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The 55-carbon isoprenoid, undecaprenyl-phosphate (UndP), is a universal carrier lipid that ferries most glycans and glycopolymers across the cytoplasmic membrane in bacteria. In addition to peptidoglycan precursors, UndP transports O-antigen, capsule, wall teichoic acids, and sugar modifications. How this shared but limited lipid is distributed among competing pathways is just beginning to be elucidated. We recently reported that in the bacterium Bacillus subtilis, the stress-response sigma factor SigM and its cognate anti-sigma factor complex respond to changes in the free UndP pool. When levels are low, SigM activates genes that increase flux through the essential cell wall synthesis pathway, promote the recycling of the lipid carrier, and liberate the carrier from other polymer pathways. Here, we report that two additional enzymes under SigM control help maintain the free pool of UndP. One, UshA (YqjL), resembles alpha-beta hydrolases and liberates UndP from undecaprenyl-monophosphate-linked sugars. The other, UpsH (YpbG), resembles metallophosphoesterases and releases UndP from undecaprenyl-diphosphate-linked wall teichoic acids polymers but not lipid-linked peptidoglycan precursors. UshA becomes critical for growth when UndP-linked sugars are sequestered, and the carrier lipid pool is depleted. Similarly, UpsH becomes essential for viability when UndPP-linked intermediates accumulate. Mutations in the predicted catalytic residues of both putative hydrolases abrogate their function arguing that they act directly to release UndP. These findings define two new enzymes that liberate the carrier lipid from UndP- and UndPP-linked intermediates and bolster the model that the SigM stress-response pathway maintains the UndP pool and prioritizes its use for peptidoglycan synthesis.IMPORTANCEMotivated by the success of naturally occurring glycopeptide antibiotics like vancomycin, one arm of recent antibiotic discovery efforts has focused on compounds that bind lipid-linked precursors used to build extracytoplasmic polymers. Trapping these precursors depletes the universal carrier lipid undecaprenyl-phosphate, which is required for the synthesis of virtually all surface polymers, including peptidoglycan. Understanding how cells respond to this stress to restore the carrier lipid pool is critical to identifying effective drugs. Here, we report the identification of two new enzymes that are produced in response to the depletion of the carrier lipid pool. These enzymes recover the carrier lipid but cleave distinct lipid-linked precursors to do so.

All organisms produce an intracellular Zn²⁺-dependent enzyme, phosphomannose isomerase (PMI) or mannose-6 phosphate isomerase, that catalyzes the reversible conversion of mannose-6-phosphate and fructose-6-phosphate during sugar metabolism and polysaccharide biosynthesis. Unexpectedly, we discovered an additional PMI function in Borrelia burgdorferi, the pathogen of Lyme disease, where the enzyme is localized on the cell surface and binds to collagen IV-a host extracellular matrix component predominantly found in the skin. The AlphaFold 3-based structural model of B. burgdorferi PMI (BbPMI) retains the active site with tetrahedrally-coordinated Zn²⁺ seen in other PMIs of known structure, residing in an elongated crevice. Ligand docking shows that the crevice can accommodate the tip trisaccharide moiety of a glycosylated asparagine residue on the collagen IV 7S domain. Low doses of a well-known PMI benzoisothiazolone inhibitor impair the growth of diverse strains of B. burgdorferi in culture, but not other tested Gram-negative or Gram-positive pathogens. Borrelia cells are even more susceptible to several other structurally related benzoisothiazolone analogs. The passive transfer of anti-BbPMI antibodies in ticks can impact spirochete transmission to mice, while the treatment of collagen IV-containing murine skin with PMI inhibitors impairs spirochete infectivity. Taken together, these results highlight a newly discovered role for BbPMI in mediating host-pathogen interactions during the spirochete infectivity process. In turn, this discovery offers an opportunity for the development of a novel therapeutic strategy to combat Lyme disease by preventing the BbPMI interaction with its host receptor, collagen IV.

