Tuberculostearic Acid-Containing Phosphatidylinositols as Markers of Bacterial Burden in Tuberculosis.

One-fourth of the global human population is estimated to be infected with strains of the Mycobacterium tuberculosis complex (MTBC), the causative agent of tuberculosis (TB). Using lipidomic approaches, we show that tuberculostearic acid (TSA)-containing phosphatidylinositols (PIs) are molecular markers for infection with clinically relevant MTBC strains and signify bacterial burden. For the most abundant lipid marker, detection limits of ∼102 colony forming units (CFUs) and ∼103 CFUs for bacterial and cell culture systems were determined, respectively. We developed a targeted lipid assay, which can be performed within a day including sample preparation─roughly 30-fold faster than in conventional methods based on bacterial culture. This indirect and culture-free detection approach allowed us to determine pathogen loads in infected murine macrophages, human neutrophils, and murine lung tissue. These marker lipids inferred from mycobacterial PIs were found in higher levels in peripheral blood mononuclear cells of TB patients compared to healthy individuals. Moreover, in a small cohort of drug-susceptible TB patients, elevated levels of these molecular markers were detected at the start of therapy and declined upon successful anti-TB treatment. Thus, the concentration of TSA-containing PIs can be used as a correlate for the mycobacterial burden in experimental models and in vitro systems and may prospectively also provide a clinically relevant tool to monitor TB severity.

The human LL-37 peptide exerts antimicrobial activity against Legionella micdadei interacting with membrane phospholipids.

Legionella micdadei is responsible for community- or nosocomial-acquired pneumonia as well as the influenza-like illness Pontiac fever. The aim of this study was to investigate the ability of L. micdadei to utilize extracellular choline for phosphatidylcholine (PC) synthesis and its consequences for the phospholipid composition of its membrane system and the interaction with the human LL-37 peptide. Comparative analysis of the PC content using isotopic labeling revealed that in presence of exogenous choline 98% of the total PC was synthesized via the Pcs pathway while the remaining 2% were generated via the PE-methylation (PmtA) pathway. PC species were to a greater extent defined by the Pcs pathway in the outer membrane than in the inner membrane. While no major changes in the bacterial lipid content were observed using 31P NMR, indication for utilization of longer acyl chains and slight increase of PG in response to choline addition was observed by a top-down lipidomics screen. The LL-37 peptide inhibited L. micdadei growth in a dose-dependent manner. Bacteria cultured with exogenous choline were more sensitive to the LL-37 peptide when compared to the standard culture condition. Our biophysical investigations show that the peptide perturbs bacterial-derived phospholipid monolayers and this interaction is dependent on the molar portion of PC. This interaction is responsible for the observed changes in the anti-L. micdadei activity of the LL-37 peptide.

Isoniazid@Fe2 O3 Nanocontainers and Their Antibacterial Effect on Tuberculosis Mycobacteria.

Isoniazid-filled Fe2 O3 hollow nanospheres (INH@Fe2 O3 , diameter <30 nm, 48 wt % INH-load) are prepared for the first time and suggested for tuberculosis therapy. After dextran-functionalization, the INH@Fe2 O3 @DEX nanocontainers show strong activity against Mycobacterium tuberculosis (M.tb.) and M.tb.-infected macrophages. The nanocontainers can be considered as "Trojan horses" and show efficient, active uptake into both M.tb.-infected macrophages and even into mycobacterial cells.

Phosphatidylinositol 4-phosphate and phosphatidylinositol 3-phosphate regulate phagolysosome biogenesis.

Professional phagocytic cells ingest microbial intruders by engulfing them into phagosomes, which subsequently mature into microbicidal phagolysosomes. Phagosome maturation requires sequential fusion of the phagosome with early endosomes, late endosomes, and lysosomes. Although various phosphoinositides (PIPs) have been detected on phagosomes, it remained unclear which PIPs actually govern phagosome maturation. Here, we analyzed the involvement of PIPs in fusion of phagosomes with various endocytic compartments and identified phosphatidylinositol 4-phosphate [PI(4)P], phosphatidylinositol 3-phosphate [PI(3)P], and the lipid kinases that generate these PIPs, as mediators of phagosome-lysosome fusion. Phagosome-early endosome fusion required PI(3)P, yet did not depend on PI(4)P. Thus, PI(3)P regulates phagosome maturation at early and late stages, whereas PI(4)P is selectively required late in the pathway.

