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The ISME Journal Jun 2024Although enteric bacteria normally reside within the animal intestine, the ability to persist extraintestinally is an essential part of their overall lifestyle, and it...
Although enteric bacteria normally reside within the animal intestine, the ability to persist extraintestinally is an essential part of their overall lifestyle, and it might contribute to transmission between hosts. Despite this potential importance, few genetic determinants of extraintestinal growth and survival have been identified, even for the best-studied model, Escherichia coli. In this work, we thus used a genome-wide library of barcoded transposon insertions to systematically identify functional clusters of genes that are crucial for E. coli fitness in lake water. Our results revealed that inactivation of pathways involved in maintaining outer membrane integrity, nucleotide biosynthesis, and chemotaxis negatively affected E. coli growth or survival in this extraintestinal environment. In contrast, inactivation of another group of genes apparently benefited E. coli growth or persistence in filtered lake water, resulting in higher abundance of these mutants. This group included rpoS, which encodes the general stress response sigma factor, and genes encoding several other global transcriptional regulators and RNA chaperones, as well as several poorly annotated genes. Based on this co-enrichment, we identified these gene products as novel positive regulators of RpoS activity. We further observed that, despite their enhanced growth, E. coli mutants with inactive RpoS had reduced viability in lake water, and they were not enriched in the presence of the autochthonous microbiota. This highlights the duality of the general stress response pathway for E. coli growth outside the host.
PubMed: 38874171
DOI: 10.1093/ismejo/wrae096 -
Microbiology Spectrum Jun 2024CSV86 displays the unique property of preferential utilization of aromatic compounds over simple carbon sources like glucose and glycerol and their co-metabolism with...
CSV86 displays the unique property of preferential utilization of aromatic compounds over simple carbon sources like glucose and glycerol and their co-metabolism with organic acids. Well-characterized growth conditions, aromatic compound metabolic pathways and their regulation, genome sequence, and advantageous eco-physiological traits (indole acetic acid production, alginate production, fusaric acid resistance, organic sulfur utilization, and siderophore production) make it an ideal host for metabolic engineering. Strain CSV86 was engineered for Carbaryl (1-naphthyl--methylcarbamate) degradation via salicylate-catechol route by expression of a Carbaryl hydrolase (CH) and a 1-naphthol 2-hydroxylase (1NH). Additionally, the engineered strain exhibited faster growth on Carbaryl upon expression of the McbT protein (encoded by the T gene, a part of Carbaryl degradation upper operon of sp. C5pp). Bioinformatic analyses predict McbT to be an outer membrane protein, and Carbaryl-dependent expression suggests its probable role in Carbaryl uptake. Enzyme activity and protein analyses suggested periplasmic localization of CH (carrying transmembrane domain plus signal peptide sequence at the N-terminus) and 1NH, enabling compartmentalization of the pathway. Enzyme activity, whole-cell oxygen uptake, spent media analyses, and qPCR results suggest that the engineered strain preferentially utilizes Carbaryl over glucose. The plasmid-encoded degradation property was stable for 75-90 generations even in the absence of selection pressure (kanamycin or Carbaryl). These results indicate the utility of CSV86 as a potential host for engineering various aromatic compound degradation pathways.IMPORTANCEThe current study describes engineering of Carbaryl metabolic pathway in CSV86. Carbaryl, a naphthalene-derived carbamate pesticide, is known to act as an endocrine disruptor, mutagen, cytotoxin, and carcinogen. Removal of xenobiotics from the environment using bioremediation faces challenges, such as slow degradation rates, instability of the degradation phenotype, and presence of simple carbon sources in the environment. The engineered CSV86-MEC2 overcomes these disadvantages as Carbaryl was degraded preferentially over glucose. Furthermore, the plasmid-borne degradation phenotype is stable, and presence of glucose and organic acids does not repress Carbaryl metabolism in the strain. The study suggests the role of outer membrane protein McbT in Carbaryl transport. This work highlights the suitability of CSV86 as an ideal host for engineering aromatic pollutant degradation pathways.
