| Background:Lyme disease,which is caused by the tick-borne spirochete Borrelia burgdorferi(Bb),is identified as a multistage and multisystem disease which results in the neurological manifestation of systemic infection at a later period,which is called Lyme neuroborreliosis(LNB).The LNB patients often show lymphocyte meningitis,radiculitis,and may have an impairment in cognition and memory.This spirochete,with characteristic features such as neural invasion,can be lurking in the central nervous system for a long time.It is acknowledged that immune responses and inflammatory responses could be an important cause of the damage to the nervous system after being infected with spirochetes.Moreover,the infection in nervous system is accompanied by complex pathophysiological processes,with thousands of changes in the expression of genes and proteins,and complex regulatory networks exist.Previous studies have studied the mechanism in the rhesus brain tissues upon exposure to Bb.Objective:The current research has aimed at providing the rationale for transcriptome and proteome analysis of frontal cortex tissues as a means for gaining insight into overall responses after infection with Bb,screening possible biomarkers,elucidating the occurrence and development of disease,and gaining novel insights to the research on the molecular mechanism of spirochetes infection in nervous system.Methods:The B31 strain was grown in BSK Ⅱ medium to the late logarithmic phase,and the plate count was examined by dark-field microscopy.Freshly harvested frontal cortex tissues were collected with 18 sections weighed 0.5 g placing in separate T-25 flasks.Half flasks(Bb)contained 4 mL of 10%FBS-RPMI 1640 medium and half flasks(control)contained 4 mL of Bb bacteria suspension of 1× 107 bacteria/mL.Then they were incubated in a humidified incubator with 5%CO2 at 37℃ for 6,12 or 24 h.Each group collects three samples at each time points,and the total RNA and protein of samples were taken.After cDNA libraries was constructed,sequencing was performed on an Illumina HiSeq 2500 sequencing platform.The gene profiles were obtained and screening criteria for significant differences were adjusted p-value(FDR)<0.05 and | log2(fold change)|>1.The peptides were labeled according to the iTRAQ-8 standard kit instructions and were then graded and separated by an Ultimate 3000 HPLC system for further LC-MS/MS analysis.Differentially expressed proteins were identified with a fold change of≥ 1.5(upregulated)or ≤0.67(downregulated)&p-value<0.05.In addition,hierarchical clustering was utilized to analysis the expression mode,and the mRNA as well as protein levels were annotated and later validated by real-time PCR and western blotting.Results:The correlation of gene expression in the sample was high.Compared to the control,there were 2249(60.2%),1064(28.5%),and 420(11.3%)differentially expressed genes(DEGs)and 43(7.5%),164(28.5%),and 368(64%)differentially expressed proteins(DEPs)at 6 h,12 h and 24 h,respectively.The profiles of the enriched terms and pathways were quite different on responses to Bb infection at different time points.At the same time point,significant differences were seen among upregulated DEGs/DEPs and downregulated DEGs/DEPs.In total,pathways were significantly enriched including the MAPK signaling pathway,NF-kappa B signaling pathway,Rapl signaling pathway,Ras signaling pathway and complement and coagulation cascades,which were closely interrelated as components of the immune response in the progression of the Bb infection.Our results also showed that the DEGs related to immune response were significantly upregulated following Bb infection such as IL6ST,CSF1R,C1QTNF7,CX3CR1,and CCL24.A linear regression analysis revealed that a weak correlation between the transcriptome and proteome profiles,which could be attributed to intricate regulation networks of transcription and translation.Furthermore,based on the analysis of omics data,translational regulation,glycosaminoglycan/proteoglycan-binding activity in colonization and dissemination to tissues,disease-associated genes and synaptic function were enriched.We also validated the mRNA expression level of eight hub genes by qPCR and the result is consistent with the transcriptome result.Indeed,FOLR2 was the most significantly altered gene and was validated by qPCR and western blotting with significant increase in expression after infection.Conclusion:1.Based on the data of transcriptome and proteome analysis of frontal cortex tissues co-cultured with Bb,inflammation and immune responses play a role in development of diseases after Bb infection.2.During the interaction between frontal cortex tissues and spirochetes,bioinformatics analysis reveals that the diversity of translational regulation,glycosaminoglycan/proteoglycan-binding activity in colonization and dissemination to tissues,the association of central nervous system infection and neurodegenerative diseases,and synaptic functions closely relate to the pathogenesis of the nervous system infection in Lyme disease.3.FOLR2 is a promising target and may represent a new diagnostic or therapeutic marker in Lyme neuroborreliosis.However,further in-depth mechanism research is still needed. |
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