Dataset Information

Two distinct loci affecting conversion to mucoidy in Pseudomonas aeruginosa in cystic fibrosis encode homologs of the serine protease HtrA.

ABSTRACT: Conversion to a mucoid, exopolysaccharide alginate-overproducing phenotype in Pseudomonas aeruginosa is associated with chronic respiratory infections in cystic fibrosis. Mucoidy is caused by muc mutations that derepress the alternative sigma factor AlgU, which in turn activates alginate biosynthetic and ancillary regulatory genes. Here we report the molecular characterization of two newly identified genes, algW and mucD, that affect expression of mucoidy. The algW gene, mapping at 69 min, was isolated on the basis of its ability to suppress mucoidy and reduce transcription of the alginate biosynthetic gene algD. The predicted primary structure of AlgW displayed similarity to HtrA (DegP), a serine protease involved in proteolysis of abnormal proteins and required for resistance to oxidative and heat stress in enteric bacteria. Inactivation of algW on the chromosome of the wild-type nonmucoid strain PAO1 caused increased sensitivity to heat, H2O2, and paraquat, a redox cycling compound inducing intracellular levels of superoxide. This mutation also permitted significant induction of alginate production in the presence of subinhibitory concentrations of paraquat. Two new genes, mucC and mucD, were identified immediately downstream of the previously characterized portion (algU mucA mucB) of the gene cluster at 67.5 min encoding the alternative sigma factor AlgU and its regulators. Interestingly, the predicted gene product of mucD also showed similarities to HtrA. Inactivation of mucD on the PAO1 chromosome resulted in conversion to the mucoid phenotype. The mutation in mucD also caused increased sensitivity to H2O2 and heat killing. However, in contrast to algW mutants, no increase in susceptibility to paraquat was observed in mucD mutants. These findings indicate that algW and mucD play partially overlapping but distinct roles in P. aeruginosa resistance to reactive oxygen intermediates and heat. In addition, since mutations in mucD and algW cause conversion to mucoidy or lower the threshold for its induction by reactive oxygen intermediates, these factors may repress alginate synthesis either directly by acting on AlgU or its regulators or indirectly by removing physiological signals that may activate this stress response system.

SUBMITTER: Boucher JC

PROVIDER: S-EPMC177686 | biostudies-other | 1996 Jan

REPOSITORIES: biostudies-other

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Two distinct loci affecting conversion to mucoidy in Pseudomonas aeruginosa in cystic fibrosis encode homologs of the serine protease HtrA.

Boucher J C JC Martinez-Salazar J J Schurr M J MJ Mudd M H MH Yu H H Deretic V V

Journal of bacteriology 19960101 2

Conversion to a mucoid, exopolysaccharide alginate-overproducing phenotype in Pseudomonas aeruginosa is associated with chronic respiratory infections in cystic fibrosis. Mucoidy is caused by muc mutations that derepress the alternative sigma factor AlgU, which in turn activates alginate biosynthetic and ancillary regulatory genes. Here we report the molecular characterization of two newly identified genes, algW and mucD, that affect expression of mucoidy. The algW gene, mapping at 69 min, was i ...[more]

