François Sanschagrin

Laval University, Quebec City, Quebec, Canada

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Publications (55)218.18 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Estrogen receptor (ER) tumor's status is critical for breast cancer management. A new rabbit antibody clone, EP1, is now available for ER status determination. The objective was to validate the EP1 antibody clone for its use in breast cancer ER status determination in a clinical setting against the previous standard, SP1. EP1 clone was assessed in 130 consecutive cases, including 50 ER-negative (<1% ER expression), 13 ER-low-positive (1% to 9% ER expression), and 67 ER-positive (≥10% ER expression). Using EP1 versus SP1, positive agreement (sensibility) was 92.5% and negative agreement (specificity) was 100%, leading to an overall agreement of 95.4%. All discordant cases (n=6) were ER-low-positive. SP1 was remeasured in 13 ER-low-positive and in 11 ER-negative cases. Overall agreement between SP1 initial tumor status and reassessment was 70.8% in those negative and low-positive cases. In conclusion, EP1 antibody has been validated for use in breast cancer with a positive agreement ≥90% and a negative agreement ≥95%, as recommended. Also, overall agreement between EP1 and SP1 was as good as between the SP1 initial status and SP1 reassessment.
    Applied immunohistochemistry & molecular morphology: AIMM / official publication of the Society for Applied Immunohistochemistry 06/2014; 22(8). DOI:10.1097/PAI.0000000000000001 · 2.06 Impact Factor
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    ABSTRACT: Purpose. Overexpression of hypoxia inducible factor-1 alpha (HIF-1α) has been found in several cancers and is thought to correlate with aggressive disease. The purpose of our study was to investigate the influence of HIF-1α on clinical outcome in uveal melanoma (UM) along with proliferative (MIB-1) and vascular (CD31, VEGF-A) markers. Methods. A retrospective analysis was carried out on UM tumors from 88 patients. HIF-1α, MIB-1, CD31 and VEGF-A expression, as well as necrosis were assessed by immunohistochemistry and hematoxylin/eosin (H&E) on paraffin-embedded UM tumor sections using a Tissue Microarray (TMA). The bivariate analysis involving HIF-1α expression and clinicopathological covariates was performed using the Chi-square test. The association of clinicopathological covariates and HIF-1α expression with patient survival was evaluated using the Kaplan-Meier approach and Cox proportional-hazards regression analysis. Results. Among our study population, 56 patients (63.6%) had high levels of HIF-1α expression. High expression of HIF-1α was associated with high expression of MIB-1 (P=0.04), CD31 (P=0.03) and VEGF-A (P<0.0001), as well as necrosis (P=0.04). However, high HIF-1α expression was not correlated with cell type, largest macroscopic tumor dimension or thickness, anterior margin, pigmentation or mitotic figures. Patients with high HIF-1α expression did not showed a reduced survival when compared to patients with low HIF-1α expression (P=0.92). Finally, HIF-1α expression was not increased after irradiation. Conclusions. An increase in HIF-1α expression is significantly associated with proliferative (MIB-1) and vascular (CD31 and VEGF-A) markers, as well as necrosis in UM. However, there is no correlation between high HIF-1α expression and patient survival.