Lipid Analysis of Airway Epithelial Cells for Studying Respiratory Diseases.

Airway epithelial cells play an important role in the pathogenesis of inflammatory lung diseases such as asthma, cystic fibrosis and COPD. Studies concerning the function of the lipid metabolism of the airway epithelium are so far based only on the detection of lipids by immunohistochemistry but quantitative analyses have not been performed. Although recent advances in mass spectrometry have allowed to identify a variety of lipid classes simultaneously in isolated tissue samples, up until now, these methods were not suitable to analyze lipids in the airway epithelium. To determine all major lipid classes in airway epithelial cells, we used an LC-MS-based approach that can easily be combined with the specific isolation procedure to obtain epithelial cells. We tested the suitability of this method with a mouse model of experimental asthma. In response to allergen challenge, perturbations in the sphingolipids were detected, which led to increased levels of ceramides. We expanded the scope of this approach analysing human bronchus samples without pathological findings of adenocarcinoma patients. For the human lung epithelium an unusual lipid class distribution was found in which ceramide was the predominant sphingolipid. In summary, we show that disease progression and lipid metabolism perturbation can be monitored in animal models and that the method can be used for the analysis of clinical samples.

Bioavailability and allergoprotective capacity of milk-associated conjugated linoleic acid in a murine model of allergic airway inflammation.

BACKGROUND: Cross-sectional epidemiological studies have demonstrated that farm milk from traditional farm settings possesses allergoprotective properties. Up to now, it has not been clarified which milk ingredient is responsible for protection against allergic diseases. As farm milk is rich in conjugated linoleic acids (CLA), it is hypothesized that this n-3 polyunsaturated fatty acid family contributes to the allergoprotective capacity of farm milk. We aim to prove this hypothesis in a murine model of allergic airway inflammation. METHODS: To prove the bioavailability and allergoprotective capacity of milk-associated CLA in a standardized protocol, milk batches that differed significantly in terms of their CLA content were spray dried and incorporated into a basic diet by substituting the regular sunflower fat fraction. Initially, the milk CLA uptake from the diet was monitored via measurement of the CLA content in plasma and erythrocyte membranes obtained from supplemented mice. To determine whether a milk CLA-enriched diet possesses allergoprotective properties, female Balb/c mice were fed the milk CLA-enriched diet ahead of sensitization and a challenge with ovalbumin (OVA) and the parameters of airway inflammation and eisosanoid pattern were measured. RESULTS: In animals, supplementation with a diet rich in milk CLA resulted in elevated CLA levels in plasma and erythrocyte membranes, indicating bioavailability of milk fatty acids. Though membrane-associated phospholipid patterns were affected by supplementation with milk CLA, this application neither reduced the hallmarks of allergic airway inflammation in sensitized and OVA-challenged mice nor modified the eiconsanoid pattern in the bronchoalveolar lavage fluid of these animals. CONCLUSION: Milk-associated CLA was not capable of preventing murine allergic airway inflammation in an animal model of OVA-induced allergic airway inflammation.

Ingestion of (n-3) fatty acids augments basal and platelet activating factor-induced permeability to dextran in the rat mesenteric vascular bed.

