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.     

Expression of extracellular polysaccharides and proteins by clinical isolates of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> in response to environmental conditions

The opportunistic pathogen Pseudomonas aeruginosa causes chronic respiratory infections in patients with cystic fibrosis (CF). Persistence of this bacterium is attributed to its ability to form biofilms which rely on an extracellular polymeric substance matrix. Extracellular polysaccharides (EPS) and secreted proteins are key matrix components of P. aeruginosa biofilms. Recently, nebulized magnesium sulfate has been reported as a significant bronchodilator for asthmatic patients including CF. However, the impact of magnesium sulfate on the virulence effect of P. aeruginosa is lacking. In this report, we investigated the influence of magnesium sulfate and other environmental factors on the synthesis of alginate and secretion of proteins by a mucoid and a non-mucoid strain of P. aeruginosa, respectively. By applying the Plackett-Burman and Box-Behnken experimental designs, we found that phosphates (6.0 g/l), ammonium sulfate (4.0 g/l), and trace elements (0.6 mg/l) markedly supported alginate production by the mucoid strain. However, ferrous sulfate (0.3 mg/l), magnesium sulfate (0.02 g/l), and phosphates (6.0 g/l) reinforced the secretion of proteins by the non-mucoid strain.     

Controlling persister cells of Pseudomonas aeruginosa PDO300 by (Z)-4-bromo-5-(bromomethylene)-3-methylfuran-2(5H)-one

Pseudomonas aeruginosa is a major pathogen causing chronic pulmonary infections; for example, 80% of cystic fibrosis patients get infected by this bacterium as the disease progresses. Such chronic infections are challenging because P. aeruginosa exhibits high-level tolerance to antibiotics by forming biofilms (multicellular structures attached to surfaces), by entering dormancy and forming antibiotic tolerant persister cells, and by conversion to the mucoid phenotype. Recently, we reported that a synthetic quorum sensing inhibitor, (Z)-4-bromo-5-(bromomethylene)-3-methylfuran-2(5H)-one (BF8), can sensitize both planktonic and biofilm-associated persister cells of P. aeruginosa PAO1 to antibiotics at the concentrations non-inhibitory to its growth. In this study, we further characterized the effects of this compound on the mucoid strain P. aeruginosa PDO300. BF8 was found to reduce persistence during the growth of PDO300 and effectively kill the persister cells isolated from PDO300 cultures. In addition to planktonic cells, BF8 was also found to inhibit biofilm formation of PDO300 and reduce associated persistence. These findings broaden the activities of this class of compounds and indicate that BF8 also has other targets in P. aeruginosa in addition to quorum sensing.     

Impact of Alginate Overproduction on Attachment and Biofilm Architecture of a Supermucoid Pseudomonas aeruginosa Strain

The supermucoid Pseudomonas aeruginosa strain PDO300Δalg8(pBBR1MCS-5:alg8) showed strongly impaired attachment compared with the respective mucoid or nonmucoid strains and formed a thicker biofilm with large extended mushroom-like microcolonies. Alginate lyase treatment dissolved microcolonies. The data suggested that alginate overproduction impairs attachment but plays a structural role in microcolony formation.Alginate is an important virulence factor for Pseudomonas aeruginosa, and the conversion of nonmucoid strains to alginate-overproducing mucoid strains early after the infection of cystic fibrosis patients is associated with a decline of pulmonary function and survival rate (11, 13). Alginate functions as extracellular matrix material, enabling the formation of differentiated biofilms in which the diffusion of clinical antibiotics is decreased and the embedded cells are protected against human antibacterial defense mechanisms (9, 12). Although alginate is not required for P. aeruginosa biofilm formation (15), previous studies have provided evidence that it plays a role in the formation of thick and three-dimensional biofilms (5, 9). To further investigate the impact of alginate on attachment and biofilm architecture, we used a recently generated supermucoid strain, PDO300Δalg8(pBBR1MCS-5:alg8) (14). This strain showed about 15-fold alginate overproduction compared to alginate-producing mucoid P. aeruginosa. The gene alg8 encodes the proposed catalytic subunit of alginate polymerase and is essential for alginate biosynthesis (14).     

