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Intrastrain genetic and phenotypic heterogeneity of Pseudomonas aeruginosa is a hallmark of chronic lung infections in individuals with cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD). Although the coexistence of multiple P. aeruginosa lineages within a single host is well documented, the impact of this heterogeneity on infection microbiogeography remains poorly understood. We previously showed that loss of the lipopolysaccharide (LPS) O-specific antigen (OSA) alters P. aeruginosa aggregate assembly. Since OSA-deficient variants are common in chronic pulmonary infections and associated with increased pathogenesis and immune evasion, we investigated whether intrastrain OSA diversity shapes infection microbiogeography. We constructed mixed populations containing equal ratios of OSA-deficient variants and wild-type (WT) cells and examined aggregate assembly and population structures in a synthetic CF sputum model (SCFM2). To assess OSA heterogeneity in vivo, we used a murine pneumonia model combined with hybridization chain reaction (HCR) RNA-FISH and whole-tissue clearing to visualize spatial organization in the airways. In SCFM2, OSA-deficient variants increased total population size, reduced WT aggregate size, and altered spatial organization. We employed 2-plex HCR RNA-FISH to distinguish WT and OSA-deficient variants in murine lungs. Interestingly, in contrast to in vitro conditions, OSA-deficient cells led to significantly larger WT aggregates in the airways. These findings highlight the role of intrastrain genetic heterogeneity in shaping infection microbiogeography and provide a framework for understanding how population dynamics influence microbial physiology and host-pathogen interactions at the micron scale.IMPORTANCEIntrastrain genetic and phenotypic diversity within Pseudomonas aeruginosa populations is common in chronic pulmonary infections. While this intrastrain heterogeneity is a hallmark of chronic infection, its consequences for the spatial organization of P. aeruginosa within the airways remain unclear. Here, we demonstrate that the loss of O-specific antigen in a subpopulation of P. aeruginosa significantly alters the spatial architecture of P. aeruginosa, without changing the total population size or composition. Using a combination of tissue clearing and hybridization chain reaction RNA-FISH in a murine lung infection model, we mapped the localization of genetically distinct P. aeruginosa variants in mixed populations in vivo. These findings reveal that genetic diversification within a strain can reshape the infection landscape at the micron scale, highlighting the overlooked role of intrastrain dynamics in shaping the microbiogeography of infections and influencing host-pathogen interactions.

When susceptible bacterial cultures are treated with antibiotics, some cells can survive treatment without heritable resistance, giving rise to susceptible daughter cells in a phenomenon termed antibiotic persistence. Current models of fluoroquinolone (FQ) persistence in stationary-phase cultures posit that post-treatment resuscitation is dependent on double-stranded break (DSB) repair through RecA-mediated homology-directed repair. Previously, we reported that stationary-phase P. aeruginosa does not depend on RecA to persist. In this work, we ask whether P. aeruginosa FQ persisters from stationary-phase cultures suffer DSBs at all. We measured DSB formation in Levofloxacin (LVX)-treated cells recovering from treatment using strains expressing fluorescently labeled DSB-binding protein, Gam. We find that, surprisingly, the majority of P. aeruginosa LVX persisters survive treatment without apparent DSBs. Persisters that have evidence of DSBs take longer until their first division compared to persisters without DSBs. Additionally, the fates of their progenies suggest that persisters may cope with DSBs by repair or damage sequestration. These observations pave the way for mechanistic studies into P. aeruginosa FQ persistence and highlight the need for single-cell tools to track FQ-induced damage.

Importance: Pseudomonas aeruginosa is an opportunistic pathogen of significant clinical interest. When susceptible cultures of P. aeruginosa are treated with fluoroquinolone (FQ) antibiotics, some cells survive treatment and regrow in a phenomenon termed antibiotic persistence. Studies in Escherichia coli and other bacterial species suggest that FQ persisters survive by repairing DNA double-stranded breaks (DSBs) after antibiotic removal. In this study, we show that most stationary-phase P. aeruginosa survive by avoiding DSBs rather than repairing them.

