ABSTRACT
BACKGROUND: Head and neck lesions of tuberculosis, though not uncommon are often difficult to diagnose and require a unique management protocol. These lesions are often misdiagnosed as bacterial infections, malignancies or other granulomatous diseases. Hence in our study we endeavor to gain a better understanding of the diagnostic and management protocols of tuberculosis in otorhinolaryngology. METHODS: We have performed an observational study at our institute, the patient's details were obtained from patient record forms and noted in a standard proforma. Results were calculated as percentage and Chi square analysis was performed. RESULTS: We found cervical tuberculous lymphadenitis to be the most common manifestation 76.97%, with a significant association with pulmonary tuberculosis. Neck swelling was the most common presenting complaint, 65.35%. 26-50 years of age was the most commonly involved age group. CONCLUSION: FNAC, PCR and histopathology are the modalities for bacteriological diagnosis for tuberculosis of Head and Neck. Anti-tuberculous therapy is uniformly found to be useful in all the patients, with surgical intervention used as and when required.
Subject(s)
Neoplasms , Tuberculosis, Lymph Node , Tuberculosis, Pulmonary , Humans , Tertiary Care Centers , Neck/pathology , Tuberculosis, Lymph Node/diagnosis , Tuberculosis, Lymph Node/drug therapy , Tuberculosis, Lymph Node/epidemiology , Tuberculosis, Pulmonary/diagnosis , Tuberculosis, Pulmonary/drug therapy , Tuberculosis, Pulmonary/epidemiologyABSTRACT
Staphylococcus aureus is an important clinical bacterium prevalent in human-associated microbiomes and the cause of many diseases. However, S. aureus has been intractable to synthetic biology approaches due to limited characterized genetic parts for this nonmodel Gram-positive bacterium. Moreover, genetic manipulation of S. aureus has relied on cumbersome and inefficient cloning strategies. Here, we report the first standardized genetic parts toolbox for S. aureus, which includes characterized promoters, ribosome binding sites, terminators, and plasmid replicons from a variety of bacteria for precise control of gene expression. We established a standard relative expression unit (REU) for S. aureus using a plasmid reference and characterized genetic parts in standardized REUs using S. aureus ATCC 12600. We constructed promoter and terminator part plasmids that are compatible with an efficient Type IIS DNA assembly strategy to effectively build multipart DNA constructs. A library of 24 constitutive promoters was built and characterized in S. aureus, which showed a 380-fold activity range. This promoter library was also assayed in Bacillus subtilis (122-fold activity range) to demonstrate the transferability of the constitutive promoters between these Gram-positive bacteria. By applying an iterative design-build-test-learn cycle, we demonstrated the use of our toolbox for the rational design and engineering of a tetracycline sensor in S. aureus using the PXyl-TetO aTc-inducible promoter that achieved 25.8-fold induction. This toolbox greatly expands the growing number of genetic parts for Gram-positive bacteria and will allow researchers to leverage synthetic biology approaches to study and engineer cellular processes in S. aureus.
Subject(s)
DNA , Staphylococcus aureus , Humans , Staphylococcus aureus/genetics , Plasmids/genetics , Promoter Regions, Genetic/genetics , Gene LibraryABSTRACT
A universal biochemical signal for bacterial cell-cell communication could facilitate programming dynamic responses in diverse bacterial consortia. However, the classical quorum sensing paradigm is that Gram-negative and Gram-positive bacteria generally communicate via homoserine lactones (HSLs) or oligopeptide molecular signals, respectively, to elicit population responses. Here, we create synthetic HSL sensors for Gram-positive Bacillus subtilis 168 using allosteric LuxR-type regulators (RpaR, LuxR, RhlR, and CinR) and synthetic promoters. Promoters were combinatorially designed from different sequence elements (-35, -16, -10, and transcriptional start regions). We quantified the effects of these combinatorial promoters on sensor activity and determined how regulator expression affects its activation, achieving up to 293-fold activation. Using the statistical design of experiments, we identified significant effects of promoter regions and pairwise interactions on sensor activity, which helped to understand the sequence-function relationships for synthetic promoter design. We present the first known set of functional HSL sensors (≥20-fold dynamic range) in B. subtilis for four different HSL chemical signals: p-coumaroyl-HSL, 3-oxohexanoyl-HSL, n-butyryl-HSL, and n-(3-hydroxytetradecanoyl)-HSL. This set of synthetic HSL sensors for a Gram-positive bacterium can pave the way for designable interspecies communication within microbial consortia.