The exopolysaccharide alginate protects Pseudomonas aeruginosa biofilm bacteria from IFN-gamma-mediated macrophage killing

The ability of Pseudomonas aeruginosa to form biofilms and cause chronic infections in the lungs of cystic fibrosis patients is well documented. Numerous studies have revealed that P. aeruginosa biofilms are highly refractory to antibiotics. However, dramatically fewer studies have addressed P. aeruginosa biofilm resistance to the host's immune system. In planktonic, unattached (nonbiofilm) P. aeruginosa, the exopolysaccharide alginate provides protection against a variety of host factors yet the role of alginate in protection of biofilm bacteria is unclear. To address this issue, we tested wild-type strains PAO1, PA14, the mucoid cystic fibrosis isolate, FRD1 (mucA22+), and the respective isogenic mutants which lacked the ability to produce alginate, for their susceptibility to human leukocytes in the presence and absence of IFN-gamma. Human leukocytes, in the presence of recombinant human IFN-gamma, killed biofilm bacteria lacking alginate after a 4-h challenge at 37 degrees C. Bacterial killing was dependent on the presence of IFN-gamma. Killing of the alginate-negative biofilm bacteria was mediated through mononuclear cell phagocytosis since treatment with cytochalasin B, which prevents actin polymerization, inhibited leukocyte-specific bacterial killing. By direct microscopic observation, phagocytosis of alginate-negative biofilm bacteria was significantly increased in the presence of IFN-gamma vs all other treatments. Addition of exogenous, purified alginate to the alginate-negative biofilms restored resistance to human leukocyte killing. Our results suggest that although alginate may not play a significant role in bacterial attachment, biofilm development, and formation, it may play an important role in protecting mucoid P. aeruginosa biofilm bacteria from the human immune system.     

Diffusion Retardation by Binding of Tobramycin in an Alginate Biofilm Model

Microbial cells embedded in a self-produced extracellular biofilm matrix cause chronic infections, e. g. by Pseudomonas aeruginosa in the lungs of cystic fibrosis patients. The antibiotic killing of bacteria in biofilms is generally known to be reduced by 100–1000 times relative to planktonic bacteria. This makes such infections difficult to treat. We have therefore proposed that biofilms can be regarded as an independent compartment with distinct pharmacokinetics. To elucidate this pharmacokinetics we have measured the penetration of the tobramycin into seaweed alginate beads which serve as a model of the extracellular polysaccharide matrix in P. aeruginosa biofilm. We find that, rather than a normal first order saturation curve, the concentration of tobramycin in the alginate beads follows a power-law as a function of the external concentration. Further, the tobramycin is observed to be uniformly distributed throughout the volume of the alginate bead. The power-law appears to be a consequence of binding to a multitude of different binding sites. In a diffusion model these results are shown to produce pronounced retardation of the penetration of tobramycin into the biofilm. This filtering of the free tobramycin concentration inside biofilm beads is expected to aid in augmenting the survival probability of bacteria residing in the biofilm.     

Investigating the Link Between Imipenem Resistance and Biofilm Formation by Pseudomonas aeruginosa

Pseudomonas aeruginosa, a ubiquitous environmental organism, is a difficult-to-treat opportunistic pathogen due to its broad-spectrum antibiotic resistance and its ability to form biofilms. In this study, we investigate the link between resistance to a clinically important antibiotic, imipenem, and biofilm formation. First, we observed that the laboratory strain P. aeruginosa PAO1 carrying a mutation in the oprD gene, which confers resistance to imipenem, showed a modest reduction in biofilm formation. We also observed an inverse relationship between imipenem resistance and biofilm formation for imipenem-resistant strains selected in vitro, as well as for clinical isolates. We identified two clinical isolates of P. aeruginosa from the sputum of cystic fibrosis patients that formed robust biofilms, but were sensitive to imipenem (MIC?≤?2 μg/ml). To test the hypothesis that there is a general link between imipenem resistance and biofilm formation, we performed transposon mutagenesis of these two clinical strains to identify mutants defective in biofilm formation, and then tested these mutants for imipenem resistance. Analysis of the transposon mutants revealed a role for previously described biofilm factors in these clinical isolates of P. aeruginosa, including mutations in the pilY1, pilX, pilW, algC, and pslI genes, but none of the biofilm-deficient mutants became imipenem resistant (MIC?≥?8 μg/ml), arguing against a general link between biofilm formation and resistance to imipenem. Thus, assessing biofilm formation capabilities of environmental isolates is unlikely to serve as a good predictor of imipenem resistance. We also discuss our findings in light of the limited literature addressing planktonic antibiotic resistance factors that impact biofilm formation.     

