Proteomic analysis of three Borrelia burgdorferi sensu lato native species and disseminating clones: Relevance for Lyme vaccine design

Lyme borreliosis is the most important vector‐borne disease in the Northern hemisphere. It is caused by Borrelia burgdorferi sensu lato bacteria transmitted to humans by the bite of hard ticks, Ixodes spp. Although antibiotic treatments are efficient in the early stage of the infection, a significant number of patients develop disseminated manifestations (articular, neurological, and cutaneous) due to unnoticed or absence of erythema migrans, or to inappropriate treatment. Vaccine could be an efficient approach to decrease Lyme disease incidence. We have developed a proteomic approach based on a one dimensional gel electrophoresis followed by LC‐MS/MS strategy to identify new vaccine candidates. We analyzed a disseminating clone and the associated wild‐type strain for each major pathogenic Borrelia species: B. burgdorferi sensu stricto, B. garinii, and B. afzelii. We identified specific proteins and common proteins to the disseminating clones of the three main species. In parallel, we used a spectral counting strategy to identify upregulated proteins common to the clones. Finally, 40 proteins were found that could potentially be involved in bacterial virulence and of interest in the development of a new vaccine. We selected the three proteins specifically detected in the disseminating clones of the three Borrelia species and checked by RT‐PCR whether they are expressed in mouse skin upon B. burgdorferi ss inoculation. Interestingly, BB0566 appears as a potential vaccine candidate. All MS data have been deposited in the ProteomeXchange with identifier PXD000876 ( http://proteomecentral.proteomexchange.org/dataset/PXD000876 ).     

The lipid raft proteome of Borrelia burgdorferi

Eukaryotic lipid rafts are membrane microdomains that have significant amounts of cholesterol and a selective set of proteins that have been associated with multiple biological functions. The Lyme disease agent, Borrelia burgdorferi, is one of an increasing number of bacterial pathogens that incorporates cholesterol onto its membrane, and form cholesterol glycolipid domains that possess all the hallmarks of eukaryotic lipid rafts. In this study, we isolated lipid rafts from cultured B. burgdorferi as a detergent resistant membrane (DRM) fraction on density gradients, and characterized those molecules that partitioned exclusively or are highly enriched in these domains. Cholesterol glycolipids, the previously known raft‐associated lipoproteins OspA and OpsB, and cholera toxin partitioned into the lipid rafts fraction indicating compatibility with components of the DRM. The proteome of lipid rafts was analyzed by a combination of LC‐MS/MS or MudPIT. Identified proteins were analyzed in silico for parameters that included localization, isoelectric point, molecular mass and biological function. The proteome provided a consistent pattern of lipoproteins, proteases and their substrates, sensing molecules and prokaryotic homologs of eukaryotic lipid rafts. This study provides the first analysis of a prokaryotic lipid raft and has relevance for the biology of Borrelia, other pathogenic bacteria, as well as for the evolution of these structures. All MS data have been deposited in the ProteomeXchange with identifier PXD002365 ( http://proteomecentral.proteomexchange.org/dataset/PXD002365 ).     

Comparison of Borrelia isolated from UK foci of Lyme disease

Abstract Restriction endonuclease digestion of linear borrelial chromosomal DNA showed that three isolates of UK Lyme disease spirochaetes differed markedly from each other and from published data for other isolates from North America and continental Europe. Analysis of linear plasmid bands revealed that UK isolates differed from each other in the number and sizes of the plasmids in isolates from different foci of UK Lyme disease. Fatty acid analysis (of fatty acid methyl ester (FAME) profiles) showed the UK isolates clustering together with the relapsing fever spirochaetes, Borrelia turicatae and Borrelia parkeri . These data are discussed in respect of current knowledge of Lyme borreliosis in the UK.     

