[show abstract][hide abstract] 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. · 1.94 Impact Factor
[show abstract][hide abstract] 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.
[show abstract][hide abstract] 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.
[show abstract][hide abstract] 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. · 14.40 Impact Factor
[show abstract][hide abstract] 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.
[show abstract][hide abstract] 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.
[show abstract][hide abstract] 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. · 3.94 Impact Factor
[show abstract][hide abstract] 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 (Clifton, N.J.) 02/2008; 416:61-82.
[show abstract][hide abstract] 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.
[show abstract][hide abstract] 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.
[show abstract][hide abstract] 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.
[show abstract][hide abstract] 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.
[show abstract][hide abstract] 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.
[show abstract][hide abstract] ABSTRACT: The pulmonary collectin, surfactant protein A (SP-A), is a broad spectrum opsonin with microbicidal membrane permeabilization properties that plays a role in the innate immune response of the lung. However, the factors that govern SP-A's microbial specificity and the mechanisms by which it mediates membrane permeabilization and opsonization are not fully understood. In an effort to identify bacterial factors that confer susceptibility or resistance to SP-A, we used comparative signature-tagged mutagenesis to screen a library of 1,680 Pseudomonas aeruginosa mutants for evidence of differential pulmonary clearance in SP-A-sufficient (SP-A) and SP-A-deficient (SP-A) mice. Two SP-A-sensitive P. aeruginosa mutants harboring transposon insertions in genes required for salicylate biosynthesis (pch) and phosphoenolpyruvate-protein-phosphotransferase (ptsP) were recovered. The mutants were indistinguishable from the parental wild-type PA01 with regard to opsonization by SP-A, but they exhibited increased susceptibility to SP-A-mediated membrane permeabilization. These results suggest that bacterial gene functions that are required to maintain membrane integrity play crucial roles in resistance of P. aeruginosa to the permeabilizing effects of SP-A.
[show abstract][hide abstract] ABSTRACT: The revolutionary era of antibiotics has been overwhelmed by the evolutionary capacity of microorganisms such as Pseudomonas aeruginosa to develop resistance to all classes of antibiotics. In the perspective of identifying new antimicrobials using novel strategies, we targeted the essential and highly conserved FtsA protein from the bacterial cell division machinery of P.aeruginosa. In a series of experiments we cloned, overproduced and purified the FtsA and FtsZ proteins. Expression of FtsA into Escherichia coli cells led to its accumulation in inclusion bodies. We developed a protocol permitting the purification and refolding of enzymatically active FtsA hydrolysing ATP. The purified enzyme was used to screen for peptide inhibitors of ATPase activity using phage display. Selective biopanning assays were done and phages were eluted using ATP, a non-hydrolysable ATP analogue and the protein FtsZ known to interact with FtsA in the divisome during the process of bacterial cell division. We identified two consensus peptide sequences interacting with FtsA and a competitive ELISA was used to identify peptides having high affinity for the target protein. Five of the six peptides synthesized showed specific inhibition of ATPase activity of FtsA with IC50 values between 0.7 and 35 mM. Discovery of peptides inhibiting the essential cell division machinery in bacteria is the first step for the future development of antimicrobial agents via peptidomimetism.
Protein Engineering Design and Selection 03/2005; 18(2):85-91. · 2.59 Impact Factor
[show abstract][hide abstract] ABSTRACT: In Gram-negative bacteria, resistance to beta-lactam antibiotics and to known inhibitors mediated by metallo-beta-lactamases is a major concern and a serious threat to public health. Since no clinically useful inhibitors are available against class B metallo-beta-lactamases, the aim of the study was to identify peptides as inhibitors.
The L-1 metalloenzyme from Stenotrophomonas maltophilia was cloned, over-expressed, purified to homogeneity and used in screening of peptide libraries by phage display with a selective and competitive biopanning assay. This was based upon the high affinity of L-1 for cefoxitin and its slow hydrolysis.