Importance: All organisms produce an intracellular enzyme, phosphomannose isomerase (PMI), that converts specific sugars during metabolism. Unexpectedly, we discovered an additional PMI function in Borrelia burgdorferi, the Lyme disease pathogen, where the enzyme is localized on the cell surface and binds to collagen IV-a host extracellular molecule mainly found in the skin. Low doses of PMI chemical inhibitors impair the growth of diverse strains of B. burgdorferi in culture, but not other tested bacterial pathogens. The passive transfer of anti-BbPMI antibodies in ticks can impact B. burgdorferi transmission to mice, while the treatment of collagen IV-containing murine skin with PMI inhibitors impairs infectivity. Taken together, these results highlight a newly discovered role for BbPMI in mediating host-pathogen interactions during infection. In turn, this discovery offers an opportunity for the development of a novel therapeutic strategy to combat Lyme disease by preventing BbPMI function and interaction with host collagen IV.

The healthy human infant gut microbiome undergoes stereotypical changes in taxonomic composition between birth and maturation to an adult-like stable state. During this time, extensive communication between microbiota and the host immune system contributes to health status later in life. Although there are many reported associations between microbiota compositional alterations and disease in adults, less is known about how microbiome development is altered in pediatric diseases. One pediatric disease linked to altered gut microbiota composition is cystic fibrosis (CF), a multi-organ genetic disease involving impaired chloride secretion across epithelia and heightened inflammation both in the gut and at other body sites. Here, we use shotgun metagenomics to profile the strain-level composition and developmental dynamics of the infant fecal microbiota from several CF and non-CF longitudinal cohorts spanning from birth to greater than 36 months of life. We identify a set of keystone species that define microbiota development in early life in non-CF infants but are missing or decreased in relative abundance in infants with CF, resulting in a delayed pattern of microbiota maturation, persistent entrenchment in a transitional developmental phase, and subsequent failure to attain an adult-like stable microbiota. Delayed maturation is strongly associated with cumulative antibiotic treatments, and we also detect the increased relative abundance of oral-derived bacteria and higher levels of fungi in infants with CF, features that are associated with decreased gut bacterial density. These findings suggest the potential for future directed therapies targeted at overcoming developmental delays in microbiota maturation for infants with CF.IMPORTANCEThe human gastrointestinal tract harbors a diversity of microbes that colonize upon birth and collectively contribute to host health throughout life. Infants with the disease cystic fibrosis (CF) harbor altered gut microbiota compared to non-CF counterparts, with lower levels of beneficial bacteria. How this altered population is established in infants with CF and how it develops over the first years of life is not well understood. By leveraging multiple large non-CF infant fecal metagenomic data sets and samples from a CF cohort collected prior to highly effective modulator therapy, we define microbiome maturation in infants up to 3 years of age. Our findings identify conserved age-diagnostic species in the non-CF infant microbiome that are diminished in abundance in CF counterparts that instead exhibit an enrichment of oral-derived bacteria and fungi associated with antibiotic exposure. Together, our study builds toward microbiota-targeted therapy to restore healthy microbiota dynamics in infants with CF.

Soil microbial diversity and community life strategies are crucial for nutrient cycling during vegetation restoration. Although the changes in topsoil microbial communities during restoration have been extensively studied, the structure, life strategies, and function of microbial communities in the subsoil remain poorly understood, especially regarding their role in nutrient cycling during vegetation restoration. In this study, we conducted a comprehensive investigation of the changes in the soil microbial community, assembly process, life strategies, and nutrient cycling functional genes in soil profiles (0-100 cm) across a 36 year chronosequence (5, 15, 28, and 36 years) of fenced grassland and one grazing grassland on the Loess Plateau of China. Our results revealed that soil organic carbon increased by 76.0% in topsoil and 91.6% in subsoil after 36 years of restoration. The bacterial communities were influenced primarily by soil depth, while the fungal communities were highly sensitive to the years of restoration. Microbes in the subsoil recovered faster, and the microbial community structure and functional genes in the soil profiles gradually became more consistent following restoration. In addition, we observed a transition in microbial life history strategies from a persistent K-strategy to a rapid r-strategy during restoration. Notably, the fungal community assembly process played an important role in changes in nutrient cycling genes, which were accompanied by increased carbon fixation and nitrogen mineralization function. Overall, our findings provide several novel insights into the impact of changes in the fungal community on soil nutrient cycling in the soil profile during vegetation restoration.IMPORTANCEOur study revealed that microbes in the subsoil recovered faster than those in the topsoil, which contributed to the reduction in differences in microbial community structure and the distribution of functional genes throughout the soil profile during the restoration process. Importantly, the assembly of fungal communities plays a pivotal role in driving changes in nutrient cycling genes, such as increased carbon fixation and nitrogen mineralization, alongside a reduction in carbon degradation gene abundance. These alterations increase soil organic carbon and nutrient availability during restoration. Our results increase the understanding of the critical role of fungal communities in soil nutrient cycling genes, which facilitate nutrient accumulation in soil profiles during grassland restoration. This insight can guide the development of strategies for manipulating fungal communities to increase soil nutrients in grasslands.