DNA ligases are a fundamental class of enzymes required for DNA replication and repair. They catalyze the formation of phosphodiester bonds, specifically at single-strand breaks in double-stranded DNA. The nuclear genome of malaria parasites encodes a single DNA ligase that is likely involved in nuclear and organellar DNA replication and repair. DNA ligase I from Plasmodium falciparum (PfLig1) has been biochemically characterized and shown to possess nick-sealing activity. However, its localization and function in the three genome-containing compartments-the nucleus, apicoplast, and mitochondrion-of the malaria parasites remain unknown. Here, we found that Lig1 is located primarily in the nucleus in both human and rodent malaria parasites throughout the parasite life cycle. Furthermore, we detected its presence in organelles via a chromatin immunoprecipitation-PCR assay. Our attempts to disrupt Plasmodium berghei Lig1 (PbLig1) in the blood stages have failed, indicating that the gene is likely essential. Next, we used an Flp/FRT-based conditional mutagenesis system that silences gene function in sporozoites. We demonstrated that PbLig1 is essential for parasite liver-stage development. Sporozoites lacking PbLig1 invade hepatocytes but arrest growth during mid-liver-stage development. PbLig1 cKO parasites undergo limited nuclear division and present a reduced DNA content that fails to increase beyond mid-liver stage of development. These data suggest that Lig1 is an essential enzyme for parasite blood- and liver-stage development.IMPORTANCEUnlike mammalian cells that possess multiple DNA ligases, the malaria parasite's nuclear genome encodes a single DNA ligase. This single DNA ligase is likely involved in both DNA replication and DNA repair. However, the importance of parasite DNA ligase remains largely unknown. Here, we show that Plasmodium Lig1 is primarily found within the nucleus, but it also exhibits a distribution across parasite organelles. Knockout of PbLig1 in sporozoites abolishes parasite liver-stage development, preventing the formation of hepatic merozoites and ultimately blocking the transition from the liver to the blood stage of infection. More specifically, PbLig1 is essential for nuclear division during hepatic schizogony. These findings enhance our understanding of the role of DNA ligase I in malaria parasite liver-stage development.

In immunosuppressed humans with oropharyngeal candidiasis (OPC) and in mice with experimental OPC, Candida albicans infection is associated with a bacterial imbalance characterized by significantly reduced oral microbiome diversity and the expansion of enterococcal and streptococcal species, which may exacerbate oral mucosal pathology. In this study, we applied an unbiased genome-wide transcriptomic profiling approach to shed further mechanistic light on the role of indigenous enterococcal communities in mucosal infection in a mouse model of cancer chemotherapy-associated OPC. Transcriptomic profiling of tongue tissues revealed a wide-ranging, barrier-compromising molecular activity of resident enterococci that explains the previously observed attenuation of fungal mucosal invasion with antibiotic treatment in this mouse model. Mechanistically, we validated the pathogenic potential of resident bacteria by showing that enterococci isolated from mice with OPC produce hydrogen peroxide (H₂O₂) and induce oral epithelial cell death through apoptosis and necrosis in vitro. We also discovered that C. albicans increased enterococcal H₂O₂ production. These findings uncover a novel mechanism of pathogenic synergy between C. albicans and Enterococcus faecalis, which may be responsible for increased epithelial barrier damage and mucosal invasion by C. albicans hyphae during cancer chemotherapy.

Importance: Chemotherapy-induced mucosal barrier injury and immune suppression increase susceptibility to oropharyngeal candidiasis (OPC), a debilitating fungal infection. Our study uncovers a previously unknown pathogenic interaction between Candida albicans and Enterococcus faecalis, by showing that indigenous enterococci produce H₂O₂, which contributes to oral epithelial cell death during fungal infection. By integrating transcriptomics with functional assays, we demonstrate that enterococci compromise epithelial integrity independently of fungal burdens, highlighting the role of the bacterial microbiota in driving tissue damage. These findings emphasize the need to consider bacterial-fungal interactions in managing OPC and suggest that targeting the microbial crosstalk could be a promising adjunctive strategy in immunocompromised hosts.

Recent studies reveal that a suboptimal vaginal microbiome (VMB), including the enrichment of anaerobic bacteria associated with multiple female genital disorders, is linked to adverse pregnancy and birth outcomes in pregnant people. Problematically, however, the majority of the available data, to date, is biased toward highly developed, Global North countries, leaving underrepresented populations like the Democratic Republic of the Congo (DRC) poorly characterized. Here, we investigate the VMB from a cohort of 82 pregnant people living with human immunodeficiency virus (PLWH) on antiretroviral therapy (ART) from the DRC. Specifically, we explore the associations between the VMB via 16S rRNA gene sequencing and maternal peripheral immune factors. Additionally, we compare the VMB of pregnant PLWH-ART from DRC with publicly available VMB data (5 studies, 1861 samples) in a meta-analysis to elucidate the impact of HIV on the VMB. Combined, these analyses revealed the differences in community structure and predicted function of the microbiota between pregnant PLWH-ART and pregnant people without HIV (PWoH). Taxonomically, the VMB of DRC PLWH-ART were enriched for Lactobacillus iners-dominated VMBs (53%) or a diverse, polymicrobial VMB, that is, bacterial vaginosis (BV) (43%). Functional predictions made from these taxa suggested that protein-coupled receptors, amino sugar and nucleotide sugar metabolism, fatty acid metabolism, and polycyclic aromatic hydrocarbon degradation pathways were differentially abundant between the communities. Correlation with host plasma immune factors revealed putative links between some VMB metrics (e.g., alpha diversity and species abundance) that have been linked to adverse pregnancy and birth outcomes.

