Chemie | Biochemie | Medizin
Eduard Basler, 2001 | Oberwil, BL
Pathogenic bacteria are subjected to the defense mechanisms of the infected mammalian host. To combat these defense mechanisms, bacteria produce so-called virulence factors. Regulatory mechanisms allow bacteria to conserve energy by expressing the virulence factors only during infection. One mechanism of regulation uses a change in temperature to quickly increase protein translation. At lower temperatures RNA molecules tend to fold into loops, sometimes inhibiting the ribosome binding site (RBS) and thus repressing the translation of the downstream gene. At higher temperatures, i.e., during the infection of a host, these 5’ untranslated regions (5’UTRs) of RNA molecules unfold, revealing the RBS and inducing translation. Such 5’UTRs are called RNA-thermosensors. In my thesis, I analyzed the genome of the pathogen Pseudomonas aeruginosa and identified putative RNA-thermosensors. I then ordered the putative RNA-thermosensors as synthetic DNA fragments and tested their role in regulation of a reporter gene under different temperatures. I was able to identify two novel RNA thermosensors. While discrepancies were found between the in-silico prediction of RNA folding and the measurement of regulation of gene expression, I showed that such an approach can identify novel RNA-thermosensors in P. aeruginosa.
Can novel RNA-thermosensors be identified by combining an in-silico approach with an in-vitro approach?
To find novel putative RNA thermosensors within the genome of P. aeruginosa, I first identified 5’ UTRs within the P. aeruginosa genome and analyzed their predicted 2D-structures under low and high temperatures in silico. The 28 sequences predicted to change the most during a temperature increase were ordered as synthetic DNA fragments and cloned into a plasmid in front of a gene encoding the fluorescent protein mNeonGreen. These plasmids were then transformed into competent E. coli bacteria. The E. coli bacteria were then grown at 30 °C for 5.5 hours, at 37 °C for 1.5 hours followed by an increase to 42 °C for 4 hours and for 5.5 hours at a constant temperatures of 30 °C, 35 °C, and 40 °C. During the incubations, bacterial growth and the level of expression of the fluorescent protein mNeonGreen was measured for each clone. To identify whether the RNA-thermosensors functioned as predicted, the expression levels of mNeonGreen, were normalized to the measured bacterial growth. These values were then compared between the different temperatures to identify sequences that specifically regulated level of translation upon temperature increase.
25 predicted RNA thermosensors failed to regulate expression of the reporter gene, however, three sequences encoding putative RNA-thermosensors were identified to induce gene expression. Two of these performed as predicted in silico. The first one was the 5’UTR of the gene PA1240 encoding an enzyme essential for metabolizing fatty acids to produce energy in the form of ATP. The second one was the 5’UTR of the gene PA2713 encoding a protein with an unknown function with a predicted DNA binding domain.
The 5’UTR of the gene PA1240 is a putative RNA-thermosensor regulating a gene that might be relevant in metabolizing lipids present in lung fluid. This might be interesting as it suggests a link between the production of an enzyme needed to produce ATP and a rise in temperature i.e. infection of a host. The 5’UTR of the gene PA2713 is a putative RNA-thermosensor regulating a predicted DNA binding domain possibly involved in the adaptation to host conditions. An RNA-thermosensor regulating a transcriptional regulator has previously been identified in E. coli.
In my thesis, I showed that a combination of in-silico and in-vitro approach can identify novel putative RNA-thermosensors in P. aeruginosa. While some discrepancies were found between the in-silico prediction of RNA folding and the measurement of regulation of gene expression of the same sequence, several putative RNA thermosensors behaved as predicted in silico. I am confident that with additional experiments I could further expand the analysis of the identified sequences and confirm that some regulate protein translation in P. aeruginosa. Additionally, the method used in my thesis could also be applicable for finding RNA-thermosensors in other bacterial pathogens and could thus potentially point to genes that are needed under increased temperature in the host. Understanding the needs of bacterial pathogens during infection may help to identify novel targets for therapy.
Würdigung durch den Experten
Dr. Tim Roloff Handschin
Eduard Basler verwendet in seiner Arbeit eine bioinformatische Analyse um funktionelle RNA Thermosensoren im pathogenen Bakterium Pseudomonas aeruginosa vorherzusagen und diese anschliessend mit Kontrollexperimenten im Labor zu untersuchen. Trotz technischer Herausforderungen gelang es ihm funktionelle RNA Thermosensoren zu beschreiben und im Labor zu validieren. Diese Methode ist zudem zukünftig auf andere Bakterien anwendbar. Ein besseres Verständnis von RNA Thermosensoren eröffnet neue Möglichkeiten die Antibiotika-Krise zu bekämpfen, was diese Arbeit hoch interessant und relevant macht.
Sonderpreis Gamil Stiftung – Sail & Explore
Lehrer: Dr. Samuel Zschokke