mBio最新文献

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.

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.

Post-acute sequelae of COVID-19 involves several organs, but its basis remains poorly understood. Some infected cells in mice survive the acute infection and persist for extended periods in the respiratory tract but not in other tissues. Here, we describe two experimental models of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection to assess the effect of viral virulence on previously infected cells. Both approaches use lineage tracking of previously infected cells. In mice infected with a highly pathogenic mouse-adapted SARS-CoV-2, alveolar type 2 cells (AT2) but not alveolar type 1 (AT1) cells survived the acute infection. These cells became activated, differentiated into an AT2-to-AT1 transitional cell state (KRT8⁺ pre-alveolar type 1 transitional cell state). Additionally, nearby uninfected AT2 cells upregulated the transitional marker KRT8, thereby contributing to lung regeneration. In mice sensitized to infection by transduction with Ad5-hACE2, the infection is nonlethal, and AT1 cells survived the infection. Consequently, recovery in these mice was more rapid. Taken together, these results provide an explanation for how SARS-CoV-2 virulence contributes to poor outcomes and affects clinical recovery and lung regeneration. We also identified a new mechanism by which SARS-CoV-2 impacts lung recovery, even at times when infectious virus cannot be detected.

Importance: A major consequence of the COVID-19 pandemic is that many survivors have long-term sequelae, which are not well understood. These involve many organs, with the respiratory tract being a common site of long-term effects. Many of these sequelae can be found in mice infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). In this study, we have focused on the lungs, with particular interest in the fate and role of cells that were infected with SARS-CoV-2 and survived the acute infection. We found that some infected cells survive acute SARS-CoV-2 infection and that these surviving cells both contribute to the immune response in the lungs and are involved in lung recovery. These findings illustrate previously unexplored aspects of recovery from SARS-CoV-2 induced pneumonia and may be relevant for understanding aspects of post-acute sequelae of COVID-19.

The composition of the gut microbiome is determined by a complex interplay of diet, host genetics, microbe-microbe interactions, abiotic factors, and stochasticity. Previous studies have demonstrated the importance of host genetics in community assembly of the Caenorhabditis elegans gut microbiome and identified a central role for DBL-1/BMP immune signaling in determining the abundance of gut Enterobacteriaceae. However, the effects of DBL-1 signaling on gut bacteria were found to depend on its activation in extra-intestinal tissues, highlighting a gap in our understanding of the proximal factors that determine microbiome composition. In the present study, we used RNA-seq gene expression analysis of wildtype, dbl-1 and sma-3 mutants, and dbl-1 over-expressors to identify candidate DBL-1/BMP targets that may mediate the pathway's effects on gut commensals. Bacterial colonization experiments in mutants, or following RNAi-mediated knock-down of candidate genes specifically in the intestine, demonstrated their local contribution to intestinal control of Enterobacteriaceae abundance. Furthermore, epistasis analysis suggested that these contributions were downstream of the DBL-1 pathway, together suggesting that examined candidates were intestinal effectors and mediators of DBL-1 signaling, contributing to the shaping of gut microbiome composition.IMPORTANCECompared to the roles of diet, environmental availability, or lifestyle in determining gut microbiome composition, that of genetic factors is the least understood and often underestimated. The identification of intestinal effectors of distinct molecular functions that control enteric bacteria offers a glimpse into the genetic logic of microbiome control as well as a list of targets for future exploration of this logic.

The emerging fungal pathogen Candida auris is known for its strong skin tropism and resilience against antifungal and disinfection treatment, posing a significant challenge for healthcare units. Although efforts to identify the effectors of its unique pathogenic behavior have been insightful, the role of the high-osmolarity glycerol (HOG) pathway in this context remains unexplored. The study by Shivarathri and co-workers (R. Shivarathri, M. Chauhan, A. Datta, D. Das et al., mBio 15:e02748-24, 2024, https://doi.org/10.1128/mbio.02748-24) sought to address this gap. This report indeed advances our understanding of the critical role of the HOG pathway in C. auris pathogenicity by emphasizing its involvement in skin colonization, biofilm formation, and evasion of phagocyte attack.

