterol breakdown, and β-oxidation enzymes (limiting the PDIM precursor methylmalonyl-CoA). Thus, increased catalytic efficiency of the PDIM pathway appears to offset substrate starvation to ensure adequate production of PDIMs essential for Mtb early immune escape and virulence. Finally, isocitrate lyase 1 levels also increased, which in this context are expected to primarily catalyze the glyoxylate shunt to sustain central carbon metabolism while minimizing carbon loss. These exacting proteomic signatures are paralleled within the bedaquiline-treated Mtb transcriptome, highlighting a critical role for MazF-mt9 in orchestrating Mtb stress survival.IMPORTANCEThe bacterial pathogen that causes tuberculosis, Mycobacterium tuberculosis (Mtb), must survive a gauntlet of immune assaults to establish an infection. Here, we determined that in response to host-imposed stresses, this pathogen enlists the action of a tRNase, the MazF-mt9 toxin, to reprogram the translatome and orchestrate metabolic remodeling to ensure adequate production of specialized phthiocerol dimycocerosate (PDIM) lipids on the cell surface, which contribute to early immune evasion. This toxin also upregulates isocitrate lyase 1 as a complementary survival-oriented adaptation that conserves carbon and sustains central metabolism for essential cellular functions. Thus, this toxin-mediated cooperative reprogramming toward preservation of PDIMs and central metabolism under lipid precursor-limiting conditions likely enables Mtb to successfully infect and survive in the host lung. Overall, the MazF-mt9-mediated protein expression signatures align with the transcriptome signatures of Mtb cells during bedaquiline treatment, suggesting a precise and essential role for this toxin in Mtb stress survival.

Fecal microbiota transplantation (FMT) is an emerging therapy for inflammatory bowel disease (IBD), yet its efficacy in patients refractory to conventional treatments and its underlying mechanisms require further elucidation. We studied 37 IBD patients (15 ulcerative colitis [UC], 22 Crohn's disease [CD]) refractory to conventional therapies and 16 healthy donors. FMT monotherapy from a single donor induced week-4 clinical response in 12 UC and 9 biologic-naïve CD patients, with all responders sustaining remission and most achieving endoscopic remission by week 14. Integrated multi-omics revealed FMT restored microbial diversity and profoundly reorganized host-microbiota-metabolite networks. In nine refractory CD patients (7 infliximab [IFX] non-responders, 2 FMT non-responders), IFX-FMT combination led to week-4 response in 6 patients, all of whom attained clinical and endoscopic remission by week 14, with more complete microbial-metabolic restoration than monotherapy. Our findings establish that FMT induces remission in refractory IBD via ecosystem network rewiring, and that IFX-FMT exhibits additive effects, supporting further trials of microbiome-directed adjunctive strategies.

Importance: This study provides mechanistic and clinical insights into the therapeutic effects of fecal microbiota transplantation (FMT) in inflammatory bowel disease (IBD), particularly when combined with the anti-tumor necrosis factor (anti-TNF) biologic infliximab (IFX). While both FMT and IFX achieve response in approximately 60% of IBD patients, their combined influence on the gut microbial and metabolic landscape in refractory disease has been poorly understood. Here, we demonstrate that FMT monotherapy restores gut microbial diversity and reconfigures host-microbiota-metabolite networks, correlating with clinical and endoscopic remission in patients refractory to conventional treatments. Furthermore, in Crohn's disease patients unresponsive to either therapy alone, combined IFX-FMT induced more complete microbial and metabolic normalization and achieved remission where monotherapy had failed. These findings reveal ecosystem-level network rewiring as a central mechanism of FMT efficacy and establish the additive potential of combining microbiome-targeted and immunomodulatory therapies. This work supports the development of microbiome-informed adjunctive strategies for severe or refractory IBD, highlighting an actionable path toward personalized, mechanism-based treatment regimens.

Clinical trials: This study is registered with ClinicalTrials.gov as NCT07149441.