    Investigative ophthalmology & visual science 01/2014; 55(3). DOI:10.1167/iovs.13-13345 · 3.66 Impact Factor
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    ABSTRACT: Amplification of the human epidermal growth factor receptor 2 (HER2) is a prognostic marker for poor clinical outcome and a predictive marker for therapeutic response to targeted therapies in breast cancer patients. With the introduction of anti-HER2 therapies, accurate assessment of HER2 status has become essential. Fluorescence in situ hybridization (FISH) is a widely used technique for the determination of HER2 status in breast cancer. However, the manual signal enumeration is time-consuming. Therefore, several companies have developed automated image analysis software like MetaSystems. Some of these signal enumeration software employ the so called "tile-sampling classifier", a programming algorithm through which the software quantifies fluorescent signals in images on the basis of square tiles of fixed dimensions. Considering that the size of tile does not always correspond to the size of a single tumor cell nucleus, some users argue that this analysis method might not completely reflect the biology of cells. For that reason, MetaSystems has developed a new classifier which is able to recognize nuclei within tissue sections in order to determine the HER2 amplification status on nuclei basis. We call this new programming algorithm "nucleisampling classifier". In this study, we evaluated the accuracy of the "nuclei-sampling classifier" in determining HER2 gene amplification by FISH in nuclei of breast cancer cells. To this aim, we randomly selected from our cohort 64 breast cancer specimens (32 nonamplified and 32 amplified) and we compared results obtained through manual scoring and through this new classifier. The new classifier automatically recognized individual nuclei. The automated analysis was followed by an optional human correction, during which the user interacted with the software in order to improve the selection of cell nuclei automatically selected. Overall concordance between manual scoring and automated nucleisampling analysis was 98.4% (100% for nonamplified cases and 96.9% for amplified cases). However, after human correction, concordance between the two methods was 100%. We conclude that the nuclei-based classifier is a new available tool for automated quantitative HER2 FISH signals analysis in nuclei in breast cancer specimen and it can be used for clinical purposes. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1755654848482838.
    Diagnostic Pathology 02/2013; 8(1):17. DOI:10.1186/1746-1596-8-17 · 2.41 Impact Factor
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    ABSTRACT: Liquid chromatography mass spectrometry (LCMS) is a powerful technique that could serve to rapidly characterize cell culture protein expression profile and be used as a process monitoring and control tool. However, this application is often hampered by both the sample proteome and the LCMS signal complexities as well as the variability of this signal. To alleviate this problem, culture samples are usually extensively fractionated and pretreated before being analyzed by top-end instruments. Such an approach precludes LCMS usage for routine on-line or at-line application. In this work, by applying multivariate analysis (MA) directly on raw LCMS signals, we were able to extract relevant information from cell culture samples that were simply lyzed. By using the recombinant adenovirus production process as a model, we were able to follow the accumulation of the three major proteins produced, identified their accumulation dynamics, and draw useful conclusions from these results. The combination of LCMS and MA provides a simple, rapid, and precise means to monitor cell culture.
    Applied biochemistry and biotechnology 05/2012; 167(3):474-88. DOI:10.1007/s12010-012-9661-4 · 1.69 Impact Factor
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    ABSTRACT: Neutral-buffered formalin is the most commonly used tissue fixative in pathology laboratory. Among other fixatives, Bouin's solution has been used in several laboratories and is still in use for particular tissues. In this project, we determine if we can study breast clinical markers on archived Bouin-fixed tissue samples with immunohistochemistry (IHC) protocols optimized for tissue fixed in neutral-buffered formalin. To evaluate the concordance of IHC results between formalin-fixed and Bouin-fixed tissues, we calculated the concordance percentage and the κ statistic of 12 clinical IHC markers quantified by an automated system on breast cancer tissues fixed in neutral-buffered formalin and their corresponding tissues fixed in Bouin's solution. When positivity threshold of immunostaining was setup at ≥10% for both fixation conditions, we observed a concordance percentage of 83.9% (κ=0.65). However, when positivity threshold of immunostaining was lowered to 3% to 4% for Bouin-fixed tissues, concordance percentage was then of 96.8% (κ=0.92). Our data demonstrate that we can study IHC markers on archived Bouin-fixed tissue from patients with long clinical follow-up using IHC protocols optimized for formalin-fixed tissues after an adjustment of the positivity threshold of immunostaining quantified by an automated system.
