Microarray analysis of global gene expression in mucoid Pseudomonas aeruginosa

Pseudomonas aeruginosa is the dominant pathogen causing chronic respiratory infections in cystic fibrosis (CF). After an initial phase characterized by intermittent infections, a chronic colonization is established in CF upon the conversion of P. aeruginosa to the mucoid, exopolysaccharide alginate-overproducing phenotype. The emergence of mucoid P. aeruginosa in CF is associated with respiratory decline and poor prognosis. The switch to mucoidy in most CF isolates is caused by mutations in the mucA gene encoding an anti-sigma factor. The mutations in mucA result in the activation of the alternative sigma factor AlgU, the P. aeruginosa ortholog of Escherichia coli extreme stress sigma factor sigma(E). Because of the global nature of the regulators of mucoidy, we have hypothesized that other genes, in addition to those specific for alginate production, must be induced upon conversion to mucoidy, and their production may contribute to the pathogenesis in CF. Here we applied microarray analysis to identify on the whole-genome scale those genes that are coinduced with the AlgU sigmulon upon conversion to mucoidy. Gene expression profiles of AlgU-dependent conversion to mucoidy revealed coinduction of a specific subset of known virulence determinants (the major protease elastase gene, alkaline metalloproteinase gene aprA, and the protease secretion factor genes aprE and aprF) or toxic factors (cyanide synthase) that may have implications for disease in CF. Analysis of promoter regions of the most highly induced genes (>40-fold, P < or = 10(-4)) revealed a previously unrecognized, putative AlgU promoter upstream of the osmotically inducible gene osmE. This newly identified AlgU-dependent promoter of osmE was confirmed by mapping the mRNA 5' end by primer extension. The recognition of genes induced in mucoid P. aeruginosa, other than those associated with alginate biosynthesis, reported here revealed the identity of previously unappreciated factors potentially contributing to the morbidity and mortality caused by mucoid P. aeruginosa in CF.     

Analysis of the Pseudomonas aeruginosa regulon controlled by the sensor kinase KinB and sigma factor RpoN

Alginate overproduction by Pseudomonas aeruginosa, also known as mucoidy, is associated with chronic endobronchial infections in cystic fibrosis. Alginate biosynthesis is initiated by the extracytoplasmic function sigma factor (σ(22); AlgU/AlgT). In the wild-type (wt) nonmucoid strains, such as PAO1, AlgU is sequestered to the cytoplasmic membrane by the anti-sigma factor MucA that inhibits alginate production. One mechanism underlying the conversion to mucoidy is mutation of mucA. However, the mucoid conversion can occur in wt mucA strains via the degradation of MucA by activated intramembrane proteases AlgW and/or MucP. Previously, we reported that the deletion of the sensor kinase KinB in PAO1 induces an AlgW-dependent proteolysis of MucA, resulting in alginate overproduction. This type of mucoid induction requires the alternate sigma factor RpoN (σ(54)). To determine the RpoN-dependent KinB regulon, microarray and proteomic analyses were performed on a mucoid kinB mutant and an isogenic nonmucoid kinB rpoN double mutant. In the kinB mutant of PAO1, RpoN controlled the expression of approximately 20% of the genome. In addition to alginate biosynthetic and regulatory genes, KinB and RpoN also control a large number of genes including those involved in carbohydrate metabolism, quorum sensing, iron regulation, rhamnolipid production, and motility. In an acute pneumonia murine infection model, BALB/c mice exhibited increased survival when challenged with the kinB mutant relative to survival with PAO1 challenge. Together, these data strongly suggest that KinB regulates virulence factors important for the development of acute pneumonia and conversion to mucoidy.     

Vanadate and triclosan synergistically induce alginate production by Pseudomonas aeruginosa strain PAO1

Alginate overproduction by P. aeruginosa strains, also known as mucoidy, is associated with chronic lung infections in cystic fibrosis (CF). It is not clear how alginate induction occurs in the wild-type (wt) mucA strains. When grown on Pseudomonas isolation agar (PIA), P. aeruginosa strains PAO1 and PA14 are non-mucoid, producing minimal amounts of alginate. Here we report the addition of ammonium metavanadate (AMV), a phosphatase inhibitor, to PIA (PIA-AMV) induced mucoidy in both these laboratory strains and early lung colonizing non-mucoid isolates with a wt mucA. This phenotypic switch was reversible depending on the availability of vanadate salts and triclosan, a component of PIA. Alginate induction in PAO1 on PIA-AMV was correlated with increased proteolytic degradation of MucA, and required envelope proteases AlgW or MucP, and a two-component phosphate regulator, PhoP. Other changes included the addition of palmitate to lipid A, a phenotype also observed in chronic CF isolates. Proteomic analysis revealed the upregulation of stress chaperones, which was confirmed by increased expression of the chaperone/protease MucD. Altogether, these findings suggest a model of alginate induction and the PIA-AMV medium may be suitable for examining early lung colonization phenotypes in CF before the selection of the mucA mutants.     

