Biology subjects

Sierra, R.

Publications and source records attributed to Sierra, R..

7 recordsLinked to original sources

bioRxiv2026

Early metabolic reprogramming licenses Streptococcus pneumoniae for Influenza-driven superinfection

Bacterial pneumonia remains a major cause of morbidity and mortality following influenza A virus (IAV) infection. However, the adaptive mechanisms that enable pathogen expansion in the post-viral lung remain poorly defined. Here, using a mouse model of IAV-Streptococcus pneumoniae superinfection, we characterize bacterial transcriptional reprogramming in vivo. We identify alcohol dehydrogenases (AdhA and AdhE), that support NAD regeneration during mixed-acid fermentation, as key determinants of bacterial fitness specifically in the IAV-primed lung. Genetic deletion of these results in a pronounced fitness defect during superinfection but not in primary bacterial pneumonia. Consistent with this requirement, pharmacological inhibition of alcohol dehydrogenases limits bacterial expansion and dissemination following IAV infection. Mechanistically, we show that IAV infection profoundly remodels the lung environment, inducing hypoxia and increasing the availability of alternative carbon sources, which together impose a metabolic dependency on Adh for bacterial expansion. Our findings place metabolic adaptation as a central driver of pneumococcal outgrowth following viral infection and reveal exploitable vulnerabilities for therapeutic intervention.

microbiology↗

bioRxiv2025

A two-component system signaling hub controls enterococcal membrane remodeling in response to daptomycin

Daptomycin is a last resort antibiotic used to treat vancomycin-resistant enterococcal infections, but daptomycin resistance (DAPR) arises quickly during treatment. Resistance is due to sequential acquisition of point mutations in the two-component system LiaFSR and in cardiolipin synthases and is associated with alteration of phospholipid and glycolipid membrane composition. The molecular mechanisms underlying these lipid changes are currently unknown. Similarly, it is unclear why mutations in liaFSR occur prior to mutations in cls. We discovered that Enterococcus faecalis remodels membrane composition as a phenotypic response to daptomycin that parallels the membrane composition of DAPR strains. The enrichment in glycolipids that follows antibiotic exposure is due to LtaS1, the main LTA synthase of E. faecalis. Moreover, LtaS1 activity is governed by a network of two-component systems formed by LiaFSR, SapRS, and BsrRS that couple antibiotic sensing with membrane lipid remodeling. Together, our results provide a unifying mechanism that drives phenotypic membrane fortification in a Gram-positive pathogen which simultaneously predisposes the cell to acquire genetic high-level daptomycin resistance. Significance StatementDaptomycin is the preferred alternative to treat vancomycin-resistant Enterococcus infections. However, the efficacy of daptomycin is limited by the acquisition of daptomycin resistance. Alterations in lipid membrane composition represent a conserved strategy to fortify the Gram-positive membrane. We discovered that E. faecalis phenotypically remodels membrane composition in response to daptomycin and that the enrichment in glycolipids is dependent on LTA synthesis. We expand our limited knowledge of enterococcal LTA synthases, confirming LtaS1 as the main LTA synthase and placing LTA biogenesis under control of a network of antibiotic-responsive two-component systems. Our work provides a molecular explanation for the sequential evolution of daptomycin resistance and may support the use of existing LtaS inhibitors to prevent acquisition of daptomycin resistance.

microbiology↗

bioRxiv2025

Time-resolved structures of β2-adrenergic receptor modulation by a photoswitchable beta-blocker

G protein-coupled receptors (GPCRs) regulate essential physiological responses and are important drug targets, yet their ligand-induced conformational dynamics remain poorly understood. The {beta}2-adrenergic receptor ({beta}2AR) is a prominent member of the GPCR family. It regulates bronchial and vascular function and is a significant drug target, particularly in respiratory and smooth muscle-related disorders. We employed time-resolved crystallography at X-ray free-electron lasers (XFELs) to capture the conformational dynamics of {beta}2AR bound to photoazolol-1, a beta-blocker derivative developed for photopharmacological applications. Structural snapshots of the receptor bound to trans-photoazolol-1 (pre-photoconversion), a strained intermediate, and the fully photoisomerized cis-photoazolol-1 reveal an intricate interplay between ligand chemistry and receptor plasticity. Isomerization of the azobenzene moiety induces distinct conformational changes within the orthosteric pocket, altering interactions with the extracellular loop 2 and transmembrane helices 5 and 6. Supported by functional assays, these structural shifts suggest that photoazolol-1 transitions from an inverse agonist to a neutral antagonist upon photoactivation. Our findings uncover a mechanism of GPCR modulation reminiscent of rhodopsin activation and offer a framework for designing ligands that harness light-driven transitions to achieve spatiotemporal control of receptor function.

biochemistry↗

bioRxiv2025

Ambient temperature structural studies of Penicillin-binding Protein 2a of Methicillin-Resistant Staphylococcus aureus with XFELs and synchrotrons