Importance: Human immunodeficiency virus (HIV) remains prevalent in sub-Saharan Africa, where it has been linked to adverse birth outcomes. Suboptimal vaginal microbiomes (VMBs) have shown similar links. This pilot study fills critical gaps in understanding how HIV interacts with the pregnant VMB in populations underrepresented in microbiome research, like the Democratic Republic of the Congo (DRC). We identified maternal systemic immune factors associated with suboptimal VMBs that have been linked to poor birth outcomes. In a global meta-analysis, we found significant taxonomic and functional differences in the VMBs between pregnant people living with and without HIV, revealing potential biomarkers that increase the risk of adverse birth outcomes. These findings provide crucial insights into VMB features that may influence pregnancy health in PLWH-ART, guiding future research and tailored interventions to support safer pregnancies in the DRC and similar populations.This study is registered with NCT03048669.

Zoonotic Onchocerca lupi (Spirurida, Onchocercidae) has attracted the interest of the scientific community worldwide, by causing severe ocular infections in domestic animals (dogs, cats) and can infect wild carnivores (wolves, coyotes), as well as humans. Though recent advancements in scientific knowledge have been gained, gaps still remain about the biology of this filarioid, as well as its genetic structure. Based on mitochondrial genes, two highly divergent genotypes were identified, in the Iberian Peninsula (genotype 2) and Europe, Asia, and the United States (genotype 1), meanwhile only a draft nuclear genome of O. lupi from the United States is available. This study aimed to fill knowledge gaps about the genomic characterization of this filarioid and its Wolbachia endosymbiont. This study described the shotgun sequencing of an adult specimen of O. lupi isolated from a dog living in Portugal using the PacBio long-read sequencing technology. Three distinct genomes, such as the nuclear, mitochondrial, and Wolbachia endosymbiont, were assembled and analyzed. The assembled nuclear genome, Olupi_PT2024, exhibited high contiguity, accuracy, and completeness. Pairwise mitogenome comparative analyses among several Onchocerca species corroborated the high divergence between the two genotypes from Portugal and the USA, although the observed differences remained within the range of intra-species variation. The complete genome of the Wolbachia endosymbiont of O. lupi confirmed its classification within supergroup C and its close phylogenetic relationship with Wolbachia endosymbionts associated with the genus Onchocerca. The data on these three genomes may provide valuable resources for understanding the biology, population genetics, and phylogeography of this parasite.IMPORTANCEOnchocerca lupi, a zoonotic parasite, causes ocular onchocerciasis in both domestic and wild carnivores, as well as humans. Despite recent scientific advances, gaps remain in both the biology and genetic structure of this parasite. To date, two genotypes have been described (genotype 1 distributed in Europe, Asia, and the United States, and genotype 2 circulating in the Iberian Peninsula) based on mitochondrial gene analysis. This study provided three distinct genomes (nuclear, mitochondrial, and Wolbachia endosymbiont) of O. lupi isolated from a dog living in Portugal. Overall, the data presented here corroborate the divergence between the two genotypes and provide new insights into the identification of genes that could serve as novel therapeutic targets for this filarial disease.

Human milk oligosaccharide (HMO)-degrading Bifidobacterium species are key early colonizers of the gut and influence gut and immune maturation. Loss of these taxa, particularly Bifidobacterium infantis, in many industrialized populations has raised concern. O'Brien et al. showed that supplementation with B. infantis EVC001 in exclusively breastfed U.S. infants aged 2-4 months leads to rapid and abundant colonization that persists 1 month after supplementation, demonstrating effective colonization beyond the neonatal period (C. E. O'Brien, S. A. Frese, K. Cernioglo, K. Damian-Medina, et al., mSphere e00518-25, 2025, https://doi.org/10.1128/msphere.00518-25). These findings align with observational cohort data showing that B. infantis can overcome priority effects and dominate the gut microbiome in breastfed infants by 2-3 months of age. Key questions remain regarding colonization in mixed- or formula-fed infants, the HMO thresholds required to sustain dominance during milk- and complementary feeding, and the critical developmental windows of B. infantis colonization for beneficial immune effects. Ongoing clinical trials with B. infantis will further clarify its role in disease prevention.