PubMed: 38869268
DOI: 10.1128/spectrum.00284-24 -
Heliyon Jun 2024Multi-drug resistant ESKAPE pathogens (, aureus, , , , and Enterobacter species) are a global health threat. The severity of the problem lies in its impact on... (Review)
Review
Multi-drug resistant ESKAPE pathogens (, aureus, , , , and Enterobacter species) are a global health threat. The severity of the problem lies in its impact on mortality, therapeutic limitations, the threat to public health, and the costs associated with managing infections caused by these resistant strains. Effectively addressing this challenge requires innovative approaches to research, the development of new antimicrobials, and more responsible antibiotic use practices globally. Antimicrobial peptides (AMPs) are a part of the innate immune system of all higher organisms. They are short, cationic and amphipathic molecules with broad-spectrum activity. AMPs interact with the negatively charged bacterial membrane. In recent years, AMPs have attracted considerable interest as potential antibiotics. However, AMPs have low bioavailability and short half-lives, which may be circumvented by chemical modification. This review presents recent and strategies for the modification of AMPs to improve their stability and application in preclinical experiments.
PubMed: 38868046
DOI: 10.1016/j.heliyon.2024.e31958 -
Life Science Alliance Aug 2024Multispanning membrane proteins are inserted into the endoplasmic reticulum membrane by the ribosome-bound multipass translocon (MPT) machinery. Based on cryo-electron...
Multispanning membrane proteins are inserted into the endoplasmic reticulum membrane by the ribosome-bound multipass translocon (MPT) machinery. Based on cryo-electron tomography and extensive subtomogram analysis, we reveal the composition and arrangement of ribosome-bound MPT components in their native membrane environment. The intramembrane chaperone complex PAT and the translocon-associated protein (TRAP) complex associate substoichiometrically with the MPT in a translation-dependent manner. Although PAT is preferentially part of MPTs bound to translating ribosomes, the abundance of TRAP is highest in MPTs associated with non-translating ribosomes. The subtomogram average of the TRAP-containing MPT reveals intermolecular contacts between the luminal domains of TRAP and an unknown subunit of the back-of-Sec61 complex. AlphaFold modeling suggests this protein is nodal modulator, bridging the luminal domains of nicalin and TRAPα. Collectively, our results visualize the variability of MPT factors in the native membrane environment dependent on the translational activity of the bound ribosome.
Topics: Ribosomes; Membrane Proteins; Endoplasmic Reticulum; Protein Biosynthesis; Cryoelectron Microscopy; SEC Translocation Channels; Molecular Chaperones; Protein Transport; Models, Molecular
PubMed: 38866426
DOI: 10.26508/lsa.202302496 -
Biomarker Insights 2024Although, several studies have assessed the association of the phospholipase A2 receptor (PLA2R) and HLA-DQA1 SNPs with primary membranous nephropathy (PMN), results...
BACKGROUND
Although, several studies have assessed the association of the phospholipase A2 receptor (PLA2R) and HLA-DQA1 SNPs with primary membranous nephropathy (PMN), results were inconsistent and between-studies heterogeneity needs to be investigated.
OBJECTIVES
The aim of this review was to summarize existing data on the contribution of 10 SNPs in the PLA2R and HLA-DQA1 genes to PMN susceptibility and to investigate the between-studies heterogeneity by subgroup analyses and meta-regressions.
DESIGN
This study was performed according to the PRISMA guidelines for systematic reviews and meta-analyses.
DATA SOURCES AND METHODS
An electronic literature search for eligible studies among all papers published prior to January 10, 2024, was conducted through PubMed, EMBASE, Web of science and Scopus databases. Meta-analyses together with subgroup analyses and meta-regressions were performed for the 10 following SNPs: rs4664308, rs3749117, rs3749119, rs35771982, rs3828323, rs16844715, rs1511223, rs6757188, rs2715918, and rs2187668.
RESULTS
Combined analyses revealed a significant increase in PMN risk conferred by the following alleles: rs4664308*A, rs3749117*T, rs3749119*C, rs35771982*G, rs3828323*C, rs16844715*C, rs1511223*A, rs2715918*A, and rs2187668*A, all -values < .001. Moreover, the PLA2R-rs4664308/HLA-DQA1-rs2187668 interaction was significantly associated with an increased PMN risk, < .001. However, there was a substantial between-studies heterogeneity for some SNPs. Subgroup analyses by ethnicity for the 9 PLA2R SNPs did not show any cross-ethnic disparity. Inversely, the risk conferred by the HLA-DQA1 rs2187668*A allele was significantly higher in Caucasians (OR [95% CI] = 3.929 [3.251-4.748]) than in Asians (OR [95% CI] = 2.537 [1.94-3.318], = .007. Besides, meta-regressions revealed for the majority of investigated SNPs significant correlations of the effect size with albumin, 24-hours proteinuria, serum creatinine, and eGFR levels. Hence, the influence on PMN risk conferred by the PLA2R and HLA-DQA1 SNPs was rather noted in patients with a severe disease.
