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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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Leptospirosis patient serum reactivity against modified and unmodified versions of synthetic LipL32 peptides.Shown is the reactivity of pooled sera from laboratory-confirmed leptospirosis patients (black bars) and LipL32-specific polyclonal rabbit serum (white bars) to unmodified and tri-methylated (KMe3) versions of P1 and P2 peptides comprising experimentally-determined B-cell epitopes of LipL32 [36]. Two independent experiments were performed with reproducible results; the results from one representative experiment are shown. Error bars represent standard error of measurement from triplicate samples. For statistical analyses, the level of reactivity of patient sera against the unmodified version of P1 was compared to the level of reactivity against the P1 peptide containing a tri-methylation at position K152 (P1-K152Me3) using the Student's two-tailed t-test (*p<0.0001).
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pntd-0003280-g002: Leptospirosis patient serum reactivity against modified and unmodified versions of synthetic LipL32 peptides.Shown is the reactivity of pooled sera from laboratory-confirmed leptospirosis patients (black bars) and LipL32-specific polyclonal rabbit serum (white bars) to unmodified and tri-methylated (KMe3) versions of P1 and P2 peptides comprising experimentally-determined B-cell epitopes of LipL32 [36]. Two independent experiments were performed with reproducible results; the results from one representative experiment are shown. Error bars represent standard error of measurement from triplicate samples. For statistical analyses, the level of reactivity of patient sera against the unmodified version of P1 was compared to the level of reactivity against the P1 peptide containing a tri-methylation at position K152 (P1-K152Me3) using the Student's two-tailed t-test (*p<0.0001).

Mentions: Analysis of the modified LipL32 peptides detected within the RUIL samples highlighted four peptides that overlap with regions previously identified through epitope mapping studies as antigenic regions of LipL32 [36]. Within the reported LipL32 epitope spanning residues 132–158 of the mature protein (designated as P1; AAKAKPVQKLDDDDDGDDTYKEERHNK) we detected tri-methylation of residue K152. For the LipL32 epitope encompassing residues 162–185 of the mature protein (designated as P2; LTRIKIPNPPKSFDDLKNIDTKKL) we detected modifications on K166, K172 and K178, with the latter constituting a confirmed tri-methylation. In order to investigate if the modifications altered immune recognition of these epitopes, synthetic peptides corresponding to the two previously identified epitopes were prepared; specifically, unmodified and K152 tri-methylated versions of P1 were synthesized, while for P2 an unmodified and a representative modified peptide containing a tri-methylated K178 residue were synthesized (Table 1). When tested in an ELISA assay (Figure 2), pooled sera obtained from patients with laboratory-confirmed leptospirosis reacted strongly to the unmodified version of P1, while reactivity against the K152 tri-methylated peptide version was significantly decreased (p<0.0001). As a control, polyclonal antiserum raised against recombinant LipL32 was tested for reactivity against the unmodified and modified versions of P1; interestingly, no difference in reactivity was observed.


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)

Leptospirosis patient serum reactivity against modified and unmodified versions of synthetic LipL32 peptides.Shown is the reactivity of pooled sera from laboratory-confirmed leptospirosis patients (black bars) and LipL32-specific polyclonal rabbit serum (white bars) to unmodified and tri-methylated (KMe3) versions of P1 and P2 peptides comprising experimentally-determined B-cell epitopes of LipL32 [36]. Two independent experiments were performed with reproducible results; the results from one representative experiment are shown. Error bars represent standard error of measurement from triplicate samples. For statistical analyses, the level of reactivity of patient sera against the unmodified version of P1 was compared to the level of reactivity against the P1 peptide containing a tri-methylation at position K152 (P1-K152Me3) using the Student's two-tailed t-test (*p<0.0001).
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0003280-g002: Leptospirosis patient serum reactivity against modified and unmodified versions of synthetic LipL32 peptides.Shown is the reactivity of pooled sera from laboratory-confirmed leptospirosis patients (black bars) and LipL32-specific polyclonal rabbit serum (white bars) to unmodified and tri-methylated (KMe3) versions of P1 and P2 peptides comprising experimentally-determined B-cell epitopes of LipL32 [36]. Two independent experiments were performed with reproducible results; the results from one representative experiment are shown. Error bars represent standard error of measurement from triplicate samples. For statistical analyses, the level of reactivity of patient sera against the unmodified version of P1 was compared to the level of reactivity against the P1 peptide containing a tri-methylation at position K152 (P1-K152Me3) using the Student's two-tailed t-test (*p<0.0001).
Mentions: Analysis of the modified LipL32 peptides detected within the RUIL samples highlighted four peptides that overlap with regions previously identified through epitope mapping studies as antigenic regions of LipL32 [36]. Within the reported LipL32 epitope spanning residues 132–158 of the mature protein (designated as P1; AAKAKPVQKLDDDDDGDDTYKEERHNK) we detected tri-methylation of residue K152. For the LipL32 epitope encompassing residues 162–185 of the mature protein (designated as P2; LTRIKIPNPPKSFDDLKNIDTKKL) we detected modifications on K166, K172 and K178, with the latter constituting a confirmed tri-methylation. In order to investigate if the modifications altered immune recognition of these epitopes, synthetic peptides corresponding to the two previously identified epitopes were prepared; specifically, unmodified and K152 tri-methylated versions of P1 were synthesized, while for P2 an unmodified and a representative modified peptide containing a tri-methylated K178 residue were synthesized (Table 1). When tested in an ELISA assay (Figure 2), pooled sera obtained from patients with laboratory-confirmed leptospirosis reacted strongly to the unmodified version of P1, while reactivity against the K152 tri-methylated peptide version was significantly decreased (p<0.0001). As a control, polyclonal antiserum raised against recombinant LipL32 was tested for reactivity against the unmodified and modified versions of P1; interestingly, no difference in reactivity was observed.

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