Peptidoglycan (PG) is an important bacterial macromolecule that confers cell shape and structural integrity, and is a key antibiotic target. Its synthesis and turnover are carefully coordinated with other cellular processes and pathways. Despite established connections between the biosynthesis of PG and the outer membrane, or PG and DNA replication, links between PG and folate metabolism remain comparatively unexplored. Folate is an essential cofactor for bacterial growth and is required for the synthesis of many important metabolites. Here we show that inhibition of folate synthesis in the important Gram-negative pathogen Pseudomonas aeruginosa has downstream effects on PG metabolism and integrity that can manifest as the formation of a subpopulation of round cells that can undergo explosive lysis. Folate inhibitors potentiated β-lactams by perturbation of PG recycling, reducing expression of the AmpC β-lactamase. Supporting this mechanism, folate inhibitors also synergized with fosfomycin, an inhibitor of MurA, the first committed step in PG synthesis that can be bypassed by PG recycling. These insights led to the design of a dual-active inhibitor that overcomes NDM-1 metallo-β lactamase-mediated meropenem resistance and synergizes with the folate inhibitor, trimethoprim. We show that folate and PG metabolism are intimately connected, and targeting this connection can overcome antibiotic resistance in Gram-negative pathogens.

Importance: To combat the alarming global increase in superbugs amid the simultaneous scarcity of new drugs, we can create synergistic combinations of currently available antibiotics or chimeric molecules with dual activities, to minimize resistance. Here we show that older anti-folate drugs synergize with specific cell wall biosynthesis inhibitors to kill the priority pathogen, Pseudomonas aeruginosa. Anti-folate drugs caused a dose-dependent loss of rod cell shape followed by explosive lysis, and synergized with β-lactams that target D,D-carboxypeptidases required to tailor the cell wall. Anti-folates impaired cell wall recycling and subsequent downstream expression of the chromosomally encoded β-lactamase, AmpC, which normally destroys β-lactam antibiotics. Building on the anti-folate-like scaffold of a metallo-β-lactamase inhibitor, we created a new molecule, MLLB-2201, that potentiates β-lactams and anti-folates and restores meropenem activity against metallo-β-lactamase-expressing Escherichia coli. These strategies are useful ways to tackle the ongoing rise in dangerous bacterial pathogens.

The emergence and global spread of carbapenem-resistant Enterobacter cloacae complex species present a pressing public health challenge. Carbapenem-resistant Enterobacter spp. cause a wide variety of infections, including septic shock fatalities in newborns and immunocompromised adults. The intestine may be a major reservoir for these resistant strains, either by facilitating contamination of fomites and transfer to susceptible individuals, or through translocation from the gut to the bloodstream. For this reason, we sought to establish a neonatal mouse model to investigate the mechanisms underpinning gut colonization by carbapenem-resistant Enterobacter hormaechei. We describe a new mouse model to study gut colonization by Enterobacter spp., leading to vital insights into the adaptation of carbapenem-resistant E. hormaechei to the gut environment during the early stages of intestinal colonization. We observed successful colonization and proliferation of E. hormaechei in the 5-day-old infant mouse gut, with primary localization to the colon following oral inoculation. We also uncovered evidence that E. hormaechei uses mucus as a carbon source during colonization of the colon. Our findings underscore the importance of oxygen-dependent metabolic pathways, including the pyruvate dehydrogenase complex and N-acetyl-D-glucosamine metabolism, in gut colonization and proliferation, which aligns with previous human studies. These insights are essential for developing novel therapeutic strategies that can serve as decolonization therapies in at-risk populations.IMPORTANCEBloodstream infections caused by Enterobacter spp. pose a significant clinical threat. The intestine acts as the primary site for colonization and serves as a reservoir for infection. To combat this pathogen, it is crucial to understand how carbapenem-resistant Enterobacter spp. colonize the gut, as such knowledge can pave the way for alternative therapeutic targets. In this study, we developed a novel neonatal mouse model for gastrointestinal colonization by Enterobacter spp. and discovered that mucus plays a key role as a carbon source during colonization. Additionally, we identified two mucus catabolism pathways that contribute to intestinal colonization by carbapenem-resistant E. hormaechei. This new mouse model offers valuable insights into host-pathogen interactions and helps identify critical gastrointestinal fitness factors of Enterobacter, potentially guiding the development of vaccines and alternative therapeutic strategies to minimize intestinal carriage in patient populations at risk of infection with Enterobacter spp.