CONCLUSION
This meta-analysis showed that 9 out of the 10 investigated SNPs in PLA2R and HLA-DQA1 genes were associated with increased PMN risk. Heterogeneity could be due to disparate patient groups in terms of disease presentation for almost all SNPs, and ethnicity for the HLA-DQA1 rs2187668 SNP.
REGISTRATION
This review has been registered on PROSPERO: CRD42024506729. Available from: https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42024506729.
PubMed: 38863528
DOI: 10.1177/11772719241259602 -
Cell Communication and Signaling : CCS Jun 2024Bone resorption is driven through osteoclast differentiation by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-Β ligand...
Bone resorption is driven through osteoclast differentiation by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-Β ligand (RANKL). We noted that a disintegrin and metalloproteinase (ADAM) 10 and ADAM17 are downregulated at the expression level during osteoclast differentiation of the murine monocytic cell line RAW264.7 in response to RANKL. Both proteinases are well known to shed a variety of single-pass transmembrane molecules from the cell surface. We further showed that inhibitors of ADAM10 or ADAM17 promote osteoclastic differentiation and furthermore enhance the surface expression of receptors for RANKL and M-CSF on RAW264.7 cells. Using murine bone marrow-derived monocytic cells (BMDMCs), we demonstrated that a genetic deficiency of ADAM17 or its required regulator iRhom2 leads to increased osteoclast development in response to M-CSF and RANKL stimulation. Moreover, ADAM17-deficient osteoclast precursor cells express increased levels of the receptors for RANKL and M-CSF. Thus, ADAM17 negatively regulates osteoclast differentiation, most likely through shedding of these receptors. To assess the time-dependent contribution of ADAM10, we blocked this proteinase by adding a specific inhibitor on day 0 of BMDMC stimulation with M-CSF or on day 7 of subsequent stimulation with RANKL. Only ADAM10 inhibition beginning on day 7 increased the size of developing osteoclasts indicating that ADAM10 suppresses osteoclast differentiation at a later stage. Finally, we could confirm our findings in human peripheral blood mononuclear cells (PBMCs). Thus, downregulation of either ADAM10 or ADAM17 during osteoclast differentiation may represent a novel regulatory mechanism to enhance their differentiation process. Enhanced bone resorption is a critical issue in osteoporosis and is driven through osteoclast differentiation by specific osteogenic mediators. The present study demonstrated that the metalloproteinases ADAM17 and ADAM10 critically suppress osteoclast development. This was observed for a murine cell line, for isolated murine bone marrow cells and for human blood cells by either preferential inhibition of the proteinases or by gene knockout. As a possible mechanism, we studied the surface expression of critical receptors for osteogenic mediators on developing osteoclasts. Our findings revealed that the suppressive effects of ADAM17 and ADAM10 on osteoclastogenesis can be explained in part by the proteolytic cleavage of surface receptors by ADAM10 and ADAM17, which reduces the sensitivity of these cells to osteogenic mediators. We also observed that osteoclast differentiation was associated with the downregulation of ADAM10 and ADAM17, which reduced their suppressive effects. We therefore propose that this downregulation serves as a feedback loop for enhancing osteoclast development.
Topics: ADAM17 Protein; ADAM10 Protein; Osteoclasts; Animals; Cell Differentiation; Mice; Down-Regulation; Amyloid Precursor Protein Secretases; Membrane Proteins; Humans; RANK Ligand; RAW 264.7 Cells; Macrophage Colony-Stimulating Factor; Mice, Inbred C57BL
PubMed: 38863060
DOI: 10.1186/s12964-024-01690-y -
BMC Plant Biology Jun 2024Waterlogging stress (WS) negatively impacts crop growth and productivity, making it important to understand crop resistance processes and discover useful WS resistance...
Transcriptomic analysis reveals the regulatory mechanisms of messenger RNA (mRNA) and long non-coding RNA (lncRNA) in response to waterlogging stress in rye (Secale cereale L.).
BACKGROUND
Waterlogging stress (WS) negatively impacts crop growth and productivity, making it important to understand crop resistance processes and discover useful WS resistance genes. In this study, rye cultivars and wild rye species were subjected to 12-day WS treatment, and the cultivar Secale cereale L. Imperil showed higher tolerance. Whole transcriptome sequencing was performed on this cultivar to identify differentially expressed (DE) messenger RNAs (DE-mRNAs) and long non-coding RNAs (DE-lncRNAs) involved in WS response.
