Du Toit Loots Lung-2015 Baltimore, USA - PowerPoint Presentation

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Du Toit Loots

Lung-2015Baltimore, USAJuly 13 - 15, 2015

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Prof Du

Toit

Loots

Tuberculosis: adaptations of man and microbe in order to outcompete and survive

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History of TBRobert Koch - 1882

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History of TB

150 million years ago -

Jurassic period 3 million years ago – infection in early hominids (TB meningitis in Homo erectus)

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1.7 million years ago - Migration out of East Africa

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Evolutionary timeline = TB timeline

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Tuberculosis: Shocking StatisticsInfectious disease of primarily lungs – M. tuberculosis2nd most deadliest infectious disease (after HIV)⅓ of global population infected9 million new cases per year1,4 million deaths per year of which 25% are HIV co-infectedMDR-TB, XDR-TB is on the rise in many 3rd world countriesDespite fervent research efforts since it's discovery in 1882 – TB is still considered a global epidemicNew approaches are needed = Metabolomics?

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MetabolomicsUnbiased, identification and quantification of ALL intra- and extra-cellular metabolites (small molecule intermediates and products of metabolism) present in a biological system/sample (cell, body fluid, tissue, organism), using highly selective and sensitive analytical methods (such as liquid/gas chromatography and mass spectrometry), in conjunction with bioinformatics for identifying new metabolite markers, at a specific point in time and under certain conditions.Uses: Identifying compounds / markers of a perturbation

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Systems Biology

Systems / Integrative BiologyDNA

RNAProteinsMetabolites

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NWU-Human MetabolomicsLC-MS:Agilent 6410 LC-QQQ

Agilent 6460 LC-QQQAgilent 6220 LC-TOF

Agilent 6510 LC-QTOFAgilent LC-MSD XCT Plus IonTrapAgilent LC-UV(DAD)Synapt G2 SI UPLC QTOF Plus Ion MobilityGC-MS:Agilent GC-MSDLeco Pegasus 4D GCxGC-TOFMSLeco GC-TOFMSCE:Agilent CENMR:Bruker 500Mhz

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Untargeted and Semi-targeted metabolomicsUntargeted:GCxGC-TOFMSUPLC-TOFMS Ion MobilityNMR

Semi-Targeted GC-MS/LC-MS:Free Fatty Acids

Long Chain Fatty AcidsOrganic AcidsAcylcarnitinesAmino AcidsSamplesBloodUrineTissueSputumCell CulturesPlantSkin Secretions (Frogs)Etc…

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Metabolomics workflowMetabolite Markers

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2) TB Diagnostics:I. Olivier & Du T. Loots. (2011). An overview of tuberculosis treatments and diagnostics. What role could metabolomics play? J Cell Tissue Research, 11(1): 2655-2671.

1) TB Characterization:J.C. Schoeman & Du T. Loots. (2011). Improved disease characterization and diagnostics using metabolomics: A review. J Cell Tissue Research, 11(1): 2673-2683.Our Journey

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Cell

Cultures

1) Total Lipid Extraction Method2) Total Metabolome Extraction MethodApplications?a) DiagnosticsOlivier & Loots. (2012). Journal of Microbiological MethodsPatent: PCT/IB2012051995b) Drug ResistanceRif: Olivier & Loots. (2012). Omics.a) VirulenceMeissner-Roloff et al. (2012). Metabolomics (hyper vs hypo)Swanepoel

et al. (2013). Metabolomics (ESX-1)b) GrowthLoots et al. (2013). Metabolomics (ESX-3)Loots et al., submitted, Metabolomics (Iron)c) Drug ResistanceINH: Loots 2014. Antimicrobial Agents and Chemotherapyb) DiagnosticsPatient sputum samplesApplications of Methodologies

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Sputum:Homogenization: Schoeman et al. 2012

. Journal of Microbiological MethodsApplied to 95 Patient sputum samples: du Preez & Loots. (2013). Tuberculosis

Urine:Adaptations of Host and Microbe: de Villiers & Loots., Nature Communications (submitted)Early Prediction of Treatment Outcome: de Villiers et al. Blood: Sample collectionPatient samples

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Adaptations of M. tuberculosis to Host - Sputum

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Adaptations of Host to M.tuberculosis - Sputum

Glucose oxidase

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Adaptations of Host to M. tuberculosis - UrineKAT

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Adaptations of Host to Microbein Competition to Survive - UrineAutophagy: During nutrient deprived conditions/various disease states: vesicles containing long lived cytoplasmic constituents and organelles are fused with lysosomes, and degraded in order to supply components for cell anabolism, by releasing monomeric units of macromolecules: amino acids, fatty acids, DNA breakdown products etc.Host uses autophagy in it's defence against M. tuberculosis also.Decreased insulin may also trigger this process.

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Adaptations of Host to M. tuberculosis - Urine

KAT

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Side effects associated with TBEpinephine / increase fatty acids: weight loss, insomnia, glucose intolerance (further confirmed by d-gluconic acid δ-lactone) and the link btw TB and diabetes.

Elevated tyrosine and homovanilic acid – precursors to epinephrine (confirming sputum results)Quinolinic acid: NMDA agonist associated with neurological abnormalities. Phenylacetic/Phenyllactic acid

: vomiting, nausea, diarrhoea, drowsiness, fatigue, loss of appetite and weight loss.

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Co-treatment SuggestionsMelatonin + Anti-TB co-treatmentEliminates neurotoxicity associated with quinolinic acidIncrease the efficacy of anti-TB drugs and reduce side effects (Loots et al., 2004)BH4+ Anti-TB co-treatment

Treatment for PKU for preventing phenylacetic acid Metformin + Anti-TB co-treatmentAnti-diabetic drug lowering blood sugar levels and increasing insulin sensitivityIn TB patients - ameliorated lung pathology, reduced chronic inflammation, enhanced the specific immune response and increased the efficacy of conventional TB drugs.

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Metabolomics Contact:Email: dutoit.loots@nwu.ac.za

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Lung & Respiratory Care – 2016 Website

: lung.conferenceseries.comMeet the eminent gathering once again at

Lung & Respiratory Care -2016Manchester, UKAugust 01 - 03, 2016