Dry eye is a prevalent ocular disorder characterized by tear film instability, inflammation, and ocular discomfort. Although the ocular surface (OS) microbiome contributes to immune regulation and pathogen defense, its role in dry eye pathophysiology remains unclear. Therefore, the present study aimed to characterize alterations in the OS microbiome of patients with dry eye undergoing cyclosporin A or NewHyalUni treatment and to identify their potential roles related to clinical improvement. Patients with dry eye were treated with either cyclosporin A and NewHyalUni drop combination or NewHyalUni alone. OS samples were collected before and after treatment, and the microbiome was analyzed by whole metagenome sequencing. Potential contaminants were removed before downstream analysis to account for the low-biomass nature of OS samples. Clinical evaluations included symptom scores and the assessment of meibomian gland dysfunction (MGD). No significant differences in the overall microbial composition were observed between the treatment groups. Nevertheless, both groups demonstrated symptomatic improvement. OS microbiome alterations were strongly correlated with improvements in MGD scores. Moreover, microbial interactions were found to shift following treatment. Key species (Staphylococcus epidermidis, Staphylococcus pseudintermedius, Streptomyces lividans, and Edwardsiella tarda) were identified as potential mediators of MGD score improvement by modulating microbiome functions and suppressing inflammation-associated species. Although distinct treatment regimens did not lead to divergent microbiome profiles, symptomatic improvement was associated with alterations in a specific microbiome. These findings highlight the OS microbiome's potential role in dry eye and support the development of microbiome-based therapeutic strategies.IMPORTANCEDry eye is a common ocular disorder with complex pathophysiology that extends beyond tear deficiency and inflammation. Despite growing evidence of host-microbiome interactions at mucosal surfaces, the contribution of the ocular surface (OS) microbiome to dry eye remains poorly understood. Our findings in this study reveal that shifts in specific taxa and ecological interactions correlate with improvements in meibomian gland function and dry eye symptoms, even in the absence of major changes in overall microbiota. By identifying microbial signatures potentially linked to clinical improvement, we provide systems-level insight into the role of low-biomass microbiomes in ocular health. This work expands the current understanding of microbiome-host dynamics in non-gut environments and supports future development of microbiome-informed therapeutic strategies.CLINICAL TRIALSThis study is registered with ClinicalTrials.gov as NCT06936462.

Heterotrophic bacteria rapidly deplete essential macronutrients during growth and must navigate subsequent periods of growth arrest imposed by starvation. Nutrient limitations can be dynamic in nature, requiring ongoing regulatory adjustments involving new protein synthesis despite total biosynthetic activities being dramatically lower than during growth. Here, we have characterized the responses of the opportunistic pathogen Pseudomonas aeruginosa to prolonged starvation for carbon or nitrogen sources and to transitions between these states. We find that most cells survive both types of starvation for more than a week and maintain low but robustly detectable levels of protein synthesis in the absence of growth. Nitrogen-starved cells are larger, make more proteins, and retain fewer ribosomes than carbon-starved cells, indicating that distinct physiological strategies are adopted during the two starvation types. We found that the newly synthesized proteomes of each starvation type are distinct although many of the most highly synthesized proteins are shared between both conditions. Interestingly, we observed a temporary burst of protein synthesis as cells were transitioned between the two starvation conditions, which may reflect active remodeling of the proteome during growth arrest. We also used transposon insertion sequencing to identify genes impacting fitness in both starvation conditions and during transitions between the two and found that a highly overlapping set of global regulators most strongly influenced survival. Combining these data sets, we highlight proteases and chaperones, flagellar motility, and the nitrogen-related phosphotransferase system as key fitness-impacting functions that are actively maintained by growth-arrested Pseudomonas aeruginosa.