    Applied immunohistochemistry & molecular morphology: AIMM / official publication of the Society for Applied Immunohistochemistry 10/2010; 19(2):168-72. DOI:10.1097/PAI.0b013e3181f104a1 · 2.06 Impact Factor
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    ABSTRACT: We developed an assay to quantify DNA methylation in breast cancer cells isolated by laser capture microdissection (LCM). The assay uses methylation sensitive restriction enzyme (MSRE) digestion and quantitative polymerase chain reaction (qPCR). To assess the validity and precision of the assay, we prepared standard samples with expected methylation percentage (MP) for two gene promoters (PLAU (plasminogen inhibitor, urokinase) and TIMP3 (TIMP metallopeptidase inhibitor 3)) that we compared with measured MPs. We found good linearity of MSRE digestion and qPCR procedures for both promoters (beta=0.90-1.19+/-0.05-0.10 and r=0.95-0.98; all P<0.0001). Moreover, results remained similar after addition of a purification step between MSRE digestion and qPCR procedures. The validity of this technique was also confirmed by successfully replicating previously published MPs of four cell lines for PLAU and TIMP3 promoters. We assessed the consistency of our approach by comparing MPs of PLAU and TIMP3 promoters from nine breast cancer patients and two cell lines using LCM frozen tissues and their corresponding formalin-fixed paraffin-embedded tissues. We found good consistency (intraclass correlation coefficient=0.93) of MPs between frozen tissues and formalin-fixed paraffin-embedded tissues. Our data demonstrate that this assay based on digestion with MSRE and qPCR procedures is a good technique to quantify MP on limited amounts of DNA and may find clinical applications.
    Experimental and Molecular Pathology 10/2009; 88(1):184-9. DOI:10.1016/j.yexmp.2009.09.020 · 2.88 Impact Factor
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    ABSTRACT: The enzyme kinetics of the amide ligase MurE, a cell wall biosynthesis enzyme, from Pseudomonas aeruginosa were determined using the synthesized nucleotide substrate UDP-MurNAc-Ala-Glu (uridine 5'-diphosphoryl N-acetylmuramoyl-L-alanyl-D-glutamate). When coupled to a competitive bio-panning technique using a M13 phage display library encoding approximately 2.7 x 10(9) random peptide permutations and the specific substrates meso-A2pm (meso-diaminopimelic acid) and ATP, a peptide inhibitor of MurE was identified. The MurEp1 dodecamer selected and synthesized inhibited MurE ATPase activity with an IC(50) value of 500 microM. The inhibition was shown to be time-dependent and was reversed by the addition of meso-A2pm or UDP-MurNAc-Ala-Glu during the pre-incubation step. Kinetic analysis defined MurEp1 as a mixed inhibitor against both substrates with K(i) values of 160 and 80 microM respectively. MurEp1 was found to interfere in meso-A2pm and UDP-MurNAc-Ala-Glu binding necessary for amide bond formation. Modelling of Ps. aeruginosa MurE and docking of MurEp1 on the Ps. aeruginosa MurE surface indicated that MurEp1 binds at the juxtaposition of both meso-A2pm- and UDP-MurNAc-Ala-Glu-binding sites in the closed conformational state of the enzyme. Identification of the MurEp1 residues involved in MurE binding and inhibition will allow the development of a novel class of inhibitors having a novel mode of action against MurE.
    Biochemical Journal 05/2009; 421(2):263-72. DOI:10.1042/BJ20081395 · 4.78 Impact Factor
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    ABSTRACT: Pseudomonas aeruginosa is a ubiquitous environmental Gram-negative bacterium that is also a major opportunistic human pathogen in nosocomial infections and cystic fibrosis chronic lung infections. PhoP-PhoQ is a two-component regulatory system that has been identified as essential for virulence and cationic antimicrobial peptide resistance in several other Gram-negative bacteria. This study demonstrated that mutation of phoQ caused reduced twitching motility, biofilm formation and rapid attachment to surfaces, 2.2-fold reduced cytotoxicity to human lung epithelial cells, substantially reduced lettuce leaf virulence, and a major, 10 000-fold reduction in competitiveness in chronic rat lung infections. Microarray analysis revealed that PhoQ controlled the expression of many genes consistent with these phenotypes and with its known role in polymyxin B resistance. It was also demonstrated that PhoQ controls the expression of many genes outside the known PhoP regulon.
