Cell wall stress activates expression of a novel stress response facilitator (SrfA) under σ22 (AlgT/U) control in Pseudomonas aeruginosa.

The ECF (extracytoplasmic function) alternative sigma factor, σ(22) (AlgT/U), is required for expression of the algD promoter of the operon for alginate biosynthesis in Pseudomonas aeruginosa. Alginate production promotes chronic pulmonary infections by this opportunistic pathogen in patients with cystic fibrosis and chronic obstructive pulmonary disease. σ(22) is normally sequestered, but its deregulation and activation occur either by mutation in mucA (encoding an anti-sigma factor) or in response to envelope stress, such as inhibition of peptidoglycan synthesis. The σ(22) stress response system includes many genes in addition to those for alginate. In the present study, we characterized an intergenic region between ORFs PA2559 and PA2560 in PAO1 for a σ(22)-dependent, stress-responsive transcript, described here as PA2559.1. Northern analysis and transcript end-mapping indicated the PA2559.1 transcript was ~310 nt in length. Examination of the DNA sequence upstream of +1 revealed a σ(22) core promoter motif, GAATTT-N16-TCTGT, and site-directed mutagenesis confirmed this to be a σ(22)-dependent promoter that was highly activated during cell wall stress. PA2559.1 also contained an ORF that demonstrated increased translational activity upon cell wall stress. As determined by mutant analysis, the protein encoded by PA2559.1 was shown to play a positive role in the σ(22)-dependent activation of the algD promoter under stress in both sessile (i.e. biofilm) and planktonic conditions. Thus, it appeared to act as a stress response facilitator and so was named SrfA. The sequence of SrfA was found to be novel in nature and extremely well conserved only in P. aeruginosa, suggesting that it is of high evolutionary importance in this species.

High antimicrobial effectiveness with low hemolytic and cytotoxic activity for PEG/quaternary copolyoxetanes.

The alkyl chain length of quaternary ammonium/PEG copolyoxetanes has been varied to discern effects on solution antimicrobial efficacy, hemolytic activity and cytotoxicity. Monomers 3-((4-bromobutoxy)methyl)-3-methyloxetane (BBOx) and 3-((2-(2-methoxyethoxy)ethoxy)methyl)-3-methyloxetane (ME2Ox) were used to prepare precursor P[(BBOx)(ME2Ox)-50:50-4 kDa] copolyoxetane via cationic ring opening polymerization. The 1:1 copolymer composition and Mn (4 kDa) were confirmed by (1)H NMR spectroscopy. After C-Br substitution by a series of tertiary amines, ionic liquid Cx-50 copolyoxetanes were obtained, where 50 is the mole percent of quaternary repeat units and "x" is quaternary alkyl chain length (2, 6, 8, 10, 12, 14, or 16 carbons). Modulated differential scanning calorimetry (MDSC) studies showed Tgs between -40 and -60 °C and melting endotherms for C14-50 and C16-50. Minimum inhibitory concentrations (MIC) were determined for Escherichia coli , Staphylococcus aureus , and Pseudomonas aeruginosa . A systematic dependence of MIC on alkyl chain length was found. The most effective antimicrobials were in the C6-50 to C12-50 range. C8-50 had better overall performance with MICs of 4 µg/mL, E. coli ; 2 µg/mL, S. aureus ; and 24 µg/mL, P. aeruginosa . At 5 × MIC, C8-50 effected >99% kill in 1 h against S. aureus , E. coli , and P. aeruginosa challenges of 10(8) cfu/mL; log reductions (1 h) were 7, 3, and 5, respectively. To provide additional insight into polycation interactions with bacterial membranes, a geometric model based on the dimensions of E. coli is described that provides an estimate of the maximum number of polycations that can chemisorb. Chain dimensions were estimated for polycation C8-50 with a molecular weight of 5 kDa. Considering the approximations for polycation chemisorption (PCC), it is surprising that a calculation based on geometric considerations gives a C8-50 concentration within a factor of 2 of the MIC, 4.0 (±1.2) µg/mL for E. coli . Cx-50 copolyoxetane cytotoxicity was low for human red blood cells, human dermal fibroblasts (HDF), and human foreskin fibroblasts (HFF). Selectivities for bacterial kill over cell lysis were among the highest ever reported for polycations indicating good prospects for biocompatibility.

Identification of genes in the σ²² regulon of Pseudomonas aeruginosa required for cell envelope homeostasis in either the planktonic or the sessile mode of growth.

