For!reactions!and!pathways!to!be!labelled!as!potential!drug!targets,!they!must!not!be! catalysed!by!the!same!enzymes!in!Mtb#and!H.#sapiens.!Using!the!phylogenetic!profile!of! Figure! 3.1! and! 3.2,! pathways! were! noted! which! showed! few! orthologous! proteins! between! these! two! organisms.! Certain! pathways! were! noted! in! this! regard! including! ‘biotin! metabolism’,! ‘glyoxylate! and! dicarboxylate! metabolism’,! ‘sulfur! metabolism’,! ‘geraniol!degradation’!and!‘polycyclic!aromatic!hydrocarbon!degradation’.!
The! ‘biotin! metabolism’! pathway! has! only! two! reactions! added;! however,! these! reactions!partially!complete!the!biosynthesis!of!biotin,!an!essential!cofactor!for!carboxyl! group!transfer!enzymes!required!by!all!forms!of!life!(Caspi!et!al.,!2012).!In!E.#coli!biotin! is!synthesised!through!a!number!of!reactions!belonging!to!a!slightly!altered!fatty!acid! synthetic!pathway.!These!reactions!add!a!methyl!ester!to!the!intermediates!to!disguise! them! so! that! they! can! be! substrates! for! the! fatty! acid! synthesis! reactions.! After! two!
reiterations!of!these!fatty!acid!elongation!cycles,!pimeloylJ[acp]!is!produced,!which!then! proceeds!via!8JaminoJ7Joxononanoate!to!produce!biotin!in!reactions!that!have!already! been!characterised!in!KEGG!for!Mtb!(Lin!et!al.,!2010).!In!the!phylogenetic!profile!this! pathway! was! found! to! share! few! orthologs! with! H.#sapiens,! although! it! shares! a! large! proportion!with!both!M.#leprae!and!E.#coli.!The!high!proportion!of!shared!orthologs!with! M.# leprae! suggests! that! this! pathway! is! important! for! growth! and! survival,! a! finding! supported!by!the!literature!(Caspi!et!al.,!2012;!Lin!et!al.,!2010).!The!low!proportion!of! shared!orthologs!with!H.#sapiens!suggests!these!organisms!share!few!similarities!with! regards! to! the! biosynthesis! of! biotin.! These! results! indicate! that! reactions! of! this! pathway!might!be!potential!drug!targets!for!future!research.!
Another! pathway! that! deserves! further! investigation! is! ‘glyoxylate! and! dicarboxylate! metabolism’.! As! mentioned! previously! this! pathway! is! incredibly! important! for! the! metabolism! of! fatty! acids! as! a! source! of! carbon! and! energy! (Moat! et! al.,! 2003).! Additionally,! these! reactions! are! important! for! the! use! of! glycolate! and! glyoxylate! as! sources! of! carbon! and! energy! (Clark! and! Cronan,! 2005).! Figure! 3.3! shows! that! only! about! one! quarter! of! the! Mtb#proteins! involved! in! this! pathway! have! orthologs! in! H.# sapiens.! Those! proteins! shared! between! the! two! organisms! mostly! comprise! the! ‘methylmalonyl!pathway’,!‘glyoxylate!cycle’!and!‘glycolate!and!glyoxylate!degradation!I’! pathway.! Of! these! three! pathways,! only! the! ‘methylmalonyl! pathway’! is! predicted! by! MetaCyc! to! function! in! H.# sapiens.! However,! when! viewing! the! ‘glyoxylate! and! dicarboxylate!metabolism’!pathway!across!the!phylogenetic!profile!shown!in!Figure!3.1,! it!seems!to!be!relatively!well!conserved!among!bacteria.!While!the!lack!of!orthologous! proteins! in! H.# sapiens# might! warrant! further! examination! of! these! proteins! as! drug! targets,! their! conservation! in! other! bacteria! might! prove! difficult! in! the! actual! application!of!drugs.!
The! reactions! belonging! to! ‘sulfur! metabolism’! also! show! few! orthologs! between! Mtb# and! H.# sapiens.! Sulfur! and! sulfite! can! be! used! as! sources! of! energy! and! electrons! in! organisms!able!to!metabolise!it!(Kappler!and!Dahl,!2001).!The!bars!representing!‘sulfur! metabolism’! in! Figure! 3.3! show! close! to! even! numbers! of! orthologs! in! C.#glutamicum# and!E.#coli.!This!also!shows!very!few!shared!orthologs!in!H.#sapiens.!Lastly,!many!of!the! proteins! added! for! Mtb# also! have! orthologs! in! M.# leprae.! The! heat! map! of! the!
phylogenetic!profile!shown!in!Figure!3.1!shows!that!is!pathway!is!relatively!conserved! across!the!genome.!However,!most!of!those!proteins!that!were!added!to!this!pathway! using!BLASTP!matches!do!not!have!orthologs!across!the!phylogenetic!profile.!Most!of! these!added!proteins!are!involved!in!first!the!transport!of!taurine!and!alkanesulfonate! from!extracellular!sources!and!second!their!conversions!into!sulfite.!This!would!suggest! that! these! proteins! involved! in! transport! and! conversion! of! compounds! that! are! precursors! of! sulfite! might! require! further! examination! as! drug! targets,! particularly! since!these!proteins!are!not!shared!across!the!phylogenetic!profile!but!have!orthologs! in!the!genome!of!M.#leprae.!