    Microbiology 04/2009; 155(Pt 3):699-711. DOI:10.1099/mic.0.024554-0 · 2.84 Impact Factor
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    ABSTRACT: Pseudomonas aeruginosa isolates have a highly conserved core genome representing up to 90% of the total genomic sequence with additional variable accessory genes, many of which are found in genomic islands or islets. The identification of the Liverpool Epidemic Strain (LES) in a children's cystic fibrosis (CF) unit in 1996 and its subsequent observation in several centers in the United Kingdom challenged the previous widespread assumption that CF patients acquire only unique strains of P. aeruginosa from the environment. To learn about the forces that shaped the development of this important epidemic strain, the genome of the earliest archived LES isolate, LESB58, was sequenced. The sequence revealed the presence of many large genomic islands, including five prophage clusters, one defective (pyocin) prophage cluster, and five non-phage islands. To determine the role of these clusters, an unbiased signature tagged mutagenesis study was performed, followed by selection in the chronic rat lung infection model. Forty-seven mutants were identified by sequencing, including mutants in several genes known to be involved in Pseudomonas infection. Furthermore, genes from four prophage clusters and one genomic island were identified and in direct competition studies with the parent isolate; four were demonstrated to strongly impact on competitiveness in the chronic rat lung infection model. This strongly indicates that enhanced in vivo competitiveness is a major driver for maintenance and diversifying selection of these genomic prophage genes.
    Genome Research 01/2009; 19(1):12-23. DOI:10.1101/gr.086082.108 · 13.85 Impact Factor
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    ABSTRACT: To develop antibacterial agents having novel modes of action against bacterial cell wall biosynthesis, we targeted the essential MurF enzyme of the antibiotic resistant pathogen Pseudomonas aeruginosa. MurF catalyzes the formation of a peptide bond between D-Alanyl-D-Alanine (D-Ala-D-Ala) and the cell wall precursor uridine 5'-diphosphoryl N-acetylmuramoyl-L-alanyl-D-glutamyl-meso-diaminopimelic acid (UDP-MurNAc-Ala-Glu-meso-A2pm) with the concomitant hydrolysis of ATP to ADP and inorganic phosphate, yielding UDP-N-acetylmuramyl-pentapeptide. As MurF acts on a dipeptide, we exploited a phage display approach to identify peptide ligands having high binding affinities for the enzyme. Screening of a phage display 12-mer library using purified P. aeruginosa MurF yielded to the identification of the MurFp1 peptide. The MurF substrate UDP-MurNAc-Ala-Glumeso-A2pm was synthesized and used to develop a sensitive spectrophotometric assay to quantify MurF kinetics and inhibition. MurFp1 acted as a weak, time-dependent inhibitor of MurF activity but was a potent inhibitor when MurF was pre-incubated with UDP-MurNAc-Ala-Glu-meso-A2pm or ATP. In contrast, adding the substrate D-Ala-D-Ala during the pre-incubation nullified the inhibition. The IC50 value of MurFp1 was evaluated at 250 microM, and the Ki was established at 420 microM with respect to the mixed type of inhibition against D-Ala-D-Ala. MurFp1 exerts its inhibitory action by interfering with the utilization of D-Ala-D-Ala by the MurF amide ligase enzyme. We propose that MurFp1 exploits UDP-MurNAc-Ala-Glu-meso-A2pm-induced structural changes for better interaction with the enzyme. We present the first peptide inhibitor of MurF, an enzyme that should be exploited as a target for antimicrobial drug development.
    BMC Biochemistry 01/2009; 9:33. DOI:10.1186/1471-2091-9-33 · 1.94 Impact Factor
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    ABSTRACT: The human opportunistic pathogen Pseudomonas aeruginosa is the major cause of morbidity and mortality of cystic fibrosis patients and is responsible for a variety of infections in compromised hosts. Using PCR-based signature-tagged mutagenesis, we identified a P. aeruginosa STM5437 mutant with an insertion into the PA5437 gene (called pycR for putative pyruvate carboxylase regulator). PycR inactivation results in 100,000-fold attenuation of virulence in the rat lung in vivo. PycR has the signature of a transcriptional regulator with a predicted helix-turn-helix motif binding to a typical LysR DNA binding site in the PA5436 (pycA)-PA5437 (pycR) intercistronic region. Two pyruvate carboxylase subunits (pycA and pycB) are divergently transcribed upstream of pycR. Transcriptional start sites of pycR and pycA are located at -127 and -88 bp upstream of their initiation codons with Shine-Dalgarno and putative promoter sequences containing -10 and -35 sequences. The DNA binding of PycR was confirmed by DNA mobility shift assay. Genome-wide transcriptional profiling and quantitative real-time PCR (qRT-PCR) indicated that the genes differentially regulated by PycR include two pyruvate carboxylase genes and genes necessary for lipid metabolism, lipolytic activity, anaerobic respiration and biofilm formation. PycR is a regulator with pleiotropic effects on virulence factors, such as lipase and esterase expression and biofilm formation, which are important for maintenance of P. aeruginosa in chronic lung infection.
