Ribosome-targeting antibiotics and mechanisms of antibiotic resistance
Date
2017-04-17
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Abstract
Valgusüntees võib olla reguleeritud polüpeptiidahela poolt, mida ribosoom parajasti sünteesib. Teatud peptiidide transleerimine põhjustab ribosoomi seiskumist, mis omakorda takistab allavoolu paiknevate geenide ekspresseerumist. Teatud juhtudel vajatakse seisaku toimumiseks väikese ligandmolekuli (antibiootikum, aminohape vms) juuresolekut. Tuntakse mitmeid ribosoomi seisakut põhjustavaid peptiide, kuid nende võrdlemisel pole leitud ühtset konsensusjärjestust. Samas võib oletada, et reeglite tuvastamine on võimalik, kui analüüsida suuremat hulka peptiide. Seetõttu töötasime välja selektsioonimeetodi ribosoomi peatavate peptiide leidmiseks.
Oma meetodit kasutades identifitseerisime järjestused, mis peatasid translatsiooni erütromütsiini, troleandomütsiini, klooramfenikooli, meta-toluaadi või homoseriinlaktooni juuresolekul. Need järjestused olid aktiivsed peptiidi tasemel. Me ei tuvastanud peptiidide aminohappeliste järjestuste võrdlemisel universaalset konsensusjärjestust. Siiski märkasime teatud seaduspärasusi. Näiteks selgus, et erütromütsiini juuresolekul toimuv ribosoomi seiskumine vajab eelistatult hüdrofoobset kasvavat peptiidi. On võimalik, et ribosoomi peatumist põhjustavate järjestusmotiivide leidmiseks tuleb analüüsida suuremat peptiidide valimit. Samuti avastasime, et seisakut põhjustavate peptiidide funktsionaalsus võib olla mõjutatud vastava mRNA omaduste poolt.
Meie meetod võib olla rakendatav biotehnoloogias. Teoreetiliselt võiks ribosoomi peatumist põhjustavaid peptiide kasutada selleks, et luua uudseid geeniekspressiooni süsteeme, milles regulatsioon toimub translatsiooni tasemel.
Väitekirja teine publikatsioon kirjeldab antibiootikumiresistentsuse mehhanismi, mis toimub ribsoomi kaitsevalkude vahendusel ja tagab resistentsuse tetratsükliinile. Töö aluseks oli ribosoomi ja kaitsevalgu Tet(O) kompleksi atomaarne mudel. Struktuuriuuringud näitasid, et Tet(O) interakteerub nii väikese kui suure ribosoomi alaühikuga. Me tegime asendus- ja deletsioonimutatsioone Tet(O) domään IV lingudesse 465, 438 ja 507, mis mudeli kohaselt interakteeruvad otseselt tetratsükliini seondumiskohaga või asuvad selle lähedal. Mutantsete ja metsik-tüüpi Tet(O) variantide analüüs näitas, et kõik kolm lingu on Tet(O) tööks hädavajalikud. Asendusmutatsioonid lingudes vähendasid Tet(O) aktiivsust ja deletsioonid kaotasid aktiivsuse täielikult. Struktuursete uuringute ja mutatsioonanalüüsi tulemusi kõrvutades pakkusime välja mudeli Tet(O) vahendusel toimuva tetratsükliiniresistentsuse mehhanismi selgitamiseks. Meie arvates muudab Tet(O) ling 465 tetratsükliini seondumiskoha juures 16S rRNA struktuuri. See nõrgendab interaktsiooni tetratsükliini molekuli ja 16S rRNA vahel ning võimaldab lingul 507 ravim ribosoomilt eemale tõugata. Me oletasime, et ling 438 koos 16S rRNA nukleotiididega moodustab kanali, mille kaudu tetratsükliin ribosoomilt lahkub.
Väitekirja kolmas publikatsioon puudutab probleeme, mis on seotud uute turule ilmuvate antibiootikumidega. Nende hulgas leidub ravimeid, mille toimemehhanism pole veel täiesti selge. Üheks selliseks näiteks on nitrovinüülfuraanide hulka kuuluv G1 ehk Furvina®.