UNLABELLED: The Pseudomonas aeruginosa extracytoplasmic functioning (ECF) sigma factor σ(22) is encoded by algT/algU and is inhibited by anti-sigma factor MucA. σ(22) was originally discovered for its essential role in the expression of the exopolysaccharide alginate by mucoid strains associated with chronic pulmonary infection. However, σ(22) is now known to also have a large regulon associated with the response to cell wall stress. Our recent transcriptome analysis identified 293 open reading frames (ORFs) in the σ(22) stress stimulon that include genes for outer envelope biogenesis and remodeling, although most of the genes have undefined functions. To better understand the σ(22)-dependent stress response, mutants affected in 27 genes of the σ(22) stimulon were examined and expression was studied with lacZ fusions. Mutants constructed in the 27 genes showed no major change in response to cell wall-acting antibiotics or growth at elevated temperatures nor in alginate production. The mutants were examined for their effects on the expression of the σ(22)-dependent promoter of the alginate biosynthetic operon (PalgD) as a measure of σ(22) derepression from MucA. By testing PalgD expression under both planktonic and sessile growth conditions, 11 genes were found to play a role in the stress response that activates σ(22). Some mutations caused an increase or a decrease in the response to cell wall stress. Interestingly, mutations in 7 of the 11 genes caused constitutive PalgD expression under nonstressed conditions and thus showed that these genes are involved in maintaining envelope homeostasis. Mutations in PA0062 and PA1324 showed constitutive PalgD expression during both the planktonic and the sessile modes of growth. However, the PA5178 mutation caused constitutive PalgD expression only during planktonic growth. In contrast, mutations in PA2717, PA0567, PA3040, and PA0920 caused constitutive PalgD expression only in the sessile/biofilm mode of growth. This provides evidence that the σ(22) stimulon for cell envelope homeostasis overlaps with biofilm control mechanisms. IMPORTANCE: During chronic lung infections, such as in cystic fibrosis patients, Pseudomonas aeruginosa produces the exopolysaccharide alginate and forms biofilms that shield the organisms from the immune response and increase resistance to antibiotics. Activation of alginate genes is under the control of an extracytoplasmic stress response system that releases an alternative sigma factor (σ(22)) in response to cell wall stress and then activates expression of a large regulon. In this study, a mutant analysis of 27 members of the regulon showed that 11 play a role in envelope homeostasis and affect the stress response system itself. Interestingly, some genes demonstrate effects only in either the planktonic (free-swimming) or the sessile (biofilm) mode of growth, which leads to persistence and antibiotic tolerance. The studies presented here provide an important initial step in dissecting the mechanisms that regulate a critical signal transduction pathway that impacts P. aeruginosa pathogenesis.

Structural basis for alginate secretion across the bacterial outer membrane.

Pseudomonas aeruginosa is the predominant pathogen associated with chronic lung infection among cystic fibrosis patients. During colonization of the lung, P. aeruginosa converts to a mucoid phenotype characterized by the overproduction of the exopolysaccharide alginate. Secretion of newly synthesized alginate across the outer membrane is believed to occur through the outer membrane protein AlgE. Here we report the 2.3 Å crystal structure of AlgE, which reveals a monomeric 18-stranded ß-barrel characterized by a highly electropositive pore constriction formed by an arginine-rich conduit that likely acts as a selectivity filter for the negatively charged alginate polymer. Interestingly, the pore constriction is occluded on either side by extracellular loop L2 and an unusually long periplasmic loop, T8. In halide efflux assays, deletion of loop T8 (ΔT8-AlgE) resulted in a threefold increase in anion flux compared to the wild-type or ΔL2-AlgE supporting the idea that AlgE forms a transport pathway through the membrane and suggesting that transport is regulated by T8. This model is further supported by in vivo experiments showing that complementation of an algE deletion mutant with ΔT8-AlgE impairs alginate production. Taken together, these studies support a mechanism for exopolysaccharide export across the outer membrane that is distinct from the Wza-mediated translocation observed in canonical capsular polysaccharide export systems.

Highly effective, water-soluble, hemocompatible 1,3-propylene oxide-based antimicrobials: poly[(3,3-quaternary/PEG)-copolyoxetanes].

This study focuses on the solution antimicrobial effectiveness of a novel class of copolyoxetanes with quaternary ammonium and PEG-like side chains. A precursor P[(BBOx-m)(ME2Ox)] copolyoxetane was prepared by cationic ring-opening copolymerization of 3-((4-bromobutoxy)methyl)-3-methyloxetane (BBOx) and 3-((2-(2-methoxyethoxy)ethoxy)methyl)-3-methyloxetane (ME2Ox) to give random copolymers with 14-100 (m) mol % BBOx. Reaction of P[(BBOx-m)(ME2Ox)] with dodecyl dimethylamine gave the corresponding quaternary P[(C12-m)(ME2Ox)] polycation salts, designated C12-m, as viscous liquids in 100% yield. BBOx/ME2Ox and C12/ME2Ox ratios were obtained by (1)H NMR spectroscopy. C12-m molecular weights (M(n), 3.5-21.9 kDa) were obtained from (1)H NMR end group analysis. DSC studies up to 150 °C showed only thermal transitions between -69 and -34 °C assigned to T(g) values. Antibacterial activity for the C12-m copolyoxetanes was tested by determining minimum inhibitory concentrations (MICs) against Gram(+) Staphylococcus aureus and Gram(-) Escherichia coli and Pseudomonas aeruginosa . MIC decreased with increasing C12 mol percent, reaching a minimum in the range C12-43 to C12-60. Overall, the antimicrobial with consistently low MICs for the three tested pathogenic bacteria was C12-43: (bacteria, MIC, µg/mL) E. coli (6), S. aureus (5), and P. aeruginosa (33). For C12-43, minimum biocidal concentration (MBC) to reach 99.99% kill in 24 h required 1.5× MIC for S. aureus and 2× MIC for E. coli and P. aeruginosa . At 5× MIC against a challenge of 10(8) cfu/mL, C12-43 kills ≥99% S. aureus , E. coli , and P. aeruginosa within 1 h. C12-m copolyoxetane cytotoxicity toward human red blood cells was low, indicating good prospects for biocompatibility. The tunability of C12-m copolyoxetane compositions, effective antimicrobial behavior against Gram(+) and Gram(-) bacteria, and promising biocompatibility offer opportunities for further modification and potential applications as therapeutic agents.