The! ‘geraniol! degradation’! pathway! also! shows! few! orthologs! between! Mtb# and! H.# sapiens.!In!fact,!Mtb#proteins!involved!in!this!pathway!share!orthologies!with!very!few! proteins!across!the!entire!phylogenetic!profile!and!the!pathway!is!in!the!bottom!third!in! terms! of! average! frequency! as! shown! in! Figure! 3.2.! This! pathway! involves! the! degradation! of! citronellol,! a! compound! produced! by! plants! and! found! in! the! environment!(Caspi!et!al.,!2012).!Pseudomonas#citronellolis!can!use!citronellol!as!a!sole! source!of!carbon!and!energy!(Seubert,!1960).!Mtb#shows!many!more!proteins!assigned! to!this!pathway!than!any!of!the!organisms!compared!in!Figure!3.3!suggesting!that!this! pathway!may!be!important!for!the!organism.!On!the!other!hand,!Mtb#also!shares!very! few!orthologs!for!this!pathway!with!M.#leprae,!indicating!that!perhaps!these!reactions! are! not! essential! for! central! metabolism.! However,! the! much! greater! numbers! of! Mtb! proteins! belonging! to! this! pathway,! compared! to! other! organisms,! warrants! investigation! into! the! potential! competitive! advantages! this! pathway! might! provide! within! the! host.! Since! citronellol! is! a! common! plantJderived! compound! in! the! environment,! the! existence! of! proteins! catalysing! reactions! within! this! pathway! may! simply! be! an! evolutionary! artefact! of! a! soilJdwelling! ancestor.! However,! the! large! differences!in!numbers!of!orthologous!proteins!seen!between!Mtb#and!other!organisms! warrant!further!attention.!
Lastly! the! ‘polycyclic! aromatic! hydrocarbon! degradation’! pathway,! along! with! many! other! pathways! involved! in! ‘xenobiotics! biodegradation! and! metabolism’,! shows! few! orthologs!between!Mtb#and!H.#sapiens.!Additionally!Mtb#shares!few!orthologs!with!any! other! examined! organism! for! this! pathway! (Figure! 3.3);! this! pathway! ranks! in! the!
bottom! 20%! of! pathways,! signifying! that! it! is! one! of! the! least! conserved! as! shown! in! Figure!3.2.!These!results!identify!this!pathway!as!being!notable!and!worthy!of!further! investigation.! Many! of! the! added! proteins! to! this! pathway! are! involved! in! the! degradation!of!phenanthrene,!a!compound!found!in!cigarette!smoke.!The!most!common! way!by!which!phenanthrene,!along!with!many!other!polycyclic!aromatic!hydrocarbons! (PAHs),!enters!the!body!is!by!breathing!air!contaminated!with!fumes!from!coal,!asphalt,! wildfires,!vehicle!exhaust!or!even!grilled!food!(Phenanthrene).!A!number!of!species!of! mycobacteria!have!been!shown!to!be!able!to!grow!on!phenanthrene!(and!other!PAHs)! as! a! sole! source! of! carbon! and! energy! (Boldrin! et! al.,! 1993;! Moody! et! al.,! 2001;! Willumsen!et!al.,!2001).!Interestingly,!exposure!to!tobacco!has!been!shown!to!increase! TB! infection,! active! disease! and! mortality! (Bates! et! al.,! 2007;! Wen! et! al.,! 2010).! This! correlation!is!thought!to!occur!because!smoking!suppresses!the!immune!system,!thus! allowing!greater!chance!of!infection!and!progression!of!the!disease!as!well!as!a!reduced! ability! of! the! body! to! fight! back! against! infection.! However,! these! newly! annotated! proteins! in! Mtb! offer! another! possibility:! perhaps! smoking! increases! virulence! and! survival! of! Mtb! by! providing! a! source! of! carbon! and! energy.! The! findings! of! the! phylogenetic! profile! thus! suggest! that! reactions! within! this! pathway! could! represent! potential!drug!targets!that!must!be!examined!further.!
An!examination!of!the!number!of!H.#sapiens!proteins!that!have!orthologs!in!Mtb#results! in! the! identification! of! a! number! of! interesting! pathways.! These! pathways,! including! ‘biotin! metabolism’,! ‘glyoxylate! and! dicarboxylate! metabolism’,! ‘sulfur! metabolism’,! ‘geraniol! degradation’! and! ‘polycyclic! aromatic! hydrocarbon! degradation,! signify! pathways! that! could! be! targeted! by! novel! drugs! without! adversely! affecting! human! cells.!