Identification of immunogenic candidate for new serological tests for Brucella melitensis by a proteomic approach.

Background: The diagnosis of brucellosis by serological tests is based on antigen suspensions derived from smooth lipopolysaccharide extracts, which can give false-positive results linked to cross-reactivity with other Gram-negative microorganisms, especially Yersinia enterocolitica O:9 and Escherichia coli O157:H7. Objective: The objective of the present study was the characterization by proteomic analysis of specific immunogenic proteins not associated with smooth lipopolysaccharide to improve the diagnostic tests used in the ovine brucellosis eradication programs. Methods: The serum from a sheep positive to Brucella melitensis was treated to eliminate all antibodies against such lipopolysaccharides and highlight the reaction towards the immunoreactive proteins in western blotting. Results: The immunoreactive bands were identified by nLC-MS/MS, and through bioinformatics tools, it was possible to select 12 potential candidates as protein antigens specific for Brucella melitensis. Conclusion: The detection of new antigens not subjected to cross-reactivity with other Gram-negative microorganisms can offer additional tools for the serological diagnosis of such diseases

IgG antibodies from dourine infected horses identify a distinctive Trypanosoma equiperdum antigenic pattern of low molecular weight molecules

Diagnosis and control of dourine is strongly based on serological evidence, but knowledge of the humoral response of horses during infection is limited. In this study we developed a chemiluminescent immunoblotting (cIB) assay to characterise the Trypanosoma equiperdum antigen pattern recognised by IgGs from naturally or experimentally dourine-infected horses and analyse the kinetics of IgG humoral response following the infection. One compounding factor is that sera from uninfected animals often cross-react with T. equiperdum antigens. Development of the cIB assay was based on the hypothesis that serum IgGs from healthy and infected animals recognise different T. equiperdum antigen patterns. We used sera from 8 naturally infected horses which had recovered from Italian outbreaks and 2 experimentally infected mares. In addition, sera from 10 healthy control animals, eight of which were CFT positive but IFA negative for dourine, were collected from disease free regions. Sera were compared by the complement fixation test (CFT), indirect immune fluorescence (IFA) and the cIB assay. cIB analysis revealed that IgGs from infected horses, in contrast to IgGs from healhty horses, specifically recognise a T. equiperdum antigenic profile with low molecular weight bands ranging between 16 and 35 kDa. A time course experiment indicated that IgGs specific for the 16-35 kDa parasite protein fraction appear 17 days post-infection. The cIB assay confirmed all ten infected animals as positive and all controls as negative. This study demonstrated that analysis of IgGs by cIB can provide clear confirmation of trypanosome infection in horses, suggesting that this technique can be applied as a confirmatory serological test for dourine infection

SMALL ANGLE X-RAY SCATTERING STUDIES REVEAL IMPORTANT CLUES FOR MEMBRANE BINDING AND ACTIVITY OF FATTY ACID AMIDE HYDROLASE (FAAH)

Amides, esters, and ethers of long-chain polyunsaturated fatty acids, collectively referred to as “endocannabinoids”, represent a growing family of lipid signaling mediators found in several tissues with a wide variety of biological actions. The main members of this group of molecules are anandamide (N-arachidonoylethanolamine, AEA) and 2-arachidonoylglycerol. Fatty acid amide hydrolase (FAAH) is a typically membrane enzyme that catalyzes the conversion of AEA into arachidonic acid and ethanolamine, thus terminating the signaling of this endocannabinoid1. The homodimeric structure of FAAH has been deducted as a biological unit from the crystallographic structure of a mutant rat enzyme (TM-FAAH), a catalytically active form still able to bind the membranes even if missing the -helices mainly responsible for this interaction. Until now, direct information on FAAH aggregation state in solution and in the presence of inhibitors, substrate analogues, or lipids is still lacking.In this study we analyzed the oligomerization state of TM-FAAH in solution by small angle X-ray scattering (SAXS). We found that, among several effectors, high concentrations of Tris buffer (≥ 0.5 M) are able to stabilize monodisperse oligomers. Data analysis shows that these oligomeric structures have a value of radius of gyration of 129±13 Å, as calculated from the p(r) function (in accordance with the Guinier analysis) and a value of the maximum dimension of the particle (Dmax) of about 410±10 Å. Superimposing the crystallographic unit of FAAH (pdb entry 1MT5.pdb), which is an octamer of dimers, to the low resolution DAM model of our oligomers, we observed that these last are composed of three octamers revealing an unprecedented oligomerization state of FAAH in solution. Furthermore, we studied the structural effects of different FAAH inhibitors observing an increase of the oligomerization state of the protein in the presence of these molecules. Then we paralleled this structural information with a functional analysis of the enzyme investigating the role of these inhibitors in modulating the membrane association of FAAH using both fluorescence resonance energy transfer measurements (FRET) and Laurdan fluorescence. Taken together, the complementary information collected allowed to understand the molecular mechanisms leading to FAAH function. It also revealed the presence of an unprecedented oligomerization state of FAAH and a key role of inhibitors in favouring the subunit-subunit interactions that may impair the FAAH activity.1. Mei G. Di Venere A. Gasperi V. Nicolai E. Masuda K. R. Finazzi Agrò A. Cravatt B. F. and Maccarrone M. (2007) J. Biol. Chem. 282, 3829-3836.[...