In the gut, microRNAs (miRNAs) produced by intestinal epithelial cells are secreted into the lumen and can shape the composition and function of the gut microbiome. Crosstalk between gut microbes and the host plays a key role in irritable bowel syndrome (IBS) and inflammatory bowel diseases, yet little is known about how the miRNA-gut microbiome axis contributes to the pathogenesis of these conditions. Here, we investigate the ability of miR-21, a miRNA that we found decreased in fecal samples from IBS patients, to associate with and regulate gut microbiome function. When incubated with the human fecal microbiota, miR-21 revealed a rapid internalization or binding to microbial cells, which varied in extent across different donor samples. Fluorescence-activated cell sorting and sequencing of microbial cells incubated with fluorescently labeled miR-21 identified organisms belonging to the genera Bacteroides, Limosilactobacillus, Ruminococcus, or Coprococcus, which predominantly interacted with miR-21. Surprisingly, these and other genera also interacted with a miRNA scramble control, suggesting that physical interaction and/or uptake of these miRNAs by gut microbiota is not sequence-dependent. Nevertheless, transcriptomic analysis of the gut commensal Bacteroides thetaiotaomicron revealed a miRNA sequence-specific effect on bacterial transcript levels. Supplementation of miR-21, but not of small RNA controls, resulted in significantly altered levels of many cellular transcripts and increased transcription of a biosynthetic operon for indole and L-tryptophan, metabolites known to regulate host inflammation and colonic motility. Our study identifies a novel putative miR-21-dependent pathway of regulation of intestinal function through the gut microbiome with implications for gastrointestinal conditions.

Importance: The mammalian gut represents one of the largest and most dynamic host-microbe interfaces. Host-derived microRNAs (miRNAs), released from the gut epithelium into the lumen, have emerged as important contributors to host-microbe crosstalk. Levels of several miRNAs are altered in the stool of patients with irritable bowel syndrome or inflammatory bowel disease. Understanding how miRNAs interact with and shape gut microbiota function is crucial as it may enable the development of new targeted treatments for intestinal diseases. This study provides evidence that the miRNA miR-21 can rapidly associate with diverse microbial cells form the gut and increase levels of transcripts involved in tryptophan synthesis in a ubiquitous gut microbe. Tryptophan catabolites regulate key functions, such as gut immune response or permeability. Therefore, this mechanism represents an unexpected host-microbe interaction and suggests that host-derived miR-21 may help regulate gut function via the gut microbiota.

Invasive mold-associated cutaneous disease is a rare but potentially catastrophic consequence of trauma. However, invertebrate bites are not well recognized as a mechanism for the inoculation of fungi into subcutaneous tissue that can also result in severe infections. Invertebrates often carry fungi with human pathogenic potential as part of their microbiome, and bites break the skin, providing a conduit for them to penetrate subcutaneous tissues where the establishment of infection can produce serious skin and soft tissue fungal diseases. In this essay, we review the existing data for invertebrate bite-associated cutaneous invasive fungal infections (IBA-cIFIs) and consider the potential consequences of global warming on their epidemiology. Climate changes will be associated with changes in the range of invertebrates and adaptation of their associated microbes to warmer temperatures. Fungal adaptation to higher temperatures can defeat the mammalian protective barrier and be associated with both more and different IBA-cIFIs.

