Small fine-tuners of the bacterial stringent response – a glimpse into the working principles of Small Alarmone Synthetases

Abstract

Bakterid elavad pidevalt muutuvates ja tihtipeale karmides tingimustes. Nad asustavad kõige ligipääsmatuid paiku maailmas ja suudavad ellu jääda äärmuslikult külmades või kuumades kohtades, taluvad liigset soolsust või hapnikuvaegust. Samuti ka patogeensete bakterite jaoks on peremeesorganismi immuunsüsteem väljakutse millele vastu panna. Selleks, et kiiresti reageerida ja edukalt toime tulla muutlike ja ebasoodsate oludega on bakterirakkudel evolutsioneerunud spetsiaalsed keerukad regulatoorsed mehhanismid. Üheks kõige levinumaks selliseks mehhanismiks on “bakteriaalne poomisvastus”. Poomisvastus on vahendatud alarmoonmolekuli (p)ppGpp poolt, mida sünteesitakse vastuseks aminohappevaegusele ja muudele stressitingimustele. Selle akumuleerumine rakus soodustab bakterite ellujäämist mõjutades erinevaid rakulisi protsesse nagu transkriptisoon, translatsioon, replikatsioon. (p)ppGpp mängib olulist rolli ka bakterite virulentsuses, biofilmi tekkes ja ka antibiootikumresistentsuses. Seega poomisvastuse uurimine on ka kliinilisest aspektist väga oluline. (p)ppGpp molekuli tase rakus on reguleeritud bakterites konserveerunud RelA-SpoT homoloogsete (RSH) valkude poolt. Käesolev käsikiri on pühendatud RSH valguperekonna vähemtuntud liikmete uurimisele – E. faecalis RelQ ja S. aureus RelP. Nimelt uurisin nende valkude ensümaatilisi omadusi ja tõepoolest leidsin, et need omavad erilisi tunnuseid võrreldes teiste uuritud RSH valkudega. Erinevused nende valkude funktsioonides rõhutavad nende rolli poomisvastuse häälestamises ja seeläbi viitavad bakterite mitmekülgsele võimele reguleerida oma elutegevust erinevates tingimustes.Bacteria, being the most abundant organisms in the world, live and survive in the most extreme and unreachable places of our planet. They are able to withstand such conditions as extreme cold or heat, excessive salinity, lack of oxygen etc. Moreover, for pathogenic bacteria, immune system of the host organism is also a challenge to put up with. In order to combat hostile and continuosly changing conditions bacteria have evolved regulatory mechanisms to sense the environmental cues and change their physiology in response. One of the most global such mechanism is the stringent response. This process is mediated by the alarmone molecule named (p)ppGpp which is synthesized in bacterial cell in response to nutrient, heat and other stresses. Accumulation of this molecule leads to reprogramming of bacterial physiology such that growth and division are repressed and survival mechanisms are upregulated. This leads to increased viability of bacterium in adverse conditions. Stringent response is also implicated in virulence, antibacterial resistance and biofilm formation, therefore making this process an important study subject from the clinical point of view. (p)ppGpp levels are regulated by the representatives of RelA-SpoT Homolog (RSH) family of proteins. These enzymes are highly conserved among bacteria due to their evolutionary importance and the universal nature of the stringent response in bacteria. In this thesis, I contribute to knowledge on RSH enzymes by taking a glimpse into working mechanisms of Small Alarmone Synthetases (SAS) – so far not so well studied RSH representatives. Specifically, I have investigated the enzymatic characteristics of two SAS: Enterococcus faecalis RelQ and Staphylococcus aureus RelP. The distinctive features of the working principles found for these proteins underlay their role in fine-tuning the stringent response.