A novel immunoproteomics method for identifying in vivo-induced Campylobacter jejuni antigens using pre-adsorbed sera from infected patients

Background

Campylobacter jejuni is an important food-borne and zoonotic pathogen with a worldwide distribution. Humans and chickens are hosts of this pathogen. At present, there is no ideal vaccine for controlling human campylobacteriosis or the carriage of C. jejuni by chickens. Bacterial in vivo-induced antigens are useful as potential vaccine candidates and biomarkers of virulence.

Methods

In this study, we developed a novel systematic immunoproteomics approach to identify in vivo-induced antigens among the total cell proteins of C. jejuni using pre-adsorbed sera from patients infected with C. jejuni.

Results

Overall, 14 immunoreactive spots were probed on a PVDF membrane using pre-adsorbed human sera against C. jejuni. Then, we excised these protein spots from a duplicate gel and identified using MALDI–TOF MS. In total, 14 in vivo-induced antigens were identified using PMF and BLAST analysis. The identified proteins include CadF (CadF-1 and CadF-2), CheW, TufB, DnaK, MetK, LpxB, HslU, DmsA, PorA, ProS, CJBH_0976, CSU_0396 and hypothetical protein cje135_05017. Real-time RT-PCR was performed on 9 genes to compare their expression levels in vivo and in vitro. The data showed that 8 of the 9 analyzed genes were significantly upregulated in vivo relative to in vitro.

Conclusion

We successfully developed a novel immunoproteomics method for identifying in vivo-induced Campylobacter jejuni antigens by using pre-adsorbed sera from infected patients.

General significance

This new analysis method may prove to be useful for identifying in vivo-induced antigens within any host infected by bacteria and will contribute to the development of new subunit vaccines.     

Properties of Crystalline α-Glucosidase from Mucor javanicus

Substrate and inhibitor specificities, and transglucosylation action of crystalline α-glucosidase from the mycelia of Mucor javanicus have been investigated. The enzyme hydrolyzed maltose, methyl-α-maltoside, and soluble starch liberating glucose, but little or not phenyl-α-glucoside, methyl-α-glucoside, sucrose, isomaltose, panose and dextran. The enzyme hydrolyzed phenyl-α-maltoside to glucose and phenyl-α-glucoside. The enzyme acted also as a glucosyltransferase when it was incubated with glucosyl donor such as maltose. Maltotriose was the principal transglucosylation product formed from maltose. The enzyme also catalyzed transglucosylation from maltose to riboflavin, pyridoxine, esculin and rutin. Tris and turanose inhibited the enzyme activity, but PCMB and EDTA did not. It is suggested that the enzyme activity is closely related to the histidine residue in the active center, from the inhibition experiments using diazonium-1-H-tetrazole and rose bengal.     

Entamoeba histolytica: ADP-ribosylation of secreted glyceraldehyde-3-phosphate dehydrogenase

In addition to its classic glycolytic role, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been implicated in many activities unrelated to glycolysis, such as membrane fusion, binding to host proteins and signal transduction. GAPDH can be the target of several modifications that allow incorporation to membranes and possible regulation of its activity; among these modifications is mono-ADP-ribosylation. This post-translational modification is important for the regulation of many cellular processes and is the mechanism of action of several bacterial toxins. In a previous study, we observed the extracellular ADP-ribosylation of a 37-kDa ameba protein. We report here that GAPDH and cysteine synthase A are the main ADP-ribosylated proteins in Entamoeba histolytica extracellular medium, GAPDH is secreted from ameba at 37 degrees C in a time-dependent manner, and its enzymatic activity is not inhibited by ADP-ribosylation. Extracellular GAPDH from ameba may play an important role in the survival of this human pathogen or in interaction with host molecules, as occurs in other organisms.     

iTRAQ compatibility of peptide immobilized pH gradient isoelectric focusing

Immobilized pH gradient isoelectric focusing (IPG-IEF) has emerged as a highly promising alternative to strong-cation exchange fractionation as the first dimension in shot-gun proteomics. Herein is shown the compatibility of this method with iTRAQ isotope labeling for relative quantitation and validation of sequence matches from database searching.     

