Poomisvastus bakterite kasvus ja elus püsimises

Väitekirja elektrooniline versioon ei sisalda publikatsiooneBakterid peavad ellu jäämiseks pidevalt kohanema oma väliskeskkonnaga. Sobivates tingimustes kasvavad paljud bakteriliigid väga kiiresti. Kiire kasv iseenesest viib aga kasvutingimuste muutumiseni. Nüüd kohtame järgmist bakteritele iseloomulikku omadust — jaksu pikka aega elus püsida kasvuks mittesobivates tingimustes. Seejuures säilitavad nad olulise võime kiiresti taas kasvama hakata, kui keskkonnatingimused paranevad. Muutuvate keskkonnatingimustega kohanemiseks on bakteritel evolutsiooni käigus välja kujunenud hulganisti mehhanisme. Üks selline, keskne ja pea kõigis bakterites esinev mehhanism on poomisvastus. Poomisvastust kutsuvad esile järsud muutused keskkonnas, mis nõuavad kasvu aeglustumist, sageli peatub kasv esialgu täielikult, rakk kohaneb ja kui võimalik, jätkab kasvamist muutunud tingimustes paraja tempoga. Vähemaks reguleeritakse näiteks valgusünteesi masinavärk ning rohkemaks elus püsimise ja autonoomsuse tarbeks oluline — hulganisti kahjustuste eest kaitsevaid süsteeme ja tarvilikud anaboolsed protsessid. Poomisvastust orkestreerivad signaalmolekulid, guanosiin nukleotiidid pppGpp ja ppGpp, koondnimega (p)ppGpp. Nende nukleotiidide sünteesi eest vastutavad Escherichia coli-s kaks valku, RelA ja SpoT. Viimane neist hoolitseb ka selle eest, et (p)ppGpp-d oleks rakus parasjagu, s.t SpoT on kahefunktsionaalne, omab ka (p)ppGpp-d lagundavat aktiivsust. Mitmetes teistes bakterites (näiteks Bacillus subtilis) on poomisvastuse tarbeks vaid üks peamine kahefunktsionaalne ensüüm (RelBsu), aga ka hiljuti avastatud väikesed valgud, millest on veel vähe teada ja mis omavad kas sünteesi või hüdrolüüsi aktiivsust. Arvestades poomisvastuse ulatuslikku mõju bakteriraku füsioloogiale, ei tule vast üllatusena, et see protsess mõjutab bakterite võimet põhjustada haigust ja antibiootikumide võimet infektsiooni ravida. Antibiootikumide kasutamise algusaegadest peale pandi tähele, et sugugi mitte kõik bakterirakud ei sure baktereid tapva antibiootikumi toimel, üksikud bakterid jäävad ikka elama. Erinevalt antibiootikumi resistentsusest ei kasva sellised rakud antibiootikumi juuresolekul, nad lihtsalt taluvad, elavad üle, ja neid nimetatakse persistoriteks. Ka persistorite moodustumises on nähtud poomisvastuse rolli — kui suurem osa bakteritest kasvab jõudsalt, lülitub üksikutes siiski millegipärast sisse poomisvastus. Oletatakse, et persistorid võivad antibiootikumi kuuri lõppedes põhjustada haiguse taastekkimist. Haigusest jagu saamisel on aga antibiootikumiga võrdväärne roll kanda immuunsüsteemil, mis võiks ju jagu saada sellistest mittejagunevatest persistoritest. Samas on vähe teada selliste persistor-rakkude ja immuunsüsteemi vahelistest seostest, mida asutigi käesolevas töös kõigepealt uurima. Selgus, et nn kaasasündinud immuunsüsteem inimese vere seerumi komplemendi näol ei tapa sugugi kõiki uropatogeense E. coli rakke. Kui nüüd samaaegselt seerumile rakendati ka antibiootikumi töötlust, sõltus tulemus konkreetsest antibiootikumist. Ampitsilliini (rakukesta sünteesi inhibiitor) või amikatsiini (translatsiooni inhibiitor) lisamisel vähenes seerumis ellujäävate bakterirakkude hulk ühe-kahe suurusjärgu võrra, mis lubab oletada, et mõned rakud, mida seerum ei hävita, tapeti antibiootikumi poolt. Lisaks võimendas seerum amikatsiini toimet subinhibitoorsete kontsentratsioonide puhul. Norfloksatsiini (DNA replikatsiooni inhibiitor) lisamine seerumile ei põhjustanud mingit muutust ellu jäänud bakterirakkude arvukuses, mistõttu võib spekuleerida, et komplement ja norfloksatsiin tapavad ühesuguseid rakke. Uurimaks bakteripopulatsiooni võimaliku heterogeensuse mõju komplemendi süsteemi vahendatud tapmisele, analüüsiti järgmiseks bakterirakkude jagunemist üksikraku tasemel. Katsetulemused näitasid, et kuigi komplement tunneb ära kõik bakterirakud, ja suurem osa bakterirakkudest sureb, jäävad elama keskmisest oluliselt kiiremini kasvavad ja mittekasvavad rakud. Kui nüüd samal ajal rakendada antibiootikumi töötlust (ampitsilliini, amikatsiini või norfloksatsiiniga), jäävad