    Microbiology 08/2008; 154(Pt 7):2106-18. DOI:10.1099/mic.0.2007/011239-0 · 2.84 Impact Factor
  • Journal of Cystic Fibrosis 06/2008; 7. DOI:10.1016/S1569-1993(08)60129-0 · 3.82 Impact Factor
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    ABSTRACT: Pseudomonas aeruginosa chronic lung infections are the major cause of morbidity and mortality in cystic fibrosis (CF) patients. The P. aeruginosa strains PAO1 and PA14 were compared with the Liverpool epidemic strain LESB58 to assess in vivo growth, infection kinetics, and bacterial persistence and localization within tissues in a rat model of chronic lung infection. The three P. aeruginosa strains demonstrated similar growth curves in vivo but differences in tissue distribution. The LESB58 strain persisted in the bronchial lumen, while the PAO1 and PA14 strains were found localized in the alveolar regions and grew as macrocolonies after day 7 postinfection. Bacterial strains were compared for swimming and twitching motility and for the production of biofilm. The P. aeruginosa LESB58 strain produced more biofilm than PAO1 and PA14. Competitive index (CI) analysis of PAO1, PA14, and LESB58 in vivo indicated CI values of 0.002, 0.0002, and 0.14 between PAO1-PA14, PAO1-LESB58, and LESB58-PA14, respectively. CI analysis comparing the in vivo growth of the PAO1 DeltaPA5441 mutant and four PA14 surface attachment-defective (sad) mutants gave CI values 10 to 1,000 times lower in competitions with their respective wild-type strains PAO1 and PA14. P. aeruginosa strains studied in the rat model of chronic lung infection demonstrated similar in vivo growth but differences in virulence as shown with a competitive in vivo assay. These differences were further confirmed with biofilm and motility in vitro assays, where strain LESB58 produced more biofilm but had less capacity for motility than PAO1 and PA14.
    Journal of bacteriology 05/2008; 190(8):2804-13. DOI:10.1128/JB.01572-07 · 2.69 Impact Factor
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    Eric Potvin, François Sanschagrin, Roger C Levesque
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    ABSTRACT: In Pseudomonas aeruginosa, as in most bacterial species, the expression of genes is tightly controlled by a repertoire of transcriptional regulators, particularly the so-called sigma (sigma) factors. The basic understanding of these proteins in bacteria has initially been described in Escherichia coli where seven sigma factors are involved in core RNA polymerase interactions and promoter recognition. Now, 7 years have passed since the completion of the first genome sequence of the opportunistic pathogen P. aeruginosa. Information from the genome of P. aeruginosa PAO1 identified 550 transcriptional regulators and 24 putative sigma factors. Of the 24 sigma, 19 were of extracytoplasmic function (ECF). Here, basic knowledge of sigma and ECF proteins was reviewed with particular emphasis on their role in P. aeruginosa global gene regulation. Summarized data are obtained from in silico analysis of P. aeruginosasigma and ECF including rpoD (sigma(70)), RpoH (sigma(32)), RpoF (FliA or sigma(28)), RpoS (sigma(S) or sigma(38)), RpoN (NtrA, sigma(54) or sigma(N)), ECF including AlgU (RpoE or sigma(22)), PvdS, SigX and a collection of uncharacterized sigma ECF, some of which are implicated in iron transport. Coupled to systems biology, identification and functional genomics analysis of P. aeruginosasigma and ECF are expected to provide new means to prevent infection, new targets for antimicrobial therapy, as well as new insights into the infection process.
