Researchers from the University of Geneva (UNIGE), Switzerland, say they have identified a previously unknown regulator of gene expression in this bacterium Pseudomonas aeruginosa, the absence of which significantly reduces the infectious power of P. aeruginosa and its dangerous nature. The findings could constitute an innovative target in the fight against this pathogenic bacterium.
P. aeruginosa is an opportunistic microbe present in many ecological niches, such as plant roots, stagnant water or even the pipes of our homes. Naturally versatile, it can cause acute and chronic infections that are potentially fatal for people with weakened immune systems.
The presence of P. aeruginosa in clinical settings, where it can colonize on respirators and catheters, is a serious threat. In addition, its adaptability and resistance to many antibiotics make infections by P. aeruginosa increasingly difficult to treat.
The UNIGE team published its study (“The DEAD-box RNA helicase RhlE2 is a global regulator of Pseudomonas aeruginosa lifestyle and pathogenesis”) in Nucleic Acids Research.
“RNA helicases perform essential housekeeping and regulatory functions in all domains of life by binding and unwinding RNA molecules. The bacterial RhlE-like DEAD-box RNA helicases are among the least well studied of these enzymes. They are widespread especially among Proteobacteria, whose genomes often encode multiple homologs. The significance of the expansion and diversification of RhlE-like proteins for bacterial fitness has not yet been established. Here, we study the two RhlE homologs present in the opportunistic pathogen Pseudomonas aeruginosa,” write the investigators.
“We show that, in the course of evolution, RhlE1 and RhlE2 have diverged in their biological functions, molecular partners and RNA-dependent enzymatic activities. Whereas RhlE1 is mainly needed for growth in the cold, RhlE2 also acts as global post-transcriptional regulator, affecting the level of hundreds of cellular transcripts indispensable for both environmental adaptation and virulence. The global impact of RhlE2 is mediated by its unique C-terminal extension, which supports the RNA unwinding activity of the N-terminal domain as well as an RNA-dependent interaction with the RNase E endonuclease and the cellular RNA degradation machinery.
“Overall, our work reveals how the functional and molecular divergence between two homologous RNA helicases can contribute to bacterial fitness and pathogenesis.
RNA helicases are present in the genomes of almost all known living organisms, including bacteria, yeast, plants, and humans. However, they have acquired specific properties depending on the organism in which they are found.
“Pseudomonas aeruginosa has an RNA helicase whose function was unknown, but which was found in other pathogens,” explains Martina Valentini, PhD, a researcher leading this research in the department of microbiology and molecular medicine at UNIGE Faculty of Medicine, and holder of an SNSF Ambizione grant. “We wanted to understand what its role was, in particular in relation to the pathogenesis of the bacteria and their environmental adaptation.”
A severely reduced virulence
To do this, the Geneva team combined biochemical and molecular genetic approaches to determine the function of this protein.
“In the absence of this RNA helicase, P. aeruginosa multiplies normally in vitro, both in a liquid medium and on a semi-solid medium at 37°C,” reports Stéphane Hausmann, a researcher associate in the department of microbiology and molecular medicine at UNIGE Faculty of Medicine and first author of this study. “To determine whether the infection capacity of the bacteria was affected, we had to observe it in vivo in a living organism.”
The scientists then continued their research using Galleria mellonella larvae, a model insect for studying host-pathogen interactions. Indeed, the innate immune system of insects has important similarities with that of mammals. Moreover, these larvae can live at temperatures between 5°C and 45°C, which makes it possible to study bacterial growth at different temperatures, including that of the human body.
Three groups of larvae were observed; the first, after injection of a saline solution, saw 100% of its population survive. In the presence of a normal strain of P. aeruginosa, less than 20% survived at 20 hours after infection. In contrast, when P. aeruginosa no longer possessed the RNA helicase gene, over 90% of the larvae remained alive.
“The modified bacteria became almost harmless, while remaining very much alive,” notes Hausmann.
The results of this work show that this regulator affects the production of several virulence factors in the bacteria.
“In fact, this protein controls the degradation of numerous messenger RNAs coding for virulence factors,” continues Valentini. “From an antimicrobial drug strategy point of view, switching off the pathogen’s virulence factors rather than trying to eliminate the pathogen completely, means allowing the host immune system to naturally neutralize the bacterium and potentially reduces the risk for the development of resistance. Indeed, if we try to kill the bacteria at all costs, the bacteria will adapt to survive, which favors the appearance of resistant strains.”
The Geneva team is currently continuing its work by screening a series of known drug molecules in order to determine whether any of them have the capacity to selectively block this protein, and to study in detail the inhibition mechanisms on which the development of an effective therapeutic strategy could be based.