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Microbial Genomics May 2024is an emerging human enteric pathogen. However, the genomic features and virulence genes of strains from human gastroenteritis and other sources have not been fully...
Analysis of global genomes revealed that strains carrying T6SS are more common in human gastroenteritis than in environmental sources and are often phylogenetically related.
is an emerging human enteric pathogen. However, the genomic features and virulence genes of strains from human gastroenteritis and other sources have not been fully elucidated. Here, we conducted a genomic analysis of 565 global strains isolated from different sources, including 261 strains isolated from faecal samples of gastroenteritis patients, of which 18 genomes were sequenced in this study. The presence of bacterial virulence genes and secretion systems in strains from different sources was compared, and the phylogenetic relationship of strains was assessed based on the core genome. The complete genome of strain A20-9 isolated from a gastroenteritis patient was obtained in this study, from which 300 putative virulence factors and a T4SS-encoding plasmid, pAC, were identified. Genes encoding T4SS were also identified in a novel genomic island, ACI-1, from other T4SS-positive strains. The prevalence of T4SS was significantly lower in strains from gastroenteritis patients than in environmental strains (3 %, <0.0001 vs 14 %, <0.01). Conversely, the prevalence of T6SS was significantly higher in strains isolated from gastroenteritis patients than in environmental strains (25 %, <0.05 vs 13 %, <0.01). Four phylogenetic clusters were formed based on the core genome of 565 . strains, and strains carrying T6SS often showed close phylogenetic relationships. T3SS, aerolysin and thermostable cytotonic enterotoxin were absent in all 565 . strains. Our findings provide novel information on the genomic features of and suggest that T6SS may play a role in -induced human gastroenteritis.
Topics: Gastroenteritis; Humans; Phylogeny; Genome, Bacterial; Aeromonas caviae; Virulence Factors; Type VI Secretion Systems; Feces; Gram-Negative Bacterial Infections; Genomic Islands; Plasmids
PubMed: 38814176
DOI: 10.1099/mgen.0.001258 -
Disease Models & Mechanisms May 2024Evidence suggests the presence of microglial activation and microRNA (miRNA) dysregulation in amyotrophic lateral sclerosis (ALS), the most common form of adult motor...
Evidence suggests the presence of microglial activation and microRNA (miRNA) dysregulation in amyotrophic lateral sclerosis (ALS), the most common form of adult motor neuron disease. However, few studies have investigated whether the miRNA dysregulation originates from microglia. Furthermore, TDP-43 (encoded by TARDBP), involved in miRNA biogenesis, aggregates in tissues of ∼98% of ALS cases. Thus, this study aimed to determine whether expression of the ALS-linked TDP-43M337V mutation in a transgenic mouse model dysregulates microglia-derived miRNAs. RNA sequencing identified several dysregulated miRNAs released by transgenic microglia and a differential miRNA release by lipopolysaccharide-stimulated microglia, which was more pronounced in cells from female mice. We validated the downregulation of three candidate miRNAs, namely, miR-16-5p, miR-99a-5p and miR-191-5p, by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and identified their predicted targets, which primarily include genes involved in neuronal development and function. These results suggest that altered TDP-43 function leads to changes in the miRNA population released by microglia, which may in turn be a source of the miRNA dysregulation observed in the disease. This has important implications for the role of neuroinflammation in ALS pathology and could provide potential therapeutic targets.
Topics: Microglia; Amyotrophic Lateral Sclerosis; MicroRNAs; Animals; Female; Mice, Transgenic; Male; Mutation; Sex Characteristics; DNA-Binding Proteins; Mice; Extracellular Space; Humans; Lipopolysaccharides; Gene Expression Regulation
PubMed: 38813848
DOI: 10.1242/dmm.050638 -
Frontiers in Immunology 2024Innate immune training is a metabolic, functional, and epigenetic long-term reprogramming of innate cells triggered by different stimuli. This imprinting also reaches...