Partial purification and characterization of a mannuronan-specific alginate lyase fromPseudomonas aeruginosa

Production of a thick exopolysaccharide coat (alginate) by mucoid strains ofPseudomonas aeruginosa has been shown to contribute to the pathogenicity and persistence of these bacteria in the lungs of patients with cystic fibrosis. Previous studies have shown that some mucoidP. aeruginosa strains produce an enzyme(s) capable of degrading this alginate coat. In this study, an alginate lyase from mucoidP. aeruginosa strain WcM#2 was isolated and characterized. Lyase production was enhanced by the addition of 0.2–0.3m NaCl to the growth media. The lyase was eluted from an alginate-Sepharose affinity column with 0.5m NaCl, which can serve as a simple one-step purification protocol for obtaining semi-pure functional alginate lyase. Fractionation of the enzyme preparation on a Sephadex G-75 sizing column showed that the enzyme has an apparent molecular weight of 40,000, whereas sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) suggested a molecular weight of approximately 43,000. The affinity-purified enzyme had a pH optimum of 9.0, its activity was enhanced in the presence of 0.3m NaCl, and it showed substrate specificity for polymannuronic acid blocks. These results demonstrate the presence of a mannuronan-specific alginate lyase inP. aeruginosa that differs in several respects from previous reports ofP. aeruginosa alginate lyases.     

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.     

Role of the flagellar hook in the structural development and antibiotic tolerance of Pseudomonas aeruginosa biofilms

Pseudomonas aeruginosa biofilms exhibit an intrinsic resistance to antibiotics and constitute a considerable clinical threat. In cystic fibrosis, a common feature of biofilms formed by P. aeruginosa in the airway is the occurrence of mutants deficient in flagellar motility. This study investigates the impact of flagellum deletion on the structure and antibiotic tolerance of P. aeruginosa biofilms, and highlights a role for the flagellum in adaptation and cell survival during biofilm development. Mutations in the flagellar hook protein FlgE influence greatly P. aeruginosa biofilm structuring and antibiotic tolerance. Phenotypic analysis of the flgE knockout mutant compared to the wild type (WT) reveal increased fitness under planktonic conditions, reduced initial adhesion but enhanced formation of microcolony aggregates in a microfluidic environment, and decreased expression of genes involved in exopolysaccharide formation. Biofilm cells of the flgE knock-out mutant display enhanced tolerance towards multiple antibiotics, whereas its planktonic cells show similar resistance to the WT. Confocal microscopy of biofilms demonstrates that gentamicin does not affect the viability of cells located in the inner part of the flgE knock-out mutant biofilms due to reduced penetration. These findings suggest that deficiency in flagellar proteins like FlgE in biofilms and in cystic fibrosis infections represent phenotypic and evolutionary adaptations that alter the structure of P. aeruginosa biofilms conferring increased antibiotic tolerance.Subject terms: Microbiology, Diseases     

Mannitol Enhances Antibiotic Sensitivity of Persister Bacteria in Pseudomonas aeruginosa Biofilms

The failure of antibiotic therapies to clear Pseudomonas aeruginosa lung infection, the key mortality factor for cystic fibrosis (CF) patients, is partly attributed to the high tolerance of P. aeruginosa biofilms. Mannitol has previously been found to restore aminoglycoside sensitivity in Escherichia coli by generating a proton-motive force (PMF), suggesting a potential new strategy to improve antibiotic therapy and reduce disease progression in CF. Here, we used the commonly prescribed aminoglycoside tobramycin to select for P. aeruginosa persister cells during biofilm growth. Incubation with mannitol (10–40 mM) increased tobramycin sensitivity of persister cells up to 1,000-fold. Addition of mannitol to pre-grown biofilms was able to revert the persister phenotype and improve the efficacy of tobramycin. This effect was blocked by the addition of a PMF inhibitor or in a P. aeruginosa mutant strain unable to metabolise mannitol. Addition of glucose and NaCl at high osmolarity also improved the efficacy of tobramycin although to a lesser extent compared to mannitol. Therefore, the primary effect of mannitol in reverting biofilm associated persister cells appears to be an active, physiological response, associated with a minor contribution of osmotic stress. Mannitol was tested against clinically relevant strains, showing that biofilms containing a subpopulation of persister cells are better killed in the presence of mannitol, but a clinical strain with a high resistance to tobramycin was not affected by mannitol. Overall, these results suggest that in addition to improvements in lung function by facilitating mucus clearance in CF, mannitol also affects antibiotic sensitivity in biofilms and does so through an active, physiological response.     

