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1.
J Biol Chem ; 287(28): 23464-78, 2012 Jul 06.
Article in English | MEDLINE | ID: mdl-22582391

ABSTRACT

Enzymes secreted by Legionella pneumophila, such as phospholipases A (PLAs) and glycerophospholipid:cholesterol acyltransferases (GCATs), may target host cell lipids and therefore contribute to the establishment of Legionnaires disease. L. pneumophila possesses three proteins, PlaA, PlaC, and PlaD, belonging to the GDSL family of lipases/acyltransferases. We have shown previously that PlaC is the major GCAT secreted by L. pneumophila and that the zinc metalloproteinase ProA is essential for GCAT activity. Here we characterized the mode of PlaC GCAT activation and determined that ProA directly processes PlaC. We further found that not only cholesterol but also ergosterol present in protozoa was palmitoylated by PlaC. Such ester formations were not induced by either PlaA or PlaD. PlaD was shown here to possess lysophospholipase A activity, and interestingly, all three GDSL enzymes transferred short chain fatty acids to sterols. The three single putative catalytic amino acids (Ser-37, Asp-398, and His-401) proved essential for all PlaC-associated PLA, lysophospholipase A, and GCAT activities. A further four cysteine residues are important for the PLA/GCAT activities as well as their oxidized state, and we therefore conclude that PlaC likely forms at least one disulfide loop. Analysis of cleavage site and loop deletion mutants suggested that for GCAT activation deletion of several amino acids within the loop is necessary rather than cleavage at a single site. Our data therefore suggest a novel enzyme inhibition/activation mechanism where a disulfide loop inhibits PlaC GCAT activity until the protein is exported to the external space where it is ProA-activated.


Subject(s)
Acyltransferases/metabolism , Bacterial Proteins/metabolism , Legionella pneumophila/enzymology , Metalloproteases/metabolism , Acyltransferases/genetics , Amino Acid Sequence , Bacterial Proteins/genetics , Binding Sites/genetics , Blotting, Western , Catalytic Domain/genetics , Cholesterol/metabolism , Cysteine/genetics , Cysteine/metabolism , Enzyme Activation , Gene Knockout Techniques , Legionella pneumophila/genetics , Legionella pneumophila/metabolism , Metalloproteases/genetics , Molecular Sequence Data , Mutation , Palmitic Acid/metabolism , Propionates/metabolism , Sequence Homology, Amino Acid , Sterols/metabolism , Substrate Specificity , Zinc/metabolism
2.
Appl Environ Microbiol ; 75(22): 7229-42, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19767470

ABSTRACT

This report demonstrates the applicability of a combination of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) and chemometrics for rapid and reliable identification of vegetative cells of the causative agent of anthrax, Bacillus anthracis. Bacillus cultures were prepared under standardized conditions and inactivated according to a recently developed MS-compatible inactivation protocol for highly pathogenic microorganisms. MALDI-TOF MS was then employed to collect spectra from the microbial samples and to build up a database of bacterial reference spectra. This database comprised mass peak profiles of 374 strains from Bacillus and related genera, among them 102 strains of B. anthracis and 121 strains of B. cereus. The information contained in the database was investigated by means of visual inspection of gel view representations, univariate t tests for biomarker identification, unsupervised hierarchical clustering, and artificial neural networks (ANNs). Analysis of gel views and independent t tests suggested B. anthracis- and B. cereus group-specific signals. For example, mass spectra of B. anthracis exhibited discriminating biomarkers at 4,606, 5,413, and 6,679 Da. A systematic search in proteomic databases allowed tentative assignment of some of the biomarkers to ribosomal protein or small acid-soluble proteins. Multivariate pattern analysis by unsupervised hierarchical cluster analysis further revealed a subproteome-based taxonomy of the genus Bacillus. Superior classification accuracy was achieved when supervised ANNs were employed. For the identification of B. anthracis, independent validation of optimized ANN models yielded a diagnostic sensitivity of 100% and a specificity of 100%.


Subject(s)
Bacillus anthracis/classification , Bacterial Typing Techniques/methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Bacillus/chemistry , Bacillus/classification , Bacillus/metabolism , Bacillus anthracis/chemistry , Bacillus anthracis/metabolism , Bacillus cereus/chemistry , Bacillus cereus/classification , Bacillus cereus/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Biomarkers/analysis , Cluster Analysis , Databases, Protein , Neural Networks, Computer , Proteome , Sensitivity and Specificity , Spores, Bacterial/chemistry , Spores, Bacterial/metabolism
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