Purification and proteomic analysis of outer membrane vesicles from a clinical isolate ofLeptospira interrogans serovar Copenhageni

Division of Infectious Diseases, Department of Medicine, University of California, Los angeles, CA 90095, USA. .
PROTEOMICS (Impact Factor: 3.97). 01/2005; 5(1):144-52. DOI: 10.1002/pmic.200400880
Source: PubMed

ABSTRACT The severe pulmonary form of leptospirosis (SPFL) is an especially serious and rapid disease process characterized by alveolar hemorrhage and acute respiratory failure. The outer membrane of Leptospira facilitates direct interactions with the environs and likely contains important constituents involved during infection, transmission, survival, and adaptation to environmental conditions, including putative vaccinogen and diagnostic candidates. Outer membrane vesicles (OMVs) were purified by incubation in low-pH citrate buffer, treatment in a French press, and centrifugation over a continuous sucrose gradient. OMVs characterized by two-dimensional gel electrophoresis (2-DE) contained the previously described outer membrane proteins OmpL1, Qlp42, LipL32, LipL41, LipL36 and Loa22. In addition, unknown, hypothetical and putative outer membrane proteins were identified. High-performance liquid chromatography (HPLC) coupled with mass spectrometry and fraction collection (LC-MS+) measured the intact mass profile of the major outer membrane protein, LipL32, and the putative lipoprotein Qlp42. In contrast to a predicted molecularmass of 27,653.5 Da for LipL32 after cleavage of its signal peptide, intact mass proteomics measured the mass as ranging from 28,468 to 28,583 Da, consistent with lipidation of LipL32. In contrast to a predicted molecular mass of 39.8 kDa for Qlp42, the actual mass was measured as 24,811 and 26,461 Da consistent with a 30 kDa doublet observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and processing of the N-terminus of the mature protein. These studies indicate that purified OMVs are highly compatible with proteomics technologies including 2-DE and intact mass proteomics using LC-MS+ that facilitates definition of actual molecular masses of intact outer membrane proteins, and heterogeneity associated with them.

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Available from: Martha Maria Pereira, Aug 15, 2015
    • "HlyA is secreted by the secindependent Type I secretion process that is a multi-component energy driven pump, in which a continuous channel spanning the periplasmic space is created between the inner and outer membranes by the assembly of three proteins, namely the TolC, HlyB and HlyD (Koronakis et al., 2000). OMVs have been demonstrated in Leptospira (Haake and Matsunaga, 2002; Nally et al., 2005) and shown to contain several outer membrane proteins, including the OmpL1, LipL32, LipL36, and LipL41. "
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    • "Due to the lack of a clear understanding for the biological roles of OMVs, the last decade has seen proteomic analysis of OMVs from a variety of pathogenic bacterial species such as Escherichia coli, Helicobacter pylori, Legionella pneumophila, N. meningitidis, Pseudomonas aeruginosa etc. [17] [18] [19] [20] [21] [22] [23]. The vesicular proteome of C. jejuni, however, has yet to be sufficiently investigated, despite the likelihood of their role in pathogenesis and virulence. "
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