Genome-wide identification of spliced introns using a tiling microarray

The prediction of gene models from genome sequence remains an unsolved problem. One hallmark of eukaryotic gene structure is the presence of introns, which are spliced out of pre-mRNAs prior to translation. The excised introns are released in the form of lariats, which must be debranched prior to their turnover. In the yeast Saccharomyces cerevisiae, the absence of the debranching enzyme causes these lariat RNAs to accumulate. This accumulation allows a comparison of tiling array signals of RNA from the debranching mutant to the wild-type parent strain, and thus the identification of lariats on a genome-wide scale. This approach identified 141 of 272 known introns, confirmed three previously predicted introns, predicted four novel introns (of which two were experimentally confirmed), and led to the reannotation of four others. In many instances, signals from the tiling array delineated the 5' splice site and branchpoint site, confirming predicted gene structures. Nearly all introns that went undetected are present in mRNAs expressed at low levels. Overall, 97% of the significant probes could be attributed either to spliced introns or to genes up-regulated by deletion of the debranching enzyme. Because the debranching enzyme is conserved among eukaryotes, this approach could be generally applicable for the annotation of eukaryotic genes and the detection of novel and alternative splice forms.     

Rapid identification of mutations in a multidrug efflux pump in Pseudomonas aeruginosa

The gene mexR regulates negatively the expression of the MexA-MexB-OprM efflux pump in Pseudomonas aeruginosa, and mutations in mexR cause a multiple antibiotic resistance phenotype. Five hundred and forty resistant clones of P. aeruginosa PAO503 were isolated after selection for resistance to chloramphenicol or tetracycline. All isolates showed similar phenotypes and were resistant to tetracycline, chloramphenicol and norfloxacin. Nineteen randomly selected isolates were analyzed. Since mutational analysis by direct sequencing of all regions of interest in several strains is time-consuming and expensive, a screening method, Non-Isotopic RNase Cleavage Assay (NIRCA), was applied to identify mutant genes so that they could be targeted for DNA sequencing. NIRCA is a simple but rapid method for mutational analysis and can be performed in 3-4 h. Results of NIRCA analysis were compared with DNA sequencing. Both NIRCA and DNA sequencing analysis showed mexR gene mutations in 11 of 19 isolates but no alterations in 8 strains. An immunoblot assay showed overexpression of OprN, a component of another multidrug efflux pump, MexE-MexF-OprN, in those eight isolates. Nucleotide sequencing of quinolone resistance-determining regions of DNA gyrase (gyrA) or topoisomerase IV (parC) showed no alterations in any of the 19 mutants. The data indicate that two efflux pump systems, MexA-MexB-OprM and MexE-MexF-OprN, were involved in multidrug resistance including quinolones and that NIRCA is a sensitive method for screening mutations.     

Interaction of macrophage cationic proteins with the outer membrane of Pseudomonas aeruginosa.

The interaction of the polycationic rabbit alveolar macrophage cationic proteins MCP-1 and MCP-2 (or their identical neutrophil equivalents NP-1 and NP-2) with the surface of Pseudomonas aeruginosa was investigated. Both proteins bound avidly to purified lipopolysaccharide, as judged by their ability to competitively displace the probe dansyl polymyxin with 50% inhibition (I50) values of 2 to 3 microM. Similar I50 were measured with dansyl polymyxin as a probe for cell surface binding, suggesting that the initial binding site for MCP-1 and MCP-2 on the surface of cells was lipopolysaccharide. Both MCP-1 and MCP-2 permeabilized outer membranes to the hydrophobic fluorescent probe 1-N-phenylnaphthylamine (NPN). The initial rate of NPN uptake plotted against the concentration of MCP-1 or MCP-2 gave sigmoidal curves, suggesting cooperative permeabilization of the outer membrane. Replotting the data as a Hill plot gave an affinity parameter, S0.5, the concentration of MCP giving a half-maximal increase in the rate of NPN uptake, of 5 and 25 microM for MCP-1 and MCP-2, respectively, and thus subsequent studies concentrated on the more active permeabilizer MCP-1. Permeabilization of outer membranes to NPN was a function of buffer pH, with lower pH considerably favoring the permeabilizing effects of MCP-1. Thin-section electron microscopic visualization of MCP-1-treated cells showed production of extended blebs. Further evidence of an altered cell surface after MCP-1 treatment was obtained by demonstrating that treated unopsonized cells were more efficiently phagocytosed by unelicited rabbit alveolar macrophages. The data overall suggest that macrophage cationic proteins interact with the P. aeruginosa outer membrane in a manner typical of other polycations and suggest that one of their major functions may be to permeabilize the outer membrane.     

