Role of the sigma factor algT and its anti-sigma mucA in Pseudomonas aeruginosa

HEDEMANN LG; LOPEZ VA; MOYANO AJ; SMANIA AM

Salta

Congreso; LV Annual SAIB, LIV PABMB 2019; 2019

Pseudomonas aeruginosa is an opportunistic pathogen that chronically infects the airways of cystic fibrosis (CF) patients. In P. aeruginosa, conversion to the mucoid (exopolysaccharide alginate-overproducing) phenotype marks the onset of chronic infection and constitutes a sign of poor prognosis. Mucoid conversion frequently involves the acquisition of mutations in the mucA gene. MucA is an anti-σ factor that negatively regulates alginate production by sequestration of AlgT, an alternative σ factor responsible for the transcription of the alginate biosynthetic operon. Mutations in mucA can also affect Quorum-Sensing (QS) signals, flagellum biosynthesis or survival under anaerobic and osmotic stress conditions, revealing the existence of an extensive regulon controlled by MucA and AlgT, which is not completely understood. The most frequent mutation responsible for mucoid conversion is a deletion of a G residue within a homopolymeric track of five Gs (G5426), also known as mucA22 allele, causing the truncation of MucA C-terminal periplasmic domain. By engineering different mucA alleles, we previously showed that deletion of G5426, although severely reduces mucoid conversion frequency and mucA mutations prevalence, the few remaining mutations that still occur in mucA keep targeting the gene periplasmic coding region , thus leading to truncated versions of MucA equivalent to mucA22. We advance in the knowledge of this regulatory network, by exploring whether MucA truncated proteins can still exert their regulatory function on AlgT. Moreover, we wondered whether MucA has biological roles independent of the known AlgT regulatory pathway. Here, we constructed and characterized a set of mutants containing different combinations of mucA and algT composition, namely, ΔalgT, ΔalgTΔmucA, ΔalgTmucA22, mucA22 and partially deleted mucA mutants. Phenotypic characterizations included alginate overproduction, measurement of NO2? sensitivity under anaerobic conditions, osmotic stress tolerance, and production of acyl-homoserine lactones (AHLs). Overexpression of the mucA22 allele was able to suppress the mucoid phenotype, confirming that it partially maintains its anti-σ regulatory function. Whereas the mucA22 strain showed a mucoid phenotype and AHLs suppression, ΔalgT, ΔalgTΔmucA, and ΔalgTmucA22 remained non-mucoid and showed AHLs wild type levels. The mucA22 strain was highly sensitive to NO2- under anaerobic conditions. Surprisingly, ΔalgT mutants were resistant to NO2?like the wild-type strain. These results suggest that AlgT deregulation might be toxic under certain conditions. On the other hand, unlike the wild-type strain, all mutants failed to grow in high salt media, which suggests that tightly controlled AlgT levels are necessary to achieve tolerance to osmotic stress. Our results shed light on the regulatory pathways underlain mucoid conversion, providing potential targets for future therapeutic strategies to control chronic P. aeruginosa infections.