Brazilian artisanal ripened cheeses as sources of proteolytic lactic acid bacteria capable of reducing cow milk allergy.

AIM: The objective was to obtain lactic acid bacteria (LAB) capable of hydrolysing immunoreactive proteins in milk, to optimize the hydrolysis, to determine the proteolysis kinetics and to test the safety of the best hydrolytic strain. METHODS AND RESULTS: Brazilian cheese was used as source of LAB capable of hydrolysing main milk allergens. Proteolytic isolates were submitted to RAPD-PCR for the characterization of clonal diversity. Optimized hydrolysis was strain and protein fraction dependent. 16S rDNA sequencing identified three proteolytic strains: Enterococcus faecalis VB43, that hydrolysed αS1 -, αS2 - and ß-caseins, α-lactalbumin and ß-lactoglobulin (partial hydrolysis), and Pediococcus acidilactici VB90 and Weissella viridescens VB111, that caused partial hydrolysis of αS1 - and αS2 -caseins. Enterococcus faecalis VB43 tested negative for virulence genes asa1, agg, efaA, hyl, esp, cylLL and cylLS but positive for genes ace and gelE. Ethylenediamine tetra-acetic acid inhibited the proteolysis, indicating that the main proteases of E. faecalis VB43 are metalloproteases. CONCLUSION: Brazilian artisanal cheese is a good source of LAB capable of hydrolysing allergenic proteins in milk. One isolate (E. faecalis VB43) presented outstanding activity against these proteins and lacked most of the tested virulence genes. SIGNIFICANCE AND IMPACT OF THE STUDY: Enterococcus faecalis VB43 presents good potential for the manufacture of hypoallergenic dairy products.

Coxiella burnetii immunogenic proteins as a basis for new Q fever diagnostic and vaccine development.

Coxiella burnetii is the etiological agent of the zoonosis Q fever, which can cause an acute or a chronic, life-threatening disease in humans. It presents a highly stable cell form, which persists in the environment and is transmitted via contaminated aerosols. Ruminants are considered as the main reservoir for human infections but are usually asymptomatic. Subclinical infection in these animals and the occurrence of serologically negative shedders hamper the identification of infected animals with the currently used diagnostic techniques. This suboptimal sensitivity limits reliable identification of infected animals as well as the well-timed implementation of countermeasures. This review summarizes compounds, focusing on C. burnetii seroreactive proteins, which were discovered in recent immunoproteomic studies. We analyzed these proteins regarding their localization, function, frequency of citation, differences seen in various host species as well as sensitivity and specificity. Finally, proteins useful for the development of new diagnostic test systems as well as subunit vaccines were discussed.

Identification of immunoreactive proteins of Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (MAP) is the cause of a chronic enteritis of ruminants (bovine paratuberculosis (PTB)--Johne's disease) that is associated with enormous worldwide economic losses for the animal production. Diagnosis is based on observation of clinical signs, the detection of antibodies in milk or serum, or evaluation of bacterial culture from feces. The limit of these methods is that they are not able to detect the disease in the subclinical stage and are applicable only when the disease is already advanced. For this reason, the main purpose of this study is to use the MAP proteome to detect novel immunoreactive proteins that may be helpful for PTB diagnoses. 2DE and 2D immunoblotting of MAP proteins were performed using sera of control cattle and PTB-infected cattle in order to highlight the specific immunoreactive proteins. Among the assigned identifiers to immunoreactive spots it was found that most of them correspond to surface-located proteins while three of them have never been described before as antigens. The identification of these proteins improves scientific knowledge that could be useful for PTB diagnoses. The sequence of the identified protein can be used for the synthesis of immunoreactive peptides that could be screened for their immunoreaction against bovine sera infected with MAP. All MS data have been deposited in the ProteomeXchange consortium with identifier PXD001159 and DOI 10.6019/PXD001159.

Further characterization of Tsol-p27 as a diagnostic antigen in sub-Saharan Africa.