PMID: 8550474

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Project description:Survival of Pseudomonas aeruginosa in cystic fibrosis (CF) chronic infections is based on a genetic adaptation process consisting of mutations in specific genes, which can produce advantageous phenotypic switches and ensure its persistence in the lung. Among these, mutations inactivating the regulators MucA (alginate biosynthesis), LasR (quorum sensing) and MexZ (multidrug-efflux pump MexXY) are the most frequently observed, with those inactivating the DNA mismatch repair system (MRS) being also highly prevalent in P. aeruginosa CF isolates, leading to hypermutator phenotypes that could contribute to this adaptive mutagenesis by virtue of an increased mutation rate. Here, we characterized the mutations found in the mucA, lasR, mexZ and MRS genes in P. aeruginosa isolates obtained from Argentinean CF patients, and analyzed the potential association of mucA, lasR and mexZ mutagenesis with MRS-deficiency and antibiotic resistance. Thus, 38 isolates from 26 chronically infected CF patients were characterized for their phenotypic traits, PFGE genotypic patterns, mutations in the mucA, lasR, mexZ, mutS and mutL gene coding sequences and antibiotic resistance profiles. The most frequently mutated gene was mexZ (79%), followed by mucA (63%) and lasR (39%) as well as a high prevalence (42%) of hypermutators being observed due to loss-of-function mutations in mutL (60%) followed by mutS (40%). Interestingly, mutational spectra were particular to each gene, suggesting that several mechanisms are responsible for mutations during chronic infection. However, no link could be established between hypermutability and mutagenesis in mucA, lasR and mexZ, indicating that MRS-deficiency was not involved in the acquisition of these mutations. Finally, although inactivation of mucA, lasR and mexZ has been previously shown to confer resistance/tolerance to antibiotics, only mutations in MRS genes could be related to an antibiotic resistance increase. These results help to unravel the mutational dynamics that lead to the adaptation of P. aeruginosa to the CF lung.

Project description:AlgW, a membrane-bound periplasmic serine protease belonging to the HtrA protein family, is a key regulator of the regulated intramembrane proteolysis (RIP) pathway and is responsible for transmitting the envelope stress signals in Pseudomonas aeruginosa The AlgW PDZ domain senses and binds the C-terminal of mis-localized outer membrane proteins (OMPs) or periplasmic protein MucE, leading to catalytic activation of the protease domain. While AlgW is functionally well studied, its exact activation mechanism remains to be elucidated. Here, we show that AlgW is a novel HtrA protease that can be biochemically activated by both peptide and lipid signals. Compared with the corresponding homologue DegS in Escherichia coli, AlgW exhibits a distinct substrate specificity and regulation mechanism. Structural, biochemical, and mutagenic analyses revealed that, by specifically binding to the C-terminal decapeptide of MucE, AlgW could adopt more relaxed conformation and obtain higher activity than with tripeptide activation. We also investigated the regulatory mechanism of the LA loop in AlgW and proved that the unique structural feature of this region was responsible for the distinct enzymatic property of AlgW. These results demonstrate the unique and diverse activation mechanism of AlgW, which P. aeruginosa may utilize to enhance its adaptability to environmental stress.IMPORTANCE HtrA-family proteases are commonly employed to sense the protein folding stress and activate the regulated intramembrane proteolysis (RIP) cascade in Gram-negative bacteria. Here, we reveal the unique dual-signal activation and dynamic regulation properties of AlgW, an HtrA-type protease triggering the AlgU stress-response pathway, which controls alginate production and mucoid conversion in Pseudomonas aeruginosa The structural and functional data offer insights into the molecular basis underlying the transition of different activation states of AlgW in response to different effectors. Probing these unique features provides an opportunity to correlate the diverse regulation mechanism of AlgW with the high adaptability of P. aeruginosa to environmental changes during infection.

