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Multi-step Preparation Technique to Recover Multiple Metabolite Compound Classes for In-depth and Informative Metabolomic Analysis

Metabolomics is an emerging field which enables profiling of samples from living organisms in order to obtain insight into biological processes. A vital ...

University of Colorado Denver

Mycobacterium tuberculosis (M. tb) uses the glyoxalate bypass for intracellular survival in vivo. These studies provide evidence that the M. tb malate synthase (MS) has adapted to function as an adhesin which binds to laminin and fibronectin. This binding is achieved via the unique C-terminal region of the M. tb MS. The ability to function as an adhesin necessitates extracellular localization. We provide evidence that despite the absence of a Sec-translocation signal sequence the M. tb MS is secreted/excreted, and is anchored on the cell wall by an undefined mechanism. The MS of Mycobacterium smegmatis is cytoplasmic but the M. tb MS expressed in M. smegmatis localizes to the cell wall and enhances the adherence of the bacteria to lung epithelial A549 cells. Antibodies to the C-terminal laminin/fibronectin-binding domain interfere with the binding of the M. tb MS to laminin and fibronectin and reduce the adherence of M. tb to A549 cells. Coupled to the earlier evidence of in vivo expression of M. tb MS during active but not latent infection in humans, these studies show that a housekeeping enzyme of M. tb contributes to its armamentarium of virulence promoting factors.

Mycobacterium tuberculosis malate synthase is a laminin-binding adhesin.

Peeling off the layers: skin taping and a novel proteomics approach to study atopic dermatitis.

A myriad of methods has been reported for the isolation of genomic DNA from Mycobacterium spp.; some methods use mechanical disruption of the bacterial cells, whereas others use some form of chemical or enzymatic lysis. Regardless of the approach, the end points remain efficient breaking of the complex mycobacterial cell wall and release of high-quality DNA that is suitable for manipulation and analyses by molecular genetic techniques. This chapter providers detailed methods for the large and small isolation of mycobacterial genomic DNA.

Isolation of mycobacterium species genomic DNA.

The cell envelope of Mycobacterium tuberculosis (Mtb) is complex and diverse; composed of proteins intermingled in a matrix of peptidoglycan, mycolic acids, lipids, and carbohydrates. Proteomic studies of the Mtb cell wall have been limited; nonetheless, the characterization of resident and secreted proteins associated with the cell wall are critical to understanding bacterial survival and immune modulation in the host. In this study, the cell wall proteome was defined in order to better understand its unique biosynthetic and secretion processes. Mtb cell wall was subjected to extraction with organic solvents to remove noncovalently bound lipids and lipoglycans and remaining proteins were solubilized with either SDS, Guanidine-HCl, or TX-114. These extracts were analyzed by two-dimensional gel electrophoresis and mass-spectrometry and resulted in the identification of 234 total proteins. The lipoproteome of Mtb, enriched in the TX-114 extract, was further resolved by multidimensional chromatography and mass spectrometry to identify an additional 294 proteins. A query of the 528 total protein identifications against Neural Network or Hidden Markov model algorithms predicted secretion signals in 87 proteins. Classification of these 528 proteins also demonstrated that 35% are involved in small molecule metabolism and 25% are involved in macromolecule synthesis and degradation building upon evidence that the Mtb cell wall is actively engaged in mycobacterial survival and remodeling.

Proteomic definition of the cell wall of Mycobacterium tuberculosis.

Atopic dermatitis (AD) is the most common inflammatory skin disorder in the general population worldwide, and the majority of patients are colonized with Staphylococcus aureus. Eczema herpeticum is a disseminated herpes simplex virus infection that occurs in a small subset of patients.

Comparative proteomic profiling of patients with atopic dermatitis based on history of eczema herpeticum infection and Staphylococcus aureus colonization.

Galectin-9 is a pleiotropic immune modulator affecting numerous cell types of innate and adaptive immunity. Patients with chronic infection with either hepatitis C virus (HCV) or HIV have elevated circulating levels. Limited data exist on the regulation of natural killer (NK) cell function through interaction with galectin-9. We found that galectin-9 ligation downregulates multiple immune-activating genes, including eight involved in the NK cell-mediated cytotoxicity pathway, impairs lymphokine-activated killing, and decreases the proportion of gamma interferon (IFN-γ)-producing NK cells that had been stimulated with interleukin-12 (IL-12)/IL-15. We demonstrate that the transcriptional and functional changes induced by galectin-9 are independent of Tim-3. Consistent with these results for humans, we find that the genetic absence of galectin-9 in mice is associated with greater IFN-γ production by NK cells and enhanced degranulation. We also show that in the setting of a short-term (4-day) murine cytomegalovirus infection, terminally differentiated NKs accumulate in the livers of galectin-9 knockout mice, and that hepatic NKs spontaneously produce significantly more IFN-γ in this setting. Taken together, our results indicate that galectin-9 engagement impairs the function of NK cells, including cytotoxicity and cytokine production.

Galectin-9 functionally impairs natural killer cells in humans and mice.