From six peptides, the consensus sequence Cys-Val-His-Ser-Pro-Asn-Arg-Glu-Cys was identified as a promising inhibitor of L-1 hydrolytic activity. This peptide showed a mixed inhibition of L-1 with a K(i competitive) of 16 +/- 4 microM and a K(i uncompetitive) of 9 +/- 1 microM. The same peptide was prepared without flanking Cys residues and demonstrated no detectable inhibition of L-1 hydrolytic activity with nitrocefin as a substrate. These data confirmed the importance of the peptide conformation for the inhibition of L-1 hydrolytic activity. Further analysis revealed rescue by Zn2+ ions. The mixed inhibition indicated peptide binding near the active site of L-1 and blocking of zinc atoms for optimal conformation in the pocket of the active site.
This is the first report of a peptide inhibitor for Class B metallo-beta-lactamases. It will be used as a lead to identify more potent small molecule inhibitors via peptidomimetics.
Journal of Antimicrobial Chemotherapy 03/2005; 55(2):252-5. · 5.34 Impact Factor
[show abstract][hide abstract] ABSTRACT: Extraintestinal pathogenic Escherichia coli (ExPEC) isolates collected from different infected animals and from human patients with extraintestinal infections in 2001 were characterized for their phenotypic and genotypic antimicrobial resistance profiles, genotypes, and key virulence factors. Among the 10 antimicrobial agents tested, resistance to ampicillin, tetracycline, and sulfonamides was most frequent. Multiresistant strains were found in both the animal and the human groups of isolates. Resistance gene distribution was assessed by colony hybridization. Similar antibiotic resistance patterns could be observed in the animal and the human isolates. Although some resistance genes, such as bla(TEM), sulI, and sulII, were equally represented in the animal and human ExPEC isolates, differences in the distributions of tetracycline [tet(D)], chloramphenicol (catI, catIII, and floR), and trimethoprim (dhfrI, dhfrV, dhfrVII, and dhfrXIII) resistance genes were observed between the animal and the human isolates. Approximately one-third of the ExPEC isolates possessed a class 1 integron. The four major different variable regions of the class 1 integron contained aminoglycoside (aadA1, aadA2, aadA5, and aadA6) and/or trimethoprim (dhfrIb, dhfrXII, and dhfrXVII) resistance genes. The ExPEC strains belonged to different phylogenetic groups, depending on their host origin. Strains isolated from animal tissues belonged to either a commensal group (group A or B1) or a virulent group (group B2 or D), while the majority of the human isolates belonged to a virulent group (group B2 or D). Although the limited number of isolates evaluated in the present study prevents firm epidemiological conclusions from being made, on a more global scale, these data demonstrate that extraintestinal isolates of E. coli can possess relatively distinct intra- and intergroup resistance gene profiles, with animal isolates presenting a more heterogeneous group than human isolates.
Journal of Clinical Microbiology 01/2005; 42(12):5444-52. · 4.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: Microbial pathogens possess a repertoire of virulence determinants that make unique contributions to bacterial fitness during infection. In this chapter, we focus on the recent progress and adaptations of signature-tagged mutagenesis (STM) by PCR instead of hybridization. This is a PCR-based STM mutation-based screening method using a population of bacterial mutants for the simultaneous identification of multiple virulence genes in microbial pathogens by negative selection. Modifications of STM developed in our laboratory have been applied to Pseudomonas aeruginosa PAO1. Screening of a collection of 6912 STM mutants in the rat chronic lung model of infection identified 214 P. aeruginosa STM mutants defective in virulence. For further studies, and to illustrate better the strategies that need to be utilized, we present detailed analysis of nine selected STM mutants. The data obtained indicate that in vivo, defects in virulence give a wide variety of phenotypes: defects in known virulence factors have been found, thereby validating the method; defects have also been found in orthologs with predicted functions, and in some genes whose functions cannot be predicted from databases. A general strategy and a simple scenario is discussed using the nine STM mutants selected for further characterization. PCR-based STM represent a genomics-based method for in vivo high-throughput screening of new virulence factors.
Methods in molecular biology (Clifton, N.J.) 02/2004; 266:289-304.