HPLC analysis of bacterial alarmone nucleotide (p)ppGpp and its toxic analogue ppApp
Kuupäev
2020-09-11
Autorid
Ajakirja pealkiri
Ajakirja ISSN
Köite pealkiri
Kirjastaja
Abstrakt
Bakteritel on evolutsiooni käigus välja kujunenud arvukalt kohanemismehhanisme, mis aitavad neil ellu jääda ka karmides keskkonnatingimustes. Keerukad molekulaarsed võrgustikud kontrollivad adaptiivseid füsioloogilisi vastuseid, näiteks antibiootikumiresistentsust, biokile moodustumist ja bakterite minekut uinunud olekusse. Sellised kohanemismehhanismid sõltuvad stressi tajuvate ja sellele reageerivate valkude ensümaatilistest aktiivsustest. Üheks oluliseks komponendiks stressivastuses on signaalmolekulide süntees ja lagundamine. Käesolevas töös uuriti ühte kõige laiemalt levinud adaptiivset mehhanismi, mida nimetatakse poomisvastuseks. Selle mehhanismi puhul on võtmetähtsusega RelA / SpoT homoloogsed (RSH) ensüümid, mis sünteesivad ja lagundavad alarmoon-nukleotiide ppGpp ja ppp(G)pp. Nende nukleotiidide ühiseks nimetamiseks kasutatakse tähistust (p)ppGpp. Need molekulid mõjutavad mitmeid protsesse bakterirakus, näiteks virulentsust ja antibiootikumitolerantsust. Käesoleva töö eesmärgiks oli välja töötada metoodika nukleotiidide, sealhulgas (p)ppGpp, tasemete kvantifitseerimiseks. Rakendades seda metoodikat uuriti nukleotiidide taset bakterite kasvul ning antibiootikumitöötluse käigus.
Nukleotiidide, sealhulgas (p)ppGpp taseme kvantifitseerimiseks töötati välja HPLC-l põhinev meetod. Nukleotiidide kvantifitseerimise meetodid sisaldavad kolme etappi: proovi kogumine, nukleotiidide ekstraheerimine ja kvantifitseerimine. Kogumisetapis filtreeriti bakterikultuur ja nukleotiidide ekstraheerimiseks viidi filter äädikhappesse. (p)ppGpp kvantifitseerimiseks rakendati HPLC metoodikat 5 µm 4,6 x 150 mm tugeval anioonvahetuskolonnil. Teiste nukleotiidide tuvastamiseks ja kvantifitseerimiseks kasutati ioon-paar pöördfaasi (IPRP) kromatograafiat Kinetex C18 2,6 µm 4,6 x 150 mm kolonnil. Kasutades väljatöötatud metoodikaid uuriti nukleotiidide tasemete muutust bakterite stressivastuse korral. Soolekepikesel (Escherichia coli) analüüsiti nukleotiidide tasemeid kasvukõvera erinevates faasides ja aminohapete nälja puhul. Aminohapete nälja puhul täheldati kiiret (p)ppGpp taseme tõusu.
Translatsiooni inhibeerivate antibiootikumide (tiostreptooni, klooramfenikooli ja tetratsükliini) mõju (p)ppGpp ja teiste nukleotiidide tasemetele bakterirakus uuriti nii Gram-negatiivsetes kui ka Gram-positiivsetes bakterites, esindajateks vastavalt E.coli ja Bacillus subtilis. (p)ppGpp kuhjumise indutseerimiseks kasutati eeltöötlust muprirotsiiniga. Seejärel lisati uuritav antibiootikum subinhibeerivas kontsentratsioonis. Mõlema bakteriliigi korral pidurdasid kõik testitud translatsiooni inhibiitorid (p)ppGpp kuhjumist.
Meie uurimisrühma bioinformaatiline analüüs tuvastas, et mõnedes bakteriliikides on RSH ensüümid, millel on ainult (p)ppGpp sünteesi eest vastutav osa. Leiti, et selline ensüüm bakteris Cellulomonas marina võib fosforüleerida ka adenosiini, tekitades molekuli ppApp. Koos paralleelselt ilmunud töödega teistest laboritest on alust arvata, et tegemist on uudse regulaatornukleotiidiga. Selle nukleotiidi täpse rolli kindlakstegemine nõuab edasisi uuringuid.
