Comprehensive proteome analysis of Mycobacterium ulcerans and quantitative comparison of mycolactone biosynthesis

Abstract

Mycobacterium ulcerans is the causative agent of Buruli ulcer, a rapidly emerging human disease in which mycolactone, a cytotoxic and immunosuppressive macrocyclic polyketide, is responsible for massive skin destruction. The genome sequencing of M. ulcerans has recently been accomplished (http://genolist.pasteur.fr/BuruList/) enabling the first proteome study of this important human pathogen. Here, we present a comprehensive proteome analysis of different subcellular fractions and culture supernatant of in vitro grown M. ulcerans. By a combination of gel-based and gel-free techniques for protein and peptide separation with subsequent analysis by MS, we identified 1074 different proteins, corresponding to 25% of the protein-coding DNA sequence. Interestingly, new information was obtained about central metabolism and lipid biosynthesis, and as many as 192 conserved hypothetical proteins were found. Comparative analysis of the wild-type strain and an isogenic mycolactone-deficient mutant, by 2-DE and iTRAQ labeling of the cytoplasmic fraction, revealed differences in the expression profiles of proteins involved in lipid metabolism and information pathways, as well as stress responses.

The Cell Wall-Associated Mycolactone Polyketide Synthases Are Necessary but Not Sufficient for Mycolactone Biosynthesis

“…Future research to explore our initial observations will require Mls PKS antibodies with improved specificity. Nevertheless, the data we present here suggest that mycolactone synthesis is occurring at or within the cell wall, consistent with previous reports describing mycolactone blebbing from the bacterial cell in lipid and protein-rich vesicles [31] and an M. ulcerans proteomic investigation that also detected the Mls PKS in the cell wall [32]. It is interesting to speculate how these megadalton-sized molecular machines with their predicted homodimeric and interconnected structure are arranged within the mycobacterial cell wall.…”

Serological Evaluation of Mycobacterium ulcerans Antigens Identified by Comparative Genomics

“…We then excluded CDS <50 codons in length and the insertion sequence elements IS 2404 and IS 2606 and also applied the following criteria to include only those CDS likely to generate an immune response: i) predicted membrane association; ii) predicted secretion signal; or iii) previously confirmed as expressed in a proteomic study [32], resulting in 34 chromosomal CDS of interest. We also identified 33 unique CDS on the pMUM001 plasmid and in addition we included in our analysis the sequences encoding the 12 unique functional domains of the mycolactone polyketide synthases (MlsA1, MlsA2 & MlsB) as representative of the entire genetic variability present in this locus [9].…”

Mycobacterium tuberculosis Rv1096 protein: gene cloning, protein expression, and peptidoglycan deacetylase activity

“…Bacteria that were treated with lysozyme for 9 h were harvested by centrifugation at 4,500 × g at 4°C for 10 min, after which the pellets were washed with sterilized 1 M PBS (pH 7.0), three times. Samples were prepared for Ziehl-Neelsen acid-fast staining as described previously [24], and observed under a light microscope (Olympus CHB, Japan). The cells for electron microscopic analysis were fixed with 2.5% glutaraldehyde, followed by post-fixation at room temperature for 2 h with 1% osmium tetroxide.…”