Molecular analysis of a 66-kDa protein associated with the outer membrane of Lyme disease Borrelia

Abstract A 66-kDa protein (p66) associated with the outer membrane of Lyme disease Borrelia was analysed at the molecular level. The chromosomal genes encoding p66 in B. burgdorferi B31, B. afzelii ACAI, and B. garinii Ip90 were sequenced. Database searches revealed that the p66 gene sequences were homologous to a previously reported gene fragment of unknown function. The deduced amino acid sequences of p66 in different Lyme disease borreliae were 92–94% identical and had no homologs in the databases. Proteolytic cleavage patterns of p66 and a computer-predicted single trans-membrane helix suggested the presence of surface-exposed epitopes on the C-terminus.     

The membrane proteome of Halobacterium salinarum

The identification of 114 integral membrane proteins from Halobacterium salinarum was achieved using liquid chromatography/tandem mass spectrometric (LC/MS/MS) techniques, representing 20% of the predicted alpha-helical transmembrane proteins of the genome. For this experiment, a membrane preparation with only minor contamination by soluble proteins was prepared. From this membrane preparation a number of peripheral membrane proteins were identified by the classical two dimensional gel electrophoresis (2-DE) approach, but identification of integral membrane proteins largely failed with only a very few being identified. By use of a fluorescently labeled membrane preparation, we document that this is caused by an irreversible precipitation of the membrane proteins upon isoelectric focusing (IEF). Attempts to overcome this problem by using alternative IEF methods and IEF strip solubilisation techniques were not successful, and we conclude that the classical 2-DE approach is not suited for the identification of integral membrane proteins. Computational analysis showed that the identification of integral membrane proteins is further complicated by the generation of tryptic peptides, which are unfavorable for matrix assisted laser desorption/ionization time of flight mass spectrometric peptide mass fingerprint analysis. Together with the result from the analysis of the cytosolic proteome (see preceding paper), we could identify 34% (943) of all gene products in H. salinarum which can be theoretically expressed. This is a cautious estimate as very stringent criteria were applied for identification. These results are available under www.halolex.mpg.de.     

Prevalence of IgG reactivity in Lyme borreliosis patients versus Borrelia garinii and Borrelia afzelii in a restricted area of Northern Italy

Abstract This survey evaluates the antibody band patterns of sera taken from clinically defined cases of Lyme borreliosis, towards three locally isolated strains of Borrelia burgdorferi , belonging to the three species: Borrelia sensu stricto, Borrelia garinii and Borrelia afzelii , by means of Western blot. The sera were taken from patients resident in a limited area of Friuli Venezia Giulia (FVG) region. The data indicated that, besides a different feature of the band reactivity which correlated to the different stages of Lyme borreliosis, there was a preferential reactivity to the species Borrelia afzelii and Borrelia garinii . An immunodominant band at 51 kDa, corresponding to a protein visible in the electrophoretic profile of strain BL3 ( B. afzelii ), behaved like a marker of an early infection, because it was present exclusively in the sera of patient with ECM. The overall findings would indicate that B. afzelii and B. garinii are the prevalent genospecies in the FVG area, even if strains belonging to B. sensu stricto have been also isolated in this area. Consequently strains representative of these two species must be used as antigens in Western blot.     

Gel absorption-based sample preparation for the analysis of membrane proteome by mass spectrometry

A gel absorption-based sample preparation method for shotgun analysis of membrane proteome has been developed. In this new method, membrane proteins solubilized in a starting buffer containing a high concentration of sodium dodecyl sulfate (SDS) were directly entrapped and immobilized into gel matrix when the membrane protein solution was absorbed by the vacuum-dried polyacrylamide gel. After the detergent and other salts were removed by washing, the proteins were subjected to in-gel digestion and the tryptic peptides were extracted and analyzed by capillary liquid chromatography coupled with tandem mass spectrometry (CapLC-MS/MS). The results showed that the newly developed method not only avoided the protein loss and the adverse protein modifications during gel embedment but also improved the subsequent in-gel digestion and the recovery of tryptic peptides, particularly the hydrophobic peptides, thereby facilitating the identification of membrane proteins, especially the integral membrane proteins. Compared with the conventional tube-gel digestion method, the newly developed method increased the numbers of identified membrane proteins and integral membrane proteins by 25.0% and 30.2%, respectively, demonstrating that the method is of broad practicability in gel-based shotgun analysis of membrane proteome.     