Loss of intestinal barrier function and subsequent edema formation remains a serious clinical problem leading to hypoperfusion, anastomotic leakage, bacterial translocation, and inflammatory mediator liberation. The inflammatory mediator platelet activating factor (PAF) promotes eicosanoid-mediated edema formation and vasoconstriction. Fish oil-derived (n-3) fatty acids (FA) favor the production of less injurious eicosanoids but may also increase intestinal paracellular permeability. We hypothesized that dietary (n-3) FA would ameliorate PAF-induced vasoconstriction and enhance vascular leakage of dextran tracers. Rats were fed either an (n-3) FA-rich diet (EPA-rich diet; 4.0 g/kg EPA, 2.8 g/kg DHA) or a control diet (CON diet; 0.0 g/kg EPA and DHA) for 3 wk. Subsequently, isolated and perfused small intestines were stimulated with PAF and arterial pressure and the translocation of fluid and macromolecules from the vasculature to lumen and lymphatics were analyzed. In intestines of rats fed the EPA-rich diet, intestinal phospholipids contained up to 470% more EPA and DHA at the expense of arachidonic acid (AA). The PAF-induced increase in arterial pressure was not affected by the EPA-rich diet. However, PAF-induced fluid loss from the vascular perfusate was higher in intestines of rats fed the EPA-rich diet. This was accompanied by a greater basal loss of dextran from the vascular perfusate and a higher PAF-induced transfer of dextran from the vasculature to the lumen (P = 0.058) and lymphatics. Our data suggest that augmented intestinal barrier permeability to fluid and macromolecules is a possible side effect of (n-3) FA-rich diet supplementation.

Elucidation of the fragmentation pathways of different phosphatidylinositol phosphate species (PIPx) using IRMPD implemented on a FT-ICR MS.

This work reports on the fragmentation of phosphoinositides by tandem mass spectrometry (MS/MS) and MS³ experiments on a hybrid apex-Qe Fourier transform-ion cyclotron resonance mass spectrometer (FT-ICR MS) using internal infrared multiphoton dissociation (IRMPD). The fragmentation behavior of diacylphophatidylinositol triphosphate was intensively studied since an abundant loss of inositol biphosphate was observed. This loss was suggested to occur via phosphate migration along the inositol head group. Substantiation by MS³ experiments showed that this neutral loss is formed after the loss of water from the precursor ion, indicating phosphate migration along the inositol ring to the glycerol backbone. Further fragmentation of the ion formed by the loss of inositol biphosphate from diacylphophatidylinositol triphosphate resulted in the formation of a product ion with a molecular formula of C(3)H(5)O(7)P(2), corresponding to a glycerol backbone linked to two phosphate groups. We suggested different structures for this ion and compared their stability using modeling experiments.

Recent developments in tandem mass spectrometry for lipidomic analysis.

This review will focus on the role of mass spectrometry in the emerging field of lipidomics. Particular emphasis will be placed on recent developments in the use of tandem mass spectrometry methods in lipid analysis using low-energy collision induced dissociation (CID). After a brief discussion on ionization techniques, novel ion-activation methods that allow for increased sensitivity and selectivity will be critically discussed. Examples will be drawn from the analysis of higher order lipids, specifically triacylglycerols (TAGs) and glycerophospholipids, as the numerous positional isomers and head groups present in these classes of lipids continue to pose a significant analytical challenge to the field of lipidomics. The role of bioinformatics in the development of lipidomics will also be discussed.

Plasma free fatty acid profiling in a fish oil human intervention study using ultra-performance liquid chromatography/electrospray ionization tandem mass spectrometry.

A rapid method was developed for the simultaneous profiling of 29 free fatty acids in plasma using ultra-performance liquid chromatography/electrospray ionization tandem mass spectrometry (UPLC/ESI-MS/MS). Barium acetate was used as the cationization agent in the positive ion mode for sensitive multiple reaction monitoring (MRM) experiments. The cis- and trans-C18:1 and -C18:2 isomers were baseline-separated using two tandem reversed-phase C18 UPLC columns, while identification of two pairs of positional isomers of C18:3 and C20:3 required isomer-specific product ions, as the analytes were not chromatographically resolved. The assay linearity was greater than three orders of magnitude and correlation coefficients were >0.99; the limits of detections were typically less than 0.2 microM. The method was successfully applied to plasma free fatty acid profiling of samples from volunteers who participated in a randomized crossover study involving the administration of either placebo or fish oil capsules. The results clearly indicate the ability to measure the time profiles of the n-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in plasma for the volunteers given fish oil capsules while the concentrations of the other free fatty acids and the total free fatty acid concentration in plasma remained virtually constant.