Subject(s)
Repressor Proteins , Trans-Activators , Trans-Activators/genetics , Trans-Activators/metabolism , Repressor Proteins/metabolism , Bacillus subtilis/genetics , Bacillus subtilis/metabolism , 4-Butyrolactone/metabolism , Quorum Sensing/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial/genetics , Homoserine/metabolismABSTRACT
The aim of our study is to analyze the efficacy of nasal septal cartilage as cap-graft in laryngo-tracheoplasty in cases of Laryngotracheal stenosis. This was a prospective observational study carried out at a tertiary care hospital from March 2020 to March 2023. Total 8 patients who underwent laryngo-tracheoplasty using nasal septal cartilage as anterior Cap-graft were included in the study. Detailed history and clinical evaluation followed by diagnostic Flexible Fiber-optic Laryngoscopy and radiological investigations were done for all patients with post operative follow up for at least 1 year. Our study had maximum patients in age group of 11-30 years with male predominance, unknown compound ingestion being most common cause of intubation which was followed by tracheostomy. All patients had Cotton Mayer Grade III or IV subglottic stenosis. Out of 8 patients, 5 patients are decannulated, 1 patients still have T-tube in-situ whereas 2 patients didn't tolerate decannulation and required re-exploration. No donor site complication was seen during the study period. Nasal septal cartilage is a viable option for being used as anterior cap graft in laryngo-tracheoplasty. It can be a game changer, as can be done by E.N.T surgeon himself. No separate learning skills are required. It's cosmetically better with minimal complications; compared to life threatening complications like pneumothorax on using costal cartilage. Laryngeal framework is preserved as opposed to thyroid alar cartilage graft. Faster healing along with better postoperative donor site recovery are significant advantages.
ABSTRACT
Grape pomace is the primary wine coproduct consisting primarily of grape seeds and skins. Grape pomace holds immense potential as a functional ingredient to improve human health while its valorization can be beneficial for industrial sustainability. Pomace contains bioactive compounds, including phenols and oligosaccharides, most of which reach the colon intact, enabling interaction with the gut microbiome. Microbial analysis found that grape pomace selectively promotes the growth of many commensal bacteria strains, while other types of bacteria, including various pathogens, are highly sensitive to the pomace and its components and are inactivated. In vitro studies showed that grape pomace and its extracts inhibit the growth of pathogenic bacteria in Enterobacteriaceae family while increasing the growth and survival of some beneficial bacteria, including Bifidobacterium spp. and Lactobacillus spp. Grape pomace supplementation in mice and rats improves their gut microbiome complexity and decreases diet-induced obesity as well as related illnesses, including insulin resistance, indicating grape pomace could improve human health. A human clinical trial found that pomace, regardless of its phenolic content, had cardioprotective effects, suggesting that dietary fiber induced those health benefits. To shed light on the active components, this review explores the potential prebiotic capacity of select bioactive compounds in grape pomace.
ABSTRACT
Mammalian milk is a source of antimicrobial compounds such as xanthine oxidase (XO). The interplay of infant saliva, which contains the substrates for XO activity, and human milk containing XO has been recently shown to inhibit the growth of pathogenic bacteria. Based on the complex and protective mechanism observed in human milk, we hypothesized that bovine milk XO operates similarly, thus representing an opportunity to investigate its functionality in broader health implications. We demonstrated that bovine milk-hypoxanthine mixture (0 to 400 µM) inhibited several Gram-negative and -positive bacterial pathogens in a dose-dependent manner. Kinetic experiments revealed that XO catalyzed hypoxanthine reduction (Km, 58.0 µM; Vmax, 5.1 µmol-1 min-1 mg) resulted in the production of antimicrobial hydrogen peroxide. These results demonstrate that the antimicrobial properties of bovine milk XO are similar to those of human milk XO with significant implications for the development of novel products targeting infant health.