In-depth analysis of the human tear proteome

The tears, a critical body fluid of the surface of the eye, contain an unknown number of molecules including proteins/peptides, lipids, small molecule metabolites, and electrolytes. There have been continued efforts for exploring the human tear proteome to develop biomarkers of disease. In this study, we used the high speed TripleTOF 5600 system as the platform to analyze the human tear proteome from healthy subjects (3 females and 1 male, average age: 36±14). We have identified 1543 proteins in the tears with less than 1% false discovery rate, which represents the largest number of human tear proteins reported to date. The data set was analyzed for gene ontology (GO) and compared with the human plasma proteome, NEIBank lacrimal gland gene dataset and NEIBank cornea gene dataset. This comprehensive tear protein list may serve as a reference list of human tear proteome for biomarker research of ocular diseases or establishment of MRM (Multiple Reaction Monitoring) assays for targeted analysis. Tear fluid is a useful and an accessible source not only for evaluating ocular surface tissues (cornea and conjunctiva), inflammation, lacrimal gland function and a number of disease conditions, such as dry eye as well as response to treatment.     

Comparative proteome analysis reveals pathogen specific outer membrane proteins of Leptospira

Proteomes of pathogenic Leptospira interrogans and L. borgpetersenii and the saprophytic L. biflexa were filtered through computational tools to identify Outer Membrane Proteins (OMPs) that satisfy the required biophysical parameters for their presence on the outer membrane. A total of 133, 130, and 144 OMPs were identified in L. interrogans, L. borgpetersenii, and L. biflexa, respectively, which forms approximately 4% of proteomes. A holistic analysis of transporting and pathogenic characteristics of OMPs together with Clusters of Orthologous Groups (COGs) among the OMPs and their distribution across 3 species was made and put forward a set of 21 candidate OMPs specific to pathogenic leptospires. It is also found that proteins homologous to the candidate OMPs were also present in other pathogenic species of leptospires. Six OMPs from L. interrogans and 2 from L. borgpetersenii observed to have similar COGs while those were not found in any intermediate or saprophytic forms. These OMPs appears to have role in infection and pathogenesis and useful for anti‐leptospiral strategies.     

2-D differential membrane proteome analysis of scarce protein samples

Proteome studies with small sample amounts are difficult to perform, especially when membrane proteins are the focus of interest. In our study a new method for the analysis of scarce membrane protein samples combining large gel 2-D-CTAB/SDS-PAGE with fluorescence dye saturation labelling (satDIGE) was developed, allowing a highly sensitive differential analysis of different cell states. After Triton X-114 phase partitioning, enriched membrane protein samples of T cells were labelled at cysteine residues using fluorescence dyes and separated by large gel 2D-CTAB/SDS-PAGE. For a differential analysis 3 mug protein was found to be sufficient to detect proteins in a widespread well-separated diagonal spot pattern.     

Investigation of solubilization and digestion methods for microsomal membrane proteome analysis using data-independent LC-MSE

To evaluate the implementation of various denaturants and their efficacy in bottom-up membrane proteomic methods using LC-MS analysis, microsomes isolated from tomato roots were treated with MS-compatible surfactants (RapiGest SF Surfactant from Waters and PPS Silent Surfactant from Protein Discovery), a chaotropic reagent (guanidine hydrochloride), and an organic solvent (methanol). Peptides were analyzed in triplicate sample and technical replicates by data-independent LC-MS(E) analysis. Overall, 2333 unique peptides matching to 662 unique proteins were detected with the order of denaturant method efficacy being RapiGest SF Surfactant, PPS Silent Surfactant, guanidine hydrochloride, and methanol. Using bioinformatic analysis, 103 proteins were determined to be integral membrane proteins. When normalizing the data as a percentage of the overall number of peptides and proteins identified for each method, the order for integral membrane protein identification efficacy was methanol, guanidine hydrochloride, RapiGest SF Surfactant, and PPS Silent Surfactant. Interestingly, only 8% of the proteins were identified in all four methods with the silent surfactants having the greatest overlap at 17%. GRAVY analysis at the protein and peptide level indicated that methanol and guanidine hydrochloride promoted detection of hydrophobic proteins and peptides, respectively; however, trypsin activity in the presence of each denaturant was determined as a major factor contributing to peptide identification by LC-MS(E) . These results reveal the complementary nature of each denaturant method, which can be used in an integrated approach to provide a more effective bottom-up analysis of membrane proteomes than can be achieved using only a single denaturant.     