    FEMS Microbiology Reviews 02/2008; 32(1):38-55. DOI:10.1111/j.1574-6976.2007.00092.x · 13.81 Impact Factor
  • François Sanschagrin, Irena Kukavica-Ibrulj, Roger C Levesque
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    ABSTRACT: PCR-based signature tagged mutagenesis is an "en masse" screening technique based upon unique oligonucleotide tags (molecular barcodes) for identification of genes that will diminish or enhance maintenance of an organism in a specific ecological niche or environment. PCR-based STM applied to Pseudomonas aeruginosa permitted the identification of genes essential or in vivo maintenance by transposon insertion and negative selection in a mixed population of bacterial mutants. The innovative adaptations and refinement of the technology presented here with P. aeruginosa STM mutants selected in the rat lung have given critical information about genes essential for causing a chronic infection and a wealth of information about biological processes in vivo. The additional use of competitive index analysis for measurement of the level of virulence in vivo, microarray-based screening of selected prioritized STM mutants coupled to metabolomics analysis can now be attempted systematically on a genomic scale. PCR-based STM and combined whole-genome methods can also be applied to any organism having selectable phenotypes for screening.
    Methods in Molecular Biology 02/2008; 416:61-82. DOI:10.1007/978-1-59745-321-9_5 · 1.29 Impact Factor
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    ABSTRACT: As a model system for designing new inhibitors of bacterial cell division, we studied the essential and highly conserved FtsZ GTPase from Pseudomonas aeruginosa. A collection of GTP analogues were prepared using the solid-phase parallel synthesis approach. The synthesized GTP analogues inhibited the GTPase activity of FtsZ with IC(50) values between 450microM and 2.6mM, and 5 compounds inhibited Staphylococcus aureus growth in a biological assay. The FtsZ spectrophotometric assay developed for screening of synthesized compounds is the first step in identification of antibacterials targeting the bacterial cell division essential proteins.
    Bioorganic & Medicinal Chemistry 03/2007; 15(3):1330-40. DOI:10.1016/j.bmc.2006.11.015 · 2.95 Impact Factor
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    ABSTRACT: The gp144 endolysin gene from the Pseudomonas aeruginosa phage phiKZ was cloned and studies of gp144 expression into Escherichia coli showed host cell lysis. The gp144 protein was purified directly from the culture supernatant and from the bacterial cell pellet and showed in vitro antibacterial lytic activity against P. aeruginosa bacteria and degraded purified peptidoglycan of Gram-negative bacteria. MS analysis identified the gp144 peptidoglycan cleavage site and confirmed a lytic transglycosylase enzyme. Studies of gp144 expression in the presence of sodium azide (NaN(3)), an inhibitor of the protein export machinery, and into an E. coli MM52 secA(ts) mutant at permissive and restrictive temperatures showed that gp144 was secreted independently of the Sec system. The solution conformation of purified gp144 analyzed by circular dichroism spectroscopy was 61% in alpha-helical content, and showed a 72% decrease when interacting with dimyristoylphosphatidylglycerol (DMPG), one of the major components of bacterial membranes and less than 10% with dimyristoylphosphatidylcholine (DMPC) found in eukaryotic membranes. Membrane vesicles of DMPG anionic lipids containing calcein indicated that gp144 caused a rapid release of fluorescent calcein when interacting with synthetic membranes. These results indicated that gp144 from phiKZ is a lytic transglycosylase capable of interacting with and disorganizing bacterial membranes and has potential as an antipseudomonal in phage therapy.
