The effects of mutations on ribosomal A site on Sgm and RmtB methyltransferase activity

Aminoglikozidi se koriste u liječenju raznih infekcija uzrokovanih najčešće gram-negativnim aerobnim bakterijama.No, u današnje vrijeme javlja se problem sve češće pojave rezistencije na antibiotike. Jedan od mehanizama rezistencije na aminoglikozide je modifikacija veznog mjesta djelovanjem specifičnih metil-transferaza – enzima kojisu se najprije razvili kod bakterija vrsta Streptomices i Micromonospora koje su prirodni prizvođači aminoglikozidnih antibiotika. Zadnje desetljeće dolazi do sve češće pojave srodnih metil-transferaza i kod patogenih sojeva. Mehanizam djelovanja metil-transferaza je metilacija A-mjesta u ribosomskoj podjedinici 30S, mjesta vezanja aminoglikozidnih antibiotika. Dva su poznata mjesta djelovanja ovih enzima: metilacija G1405 na položaju N7 i A1408 na položaju N1. Metil-transferaze proučavane u ovom diplomskom radu su Sgm iz bakterije Micromonospora zionensis, prirodnog proizvođača antibiotika i RmtB iz patogena Serratia marcescens. Oba enzima modificiraju N7 položaj G1405 i ostvaruju rezistenciju bakterija na 4,6-2-deoksistreptaminske aminoglikozidne antibiotike. Cilj rada bio je proučiti utjecaj mutacija u A-mjestu 16S rRNA na aktivnost metil-transferaza i njihovu sposobnost metilacije G1405 te na taj način dobiti uvid u prirodu vezanja enzima na ciljno mjesto djelovanja. U laboratoriju su uzgojeni sojevi bakterijeEscherichia coli MC338 sa 16S rRNA divljeg tipa ili s pojedinačnim mutacijama U1406A, A1408G, G1491C ili U1495A. Sojevi su eksprimirali enzime Sgm ili RmtB. Nakon izolacije rRNA iz E. coli provedena je reakcija produljenja početnice reverznom transkriptazom na uzorcima prethodno tretiranim anilinom i natrijevim borhidridom za detekciju modifikacije m7G1405. Dobiveni produkti analizirani su elektroforezom u 9% denaturirajućem poliakrilamidnom gelu.Rezultati upućuju na to da bi za vezanje enzima metil-transferaze Sgm mogao biti važan nukleotid A1408, dok se za RmtB pokazao važniji nukleotid U1406. Rezultati upućuju i na činjenicu da bi Sgm izoliran iz proizvođača antibiotika, bakterije M. zionensis i RmtB iz patogenog soja S. marcescens mogli imati različito mjesto vezanja na molekuli 16S rRNA i s njom stupati u različite interakcije. Ovaj rad daje doprinos u proučavanju rezistencije na aminoglikozidne antibiotike putem ribosomskih metil-transferaza, kao i proučavanju bakterijske 16S rRNA i evolucijski očuvanog dekodirajućeg mjesta na ribosomu. Uvid u interakcije između metiltransferaza Sgm i RmtB i podjedinice 30S daju temelj za nastavak istraživanja koja će omogućiti razvoj specifičnog inhibitora ovih enzima i suzbiti bakterijsku rezistenciju na aminoglikozidne antibiotike.Aminoglycosides are used for treatment of various infections usually caused by aerobic gram-negative bacteria. However, the successful use of antibiotics is hampered by the growing worldwide problem of bacterial resistance to antibiotics. One of the mechanisms of resistance to aminoglycoside antibiotics is modification of the target site via 16S rRNA methyltransferase enzymes that were initially found in the Streptomyces and Micromonospora species, which are natural producers of aminoglycoside antibiotics. During the last decade similar enzymes have emerged and are rapidly spreading even in bacterial pathogens. Mechanism of action of aminogycoside resistance rRNA methyltransferases is a modification (methylation) of the 30S subunit of the ribosome, the site where aminoacyl-tRNA attaches to ribosome and the binding site of aminoglycoside antibiotics. There are two known sites of action for these enzymes in 16S rRNA: N7 position of nucleotide G1405 and N1 position of nucleotide A1408. Methyltransferases used in this work are Sgm from Micromonospora zionensis, natural antibiotic producer and RmtB from the pathogen Serratia marcescens. Both enzymes modify N7 position of G1405, and thus generate bacterial resistance to 4, 6 - 2-deoxystreptamine aminoglycoside antibiotics. The aim of this work was to study the effect of mutations in the 16SrRNA molecule on the methyltransferase activity and on their ability to modify G1405, thus gaining an insight into the nature of the enzyme binding to its target site. The RNA was isolated from Escherichia coli MC338 expressing methyltransferases Sgm or RmtB with either wild type 16S rRNA or mutant versions with single mutations U1406A, A1408G, G1491C or U1495A. For detection of m7G1405 rRNA samples were pretreated with aniline and sodium borhidride and then subjected to a reverse transcriptase primer extension reaction. Obtained products were analyzed by electrophoresis in 9% denaturing polyacrylamide gel. The results indicate that nucleotide A1408 could be important for binding of the Sgm methyltransferase, while the important nucleotide for RmtB binding is U1406. Results also suggest that the Sgm isolated from antibiotic-producing bacteria M. zionensis and RmtB from pathogenic strains S. Marcescens could have a different bindingpattern to 16S rRNA molecules and hence different interactions with 16S rRNA. This work provides a contribution to the study of aminoglycoside resistance methyltransferases, as well as to the study of bacterial 16S rRNA and evolutionarily conserved decoding site of prokaryotic ribosome. The insight into the Sgm and RmtB interaction with the 30S subunit will support further studies that could contribute to the development of specific inhibitors that would block bacterial resistance to aminoglycoside antibiotics