G1 laguneb vees ja tioolrühmi sisaldavate ühendite (näiteks tsüsteiini) olemasolu keskkonnas kiirendab seda protsessi. Tuumamagnetresonantsi ja kõrgefektiivse vedelikukromatograafia meetodeid kasutades selgitasime välja G1 lagunemise reaktsiooniskeemi ja selle käigus tekkivad ühendid. Leidsime, et G1 antibakteriaalne toime põhineb valkudes sisalduvate tioolrühmade modifitseerimisel.
On teada, et vähemalt mõned G1 lagunemise käigus tekkivatest ühenditest säilitavad antimikroobse aktiivsuse. Selleks, et teha vahet G1 ja selle laguproduktide põhjustatud antimikroobsetel efektidel, inkubeerisime uuritavat ainet enne antimikroobse aktiivsuse mõõtmist erinevates vedelsöötmetes. Selgus, et tsüsteiinivabas söötmes on G1 antimikroobne võimekus suurem kui tioolrühmi sisaldavas söötmes. Samas jäid antimikroobsed omadused alles isegi pärast kahe tunni pikkust testile eelnenud inkubatsiooniaega. Me järeldame, et G1 aktiivsus on aine kohese reaktiivsuse ja laguproduktide antimikroobsete aktiivsuste summa. Tulemused näitavad, et G1 võib sobida jagunevate bakterirakkude kasvu takistamiseks, kuid ei ole efektiivne vahend mittejagunevate bakterite vastu.
Protein synthesis can be regulated by the nascent polypeptide chain currently translated by the ribosome. Translation of certain peptides causes ribosome stalling and, thus, inhibits expression of downstream genes. In some cases, stalling requires the presence of a small ligand molecule (an antibiotic, an amino acid, etc). Several peptides are known that can induce ribosome stalling, but their comparison has not revealed a common consensus motif. However, it can be assumed that the identification of rules is possible by analysing a larger number of peptides. Therefore, we developed a selection method for identifying peptides capable of stalling the ribosome. By using our method, we identified sequences that stalled translation in response to erythromycin, troleandomycin, chloramphenicol, meta-toluate or homoserine lactone. These sequences were active on the peptide level. Comparison of amino acid sequences of peptides did not reveal any universal consensus motif. However, we noticed certain tendencies. For instance, it was found that ribosome stalling in the presence of erythromycin preferably requires a hydrophobic nascent peptide. We also discovered that the functionality of stalling peptides can be affected by properties of corresponding mRNA. Our method may be applicable in biotechnology. In theory, stalling peptides could be used for designing novel gene expression systems that are regulated at the level of translation. The second publication of dissertation describes the mechanism of antibiotic resistance that is mediated by ribosome protection proteins and confers resistance to tetracycline. The work was based on the atomic model of the complex of ribosome and protection protein Tet(O). Structural studies showed that Tet(O) interacts with both small and large ribosomal subunit. We introduced substitution and deletion mutations into Tet(O) domain IV loops 465, 438 and 507 which, according to the model, interact directly with the tetracycline binding site or are located in the vicinity. Analysis of mutant and wild-type Tet(O) variants showed that all three loops are essential for Tet(O) function. Substitution mutations reduced the activity of Tet(O) and deletions abolished the activity completely. Considering the results of structural studies and mutation analysis, the model of Tet(O)-mediated tetracycline resistance mechanism was proposed. We suggest that the loop 465 of Tet(O) distorts the backbone shape of the 16S rRNA at the tetracycline-binding site. This weakens the interaction between the tetracycline molecule and 16S rRNA and enables the 507-loop to dislodge the drug from the ribosome. We hypothesized that the 438-loop along with nucleotides of 16S rRNA forms a corridor allowing tetracycline to leave the ribosome. The third part of thesis concerns problems that are related to new antibiotics emerging on the market. Among them, there are drugs whose mechanism of action is not yet completely understood. One such example is a nitrovinylfuran derivative G1, also known as Furvina®. G1 decomposes in aqueous media and the presence of thiol-containing compounds (e.g. cysteine) accelerates this process. Nuclear magnetic resonance and high performance liquid chromatography were used to clarify the reaction scheme of G1 decomposition along with the decomposition products. We found that the antibacterial effect of G1 is based on the modification of thiol groups present in proteins. It is known that at least some of G1 decomposition products retain antimicrobial activity. In order to distinguish between the effects of G1 and its conversion products, we preincubated the test substance in different liquid growth media prior to measurement of microbial activity. We found that the antimicrobial capability of G1 is greater in cysteine-free medium when compared to media containing thiol groups. However, the antimicrobial properties were not lost even after two hours of preincubation. We conclude that the activity of G1 is a sum of its immediate reactivity and effects of its breakdown products. Our data suggest that G1 can be suitable for preventing the growth of proliferating bacterial cells, but it is not an effective tool against non-growing bacteria.