ABSTRACT
Consumption of mothers' milk is associated with reduced incidence and severity of enteric infections, leading to reduced morbidity in breastfed infants. Fucosylated and sialylated human milk oligosaccharides (HMO) are important for both direct antimicrobial action - likely via a decoy effect - and indirect antimicrobial action through commensal growth enhancement. Bovine milk oligosaccharides (BMO) are a potential source of HMO-mimics as BMO resemble HMO; however, they have simpler and less fucosylated structures. BMO isolated at large scales from bovine whey permeate were modified by the addition of fucose and/or sialic acid to generate HMO-like glycans using high-yield and cost-effective one-pot multienzyme approaches. Quadrupole time-of-flight LC/MS analysis revealed that 22 oligosaccharides were synthesized and 9 had identical composition to known HMO. Preliminary anti-adherence activity assays indicated that fucosylated BMO decreased the uptake of enterohemorrhagic Escherichia coli O157:H7 by human intestinal epithelial Caco-2 cells more effectively than native BMO.
ABSTRACT
Human milk is a unique and complex fluid that provides infant nutrition and delivers an array of bioactive molecules that serve various functions. Glycans, abundant in milk, can be found as free oligosaccharides or as glycoconjugates. Milk glycans are increasingly linked to beneficial outcomes in neonates through protection from pathogens and modulation of the immune system. Indeed, these glycans influence the development of the infant and the infant-gut microbiota. Bifidobacterium species commonly are enriched in breastfed infants and are among a limited group of bacteria that readily consume human milk oligosaccharides (HMOs) and milk glycoconjugates. Given the importance of bifidobacteria in infant health, numerous studies have examined the molecular mechanisms they employ to consume HMOs and milk glycans, thus providing insight into this unique enrichment and shedding light on a range of translational opportunities to benefit at-risk infants.
Subject(s)
Gastrointestinal Microbiome , Milk, Human/chemistry , Milk/chemistry , Polysaccharides/metabolism , Animals , Bifidobacterium/metabolism , Breast Feeding , Carbohydrate Conformation , Cattle , Dietary Supplements , Glycoconjugates/metabolism , Humans , Infant, Newborn , Oligosaccharides/administration & dosage , Oligosaccharides/chemistry , Oligosaccharides/metabolism , Polysaccharides/chemistry , Structure-Activity RelationshipABSTRACT
Epithelial cells in the lining of the intestines play critical roles in maintaining homeostasis while challenged by dynamic and sudden changes in luminal contents. Given the high density of glycosylation that encompasses their extracellular surface, environmental changes may lead to extensive reorganization of membrane-associated glycans. However, neither the molecular details nor the consequences of conditional glycan changes are well understood. Here we assessed the sensitivity of Caco-2 and HT-29 membrane N-glycosylation to variations in (i) dietary elements, (ii) microbial fermentation products and (iii) cell culture parameters relevant to intestinal epithelial cell growth and survival. Based on global LC-MS glycomic and statistical analyses, the resulting glycan expression changes were systematic, dependent upon the conditions of each controlled environment. Exposure to short chain fatty acids produced significant increases in fucosylation while further acidification promoted hypersialylation. Notably, among all conditions, increases of high mannose type glycans were identified as a major response when extracellular fructose, galactose and glutamine were independently elevated. To examine the functional consequences of this discrete shift in the displayed glycome, we applied a chemical inhibitor of the glycan processing mannosidase, globally intensifying high mannose expression. The data reveal that upregulation of high mannose glycosylation has detrimental effects on basic intestinal epithelium functions by altering permeability, host-microbe associations and membrane protein activities.
