Proteomics profile of Hanseniaspora uvarum enhanced with trehalose involved in the biocontrol efficacy of grape berry.

This present study tested the extent to which 2% w/v trehalose enhanced the proteins expression profile of Hanseniaspora uvarum Y3. Furthermore, it explored the relative gene expression of stilbene synthase (StSy), one of the vital defense-related genes found in the skin of grapes. The proteomics profile revealed that 29 proteins were differentially expressed out of which 26 were significantly up-regulated and 3 were download-regulated. The pathogenesis related (PR) and other protein spots were visible at 97.4 kDa and 14.4 kDa. Peroxiredoxin TSA1 and superoxide dismutase were the main proteins involved in defense response and both proteins were significantly up-regulated. The carbohydrate and energy metabolism proteins were also significantly up-regulated. The results revealed that the treatments were associated with substantial increase in peroxidase activity compared to the control. StSy relative gene expression level was observed to increase by 2.5-fold in grapes treated with the pre-enhanced H. uvarum compared to the control.

Blood coagulation abnormalities in multibacillary leprosy patients.

BACKGROUND: Leprosy is a chronic dermato-neurological disease caused by Mycobacterium leprae infection. In 2016, more than 200,000 new cases of leprosy were detected around the world, representing the most frequent cause of infectious irreversible deformities and disabilities. PRINCIPAL FINDINGS: In the present work, we demonstrate a consistent procoagulant profile on 40 reactional and non-reactional multibacillary leprosy patients. A retrospective analysis in search of signs of coagulation abnormalities among 638 leprosy patients identified 35 leprosy patients (5.48%) which displayed a characteristic lipid-like clot formed between blood clot and serum during serum harvesting, herein named 'leprosum clot'. Most of these patients (n = 16, 45.7%) belonged to the lepromatous leprosy pole of the disease. In addition, formation of the leprosum clot was directly correlated with increased plasma levels of soluble tissue factor and von Willebrand factor. High performance thin layer chromatography demonstrated a high content of neutral lipids in the leprosum clot, and proteomic analysis demonstrated that the leprosum clot presented in these patients is highly enriched in fibrin. Remarkably, differential 2D-proteomics analysis between leprosum clots and control clots identified two proteins present only in leprosy patients clots: complement component 3 and 4 and inter-alpha-trypsin inhibitor family heavy chain-related protein (IHRP). In agreement with those observations we demonstrated that M. leprae induces hepatocytes release of IHRP in vitro. CONCLUSIONS: We demonstrated that leprosy MB patients develop a procoagulant status due to high levels of plasmatic fibrinogen, anti-cardiolipin antibodies, von Willebrand factor and soluble tissue factor. We propose that some of these components, fibrinogen for example, presents potential as predictive biomarkers of leprosy reactions, generating tools for earlier diagnosis and treatment of these events.

Blood coagulation abnormalities in multibacilary leprosy patients

Hemostatic illnesses are frequently associated with acute and chronic infections. In the present work we demonstrated that leprosy patients developed hemostatic abnormalities, like the formation of an atypical lipid clot mass during sera harvesting, a phenomenon previously observed and never unraveled. We characterize the nature of the "leprosum clot", formed during a protrombotic state developed by some patients. During the proteomic analysis of the leprosum clot we discovered a set of potential serum biomarkers to leprosy reactional episodes diagnosis, which at this moment is based only in clinical features. Taking together, our data suggest that leprosy patients are suffering from a procoagulant status, being beneficiated by the introduction of routine coagulation tests during their treatment, which will aloud physicians to prevent some of the acute clinical symptoms related with superficial vein thrombosis such as cyanosis and tissue necrosis observed during severe cases of leprosy reactional episodes. (AU)

Advances in Proteomics of Mycobacterium leprae.

Although Mycobacterium leprae was the first bacterial pathogen identified causing human disease, it remains one of the few that is non-cultivable. Understanding the biology of M. leprae is one of the primary challenges in current leprosy research. Genomics has been extremely valuable, nonetheless, functional proteins are ultimately responsible for controlling most aspects of cellular functions, which in turn could facilitate parasitizing the host. Furthermore, bacterial proteins provide targets for most of the vaccines and immunodiagnostic tools. Better understanding of the proteomics of M. leprae could also help in developing new drugs against M. leprae. During the past nearly 15 years, there have been several developments towards the identification of M. leprae proteins employing contemporary proteomics tools. In this review, we discuss the knowledge gained on the biology and pathogenesis of M. leprae from current proteomic studies.

A CTAB based method for the preparation of total protein extract of wine spoilage microrganisms for proteomic analysis.

Mapping the proteome of microrganisms by 2D-electrophoresis is often a hard task, because many contaminants, e.g. polysaccharides of the cell wall and nucleic acid, can obstruct the pores of the IEF gel resulting in streaks and smears. A protocol based on the use of the cationic detergent cetyl-trimethylammonium bromide (CTAB) and its salt-dependent solubility was developed. The cellulose-producing strain Gluconoacetobacter hansenii AAB0248 was resolved on 7cm Minigels in over 500 protein spots (a hundred more than with protocols reported in literature). The method was further employed for mapping the proteome of some acid adapted, wine spoilage microrganisms e.g. acetic acid bacteria and a yeast.

Deciphering the proteomic profile of Mycobacterium leprae cell envelope.