Identification of Francisella tularensis genes encoding exported membrane-associated proteins using TnphoA mutagenesis of a genomic library

Francisella tularensis, the causative agent of tularemia, is a highly infectious pathogen of humans and animals, yet little is known about the surface proteins of this organism that mediate mechanisms of pathogenicity. lambdaTnphoA was used to generate random alkaline phosphatase gene fusions in a F. tularensis subsp. tularensis (strain Schu S4) genomic library to identify genes encoding exported extracytoplasmic proteins. Eleven genes encoding membrane-associated proteins were identified by this method and their respective signal peptides were characterized. Three of the genes encoded conserved 'housekeeping' enzymes, while the other eight genes were unique to F. tularensis, encoding proteins with molecular masses ranging from 11 to 78kDa as deduced from the amino acid sequences. Two genes putatively encoded lipoproteins based on the presence of characteristic signal peptidase II cleavage sites. Four selected proteins were found associated with outer membranes from Schu S4 and LVS strains by Western blotting. Indirect immunofluorescence of strain Schu S4 cells also showed evidence of protein localization to the outer membrane. Protein database searches produced significant alignments with proteins from other bacteria involved in carbohydrate transport, lipid metabolism, and cell envelope biogenesis, thereby providing clues for putative functions. These findings demonstrated that TnphoA mutagenesis can be used in conjunction with F. tularensis genome sequence data to provide a foundation for studies to identify and define cellular surface protein virulence factors of this pathogen.     

Protection of immunocompromised mice against lethal infection with Pseudomonas aeruginosa by active or passive immunization with recombinant P. aeruginosa outer membrane protein F and outer membrane protein I fusion proteins.

Recombinant outer membrane proteins (Oprs) of Pseudomonas aeruginosa were expressed in Escherichia coli as glutathione S-transferase (GST)-linked fusion proteins. GST-linked Oprs F and I (GST-OprF190-350 [GST linked to OprF spanning amino acids 190 to 350] and GST-OprI21-83, respectively) and recombinant hybrid Oprs (GST-OprF190-342-OprI21-83 and GST-OprI21-83-OprF190-350) were isolated and tested for their efficacy as vaccines in immunodeficient mice. GST-OprF-OprI protected the mice against a 975-fold 50% lethal dose of P. aeruginosa. Expression of GST-unfused OprF-OprI failed in E. coli, although this hybrid protein has been expressed without a fusion part in Saccharomyces cerevisiae and used for immunizing rabbits. The immune rabbit sera protected severe combined deficient (SCID) mice against a 1,000-fold 50% lethal dose of P. aeruginosa. Evidence is provided to show that the most C-terminal part of OprF (i.e., amino acids 332 to 350) carries an important protective epitope. Opr-based hybrid proteins may have implications for a clinical vaccine against P. aeruginosa.     

Neutralization of Pseudomonas aeruginosa enzymatic activity by antibodies elicited with proteins of Larrea divaricata Cav

Larrea divaricata Cav. (Jarilla) is a bush widely used in folk therapy for the treatment of several pathologies. Partially purified proteins of crude extract (JPCE) cross-react with proteins of Gram-negative bacteria, including Pseudomonas aeruginosa, which is an opportunistic pathogen that causes several intrahospitalary infections. This bacterium produces many proteins with enzymatic activity, including hemolysins and proteases that play a major role in acute infection caused by this bacterium. The aim of our work was to investigate if antibodies against with L. divaricata neutralize the hemolytic and proteolytic activity of P. aeruginosa. The hemolytic activity of soluble cellular proteins was inhibited 100% and extracellular proteins (EP) showed an inhibition between 44 and 95% when both bacterial fractions were treated with anti-JPCE serum. Also, in EP the neutralization was directed towards the active site of the hemolysin. When protease activity of extracellular products was tested, bands of 217, 155, 121, 47 and 27?kDa were observed in native zymograms. Neutralization between 55 and 70% of the bands of 217, 155 and 121?kDa was observed when EP were treated with anti-JPCE serum. In conclusion, our data clearly demonstrate that antibodies elicited with L. divaricata' proteins are able to neutralize the hemolytic and proteolytic activity of P. aeruginosa cellular and extracellular proteins. Our study constitutes the first report that associates the immunogenicity of plant proteins and bacterial proteins with enzymatic activity. These findings could be relevant in the development of alternatives therapies for patients suffering intrahospitalary opportunistic infections with P. aeruginosa.     

Neutralization of Pseudomonas aeruginosa enzymatic activity by antibodies elicited with proteins of Larrea divaricata Cav.