Nitrosative stress inhibits production of the virulence factor alginate in mucoid Pseudomonas aeruginosa

Alginate is a critical virulence factor contributing to the poor clinical prognosis associated with the conversion of Pseudomonas aeruginosa to mucoid phenotypes in cystic fibrosis (CF). An important mechanism of action is its ability to scavenge host innate-immune reactive species. We have previously analyzed the bacterial response to nitrosative stress by S-nitrosoglutathione (GSNO), a physiological NO√ donor with diminished levels in the CF lung. GSNO substantially increased bacterial nitrosative and oxidative defenses and so we hypothesized a similar increase in alginate production would occur. However, in mucoid P. aeruginosa, there was decreased expression of the majority of alginate synthetic genes. This microarray data was confirmed both by RT-PCR and at the functional level by direct measurements of alginate production. Our data suggest that the lowered levels of innate-immune nitrosative mediators (such as GSNO) in the CF lung exacerbate the effects of mucoid P. aeruginosa, by failing to suppress alginate biosynthesis.     

Molecular modeling and redesign of alginate lyase from Pseudomonas aeruginosa for accelerating CRPA biofilm degradation

Administration of an efficient alginate lyase (AlgL) or AlgL mutant may be a promising therapeutic strategy for treatment of cystic fibrosis patients with Pseudomonas aeruginosa infections. Nevertheless, the catalytic activity of wild‐type AlgL is not sufficiently high. It is highly desired to design and discover an AlgL mutant with significantly improved catalytic efficiency against alginate substrates. For the purpose of identifying an AlgL mutant with significantly improved catalytic activity, in this study, we first constructed and validated a structural model of AlgL interacting with substrate, providing a better understanding of the interactions between AlgL and its substrate. Based on the modeling insights, further enzyme redesign and experimental testing led to discovery of AlgL mutants, including the K197D/K321A mutant, with significantly improved catalytic activities against alginate and acetylated alginate in ciprofloxacin‐resistant P. aeruginosa (CRPA) biofilms. Further anti‐biofilm activity assays have confirmed that the K197D/K321A mutant with piperacillin/tazobactam is indeed effective in degrading the CRPA biofilms. Co‐administration of the potent mutant AlgL and an antibiotic (such as a nebulizer) could be effective for therapeutic treatment of CRPA‐infected patients with cystic fibrosis. Proteins 2016; 84:1875–1887. © 2016 Wiley Periodicals, Inc.     

Optimized plant compound with potent anti-biofilm activity across gram-negative species

Many human diseases, including cystic fibrosis lung infections, are caused or exacerbated by bacterial biofilms. Specialized modes of motility, including swarming and twitching, allow gram-negative bacteria to spread across surfaces and form biofilms. Compounds that inhibit these motilities could slow the spread of biofilms, thereby allowing antibiotics to work better. We previously demonstrated that a set of plant-derived triterpenes, including oleanolic acid and ursolic acid, inhibit formation of Escherichia coli and Pseudomonas aeruginosa biofilms, and alter expression of genes involved in chemotaxis and motility. In the present study, we have prepared a series of analogs of oleanolic acid. The analogs were evaluated against clinical isolates of E. coli and P. aeruginosa in biofilm formation assays and swarming assays. From these analogs, compound 9 was selected as a lead compound for further development. Compound 9 inhibits E. coli biofilm formation at 4 µg/mL; it also inhibits swarming at ≤1 µg/mL across multiple clinical isolates of P. aeruginosa, E. coli, Burkholderia cepacia, and Salmonella enterica, and at <0.5 µg/mL against multiple agricultural strains. Compound 9 also potentiates the activity of the antibiotics tobramycin and colistin against swarming P. aeruginosa; this is notable, as tobramycin and colistin are inhaled antibiotics commonly used to treat P. aeruginosa lung infections in people with cystic fibrosis. qPCR experiments suggested that 9 alters expression of genes involved in regulating Type IV pili; western blots confirmed that expression of Type IV pili components PilA and PilY1 decreases in P. aeruginosa in the presence of 9.     

