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: 3.81). 01/2005; 5(1):144-52. DOI: 10.1002/pmic.200400880
Source: PubMed


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, Oct 10, 2015
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    • "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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    ABSTRACT: Unlabelled: Gram-negative bacteria constitutively release outer membrane vesicles (OMVs) during cell growth that play significant roles in bacterial survival, virulence and pathogenesis. In this study, comprehensive proteomic analysis of OMVs from a human gastrointestinal pathogen Campylobacter jejuni NCTC11168 was performed using high-resolution mass spectrometry. The OMVs of C. jejuni NCTC11168 were isolated from culture supernatants then characterized using electron microscopy and dynamic light scattering revealing spherical OMVs of an average diameter of 50nm. We then identified 134 vesicular proteins using high-resolution LTQ-Orbitrap mass spectrometry. Subsequent functional analysis of the genes revealed the relationships of the vesicular proteins. Furthermore, known N-glycoproteins were identified from the list of the vesicular proteome, implying the potential role of the OMVs as a delivery means for biologically relevant bacterial glycoproteins. These results enabled us to elucidate the overall proteome profile of pathogenic bacterium C. jejuni and to speculate on the function of OMVs in bacterial infections and communication. Biological significance: This work demonstrates the importance of understanding vesicular proteomes from a human pathogen Campylobacter jejuni. From the secreted outer membrane vesicles (OMVs) of C. jejuni NCTC11168, we found a variety of virulence factors and essential proteins for bacterial survival. Bioinformatics analysis of these proteins predicted functional enrichment and localization. The most highly enriched were redox enzymes, which are considered to be essential for survival in oxygen-limiting environments and are predicted to be on the twin-arginine translocation (Tat) pathway suggesting a role for this pathway in the biogenesis of OMVs. This study additionally implicates a biological role for N-linked glycoproteins in OMVs. These approaches allow for a better understanding of the physiology of this important human pathogen.
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    • "fy antigens of the same molecular mass and isoelectric point . Antigens were excised with a One Touch 2d Gel Spot Picker 3 mm ( Web Scientific , U . K . ) . In order to detect proteins of 26 – 32 kDa , it was necessary to separate 500 ␮g of EB pro - teins . In - gel trypsin digestion was performed as previously described ( Marques et al . , 2010 ; Nally et al . , 2005 ) . Sam - ples were analyzed by nano - liquid chromatography – mass spectrometry ( nLC – MSMS ) using data - dependent acquisi - tion mode on LTQ FT Ultra mass spectrometer ( Thermo Fisher Scientific , San Jose , California , USA ) as previously described ( Marques et al . , 2010 ) . Protein identifications were made using the protein s"
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