Sample preparation remains a challenge in untargeted metabolomics studies and no method currently results in complete extraction of all metabolite classes in human plasma. Because a large variety of molecules, with vast differences in dynamic range, could be involved in human disease, there is an urgent need to develop analytical techniques that result in comprehensive coverage of metabolites. Furthermore, analysis of more focused molecular classes could be necessary to more fully interrogate markers of human disease. However, such techniques, which generally involve multiple steps, often result in high variability. We have optimized a combined liquid-liquid and solid phase extraction method for plasma and have compared that to traditional methanol precipitation using spiked internal standards as controls. This method, based largely on previously published methods, results in 5 separate fractions enriched for aqueous species, phospholipids, fatty acids, neutral lipids, and hydrophobic lipids. Using liquid chromatography mass spectrometry as the analytical method, we detect over 3806 metabolites using the new method versus 1851 metabolites using methanol alone. Qualitative analysis and quantitative analysis of both internal standards (ISTDs) and endogenous metabolites demonstrate excellent reproducibility with CV's below 15% for the combined method compared to 30% using the methanol method. While both methods have applications for clinical metabolomics, fractionation resulted in greater overall coverage and can be used for initial classification of molecular species.

New sample preparation approach for mass spectrometry-based profiling of plasma results in improved coverage of metabolome.

The role of Group IVA cytosolic phospholipase A2 (cPLA2α) activation in regulating macrophage transcriptional responses to Candida albicans infection was investigated. cPLA2α releases arachidonic acid for the production of eicosanoids. In mouse resident peritoneal macrophages, prostacyclin, prostaglandin E2 and leukotriene C4 were produced within minutes of C. albicans addition before cyclooxygenase 2 expression. The production of TNFα was lower in C. albicans-stimulated cPLA2α(+/+) than cPLA2α(-/-) macrophages due to an autocrine effect of prostaglandins that increased cAMP to a greater extent in cPLA2α(+/+) than cPLA2α(-/-) macrophages. For global insight, differential gene expression in C. albicans-stimulated cPLA2α(+/+) and cPLA2α(-/-) macrophages (3 h) was compared by microarray. cPLA2α(+/+) macrophages expressed 86 genes at lower levels and 181 genes at higher levels than cPLA2α(-/-) macrophages (≥2-fold, p<0.05). Several pro-inflammatory genes were expressed at lower levels (Tnfα, Cx3cl1, Cd40, Ccl5, Csf1, Edn1, CxCr7, Irf1, Irf4, Akna, Ifnγ, several IFNγ-inducible GTPases). Genes that dampen inflammation (Socs3, Il10, Crem, Stat3, Thbd, Thbs1, Abca1) and genes involved in host defense (Gja1, Csf3, Trem1, Hdc) were expressed at higher levels in cPLA2α(+/+) macrophages. Representative genes expressed lower in cPLA2α(+/+) macrophages (Tnfα, Csf1) were increased by treatment with a prostacyclin receptor antagonist and protein kinase A inhibitor, whereas genes expressed at higher levels (Crem, Nr4a2, Il10, Csf3) were suppressed. The results suggest that C. albicans stimulates an autocrine loop in macrophages involving cPLA2α, cyclooxygenase 1-derived prostaglandins and increased cAMP that globally effects expression of genes involved in host defense and inflammation.

Cytosolic phospholipase A(2)α and eicosanoids regulate expression of genes in macrophages involved in host defense and inflammation.

Cigarette smoke exposure is linked to the development of a variety of chronic lung and systemic diseases in susceptible individuals. Metabolomics approaches may aid in defining disease phenotypes, may help predict responses to treatment, and could identify biomarkers of risk for developing disease. Using a mouse model of chronic cigarette smoke exposure sufficient to cause mild emphysema, we investigated whether cigarette smoke induces distinct metabolic profiles and determined their persistence following smoking cessation. Metabolites were extracted from plasma and fractionated based on chemical class using liquid-liquid and solid-phase extraction prior to performing liquid chromatography mass spectrometry-based metabolomics. Metabolites were evaluated for statistically significant differences among group means (p-value≤0.05) and fold change ≥1.5). Cigarette smoke exposure was associated with significant differences in amino acid, purine, lipid, fatty acid, and steroid metabolite levels compared to air exposed animals. Whereas 60% of the metabolite changes were reversible, 40% of metabolites remained persistently altered even following 2 months of smoking cessation, including nicotine metabolites. Validation of metabolite species and translation of these findings to human plasma metabolite signatures induced by cigarette smoking may lead to the discovery of biomarkers or pathogenic pathways of smoking-induced disease.

Spencer Mahaffey

Department of Pharmacology,
School of Medicine,
Department of Pharmacology, School of Medicine

University of Colorado Denver

Multi-step Preparation Technique to Recover Multiple Metabolite Compound Classes for In-depth and Informative Metabolomic Analysis

Spencer Mahaffey

Department of Pharmacology,School of Medicine,Department of Pharmacology, School of Medicine

University of Colorado Denver

Multi-step Preparation Technique to Recover Multiple Metabolite Compound Classes for In-depth and Informative Metabolomic Analysis

Department of Pharmacology,
School of Medicine,
Department of Pharmacology, School of Medicine