The emergence and global dissemination of antimicrobial resistant pathogens is one of the most serious global public health threats in the 21st century. Due to the limited success in developing novel antibiotics, alternative therapeutic approaches such as antisense antimicrobial therapies are being explored as promising strategies to address bacterial resistance. To achieve this goal, it will be crucial to understand how bacteria regulate gene expression and resist antimicrobial treatments. This involves accessing their regulatory networks, in which small noncoding RNAs (sRNAs) play a prominent role.

Bacterial sRNAs, as an important class of posttranscriptional regulators, play critical roles in influencing various biological processes, including antibiotic resistance. These regulators enable rapid responses to fluctuating environmental conditions and antibiotic exposure by altering mRNA accessibility or degradation. While several sRNAs in certain pathogens have been demonstrated to influence intrinsic antibiotic resistance mechanisms by affecting processes such as antibiotic uptake, cell wall synthesis, drug efflux, and biofilm formation, our understanding of riboregulatory sRNAs and their specific pathways in other pathogens, such as those within the Burkholderia cepacia complex (Bcc), is still limited.

Bcc bacteria are a group of opportunistic pathogens that are particularly feared among the cystic fibrosis (CF) community due to their resistance to multiple antibiotics, and the unpredictable outcome of infections, ranging from asymptomatic cases to the cepacia syndrome, a fast and fatal necrotizing pneumonia often associated with septicemia. Currently there is no evidence of an effective eradication protocol for Bcc bacteria, and some combinations of antibiotics are becoming less effective against these pathogens.

Recently, it was demonstrated that the B. cenocepacia susceptibility to antimicrobials is modulated by the acidic pH and the CF nutritional environment, suggesting that Bcc resistance to antibiotics is dependent on gene expression regulation.

Therefore, this exploratory project aims to identify and characterize the regulatory sRNAs involved in Bcc antibiotic resistance under clinically relevant conditions, and to assess their regulatory networks to find potential targets for RNA-based antimicrobial strategies against Bcc infections.

To achieve this goal, we will leverage the expertise and experience of team members in the identification and characterization of sRNAs from B. cenocepacia, as well as in the study of the targetome of three important RNA-binding proteins in these bacteria: the chaperones Hfq, Hfq2, and ProQ.

To identify sRNAs that modulate antibiotic resistance in B. cenocepacia, the expression profile of the 264 sRNAs already identified in B. cenocepacia will be accessed under conditions that mimic the acidic CF nutritional environment and with subinhibitory concentrations of clinically relevant antibiotics. The differentially expressed sRNAs, whose predicted targets include genes involved in antimicrobial resistance and are conserved only among B. cenocepacia or among Bcc species, will be selected. These sRNAs will be overexpressed and silenced in B. cenocepacia using appropriate plasmids, and the effect of this silencing or overexpression on B. cenocepacia resistance to certain antibiotics will be tested in vitro using minimum inhibitory concentration assays (MICs). The sRNAs that exhibit an effect on the intrinsic drug resistance of B. cenocepacia will be selected, and the global transcriptomic changes induced by their altered expression will be assessed using RNA-sequencing.

The data obtained will enable the prediction of the differentially expressed targets of each sRNA, as well as the exploration of their regulatory networks under the various conditions tested. This knowledge will be crucial for the subsequent development of an antisense therapy against Bcc infections. The impact of manipulating the selected sRNAs on the efficacy of antibiotic treatments will finally be assessed in vivo using the larva of the greater wax moth Galleria mellonella.

Overall, this project will not only contribute to identifying sRNAs involved in Bcc resistance to antibiotics, but also to deciphering the antibiotic-responsive sRNA networks. This will provide an opportunity to enhance the efficacy of existing antibiotics against Bcc bacteria by silencing resistance genes in combined therapy, as well as to improve antibiotic efficacy by manipulating the levels of sRNAs involved in resistance. The rational development of novel strategies to combat infections caused by Bcc bacteria in patients suffering from Cystic Fibrosis and other diseases is aligned with the 2030 Agenda and its sustainable development goals for health.