Larrea divaricata Cav. (Jarilla) is a bush widely used in folk therapy for the treatment of several pathologies. Partially purified proteins of crude extract (JPCE) cross-react with proteins of Gram-negative bacteria, including Pseudomonas aeruginosa, which is an opportunistic pathogen that causes several intrahospitalary infections. This bacterium produces many proteins with enzymatic activity, including hemolysins and proteases that play a major role in acute infection caused by this bacterium. The aim of our work was to investigate if antibodies against with L. divaricata neutralize the hemolytic and proteolytic activity of P. aeruginosa. The hemolytic activity of soluble cellular proteins was inhibited 100% and extracellular proteins (EP) showed an inhibition between 44 and 95% when both bacterial fractions were treated with anti-JPCE serum. Also, in EP the neutralization was directed towards the active site of the hemolysin. When protease activity of extracellular products was tested, bands of 217, 155, 121, 47 and 27?kDa were observed in native zymograms. Neutralization between 55 and 70% of the bands of 217, 155 and 121?kDa was observed when EP were treated with anti-JPCE serum. In conclusion, our data clearly demonstrate that antibodies elicited with L. divaricata’ proteins are able to neutralize the hemolytic and proteolytic activity of P. aeruginosa cellular and extracellular proteins. Our study constitutes the first report that associates the immunogenicity of plant proteins and bacterial proteins with enzymatic activity. These findings could be relevant in the development of alternatives therapies for patients suffering intrahospitalary opportunistic infections with P. aeruginosa.     

Alkane biodegradation in Pseudomonas aeruginosa strains isolated from a polluted zone: identification of alkB and alkB-related genes

Pseudomonas aeruginosa strains that grow on crude oil as the sole source of carbon and energy were isolated from an environment in Morocco polluted by petroleum refinery effluents. The twenty isolates grew on saturated alkanes from C12 to C22. Three of the isolates were also able to grow on low molecular weight C6 to C10 n-alkanes, but the other 17 strains were not. The strains were tested for alkB and a/kB-related genes encoding alkane-1-monooxygenase (alkane hydroxylase). Oligonucleotide primers specific for the alkB gene of strain P. putida (GPo1 ) and for the alkB1 and alkB2 genes of P. aeruginosa strain PAO1 allowed amplification from the P. aeruginosa isolates of fragments similar to alkB1 and alkB2 genes of strain PAO1. Only 3 strains carried an alkB gene very similar to that of strain GPo1, and these strains were the same ones that could utilise C6 to C10 n-alkanes.     

Interaction of a rat lung lectin with the exopolysaccharides of Pseudomonas aeruginosa.

The specific interaction between the exopolysaccharide purified from a number of Pseudomonas aeruginosa isolates from cystic fibrosis patients and a rat lung heparin-lectin was assayed. The polysaccharide prepared from Homma serotypes M, B, I, and G did not act as hapten inhibitors of lectin activity, whereas the polymers prepared from ca. 80% of strains that did not type with Homma serum did act as hapten inhibitors. Inhibition was shown not to be due to lipopolysaccharide. The infrared spectrums of both inhibitory and noninhibitory polymers appeared very similar, although small amounts of glucose and an unidentified amino sugar were found only in the nontypable strains. This evidence suggests that rat lung lectin recognizes and distinguishes a specific type of alginate-like polymer prevalent on the Homma nontypable P. aeruginosa.     

Determination of IgG subclass antibodies to Pseudomonas aeruginosa outer membrane proteins in cystic fibrosis lung infection using immunoblotting and enzyme-linked immunosorbent assay

IgG subclass antibodies to Pseudomonas aemginosa outer membrane proteins (OMP) were investigated in serum from cystic fibrosis (CF) patients by immunoblotting and enzyme-linked immunosorbent assay (ELISA). Fifteen patients (eight in good and seven in poor clinical condition) have been followed for an average of 13 years with multiple serum samples covering the preinfection, and early and late stages of chronic infection. Laser-scanning densitometry of photographs taken from immunoblots was used to quantify antibody level and compare with ELISA titres. The earliest anti-OMP antibodies to appear were of the IgG1 subclass. There was no significant difference in IgG subclass antibody levels to OMPs between patients in relatively good and poor clinical condition. Data presented indicate a high positive correlation among measurements of IgG subclass antibodies to P. aeruginosa OMPs using ELISA and immunoblotting.     

