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Microbial physiology Microbial proteomics Microbial signaling Pathogen detectionFlow Cytometric Quantification of Fatty Acid Uptake by Mycobacterium tuberculosis in Macrophages
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Mycobacterium tuberculosis (Mtb) has evolved to assimilate fatty acids from its host. However, until recently, there was no reliable way to quantify fatty acid uptake by the bacteria during host cell infection. Here we describe a new method to quantify fatty acid uptake by intracellular bacilli. We infect macrophages with Mtb constitutively expressing mCherry and then metabolically label them with Bodipy-palmitate. Following the labeling procedure, we isolate Mtb-containing phagosomes on a sucrose cushion and disrupt the phagosomes with detergent. After extensive washes, the isolated bacteria are analyzed by flow cytometry to determine the level of Bodipy-palmitate signal associated with the bacteria. Using a Mtb mutant strain defective in fatty acid uptake in liquid culture we determined that this mutant assimilated 10-fold less Bodipy-palmitate than the wild type strain during infection in macrophages. This quantitative method of fatty acid uptake can be used to further identify pathways involved in lipid uptake by intracellular Mtb and possibly other bacteria.
Sterol Analysis in Kluyveromyces lactis
Sterols are essential lipids of most eukaryotic cells with multiple functions (structural, regulatory and developmental). Sterol profile of yeast cells is often determined during the studies of ergosterol synthesis mutants used to uncover a number of functions for various sterols in yeast cells. Molecular studies of ergosterol biosynthesis have been also employed to identify essential steps in the pathway against which antifungals might be developed. We present here a protocol for the isolation of non-saponifiable lipids (sterols) from Kluyveromyces lactis yeast cells and a chromatographic method for quantitative analysis of sterols in lipid extracts (HPLC) that can be performed in laboratories with standard equipment.
Metabolite and Fatty Acid Analysis of Yeast Cells and Culture Supernatants
Metabolite and fatty acid analysis play important roles in evaluating the metabolic state of microorganisms. To examine the growth state and metabolism response of cells to environmental stress or genetic modification, intracellular and extracellular metabolites including fatty acids are usually analyzed to help understand the cellular biochemical changes in microorganisms. In this protocol, gas chromatography-mass spectrometry based analysis was employed to investigate the fatty acids and other metabolites in yeast cells.
Metabolic Labeling of Yeast Sphingolipids with Radioactive D-erythro-[4,5-3H]dihydrosphingosine
Yeast sphingolipids can be metabolically labeled with exogenously added radioactive precursors. Here we describe a method to label ceramides and complex sphingolipids of Saccharomyces cerevisiae with a radioactive ceramide precursor, D-erythro-[4, 5-3H]dihydrosphingosine. This protocol is used to study the biosynthesis, transport and metabolism of sphingolipids in yeast.