Subject(s)
Cell Membrane/drug effects , Fatty Acids, Volatile/pharmacology , Glycomics , Mannose/pharmacology , Mannosidases/metabolism , Alkaloids/pharmacology , Caco-2 Cells , Carbohydrate Sequence , Cell Membrane/chemistry , Cell Membrane/enzymology , Enzyme Inhibitors/pharmacology , Fatty Acids, Volatile/metabolism , Fructose/metabolism , Fructose/pharmacology , Fucose/metabolism , Fucose/pharmacology , Galactose/metabolism , Galactose/pharmacology , Glutamine/metabolism , Glutamine/pharmacology , Glycosylation/drug effects , HT29 Cells , Humans , Mannose/metabolism , Mannosidases/antagonists & inhibitorsABSTRACT
UNLABELLED: The type I signal peptidase of Staphylococcus aureus, SpsB, is an attractive antibacterial target because it is essential for viability and extracellularly accessible. We synthesized compound 103, a novel arylomycin-derived inhibitor of SpsB with significant potency against various clinical S. aureus strains (MIC of ~1 µg/ml). The predominant clinical strain USA300 developed spontaneous resistance to compound 103 with high frequency, resulting from single point mutations inside or immediately upstream of cro/cI, a homolog of the lambda phage transcriptional repressor cro These cro/cI mutations led to marked (>50-fold) overexpression of three genes encoding a putative ABC transporter. Overexpression of this ABC transporter was both necessary and sufficient for resistance and, notably, circumvented the essentiality of SpsB during in vitro culture. Mutation of its predicted ATPase gene abolished resistance, suggesting a possible role for active transport; in these bacteria, resistance to compound 103 occurred with low frequency and through mutations in spsB Bacteria overexpressing the ABC transporter and lacking SpsB were capable of secreting a subset of proteins that are normally cleaved by SpsB and instead were cleaved at a site distinct from the canonical signal peptide. These bacteria secreted reduced levels of virulence-associated proteins and were unable to establish infection in mice. This study reveals the mechanism of resistance to a novel arylomycin derivative and demonstrates that the nominal essentiality of the S. aureus signal peptidase can be circumvented by the upregulation of a putative ABC transporter in vitro but not in vivo IMPORTANCE: The type I signal peptidase of Staphylococcus aureus (SpsB) enables the secretion of numerous proteins by cleavage of the signal peptide. We synthesized an SpsB inhibitor with potent activity against various clinical S. aureus strains. The predominant S. aureus strain USA300 develops resistance to this inhibitor by mutations in a novel transcriptional repressor (cro/cI), causing overexpression of a putative ABC transporter. This mechanism promotes the cleavage and secretion of various proteins independently of SpsB and compensates for the requirement of SpsB for viability in vitro However, bacteria overexpressing the ABC transporter and lacking SpsB secrete reduced levels of virulence-associated proteins and are unable to infect mice. This study describes a bacterial resistance mechanism that provides novel insights into the biology of bacterial secretion.
Subject(s)
ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Staphylococcus aureus/enzymology , Staphylococcus aureus/genetics , Animals , Anti-Bacterial Agents/pharmacology , Disease Models, Animal , Drug Resistance, Bacterial , Gene Expression , Membrane Proteins/antagonists & inhibitors , Mice , Microbial Sensitivity Tests , Mutation , Selection, Genetic , Staphylococcal Infections/microbiology , Staphylococcus aureus/drug effects , Staphylococcus aureus/metabolism , VirulenceABSTRACT
A novel α1-2-fucosyltransferase from Thermosynechococcus elongatus BP-1 (Te2FT) with high fucosyltransferase activity and low donor hydrolysis activity was discovered and characterized. It was used in an efficient one-pot multienzyme (OPME) fucosylation system for the high-yield synthesis of human blood group H antigens containing ß1-3-linked galactosides and an important human milk oligosaccharide (HMOS) lacto-N-fucopentaose I (LNFP I) on preparative and gram scales. LNFP I was shown to be selectively consumed by Bifidobacterium longum subsp. infantis but not Bifidobacterium animalis subsp. lactis and is a potential prebiotic.
