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Du Toit LootsLung-2015Baltimore, USAJuly 13 - 15, 2015

Prof Du Toit Loots Tuberculosis: adaptations of man and microbe in order to outcompete and survive

History of TBRobert Koch - 1882

History of TB

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

1.7 million years ago - Migration out of East AfricaEvolutionary timeline = TB timeline

Tuberculosis: Shocking StatisticsInfectious disease of primarily lungs M. tuberculosis2nd most deadliest infectious disease (after HIV) 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?

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 perturbationSystems Biology

Systems / Integrative BiologyDNARNAProteinsMetabolitesNWU-Human MetabolomicsLC-MS:Agilent 6410 LC-QQQAgilent 6460 LC-QQQAgilent 6220 LC-TOFAgilent 6510 LC-QTOFAgilent LC-MSD XCT Plus IonTrapAgilent LC-UV(DAD)Synapt G2 SI UPLC QTOF Plus Ion Mobility

GC-MS:Agilent GC-MSDLeco Pegasus 4D GCxGC-TOFMSLeco GC-TOFMS

CE:Agilent CE

NMR:Bruker 500Mhz

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

Semi-Targeted GC-MS/LC-MS:Free Fatty AcidsLong Chain Fatty AcidsOrganic AcidsAcylcarnitinesAmino AcidsSamplesBloodUrineTissueSputumCell CulturesPlantSkin Secretions (Frogs)EtcMetabolomics workflow

Metabolite Markers2) 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 JourneyCell Cultures1) Total Lipid Extraction Method2) Total Metabolome Extraction MethodApplications?a) DiagnosticsOlivier & Loots. (2012). Journal of Microbiological MethodsPatent: PCT/IB2012051995

b) 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 Chemotherapy

b) DiagnosticsPatient sputum samples

Applications of Methodologies

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 samplesAdaptations of M. tuberculosis to Host - Sputum

Adaptations of Host to M.tuberculosis - Sputum

Glucose oxidaseAdaptations of Host to M. tuberculosis - Urine

KATAdaptations 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.

Adaptations of Host to M. tuberculosis - Urine

KAT

Side effects associated with TBEpinephine / increase fatty acids: weight loss, insomnia, glucose intolerance (further confirmed by d-gluconic acid -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.

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-treatmentTreatment 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.

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

Lung & Respiratory Care 2016 Website: lung.conferenceseries.comMeet the eminent gathering once again atLung & Respiratory Care -2016Manchester, UKAugust 01 - 03, 2016