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

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.

Application of immunoproteomics to leptospirosis: towards clinical diagnostics and vaccine discovery

“…A set of membrane proteins that was identified in their study was not detectable in our present study. On the other hand, we have information on immunogenic cytosolic proteins, and such information could not be obtained using just the membrane fraction of leptospiral antigens in the study by Nally and colleagues [29]. Performing the immunoproteomic analysis on both membrane and cytosolic fractions, separately, would be very interesting and should provide complementary results to our present study.…”

“…First, we used bacterial whole cell lysates as the antigens and identified 13 unique proteins from totally 24 immunoreactive spots. Recently, Nally et al [29] performed proteome analysis of outer membrane vesicles of L. interrogans (serovar: Copenhageni) and identified 32 unique proteins from 40 visualized spots. A set of membrane proteins that was identified in their study was not detectable in our present study.…”

Proteomic Analysis of Leptospira interrogans Shed in Urine of Chronically Infected Hosts

“…Leptospiruria in maintenance hosts is of high intensity, constant, and of long duration compared to that in accidental hosts, where it is of low intensity, intermittent, and of short duration (4). In contrast to previous studies which focused on characterization of the proteome of in vitro-cultivated Leptospira (IVCL) (5,16), the present study has exploited leptospiruria during a chronic animal disease model to provide, for the first time, a proteomic analysis of leptospires as excreted in urine of chronically infected hosts. Results confirm that leptospires differentially express proteins in the host, which in turn likely facilitates persistence in the presence of a specific host antibody response.…”

High-coverage proteome analysis reveals the first insight of protein modification systems in the pathogenic spirochete Leptospira interrogans

“…The genome sequences of the pathogenic L. interrogans serovar Lai and serovar Copenhageni, the pathogenic L. borgpetersenii serovar Hardjo and the saprophytic L. biflexa serovar Patoc have been reported [3][4][5][6]. The accomplishment of these sequencing projects greatly facilitated the studies of Leptospira physiology and pathology at the genomic [4][5][6], transcriptomic [7,8] and proteomic levels [9][10][11][12][13][14]. However, there are discrepancies in the annotations for the same genome sequences by different institutions or by using diverse prediction methodologies, which hinders further studies [8].…”