alles vaid mittekasvavad rakud ning kiiremini kasvavad hävitatakse. Kuivõrd katsed seerumiga näitasid muuhulgas, et bakterite suremisel on positiivne korrelatsioon rakkude seerumis kasvama hakkamisega ja mittejagunevad rakud on kaitstud nii antibiootikumi toime kui komplemendi eest, uuriti järgmisena E. coli rakkude kasvama hakkamise regulatsiooni ja poomisvastuse rolli selles. Selgus, et rakud, kus puudub peamine poomisvastuse valk RelA (edaspidi ΔrelA tüvi), hakkavad soodsate kasvutingimuste saabudes kasvama neli tundi hiljem metsiktüüpi rakkudest, kui keskkonnas puuduvad aminohapped. Lisaks aminohapetele mõjutas kasvama hakkamist ka süsinikuallikas — ΔrelA tüvi toibus metsiktüüpi tüvest hiljem süsinikuallikana glükoosi sisaldaval söötmel, ent võrdväärselt glütseroolil kasvades. Selgus, et selline RelA funktsiooni puudumine ja toibumisdefekt võib mõjutada antibiootikumi toimet—ampitsilliin tappis glükoosil toibuvaid metsik-tüüpi rakke efektiivsemalt kui ΔrelA rakke. Mõnevõrra üllatuslikult elasid ΔrelA rakud paremini üle ka ampitsilliinitöötluse glütseroolil toibudes. Igatahes, teatud tingimuste korral võib poomisvastus olla vajalik rakkude kiiresti kasvama hakkamiseks, mis omakorda võib mõjutada antibiootikumi toimet neile rakkudele. Eelpool nägime, et poomisvastusel on roll bakterirakkude kasvama hakkamisel ja see mõjutab antibiootikumi toimet neile rakkudele. Tõsi küll, teatud üsnagi kitsastes tingimustes oli funktsionaalse poomisvastuse puudumine ampitsilliini toime üle elamiseks kasulik. Samas on küllaldaselt töid, mis näitavad, et poomisvastuse puudumise korral on vähenenud bakterite võime haigust põhjustada. Koos antibiootikumi resistentsuse hirmuäratava levikuga otsitakse seepärast ka spetsiifilisi poomisvastuse pärssijaid üsna palavikuliselt. Isegi kui neist ei ole peatset ja vahetut kasu meditsiinile, oleksid spetsiifilised inhibiitorid oluline töövahend bakteriraku füsioloogia uurimiseks. Sestap soovisime järgmiseks leida poomisvastuse inhibiitoreid. Neid otsiti keemiliste ühendite raamatukogust (17500 ühendit), kasutades testsüsteemina bakterit B. subtilis, sest ainete sisenemine rakku on gram-positiivsetel bakteritel hõlpsam kui gram-negatiivsetel. . Otsingu tulemusel leiti 17 uut antibakteriaalset ühendit, kahjuks polnud ükski neist piisavalt spetsiifiline poomisvastuse suhtes. Jääb üle vaid loota, et välja töötatud kõrge läbilaskevõimega poomisvastuse inhibiitorite testsüsteem annab positiivse tulemuse mõne teise keemiliselt sünteesitud ja/või loodusest isoleeritud ühendite raamatukogu puhul.One of the most remarkable features of bacteria is the speed at which they proliferate. This itself is an intricate balancing act—cells need to orchestrate myriad of processes in time scale of seconds. Rapid growth, however, inevitably leads to a change in growth environment and, then, we encounter the next remarkable feature of bacterial cells—withstand stasis and otherwise harsh environment. In rapidly growing bacterial cells, protein biosynthesis takes the largest toll on cellular energy. Sensing the status of ribosome, the molecular machinery of translation, is therefore important part of bacterial growth regulation. The process, which probes the capability of aminoacylation of tRNAs to keep up with the requirements of translation, is called the stringent response. The stringent response is elicited when uncharged tRNA binds to the ribosomal A-site. In E. coli and other beta- and gammaproteobacteria, such ribosomes are recognized by a RelA protein that binds to the ribosome and synthesizes small nucleotide alarmone molecules ppGpp and pppGpp, collectively (p)ppGpp. Accumulation of the alarmone downregulates translation, proliferation, catabolism, halts the growth and directs the cellular resources towards anabolism and stasis survival. In case of wide variety of other stresses that have apparently less in common—except that they are all prone to affect translation, though less directly—(p)ppGpp accumulates, too. In beta- and gammaproteobacteria, the enzyme responsible for this is called SpoT. SpoT, a homologue of RelA, in addition to synthesis, can also hydrolyse (p)ppGpp. Apparently, the net effect of SpoT on (p)ppGpp levels is achieved by a balance between its activity of synthesis and hydrolysis. The balance is important not only in times of trouble but also during steady state growth conditions—SpoT is responsible for fine tuning the (p)ppGpp levels necessary for balanced growth establishing a linear reciprocal relationship between (p)ppGpp levels and growth rate. In wide variety of other bacteria, however, there is just one large bifunctional protein homologous to RelA/SpoT. In addition, some bacteria contain additional small proteins with either the (p)ppGpp synthetase or hydrolase domain. According to the global nature of (p)ppGpp effect on bacterial physiology and its perceived role in stasis preparation and survival, (p)ppGpp has implications for bacterial virulence. Attenuated infection in mice, for stringent response defective strains, has been reported for several bacterial species, including Mycobacterium tuberculosis, Vibrio cholerae, Salmonella typhimurium, Yersinia pestis, Streptococcus pneumoniae, and Brucella sp. In addition to the regulation of virulence, stringent response can interfere with the action of antibiotics, too. Bactericidal antibiotics, for example, require an active target to kill the bacterial cells and are therefore inefficient in case of non-growing dormant cells. It has been also proposed that in few cells of growing bacterial population, for some reason, (p)ppGpp accumulates to high levels. Then, if the bactericidal antibiotic treatment is applied, while the rest of the bacterial population is eradicated, the few dormant cells survive and cause the recurrent infection, once the antibiotic regime is discontinued. Yet, dormant bacteria should be eliminated by the action of immune system. After all, not all antibiotics are bactericidal, some are are bacteriostatic but still effective by acting in concert with the immune system. Current work, therefore, set out to investigate the connections between bacterial growth, the action of antibiotics and the insult of immune system. We worked with (i) uropathogenic Escherichia coli (UPEC, strain CFT073, O6:K2:H1), once isolated from a patient with acute pyelonephritis i.e. a strain capable of causing bacteremia, and (ii) human serum with its complement system as a model for innate immunity. We found that serum mediated killing eradicates most of the growing population of UPEC, only the very rapidly growing and the dormant cells, despite being recognized by the complement system, survive the insult. During simultaneous application of serum and various antibiotics from different classes, however, only dormant cells survive as antibiotics result in clearance of the rapidly growing cells. Since the non-growing state in growth supporting environment was protective against both antibiotic treatment and action of the immune system, we next set out to elucidate mechanisms controlling the growth resumption of E. coli. Of several plausible target genes initially studied, the stringent response factor RelA stood out. We found that a culture of stringent response deficient E. coli, i.e. relaxed strain, turned out to be defective in growth resumption rendering cells non-growing for longer periods of time in growth supporting environment. The growth resumption defect of relaxed E. coli was a function of both the amino acid and carbon source composition of the medium. Curiously, compared to wild-type, relaxed strain survived ampicillin treatment better even if the growth resumption of the two strains was equal. As we learned that the stringent response is a key player in growth resuscitation and given its reported importance to bacterial virulence, we set up a high-throughput search for specific inhibitors of the stringent response. If not an immediate value for medicine, we reasoned, such inhibitors would be a powerful tool for studies of bacterial physiology. A screening system was established, it failed to yield the desired compound, but resulted in identification of novel class of antibacterials, derivatives of 4-(6-(phenoxy)alkyl)-3,5-dimethyl-1H-pyrazole