INTRODUCTION
Innate immune training is a metabolic, functional, and epigenetic long-term reprogramming of innate cells triggered by different stimuli. This imprinting also reaches hematopoietic precursors in the bone marrow to sustain a memory-like phenotype. Dendritic cells (DCs) can exhibit memory-like responses, enhanced upon subsequent exposure to a pathogen; however, whether this imprinting is lineage and stimulus-restricted is still being determined. Nevertheless, the functional consequences of DCs training on the adaptive and protective immune response against non-infectious diseases remain unresolved.
METHODS
We evaluated the effect of the nontoxic cholera B subunit (CTB), LPS and LTA in the induction of trained immunity in murine DCs revealed by TNFa and LDH expression, through confocal microscopy. Additionally, we obtained bone marrow DCs (BMDCs) from mice treated with CTB, LPS, and LTA and evaluated training features in DCs and their antigen-presenting cell capability using multiparametric cytometry. Finally, we design an experimental melanoma mouse model to demonstrate protection induced by CTB-trained DCs in vivo.
RESULTS
CTB-trained DCs exhibit increased expression of TNFa, and metabolic reprogramming indicated by LDH expression. Moreover, CTB training has an imprint on DC precursors, increasing the number and antigen-presenting function in BMDCs. We found that training by CTB stimulates the recruitment of DC precursors and DCs infiltration at the skin and lymph nodes. Interestingly, training-induced by CTB promotes a highly co-stimulatory phenotype in tumor-infiltrating DCs (CD86+) and a heightened functionality of exhausted CD8 T cells (Ki67+, GZMB+), which were associated with a protective response against melanoma challenge in vivo.
CONCLUSION
Our work indicates that CTB can induce innate immune training on DCs, which turns into an efficient adaptive immune response in the melanoma model and might be a potential immunotherapeutic approach for tumor growth control.
Topics: Animals; Dendritic Cells; Mice; CD8-Positive T-Lymphocytes; Cholera Toxin; Melanoma, Experimental; Mice, Inbred C57BL; Immunity, Innate; Female; Immunologic Memory; Trained Immunity
PubMed: 38812523
DOI: 10.3389/fimmu.2024.1362289 -
Scientific Reports May 2024Poly-ε-caprolactone (PCL) has been widely used as biocompatible materials in tissue engineering. They have been used in mammalian cell proliferation to polarization and...
Poly-ε-caprolactone (PCL) has been widely used as biocompatible materials in tissue engineering. They have been used in mammalian cell proliferation to polarization and differentiation. Their modified versions had regulatory activities on mammalian macrophages in vitro. There are also studies suggesting different nanofiber diameters might alter the biological activities of these materials. Based on these cues, we examined the inflammatory activities and adherence properties of mammalian macrophages on electrospun PCL nanofibrous scaffolds formed with PCL having different nanofiber diameters. Our results suggest that macrophages could easily attach and get dispersed on the scaffolds. Macrophages lost their inflammatory cytokine TNF and IL6 production capacity in the presence of LPS when they were incubated on nanofibers. These effects were independent of the mean fiber diameters. Overall, the scaffolds have potential to be used as biocompatible materials to suppress excessive inflammatory reactions during tissue and organ transplantation by caging and suppressing the inflammatory cells.
Topics: Nanofibers; Macrophages; Animals; Mice; Inflammation; Tissue Scaffolds; Polyesters; Tissue Engineering; Biocompatible Materials; Interleukin-6; Lipopolysaccharides; RAW 264.7 Cells; Tumor Necrosis Factor-alpha; Cell Adhesion
PubMed: 38811651
DOI: 10.1038/s41598-024-61450-3 -
MBio May 2024Pathogenic bacteria rely on secreted virulence factors to cause disease in susceptible hosts. However, in Gram-positive bacteria, the mechanisms underlying secreted...