Protein synthesis can be regulated by the nascent polypeptide chain currently translated by the ribosome. Translation of certain peptides causes ribosome stalling and, thus, inhibits expression of downstream genes. In some cases, stalling requires the presence of a small ligand molecule (an antibiotic, an amino acid, etc). Several peptides are known that can induce ribosome stalling, but their comparison has not revealed a common consensus motif. However, it can be assumed that the identification of rules is possible by analysing a larger number of peptides. Therefore, we developed a selection method for identifying peptides capable of stalling the ribosome. By using our method, we identified sequences that stalled translation in response to erythromycin, troleandomycin, chloramphenicol, meta-toluate or homoserine lactone. These sequences were active on the peptide level. Comparison of amino acid sequences of peptides did not reveal any universal consensus motif. However, we noticed certain tendencies. For instance, it was found that ribosome stalling in the presence of erythromycin preferably requires a hydrophobic nascent peptide. We also discovered that the functionality of stalling peptides can be affected by properties of corresponding mRNA. Our method may be applicable in biotechnology. In theory, stalling peptides could be used for designing novel gene expression systems that are regulated at the level of translation. The second publication of dissertation describes the mechanism of antibiotic resistance that is mediated by ribosome protection proteins and confers resistance to tetracycline. The work was based on the atomic model of the complex of ribosome and protection protein Tet(O). Structural studies showed that Tet(O) interacts with both small and large ribosomal subunit. We introduced substitution and deletion mutations into Tet(O) domain IV loops 465, 438 and 507 which, according to the model, interact directly with the tetracycline binding site or are located in the vicinity. Analysis of mutant and wild-type Tet(O) variants showed that all three loops are essential for Tet(O) function. Substitution mutations reduced the activity of Tet(O) and deletions abolished the activity completely. Considering the results of structural studies and mutation analysis, the model of Tet(O)-mediated tetracycline resistance mechanism was proposed. We suggest that the loop 465 of Tet(O) distorts the backbone shape of the 16S rRNA at the tetracycline-binding site. This weakens the interaction between the tetracycline molecule and 16S rRNA and enables the 507-loop to dislodge the drug from the ribosome. We hypothesized that the 438-loop along with nucleotides of 16S rRNA forms a corridor allowing tetracycline to leave the ribosome. The third part of thesis concerns problems that are related to new antibiotics emerging on the market. Among them, there are drugs whose mechanism of action is not yet completely understood. One such example is a nitrovinylfuran derivative G1, also known as Furvina®. G1 decomposes in aqueous media and the presence of thiol-containing compounds (e.g. cysteine) accelerates this process. Nuclear magnetic resonance and high performance liquid chromatography were used to clarify the reaction scheme of G1 decomposition along with the decomposition products. We found that the antibacterial effect of G1 is based on the modification of thiol groups present in proteins. It is known that at least some of G1 decomposition products retain antimicrobial activity. In order to distinguish between the effects of G1 and its conversion products, we preincubated the test substance in different liquid growth media prior to measurement of microbial activity. We found that the antimicrobial capability of G1 is greater in cysteine-free medium when compared to media containing thiol groups. However, the antimicrobial properties were not lost even after two hours of preincubation. We conclude that the activity of G1 is a sum of its immediate reactivity and effects of its breakdown products. Our data suggest that G1 can be suitable for preventing the growth of proliferating bacterial cells, but it is not an effective tool against non-growing bacteria.
Description
Väitekirja elektrooniline versioon ei sisalda publikatsioone
Keywords
ribosoomid, antibiootikumid, ravimiresistentsus, ribosomes, antibiotics, drug resistance