Subject(s)
Blood Group Antigens/biosynthesis , Cyanobacteria/enzymology , Fucosyltransferases/metabolism , Milk, Human/chemistry , Oligosaccharides/biosynthesis , Blood Group Antigens/chemistry , Carbohydrate Conformation , Enzyme Activation , Humans , Milk, Human/metabolism , Oligosaccharides/chemistry , Galactoside 2-alpha-L-fucosyltransferaseABSTRACT
Group IVA cytosolic phospholipase A2 (GIVA cPLA2) is the rate-limiting provider of pro-inflammatory mediators in many tissues and is thus an attractive target for the development of novel anti-inflammatory agents. In this work, we present the synthesis of new thiazolyl ketones and the study of their activities in vitro, in cells, and in vivo. Within this series of compounds, methyl 2-(2-(4-octylphenoxy)acetyl)thiazole-4-carboxylate (GK470) was found to be the most potent inhibitor of GIVA cPLA2, exhibiting an XI(50) value of 0.011 mole fraction in a mixed micelle assay and an IC50 of 300 nM in a vesicle assay. In a cellular assay using SW982 fibroblast-like synoviocytes, it suppressed the release of arachidonic acid with an IC50 value of 0.6 µM. In a prophylactic collagen-induced arthritis model, it exhibited an anti-inflammatory effect comparable to the reference drug methotrexate, whereas in a therapeutic model, it showed results comparable to those of the reference drug Enbrel. In both models, it significantly reduced plasma PGE2 levels.
Subject(s)
Blood Proteins/chemistry , Blood Proteins/pharmacology , Cytosol/enzymology , Group IV Phospholipases A2/antagonists & inhibitors , Ketones/chemistry , Thiazoles/chemistry , Thiazoles/pharmacology , Animals , Arachidonic Acid/metabolism , Arthritis/blood , Arthritis/chemically induced , Arthritis/drug therapy , Blood Proteins/chemical synthesis , Blood Proteins/therapeutic use , Cell Line, Tumor , Collagen/adverse effects , Dinoprostone/blood , Drug Design , Humans , Male , Mice , Oleic Acid/metabolism , Synovial Membrane/drug effects , Synovial Membrane/metabolism , Thiazoles/chemical synthesis , Thiazoles/therapeutic useABSTRACT
Eicosanoids, including prostaglandins (PG) and leukotrienes, are lipid mediators derived from arachidonic acid. A quantitative and biochemical level understanding of eicosanoid metabolism would aid in understanding the mechanisms that govern inflammatory processes. Here, we present a combined experimental and computational approach to understanding the biochemical basis of eicosanoid metabolism in macrophages. Lipidomic and transcriptomic measurements and analyses reveal temporal and dynamic changes of the eicosanoid metabolic network in mouse bone marrow-derived macrophages (BMDM) upon stimulation of the Toll-like receptor 4 with Kdo2-Lipid A (KLA) and stimulation of the P2X7 purinergic receptor with adenosine 5'-triphosphate. Kinetic models were developed for the cyclooxygenase (COX) and lipoxygenase branches of arachidonic acid metabolism, and then the rate constants were estimated with a data set from ATP-stimulated BMDM, using a two-step matrix-based approach employing a constrained least-squares method followed by nonlinear optimization. The robustness of the model was validated through parametric sensitivity, uncertainty analysis, and predicting an independent dataset from KLA-primed ATP-stimulated BMDM by allowing the parameters to vary within the uncertainty range of the calculated parameters. We analyzed the functional coupling between COX isozymes and terminal enzymes by developing a PGH2-divided model. This provided evidence for the functional coupling between COX-2 and PGE2 synthase, between COX-1/COX-2 and PGD2 synthase, and also between COX-1 and thromboxane A2 synthase. Further, these functional couplings were experimentally validated using COX-1 and COX-2 selective inhibitors. The resulting fluxomics analysis demonstrates that the "multi-omics" systems biology approach can define the complex machinery of eicosanoid networks.
Subject(s)
Eicosanoids/metabolism , Intramolecular Oxidoreductases/metabolism , Lipocalins/metabolism , Lipoxygenase/metabolism , Models, Biological , Prostaglandin-Endoperoxide Synthases/metabolism , Thromboxane-A Synthase/metabolism , Adenosine Triphosphate/pharmacology , Animals , Cells, Cultured , Cyclooxygenase 2 Inhibitors/pharmacology , Kinetics , Lipopolysaccharides/pharmacology , Macrophages/drug effects , Macrophages/metabolism , Mice , Mice, Inbred C57BLABSTRACT
Little is known about the expression of methicillin-resistant Staphylococcus aureus (MRSA) genes during infection conditions. Here, we described the transcriptome of the clinical MRSA strain USA300 derived from human cutaneous abscesses, and compared it with USA300 bacteria derived from infected kidneys in a mouse model. Remarkable similarity between the transcriptomes allowed us to identify genes encoding multiple proteases and toxins, and iron- and peptide-transporter molecules, which are upregulated in both infections and are likely important for establishment of infection. We also showed that disruption of the global transcriptional regulators agr and sae prevents in vivo upregulation of many toxins and proteases, protecting mice from lethal infection dose, and hinting at the role of these transcriptional regulators in the pathology of MRSA infection.