Commercial antigens used to diagnose human neurocysticercosis are obtained from either a soluble parasite extract or a parasite-derived glycoprotein fraction. The aim of the present study was to identify antigenic proteins as potential diagnostic candidates in Mozambique. Soluble proteins from Taenia solium cysticerci were separated by two-dimensional electrophoresis and blotted onto nitrocellulose membranes. Subtracted hybridization was performed with serum samples obtained from patients with neurocysticercosis (NCC) and from a NCC-negative control group. Six antigenic proteins were identified and sequenced by liquid chromatography-mass spectrometry. Among these we found Tsol-p27, which was previously identified as a diagnostic candidate in a study conducted in Nicaragua, Central America. Here, we evaluated Tsol-p27 and the antigen cC1 as potential recombinant diagnostic reagents, and also investigated the localization and partial function of Tsol-p27. Immunoblotting demonstrated that Tsol-p27 was recognized by all 10 serum samples from NCC-positive individuals, whereas cC1 was identified by only five of the 10 positive sera. None of the antigens were recognized by negative control sera. Despite the limited number of serum samples evaluated in this study, the results suggest that Tsol-p27 can be a suitable candidate for diagnosis of human NCC, not only in Central America but also in sub-Saharan Africa.

Immunoaffinity-based mass spectrometric characterization of immunoreactive proteins of Salmonella Typhi.

Salmonella Typhi, a human-restricted Gram negative enterobacteriaceae, is the causative agent of typhoid fever in human being. The available serodiagnostic tools for the diagnosis of typhoid fever lack sensitivity and/or specificity. This study aimed to identify the immunoreactive proteins of S. Typhi that could help to develop improved diagnostic tools. Here, we performed immunoaffinity-based proteomic approach that uses charged columns to retrieve IgG and IgM antibodies from the plasma of typhoid patients followed by capture of S. Typhi proteins. These proteins were then characterized by mass spectrometry and bioinformatics tools. Using this approach, we identified 28 immunoreactive proteins of S. Typhi, in which 14 proteins were captured by IgG charged column and 4 proteins were captured by IgM column. We also identified 10 proteins (hlyE, rfbH, dapD, argI, glyA, pflB, trxB, groEL, tufA and pepD) captured by both columns. The prediction of antigenicity and immunogenicity resulted that 22 proteins were antigenic while 6 were non-antigenic on the scale of 0.4 threshold value of VaxiJen. These proteins successfully simulated the immune system in silico and in response higher amount of antibodies' titers were recorded in C-IMMSIM, confirming the immunogenic nature of these proteins. The identified proteins are of diverse nature and functions including those involved in virulence and pathogenesis, energy metabolism, cell development, biosynthesis of amino acids, regulatory functions and biosynthesis of cofactors. The findings of this study would be helpful in the development of improved vaccines and diagnostic tools for typhoid fever.

Molecular Characteristic, Antibiotic Resistance, and Detection of Highly Immunoreactive Proteins of Group B Streptococcus Strains Isolated From Urinary Tract Infections in Polish Adults.