Composition of the outgrowth medium modulates wake-up kinetics and ampicillin sensitivity of stringent and relaxed Escherichia coli

The transition of Escherichia coli from the exponential into the stationary phase of growth induces the stringent response, which is mediated by the rapid accumulation of the alarmone nucleotide (p)ppGpp produced by the enzyme RelA. The significance of RelA's functionality during the transition in the opposite direction, i.e. from the stationary phase into new exponential growth, is less well understood. Here we show that the relaxed strain, i.e. lacking the relA gene, displays a relative delay in regrowth during the new exponential growth phase in comparison with the isogenic wild type strain. The severity of the effect is a function of both the carbon source and amino acid composition of the outgrowth media. As a result, the loss of RelA functionality increases E. coli tolerance to the bactericidal antibiotic ampicillin during growth resumption in fresh media in a medium-specific way. Taken together, our data underscore the crucial role of medium composition and growth conditions for studies of the role of individual genes and regulatory networks in bacterial phenotypic tolerance to antibiotics

HPLC-based quantification of bacterial housekeeping nucleotides and alarmone messengers ppGpp and pppGpp

Here we describe an HPLC-based method to quantify bacterial housekeeping nucleotides and the signaling messengers ppGpp and pppGpp. We have replicated and tested several previously reported HPLC-based approaches and assembled a method that can process 50 samples in three days, thus making kinetically resolved experiments feasible. The method combines cell harvesting by rapid filtration, followed by acid extraction, freeze-drying with chromatographic separation. We use a combination of C18 IPRP-HPLC (GMP unresolved and co-migrating with IMP; GDP and GTP; AMP, ADP and ATP; CTP; UTP) and SAX-HPLC in isocratic mode (ppGpp and pppGpp) with UV detection. The approach is applicable to bacteria without the requirement of metabolic labelling with 32P-labelled radioactive precursors. We applied our method to quantify nucleotide pools in Escherichia coli BW25113 K12-strain both throughout the growth curve and during acute stringent response induced by mupirocin. While ppGpp and pppGpp levels vary drastically (40-and >= 8-fold, respectively) these changes are decoupled from the quotients of the housekeeping pool and guanosine and adenosine housekeeping nucleotides: NTP/NDP/NMP ratio remains stable at 6/1/0.3 during both normal batch culture growth and upon acute amino acid starvation

Auxotrophy-based High Throughput Screening assay for the identification of Bacillus subtilis stringent response inhibitors

The stringent response is a central adaptation mechanism that allows bacteria to adjust their growth and metabolism according to environmental conditions. The functionality of the stringent response is crucial for bacterial virulence, survival during host invasion as well as antibiotic resistance and tolerance. Therefore, specific inhibitors of the stringent response hold great promise as molecular tools for disarming and pacifying bacterial pathogens. By taking advantage of the valine amino acid auxotrophy of the Bacillus subtilis stringent response-deficient strain, we have set up a High Throughput Screening assay for the identification of stringent response inhibitors. By screening 17,500 compounds, we have identified a novel class of antibacterials based on the 4-(6-(phenoxy) alkyl)-3,5-dimethyl-1H-pyrazole core. Detailed characterization of the hit compounds as well as two previously identified promising stringent response inhibitors-a ppGpp-mimic nucleotide Relacin and cationic peptide 1018 - showed that neither of the compounds is sufficiently specific, thus motivating future application of our screening assay to larger and more diverse molecular libraries

Evaluation of the characteristics of leucyl-tRNA synthetase (LeuRS) inhibitor AN3365 in combination with different antibiotic classes