Subject(s)
Gene Expression Regulation, Bacterial/physiology , Methicillin-Resistant Staphylococcus aureus/metabolism , Transcriptome , Abscess/microbiology , Animals , Humans , Methicillin-Resistant Staphylococcus aureus/classification , Methicillin-Resistant Staphylococcus aureus/genetics , Methicillin-Resistant Staphylococcus aureus/pathogenicity , Mice , Protein Array Analysis , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , Skin Diseases, Bacterial/microbiology , VirulenceABSTRACT
Infection of host tissues by Staphylococcus aureus and S. epidermidis requires an unusual family of staphylococcal adhesive proteins that contain long stretches of serine-aspartate dipeptide-repeats (SDR). The prototype member of this family is clumping factor A (ClfA), a key virulence factor that mediates adhesion to host tissues by binding to extracellular matrix proteins such as fibrinogen. However, the biological siginificance of the SDR-domain and its implication for pathogenesis remain poorly understood. Here, we identified two novel bacterial glycosyltransferases, SdgA and SdgB, which modify all SDR-proteins in these two bacterial species. Genetic and biochemical data demonstrated that these two glycosyltransferases directly bind and covalently link N-acetylglucosamine (GlcNAc) moieties to the SDR-domain in a step-wise manner, with SdgB appending the sugar residues proximal to the target Ser-Asp repeats, followed by additional modification by SdgA. GlcNAc-modification of SDR-proteins by SdgB creates an immunodominant epitope for highly opsonic human antibodies, which represent up to 1% of total human IgG. Deletion of these glycosyltransferases renders SDR-proteins vulnerable to proteolysis by human neutrophil-derived cathepsin G. Thus, SdgA and SdgB glycosylate staphylococcal SDR-proteins, which protects them against host proteolytic activity, and yet generates major eptopes for the human anti-staphylococcal antibody response, which may represent an ongoing competition between host and pathogen.
Subject(s)
Bacterial Proteins/immunology , Glycosyltransferases/immunology , Host-Pathogen Interactions/immunology , Methicillin-Resistant Staphylococcus aureus/physiology , Staphylococcal Infections/immunology , Staphylococcus epidermidis/physiology , Virulence Factors/immunology , Animals , Antibodies, Bacterial/genetics , Antibodies, Bacterial/immunology , Bacterial Adhesion/genetics , Bacterial Adhesion/immunology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cathepsin G/genetics , Cathepsin G/immunology , Cathepsin G/metabolism , Cell Line, Tumor , Cell Wall/enzymology , Cell Wall/genetics , Cell Wall/immunology , Epitopes/genetics , Epitopes/immunology , Epitopes/metabolism , Female , Glycosyltransferases/genetics , Glycosyltransferases/metabolism , Host-Pathogen Interactions/genetics , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Male , Mice , Repetitive Sequences, Amino Acid , Staphylococcal Infections/enzymology , Staphylococcal Infections/genetics , Virulence Factors/genetics , Virulence Factors/metabolismABSTRACT
Group VIA calcium-independent phospholipase A2 (GVIA iPLA2) has recently emerged as an important pharmaceutical target. Selective and potent GVIA iPLA2 inhibitors can be used to study its role in various neurological disorders. In the current work, we explore the significance of the introduction of a substituent in previously reported potent GVIA iPLA2 inhibitors. 1,1,1,2,2-Pentafluoro-7-(4-methoxyphenyl)heptan-3-one (GK187) is the most potent and selective GVIA iPLA2 inhibitor ever reported with a XI(50) value of 0.0001, and with no significant inhibition against GIVA cPLA2 or GV sPLA2. We also compare the inhibition of two difluoromethyl ketones on GVIA iPLA2, GIVA cPLA2, and GV sPLA2.
