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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.

Synergy between influenza A virus (IAV) and Streptococcus pneumoniae is a long-recognized and clinically important problem. Recent work has demonstrated that IAV particles can directly bind to the bacterial surface and that bacterial-viral complexes exhibit enhanced bacterial colonization and invasive disease, increased viral environmental survival leading to increased efficacy of airborne transmission, and enhanced vaccine response to both pathogens over simultaneous co-infection without direct interactions. However, the molecule(s) responsible for mediating the direct interaction are yet to be characterized. In this study, we demonstrate that the broadly conserved Gram-positive bacterial cell wall glycan lipoteichoic acid (LTA) is one of the molecules that can mediate this interaction. This interaction between viral particles and bacterial cell-envelope glycans is also demonstrated in interactions between enteric viruses and enteric bacteria, suggesting a conserved mechanism of trans-kingdom interactions. We show that LTA will compete for binding between IAV and S. pneumoniae, that disruption of genes responsible for LTA presentation at the cell surface will reduce viral binding, and that viral neuraminidase can bind LTA. This work adds to the growing body of literature on direct bacterial-viral interactions between human-associated bacteria and pathogenic viruses and can provide novel insights into the lethal synergy of influenza-pneumococcal co-infections.IMPORTANCECo-infection between influenza A virus (IAV) and Streptococcus pneumoniae leads to severe disease. Recently, it was demonstrated that IAV particles can bind to the surface of bacterial cells and that direct interactions will enhance both bacterial and viral pathogenesis as well as immune responses to each pathogen. However, it is unclear what bacterial and viral components are responsible for the interaction. We demonstrate that a carbohydrate component of the bacterial cell wall can bind to IAV particles. This is similar to direct interactions observed between enteric viruses and cell wall components of enteric bacteria. This work adds to the body of knowledge about trans-kingdom interactions between human-associated bacteria and human pathogenic viruses, as well as providing novel insights into the serious clinical problem of influenza-pneumococcal synergy.

The infant gut microbiome plays a critical role in the early development of the immune system, brain function, metabolism, and defense against pathogens. However, data from underrepresented populations, like Iceland, with its distinct dietary and lifestyle habits, remain limited. This paper presents the initial findings from the Icelandic Diet and the Infant Gut Microbiome Development (IceGut) study. Fecal samples were collected at multiple time points, representing 328 unique study identifiers, with one to five samples per child, from before the introduction of solid foods up to 5 years of age, and postpartum samples from 214 mothers. Microbial composition and predicted functional potential were assessed using 16S rRNA gene sequencing. Children in the cohort followed typical gut microbiome maturation, but at 1 year of age, they showed a notably higher relative abundance of Blautia than reported in comparable cohorts. This time point marked a transition in both taxonomic composition and predicted functional gene counts. By 5 years, the children had higher observed richness than their mothers but lower Shannon and Simpson diversities. At 2 and 5 years, and in the mothers, samples positive for archaea had significantly higher alpha diversity than samples that tested negative for archaea. Mothers with gestational diabetes mellitus (GDM) exhibited a higher relative abundance of Blautia but a lower alpha diversity. The variance in offspring gut microbiome explained by maternal GDM became progressively stronger over time, being significant at the age of 5 and explaining 2.5% of the variance.

Importance: This study provides the first comprehensive analysis of gut microbiome development in Icelandic children, covering the time from before the introduction of solid foods to 5 years of age. Although the overall developmental patterns of the gut microbiome in Icelandic children were similar to what has been seen in other studies, interesting differences were observed, such as a higher abundance of Blautia at an earlier age compared to other study populations. Higher alpha diversity in archaeal-positive samples, both in mothers and in children at the ages of 2 and 5, compared with archaeal-negative samples seen in the present study, is worth further investigation. Additionally, the study suggests a potential role of maternal and perinatal factors, particularly GDM, which was not evident until the age of 5 years, emphasizing the necessity of long-term studies.

