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Post-translational modification of LipL32 during Leptospira interrogans infection.

Witchell TD, Eshghi A, Nally JE, Hof R, Boulanger MJ, Wunder EA, Ko AI, Haake DA, Cameron CE - PLoS Negl Trop Dis (2014)

Bottom Line: 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.The identity of each modified lysine residue was confirmed by fragmentation pattern analysis of the peptide mass spectra.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada.

ABSTRACT

Background: Leptospirosis, 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 findings: Previous 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/significance: The 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.

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Modified lysine residues mapped onto the Ca2+ bound LipL32 structure [39].A. Secondary structure and B. surface representations. The numbering system C1-K253 corresponds to the mature LipL32 protein (lacking the 19 residue cleaved SPII signal sequence) established by Vivian et al.[40] and adopted by Tung et al.[39]. Note, the Ca2+ binding site and predicted collagen binding surface in panel B are highlighted in yellow and frame Lys199. The figure was generated using PyMOL (Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC).
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pntd-0003280-g003: Modified lysine residues mapped onto the Ca2+ bound LipL32 structure [39].A. Secondary structure and B. surface representations. The numbering system C1-K253 corresponds to the mature LipL32 protein (lacking the 19 residue cleaved SPII signal sequence) established by Vivian et al.[40] and adopted by Tung et al.[39]. Note, the Ca2+ binding site and predicted collagen binding surface in panel B are highlighted in yellow and frame Lys199. The figure was generated using PyMOL (Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC).

Mentions: The structural position of the eight modified lysines detected within the LipL32 sample originating from the rat urine-isolated leptospires was determined through examination of the Ca2+-bound LipL32 crystal structure [39]. For consistency and ease of interpretation, we have used the numbering system C1-K253 which corresponds to the mature LipL32 protein (lacking the 19 residue cleaved SPII signal sequence) set forward by Vivian et al.[40] and adopted by Tung et al.[39]. As shown in Figure 3A, the observed modifications were localized to the β2 strand (K29), the loop between the β8 and β9 strands (K152), the β9 strand (K166), the loop between the β9 strand and the α3 helix (K172), the α3 helix (K178), the loop between the β10 and β11 strands (K199) and the α4 helix (K245 and K246). All eight modified lysines localized to surface accessible regions of the structure (Figure 3B). Of note, the modified residues K152 and K199 are located in immediate proximity to the LipL32 Ca2+-binding site, which comprises the negatively charged surface formed by residues D142–D149 (encompassing a portion of the β8 strand and the β8β9 loop) and the Ca2+ ion-coordinating residues in the α1β7 loop (D113 and T114) and the β8β9 loop (D145, D146 and Y159) [39]. Lysine 199 is also in close proximity to the reported collagen-binding site encompassing residues L53, V54, Y62, W115, R117, Y151 and Y198[40] (Figure 3B). In fact, K199 is positioned on a flexible loop between strands 10 and 11 that bridges the Ca2+ and reported collagen-binding sites of LipL32, suggesting that modification of K199 may play a role in regulating protein architecture and function.


Post-translational modification of LipL32 during Leptospira interrogans infection.

Witchell TD, Eshghi A, Nally JE, Hof R, Boulanger MJ, Wunder EA, Ko AI, Haake DA, Cameron CE - PLoS Negl Trop Dis (2014)

Modified lysine residues mapped onto the Ca2+ bound LipL32 structure [39].A. Secondary structure and B. surface representations. The numbering system C1-K253 corresponds to the mature LipL32 protein (lacking the 19 residue cleaved SPII signal sequence) established by Vivian et al.[40] and adopted by Tung et al.[39]. Note, the Ca2+ binding site and predicted collagen binding surface in panel B are highlighted in yellow and frame Lys199. The figure was generated using PyMOL (Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4214626&req=5

pntd-0003280-g003: Modified lysine residues mapped onto the Ca2+ bound LipL32 structure [39].A. Secondary structure and B. surface representations. The numbering system C1-K253 corresponds to the mature LipL32 protein (lacking the 19 residue cleaved SPII signal sequence) established by Vivian et al.[40] and adopted by Tung et al.[39]. Note, the Ca2+ binding site and predicted collagen binding surface in panel B are highlighted in yellow and frame Lys199. The figure was generated using PyMOL (Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC).
Mentions: The structural position of the eight modified lysines detected within the LipL32 sample originating from the rat urine-isolated leptospires was determined through examination of the Ca2+-bound LipL32 crystal structure [39]. For consistency and ease of interpretation, we have used the numbering system C1-K253 which corresponds to the mature LipL32 protein (lacking the 19 residue cleaved SPII signal sequence) set forward by Vivian et al.[40] and adopted by Tung et al.[39]. As shown in Figure 3A, the observed modifications were localized to the β2 strand (K29), the loop between the β8 and β9 strands (K152), the β9 strand (K166), the loop between the β9 strand and the α3 helix (K172), the α3 helix (K178), the loop between the β10 and β11 strands (K199) and the α4 helix (K245 and K246). All eight modified lysines localized to surface accessible regions of the structure (Figure 3B). Of note, the modified residues K152 and K199 are located in immediate proximity to the LipL32 Ca2+-binding site, which comprises the negatively charged surface formed by residues D142–D149 (encompassing a portion of the β8 strand and the β8β9 loop) and the Ca2+ ion-coordinating residues in the α1β7 loop (D113 and T114) and the β8β9 loop (D145, D146 and Y159) [39]. Lysine 199 is also in close proximity to the reported collagen-binding site encompassing residues L53, V54, Y62, W115, R117, Y151 and Y198[40] (Figure 3B). In fact, K199 is positioned on a flexible loop between strands 10 and 11 that bridges the Ca2+ and reported collagen-binding sites of LipL32, suggesting that modification of K199 may play a role in regulating protein architecture and function.

Bottom Line: 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.The identity of each modified lysine residue was confirmed by fragmentation pattern analysis of the peptide mass spectra.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada.

ABSTRACT

Background: Leptospirosis, 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 findings: Previous 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/significance: The 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.

Show MeSH
Related in: MedlinePlus