Bacteria through evolution developed numerous adaptation mechanisms that made them survive in harsh environmental conditions. Therefore, to protect themselves from environmental challenges bacteria evolved complex molecular networks that leads to suitable physiological responses by acquiring resistance to antibiotics, forming biofilms or by entering in a dormant state. These adaptation mechanisms depend on enzymatic activity of specific proteins that sense and respond to stress. The responses of these stresses are mediated by synthesis and degradation of signaling molecules that can regulate transcription and protein activities. The PhD work comprise the study of stringent response that is one of the most widely spread adaptive mechanism in bacteria. This mechanism is orchestrated by RelA SpoT Homologue (RSH) enzymes that produce and degrade a highly charged alarmone nucleotide called guanosine(penta)tetraphosphate ((p)ppGpp), comprising guanosine pentaphosphate (pppGpp) and tetraphosphate (ppGpp), collectively referred as (p)ppGpp. The (p)ppGpp-mediated signaling is one of the master regulators of bacterial physiology and plays an important role in bacterial virulence, and tolerance to antibiotics. In order to quantify the varying levels of (p)ppGpp and housekeeping nucleotides in different stress conditions as well as during normal bacterial growth, we developed a HPLC-based quantification method. Using Escherichia coli and Bacillus subtilis as the two representatives of Gram-negative and Gram-positive bacteria, I studied the effects of antibiotic treatment on the cellular levels of ppGpp, (p)ppGpp as well as housekeeping nucleotides such as ATP and GTP. Finally, using the HPLC-based approach, I discovered that a toxic Small Alarmone Synthetase RSH from Cellulomonas marina, in addition to coproducing ppGpp alarmone synthesizes a highly toxic ppGpp analogue, ppApp. Together with the recent report by Laub and Whitney labs who described Pseudomonas aeruginosa Tas1 – a divergent RSH enzyme that acts as a toxic effector of a secretion system via production of (pp)pApp (Ahmad et al., 2019) this discovery opens up a new direction in studies of RSH enzymes.
Bacteria through evolution developed numerous adaptation mechanisms that made them survive in harsh environmental conditions. Therefore, to protect themselves from environmental challenges bacteria evolved complex molecular networks that leads to suitable physiological responses by acquiring resistance to antibiotics, forming biofilms or by entering in a dormant state. These adaptation mechanisms depend on enzymatic activity of specific proteins that sense and respond to stress. The responses of these stresses are mediated by synthesis and degradation of signaling molecules that can regulate transcription and protein activities. The PhD work comprise the study of stringent response that is one of the most widely spread adaptive mechanism in bacteria. This mechanism is orchestrated by RelA SpoT Homologue (RSH) enzymes that produce and degrade a highly charged alarmone nucleotide called guanosine(penta)tetraphosphate ((p)ppGpp), comprising guanosine pentaphosphate (pppGpp) and tetraphosphate (ppGpp), collectively referred as (p)ppGpp. The (p)ppGpp-mediated signaling is one of the master regulators of bacterial physiology and plays an important role in bacterial virulence, and tolerance to antibiotics. In order to quantify the varying levels of (p)ppGpp and housekeeping nucleotides in different stress conditions as well as during normal bacterial growth, we developed a HPLC-based quantification method. Using Escherichia coli and Bacillus subtilis as the two representatives of Gram-negative and Gram-positive bacteria, I studied the effects of antibiotic treatment on the cellular levels of ppGpp, (p)ppGpp as well as housekeeping nucleotides such as ATP and GTP. Finally, using the HPLC-based approach, I discovered that a toxic Small Alarmone Synthetase RSH from Cellulomonas marina, in addition to coproducing ppGpp alarmone synthesizes a highly toxic ppGpp analogue, ppApp. Together with the recent report by Laub and Whitney labs who described Pseudomonas aeruginosa Tas1 – a divergent RSH enzyme that acts as a toxic effector of a secretion system via production of (pp)pApp (Ahmad et al., 2019) this discovery opens up a new direction in studies of RSH enzymes.
Kirjeldus
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Märksõnad
bacteria, nucleotides, high pressure liquid chromatography