Enterococcus faecium is a member of the human gut microbiota that has evolved into a problematic nosocomial pathogen and a leading cause of infections in hospitalized patients. Treatment of E. faecium infections is complicated by antibiotic resistance, making it important to understand resistance mechanisms and their broader consequences in this pathogen. Here, we explored the collateral effects of rifampin resistance-associated mutations in the E. faecium RNA polymerase β-subunit (RpoB). Of 14,384 publicly available E. faecium genomes, nearly one-third carried a mutation in the rifampin resistance-determining region (RRDR) of RpoB. In a local population of 710 E. faecium clinical isolates collected from patients at a single hospital, we found significant associations between the presence of RRDR mutations and prior exposure to rifamycin antibiotics, as well as associations between RRDR mutations and altered daptomycin susceptibility. To investigate the phenotypic impacts of RRDR mutations, we generated and studied four isogenic strains with distinct RRDR mutations (Q473K, G482D, H486Y, and S491L) that overlapped with clinical isolate variants. Transcriptomic and phenotypic analyses revealed allele-specific effects on E. faecium gene expression, growth dynamics, antibiotic susceptibility, isopropanol tolerance, and cell wall physiology. One frequently observed mutation, H486Y, caused minimal transcriptional changes and enhanced bacterial fitness under antibiotic stress. In contrast, the S491L mutation induced extensive transcriptional changes and slowed bacterial growth but also conferred increased isopropanol tolerance, potentially enhancing bacterial survival on hospital surfaces. Overall, our findings highlight the multifaceted impacts of RRDR mutations in shaping E. faecium physiology and antibiotic resistance, two important features of this hospital-associated pathogen.IMPORTANCEUnderstanding how antimicrobial resistance affects bacterial physiology is critical for developing effective therapeutics against bacterial infections. In this study, we found that rifampin resistance-associated mutations in RpoB are widespread in Enterococcus faecium, a leading multidrug-resistant pathogen. By studying isogenic wild-type and RpoB mutant strains, we discovered that RpoB mutations, although conferring resistance to rifampin, have distinct allele-specific effects on other bacterial phenotypes. Some of these collateral effects appear to promote E. faecium resistance to antibiotics and survival in the hospital environment, raising questions about the selective pressures driving their emergence. Overall, our study underscores the importance of examining the collateral effects of resistance-associated mutations in multidrug-resistant pathogens, which could help mitigate their persistence and spread among vulnerable patients.

Nucleoid-associated proteins (NAPs) are essential in bacteria for maintaining nucleoid architecture and regulating the expression of target genes. Although some NAPs have been well studied in certain bacterial species, their specific functions and regulatory mechanisms remain poorly characterized in mycobacteria. In this study, we identified NapR as a novel nucleoid-associated protein in mycobacteria. We showed that NapR, which is highly conserved among mycobacteria, binds DNA and modulates DNA topology through bridging. Furthermore, we demonstrated that NapR acts as a positive transcriptional regulator of the ggr gene, which encodes geranylgeranyl reductase, thus regulating bacterial antioxidant defense. By controlling ggr expression, NapR modulates the level of intracellular ROS and influences the antioxidant capacity of mycobacteria. This study identifies NapR as a novel nucleoid-associated protein and defines a specific regulatory pathway involved in mycobacterial antioxidant defense, providing new insights into the mechanisms of the bacterial oxidative stress response.IMPORTANCEAs important global regulatory factors, nucleoid-associated proteins (NAPs) can help bacteria adapt to environmental stress, such as oxidative stress. However, the regulatory mechanism of NAPs in mycobacterial antioxidant defense is largely unclear and remains to be explored. Here, we identify NapR as a novel nucleoid-associated protein that modulates DNA topology by bridging. We revealed the regulatory effect of NapR on mycobacterial antioxidant defense. NapR positively regulates the expression of the geranylgeranyl reductase-encoding gene ggr. In addition, the ability of NapR to regulate the levels of intracellular ROS relies on ggr, ultimately leading to the antioxidant defense of Mycobacterium smegmatis. Our findings identify a new member of the NAP family and contribute to understanding the mechanisms of bacterial antioxidant defense.

The emergence of pathogens resistant to antimicrobials has become a forefront concern for clinicians and patients alike. Antimicrobial resistance (AMR) is exacerbated by the misuse and overuse of antibiotics. Pregnant women and their infants are an important area of focus, as antibiotic use during this vulnerable period of development may generate reservoirs of AMR genes, which would contribute to future risk. Identifying the extent of antibiotic use and its association with ARG composition and persistence within this window is crucial. We sought to characterize the gut resistomes of 3-month-old infants (n = 212) and pregnant women in their third trimester (n = 99) to assess ARG burden in these populations. For a subset of women and their infants (n = 33 pairs), we explored overlap of ARG. Preliminary analyses demonstrated that pregnant women and infants had markedly different resistome communities and identified other environmental and demographic characteristics to be associated with univariate differences in infant ARG composition. When controlling for the race of the mother, infant diet, and infant antibiotic exposure since birth, delivery by cesarean section was associated with increased diversity of ARG relative to the diversity of ARG in the samples from vaginally born infants. Cesarean-born infants had increased richness of aminoglycoside ARG and increased diversity of beta-lactamase and tetracycline ARG relative to vaginally born infants. Furthermore, infants consuming any formula had increased overall richness and diversity of ARG in multivariate analyses. This study provides further insight into how diet and method of delivery are associated with resistome composition within the first 3 months of infant microbiome development.IMPORTANCEPregnancy and the first 3 months of life are vulnerable periods for antibiotic exposure and subsequent development of antimicrobial resistance (AMR). AMR is an increasingly worrisome problem for global public health. The full repertoire of AMR genes present in the gut collectively is referred to as the resistome. Herein, the associations between a variety of demographic and environmental factors, including race of the pregnant women, sex of the infant, mode of delivery, amount of breast milk consumed in infant diet, and antibiotic exposure during the first 3 months of life, with resistome composition are reported. Infants consuming any formula had a greater richness and diversity of ARG overall, and cesarean-born infants had greater diversity of ARG within their resistomes. These findings give insight into the early seeding of the infant resistome, which is crucial to understanding how the resistome develops throughout life.