Group B streptococcus (GBS) is one of the uropathogens that causes urinary tract infections (UTIs). The aims of this article were molecular characterization, an analysis of antimicrobial susceptibility profiles, adherence to bladder endothelial cells, and the detection of immunoreactive proteins of 94 clinical strains of GBS isolated from adult Polish patients with UTI. Antibiotic susceptibilities were determined by disk diffusion. Serotyping and Alp family genes detection were studied using multiplex PCR. Genetic profiles were determined by pulsed-field gel electrophoresis. The adherence ability of the studied strains was estimated by incubation on human bladder microvascular endothelial cell line. Immunoreactive proteins were studied by immunoblotting. Antibiotic susceptibility investigation revealed that 22% of GBS strains were resistant to erythromycin, whereas 18% demonstrated resistance to clindamycin. cMLSB was present in 76% of the resistant strains, M phenotype was detected in 14%, whereas iMLSB was present for 10%. The most common serotype was serotype III (31%), followed by serotype V (27%), and serotype Ia (17%). The genes that dominated among other Alp genes were: epsilon (29%), alp2 (27%), and rib (23%). The most common co-occurring serotypes and Alp genes were: Ia and epsilon, III and rib, III and alp2, V and alp2, and V and alp3 (p < 0.001). The PFGE method showed high clonality for serotype V and cMLSB (p < 001). The PFGE method showed high clonality for serotype V. Furthermore, this serotype was significantly associated with the cMLSB phenotype (p < 0.001). The most common immunoreactive proteins demonstrated masses of 50 kDa and 45-47 kDa. Although examined GBS isolates showed high genetic diversity, immunoreactive proteins were common for most of the studied GBS isolates, which may indicate their conservation, and allows to consider them as potential immunodiagnostic markers. Although the examined GBS isolates showed high genetic diversity, immunoreactive proteins were shared by most of the studied GBS isolates. It may indicate their conservation, thus allowing to consider them as potential immunodiagnostic markers.

Characterization of the Burkholderia cenocepacia J2315 Surface-Exposed Immunoproteome.

Infections by the Burkholderia cepacia complex (Bcc) remain seriously life threatening to cystic fibrosis (CF) patients, and no effective eradication is available. A vaccine to protect patients against Bcc infections is a highly attractive therapeutic option, but none is available. A strategy combining the bioinformatics identification of putative surface-exposed proteins with an experimental approach encompassing the "shaving" of surface-exposed proteins with trypsin followed by peptide identification by liquid chromatography and mass spectrometry is here reported. The methodology allowed the bioinformatics identification of 263 potentially surface-exposed proteins, 16 of them also experimentally identified by the "shaving" approach. Of the proteins identified, 143 have a high probability of containing B-cell epitopes that are surface-exposed. The immunogenicity of three of these proteins was demonstrated using serum samples from Bcc-infected CF patients and Western blotting, validating the usefulness of this methodology in identifying potentially immunogenic surface-exposed proteins that might be used for the development of Bcc-protective vaccines.

Exoproteome Analysis of Human Pathogenic Dermatophyte Species and Identification of Immunoreactive Proteins.

PURPOSE: Increasing incidence of onychomycosis and tinea pedis in humans of industrialized countries together with deep tissue infections are a therapeutic challenge in clinical mycology. For a better understanding of the pathology and immunology of infection, the authors analyze the exoproteomes of three reference strains of the most common clinical dermatophyte species (Trichophyton rubrum, Trichophyton interdigitale, Arthroderma benhamiae) and of Trichophyton strains isolated from affected patients. EXPERIMENTAL DESIGN: Extracellular proteins of those in vitro grown strains are separated via 2D High Performance Electrophoresis and identified by mass spectrometry to find proteins with provoked host immune reactivity. RESULTS: More than 80 secreted proteins including virulence factors such as peptidases and other hydrolases are identified. By Western blotting with respective patient sera, up to 31 proteins with significant antigen-antibody reactions are detected in comparison with control sera, for example, peptidases as well as several oxidoreductases. One protein, beta-glucosidase F2SZI9 seems to be a commonly processed antigen in all Trichophyton infections. CONCLUSIONS AND CLINICAL RELEVANCE: These first global exoproteome data of three dermatophyte species can be a stepping stone on the way to further study the molecular mechanisms of Trichophyton pathogenicity-associated traits. Possible candidates for potential new diagnostic methods or vaccination have to be validated in further investigations.

Reliable tool for detection of novel Coxiella burnetii antigens, using immobilized human polyclonal antibodies.

