Project description:Serratia marcescens is a bacterium frequently found in the environment, but over the last several decades it has evolved into a concerning clinical pathogen, causing fatal bacteremia. To establish such infections, pathogens require specific nutrients; one very limited but essential nutrient is iron. We sought to characterize the iron acquisition systems in S. marcescens isolate UMH9, which was recovered from a clinical bloodstream infection. Using RNA sequencing (RNA-seq), we identified two predicted siderophore gene clusters (cbs and sch) that were regulated by iron. Mutants were constructed to delete each iron acquisition locus individually and in conjunction, generating both single and double mutants for the putative siderophore systems. Mutants lacking the sch gene cluster lost their iron-chelating ability as quantified by the chrome azurol S (CAS) assay, whereas the cbs mutant retained wild-type activity. Mass spectrometry-based analysis identified the chelating siderophore to be serratiochelin, a siderophore previously identified in Serratia plymuthica. Serratiochelin-producing mutants also displayed a decreased growth rate under iron-limited conditions created by dipyridyl added to LB medium. Additionally, mutants lacking serratiochelin were significantly outcompeted during cochallenge with wild-type UMH9 in the kidneys and spleen after inoculation via the tail vein in a bacteremia mouse model. This result was further confirmed by an independent challenge, suggesting that serratiochelin is required for full S. marcescens pathogenesis in the bloodstream. Nine other clinical isolates have at least 90% protein identity to the UMH9 serratiochelin system; therefore, our results are broadly applicable to emerging clinical isolates of S. marcescens causing bacteremia.
Project description:We investigated the function of the SNX/H-type regulator of G-protein signaling (RGS) protein RGS4 and found alterations in enzyme regulation, stress response, siderophore production and metabolism of several carbon sources in light and darkness
Project description:Therapeutic angiogenesis based on gene therapies is a potential peripheral artery disease (PAD) treatment approach. Here, we developed a graphene nanoparticle-based IL-4 plasmid delivery system (GNPs-pIL-4) to reprogram macrophage polarization and activate the OSM/GSNOR/ENG axis to improve angiogenesis and tissue repair in ischemic limbs. Single-cell RNA sequencing analysis revealed that GNPs-pIL-4 treatment significantly enhanced the number and strength of intercellular communications in ischemic tissues, with enrichment of pathways associated with endothelial sprouting and neovascularization.
Project description:The aim of the experiment was to identify genes differentially expressed between the susceptible wild type strain P. aeruginosa PAO1 (PT5) and a mutant resistant to a drug-siderophore conjugate, in order to obtain information on the resistance mechanism(s). A mutant of PT5 able to grow at 4 mg/l BAL30072, a drug-siderophore conjugate, was selected in vitro . The susceptible wild type strain PT5 and the mutant (BAL6) were grown in LB medium and the mutant also in the presence of 4 mg/l BAL30072 to mid-exponential growth phase (OD600 =2) in triplicate cultures. RNA was extracted using the RNeasy Kit (Qiagen). A total of nine Affymterix P. aeruginosa arrays were hybridized (one for each replicate) under standard conditions.
Project description:Pseudomonas aeruginosa is a common nosocomial pathogen which produces siderophores to solubilize and transport chelated Fe3+ to aid its survival in both the environment and the host. However, there is a lack of comprehensive understanding regarding the molecular mechanisms underlying siderophore synthesis, uptake, and regulation within various ecological niches. In this study, we demonstrated that the BfmRS two-component system, part of the core genome of P. aeruginosa, plays a crucial role in siderophore metabolism. We have identified BfmS as an osmosensing histidine kinase that responds to external osmolytes, then modulates the activation of the response regulator BfmR. Under high osmolality, BfmR could directly bind to the promoters of pvd, fpv, and femARI gene clusters, thereby enhancing their expression and promoting siderophore metabolism. The proteomic and phenotypic analyses confirmed that deletion of bfmRS results in reduced expression levels of siderophore-related proteins as well as siderophore production. Importantly, loss of bfmR or bfmS significantly impaired bacterial survival in both iron deficiency medium and mouse lung infection models. Furthermore, phylogenetic analysis revealed that BfmRS is highly conserved and widely distributed across Pseudomonas species, evidences also proved that the BfmR of P. putida KT2440 and P. sp. MRSN12121 activated siderophore genes in response to high osmolality. Overall, this study sheds light on the previously unexplored signal transduction pathway, BfmRS, which governs the siderophore regulation in Pseudomonas species through perceiving an osmotic upshift. Considering that siderophores serve as unique social mediators, our findings contribute to a better understanding of how siderophores facilitate bacterial interactions with their eukaryotic hosts and contribute to the establishment of stable communities.
Project description:The intermediate filament protein Nestin serves as a biomarker for stem cells and has been used to identify subsets of cancer stem-like cells. However, the mechanistic contributions of Nestin to cancer pathogenesis are not understood. Here we report that Nestin binds the hedgehog pathway transcription factor Gli3 to mediate the development of medulloblastomas of the hedgehog subtype. In a mouse model system, Nestin levels increased progressively during medulloblastoma formation resulting in enhanced tumor growth. Conversely, loss of Nestin dramatically inhibited proliferation and promoted differentiation. Mechanistic investigations revealed that the tumor-promoting effects of Nestin were mediated by binding to Gli3, a zinc finger transcription factor that negatively regulates hedgehog signaling. Nestin binding to Gli3 blocked Gli3 phosphorylation and its subsequent proteolytic processing, thereby abrogating its ability to negatively regulate the hedgehog pathway. Our findings show how Nestin drives hedgehog pathway-driven cancers and uncover in Gli3 a therapeutic target to treat these malignancies. Nestin+ and Nestin- GNPs (granule neuron precursors) were purified from Nestin-CFP/Math1-Cre/Ptch1-loxp cerebella at postnatal day 4 by FACs, and total RNA from these two cell populations were extracted, and then labeled and hybridized to Affymetrix Mouse Genome 430 2.0 arrays.