In search for the inhibitors of Escherichia coli stringent response factor RelA
Date
2017-09-27
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Abstract
Keskkonnatingimuste jälgimiseks ja muutustega kohanemiseks on bakteritel mitmeid sensoorseid süsteeme. Seejuures on levinud strateegiaks sekundaarsete signaalmolekulite kasutamine: stressisignaali ilmumisega muutub signaalmolekulide rakusisene kontsentratsioon ning see omakorda reguleerib sihtensüümide aktiivsust. Üks sellistest stressivastuse süsteemidest on poomisvastus (stringent response), mis aktiveeritakse sõltuvalt alarmoonmolekuli (p)ppGpp rakusisesest kontsentratsioonist. (p)ppGpp on võimeline reguleerima mitmete ensüümide aktiivsust, kuid peamiseks märklauaks on RNA polümeraas. (p)ppGpp rakusisest taset kontrollivad E. coli RelA-SpoT-ga homoloogsed valgud (RSH), mis kas sünteesivad või hüdrolüüsivad (p)ppGpp-d vastavalt keskkonnatingimustele. Eksperimentaalselt on tõestatud, et poomisvastus kontrollib bakterite virulentsust, persisterite moodustamist, antibiootikumide taluvust ning antibiootikumide tootmist, samuti osaleb bakterite hulgatunnetuses (quorum sensing) ja bakterite ellujäämises fagotsütoosi jooksul. Seepärast on poomisvastuse mehhanismide mõistmine väga oluline ja (p)ppGpp rakusisest taset kontrollivate ühendite loomine võiks viia meditsiini ja biotehnoloogia seisukohalt oluliste rakendusteni. Hiljuti avastati, et ppGpp struktuuril põhinev aine Relacin on võimeline inhibeerima RelA aktiivsust kuid tõhusa inhibeerimise jaoks on vaja Relacini kasutada väga kõrges kontsentratsioonis.
Käesoleva töö raames iseloomustasin (p)ppGpp struktuuri põhjal disainitud uudsete keemiliste ainete efekti E. coli RelA aktiivsusele. Samuti pakun välja uusi võimalusi RelA inhibiitorite arendamiseks. Peale uute sünteesitud ainete efektide vaatasin üle võimalusi mõjutada poomisvastust juba tuntud translatsiooni inhibiitorite abil ning iseloomustasin thiostreptooni tugevat inhibeerimisvõimet (p)ppGpp sünteesile
Bacteria are the most abundant living organisms on Earth. Through billions of years of evolution they developed numerous adaptation mechanisms that allow them to survive in constantly changing environmental conditions. Bacteria protect themselves from various environmental challenges by entering a dormant state, by acquiring resistance to antibiotics, forming biofilms etc. All these varied adaptation mechanisms rely on the enzymatic activity of specific proteins that sense and response to stress – and that renders these proteins promising targets for the development of novel antibacterial agents. The current work elucidates one of bacterial adaptive mechanisms called the stringent response that is orchestrated by RelA SpoT Homologue (RSH) enzymes in nutritionally poor environment, upon heat shock or cell wall damage. The varying level of effector-molecule of the stringent response – a highly charged nucleotide alarmone (p)ppGpp – is the key mediator of the survival program launched by bacteria during stringent response. In order to turn off the stringent response and increase the susceptibility of bacteria towards antibiotics, one can either target the RSH enzymes themselves or compromise the signaling nucleotide (p)ppGpp direclty. This dissertation discusses the possibilities of inhibiting the activity of Escherichia coli stringent factor RelA by re-examining the mechanism of action for the classical antibiotics and characterizing newly developed molecular tools based on a (p)ppGpp scaffold.
Bacteria are the most abundant living organisms on Earth. Through billions of years of evolution they developed numerous adaptation mechanisms that allow them to survive in constantly changing environmental conditions. Bacteria protect themselves from various environmental challenges by entering a dormant state, by acquiring resistance to antibiotics, forming biofilms etc. All these varied adaptation mechanisms rely on the enzymatic activity of specific proteins that sense and response to stress – and that renders these proteins promising targets for the development of novel antibacterial agents. The current work elucidates one of bacterial adaptive mechanisms called the stringent response that is orchestrated by RelA SpoT Homologue (RSH) enzymes in nutritionally poor environment, upon heat shock or cell wall damage. The varying level of effector-molecule of the stringent response – a highly charged nucleotide alarmone (p)ppGpp – is the key mediator of the survival program launched by bacteria during stringent response. In order to turn off the stringent response and increase the susceptibility of bacteria towards antibiotics, one can either target the RSH enzymes themselves or compromise the signaling nucleotide (p)ppGpp direclty. This dissertation discusses the possibilities of inhibiting the activity of Escherichia coli stringent factor RelA by re-examining the mechanism of action for the classical antibiotics and characterizing newly developed molecular tools based on a (p)ppGpp scaffold.
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Keywords
bakterid, adaptatsioonimehhanismid, signaalmolekulid, Escherichia coli, ensüümiaktiivsus, RNA polümeraas, poomisvastus, inhibitsioon, bacteria, adaptive mechanisms, signal molecules, enzyme activity, RNA polymerase, stringent response, inhibition