Importance: Molecular microbiology has traditionally focused on exponential growth in model organisms as the preferred context in which to study bacterial physiology, especially the regulation of new protein synthesis. However, in natural environments, including many infection contexts, bacteria frequently enter growth arrest due to nutrient limitation. The dynamics and regulation of protein synthesis in growth-arrested cells remain poorly understood, especially in pathogens. Furthermore, growth arrest increases tolerance to a variety of stresses, including many clinically used antimicrobials. We have conducted a comprehensive exploration of the proteins being made by growth arrested Pseudomonas aeruginosa during total nitrogen or carbon starvation and at the transition between these two starvation types, and the genes supporting fitness under these conditions. These datasets suggest dynamic redistribution of resources among important cellular functions and will serve as a resource for further investigations of starvation-induced growth arrest, a ubiquitous but understudied physiological state of heterotrophic bacteria.

Rapid tracking of emerging pathogenic microorganisms is crucial for designing effective treatment, infection control, and prevention strategies. While whole-genome sequencing (WGS) offers the necessary granularity to track emerging clones, it remains prohibitively expensive at the scales needed to monitor with high resolution in real time. We present CURED (Classification Using Restriction Enzyme Diagnostics), which uses a training set of sequenced genomes to identify unique k-mers with restriction sites specific to a clonal lineage. CURED enables fast and inexpensive PCR-based diagnostic tests for surveillance or outbreak investigations with minimal use of WGS. Benchmarking against existing tools, CURED compares favorably and scales more efficiently than other k-mer search strategies. We validated and tested CURED in five distinct data sets: (i) previously identified biomarkers described for a methicillin-resistant Staphylococcus aureus (MRSA) clone in Rio de Janeiro, (ii) diagnostic alleles for different lineages in the USA300 MRSA clone, (ii) the extensively drug-resistant Acinetobacter baumannii Global Clone 1 lineage, (iv) toxigenic versus non-toxigenic Clostridioides difficile, and (v) circulating S. aureus clones in a neonatal intensive care unit (NICU). We implemented CURED as part of NICU infection prevention efforts and report the test's speed, sensitivity, and specificity in a real-world setting. CURED is a scalable, multithreaded, memory-, and cost-efficient pipeline tailored for rapid clone detection and restriction site analysis. While particularly impactful for localized outbreak investigations and targeted surveillance, our preliminary work at the global scale suggests broader implementation is feasible. CURED is freely available at https://github.com/microbialARC/CURED.IMPORTANCETimely and cost-effective detection of emerging microbial clones is essential for infection prevention and public health surveillance. While whole-genome sequencing remains the gold standard for tracking microbial evolution and transmission, its cost, infrastructure requirements, and turnaround time limit its scalability, especially in resource-limited settings. CURED addresses this gap by enabling the development of inexpensive, PCR-based diagnostic assays informed by genomic data, without requiring further sequencing. By identifying lineage-specific restriction sites through a scalable and memory-efficient k-mer pipeline, CURED enables the translation of genome-scale insights into actionable diagnostics. This tool supports broader implementation of genomic-informed diagnostics in both local and global pathogen surveillance efforts.