Hsp42 is the general small heat shock protein in the cytosol of Saccharomyces cerevisiae

Small heat shock proteins (sHsps) are ubiquitous molecular chaperones that prevent the unspecific aggregation of proteins. So far, Hsp26 was the only unambiguously identified member of the sHsp family in Saccharomyces cerevisiae. We show here that the sHsp system in the cytosol of S. cerevisiae consists of two proteins, Hsp26 and Hsp42. Hsp42 forms large dynamic oligomers with a barrel-like structure. In contrast to Hsp26, which functions predominantly at heat shock temperatures, Hsp42 is active as a chaperone under all conditions tested in vivo and in vitro. Under heat shock conditions, both Hsp42 and Hsp26 suppress the aggregation of one-third of the cytosolic proteins. This subset is about 90% overlapping for Hsp42 and Hsp26. The sHsp substrates belong to different biochemical pathways. This indicates a general protective function of sHsps for proteome stability in S. cerevisiae. Consistent with this observation, sHsp knockout strains show phenotypical defects. Taken together, our results define Hsp42 as an important player for protein homeostasis at physiological and under stress conditions.     

Optimization of the first dimension for separation by two-dimensional gel electrophoresis of basic proteins from human brain tissue

A major cause of poor resolution in the alkaline pH range of two-dimensional electrophoresis (2-DE) gels is unsatisfactory separation of basic proteins in the first dimension. We have compared methods for the separation of basic proteins in the isoelectric focusing dimension of human brain proteins. The combined use of anodic cup-loading and the hydroxyethyldisulphide containing solution (DeStreak) produced better resolution in both analytical and micropreparative protein loaded 2-DE gels than the other methods investigated.     

Identification of phospholipase D from cabbage as N-terminally acetylated PLD2

Recently, the genes of two isoenzymes of phospholipase D from white cabbage (PLD1 and PLD2) with molecular masses of 91.7 and 91.9 kDa, respectively, have been sequenced and expressed in Escherichia coli [Schäffner, I., Rücknagel, K.-P., Mansfeld, J., and Ulbrich-Hofmann, R. (2002). Eur. J. Lipid Sci. Technol. 104: 79–87]. Both enzymes are highly homologous (91% identity) and behave very similarly. Phospholipase D purified from white cabbage leaves (PLD_cab) is compared with the two recombinant enzymes in sodium dodecylsulfate and native polyacrylamide gel electrophoresis, isoelectric focusing, N-terminal sequencing, and mass spectrometry after tryptic digestion. As a result, PLD_cab clearly can be assigned to PLD2. In contrast to recombinant PLD2, however, PLD_cab is N-terminally acetylated.     

Carboxyl-terminal modification alters the subunit interactions and assembly pathways of normal and sickle hemoglobins

Parallel isofocusing studies established that carboxypeptidase A removal of the His-146 (HC3) and Tyr-145 (HC2) residues of heme subunits affected the assembly properties of both Des (A) and Des (S) with heme chains, albeit to differing degrees. Indeed, the rate of Des (A) oligomer dissociation (k ₁), as determined by visible spectroscopy, was 4.3-fold faster than that of its native (A) counterpart. Furthermore, Soret spectral studies have affirmed distinct rates of normal (HbA), sickle (HbS), and Des HbA hemoglobin assembly (k₂) from their and [Des (A)] heme-containing monomers. Matching kinetic analysis of Des (A) and Des (S) chain assembly (with an identical chain) revealed 4.6- and 7.8-fold faster combination rates than those seen for (A) and (S) chains, respectively. This 3-fold disparity in rates strongly supports the critical role of the -6 (A3) residue, and its amino-terminal region, in chain partner recognition and subsequent human hemoglobin assembly.     

Comparison of <Emphasis Type="Italic">Escherichia coli,Saccharomyces cerevisiae,Pichia pastoris,Spodoptera frugiperda</Emphasis>, and COS7 cells for recombinant gene expression

Expression of a rabbit liver carboxylesterase has been achieved in several different model systems including Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Spodoptera frugiperda, and COS7 cells. Although, recombinant protein was observed in E. coli sonicates, little or no enzymatic activity was detected. Similarly, no activity was observed following expression in S. cerevisiae. In contrast, active protein was produced in P. pastoris from S. frugiperda, following baculoviral infection and in COS7 cells following transient transfection of plasmid DNA. For the preparation of small amounts of protein for kinetic and biochemical studies, enzyme expressed in P. pastoris has proved sufficient. However, to produce large amounts of carboxylesterase for structural studies, baculoviral-mediated expression of a secreted form of the protein in S. frugiperda was the most efficient. Using this system, we have generated and purified milligram quantities of essentially pure protein. These results demonstrate that the choice of in vitro system for the generation of large amounts of active carboxylesterase, and probably most endoplasmic reticulum processed proteins, is crucial for high level expression and subsequent purification.