UNLABELLED
Pathogenic bacteria rely on secreted virulence factors to cause disease in susceptible hosts. However, in Gram-positive bacteria, the mechanisms underlying secreted protein activation and regulation post-membrane translocation remain largely unknown. Using proteomics, we identified several proteins that are dependent on the secreted chaperone PrsA2. We followed with phenotypic, biochemical, and biophysical assays and computational analyses to examine the regulation of a detected key secreted virulence factor, listeriolysin O (LLO), and its interaction with PrsA2 from the bacterial pathogen (). Critical to virulence is internalization by host cells and the subsequent action of the cholesterol-dependent pore-forming toxin, LLO, which enables bacterial escape from the host cell phagosome. Since is a Gram-positive organism, the space between the cell membrane and wall is solvent exposed. Therefore, we hypothesized that the drop from neutral to acidic pH as the pathogen is internalized into a phagosome is critical to regulating the interaction of PrsA2 with LLO. Here, we demonstrate that PrsA2 directly interacts with LLO in a pH-dependent manner. We show that PrsA2 protects and sequesters LLO under neutral pH conditions where LLO can be observed to aggregate. In addition, we identify molecular features of PrsA2 that are required for interaction and ultimately the folding and activity of LLO. Moreover, protein-complex modeling suggests that PrsA2 interacts with LLO via its cholesterol-binding domain. These findings highlight a mechanism by which a Gram-positive secretion chaperone regulates the secretion, stability, and folding of a pore-forming toxin under conditions relevant to host cell infection.
IMPORTANCE
is a ubiquitous food-borne pathogen that can cause severe disease to vulnerable populations. During infection, relies on a wide repertoire of secreted virulence factors including the LLO that enables the bacterium to invade the host and spread from cell to cell. After membrane translocation, secreted factors must become active in the challenging bacterial cell membrane-wall interface. However, the mechanisms required for secreted protein folding and function are largely unknown. encodes a chaperone, PrsA2, that is critical for the activity of secreted factors. Here, we show that PrsA2 directly associates and protects the major virulence factor, LLO, under conditions corresponding to the host cytosol, where LLO undergoes irreversible denaturation. Additionally, we identify molecular features of PrsA2 that enable its interaction with LLO. Together, our results suggest that and perhaps other Gram-positive bacteria utilize secreted chaperones to regulate the activity of pore-forming toxins during infection.
PubMed: 38809022
DOI: 10.1128/mbio.00743-24 -
Drug Design, Development and Therapy 2024The aim of this study is to uncover the anti-inflammatory propertity of andrographolide (AGP) in acute exacerbation of chronic obstructive pulmonary disease (AECOPD) and...
PURPOSE
The aim of this study is to uncover the anti-inflammatory propertity of andrographolide (AGP) in acute exacerbation of chronic obstructive pulmonary disease (AECOPD) and the underlying mechanisms related to the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome pathway.
METHODS
An in vivo experiment was conducted on murine model of AECOPD through endotracheal atomization of elastase and lipopolysaccharide (LPS). Intraperitoneal AGP was administered four times. NLRP3 inflammasome pathway molecules were examined using real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis. By using enzyme-linked immunosorbent assay (ELISA), we tested interleukin (IL)-1β levels in bronchoalveolar lavage fluid. An in vitro study was conducted to determine how AGP impacts the NLRP3 inflammasome in THP-1 derived macrophages. The levels of molecules involved in the pathway were measured. Furthermore, molecular docking analyses were carried out to investigate the interactions between AGP and pathway targets.
RESULTS
In the in vivo study, NLRP3 inflammasome activation was observed in mice experiencing AECOPD. The administration of high-dose AGP demonstrated a mitigating effect on inflammatory cells infiltration in the lungs. Moreover, AGP administration effectively suppressed the expression of NLRP3, apoptosis associated speck-like protein that contains a CARD (PYCARD), cysteinyl aspartate-specific protease-1 (Caspase-1), IL-1β, and IL-18 at both the genetic and protein levels. In the in vitro experiment, IL-1β levels were significantly elevated in THP-1 derived macrophages with activated inflammasome compared to the control group. Furthermore, the downregulation of NLRP3, CASP1, and IL1B genes was observed upon the inhibition of NLRP3 expression through small interfering RNA (siRNA). AGP demonstrated inhibitory effects on the gene expression and protein levels of NLRP3, Caspase-1, and IL-1β. Additionally, molecular docking analysis confirmed that AGP exhibited a favorable binding affinity with all five targets of the pathway.