Penicillin-binding protein 2a (PBP2a) is a transpeptidase responsible for the {beta}-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA), posing significant challenges to antibiotic therapy. PBP2as unique structural features, including its highly flexible active site and allosteric regulation, enable it to maintain catalytic activity even in the presence of {beta}-lactam antibiotics. Despite extensive characterization using cryogenic crystallography, key questions remain about its dynamic properties and conformational changes under near-physiological conditions. Room-temperature crystallography methods, particularly serial femtosecond X-ray crystallography (SFX) at XFELs, provide a powerful approach to capture these dynamics. Unlike cryogenic conditions that can constrain protein flexibility, SFX enables the study of conformational variability and interaction networks closer to physiological states. Here, we present the first room-temperature structures of PBP2a obtained using SFX to uncover insights into the enzymes flexibility, allosteric communication, and catalytic mechanisms. These findings are built upon optimized large-scale production and crystallization protocols for PBP2a, ensuring high-quality microcrystals suitable for SFX data collection. The room-temperature structures reveal novel interaction patterns, including unique salt bridge networks and dynamic structural elements absent in cryogenic studies. Furthermore, comparative analyses highlight how environmental conditions influence the conformational states of PBP2a, providing new perspectives on its resistance mechanisms. By integrating structural data from EuXFEL and LCLS, this study not only enhances our understanding of PBP2as functional dynamics but also underscores the value of room-temperature crystallography in studying antibiotic resistance. These insights could guide the design of next-generation {beta}-lactam antibiotics capable of overcoming PBP2a-mediated resistance.

biochemistry↗

bioRxiv2024

Genomic characterization of plasmids harboring blaNDM-1,-5,-7 carbapenemase alleles in clinical Klebsiella pneumoniae in Pakistan

Klebsiella pneumoniae is notorious for causing healthcare-associated infections, which become more complicated by the acquisition of blaNDM genes via mobile genetic elements. Although Pakistan is a well-established hot spot of blaNDM-positive K. pneumoniae, detailed molecular descriptions of blaNDM-carrying plasmids are scarce. Seven K. pneumoniae isolates harboring blaNDM were recovered from clinical sample sources during a six-month period and tested for antimicrobial susceptibility. A long-read approach was used for whole genome sequencing to obtain circularized plasmids and chromosomes for typing, annotation, and comparative analysis. The isolates were susceptible to colistin and tigecycline only among the tested antibiotics. We identified five STs: ST11, ST16, ST716, ST464, and ST2856. Notably, three strains possessed the hypervirulent capsule KL2, while five were classified as O locus type O2a. Evidence of genetic diversity was further highlighted by the presence of four IncC plasmids harboring blaNDM-1, two IncX3 plasmids harboring blaNDM-5, and a single hybrid IncFIB/IncHI1B plasmid harboring blaNDM-7. These plasmids also carried additional ARGs conferring resistance to aminoglycosides, cephalosporins, and fluoroquinolones. We identified the plasmidome of the K. pneumoniae isolates and characterized the NDM-carrying plasmids. Genetic analysis confirmed the presence of blaNDM-1 and blaNDM-5 on broad host range plasmids and blaNDM-7 in a previously unreported hybrid plasmid backbone. We emphasized the critical role of plasmids in spreading blaNDM in the clinical setting in Pakistan. Hence, we stressed the urgent need for enhanced surveillance, not least in LMICs, infection control measures, and adherence to the AWaRe guidelines in antibiotics use.

microbiology↗

bioRxiv2023

P53 and BCL-2 family proteins PUMA and NOXA define competitive fitness in Pluripotent Cells

Cell Competition is a process by which neighboring cells compare their fitness. As a result, viable but suboptimal cells are selectively eliminated in the presence of fitter cells. In the early mammalian embryo, epiblast pluripotent cells undergo extensive Cell Competition, which prevents suboptimal cells from contributing to the newly forming organism. While competitive ability is regulated by MYC in the epiblast, the mechanisms that contribute to competitive fitness in this context are largely unknown. Here, we report that P53 and its pro-apoptotic targets PUMA and NOXA regulate apoptosis susceptibility and competitive fitness in pluripotent cells. PUMA is widely expressed specifically in pluripotent cells in vitro and in vivo. We show that the p53-PUMA/NOXA pathway regulates mitochondrial membrane potential and oxidative status. We found that P53 regulates MYC levels in pluripotent cells, which connects these two Cell competition pathways, however, MYC and PUMA/NOXA levels are independently regulated by P53. We propose a model that integrates a bifurcated P53 pathway regulating both MYC and PUMA/NOXA levels and determines competitive fitness through regulation of mitochondrial activity.

developmental biology↗

bioRxiv2018

Myc is dispensable for cardiac development in the mouse but can rescue Mycn-deficient hearts through functional replacement and cell competition

Myc is considered an essential transcription factor for heart development, but cardiac defects have only been studied in global Myc loss of function models. Here, we eliminated Myc by recombining a Myc floxed allele with the Nkx2.5Cre driver. We observed no anatomical, cellular or functional alterations in either fetuses or adult cardiac Myc-deficient mice. We re-examined Myc expression during development and found no expression in developing cardiomyocytes. In contrast, we confirmed that Mycn is essential for cardiomyocyte proliferation and cardiogenesis. Mosaic Myc overexpression in a Mycn-deficient background, shows that Myc can replace Mycn function, recovering heart development. We further show that this recovery involves the elimination of Mycn-deficient cells by Cell Competition. Our results indicate that Myc is dispensable during cardiogenesis and adult heart homeostasis and Mycn is exclusively responsible for cardiomyocyte proliferation during heart development. Nonetheless, our results show that Myc can functionally replace Mycn. We also show that cardiomyocytes compete according to their overall Myc+Mycn levels and that Cell Competition eliminates flawed cardiomyocytes, suggesting its relevance as a quality control mechanism in cardiac development.

developmental biology↗