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Streptococcus pyogenes (group A Streptococcus, GAS) causes various clinical complications and invasive diseases. Our previous studies have shown that GAS survives inside endothelial cells due to the insufficient acidification of lysosomes, which fuse with reactive oxygen species (ROS)-induced phagosomes of LC3-associated phagocytosis. For catalase-deficient peroxide-producing GAS to survive in hosts, GAS uses a peroxide response regulator (PerR) to modulate ROS-induced oxidative stress and metal ion regulation. However, it remains unclear whether PerR regulates zinc homeostasis during infections. We generated the GAS ΔperR isogenic mutant and conducted dual RNA-seq analysis, an endothelial cell infection model, computational predictions, and phenotypic characterization to demonstrate the protective role of PerR in GAS survival in endothelial cells. The ΔperR mutant's vulnerability to zinc deprivation demonstrated that PerR coordinates iron and zinc homeostasis, likely using PmtA's iron efflux, iron and zinc-chelating ferritin-like Dpr, the AdcR regulon (adcA, adcAII, and phtD), and zinc efflux (czcD). We also demonstrated that the wild-type strain and ΔperR mutant encounter zinc restriction inside the phagolysosome GAS-containing vacuoles of endothelial cells. This host zinc starvation severely reduces the survival of the ΔperR mutant. These results suggest that the PerR-mediated iron and zinc modulation through Dpr is more important than had been previously thought. Consequently, PerR enhances GAS fitness during its invasions of human endothelial cells.

Importance: Our study combines dual RNA-seq analysis, an endothelial cell infection model, computational predictions, and phenotypic characterization to discover the impact of group A Streptococcus (GAS) PerR on the coordination of iron and zinc homeostasis during infection. We found that PmtA's iron efflux, iron and zinc-chelating ferritin-like Dpr, the AdcR regulon, and zinc efflux are delicately modulated by PerR. We also determined that zinc limitation inside the phagolysosome GAS-containing vacuoles of endothelial cells causes host zinc starvation, resulting in reduced survival of the ΔperR mutant. Consequently, PerR enhances GAS fitness through Dpr during its invasions of human endothelial cells. Our novel findings offer new insights into how GAS combats iron-mediated oxidative stress and zinc homeostasis that may help develop new anti-GAS treatments.

The gut microbiome undergoes dynamic age-related changes shaped by diet and maternal factors. Here, we present a species-level, long-read 16S rRNA survey of the developing gut microbiome in a translational canine model, profiling 89 purebred Hungarian Pumis across early-life and reproductive stages. We collected 456 fecal samples longitudinally: 60 puppies followed from birth to 81 weeks, their mothers sampled during pregnancy and lactation, and adult controls from six kennels. We recorded detailed dietary metadata and reproductive status throughout the study. Age was the strongest determinant of alpha diversity, with a rapid increase during weaning and stabilization by 6 months of age. Beta diversity analyses revealed structured compositional transitions from early developmental phases to adulthood, including a shift toward more uniform, adult-like communities. Within-kennel variation was modest, consistent with shared environmental exposures. Mixed-effects models showed robust associations between specific taxa and age, diet, and kennel, while SparCC-inferred co-occurrence networks indicated increasing ecological complexity with age. We also demonstrated that the delivery mode-vaginal versus cesarean-impacted early-life microbiome composition: Lactobacillus spp. were significantly more abundant in cesarean-born puppies than in vaginally delivered littermates during the 8-10-week window. We also observed reproducible maternal microbiome shifts during pregnancy and lactation, with potential implications for vertical microbial transfer. Taken together, our results show that domestic dogs follow a reproducible, age-structured trajectory of microbial maturation that parallels human development, including delivery-mode effects and diet-responsive taxa.IMPORTANCEMicrobiome research is among the fastest-moving areas in biomedicine driven by major global efforts to understand how microbial communities shape human health and disease. Dogs provide an ideal translational model because their gut microbiota more closely resembles that of humans than that of other studied animals; moreover, breeds show high within-breed genetic homogeneity; diets can be tightly regulated; and longitudinal sampling across the lifespan is feasible. Mapping shifts driven by diet and maternal factors-from early-life events through later life, including senior stages-is essential to leverage microbial plasticity for prevention, with implications for inflammation, metabolic disease, and neurodegeneration. Here, we advance this goal by providing a longitudinal, high-resolution data set and demonstrating that full-length 16S rRNA sequencing is a powerful tool for resolving fine-scale patterns of gut colonization and maturation.