Proteomic and bioinformatic analysis of membrane proteome in type 2 diabetic mouse liver

Type 2 diabetes mellitus (T2DM) is the most prevalent and serious metabolic disease affecting people worldwide. T2DM results from insulin resistance of the liver, muscle, and adipose tissue. In this study, we used proteomic and bioinformatic methodologies to identify novel hepatic membrane proteins that are related to the development of hepatic insulin resistance, steatosis, and T2DM. Using FT‐ICR MS, we identified 95 significantly differentially expressed proteins in the membrane fraction of normal and T2DM db/db mouse liver. These proteins are primarily involved in energy metabolism pathways, molecular transport, and cellular signaling, and many of them have not previously been reported in diabetic studies. Bioinformatic analysis revealed that 16 proteins may be related to the regulation of insulin signaling in the liver. In addition, six proteins are associated with energy stress‐induced, nine proteins with inflammatory stress‐induced, and 14 proteins with endoplasmic reticulum stress‐induced hepatic insulin resistance. Moreover, we identified 19 proteins that may regulate hepatic insulin resistance in a c‐Jun amino‐terminal kinase‐dependent manner. In addition, three proteins, 14–3‐3 protein beta (YWHAB), Slc2a4 (GLUT4), and Dlg4 (PSD‐95), are discovered by comprehensive bioinformatic analysis, which have correlations with several proteins identified by proteomics approach. The newly identified proteins in T2DM should provide additional insight into the development and pathophysiology of hepatic steatosis and insulin resistance, and they may serve as useful diagnostic markers and/or therapeutic targets for these diseases.     

Comparative proteome profile of immature rat ovary during primordial follicle assembly and development

The assembly of primordial follicles early in ovarian development and subsequent transition to primary follicles are critical processes in ovarian biology. Inappropriate coordination of these processes contributes to ovarian pathologies such as premature ovarian failure and infertility. To better understand the molecular mechanisms involved in primordial follicle assembly and development, 2‐D PAGE and MALDI‐TOF/TOF technologies were used to construct a comparative proteome profile of the immature rat ovary at specific time‐points (0, 24, 48, and 72 h postpartum). A total of 154 differential protein spots corresponding to 134 different proteins were definitively identified between any two time‐points. Further cluster analysis showed four expression patterns, and each pattern correlated with specific cell processes that occur during early ovarian development. Seven proteins were randomly selected to verify expression patterns using Western blotting, and subsequently immunohistochemistry was performed to further investigate their cellular localization. Additionally, detailed functional analyses of these differentially expressed proteins were performed. Elucidation of how these changes in protein expression level coordinate primordial follicles assembly and development is intended to provide a better understanding of these critical biological processes early in ovarian development and will provide potential therapeutic molecular targets to regulate ovarian function and treat ovarian disease.     

Comparative analysis of proteome differential regulation during cell dedifferentiation in Arabidopsis

Cell dedifferentiation is a cell fate switching process in which differentiated cells undergo genome reprogramming to regain the competency of cell division and organ regeneration. The molecular mechanism underlying the cell dedifferentiation process remains obscure. In this report, we investigate the cell dedifferentiation process in Arabidopsis using a shotgun proteomics approach. A total of 758 proteins are identified by two or more matched peptides. Comparative analyses at four time points using two label-free methods reveal that 193 proteins display up-regulation and 183 proteins display down-regulation within 48 h. While the results of the two label-free quantification methods match well with each other, comparison with previously published 2-DE gel results reveal that label-free quantification results differ substantially from those of the 2-DE method for proteins with peptides common to multiple proteins, suggesting a limitation of the label-free methods in quantifying proteins with closely related family members in complex samples. Our results show that the shotgun approach and the traditional 2-DE gel approach complement each other in both protein identification and quantification. An interesting observation is that core histones and histone variants are subjected to extensive down-regulation, indicating that there is a dramatic change in the chromatin during cell differentiation.     

