Date Published: October 30, 2014
Publisher: Public Library of Science
Author(s): Timothy D. Witchell, Azad Eshghi, Jarlath E. Nally, Rebecca Hof, Martin J. Boulanger, Elsio A. Wunder, Albert I. Ko, David A. Haake, Caroline E. Cameron, Pamela L. C. Small. http://doi.org/10.1371/journal.pntd.0003280
Abstract: BackgroundLeptospirosis, a re-emerging disease of global importance caused by pathogenic Leptospira spp., is considered the world’s most widespread zoonotic disease. Rats serve as asymptomatic carriers of pathogenic Leptospira and are critical for disease spread. In such reservoir hosts, leptospires colonize the kidney, are shed in the urine, persist in fresh water and gain access to a new mammalian host through breaches in the skin.Methodology/Principal FindingsPrevious studies have provided evidence for post-translational modification (PTM) of leptospiral proteins. In the current study, we used proteomic analyses to determine the presence of PTMs on the highly abundant leptospiral protein, LipL32, from rat urine-isolated L. interrogans serovar Copenhageni compared to in vitro-grown organisms. We observed either acetylation or tri-methylation of lysine residues within multiple LipL32 peptides, including peptides corresponding to regions of LipL32 previously identified as epitopes. Intriguingly, the PTMs were unique to the LipL32 peptides originating from in vivo relative to in vitro grown leptospires. The identity of each modified lysine residue was confirmed by fragmentation pattern analysis of the peptide mass spectra. A synthetic peptide containing an identified tri-methylated lysine, which corresponds to a previously identified LipL32 epitope, demonstrated significantly reduced immunoreactivity with serum collected from leptospirosis patients compared to the peptide version lacking the tri-methylation. Further, a subset of the identified PTMs are in close proximity to the established calcium-binding and putative collagen-binding sites that have been identified within LipL32.Conclusions/SignificanceThe exclusive detection of PTMs on lysine residues within LipL32 from in vivo-isolated L. interrogans implies that infection-generated modification of leptospiral proteins may have a biologically relevant function during the course of infection. Although definitive determination of the role of these PTMs must await further investigations, the reduced immune recognition of a modified LipL32 epitope suggests the intriguing possibility that LipL32 modification represents a novel mechanism of immune evasion within Leptospira.
Partial Text: Pathogenic Leptospira spp. are the causative agents of leptospirosis, which is considered to be the world’s most widespread zoonotic disease –. Recent data shows the incidence of leptospirosis is increasing, with outbreaks frequently occurring within urban slum settings –, an environment in which approximately 31.6% of the world’s total urban population resides . Therefore, leptospirosis represents a significant public health threat in these communities.
Two previous proteomic studies have established the presence of post-translationally modified proteins within L. interrogans. The leptospiral protein OmpL32 (corresponding to LIC11848) was shown to be differentially methylated on glutamic acid residues from L. interrogans grown under in vitro conditions . Additionally, a global PTM analysis of in vitro-cultured L. interrogans serovar Lai detected multiple PTMs on a broad range of proteins, including 46 proteins with 54 lysine acetylation sites, 104 proteins with 135 glutamic acid/glutamine methylation sites, and 58 proteins with 64 lysine/arginine methylation sites. One of the lysine-modified proteins detected within the latter study was LipL32, which was shown to be devoid of lysine acetylations but to contain mono-, di-, and tri-methyl groups on residue K152. In the study presented herein, we similarly detected modification of residue K152, as well as seven other lysine residues, within LipL32 from in vivo-grown leptospires. Of the modified lysines that were detected an unambiguous assignment of tri-methylation could be made for only residues K152, K178 and K246. By extrapolation from these results it is plausible that the remaining five detected PTMs constitute similar lysine tri-methylations, however definitive assignment could not be made from the spectra obtained in this study.