Factors Associated with Improved Outcome of Pseudomonas aeruginosa Bacteremia in a Finnish University Hospital

 All 134 episodes of bacteremia caused solely by Pseudomonas aeruginosa in a university hospital in the periods 1976–1982 and 1992–1996 were reviewed retrospectively to determine the clinical manifestations, outcome and prognostic factors. The mortality for the 30-day interval after drawing the first positive blood culture was 41%, but dropped from 53% in the first period to 29% in the second period (P=0.006). Mortality was highest in patients treated with an aminoglycoside only, as against those treated with other appropriate antibiotics (55% versus 25%, P=0.001). Over the two decades studied, use of an aminoglycoside only decreased, use of paracetamol (=acetaminophen) increased, and removal of both urinary and blood vessel catheters became more common. The mortality was 18% in patients with catheter removal (46% in the other patients, P=0.017) and 27% in patients who received paracetamol around the time of drawing the first positive blood culture (50% for the other patients, P=0.010). Logistic regression analysis showed that shock, central nervous system involvement, preceding thromboembolism and rapidly fatal underlying disease were associated with a fatal outcome, whereas catheter removal, appropriate antibiotic therapy and paracetamol therapy were associated with survival. The improved prognosis of Pseudomonas aeruginosa bacteremia over the two decades is thus due mainly to three changes in management of the infection: the more frequent use of new anti-pseudomonal β-lactams and ciprofloxacin instead of aminoglycosides as monotherapy; the more frequent practice of removing catheters; and the increased use of paracetamol around the time of drawing the first positive blood sample.     

The adsorption of Pseudomonas aeruginosa pilus-dependent bacteriophages to a host mutant with nonretractile pili

Pseudomonas aeruginosa strain K is the host for two pilus-dependent bacteriophages (Pf, filamentous, and PO4, long noncontractile tail). This paper describes the isolation of a strain K mutant, which is resistant to both phages and yet bears many pili serologically similar to those of its parent. The efficiency of adsorption by the mutant was found to be close to that of strain K for each phage. The location of adsorbed P04 virions along the sides of the mutant's pili was consistent with the hypothesis that the pili were nonretractile. Pilus length measurements on strain K and the mutant showed no change in distribution before and after phage adsorption. This result is discussed in detail with reference to possible models for P04 penetration.     

Enhancement of Pseudomonas aeruginosa lung clearance after local immunization with a temperature-sensitive mutant.

We investigated the capacity of the temperature-sensitive mutant strain A/10/25 of Pseudomonas aeruginosa (ts-Psa) to induce enhancement of lung defenses against wild type P. aeruginosa (wt-Psa). Mice of the DBA/2J inbred strain were immunized by aerosolization with a single dose of 2 x 10(5) to 4 x 10(5) CFU of ts-Psa and were challenged 7, 14, and 21 days later with wt-Psa. The uncleared bacteria ratio was determined 4 h after aerosol exposure; significant enhancement in lung clearance of wt-Psa (P less than 0.01) was evident as early as 7 days after immunization and detectable for at least 21 days. Aerosol immunization with Staphylococcus aureus did not enhance lung clearance of wt-Psa; however, slight but significant enhancement in S. aureus clearance was observed in mice immunized 7 days before with ts-Psa. No enhancement of S. aureus clearance was seen in ts-Psa immunized animals after 14 and 21 days. Analysis of the cell composition of lung lavage fluids revealed a transient cell response characterized by rapid increase in the absolute number of polymorphonuclear leukocytes, followed later by an increase in alveolar macrophages. The characteristics of lung lavages returned to base-line values 6 days after aerosol immunization, and a second exposure to a ts-Psa aerosol produced a response of similar magnitude and quality. We conclude that aerosol immunization with a temperature-sensitive mutant of P. aeruginosa enhances specific pulmonary defense mechanisms against the parental pathogen in mice.     

A metric for the stiffness of calcified aortic valves using a combined computational and experimental approach

Calcific aortic valve disease is the most common heart valve disease. It is associated with a significant increase in cardiovascular morbidity and mortality and independently increases the cardiovascular risk. It is then important to develop parameters that can estimate the stiffness of the valve. Such parameters may contribute to early detection of the disease or track its progression and optimize the timing for therapy. In this study, we introduce a metric representing the stiffness of the native aortic calcified valve over a wide range of stenosis severities. Our approach is based on three-dimensional structural finite-element simulations and in vitro measurements. The proposed method is developed first in a pulse duplicator; its clinical applicability is then evaluated in three patients with severe aortic stenosis. Our results indicate that the value of the proposed metric varies considerably between healthy valves and valves with very severe aortic stenosis, from 0.001 to 7.38 MPa, respectively. The method introduced in this study could give useful information regarding the stiffness of the valve leaflets with potential application to the evaluation of aortic sclerosis and aortic stenosis.     

