Overproduction of the AlgT Sigma Factor Is Lethal to Mucoid Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa isolates from chronic lung infections often overproduce alginate, giving rise to the mucoid phenotype. Isolation of mucoid strains from chronic lung infections correlates with a poor patient outcome. The most common mutation that causes the mucoid phenotype is called mucA22 and results in a truncated form of the anti-sigma factor MucA that is continuously subjected to proteolysis. When a functional MucA is absent, the cognate sigma factor, AlgT, is no longer sequestered and continuously transcribes the alginate biosynthesis operon leading to alginate overproduction. In this work, we report that in the absence of wild-type MucA, providing exogenous AlgT is toxic. This is intriguing since mucoid strains endogenously possess high levels of AlgT. Furthermore, we show that suppressors of toxic AlgT production have mutations in mucP, a protease involved in MucA degradation, and provide the first atomistic model of MucP. Based on our findings, we speculate that mutations in mucP stabilize the truncated form of MucA22 rendering it functional and therefore able to reduce toxicity by properly sequestering AlgT. IMPORTANCE Pseudomonas aeruginosa is an opportunistic bacterial pathogen capable of causing chronic lung infections. Phenotypes important for the long-term persistence and adaption to this unique lung ecosystem are largely regulated by the AlgT sigma factor. Chronic infection isolates often contain mutations in the anti-sigma factor mucA resulting in uncontrolled AlgT and continuous production of alginate, in addition to the expression of ∼300 additional genes. Here we report that in the absence of wild-type MucA, AlgT overproduction is lethal and that suppressors of toxic AlgT production have mutations in the MucA protease, MucP. Since AlgT contributes to the establishment of chronic infections, understanding how AlgT is regulated will provide vital information on how P. aeruginosa is capable of causing long-term infections.

The anti-sigma factor MucA is required for viability inPseudomonas aeruginosa

“…This toxicity of algU overexpression was be rescued by overexpression of rpoD (Fig 7), presumably via decreased AlgU activity. Together, our work strongly suggests that growth defect in the absence of mucA is caused by unchecked AlgU activity (Figs 5-7), in agreement with previous studies suggesting that overproduction of AlgU is toxic (Cross et al ., 2020, Hershberger et al ., 1995, Schurr et al ., 1994).…”

“…Our work strongly suggests that lethality in the absence of mucA is caused by unchecked AlgU activity (Figs 5 and 6), in agreement with previous studies suggesting that overproduction of AlgU is toxic (Cross et al , 2020, Hershberger et al , 1995, Schurr et al , 1994). Our data suggests that sigma factor competition may play a role in this lethality (Fig 6).…”

“…We therefore reasoned that overexpression of algU should be toxic. Supporting this hypothesis, the toxic effects of algU overexpression have been previously noted (Cross et al ., 2020, Hershberger et al ., 1995, Schurr et al ., 1994). However, studies have shown that algU can be overexpressed in wild-type P. aeruginosa (Qiu et al ., 2008, Schulz et al ., 2015).…”

“…We therefore reasoned that overexpression of algU should be lethal. Supporting this hypothesis, the toxic effects of algU overexpression have been previously noted [44][45][46]. However, studies have shown that algU can be overexpressed in wild-type P. aeruginosa [16,17].…”

Pseudomonas aeruginosa: An Audacious Pathogen with an Adaptable Arsenal of Virulence Factors

“…It is a high molecular weight, random polymer with variable ratios of D-mannuronic and L-guluronic acids that are β1-4 linked and partially O-acetylated. The algD operon encodes the enzymes required for alginate synthesis, and its expression is regulated by AlgT σ-factor [ 62 ], whose overexpression is lethal to mucoid strains [ 71 ]. This exopolysaccharide is overproduced by mucoid P. aeruginosa strains, and despite not being a requirement for biofilm formation [ 72 , 73 ], it helps its maturation, architecture, and stability [ 73 , 74 ].…”