RESULTS
Among the 6 species, Secale cereale L. Imperil showed higher tolerance than wild rye species against WS. The cultivar effectively mitigated oxidative stress, and regulated hydrogen peroxide and superoxide anion. A total of 728 DE-mRNAs and 60 DE-lncRNAs were discovered. Among these, 318 DE-mRNAs and 32 DE-lncRNAs were upregulated, and 410 DE-mRNAs and 28 DE-lncRNAs were downregulated. GO enrichment analysis discovered metabolic processes, cellular processes, and single-organism processes as enriched biological processes (BP). For cellular components (CC), the enriched terms were membrane, membrane part, cell, and cell part. Enriched molecular functions (MF) terms were catalytic activity, binding, and transporter activity. LncRNA and mRNA regulatory processes were mainly related to MAPK signaling pathway-plant, plant hormone signal transduction, phenylpropanoid biosynthesis, anthocyanin biosynthesis, glutathione metabolism, ubiquitin-mediated proteolysis, ABC transporter, Cytochrome b6/f complex, secondary metabolite biosynthesis, and carotenoid biosynthesis pathways. The signalling of ethylene-related pathways was not mainly dependent on AP2/ERF and WRKY transcription factors (TF), but on other factors. Photosynthetic activity was active, and carotenoid levels increased in rye under WS. Sphingolipids, the cytochrome b6/f complex, and glutamate are involved in rye WS response. Sucrose transportation was not significantly inhibited, and sucrose breakdown occurs in rye under WS.
CONCLUSIONS
This study investigated the expression levels and regulatory functions of mRNAs and lncRNAs in 12-day waterlogged rye seedlings. The findings shed light on the genes that play a significant role in rye ability to withstand WS. The findings from this study will serve as a foundation for further investigations into the mRNA and lncRNA WS responses in rye.
Topics: RNA, Long Noncoding; RNA, Messenger; Secale; Gene Expression Profiling; Stress, Physiological; Gene Expression Regulation, Plant; RNA, Plant; Transcriptome
PubMed: 38862913
DOI: 10.1186/s12870-024-05234-x -
MSphere Jun 2024Eggs, an important part of a healthy daily diet, can protect chicken embryo development due to the shell barrier and various antibacterial components within the egg...
UNLABELLED
Eggs, an important part of a healthy daily diet, can protect chicken embryo development due to the shell barrier and various antibacterial components within the egg white. Our previous study demonstrated that Pullorum, highly adapted to chickens, can survive in the egg white and, therefore, be passed to newly hatched chicks. However, the survival strategy of Pullorum in antibacterial conditions remains unknown. The overall transcripts in the egg white showed a large-scale shift compared to LB broth. The expression of common response genes and pathways, such as those involved in iron uptake, biotin biosynthesis, and virulence, was significantly changed, consistent with the other transovarial transmission serovar Enteritidis. Notably, membrane stress response, amino acid metabolism, and carbohydrate metabolism were specifically affected. Additional upregulated functionally relevant genes (JI728_13095, JI728_13100, JI728_17960, JI728_10085, JI728_15605, and ) as mutants confirmed the susceptible phenotype. Furthermore, deletion resulted in an increased survival capacity in the egg white, consistent with the downregulated expression. The second-round RNA-Seq analysis of the Δ mutant in the egg white revealed significantly upregulated genes compared with the wild type in the egg white responsible for energy metabolism located on the and operons regulated by FhlA, indicating the Δ mutant cannot receive enough oxygen and switched to fermentative growth due to its inability to attach to the albumen surface. Together, this study provides a first estimate of the global transcriptional response of Pullorum under antibacterial egg white and highlights the new potential role of deletion in optimizing energy metabolism pathways that may assist vertical transmission.
IMPORTANCE
Pullorum disease, causing serious embryo death and chick mortality, results in substantial economic losses worldwide due to transovarial transmission. Egg-borne outbreaks are frequently reported in many countries. The present study has filled the knowledge gap regarding how the specific chicken-adapted pathogen Pullorum behaves within the challenging environment of egg white. The deletion of the fimbrial system can increase survival in the albumen, possibly by reprogramming metabolism-related gene products, which reveals a new adaptive strategy of pathogens. Moreover, the comparison, including previous research on Enteritidis, capable of vertical transmission, aims to provide diversified data sets in the field and further help to implement reasonable and effective measures to improve both food safety and animal health.
PubMed: 38860771
DOI: 10.1128/msphere.00362-24 -
Journal of Experimental & Clinical... Jun 2024Cancer-associated fibroblasts (CAFs) play a significant role in fueling prostate cancer (PCa) progression by interacting with tumor cells. A previous gene expression...
