Identification of Pseudomonas aeruginosa genetic determinants that connect redox metabolism to alginate biosynthesis

Pseudomonas aeruginosa is a well-known human pathogen that contributes significantly to chronic infections, particularly in cystic fibrosis (CF) patients. During this chronic infection, P. aeruginosa undergoes a phenotype change, the inactivation of mucA, which leads to the production of exopolysaccharide alginate, known as mucoid, a key virulence factor associated with biofilm formation. This mucoid phenotype allows the bacterium to persist in the lungs of CF patients for the duration of their lives. Previously, we identified ebselen oxide (EbO) as an inhibitor that suppresses alginate production in P. aeruginosa. In the current study, we synthesized a series of structural analogs based on EbO and ebselen (Eb) and evaluated their ability to inhibit alginate production. These analogs did have similar or lower inhibitory activity than EbO. The mechanism by which EbO inhibits alginate production remains unclear. We employed RNA sequencing analysis of P. aeruginosa treated with inhibitors and identified several candidate genes potentially involved in this inhibitory pathway. Interestingly, we observed that a transposon and in-frame deletion mutants of the candidate genes were defective for alginate production. These findings suggest there are additional requirements for optimal alginate production in conditions that mimic the CF lung beyond the algD-A operon. IMPORTANCEWhen bacteria encounter the correct conditions, they can dedicate their energy toward a specific function to maximize the function. One example is the low calcium response in Yersinia pestis in which the bacteria arrest growth when grown at 37 {degrees}C in the absence of calcium because it uses all its energy for type III secretion. Another example is production of alginate by P. aeruginosa in the lungs of CF patients that can lead to occlusion of the airways. In both cases, the dedicated use of energy toward type III secretion for Y. pestis and alginate biosynthesis for P. aeruginosa reduces the ability of the bacteria to multiply. In the lab, suppressors can be easily identified that restore bacteria growth. The suppressor mutations are often located in the operons that are up-regulated and thereby prevent the execution of the energetically costly process. While these results indicate these processes are energetically costly, we still do not understand how the bacteria dedicate their energy toward these processes over other cellular processes such as growth. Previously, we identified ebselen oxide (EbO) as an inhibitor that suppresses alginate production in P. aeruginosa, but chemical analogues fail to improve the inhibitory activity. We used RNA sequencing analysis of P. aeruginosa treated with inhibitors and identified several candidate genes potentially involved in this inhibitory pathway. Interestingly, we observed that a transposon and in-frame deletion mutants of these genes involved in redox reactions were defective for alginate production. These findings suggest there proteins may shunt energy for optimal alginate production in conditions that mimic the CF lung beyond the algD-A operon. Results from P. aeruginosa alginate production may inform how other bacteria can similarly focus energy toward specific cellular processes.