Role of alginate and its O acetylation in formation of Pseudomonas aeruginosa microcolonies and biofilms

Attenuated total reflection/Fourier transform-infrared spectrometry (ATR/FT-IR) and scanning confocal laser microscopy (SCLM) were used to study the role of alginate and alginate structure in the attachment and growth of Pseudomonas aeruginosa on surfaces. Developing biofilms of the mucoid (alginate-producing) cystic fibrosis pulmonary isolate FRD1, as well as mucoid and nonmucoid mutant strains, were monitored by ATR/FT-IR for 44 and 88 h as IR absorbance bands in the region of 2,000 to 1,000 cm(-1). All strains produced biofilms that absorbed IR radiation near 1,650 cm(-1) (amide I), 1,550 cm(-1) (amide II), 1,240 cm(-1) (P==O stretching, C---O---C stretching, and/or amide III vibrations), 1,100 to 1,000 cm(-1) (C---OH and P---O stretching) 1,450 cm(-1), and 1,400 cm(-1). The FRD1 biofilms produced spectra with an increase in relative absorbance at 1,060 cm(-1) (C---OH stretching of alginate) and 1,250 cm(-1) (C---O stretching of the O-acetyl group in alginate), as compared to biofilms of nonmucoid mutant strains. Dehydration of an 88-h FRD1 biofilm revealed other IR bands that were also found in the spectrum of purified FRD1 alginate. These results provide evidence that alginate was present within the FRD1 biofilms and at greater relative concentrations at depths exceeding 1 micrometer, the analysis range for the ATR/FT-IR technique. After 88 h, biofilms of the nonmucoid strains produced amide II absorbances that were six to eight times as intense as those of the mucoid FRD1 parent strain. However, the cell densities in biofilms were similar, suggesting that FRD1 formed biofilms with most cells at depths that exceeded the analysis range of the ATR/FT-IR technique. SCLM analysis confirmed this result, demonstrating that nonmucoid strains formed densely packed biofilms that were generally less than 6 micrometer in depth. In contrast, FRD1 produced microcolonies that were approximately 40 micrometer in depth. An algJ mutant strain that produced alginate lacking O-acetyl groups gave an amide II signal approximately fivefold weaker than that of FRD1 and produced small microcolonies. After 44 h, the algJ mutant switched to the nonmucoid phenotype and formed uniform biofilms, similar to biofilms produced by the nonmucoid strains. These results demonstrate that alginate, although not required for P. aeruginosa biofilm development, plays a role in the biofilm structure and may act as intercellular material, required for formation of thicker three-dimensional biofilms. The results also demonstrate the importance of alginate O acetylation in P. aeruginosa biofilm architecture.     

Cellular function of elastase in Pseudomonas aeruginosa: role in the cleavage of nucleoside diphosphate kinase and in alginate synthesis

Elastase is a major virulence factor in Pseudomonas aeruginosa that is believed to cause extensive tissue damage during infection in the human host. Elastase is secreted in non-mucoid P. aeruginosa. It is known that secretion of most virulence factors such as elastase, lipase, exotoxin A, etc., in P. aeruginosa is greatly reduced in alginate-secreting mucoid cells isolated from the lungs of cystic fibrosis (CF) patients. We have previously reported that in mucoid P. aeruginosaan intracellular protease cleaves the 16 kDa form of nucleoside diphosphate kinase (Ndk) to a truncated 12 kDa form. This smaller form is membrane associated and has been observed to form complexes with specific proteins to predominantly generate GTP, an important molecule in alginate synthesis. The main aim of this study was to purify and characterize this protease. The protease was purified by hydrophobic interaction chromatography of the crude extract of mucoid P. aeruginosa 8821, a CF isolate. Further analysis using a gelatin containing SDS–polyacrylamide gel detected the presence of a 103 kDa protease, which when boiled, migrated as a 33 kDa protein on a SDS–polyacrylamide gel. The first 10 amino acids from the N-terminus of the 33 kDa protease showed 100% identity to the mature form of elastase. An elastase-negative lasB ::Cm knock-out mutant in the mucoid 8821 background was constructed, and it showed a non-mucoid phenotype. This mutant showed the presence of only the 16 kDa form of Ndk both in the cytoplasm and membrane fractions. We present evidence for the retention of active elastase in the periplasm of mucoid P. aeruginosa and its role in the generation of the 12 kDa form of Ndk. Finally, we demonstrate that elastase, when overproduced in both mucoid and non-mucoid cells, stimulates alginate synthesis. This suggests that the genetic rearrangements that trigger mucoidy in P. aeruginosa also allow retention of elastase in the periplasm in an active oligomeric form that facilitates cleavage of 16 kDa Ndk to its 12 kDa form for the generation of GTP, required for alginate synthesis.