Alginate acetylation influences initial surface colonization by mucoid Pseudomonas aeruginosa

Mucoid strains of Pseudomonas aeruginosa overproduce the exopolysaccharide alginate, which is substituted with O-acetyl groups. Under non-growing conditions in phosphate buffer, a mucoid clinical strain formed microcolonies on steel surfaces, while an acetylation-defective mutant was unable to form cell clusters. Enzymatic degradation of alginate by alginate lyase prevented microcolony formation of the mucoid parent strain. In a continuous-culture flow-cell system, using gluconate minimal medium, the mucoid strain with acetylated alginate formed microcolonies and grew into heterogenous biofilms, whereas the acetylation-defective mutant produced a thinner and more homogeneous biofilm. A lowered viscosity of extracellular material from the acetylation-defective mutant indicated a weakening of exopolymer interactions by loss of acetyl groups. These results suggest that acetyl substituents are necessary for the function of high-molecular-mass alginate to mediate cell aggregation into microcolonies in the early stages of biofilm development by mucoid P. aeruginosa, thereby determining the architecture of the mature biofilm.     

The roles of biofilm matrix polysaccharide Psl in mucoid Pseudomonas aeruginosa biofilms

The opportunistic pathogen Pseudomonas aeruginosa causes life-threatening, persistent infections in patients with cystic fibrosis (CF). Persistence is attributed to the ability of these bacteria to form structured communities (biofilms). Biofilms rely on an extracellular polymeric substances matrix to maintain structure. Psl exopolysaccharide is a key matrix component of nonmucoid biofilms, yet the role of Psl in mucoid biofilms is unknown. In this report, using a variety of mutants in a mucoid P.?aeruginosa background, we found that deletion of Psl-encoding genes dramatically decreased their biofilm formation ability, indicating that Psl is also a critical matrix component of mucoid biofilms. Our data also suggest that the overproduction of alginate leads to mucoid biofilms, which occupy more space, whereas Psl-dependent biofilms are densely packed. These data suggest that Psl polysaccharide may have significant contributions in biofilm persistence in patients with CF and may be helpful for designing therapies for P.?aeruginosa CF infection.     

Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia.

Respiratory infections with Pseudomonas aeruginosa and Burkholderia cepacia play a major role in the pathogenesis of cystic fibrosis (CF). This review summarizes the latest advances in understanding host-pathogen interactions in CF with an emphasis on the role and control of conversion to mucoidy in P. aeruginosa, a phenomenon epitomizing the adaptation of this opportunistic pathogen to the chronic chourse of infection in CF, and on the innate resistance to antibiotics of B. cepacia, person-to-person spread, and sometimes rapidly fatal disease caused by this organism. While understanding the mechanism of conversion to mucoidy in P. aeruginosa has progressed to the point where this phenomenon has evolved into a model system for studying bacterial stress response in microbial pathogenesis, the more recent challenge with B. cepacia, which has emerged as a potent bona fide CF pathogen, is discussed in the context of clinical issues, taxonomy, transmission, and potential modes of pathogenicity.     

Understanding the control of Pseudomonas aeruginosa alginate synthesis and the prospects for management of chronic infections in cystic fibrosis

Decades of research have been dedicated to the study of the opportunistic pathogen Pseudomonas aeruginosa, a Gram-negative, environmental bacterium that secretes the exopolysaccharide alginate during chronic lung infection of cystic fibrosis (CF) patients. Although P. aeruginosa utilizes a variety of factors to establish a successful infection in the lungs of CF patients, alginate has stood out as one of the best-studied prognostic indicators of chronic lung infection. While the genetics, biosynthesis and regulation of alginate are well understood, questions still remain concerning its role in biofilm development and its potential as a therapeutic target. The purpose of this review is to provide a brief summary of alginate biosynthesis and regulation, and to highlight recent discoveries in the areas of alginate production, biofilm formation and vaccine design. This information is placed in context with a proposed P. aeruginosa infectious pathway, highlighting avenues for the use of existing therapies as well as the potential for novel agents to reduce or eliminate chronic infections in CF patients.     