Coxiella burnetii (C. burnetii) is the etiological agent of a Q fever-the re-emerging disease with considerable economic impact. Due to many similar symptoms with commonly occurring infections, its clinical diagnosis is very difficult. Thus, a strong effort should be taken to raise the awareness and develop a robust strategy for an accurate diagnosis. The identification of specific C. burnetii biomarkers could be valuable for a sensitive and selective diagnosis of the disease. Herein, we described a workflow to identify immunoreactive proteins of C. burnetii with a high confidence. It is based on immunocapturing of bacterial antigens by biofunctionalized magnetic microspheres, followed by tandem mass spectrometry (MS/MS) identification. We detected dozens of previously reported antigens and proposed 15 novel biomarkers, which specificity was confirmed by in silico epitope analysis. Among them, the cardiolipin synthetase participating in the synthesis of cardiolipin was recognized. This biomarker could play a critical role in the early management of acute Q fever and prevention of Q fever endocarditis.

Proteomic analysis of excretory-secretory products from young adults of Angiostrongylus cantonensis

BACKGROUND Angiostrongyliasis is caused by the nematode Angiostrongylus cantonensis and can lead to eosinophilic meningitis and meningoencephalitis in humans. The young adult worms play central pathogenic roles in the central nervous system (CNS); however, the underlying mechanism is unclear. Excretory-secretory products (ESPs) are good investigation targets for studying the relationship between a host and its parasite. OBJECTIVES We aimed to profile, identify, and characterise the proteins in the ESPs of A. cantonensis young adults. METHODS The ESPs of young adult worms were collected from culture medium after incubation ranging from 24 to 96 h. Proteomic and bioinformatics analyses were performed to characterise the ESPs. FINDINGS A total of 51 spots were identified, and the highly expressed proteins included two protein disulphide isomerases, one calreticulin, and three uncharacterised proteins. Subsequently, approximately 254 proteins were identified in the ESPs of A. cantonensis young adults via liquid chromatography-mass spectrometry (LC-MS/MS) analysis, and these were further classified according to their characteristics and biological functions. Finally, we identified the immunoreactive proteins from a reference map of ESPs from A. cantonensis young adults. Approximately eight proteins were identified, including a protein disulphide isomerase, a putative aspartic protease, annexin, and five uncharacterised proteins. The study established and identified protein reference maps for the ESPs of A. cantonensis young adults. MAIN CONCLUSIONS The identified proteins may be potential targets for the development of diagnostic or therapeutic agents for human angiostrongyliasis.

Identification of immunoreactive proteins of Streptococcus agalactiae isolated from cultured tilapia in China.

Streptococcus agalactiae (Group B streptococcus, GBS) is an important zoonotic pathogen that can cause lethal infections in humans and animals, including aquatic species. Immunoreactive proteins of the S. agalactiae strain, GD201008-001, isolated from cultured tilapia in China, were screened by immunoproteomics using hyperimmune sera, convalescent guinea pig sera and GD201008-001-infected tilapia antisera as primary detection antibodies. A total of 16 different proteins were identified including 13 novel immunoreactive proteins of S. agalactiae. Four proteins, serine-rich repeat glycoprotein 1, branched-chain alpha-keto acid dehydrogenase (BKD) subunit E2, 5'-nucleotidase family protein and ornithine carbamoyltransferase, were shown to react with the three types of sera and thus were considered to represent novel S. agalactiae vaccine candidate antigens. Our findings represent the basis for vaccine development for piscine S. agalactiae and are necessary for understanding virulence factors and immunogenicity of S. agalactiae with different hosts.

Determination of immunoreactive proteins of Babesia ovis.