Metagenomes often contain many reads derived from eukaryotes, but there is usually no reliable method for estimating their prevalence. This forces many analysis techniques to make the often-faulty assumption that all reads are prokaryotic. Here, we present SingleM prokaryotic_fraction (SPF), an algorithm that scalably and robustly estimates the number of bacterial and archaeal reads in a metagenome. It also estimates the average genome size of bacteria/archaea in a sample. SPF does not use eukaryotic reference genome data and can be applied to any modern Illumina metagenome. Based on SPF, we propose the domain-adjusted mapping rate (DAMR) as an improved metric to assess prokaryotic genome recovery from metagenomes. Applying SPF to 136,284 publicly available metagenomes, we report substantial variation in prokaryotic fractions and biome-specific patterns of prokaryotic abundance, providing insights into how microorganisms and eukaryotes are distributed across Earth. Finally, we show that substantial amounts of human host DNA sequence data have been deposited in public metagenome repositories, possibly counter to ethical directives that mandate screening of these reads prior to release. As the adoption of metagenomic sequencing continues to grow, we foresee SPF being a valuable tool for the appraisal of genome recovery efforts and for investigating global patterns of microorganism distribution.IMPORTANCEMetagenomics data sets capture DNA from all organisms in a sample, enabling the analysis of communities without relying on culture-based techniques. However, many samples include uncharacterized eukaryotic organisms and viral elements, meaning the proportion of bacterial and archaeal DNA is often unknown. This study presents SingleM prokaryotic_fraction (SPF), a robust and scalable method for estimating the prevalence of bacterial and archaeal DNA in metagenomes. Crucially, SPF is calculated independent of eukaryotic and viral reference genomes, which are often incomplete or unavailable. Applying SPF to over 136,000 public metagenomes uncovered substantial variability between microbial communities living in different environments. SPF also identified previously overlooked human genetic data contamination in public data sets, raising important ethical and privacy considerations. Building on SPF, we propose the domain-adjusted mapping rate (DAMR) metric, a new metric that improves genome recovery assessment by accounting for non-prokaryotic reads.

The gut microbiome (GM) plays an essential role in health, and its dysbiosis can increase the risk of colon cancer. While the detrimental effects of high-dose ionizing radiation on GM have been documented, little is known about the effects of low doses, including from internal exposure to tritium, which is produced by nuclear power generation and emits beta radiation, making it a public concern. We examined the effects of chronic irradiation with internal tritium beta radiation or external ⁶⁰Co gamma radiation on GM and intestinal tumorigenesis in the Apc^Min/+ mouse model of colorectal cancer. Mice were exposed to tritiated drinking water (HTO) or gamma radiation at cumulative doses of 0, 10, 100, and 2,000 mGy, followed by intestine, blood plasma, and fecal sample collections at 12, 16, and 20 weeks of age. HTO- and gamma-exposed cohorts had distinct tumor size and multiplicity patterns, with non-monotonous dose-responses. Complex patterns of blood cytokine changes with age, dose, and type of irradiation were recorded. GM analyses using 16S rRNA amplicon sequencing revealed significant changes in alpha and beta diversity in irradiated mice compared to controls, indicating altered microbial dynamics. HTO and gamma radiation induced distinct microbiome changes that did not correlate with tumor and blood cytokine readouts. Our results suggest that chronic exposure to low-dose gamma- or internal HTO beta radiation can affect GM in a radiation type and dose-dependent non-linear manner. Our results provide novel insight into the effects of low-dose gamma- and tritium beta radiation on GM and a possible association with tumorigenesis.

Importance: Low-dose ionizing radiation is one of the few environmental stressors that simultaneously reshapes host physiology and the structure-function landscape of resident microbiomes, yet mechanistic insight at ecologically relevant doses has been scarce. By integrating longitudinal 16S rRNA profiling, multiplex cytokine analyses, and quantitative tumor phenotyping in the Apc^Min/+ mouse model, our study demonstrates that continuous exposure to either external ⁶⁰Co γ-photons or tritium beta particles perturbs gut microbial community structure in radiation-quality-specific ways and that these shifts track with, and sometimes precede, complex, non-monotonic changes in intestinal tumor burden. The work expands the traditional radiobiology focus from host-centric DNA damage to a systems-level view in which microbe-host-radiation interactions form a dynamic network influencing early colorectal carcinogenesis.

