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Abstract
Alginate overproduction, or mucoidy, plays an important role in the pathogenesis of P. aeruginosa lung infection in cystic fibrosis (CF). Mucoid strains with mucA mutations predominantly populate in chronically-infected patients. However, the mucoid strains can revert to nonmucoidy in vitro through suppressor mutations. We screened a mariner transposon library using CF149, a non-mucoid clinical isolate with a misssense mutation in algU (AlgUA61V). The wild type AlgU is a stress-related sigma factor that activates transcription of alginate biosynthesis. Three mucoid mutants were identified with transposon insertions that caused 1) an overexpression of AlgUA61V, 2) an overexpression of the stringent starvation protein A (SspA), and 3) a reduced expression of the major sigma factor RpoD (σ70). Induction of AlgUA61V in trans caused conversion to mucoidy in CF149 and PAO1DalgU, suggesting that AlgUA61V is functional in activating alginate production. Furthermore, the level of AlgUA61V was increased in all three mutants relative to CF149. However, compared to the wild type AlgU, AlgUA61V had a reduced activity in promoting alginate production in PAO1ΔalgU. SspA and three other anti-σ70 orthologues, P. aeruginosa AlgQ, E. coli Rsd, and T4 phage AsiA, all induced mucoidy, suggesting that reducing activity of RpoD is linked to mucoid conversion in CF149. Conversely, RpoD overexpression resulted in suppression of mucoidy in all mucoid strains tested, indicating that sigma factor competition can regulate mucoidy. Additionally, an RpoD-dependent promoter (PssrA) was more active in non-mucoid strains than in isogenic mucoid variants. Altogether, our results indicate that the anti-σ70 factors can induce conversion to mucoidy in P. aeruginosa CF149 with algU-suppressor mutation via modulation of RpoD.
Highlights
The Gram-negative bacterium P. aeruginosa is an important opportunistic pathogen in humans, and has the potential to proliferate in a wide range of niches
CF149 displays a nonmucoid phenotype on Pseudomonas isolation agar (PIA) and PIA plus ammonium metavanadate (PIA-AMV) plates [29]
Through screening a transposon library, we found that overexpression of sspA and CF149 algU, and reduced expression of rpoD, are functionally equivalent in causing mucoid conversion in the non-mucoid clinical isolate CF149
Summary
The Gram-negative bacterium P. aeruginosa is an important opportunistic pathogen in humans, and has the potential to proliferate in a wide range of niches. P. aeruginosa is one of the major etiological agents of hospital-acquired infections and ventilator-associated pneumonia [1]. P. aeruginosa can produce a capsule-like polysaccharide called alginate. Mucoid conversion facilitates the establishment of persistent infection with P. aeruginosa in CF. Reversion to non-mucoidy is common in vitro in the absence of a selective pressure, and in vivo during the end-stage of CF disease [9]. Environmental signals such as high osmolarity, nitrogen or phosphate starvation, and ethanol-induced membrane perturbation can activate transcription of algD encoding the key enzyme for alginate biosynthesis [10], the selective pressure for mucoid conversion of P. aeruginosa in CF respiratory environment is not fully understood
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