Chemie | Biochemie | Medizin
Gabriela Basler, 2004 | Oberwil, BL
Bacteria are highly adaptable single-cell organisms, which developed mechanisms to allow them to thrive in diverse environmental conditions. This includes their ability to adapt to varying concentrations of essential elements like iron. The human pathogen P. aeruginosa infects lungs of cystic fibrosis patients and is becoming increasingly resistant to antibiotics. The pathogen uses siderophores and iron uptake systems to acquire iron from the environment or the host. Because iron is unavailable for pathogens in the host it signals that the pathogen needs to acquire iron and turn on virulence factors. Therefore, analysis of iron regulated genes can also reveal genes required for virulence. My thesis aimed to identify iron-responsive genes in P. aeruginosa. Computational analysis identified putative iron regulated promoters, which were then cloned into reporter plasmids. Twelve out of twenty-four promoters significantly changed gene expression in response to iron, including three previously uncharacterized promoters. My thesis combines computational analysis and experimental testing, revealing both known and novel iron-regulated genes in P. aeruginosa. Understanding the iron regulation offers potential to expand therapeutic approaches targeting this human pathogen.
Introduction
Which genes in the human pathogen Pseudomonas aeruginosa change their expression level in response to iron availability?
Methods
The genome of P. aeruginosa was analyzed computationally to identify iron-responsive genes by searching for known repressor binding sequences. Twenty-four iron-regulated genes were identified and cloned by into a reporter plasmid with the green fluorescent protein (GFP). These plasmids were then transformed into P. aeruginosa and GFP expression was measured over time at various iron availability. The significance of the candidate iron regulated genes was then further investigated using mutants of P. aeruginosa lacking the corresponding gene in order to verify their contribution to growth under limited iron availability.
Results
The promoters regulated by iron showed lower GFP expression in high iron concentrations than in low iron concentrations (repression in presence of iron) . Out of the twenty-four promoters, nine were previously known to be regulated by iron. I was also able to show the iron regulation of three genes, including tesG , which were not previously shown to be regulated by iron. Importantly, tesG is known to encode a virulence factor. I was also able to show that PA0976.1, which encodes a tRNA of lysine, is regulated by iron. The function of the newly discovered iron regulated gene PA2221 is unknown.
Discussion
The gene tesG which expresses a virulence factor is regulated by iron. When P. aeruginosa is outside the host, in environments where iron concentrations are high, the gene is not expressed. On the other hand, when P. aeruginosa is in the host, where iron concentrations are low, the gene is expressed. This gene may therefore be an interesting target for further experiments as it is known that this gene plays an important role in the pathogenicity of P. aeruginosa. My results also show the iron regulation of the gene PA0976.1, which is known to encode the tRNA of lysine. This suggests that the tRNA-Lys might not be produced in iron-rich media. However, tRNA-Lys is essential for the incorporation of lysine into proteins and therefore it is possible that the second gene encoding tRNA-Lys is expressed more to compensate .
Conclusions
I was able to show that the gene tesG, which was previously shown to encode a virulence factor, is regulated by iron and thus could be an interesting subject of further experiments. My data also show that it is necessary to confirm the in-silico analysis by experimentation because twelve genes predicted to be repressed by iron showed no difference in expression in response to iron concentration. On a broader scale, my findings provide a better understanding of how P. aeruginosa adapts to varying iron levels. This knowledge can provide new directions in the fight of P. aeruginosa and therefore help patients suffering from infections.
Würdigung durch den Experten
Dr. Enea Maffei
In her study, Gabriela investigated how pathogens detect and respond to iron deficiency, a crucial factor in infections. Through in silico analysis and carefully conducted experiments, Gabriela provided compelling insights into P. aeruginosa’s strategies in responding to varying iron levels. Particularly noteworthy is Gabriela’s discovery of novel genes responsive to iron scarcity, including an established virulence factor. This underscores the significance of fundamental research in guiding the discovery of novel anti-bacterial strategies.
Prädikat:
hervorragend
Sonderpreis «London International Youth Science Forum (LIYSF)» gestiftet von der Metrohm Stiftung
Gymnasium Oberwil
Lehrer: Dr. Samuel Zschokke