Electron microscopic identification of aberrant melanosomes using a combined dopa/Warthin-Starry technique

A method combining the DOPA and Warthin-Starry techniques is described in order to positively establish the nature of pleomorphic granules observed in the cytoplasm of cells of putative amelanotic melanoma. The technique identifies these granules as aberrant melanosomes by discretely depositing electron dense silver on suitably prepared sections of DOPA-treated tissue blocks.     

Genetic characterization indicates that a specific subpopulation of Pseudomonas aeruginosa is associated with keratitis infections

Pseudomonas aeruginosa is a common opportunistic bacterial pathogen that causes a variety of infections in humans. Populations of P. aeruginosa are dominated by common clones that can be isolated from diverse clinical and environmental sources. To determine whether specific clones are associated with corneal infection, we used a portable genotyping microarray system to analyze a set of 63 P. aeruginosa isolates from patients with corneal ulcers (keratitis). We then used population analysis to compare the keratitis isolates to a wider collection of P. aeruginosa from various nonocular sources. We identified various markers in a subpopulation of P. aeruginosa associated with keratitis that were in strong disequilibrium with the wider P. aeruginosa population, including oriC, exoU, katN, unmodified flagellin, and the carriage of common genomic islands. The genome sequencing of a keratitis isolate (39016; representing the dominant serotype O11), which was associated with a prolonged clinical healing time, revealed several genomic islands and prophages within the accessory genome. The PCR amplification screening of all 63 keratitis isolates, however, provided little evidence for the shared carriage of specific prophages or genomic islands between serotypes. P. aeruginosa twitching motility, due to type IV pili, is implicated in corneal virulence. We demonstrated that 46% of the O11 keratitis isolates, including 39016, carry a distinctive pilA, encoding the pilin of type IV pili. Thus, the keratitis isolates were associated with specific characteristics, indicating that a subpopulation of P. aeruginosa is adapted to cause corneal infection.     

Direct identification of major blood culture pathogens, including Pseudomonas aeruginosa and Escherichia coli, by a panel of fluorescence in situ hybridization assays using peptide nucleic acid probes

Rapid identification of four major pathogens from 1,231 positive blood cultures by fluorescence in situ hybridization with peptide nucleic acid probes (AdvanDx Inc., Woburn, Mass.) was evaluated. For Escherichia coli, Staphylococcus aureus, and Candida albicans results agreed with conventional identification. The lower sensitivity of the Pseudomonas aeruginosa assay should not compromise the utility of the four assays.     

Unveiling the early events of Pseudomonas aeruginosa adaptation in cystic fibrosis airway environment using a long-term in vitro maintenance

Pseudomonas aeruginosa chronic infections are the major cause of high morbidity and mortality in cystic fibrosis (CF) patients due to the use of sophisticated mechanisms of adaptation, including clonal diversification into specialized CF-adapted phenotypes. In contrast to chronic infections, very little is known about what occurs after CF lungs colonization and at early infection stages.This study aims to investigate the early events of P. aeruginosa adaptation to CF environment, in particular, to inspect the occurrence of clonal diversification at early stages of infection development and its impact on antibiotherapy effectiveness.To mimic CF early infections, three P. aeruginosa strains were long-term grown in artificial sputum (ASM) over 10 days and phenotypic diversity verified through colony morphology characterization. Biofilm sub- and inhibitory concentrations of ciprofloxacin were applied to non- and diversified populations to evaluate antibiotic effectiveness on P. aeruginosa eradication.Our results demonstrated that clonal diversification might occur after ASM colonization and growth. However, this phenotypic diversification did not compromise ciprofloxacin efficacy in P. aeruginosa eradication since a biofilm minimal inhibitory dosage would be applied. The expected absence of mutators in P. aeruginosa populations led us to speculate that clonal diversification in the absence of ciprofloxacin treatments could be driven by niche specialization. Yet, biofilm sub-inhibitory concentrations of ciprofloxacin seemed to overlap niche specialization as “fitter” variants emerged, such as mucoid, small colony and pinpoint variants, known to be highly resistant to antibiotics. The pathogenic potential of all emergent colony morphotypes-associated bacteria, distinct from the wild-morphotypes, revealed that P. aeruginosa evolved to a non-swimming phenotype. Impaired swimming motility seemed to be one of the first evolutionary steps of P. aeruginosa in CF lungs that could pave the way for further adaptation steps including biofilm formation and progress to chronic infection. Based on our findings, impaired swimming motility seemed to be a candidate to disease marker of P. aeruginosa infection development. Despite our in vitro CF model represents a step forward towards in vivo scenario simulation and provided valuable insights about the early events, more and distinct P. aeruginosa strains should be studied to strengthen our results.