Lung2015 Baltimore USA July 13 15 2015 Prof Du Toit Loots Tuberculosis adaptations of man and microbe in order to outcompete and survive History of TB Robert Koch 1882 History of TB ID: 489008
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Du Toit Loots
Lung-2015Baltimore, USAJuly 13 - 15, 2015Slide2
Prof Du
Toit
Loots
Tuberculosis: adaptations of man and microbe in order to outcompete and surviveSlide3
History of TBRobert Koch - 1882Slide4
History of TB
150 million years ago -
Jurassic period 3 million years ago – infection in early hominids (TB meningitis in Homo erectus)Slide5
1.7 million years ago - Migration out of East AfricaSlide6
Evolutionary timeline = TB timelineSlide7
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?Slide8
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 perturbationSlide9
Systems Biology
Systems / Integrative BiologyDNA
RNAProteinsMetabolitesSlide10
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 500MhzSlide11
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…Slide12
Metabolomics workflowMetabolite MarkersSlide13
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 JourneySlide14
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 MethodologiesSlide15
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 samplesSlide16
Adaptations of M. tuberculosis to Host - SputumSlide17
Adaptations of Host to M.tuberculosis - Sputum
Glucose oxidaseSlide18
Adaptations of Host to M. tuberculosis - UrineKATSlide19
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.Slide20
Adaptations of Host to M. tuberculosis - Urine
KATSlide21
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.Slide22
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.zaSlide24
Lung & Respiratory Care – 2016 Website
: lung.conferenceseries.comMeet the eminent gathering once again at
Lung & Respiratory Care -2016Manchester, UKAugust 01 - 03, 2016