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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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Two-dimensional gel electrophoresis (2DGE) of Leptospira whole proteome profiles indicating LipL32 protein spots analyzed by MS/MS.(A) 2DGE profile of in vitro-cultured Leptospira (IVCL) and (B) 2DGE profile of rat urine-isolated Leptospira (RUIL). Protein derived from whole cells were separated using immobilized pH gradient IPG strips (pH 3–10, non-linear) followed by SDS-PAGE. Circled protein spots corresponding to LipL32 were excised for analysis by LC-MS/MS.
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pntd-0003280-g001: Two-dimensional gel electrophoresis (2DGE) of Leptospira whole proteome profiles indicating LipL32 protein spots analyzed by MS/MS.(A) 2DGE profile of in vitro-cultured Leptospira (IVCL) and (B) 2DGE profile of rat urine-isolated Leptospira (RUIL). Protein derived from whole cells were separated using immobilized pH gradient IPG strips (pH 3–10, non-linear) followed by SDS-PAGE. Circled protein spots corresponding to LipL32 were excised for analysis by LC-MS/MS.

Mentions: In the current study we investigated the presence of PTMs, with a focus upon methylations, occurring in LipL32 from L. interrogans serovar Copenhageni cells grown under in vivo and in vitro growth conditions. Total protein was prepared from leptospires shed in the urine of rats housed in metabolic cages (rat urine-isolated Leptospira; RUIL) and from leptospires cultured in EMJH medium (in vitro-cultivated Leptospira; IVCL), and each of the whole cell proteomes was independently subjected to two-dimensional gel electrophoresis (Figure 1). Two protein spots corresponding to LipL32 were excised from each of the RUIL and IVCL samples; to allow for a direct comparison, the same LipL32 isoform was selected from each gel. The protein spots were digested with trypsin and analyzed by LC-MS/MS to verify the identity of the excised proteins. The resultant spectrometry data was subjected to manual analysis to confirm detected PTM position assignments. A list of peptides identified by LC-MS/MS, including peptides containing confirmed PTMs, is shown in Table 2, and the obtained MS/MS data and spectra for peptides containing confirmed PTMs can be found in the Supplementary Data Files (Table S1 and Figure S1, respectively).


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)

Two-dimensional gel electrophoresis (2DGE) of Leptospira whole proteome profiles indicating LipL32 protein spots analyzed by MS/MS.(A) 2DGE profile of in vitro-cultured Leptospira (IVCL) and (B) 2DGE profile of rat urine-isolated Leptospira (RUIL). Protein derived from whole cells were separated using immobilized pH gradient IPG strips (pH 3–10, non-linear) followed by SDS-PAGE. Circled protein spots corresponding to LipL32 were excised for analysis by LC-MS/MS.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0003280-g001: Two-dimensional gel electrophoresis (2DGE) of Leptospira whole proteome profiles indicating LipL32 protein spots analyzed by MS/MS.(A) 2DGE profile of in vitro-cultured Leptospira (IVCL) and (B) 2DGE profile of rat urine-isolated Leptospira (RUIL). Protein derived from whole cells were separated using immobilized pH gradient IPG strips (pH 3–10, non-linear) followed by SDS-PAGE. Circled protein spots corresponding to LipL32 were excised for analysis by LC-MS/MS.
Mentions: In the current study we investigated the presence of PTMs, with a focus upon methylations, occurring in LipL32 from L. interrogans serovar Copenhageni cells grown under in vivo and in vitro growth conditions. Total protein was prepared from leptospires shed in the urine of rats housed in metabolic cages (rat urine-isolated Leptospira; RUIL) and from leptospires cultured in EMJH medium (in vitro-cultivated Leptospira; IVCL), and each of the whole cell proteomes was independently subjected to two-dimensional gel electrophoresis (Figure 1). Two protein spots corresponding to LipL32 were excised from each of the RUIL and IVCL samples; to allow for a direct comparison, the same LipL32 isoform was selected from each gel. The protein spots were digested with trypsin and analyzed by LC-MS/MS to verify the identity of the excised proteins. The resultant spectrometry data was subjected to manual analysis to confirm detected PTM position assignments. A list of peptides identified by LC-MS/MS, including peptides containing confirmed PTMs, is shown in Table 2, and the obtained MS/MS data and spectra for peptides containing confirmed PTMs can be found in the Supplementary Data Files (Table S1 and Figure S1, respectively).

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