Comparative proteome analysis of splenic lymphocytes in long-term high-fat diet and dietary supplement with lipoic acid mice

The objective of this investigation was to explore possible molecular changes for role of a high-fat diet (HFD)-induced oxidative stress in splenic lymphocytes, and whether a dietary lipoic acid (LA) supplement could attenuate these changes. Male C57BL/6 mice were fed one of three diets 10 weeks and outcome measures centered on parameters of oxidative stress and lymphocytes apoptosis in spleen. Two-dimensional gel electrophoresis was used to compare the proteomes of splenic lymphocytes with three dietary groups. Differentially expressed spots whose expression altered over three fold were identified by MALDI-TOF MS. In this study, HFD resulted in oxidative stress in mice spleen, and significantly increased apoptotic percentage of splenic lymphocytes. Bioinformatic evaluation results of MALDI-TOF MS showed that 20 differentially expressed protein spots were known to be involved in many processes associated with cell function, such as cytoskeleton, energy metabolism and oxidative stress, signal transduction and cell defense. In conclusion, these results indicate that HFD-induced oxidative stress could lead to the functional decline of splenic lymphocytes, and LA supplement attenuates the alterations of protein expression to maintain the basic biological processes.     

Human saliva proteome analysis and disease biomarker discovery

Human saliva is an attractive body fluid for disease diagnosis and prognosis because saliva testing is simple, safe, low-cost and noninvasive. Comprehensive analysis and identification of the proteomic content in human whole and ductal saliva will not only contribute to the understanding of oral health and disease pathogenesis, but also form a foundation for the discovery of saliva protein biomarkers for human disease detection. In this article, we have summarized the proteomic technologies for comprehensive identification of proteins in human whole and ductal saliva. We have also discussed potential quantitative proteomic approaches to the discovery of saliva protein biomarkers for human oral and systemic diseases. With the fast development of mass spectrometry and proteomic technologies, we are enthusiastic that saliva protein biomarkers will be developed for clinical diagnosis and prognosis of human diseases in the future.     

Comparative analyses of three legume species reveals conserved and unique root extracellular proteins

Increasing evidence suggests that root extracellular proteins are involved in interactions between roots and their soil environment. In the present study, exudates released by 6‐day‐old roots of the three legume species white lupin (Lupinus albus), soybean (Glycine max), and cowpea (Vigna sinensis) were collected under axenic conditions, and their constitutively secreted proteomes were analyzed. Between 42 and 93 unique root extracellular proteins with 2 or more different peptide fragments per protein were identified by LC‐MS/MS. Functional annotation of these proteins classified them into 14–16 different functional categories. Among those 14 homologous proteins were identified in at least two legume species. Among the unique proteins, 58 in white lupin, 85 in soybean, and 31 in cowpea were specific for each plant species, and many of them were classified in the same functional categories. Interestingly, in contrast to soybean and cowpea, two protein bands of approximately 16 and 30 kDa were present on the SDS‐PAGE gel of white lupin. The identification of these bands revealed a class III chitinase and a thaumatin‐like protein. Both belong to the class of pathogenesis‐related proteins. The results imply that root extracellular proteins play important roles in the cross‐talk between plant roots and the rhizosphere.     

Evidence of Dbps (decorin binding proteins) among European strains of Borrelia burgdorferi sensu lato and in the immune response of LB patient sera

Decorin binding proteins DbpA and DbpB act as Borrelia burgdorferi (B. burgdorferi) adhesins to decorin, and are able to elicit a persistent antibody response in the mouse; accordingly DbpA protein would seem to be promising in immunoprofilaxis of Lyme borreliosis (LB). This study examines the distribution of Dbp epitopes in European strains of B. burgdorferi, of different genospecies and the presence of antibodies to Dbps in human sera from patients suffering from early and late LB, as revealed by immunoblotting. Different levels of expression of Dbp epitopes were found both among and within genospecies; data from human sera indicate that Dbps are expressed during infection though not as strongly as in the mouse infection.