Capsule polysaccharide (CPS) is among the most important virulence factors of Klebsiella pneumoniae. Previous studies demonstrated that CPS plays multiple functional roles, but the mechanism by which this virulence factor enhances the survival fitness of K. pneumoniae remains unclear. In this work, we demonstrate that CPS is the main cellular component that not only elicits the host immune response to K. pneumoniae but also enables this pathogen to survive for a prolonged period under adverse environmental conditions. Consistently, our in vitro experiments suggest that CPS prevents K. pneumoniae from phagocytosis, rendering the encapsulated strain more difficult to be eradicated by the host. We also found that phagocytosis of K. pneumoniae is partially mediated by LOX-1, a scavenger receptor of the host, and that CPS may impede interaction between LOX-1 and this pathogenic bacteria, therefore reducing the phagocytosis process. These findings provide insights into the pathogenic mechanisms of this important clinical pathogen and should facilitate the design of new strategies to combat K. pneumoniae infections.

Importance: Klebsiella pneumoniae has become one of the most important clinical bacterial pathogens due to its evolution into hyperresistant and hypervirulent phenotypes. The mechanism of virulence of this pathogen is not well understood, particularly because it differs from other Enterobacteriaceae pathogens such as Escherichia coli and Salmonella. The capsule polysaccharide (CPS) of this pathogen is well recognized for contributing to the virulence of K. pneumoniae, but the exact mechanisms underlying its contribution are unclear. In this study, we demonstrated that CPS does not directly contribute to the host response; rather, it forms an external coat that blocks host recognition and prevents immune cells from binding to receptor proteins on K. pneumoniae, thus inhibiting phagocytosis, which makes it more challenging for the body to fight off infections. Understanding these mechanisms is vital for developing new treatments against K. pneumoniae infections, ultimately improving patient outcomes and public health.

Acinetobacter baumannii, a prominent nosocomial pathogen renowned for its extensive resistance to antimicrobial agents, poses a significant challenge in the accurate prediction of antimicrobial resistance (AMR) from genomic data. Despite thorough researches on the molecular mechanisms of AMR, gaps remain in our understanding of key contributors. This study utilized rule-based and three machine learning models to predict AMR phenotypes, aiming to decipher key genomic factors associated with AMR. Genomes and antibiotic resistance phenotypes from 1,012 public isolates were employed for model construction and training. To validate the models, a data set comprising 164 self-collected strains underwent next-generation sequencing, nanopore long-read sequencing, and antimicrobial susceptibility testing using the broth dilution method. It was found that the presence of antibiotic resistance genes (ARGs) alone was insufficient to accurately predict AMR phenotype for the majority of antibiotics (90%, 18 out of 20) in the public data set. Conversely, it was observed that combining ARGs with insertion sequence (IS) elements significantly enhanced predictive performance. The Random Forest model was found to outperform the support vector machine (SVM), logistic regression model, and rule-based method across all 20 antibiotics, with accuracies ranging from 83.80% to 97.70%. In the validation data set, even higher accuracies were achieved, ranging from 85.63% to 99.31%. Furthermore, conserved sequence patterns between IS elements and ARGs were validated using self-collected long-read sequencing data, substantially enhancing the accuracy of AMR prediction in A. baumannii. This study underscores the pivotal role of IS elements in AMR.

Importance: The interplay between insertion sequences (ISs) and antibiotic resistance genes (ARGs) in Acinetobacter baumannii contributes to resistance against specific antibiotics. Conventionally, genetic variations and ARGs have been utilized for predicting resistance phenotypes, with the potential pivotal role of IS elements largely overlooked. Our study advances this approach by integrating both rule-based and machine learning models to predict AMR in A. baumannii. This significantly enhances the accuracy of AMR prediction, emphasizing the pivotal function of IS elements in antibiotic resistance. Notably, we uncover a series of conserved sequence patterns linking IS elements and ARGs, which outperform ARGs alone in phenotypic prediction. Our findings are crucial for bioinformatics strategies aimed at studying and tracking AMR, offering novel insights into combating the escalating AMR challenge.