Fungi are ubiquitous in natural ecosystems, and environmental reservoirs such as bat hibernacula can harbor fungal pathogens and shape disease dynamics. Beyond serving as pathogen reservoirs, these environments may also contain volatile organic compounds (VOCs) with antifungal properties that help a host resist infection. Studies have shown that various VOCs from bat caves significantly inhibit the growth of Pseudogymnoascus destructans, the pathogen responsible for white-nose syndrome (WNS), although the underlying mechanisms remain unclear. This study investigates two VOCs isolated from bat cave environments-isovaleric acid (IVA) and ethyl methyl carbonate (EMC)-to evaluate their single-agent and combination activities against P. destructans in vitro and to explore the underlying mechanisms. The results show that both IVA and EMC significantly inhibit mycelial growth in a dose-dependent manner and exhibit synergistic antifungal effects. Physiological and biochemical analyses revealed that VOC treatment disrupts cell wall and membrane integrity, induces apoptosis, elevates reactive oxygen species levels, and causes DNA damage. Concentrations of adenosine triphosphate, malondialdehyde, ergosterol, and NADPH also increased significantly. Transcriptomic and metabolomic analyses showed disruption of the mycelial structure, modulation of virulence-associated pathways, induction of oxidative stress and apoptosis, and interference with purine metabolism, cAMP signaling, and energy metabolism. Notably, combined IVA-EMC treatment enhanced DNA damage and suppressed heat shock protein expression, effectively inhibiting P. destructans growth. Taken together, our study elucidates the antifungal potential of environmental VOCs and offers new insights and application prospects for preventing and controlling WNS.IMPORTANCEWhite-nose syndrome has devastated bat populations across North America, yet effective control measures remain limited. This study highlights the potential of naturally occurring volatile organic compounds from bat cave environments as antifungal agents against Pseudogymnoascus destructans in vitro. By uncovering the physiological and molecular mechanisms of the action of isovaleric acid and ethyl methyl carbonate, individually and in combination, this work paves the way for novel, environmentally derived strategies for managing white-nose syndrome and fungal pathogens more broadly.

High-quality reference genomes are essential for comparative genomics and accurate genotype-phenotype mapping. Here, we corrected the Mycobacterium tuberculosis Erdman strain reference genome (Erdman_TI) using ultra-deep HiFi sequencing. Among the small variants (n = 275) between Erdman_TI and the current Erdman reference NC_020559.1 (Erdman_STJ), numerous are likely errors in Erdman_STJ. We identified a novel bias toward in-frame structural variations (SVs) in pe/ppe genes and 28 SVs between Erdman_TI and Erdman_STJ, half representing likely errors in Erdman_STJ. Other SVs were consistent with in vitro evolution, including copy number variation (CNV) of promoter tandem repeats (PTRs). PTR CNVs were polyphyletic and within isogenic populations (10^-2-10^-3 CNVs/chromosome), demonstrating the impact of phase-variable CNV across evolutionary timescales. These hypervariable PTRs pinpoint a genomic basis for rapidly switching nitric oxide resistance (Dop), biofilm formation (LpdA), drug tolerance (EfpA), and glycerol utilization (GlpD2) phenotypes. This work uncovers a common phase variation mechanism obscured by short-read sequencing limitations and provides an improved reference for comparative studies.

Importance: Mycobacterium tuberculosis (Mtb), the pathogen responsible for tuberculosis, is often described as genetically stable. Our findings reveal an overlooked evolutionary adaptation mechanism: phase variation driven by tandem repeat copy number changes in gene promoters. Enabled by ultra-deep, long-read sequencing, we corrected errors in the Erdman reference genome and uncovered frequent, spontaneous expansions and contractions of promoter repeats upstream of genes linked to nitric oxide resistance, drug efflux, and biofilm formation. Through altering promoter strength, these dynamic promoter variants may generate phenotypic diversity within subpopulations and across diverse clinical lineages, suggesting a conserved evolutionary advantage for navigating host-imposed stress. This reframes Mtb's evolutionary potential, highlighting how adaptive flexibility has been underestimated due to reliance on short-read sequencing and limited resolution of subpopulations at standard genomic depths. Our findings underscore the need to integrate structural variation-aware approaches into studies of Mtb pathogenesis, evolution, and drug response.