BACKGROUND
Cancer-associated fibroblasts (CAFs) play a significant role in fueling prostate cancer (PCa) progression by interacting with tumor cells. A previous gene expression analysis revealed that CAFs up-regulate genes coding for voltage-gated cation channels, as compared to normal prostate fibroblasts (NPFs). In this study, we explored the impact of antiarrhythmic drugs, known cation channel inhibitors, on the activated state of CAFs and their interaction with PCa cells.
METHODS
The effect of antiarrhythmic treatment on CAF activated phenotype was assessed in terms of cell morphology and fibroblast activation markers. CAF contractility and migration were evaluated by 3D gel collagen contraction and scratch assays, respectively. The ability of antiarrhythmics to impair CAF-PCa cell interplay was investigated in CAF-PCa cell co-cultures by assessing tumor cell growth and expression of epithelial-to-mesenchymal transition (EMT) markers. The effect on in vivo tumor growth was assessed by subcutaneously injecting PCa cells in SCID mice and intratumorally administering the medium of antiarrhythmic-treated CAFs or in co-injection experiments, where antiarrhythmic-treated CAFs were co-injected with PCa cells.
RESULTS
Activated fibroblasts show increased membrane conductance for potassium, sodium and calcium, consistently with the mRNA and protein content analysis. Antiarrhythmics modulate the expression of fibroblast activation markers. Although to a variable extent, these drugs also reduce CAF motility and hinder their ability to remodel the extracellular matrix, for example by reducing MMP-2 release. Furthermore, conditioned medium and co-culture experiments showed that antiarrhythmics can, at least in part, reverse the protumor effects exerted by CAFs on PCa cell growth and plasticity, both in androgen-sensitive and castration-resistant cell lines. Consistently, the transcriptome of antiarrhythmic-treated CAFs resembles that of tumor-suppressive NPFs. In vivo experiments confirmed that the conditioned medium or the direct coinjection of antiarrhythmic-treated CAFs reduced the tumor growth rate of PCa xenografts.
CONCLUSIONS
Collectively, such data suggest a new therapeutic strategy for PCa based on the repositioning of antiarrhythmic drugs with the aim of normalizing CAF phenotype and creating a less permissive tumor microenvironment.
Topics: Male; Humans; Prostatic Neoplasms; Anti-Arrhythmia Agents; Mice; Animals; Cancer-Associated Fibroblasts; Phenotype; Cell Line, Tumor; Drug Repositioning; Mice, SCID; Xenograft Model Antitumor Assays; Epithelial-Mesenchymal Transition; Cell Movement
PubMed: 38858661
DOI: 10.1186/s13046-024-03081-0 -
Scientific Reports Jun 2024Myocardin-related transcription factors (MRTFs: myocardin/MYOCD, MRTF-A/MRTFA, and MRTF-B/MRTFB) suppress production of pro-inflammatory cytokines and chemokines in...
Myocardin-related transcription factors (MRTFs: myocardin/MYOCD, MRTF-A/MRTFA, and MRTF-B/MRTFB) suppress production of pro-inflammatory cytokines and chemokines in human smooth muscle cells (SMCs) through sequestration of RelA in the NF-κB complex, but additional mechanisms are likely involved. The cGAS-STING pathway is activated by double-stranded DNA in the cytosolic compartment and acts through TANK-binding kinase 1 (TBK1) to spark inflammation. The present study tested if MRTFs suppress inflammation also by targeting cGAS-STING signaling. Interrogation of a transcriptomic dataset where myocardin was overexpressed using a panel of 56 cGAS-STING cytokines showed the panel to be repressed. Moreover, MYOCD, MRTFA, and SRF associated negatively with the panel in human arteries. RT-qPCR in human bronchial SMCs showed that all MRTFs reduced pro-inflammatory cytokines on the panel. MRTFs diminished phosphorylation of TBK1, while STING phosphorylation was marginally affected. The TBK1 inhibitor amlexanox, but not the STING inhibitor H-151, reduced the anti-inflammatory effect of MRTF-A. Co-immunoprecipitation and proximity ligation assays supported binding between MRTF-A and TBK1 in SMCs. MRTFs thus appear to suppress cellular inflammation in part by acting on the kinase TBK1. This may defend SMCs against pro-inflammatory insults in disease.
Topics: Humans; Protein Serine-Threonine Kinases; Nuclear Proteins; Myocytes, Smooth Muscle; Trans-Activators; Inflammation; Signal Transduction; Cytokines; Phosphorylation; Transcription Factors; Membrane Proteins; Cells, Cultured
PubMed: 38858497
DOI: 10.1038/s41598-024-63901-3