Subject(s)
Group VI Phospholipases A2/antagonists & inhibitors , Ketones/chemistry , Phospholipase A2 Inhibitors/chemistry , Fluorine/chemistry , Group VI Phospholipases A2/metabolism , Ketones/chemical synthesis , Ketones/metabolism , Phospholipase A2 Inhibitors/chemical synthesis , Phospholipase A2 Inhibitors/metabolism , Protein Binding , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/metabolismABSTRACT
UNLABELLED: The strong restriction barrier present in Staphylococcus aureus and Staphylococcus epidermidis has limited functional genomic analysis to a small subset of strains that are amenable to genetic manipulation. Recently, a conserved type IV restriction system termed SauUSI (which specifically recognizes cytosine methylated DNA) was identified as the major barrier to transformation with foreign DNA. Here we have independently corroborated these findings in a widely used laboratory strain of S. aureus. Additionally, we have constructed a DNA cytosine methyltransferase mutant in the high-efficiency Escherichia coli cloning strain DH10B (called DC10B). Plasmids isolated from DC10B can be directly transformed into clinical isolates of S. aureus and S. epidermidis. We also show that the loss of restriction (both type I and IV) in an S. aureus USA300 strain does not have an impact on virulence. Circumventing the SauUSI restriction barrier, combined with an improved deletion and transformation protocol, has allowed the genetic manipulation of previously untransformable strains of these important opportunistic pathogens. IMPORTANCE: Staphylococcal infections place a huge burden on the health care sector due both to their severity and also to the economic impact of treating the infections because of prolonged hospitalization. To improve the understanding of Staphylococcus aureus and Staphylococcus epidermidis infections, we have developed a series of improved techniques that allow the genetic manipulation of strains that were previously refractory to transformation. These developments will speed up the process of mutant construction and increase our understanding of these species as a whole, rather than just a small subset of strains that could previously be manipulated.
Subject(s)
Gene Transfer Techniques , Staphylococcus aureus/genetics , Staphylococcus epidermidis/genetics , Transformation, Bacterial , DNA Methylation , DNA Restriction-Modification Enzymes , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA-Cytosine Methylases/genetics , DNA-Cytosine Methylases/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Genetic Vectors , Humans , Molecular Sequence Data , Plasmids , Sequence Analysis, DNA , Staphylococcus aureus/pathogenicity , VirulenceABSTRACT
Eicosanoids constitute a diverse class of bioactive lipid mediators that are produced from arachidonic acid and play critical roles in cell signaling and inflammatory aspects of numerous diseases. We have previously quantified eicosanoid metabolite production in RAW264.7 macrophage cells in response to Toll-like receptor 4 signaling and analyzed the levels of transcripts coding for the enzymes involved in the eicosanoid metabolite biosynthetic pathways. We now report the quantification of changes in protein levels under similar experimental conditions in RAW264.7 macrophages by multiple reaction monitoring mass spectrometry, an accurate targeted protein quantification method. The data complete the first fully integrated genomic, proteomic, and metabolomic analysis of the eicosanoid biochemical pathway.
Subject(s)
Arachidonic Acid/metabolism , Biosynthetic Pathways/drug effects , Eicosanoids/biosynthesis , Inflammation/metabolism , Macrophages/metabolism , Adenosine Triphosphate/pharmacology , Animals , Cell Line , Inflammation/chemically induced , Lipid Metabolism/drug effects , Lipopolysaccharides/pharmacology , Macrophages/drug effects , Mass Spectrometry , Metabolomics , Mice , Proteomics , Signal Transduction/drug effectsABSTRACT
Bacteria can exist in metabolically inactive states that allow them to survive conditions that are not conducive for growth. Such dormant cells may sense when conditions have improved and re-initiate growth, lest they be outcompeted by their neighbours. Growing bacteria turn over and release large quantities of their cell walls into the environment. Drawing from recent work on the germination of Bacillus subtilis spores, we propose that many microorganisms exit dormancy in response to cell wall muropeptides.