    FEMS Microbiology Letters 02/2007; 266(2):201-9. DOI:10.1111/j.1574-6968.2006.00523.x · 2.72 Impact Factor
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    José Molina-López, François Sanschagrin, Roger C Levesque
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    ABSTRACT: The MurA enzyme from Pseudomonas aeruginosa was purified to homogeneity and found to be biologically active as a UDP-N-acetylglucosamine (UNAG) enolpyruvyl transferase in a coupled enzyme assay where ATPase activity was measured by the release of inorganic phosphate. A microtiter plate assay coupled to competitive biopanning using the UDP-N-acetylglucosamine was used to screen 10(9) C-7-C and 12-mers peptides from phage display libraries. From 60 phage-encoded peptides identified after the fourth round of biopanning, deduced amino acid sequences were aligned and two peptides were synthesized and tested for inhibition of the MurA-catalyzed reaction. The PEP 1354 peptide inhibited the ATPase activity of MurA with an IC(50) value of 200muM and was found to be a competitive inhibitor of UNAG. The pre-incubation of MurA with inhibitor indicated a time-independent inhibition. This time-dependent inhibition is the first report of peptide inhibitors of MurA, which represent the scaffold for the synthesis of inhibitory peptidomimetic molecules.
    Peptides 01/2007; 27(12):3115-21. DOI:10.1016/j.peptides.2006.08.023 · 2.61 Impact Factor
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    ABSTRACT: The purified Pseudomonas aeruginosa cell wall biosynthesis MurD amide ligase enzyme was used to screen C-7-C and 12 mers peptides from phage display libraries using competitive biopanning approaches with the specific substrates D-glutamate and ATP. From the 60 phage-encoded peptides identified, DNA was sequenced, deduced amino acid sequences aligned and two peptides were synthesized from consensus sequences identified. The UDP-N-acetylmuramyl-L-alanine MurD substrate was synthesized, purified and used to develop a spectrophotometric assay. One peptide synthesized was found to specifically inhibit ATPase activity of MurD. The IC50 value was estimated at 4 microM for the C-7-C MurDp1 peptide. The loop conformation of MurDp1 was shown to be important for the inhibition of the UDP-N-acetylmuramyl-L-alanine:D-glutamate MurD ligase. The linear 12 mers MurD2 peptide has an IC50 value of 15 mM. A conserved amino acid motif was found between MurDp2 and the bacterial glyceraldehyde 3-phosphate dehydrogenase indicating that MurDp2 binds at a protein-protein interacting site. The approach proposed and results obtained suggest that efficient peptide inhibitors as well as protein-protein interaction domains can be identified by phage display.
    Peptides 08/2006; 27(7):1693-700. DOI:10.1016/j.peptides.2006.01.017 · 2.61 Impact Factor
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    ABSTRACT: Synopsis Everyday, normal breathing deposits numerous microorganisms on the surfactant membrane that lines the air-exchanging surfaces of the lung. Surfactant protein SP-A, a component of the surfactant membrane, helps to maintain the lung in a germ-free state by aggregating inhaled microorganisms and facilitating their ingestion by immune cells, and by increasing the permeability of their cell membranes. However, the bacterial pathogen Pseudomonas aeruginosa is resistant to SP-A-mediated membrane disruption. Using a genetic tool called comparative signature-tagged mutagenesis, the authors have identified two P. aeruginosa genes, pch and ptsP, that are required to resist SP-A-mediated membrane permeabilization. Molecular analyses indicate that the pch gene encodes an enzyme that synthesizes salicylate, a compound utilized by bacteria to acquire essential metal ions. The ptsP gene encodes an enzyme called phosphoenolpyruvate-protein-phosphotransferase. The loss of salicylate and phosphoenolpyruvate-protein-phosphotransferase weakens the P. aeruginosa cell membrane, which allows SP-A to poke holes on the membrane and kill the bacteria. This is the first known report of the roles played by salicylate and phosphoenolpyruvate-protein-phosphotransferase in maintenance of bacterial membrane, and consequently, protecting bacteria from killing by SP-A, through disruption of membrane integrity.
    PLoS Pathogens 12/2005; 1(3):259-68. DOI:10.1371/journal.ppat.0010031 · 8.14 Impact Factor

Publication Stats

2k Citations
218.18 Total Impact Points

Institutions

  • 1998–2014
    • Laval University
      • Department of Medical Biology
      Quebec City, Quebec, Canada
    • Baylor College of Medicine
      Houston, Texas, United States
  • 1995–2013
    • Université du Québec
      Quebec City, Quebec, Canada