Cerebrospinal fluid proteome of patients with acute Lyme disease

Abstract

During acute Lyme disease, bacteria can disseminate to the central nervous system (CNS), leading to the development of meningitis and other neurologic symptoms. Here we have analyzed pooled cerebrospinal fluid (CSF) allowing a deep view into the proteome for patients diagnosed with early disseminated Lyme disease and CSF inflammation. Additionally, we analyzed individual patient samples and quantified differences in protein abundance employing label-free quantitative mass spectrometry-based methods. We identified 108 proteins that differ significantly in abundance in patients with acute Lyme disease from controls. Comparison between infected patients and control subjects revealed differences in proteins in the CSF associated with cell death localized to brain synapses and others that likely originate from brain parenchyma.

Figure 1

Cerebrospinal fluid proteome of pooled samples from subjects with acute Lyme disease and non-infected control subjects. Pooled CSF from Lyme disease and control samples were digested, fractionated offline by strong cation exchange, and analyzed by reversed phase LC-MS/MS. We identified peptides attributable to a total of 1458 proteins considering all sample fractions, 906 in the pooled control sample and 1104 in the pooled acute Lyme disease sample. There were 552 proteins common to both samples, 552 were uniquely identified in the acute Lyme disease pooled sample and 354 were uniquely identified in the control pooled sample. Functional analysis for proteins unique to either control or Lyme subject pooled samples was performed employing Ingenuity Pathway tools (www.ingenuity.com) revealing the presence of proteins associated with cell death in the acute Lyme disease pooled samples. In contrast proteins relating to development and amino acid metabolism were singularly identified in pooled control samples.

Figure 2

Distribution pattern for CNS proteins identified in the CSF proteome of Lyme disease and control subject samples.

Figure 3

Venn diagram with CNS compartmentalization. Comparison between the CSF proteome of combined normal subjects and acute Lyme disease patients (yellow circle) with previously reported plasma proteome (red circle) and brain postsynaptic density proteome (blue circle).

Figure 4

Analysis of individual patient samples led to the quantification of 247 proteins. An analysis of variance of the protein abundances revealed 108 proteins as being differentially abundant (p value < 0.05) (A) where 60 were increased and 48 were decreased in abundance relative to control. Unsupervised cluster analysis of the subject samples shows that the quantified protein abundances (rows in matrix) allowed for partial segregation of patient and control samples (columns) (B).

Figure 5

Network analysis of proteins found to be significantly altered in abundance in the CSF proteome by ANOVA (p value <0.05) following analysis of individual subject samples. Network analysis was performed employing ingenuity pathway tools (www.ingenuity.com) on proteins that were annotated in the Ingenuity database (101 of the 108 proteins). Illustrated are proteins, colored by ratio (Lyme/Control) of average protein abundances, with known functional and disease associated relationships that differed in abundance levels in the CSF.

Figure 6

Proteins with greatest discriminating power for Lyme disease. ROC curve analysis of all proteins identified and quantified in the CSF led to the identification of 13 proteins that had good discriminating power with areas under the curve (AUC) greater than 0.8. Shown are the scaled protein abundance values of the 13 proteins that show the greatest discriminating power in the ROC curve analysis.