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Emerging viruses remain a threat to human health; however, many aspects of their infection cycle are still poorly understood. Host lipid structures and abundances are observed to be significantly altered during infection, and the mechanisms regulating lipid synthesis and modification remain largely unknown. In this work, we analyzed a large multi-omic data set from three Middle East respiratory syndrome coronavirus (MERS-CoV)-infected primary human lung cell types, all derived from three distinct donors to investigate the changes in lipid species during infection. Analysis of lipidomics data identified perturbations of various lipid classes, and we hypothesized and confirmed that MERS-CoV infection orchestrates an increase in ceramide via sphingomyelinase pathways required for infection. We also identified a minor subset of proteins with lipid-related functions with increased differential expression among a striking majority of lipid-related proteins with decreased differential expression. The most prominent of these is ACSL3, a long-chain acyl-CoA synthetase that is key for the synthesis of triacylglycerides and is associated with lipid droplet formation, an established feature of coronavirus-infected cells. Accordingly, the inhibition of acyl-CoA synthetase activity reduced MERS-CoV replication. These results suggest a model wherein coronaviruses perturb overall cellular metabolism to shift resources to the production of ceramides and triacylglycerides, particularly through acyl-CoA synthetase activity. Our findings suggest a strategy for targeting CoV replication through the inhibition of specific subsets of lipid metabolism.

Importance: Combating emerging viral threats requires an in-depth understanding of how the virus commandeers host resources to facilitate replication. Viral particles are comprised of protein and lipids; hence, the synthesis of both is critical for virus spread. Our studies have demonstrated that the synthesis of two lipid species, ceramides and triacylglycerides, is essential for Middle East respiratory syndrome coronavirus replication and that virus replication is impaired if these synthetic pathways are blocked. These results suggest a model wherein coronaviruses perturb overall cellular metabolism to shift resources to the production of ceramides and triacylglycerides. Our findings suggest a strategy for targeting coronavirus replication through the inhibition of specific subsets of lipid metabolism.

Tomato is a staple crop and an excellent model to study host-microbiota interactions in the plant food chain. In this study, we describe a "lab-in-the-field" approach to investigate the microbiota of field-grown tomato plants. High-throughput amplicon sequencing revealed a three-microhabitat partition, phyllosphere, rhizosphere, and root interior, differentiating host-associated communities from the environmental microbiota. An individual bacterium, classified as Acinetobacter sp., emerged as a dominant member of the microbiota at the plant-soil continuum. To gain insights into the functional significance of this enrichment, we subjected rhizosphere specimens to shotgun metagenomics. Similar to the amplicon sequencing survey, a "microhabitat effect," defined by a set of rhizosphere-enriched functions, was identified. Mobilization of mineral nutrients, as well as adaptation to salinity and polymicrobial communities, including antimicrobial resistance genes (ARGs), emerged as a functional requirement sustaining metagenomic diversification. A metagenome-assembled genome representative of Acinetobacter calcoaceticus was retrieved, and metagenomic reads associated with this species identified a functional specialization for plant-growth promotion traits, such as phosphate solubilization, siderophore production, and reactive oxygen species detoxification, which were similarly represented in a tomato genotype-independent fashion. Our results revealed that the enrichment of a beneficial bacterium capable of alleviating plant abiotic stresses appears decoupled from ARGs facilitating microbiota persistence at the root-soil interface.IMPORTANCETomatoes are at center stage in global food security due to their high nutritional value, widespread cultivation, and versatility. Tomatoes provide essential vitamins and minerals, contribute to diverse diets, and support farmer livelihoods, making them a cornerstone of sustainable food systems. Beyond direct dietary benefits, the intricate relationship between tomatoes, their associated microbiota, and antimicrobial resistance gene (ARG) is increasingly recognized. Tomato plants host diverse microbial communities in association with their organs, which influence plant health and productivity. Crop management impacts the composition and function of these communities, contributing to the prevalence of ARGs in the soil and on the plants themselves. These genes can potentially transfer to human pathogens, posing a food safety and public health risk. Understanding these complex interactions is critical for developing sustainable agricultural practices capable of mitigating the impact of climatic modifications and the global threat of antimicrobial resistance.

The study of the human microbiome (mirroring broader practice across biomedical science), has historically defaulted to the use of simplified, socially constructed "boxes," such as racial and ethnic labels, that fail to accurately capture human variation and fundamentally misdirect the search for mechanisms to explain differences in health outcomes. Five years ago, I proposed a "frameshift," a fundamental conceptual shift away from relying on these categories and toward a more nuanced, careful approach to the complexity of human variation. Moving "out of the box" means tackling the difficult but essential work of analyzing microbial variation through a systems lens, connecting large-scale ecosocial drivers to individual mechanisms and outcomes. In this Full Circle review, I discuss rapid progress in the field toward this new framework and argue that by adopting transdisciplinary methods, we can generate more accurate, actionable, and equitable solutions for human health.