Candida albicans is a fungal commensal and also a prevalent pathogen of humans. p24 proteins are a family of type I membrane proteins regarded as cargo receptors for endoplasmic reticulum (ER) to Golgi transport and are thought to be involved in regulating secretion. Here, we sought to explore the impact of this family of proteins on C. albicans pathogenicity. The expression of all four members of the p24 family is upregulated during invasive candidiasis. Their expression is independent of yeast-to-hypha transition but is highly induced by tissue culture conditions. We then generated single deletion mutants for each member of the p24 family for phenotypic characterization. All these mutants exhibit significantly attenuated virulence in a mouse model of systemic infection and reduced survival in macrophages but are dispensable for vegetative growth and morphogenesis. They also show lower abundance of chitin and phosphomannan in the cell wall and enhanced sensitivity to fluconazole, an azole antifungal drug. Importantly, the absence of p24 proteins leads to defective protein secretion in C. albicans, including pathogenicity-related effectors and lipases, and reduces commensal fitness. These results suggest that p24 proteins are critical for cell wall integrity, secretion of virulence factors, and virulence in C. albicans.IMPORTANCECandida albicans is an important opportunistic fungal pathogen of immunocompromised individuals and a top-ranking WHO fungal priority pathogen due to the high frequency and mortality of invasive candidiasis. The eukaryotic p24 family of proteins has long been known to be key regulators of protein trafficking along the secretory pathway, but their potential roles regarding pathogenesis in C. albicans remain unknown. Here, we discover that all members of the p24 family are required for cell wall integrity, proper secretion of virulence factors, survival in macrophages, and virulence in a systemic infection model. However, they are dispensable for vegetative growth and yeast-to-hypha transition, the best-known virulence attribute. Our study systematically investigates C. albicans p24 proteins and highlights the critical role that the early secretory pathway plays in fungal pathogenicity.

Halogenated phenazine (HP) compounds have shown promise as antimicrobial agents, particularly against biofilm-associated Gram-positive pathogens. Among these compounds, HP-29 demonstrates potent activity against methicillin-resistant Staphylococcus aureus by inducing rapid iron starvation. As maintenance of trace metals homeostasis is critical for the survival of Streptococcus mutans, this study investigated the antimicrobial efficacy of HP-29 and the impact of metal supplementation on this major oral and occasional systemic pathogen. As anticipated, HP-29 inhibited S. mutans growth in a dose-dependent manner, with iron supplementation alleviating the antimicrobial effect. Cobalt, manganese, or nickel supplementation also mitigated the inhibitory activity of HP-29, but, unexpectedly, the addition of zinc greatly enhanced HP-29 antimicrobial activity. This zinc-driven potentiation of HP-29 extended to other Gram-positive pathogens, including Enterococcus faecalis and S. aureus. Inductively coupled plasma mass spectrometry analysis revealed that intracellular iron content decreased significantly following exposure to HP-29. When combined with zinc, HP-29 triggered a 5-fold increase in intracellular zinc and reduced manganese levels by ~50%. Transcriptome analysis showed that HP-29 treatment, with or without zinc, altered expression of genes linked to iron and manganese uptake as well as zinc efflux, suggesting broad disruption of metal ion regulation. These findings highlight HP-29 as a potent antimicrobial that broadly impairs metal homeostasis. The unexpected synergy of HP-29 with zinc points toward a promising dual-agent therapeutic strategy against Gram-positive pathogens.IMPORTANCEWidespread development of antibiotic resistance has created a constantly moving target when combating infectious microbes. Here, we further explore an antimicrobial halogenated phenazine, HP-29, which is effective against Gram-positive bacteria through disruption of intracellular trace metal equilibrium. We showed that HP-29 inhibits growth of the oral and systemic pathogen Streptococcus mutans and that its antimicrobial effect is greatly potentiated by the addition of zinc. The zinc-mediated enhancement of HP-29's efficacy was also observed in other Gram-positive pathogens, including Enterococcus faecalis and Staphylococcus aureus. Intracellular trace metal quantifications and transcriptome analysis confirmed that HP-29 treatment impairs trace metal homeostasis, an outcome that is exacerbated when S. mutans is treated with both HP-29 and zinc. The observed synergy of HP-29 with zinc supports the development of a dual-agent therapeutic strategy against Gram-positive pathogens.