CONCLUSION
AGP effectively inhibited NLRP3 inflammasome activation and mitigated the inflammatory reaction of AECOPD both in animal models and in vitro experiments, highlighting the potential of AGP as a treatment for AECOPD with anti-inflammatory properties.
Topics: NLR Family, Pyrin Domain-Containing 3 Protein; Pulmonary Disease, Chronic Obstructive; Animals; Diterpenes; Mice; Inflammasomes; Humans; Mice, Inbred C57BL; Molecular Docking Simulation; Male; Inflammation; Disease Models, Animal; Dose-Response Relationship, Drug; Anti-Inflammatory Agents; Lipopolysaccharides; Structure-Activity Relationship
PubMed: 38808326
DOI: 10.2147/DDDT.S445788 -
Italian Journal of Food Safety May 2024Notification of foodborne outbreaks has been mandatory in Europe since 2005, and surveillance is carried out along the entire food chain. Here we report the results...
Notification of foodborne outbreaks has been mandatory in Europe since 2005, and surveillance is carried out along the entire food chain. Here we report the results obtained from laboratory investigations about four cases of foodborne outbreaks that occurred in Sicily between 2009 and 2016, deemed to be related to staphylococcal enterotoxins (SEs) and coagulase-positive (CPS) by the Local Public Health Authority. cheese samples were processed by culture methods for enumeration of CPS and immunoenzymatic assays for detection and differentiation of the SEs possibly contained in food samples. In all cases, the mistrusted foods were found to be contaminated by CPS at bacterial loads between 5 and 8 log CFU/g and contained SE type C (SEC). The reported data confirm the risk of staphylococcal food poisoning associated with the consumption of raw milk cheese. SEC is the most commonly occurring SE in goat milk and dairy products and the most represented enterotoxin in Sicilian dairy products. Our results highlighted the need for improving the current monitoring efficiency and implementing the available laboratory methods to collect more faithful epidemiological data on the current prevalence of staphylococcal toxins in the food chain, including SEs currently not detectable by validated analytical methods.
PubMed: 38807740
DOI: 10.4081/ijfs.2024.11667 -
Frontiers in Immunology 2024L-carnitine, available as feed additive, is essential for the beta-oxidation of free fatty acids in the mitochondrial matrix. It provides energy to immune cells and may...
L-carnitine, available as feed additive, is essential for the beta-oxidation of free fatty acids in the mitochondrial matrix. It provides energy to immune cells and may positively impact the functionality of leukocytes during the acute phase response, a situation of high energy demand. To test this hypothesis, German Holstein cows were assigned to a control group (CON, n = 26) and an L-carnitine supplemented group (CAR, n = 27, rumen-protected L-carnitine product: 125 g/cow/d, corresponded to total L-carnitine intake: 25 g/cow/d, supplied with concentrate) and received an intravenous bolus injection of lipopolysaccharides (LPS, 0.5 µg/kg body weight, ) on day 111 as a model of standardized systemic inflammation. Blood samples were collected from day 1 until day 14 , with frequent sampling through an indwelling venous catheter from 0.5 h to 12 h . All parameters of the white blood cell count responded significantly to LPS, while only a few parameters were affected by L-carnitine supplementation. The mean eosinophil count, as well as the percentage of basophils were significantly higher in CAR than in CON over time, which may be due to an increased membrane stability. However, phagocytosis and production of reactive oxygen species by leukocytes remained unchanged following L-carnitine supplementation. In conclusion, although supplementation with 25 g L-carnitine per cow and day resulted in increased proportions of specific leukocyte populations, it had only minor effects on the functional parameters studied in mid-lactating dairy cows during LPS-induced inflammation, and there was no evidence of direct improvement of immune functionality.
Topics: Animals; Cattle; Lipopolysaccharides; Carnitine; Dietary Supplements; Female; Lactation; Inflammation; Leukocyte Count
PubMed: 38807585
DOI: 10.3389/fimmu.2024.1390137 -
Journal of Neuroinflammation May 2024Intrauterine inflammation is considered a major cause of brain injury in preterm infants, leading to long-term neurodevelopmental deficits. A potential contributor to...