The disease pyramid conceptualizes the predictors of host infection risk, linking the host, the pathogen, environmental conditions, and both host and environmental microbiomes. However, the importance of the interaction between environmental and host-associated microbiomes in shaping infectious disease dynamics remains poorly understood. While the majority of studies have focused on bacteria, the role of micro-eukaryotes has been seldom investigated. Here, we explore three axes of the disease pyramid using an 18S rRNA gene metabarcoding approach to analyze the micro-eukaryotic assemblages of biofilm, water, and skin samples from three European amphibian species. Skin bacterial communities of the investigated amphibian populations have already been shown to be impacted by the presence of the lethal fungal pathogen Batrachochytrium dendrobatidis (Bd), with a higher abundance of protective bacteria in infected populations and a greater environmental microbial contribution to the skin microbiota in Bd-positive lakes. Here, we explored the relationships between the micro-eukaryotic skin communities of these tadpole populations with their surrounding environment. Tadpoles were sampled at 22 mountain lakes located in the Pyrenees (France), 8 of which harbored amphibian populations infected by Bd. We found that biofilms from Bd-negative lakes had higher environmental micro-eukaryotic diversity and a greater abundance of putative anti-Bd fungi, both in the environment and on the skin microbiota of Bufo spinosus and Rana temporaria, but not of Alytes obstetricans. Bayesian SourceTracker analysis further showed that the environmental contribution from biofilms to amphibian skin micro-eukaryotic assemblages was higher in Bd-positive lakes for B. spinosus and R. temporaria, but not for A. obstetricans.IMPORTANCEResearch on host-associated microbiomes and infectious diseases has mostly focused on bacteria, overlooking the potential contributions of micro-eukaryotes to infection dynamics. Here, we show that environmental and skin-associated micro-eukaryotes-especially putative anti-Batrachochytrium dendrobatidis (Bd) fungi-differ between Bd-positive and Bd-negative amphibian populations in mountain lakes. Our results suggest that micro-eukaryotes influence disease resistance and microbiome assembly, similarly to bacteria. Importantly, environmental reservoirs of micro-eukaryotes appear to contribute differently across infection contexts. These findings demonstrate the importance of adopting a broader microbiome perspective that includes micro-eukaryotes when investigating the ecological mechanisms underlying infectious disease risk.

Host-microbial metabolic interactions have been recognized as an essential factor in host health and disease. Genome-scale metabolic modeling approaches have made important contributions to our understanding of the interactions in such communities. One particular such modeling approach is BacArena, in which metabolic models grow, reproduce, and interact as independent agents in a spatiotemporal metabolic environment. Here, we present a modeling application of BacArena, a virtual colonic environment, which reveals spatiotemporal metabolic interactions in a computational colonic environment. This environment resembles the crypt space together with the mucus layers, the lumen, and fluid dynamics. Our proof-of-principle experiments include mono-colonization simulations of context-specific colonic cells and simulations of context-specific colonic cells with the SIHUMIx minimal model microbiome. Our simulations propose host-microbial and microbial-microbial interactions that can be verified based on the literature. Most importantly, the Virtual Colon offers visualization of interactions through time and space, adding another dimension to the genome-scale metabolic modeling approaches. Lastly, like BacArena, it is freely available and can be easily adapted to model other spatially structured environments (http://www.github.com/maringos/VirtualColon).IMPORTANCEInteractions between the human body and gut microbes are crucial for health and disease. We present the Virtual Colon, an extension of the individual-based microbiome modeling approach BacArena that mimics key features of the colon, including the crypts, mucus layers, lumen, and fluid flow. Using this model, we simulate gut environments including host cells with bacterial species alone and with a simplified gut microbiota (SIHUMIx). These simulations reveal patterns of host-microbe and microbe-microbe interactions that align with known findings. A key strength of the Virtual Colon is its ability to show how interactions unfold over time and space, offering new insights beyond traditional modeling approaches. The Virtual Colon is freely available and can be adapted to other structured biological environments (http://www.github.com/maringos/VirtualColon).