Aminoacyl tRNA synthetases are enzymes involved in the key process of coupling an amino acid to its cognate tRNA. AN3365 is a novel antibiotic that specifically targets leucyl-tRNA synthetase, whose development was halted after evaluation in phase II clinical trials owing to the rapid selection of resistance. In an attempt to bring AN3365 back into the developmental pipeline we have evaluated the efficacy of AN3365 in combination with different classes of antibiotic and characterized its mechanism of action. Although we detect no synergy or antagonism in combination with a range of antibiotic classes, a combination of AN3365 with colistin reduces the accumulation of AN3365-resistant and colistin resistance mutations. We also demonstrate that treatment with AN3365 results in the dramatic accumulation of the alarmone (p)ppGpp, the effector of the stringent response-a key player in antibiotic tolerance

Molecular mutagenesis of ppGpp : turning a RelA activator into an inhibitor

The alarmone nucleotide (p) ppGpp is a key regulator of bacterial metabolism, growth, stress tolerance and virulence, making (p) ppGpp-mediated signaling a promising target for development of antibacterials. Although ppGpp itself is an activator of the ribosome-associated ppGpp synthetase RelA, several ppGpp mimics have been developed as RelA inhibitors. However promising, the currently available ppGpp mimics are relatively inefficient, with IC50 in the sub-mM range. In an attempt to identify a potent and specific inhibitor of RelA capable of abrogating (p) ppGpp production in live bacterial cells, we have tested a targeted nucleotide library using a biochemical test system comprised of purified Escherichia coli components. While none of the compounds fulfilled this aim, the screen has yielded several potentially useful molecular tools for biochemical and structural work

Subinhibitory Concentrations of Bacteriostatic Antibiotics Induce relA-Dependent and relA-Independent Tolerance to beta-Lactams

The nucleotide (p) ppGpp is a key regulator of bacterial metabolism, growth, stress tolerance, and virulence. During amino acid starvation, the Escherichia coli (p) ppGpp synthetase RelA is activated by deacylated tRNA in the ribosomal A-site. An increase in (p) ppGpp is believed to drive the formation of antibiotic-tolerant persister cells, prompting the development of strategies to inhibit (p) ppGpp synthesis. We show that in a biochemical system from purified E. coli components, the antibiotic thiostrepton efficiently inhibits RelA activation by the A-site tRNA. In bacterial cultures, the ribosomal inhibitors thiostrepton, chloramphenicol, and tetracycline all efficiently abolish accumulation of (p) ppGpp induced by the Ile-tRNA synthetase inhibitor mupirocin. This abolishment, however, does not reduce the persister level. In contrast, the combination of dihydrofolate reductase inhibitor trimethoprim with mupirocin, tetracycline, or chloramphenicol leads to ampicillin tolerance. The effect is independent of RelA functionality, specific to beta-lactams, and not observed with the fluoroquinolone norfloxacin. These results refine our understanding of (p) ppGpp's role in antibiotic tolerance and persistence and demonstrate unexpected drug interactions that lead to tolerance to bactericidal antibiotics

The ribosomal A-site finger is crucial for binding and activation of the stringent factor RelA

During amino acid starvation the Escherichia coli stringent response factor RelA recognizes deacylated tRNA in the ribosomal A-site. This interaction activates RelA-mediated synthesis of alarmone nucleotides pppGpp and ppGpp, collectively referred to as (p)ppGpp. These two alarmones are synthesized by addition of a pyrophosphate moiety to the 3' position of the abundant cellular nucleotide GTP and less abundant nucleotide GDP, respectively. Using untagged native RelA we show that allosteric activation of RelA by pppGpp increases the efficiency of GDP conversion to achieve the maximum rate of (p) ppGpp production. Using a panel of ribosomal RNA mutants, we show that the A-site finger structural element of 23S rRNA helix 38 is crucial for RelA binding to the ribosome and consequent activation, and deletion of the element severely compromises (p) ppGpp accumulation in E. coli upon amino acid starvation. Through binding assays and enzymology, we show that E. coli RelA does not form a stable complex with, and is not activated by, deacylated tRNA off the ribosome. This indicates that in the cell, RelA first binds the empty A-site and then recruits tRNA rather than first binding tRNA and then binding the ribosome