Project description:Pseudomonas aeruginosa induces pathways indicative of low zinc availability in the cystic fibrosis (CF) lung environment. To learn more about P. aeruginosa zinc access in CF, we grew P. aeruginosa strain PAO1 directly in expectorated CF sputum. The P. aeruginosa Zur transcriptional repressor controls the response to low intracellular zinc, and we used the NanoString methodology to monitor levels of Zur-regulated transcripts, including those encoding a zincophore system, a zinc importer, and paralogs of zinc containing proteins that do not require zinc for activity. Zur-controlled transcripts were induced in sputum-grown P. aeruginosa compared to those grown in control cultures but not if the sputum was amended with zinc. Amendment of sputum with ferrous iron did not reduce expression of Zur-regulated genes. A reporter fusion to a Zur-regulated promoter had variable activity in P. aeruginosa grown in sputum from different donors, and this variation inversely correlated with sputum zinc concentrations. Recombinant human calprotectin (CP), a divalent-metal binding protein released by neutrophils, was sufficient to induce a zinc starvation response in P. aeruginosa grown in laboratory medium or zinc-amended CF sputum, indicating that CP is functional in the sputum environment. Zinc metalloproteases comprise a large fraction of secreted zinc-binding P. aeruginosa proteins. Here, we show that recombinant CP inhibited both LasB-mediated casein degradation and LasA-mediated lysis of Staphylococcus aureus, which was reversible with added zinc. These studies reveal the potential for CP-mediated zinc chelation to posttranslationally inhibit zinc metalloprotease activity and thereby affect the protease-dependent physiology and/or virulence of P. aeruginosa in the CF lung environment. IMPORTANCE The factors that contribute to worse outcomes in individuals with cystic fibrosis (CF) with chronic Pseudomonas aeruginosa infections are not well understood. Therefore, there is a need to understand environmental factors within the CF airway that contribute to P. aeruginosa colonization and infection. We demonstrate that growing bacteria in CF sputum induces a zinc starvation response that inversely correlates with sputum zinc levels. Additionally, both calprotectin and a chemical zinc chelator inhibit the proteolytic activities of LasA and LasB proteases, suggesting that extracellular zinc chelators can influence proteolytic activity and thus P. aeruginosa virulence and nutrient acquisition in vivo.

Project description:Mucoidy due to alginate overproduction by the Gram-negative bacterium Pseudomonas aeruginosa facilitates chronic lung infections in patients with cystic fibrosis (CF). We previously reported that disruption in de novo synthesis of pyrimidines resulted in conversion to a nonmucoid small-colony variant (SCV) in the mucoid P. aeruginosa strain (PAO581), which has a truncated anti-sigma factor, MucA25, that cannot sequester sigma factor AlgU (AlgT). Here, we showed that supplementation with the nitrogenous bases uracil or cytosine in growth medium complemented the SCV to normal growth, and nonmucoidy to mucoidy, in these mucA25 mutants. This conversion was associated with an increase in intracellular levels of UMP and UTP suggesting that nucleotide restoration occurred via a salvage pathway. In addition, supplemented pyrimidines caused an increase in activity of the alginate biosynthesis promoter (P algD ), but had no effect on P algU , which controls transcription of algU Cytosolic levels of AlgU were not influenced by uracil supplementation, yet levels of RpoN, a sigma factor that regulates nitrogen metabolism, increased with disruption of pyrimidine synthesis and decreased after supplementation of uracil. This suggested that an elevated level of RpoN in SCV may block alginate biosynthesis. To support this, we observed that overexpressing rpoN resulted in a phenotypic switch to nonmucoidy in PAO581 and in mucoid clinical isolates. Furthermore, transcription of an RpoN-regulated promoter increased in the mutants and decreased after uracil supplementation. These results suggest that the balance of RpoN and AlgU levels may regulate growth from SCV to mucoidy through sigma factor competition for P algD IMPORTANCE Chronic lung infections with P. aeruginosa are the main cause of morbidity and mortality in patients with cystic fibrosis. This bacterium overproduces a capsular polysaccharide called alginate (also known as mucoidy), which aids in bacterial persistence in the lungs and in resistance to therapeutic regimens and host immune responses. The current study explores a previously unknown link between pyrimidine biosynthesis and mucoidy at the level of transcriptional regulation. Identifying/characterizing this link could provide novel targets for the control of bacterial growth and mucoidy. Inhibiting mucoidy may improve antimicrobial efficacy and facilitate host defenses to clear the noncapsulated P. aeruginosa bacteria, leading to improved prognosis for patients with cystic fibrosis.

Dataset Information

Two distinct loci affecting conversion to mucoidy in Pseudomonas aeruginosa in cystic fibrosis encode homologs of the serine protease HtrA.

Publications

Two distinct loci affecting conversion to mucoidy in Pseudomonas aeruginosa in cystic fibrosis encode homologs of the serine protease HtrA.

Similar Datasets

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