Babesia ovis, an intraerythrocytic protozoan parasite transmitted by ticks, causes severe infections in sheep in tropical and subtropical regions of the world. Parasite-specific immunoreactive proteins have been used as antigen in the serological diagnosis of babesiosis. There is no study about determination of B. ovis-specific proteins in sheep. This study was planned to determine the immunoreactive proteins of B. ovis. In this study, two splenectomized lambs, and twelve seropositive sheep and five seronegative lambs for anti-B. ovis antibodies were used as materials. Infected blood samples at 5% of parasitemia from the two splenectomized lambs experimentally infected with a virulent B. ovis field strain were analyzed for B. ovis-specific proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting (WB). B. ovis-specific five major proteins were recognized by anti-B. ovis serum but not by healthy sheep serum. They were of approximate molecular weights 154, 109, 77, 58, and 38 kDa. As the control samples, protein profiles of the blood extracts of two lambs before splenectomy operation were also blotted with the immune sera, but none of the five proteins was detected. These proteins were also immunoblotted with heterologous positive and negative sheep sera. All of twelve positive sera recognized the 109 kDa protein with 100 percent sensitivity. The 77 kDa protein reacted in 11 of 12 sera (91.6%). The sensitivities of the other 3 proteins ranged between 83.3% and 25%. The five protein bands immunoblotted with sera of the 5 negative lambs did not give any positive reaction. The results of this study revealed the presence of proteins recognized by the serum antibodies of experimentally and naturally infected sheep with B. ovis. Additional studies on the purification of these proteins and on subsequently their utilization in a serodiagnostic method are required to improve the serological diagnosis of ovine babesiosis.

Detecção de proteínas imunorreativas de Rickettsia sp. cepa Mata Atlântica / Detection of immunoreactive proteins of Rickettsia sp. Atlantic Forest strain

A Febre Maculosa Brasileira (FMB) é uma doença infecciosa, transmitida por carrapatos ao homem. Uma nova riquetsiose humana foi descrita como causadora de Febre Maculosa no Estado de São Paulo, sendo denominada de Rickettsia sp. cepa Mata Atlântica. O presente trabalho teve como objetivo detectar e identificar proteínas com potencial de estimular o sistema imune de hospedeiro mamífero, desta nova cepa descrita. Para tanto, foi realizado a extração proteica total de Rickettsia sp. cepa Mata Atlântica. As proteínas extraídas foram fracionadas por eletroforese. As bandas proteicas foram transferidas para membranas de nitrocelulose por migração elétrica e submetidas à técnica de Western-blot, para detecção proteica. Ao todo sete proteínas imunorreativas foram detectadas. Duas proteínas apresentaram maior abundancia, com peso molecular, de 200 e 130 kDa respectivamente. Através da comparação de mapas proteômicos existentes e pelo peso molecular que estas proteínas apresentaram, sugere-se que as duas proteínas detectadas representem rOmpA (200 kDa) e rOmpB (130 kDa). As demais proteínas detectadas apresentaram menor ocorrência e peso molecular inferior a 78 kDa, podendo representar membros da família de antígenos de superfície celular (Sca - Surface cell antigen). As proteínas detectadas poderão servir como base de estudo na elaboração de métodos diagnósticos sensíveis e específicos, no desenvolvimento de vacinas, além de possibilitarem novos estudos para terapias mais eficazes.(AU)

Brazilian Spotted Fever (BSF) is an infectious disease transmitted by ticks to humans. A new human rickettsial infection was reported to cause spotted fever in the State of São Paulo and was named Rickettsia sp. Strain Atlantic Forest. This study aimed to detect and identify proteins with potential to stimulate the immune system of mammalian host of this new strain described. Therefore, we performed total protein extraction Rickettsia sp. Strain Atlantic Forest. The extracted proteins were fractionated by electrophoresis. The protein bands were transferred to nitrocellulose membrane by electrical migration and subjected to Western blot for protein detection. In all, seven immunoreactive proteins were detected. Two proteins showed higher abundance, with molecular weight of 200 and 130 kDa respectively. By comparing existing proteomic maps and the molecular weight of these proteins showed that, it is suggested that the two proteins detected representing rOmpA (200 kDa) and rOmpB (130 kDa). The other detected proteins had lower occurrence and molecular weight less than 78 kDa, which may represent members of the cell surface antigens Family (Sca - Surface cell antigen). The detected proteins may serve as a study based on the development of sensitive and specific diagnostic methods in the development of vaccines and they enable further studies to more effective therapies.(AU)