Heat stress has become one of the major threats affecting animal reproductive performance. Although probiotics show potential in maintaining testicular health, their protective effects and underlying mechanisms against heat stress-induced testicular injury remain unclear. In this study, Lactiplantibacillus plantarum 082 (LP082) was administered to heat-stressed mice, and its role and mechanism in alleviating testicular damage were systematically evaluated through biochemical assays, histopathological analysis, gut microbiota diversity profiling, and gas chromatography-based quantification of short-chain fatty acids (SCFAs). The results showed that heat stress significantly reduced testicular weight in mice, caused testicular tissue damage, and triggered pronounced inflammatory responses and oxidative stress. LP082 intervention notably ameliorated heat stress-induced gut dysbiosis: on one hand, it significantly decreased the abundance of harmful bacteria such as Klebsiella and Bacteroides, repaired intestinal barrier damage, and effectively blocked the translocation of lipopolysaccharide (LPS) from the gut to the circulatory system, thereby reducing systemic LPS load; on the other hand, it significantly promoted the proliferation of beneficial bacteria, including Akkermansia, Lactiplantibacillus, Bifidobacterium, and Faecalibaculum, thus elevating SCFA levels. These improvements collectively mitigated systemic inflammation and oxidative stress, ultimately alleviating testicular tissue damage. In summary, LP082 exerts protective effects against heat stress-induced testicular damage via modulation of the "gut-testis axis," providing both theoretical insights and experimental evidence for developing probiotic-based strategies to safeguard male reproductive health under high-temperature conditions.

Importance: Global warming-induced heat stress severely impairs male reproductive health, with no effective interventions available. The "gut-testis axis" is increasingly recognized but poorly studied in heat stress-related testicular injury. This study identifies Lactiplantibacillus plantarum 082 as a viable protector, which acts by remodeling gut microbiota, repairing intestinal barriers, and regulating lipopolysaccharide and short-chain fatty acid levels. It fills gaps in probiotic-mediated gut-testis axis regulation, provides an experimental basis for probiotic-based strategies, and offers new insights to mitigate heat stress-related reproductive harm in animals and humans.

Escherichia coli sequence type (ST) 405, a high-risk international clone, has emerged as a critical lineage for the dissemination of antibiotic resistance genes (ARGs), including the bla_NDM-5 gene. This study aims to elucidate the genomic landscape of ST405 through comparative genomic analysis of both publicly available ST405 genomes and three ST405 isolates from sewage and wild-bird fecal samples in Pakistan. A total of 1,778 E. coli ST405 genomes were investigated through phylogenomic and population-structure analyses, revealing distinct clusters and subgroup lineages within this ST. Among these, subgroup B was identified as a pivotal contributor to the dissemination of the bla_NDM-5 gene within ST405. Temporal analysis indicates that subgroup B is expanding over time, raising concerns regarding its potential contributions to increased antibiotic resistance within ST405 populations. Genetic-structure analyses of bla_NDM-5-carrying plasmids of the sewage isolates uncovered the presence of nearly identical plasmids in other multidrug-resistant STs, ST156 and ST648, suggesting a genetic linkage among these STs. Additionally, a novel bla_NDM-5 genetic context was identified in the animal isolate, characterized by unique composite-transposon and inversion structures mediated by IS26 replicative transposition, highlighting the dynamic evolution of the ST405-associated plasmid. These findings underscore the intra-ST dissemination of the bla_NDM-5 gene and the ongoing diversification of its genetic context within ST405. This comprehensive analysis advances our understanding of ST405, providing insights into the genetic mechanisms underlying its enhanced antibiotic resistance and the emergence of a novel ARG-carrying plasmid within the One Health framework.

Importance: This study provides a comprehensive genomic landscape of ST405, a high-risk international clone and a carrier of the bla_NDM-5 gene, revealing phylogenetically distinct patterns in the distribution of antibiotic resistance genes and virulence factors, and a critical phylogenetic lineage that serves as a primary reservoir of the bla_NDM-5 gene. Furthermore, the genetic linkage between ST405 and other STs (ST156 and ST648) through the sharing of identical bla_NDM-5-carrying plasmids, and the emergence of a novel bla_NDM-5 genetic structure in an animal isolate, underscore the pivotal role of ST405 in the dissemination of the bla_NDM-5 gene. These findings highlight the public health significance of ST405 and its contribution to the global spread of carbapenem resistance.