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial pathogen which establishes chronic pulmonary infections in patients with cystic fibrosis (CF). The overproduction of a capsular polysaccharide called alginate, also known as mucoidy, promotes the formation of mucoid biofilms which are more resistant than planktonic cells to antibiotic chemotherapy and host defenses. Additionally, the conversion from the nonmucoid to mucoid phenotype is a clinical marker for the onset of chronic infection in CF. Alginate overproduction by P. aeruginosa is an endergonic process which heavily taxes cellular energy. Therefore, alginate production is highly regulated in P. aeruginosa. To better understand alginate regulation, we describe a protocol using the mini-himar1 transposon mutagenesis for the identification of novel alginate regulators in a prototypic strain PAO1. The procedure consists of two basic steps. First, we transferred the mini-himar1 transposon (pFAC) from host E. coli SM10/λpir into recipient P. aeruginosa PAO1 via biparental conjugation to create a high-density insertion mutant library, which were selected on Pseudomonas isolation agar plates supplemented with gentamycin. Secondly, we screened and isolated the mucoid colonies to map the insertion site through inverse PCR using DNA primers pointing outward from the gentamycin cassette and DNA sequencing. Using this protocol, we have identified two novel alginate regulators, mucE (PA4033) and kinB (PA5484), in strain PAO1 with a wild-type mucA encoding the anti-sigma factor MucA for the master alginate regulator AlgU (AlgT, σ²²). This high-throughput mutagenesis protocol can be modified for the identification of other virulence-related genes causing change in colony morphology.     

Evolution and adaptation in Pseudomonas aeruginosa biofilms driven by mismatch repair system-deficient mutators

Pseudomonas aeruginosa is an important opportunistic pathogen causing chronic airway infections, especially in cystic fibrosis (CF) patients. The majority of the CF patients acquire P. aeruginosa during early childhood, and most of them develop chronic infections resulting in severe lung disease, which are rarely eradicated despite intensive antibiotic therapy. Current knowledge indicates that three major adaptive strategies, biofilm development, phenotypic diversification, and mutator phenotypes [driven by a defective mismatch repair system (MRS)], play important roles in P. aeruginosa chronic infections, but the relationship between these strategies is still poorly understood. We have used the flow-cell biofilm model system to investigate the impact of the mutS associated mutator phenotype on development, dynamics, diversification and adaptation of P. aeruginosa biofilms. Through competition experiments we demonstrate for the first time that P. aeruginosa MRS-deficient mutators had enhanced adaptability over wild-type strains when grown in structured biofilms but not as planktonic cells. This advantage was associated with enhanced micro-colony development and increased rates of phenotypic diversification, evidenced by biofilm architecture features and by a wider range and proportion of morphotypic colony variants, respectively. Additionally, morphotypic variants generated in mutator biofilms showed increased competitiveness, providing further evidence for mutator-driven adaptive evolution in the biofilm mode of growth. This work helps to understand the basis for the specific high proportion and role of mutators in chronic infections, where P. aeruginosa develops in biofilm communities.     

Alginate as an auxiliary bacterial membrane: binding of membrane-active peptides by polysaccharides.

The chronicity of Pseudomonas aeruginosa infections in cystic fibrosis (CF) patients is characterized by overproduction of the exopolysaccharide alginate, in which biofilm bacteria are embedded. Alginate apparently contributes to the antibiotic resistance of bacteria in this form by acting as a diffusion barrier to positively charged antimicrobial agents. We have been investigating cationic antimicrobial peptides (CAPs) (prototypic sequence: KKAAAXAAAAAXAAWAAXAAAKKKK-NH(2), where X is any of the 20 commonly occurring amino acids) that were originally designed as transmembrane mimetic peptides. Peptides of this group above a specific hydrophobicity threshold insert spontaneously into membranes and have antibacterial activity at micromolar concentrations. While investigating the molecular basis of biofilm resistance to peptides, we found that the anionic alginate polysaccharide induces conformational changes in the most hydrophobic of these peptides typically associated with insertion of such peptides into membrane environments [Chan et al., J. Biol. Chem. (2004) vol. 279, pp. 38749-38754]. Through a combination of experiments measuring release of the fluorescent dye calcein from phospholipid vesicles, peptide interactions with vesicles in the presence and absence of alginate, and affinity of peptides for alginate as a function of net peptide core hydrophobicity, we show here that alginate offers a microenvironment that provides a protective mechanism for the encased bacteria by both binding and promoting the self-association of the CAPs. The overall results indicate that hydrophilic alginate polymers contain a significant hydrophobic compartment, and behave as an 'auxiliary membrane' for bacteria, thus identifying a unique protective role for biofilm exopolysaccharide matrices.     