The situation regarding drug resistance among gram-negative bacteria is becoming increasingly severe. While antimicrobial peptides are an ideal alternative to traditional antibiotics, single-target natural antimicrobial peptides exhibit limitations, including high toxicity and poor permeability. Given the numerous advantages of dual-target peptides for disease treatment, we designed and synthesized the first membrane/ribosome dual-target antimicrobial peptide, FPON, through a functional peptide splicing strategy utilizing FP-CATH and Oncocin as templates. FPON specifically targets gram-negative bacteria and possesses dual functionalities: the ability to disrupt bacterial membrane integrity and the ability to inhibit protein translation. Additionally, FPON exhibited low toxicity and demonstrated significant activity against drug-resistant bacteria in vitro and in vivo. In conclusion, the results presented in this study provide further evidence that dual-targeted antimicrobial peptides constitute an effective treatment strategy against gram-negative drug-resistant bacteria.IMPORTANCEThe issue of antibiotic drug resistance in gram-negative bacteria is one of grave urgency. While single-target antimicrobial peptides offer a potential solution to antibiotic resistance, therapeutic applications are constrained by their high toxicity and poor penetration. In this study, FP-CATH and Oncocin were used as templates for functional peptide splicing to develop FPON, a novel antimicrobial peptide. FPON was shown to disrupt bacterial membranes and inhibit protein synthesis, effectively eliminating gram-negative bacteria. Moreover, FPON exhibits low toxicity and has a significant effect against drug-resistant bacteria. Our research demonstrates that a dual-target design offers a promising avenue for addressing drug-resistant infections.

The increase in the infection caused by Acinetobacter baumannii is sustained by the selection of distinct epidemic clonal lineages, which are frequently resistant to a broad range of antimicrobials and possess virulence traits responsible for their persistence in the contaminated environment and spread among patients. The present study aimed to perform an integrated genomic and phenotypic analysis to assess the virulence features of ST25 isolates. A. baumannii isolates assigned to the ST25 epidemic clonal lineage shared high genomic similarity and clustered in four clades (I, II, III, and IV), with clade IV further subdivided into CIVa, CIVb, CIVc, and CIVd. Capsular locus (KL) KL14 was the predominant KL type (47%). Accessory genome analysis showed the presence of tartrate metabolism genes only in CII genomes. CIVb and CIVd ST25 A. baumannii isolates showed higher ability to infect Galleria mellonella larvae than CI, CII, CIII, and CIVc isolates. Hydrogen peroxide resistance was higher in CI, CII, CIVb, and CIVd isolates compared with CIII and CIVc isolates. In desiccation survival tests, CIII, CIVb, and CIVd isolates exhibited prolonged survival. In addition, CI, CII, CIII, CIVb, and CIVd isolates showed higher serum resistance than CIVc isolates. Also, KL14 type and lipooligosaccharide outer core locus (OCL) OCL6 type isolates were significantly more resistant to oxidative stress, to desiccation, and possessed a high ability to kill G. mellonella larvae. A positive and significant correlation was found between AdeB and AdeJ efflux pump expression and hydrogen peroxide resistance.IMPORTANCEIn this study, we characterized the genotypic and phenotypic features of A. baumannii strains assigned to the ST25 epidemic clonal lineage, which were isolated from humans, animals, and the environment. We found that ST25 A. baumannii isolates, irrespective of their antimicrobial resistance, showed peculiar virulence features among clades, isolates assigned to clade IVb and IVd showing the highest virulence and elevated resistance to serum and desiccation. Also, a positive significant correlation was found between the presence of KL14 and outer core locus 6 genotypes and resistance to oxidative stress, resistance to desiccation, and the ability to kill G. mellonella larvae. Phenotypic differences reflected clade identity rather than isolate origin, suggesting that specific virulence traits contribute to the environmental persistence and pathogenic potential of A. baumannii ST25 isolates.

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.

Glycosaminoglycans (GAGs), comprising uronic acids and amino sugars, are widely distributed in human tissues such as the intestine and oral cavity. Various bacteria colonize these tissues by assimilating GAGs. During GAG degradation, 4-deoxy-l-threo-5-hexosulose uronate (DHU) is produced. Pectin, an abundant plant component, is also degraded into DHU. DHU is metabolized in a stepwise manner by the isomerase KduI or its nonhomologous isofunctional enzyme DhuI, followed by the reductase KduD or DhuD, belonging to the same reductase-dehydrogenase family. Previous studies have found that the genes encoding isomerase and reductase (kduI-kduD and dhuD-dhuI, respectively) are usually organized in clusters. Therefore, it was believed that the kduI-kduD and dhuD-dhuI clusters evolved independently. However, the discovery of a hybrid kduI-dhuD cluster raised questions regarding the evolution of these clusters. This study investigated the diversity of clusters through a pan-genomic phylogenetic analysis across 3,550 bacterial strains. Among 16 possible cluster structures, 10 types were involved in DHU metabolism. Bacteroidota possessed a hybrid-type kduI-dhuD cluster, while Bacillota, but not Pseudomonadota or Bacteroidota, possessed the cluster dhuD-dhuI. Using public data sets from the human fecal microbiome and environmental habitats, we detected the prevalence of kduI-dhuD and dhuD-dhuI clusters in gut microbes. Although DHU is generated from oligomerized GAG degradation by unsaturated glucuronyl hydrolase (UGL), the UGL gene was frequently found in pathogenic strains containing kduD-kduI, dhuD-dhuI, kduI-dhuD, or dhuD-kduI, indicating that the acquisition of these clusters is advantageous for human colonization.IMPORTANCEGlycosaminoglycans (GAGs), crucial components of the extracellular matrix, play vital roles in host infection by pathogenic bacteria and host colonization by commensal bacteria. The dhuD-dhuI cluster is well conserved within certain phyla, and it appears to have a strong association with GAG metabolism. In contrast, kduI-containing clusters are more widely distributed across bacterial species. Based on the possession ratios of genes encoding the enzymes involved in the production of 4-deoxy-l-threo-5-hexosulose uronate, this study indicates that the substrates differ depending on the specific cluster type.

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.