Bio-inspired chemical reactors for growing aligned gold nanoparticle-like wires.

Bioinspired masks, created by merging sol-gel chemistry with biotemplating, were used as local chemical reactors to grow aligned arrays of gold nanoparticle-like wires.

Use of cell wall stress to characterize sigma 22 (AlgT/U) activation by regulated proteolysis and its regulon in Pseudomonas aeruginosa.

MucA sequesters extracytoplasmic function (ECF) sigma(22) (algT/U encoded) from target promoters including PalgD for alginate biosynthesis. We have shown that cell wall stress (e.g. d-cycloserine) is a potent inducer of the algD operon. Here we showed that MucB, encoded by the algT-mucABCD operon, interacts with MucA in the sigma-sequestration complex. We hypothesized that AlgW protease (a DegS homologue) is activated by cell wall stress to cleave MucA and release sigma(22). When strain PAO1 was exposed to d-cycloserine, MucA was degraded within just 10 min, and sigma(22) was activated. However, in an algW mutant, MucA was stable with no increased sigma(22) activity. Studies on a yaeL mutant, defective in an RseP/YaeL homologue, suggest that YaeL protease cleaves MucA only after cleavage by AlgW. A defect in mucD, encoding a periplasmic HtrA/DegP homologue, caused MucA instability, suggesting MucD degrades cell wall stress signals. Overall, these data indicate that cell wall stress signals release sigma(22) by regulated intramembrane proteolysis (RIP). Microarray analyses identified genes of the early and late cell wall stress stimulon, which included genes for alginate production. The subset of genes in the sigma(22) regulon was then determined, which included gene products predicted to contribute to recovery from cell wall stress.

Cell wall-inhibitory antibiotics activate the alginate biosynthesis operon in Pseudomonas aeruginosa: Roles of sigma (AlgT) and the AlgW and Prc proteases.

A bioassay was developed to identify stimuli that promote the transcriptional induction of the algD operon for alginate biosynthesis in Pseudomonas aeruginosa. Strain PAO1 carried the algD promoter fused to a chloramphenicol acetyl-transferase cartridge (PalgD-cat), and > 50 compounds were tested for promoting chloramphenicol resistance. Most compounds showing PalgD-cat induction were cell wall-active antibiotics that blocked peptidoglycan synthesis. PalgD-cat induction was blocked by mutations in the genes for sigma22 (algT/algU) or regulators AlgB and AlgR. Anti-sigma factor MucA was the primary regulator of sigma22 activity. A transcriptome analysis using microarrays verified that the algD operon undergoes high induction by D-cycloserine. A similar sigma(E)-RseAB complex in Escherichia coli responds to envelope stress, which requires DegS protease in a regulated intramembrane proteolysis (RIP) cascade to derepress the sigma. Mutant phenotypic studies in P. aeruginosa showed that AlgW (PA4446) is likely to be the DegS functional homologue. A mutation in algW resulted in a complete lack of PalgD-cat induction by D-cycloserine. Overexpression of algW in PAO1 resulted in a mucoid phenotype and alginate production, even in the absence of cell wall stress, suggesting that AlgW protease plays a role in sigma22 activation. In addition, a mutation in gene PA3257 (prc), encoding a Prc-like protease, resulted in poor induction of PalgD-cat by D-cycloserine, suggesting that it also plays a role in the response to cell wall stress.

Independent regulation of MucD, an HtrA-like protease in Pseudomonas aeruginosa, and the role of its proteolytic motif in alginate gene regulation.

Expression of mucD, encoding a homologue of the HtrA(DegP) family of endoserine proteases, was investigated in Pseudomonas aeruginosa. Expressed from the algT-mucABCD operon, MucD was detected in mucoid (FRD1) and nonmucoid (PAO1) parental strains and also when polar insertions were placed upstream in algT or mucB. A transcriptional start site for a mucD promoter (PmucD) was mapped within mucC. Expression of single-copy mucD217, encoding MucD altered in the protease motif (S217A), was defective in temperature resistance and alginate gene regulation.