Phenotypes selected during chronic lung infection in cystic fibrosis patients: implications for the treatment of Pseudomonas aeruginosa biofilm infections

During chronic lung infection of patients with cystic fibrosis, Pseudomonas aeruginosa can survive for long periods of time under the challenging selective pressure imposed by the immune system and antibiotic treatment as a result of its biofilm mode of growth and adaptive evolution mediated by genetic variation. Mucoidy, hypermutability and acquirement of mutational antibiotic resistance are important adaptive phenotypes that are selected during chronic P.?aeruginosa infection. This review dicsusses the role played by these phenotypes for the tolerance of biofilms to antibiotics and show that mucoidy and hypermutability change the architecture of in vitro formed biofilms and lead to increase tolerance to antibiotics. Production of high levels of beta-lactamase impairs penetration of beta-lactam antibiotics due to inactivation of the antibiotic. In conclusion, these data underline the importance of biofilm prevention strategies by early aggressive antibiotic prophylaxis or therapy before phenotypic diversification during chronic lung infection of patients with cystic fibrosis.     

Pseudomonas aeruginosa flagellin and alginate elicit very distinct gene expression patterns in airway epithelial cells: implications for cystic fibrosis disease

Infection with the opportunistic pathogen Pseudomonas aeruginosa remains a major health concern. Two P. aeruginosa phenotypes relevant in human disease include motility and mucoidy. Motility is characterized by the presence of flagella and is essential in the establishment of acute infections, while mucoidy, defined by the production of the exopolysaccharide alginate, is critical in the development of chronic infections, such as the infections seen in cystic fibrosis patients. Indeed, chronic infection of the lung by mucoid P. aeruginosa is a major cause of morbidity and mortality in cystic fibrosis patients. We have used Calu-3 human airway epithelial cells to investigate global responses to infection with motile and mucoid P. aeruginosa. The response of airway epithelial cells to exposure to P. aeruginosa motile strains is characterized by a specific increase in gene expression in pathways controlling inflammation and host defense. By contrast, the response of airway epithelia to the stimuli presented by mucoid P. aeruginosa is not proinflammatory and, hence, may not be conducive to the effective elimination of the pathogen. The pattern of gene expression directed by flagellin, but not alginate, includes innate host defense genes, proinflammatory cytokines, and chemokines. By contrast, infection with alginate-producing P. aeruginosa results in an overall attenuation of host responses and an antiapoptotic effect.     

Control of AlgU, a member of the sigma E-like family of stress sigma factors, by the negative regulators MucA and MucB and Pseudomonas aeruginosa conversion to mucoidy in cystic fibrosis.

The alternative sigma factor AlgU (Pseudomonas aeruginosa sigma E) is required for full resistance of P. aeruginosa to oxidative stress and extreme temperatures. AlgU also controls conversion of P. aeruginosa to the mucoid, alginate-overproducing phenotype associated with lethal infections in cystic fibrosis patients. Mutations that cause conversion to mucoidy in cystic fibrosis isolates occur frequently in mucA, the second gene within the algU mucABCD gene cluster. Here we analyze the biochemical basis of conversion to mucoidy. MucA was shown to act as an anti-sigma factor by binding to AlgU and inhibiting its activity. MucB, another negative regulator of AlgU, was localized in the periplasm. MucB exerts its function from this compartment, since deletion of the leader peptide and the cytoplasmic location of MucB abrogated its ability to inhibit mucoidy. These data support a model in which a multicomponent system, encompassing an anti-delta factor and elements in the periplasmic compartment, modulates activity of AlgU. Since factors controlling AlgU are conserved in other gram-negative bacteria, the processes controlling conversion to mucoidy in P. aeruginosa may be applicable to the regulation of AlgU (sigma E) equivalents in other organisms.     

Overexpression of CupB5 activates alginate overproduction in Pseudomonas aeruginosa by a novel AlgW‐dependent mechanism

In Pseudomonas aeruginosa, alginate overproduction, also known as mucoidy, is negatively regulated by the transmembrane protein MucA, which sequesters the alternative sigma factor AlgU. MucA is degraded via a proteolysis pathway that frees AlgU from sequestration, activating alginate biosynthesis. Initiation of this pathway normally requires two signals: peptide sequences in unassembled outer‐membrane proteins (OMPs) activate the AlgW protease, and unassembled lipopolysaccharides bind periplasmic MucB, releasing MucA and facilitating its proteolysis by activated AlgW. To search for novel alginate regulators, we screened a transposon library in the non‐mucoid reference strain PAO1, and identified a mutant that confers mucoidy through overexpression of a protein encoded by the c haperone‐u sher p athway gene cupB5. CupB5‐dependent mucoidy occurs through the AlgU pathway and can be reversed by overexpression of MucA or MucB. In the presence of activating OMP peptides, peptides corresponding to a region of CupB5 needed for mucoidy further stimulated AlgW cleavage of MucA in vitro. Moreover, the CupB5 peptide allowed OMP‐activated AlgW cleavage of MucA in the presence of the MucB inhibitor. These results support a novel mechanism for conversion to mucoidy in which the proteolytic activity of AlgW and its ability to compete with MucB for MucA is mediated by independent peptide signals.