BACKGROUND
Intrauterine inflammation is considered a major cause of brain injury in preterm infants, leading to long-term neurodevelopmental deficits. A potential contributor to this brain injury is dysregulation of neurovascular coupling. We have shown that intrauterine inflammation induced by intra-amniotic lipopolysaccharide (LPS) in preterm lambs, and postnatal dopamine administration, disrupts neurovascular coupling and the functional cerebral haemodynamic responses, potentially leading to impaired brain development. In this study, we aimed to characterise the structural changes of the neurovascular unit following intrauterine LPS exposure and postnatal dopamine administration in the brain of preterm lambs using cellular and molecular analyses.
METHODS
At 119-120 days of gestation (term = 147 days), LPS was administered into the amniotic sac in pregnant ewes. At 126-7 days of gestation, the LPS-exposed lambs were delivered, ventilated and given either a continuous intravenous infusion of dopamine at 10 µg/kg/min or isovolumetric vehicle solution for 90 min (LPS, n = 6; LPS, n = 6). Control preterm lambs not exposed to LPS were also administered vehicle or dopamine (CTL, n = 9; CTL, n = 7). Post-mortem brain tissue was collected 3-4 h after birth for immunohistochemistry and RT-qPCR analysis of components of the neurovascular unit.
RESULTS
LPS exposure increased vascular leakage in the presence of increased vascular density and remodelling with increased astrocyte "end feet" vessel coverage, together with downregulated mRNA levels of the tight junction proteins Claudin-1 and Occludin. Dopamine administration decreased vessel density and size, decreased endothelial glucose transporter, reduced neuronal dendritic coverage, increased cell proliferation within vessel walls, and increased pericyte vascular coverage particularly within the cortical and deep grey matter. Dopamine also downregulated VEGFA and Occludin tight junction mRNA, and upregulated dopamine receptor DRD1 and oxidative protein (NOX1, SOD3) mRNA levels. Dopamine administration following LPS exposure did not exacerbate any effects induced by LPS.
CONCLUSION
LPS exposure and dopamine administration independently alters the neurovascular unit in the preterm brain. Alterations to the neurovascular unit may predispose the developing brain to further injury.
Topics: Animals; Dopamine; Sheep; Female; Animals, Newborn; Lipopolysaccharides; Pregnancy; Brain; Inflammation; Blood-Brain Barrier; Premature Birth
PubMed: 38807204
DOI: 10.1186/s12974-024-03137-0 -
Nature Communications May 2024Despite major advances in linking single genetic variants to single causal genes, the significance of genetic variation on transcript-level regulation of expression,...
Despite major advances in linking single genetic variants to single causal genes, the significance of genetic variation on transcript-level regulation of expression, transcript-specific functions, and relevance to human disease has been poorly investigated. Strawberry notch homolog 2 (SBNO2) is a candidate gene in a susceptibility locus with different variants associated with Crohn's disease and bone mineral density. The SBNO2 locus is also differentially methylated in Crohn's disease but the functional mechanisms are unknown. Here we show that the isoforms of SBNO2 are differentially regulated by lipopolysaccharide and IL-10. We identify Crohn's disease associated isoform quantitative trait loci that negatively regulate the expression of the noncanonical isoform 2 corresponding with the methylation signals at the isoform 2 promoter in IBD and CD. The two isoforms of SBNO2 drive differential gene networks with isoform 2 dominantly impacting antimicrobial activity in macrophages. Our data highlight the role of isoform quantitative trait loci to understand disease susceptibility and resolve underlying mechanisms of disease.
Topics: Quantitative Trait Loci; Crohn Disease; Humans; Genetic Predisposition to Disease; Protein Isoforms; Lipopolysaccharides; Interleukin-10; Promoter Regions, Genetic; DNA Methylation; Macrophages; Gene Expression Regulation
PubMed: 38806456
DOI: 10.1038/s41467-024-47218-3