The complete sequence of the Mycobacterium leprae genome, an obligate intracellular pathogen, shows a dramatic reduction of functional genes, with a coding capacity of less than 50%. Despite this massive gene decay, the leprosy bacillus has managed to preserve a minimal gene set, most of it shared with Mycobacterium tuberculosis, allowing its survival in the host with ensuing pathological manifestations. Thus, the identification of proteins that are actually expressed in vivo by M. leprae is of high significance in understanding obligate, intracellular mycobacterial pathogenesis. In this study, a high-throughput proteomic approach was undertaken resulting in the identification of 218 new M. leprae proteins. Of these, 60 were in the soluble/cytosol fraction, 98 in the membrane and 104 in the cell wall. Although several proteins were identified in more than one subcellular fraction, the majority were unique to one. As expected, a high percentage of these included enzymes responsible for lipid biosynthesis and degradation, biosynthesis of the major components of the mycobacterial cell envelope, proteins involved in transportation across lipid barriers, and lipoproteins and transmembrane proteins with unknown functions. The data presented in this study contribute to our understanding of the in vivo composition and physiology of the mycobacterial cell envelope, a compartment known to play a major role in bacterial pathogenesis.

Serum proteome of leprosy patients undergoing erythema nodosum leprosum reaction: regulation of expression of the isoforms of haptoglobin.

Validated proteome profile allows better understanding of disease progression, subtype classification, susceptibility patterns, and disease prognosis. Leprosy is a spectral disease, with clinically, histologically, immunologically, and bacteriologically distinguishable subtypes. In addition, a significant fraction of patients undergo immune mediated reactions even after multidrug therapy (MDT). Erythema nodosum leprosum (ENL) is an immune complex mediated reactional condition in leprosy, characterized by a systemic inflammatory condition afflicting borderline lepromatous (BL) and lepromatous leprosy patients (LL). In this study, we have analyzed serum proteome of leprosy patients undergoing ENL reactions and compared it with that of healthy noncontact controls. Depletion of albumin and immunoglobulin G (IgG) was optimized using Aurum serum protein mini kit (Bio-Rad), and then two-dimensional gel electrophoresis (2-DE) of these serum samples was performed. Differentially expressed proteins were identified by MALDI-TOF and MALDI-TOF MS/MS mass spectrometry. Significant increase in one of the isoforms of alpha2 chain of haptoglobin was observed in ENL condition. In addition, haptoglobin phenotype was determined for healthy controls and leprosy patients. Hp 0-0 phenotype was detected in 21.4% of the ENL patients undergoing treatment, which on follow up examination showed typable phenotype, thus showing a condition of acquired anhaptoglobinemia. Since ENL still remains a threat to leprosy disease management, the above findings may provide new insights in understanding the development and progression of this inflammatory condition.

Immunogenic membrane-associated proteins of Mycobacterium tuberculosis revealed by proteomics.

Membrane-associated proteins of Mycobacterium tuberculosis offer a challenge, as well as an opportunity, in the quest for better therapeutic and prophylactic interventions against tuberculosis. The authors have previously reported that extraction with the detergent Triton X-114 (TX-114) is a useful step in proteomic analysis of mycobacterial cell membranes, and detergent-soluble membrane proteins of mycobacteria are potent stimulators of human T cells. In this study 1-D and 2-D gel electrophoresis-based protocols were used for the analysis of proteins in the TX-114 extract of M. tuberculosis membranes. Peptide mass mapping (using MALDI-TOF-MS, matrix assisted laser desorption/ionization time of flight mass spectrometry) of 116 samples led to the identification of 105 proteins, 9 of which were new to the M. tuberculosis proteome. Functional orthologues of 73 of these proteins were also present in Mycobacterium leprae, suggesting their relative importance. Bioinformatics predicted that as many as 73% of the proteins had a hydrophobic disposition. 1-D gel electrophoresis revealed more hydrophobic/transmembrane and basic proteins than 2-D gel electrophoresis. Identified proteins fell into the following major categories: protein synthesis, cell wall biogenesis/architecture and conserved hypotheticals/unknowns. To identify immunodominant proteins of the detergent phase (DP), 14 low-molecular-mass fractions prepared by continuous-elution gel electrophoresis were subjected to T cell activation assays using blood samples from BCG-vaccinated healthy donors from a tuberculosis endemic area. Analysis of the responses (cell proliferation and IFN-gamma production) showed that the immunodominance of certain DP fractions was most probably due to ribosomal proteins, which is consistent with both their specificity for mycobacteria and their abundance. Other membrane-associated proteins, including transmembrane proteins/lipoproteins and ESAT-6, did not appear to contribute significantly to the observed T cell responses.

Cooperative evolution in protein complexes of yeast from comparative analyses of its interaction network.

A comparative analysis among Saccharomyces cerevisiae and the other four yeasts Candida glabrata, Kluyveromyces lactis, Debaryomyces hansenii, and Yarrowia lipolytica is presented. The broad evolutionary range spanned by the organisms allows to quantitatively demonstrate novel evolutionary effects in protein complexes. The evolution rates within cliques of interlinked proteins are found to bear strong multipoint correlations, witnessing a cooperative coevolution of complex subunits. The coevolution is found to be largely independent of the tendency of the subunits to have similar abundances.

Continued proteomic analysis of Mycobacterium leprae subcellular fractions.

Recently the sequence of the Mycobacterium leprae chromosome, the only known obligate intracellular mycobacterium, was completed. It has a dramatic reduction in functional genes, with a coding capacity of only 49.5%, the lowest one so far observed among bacterial genomes. The leprosy bacillus seems to preserve a minimal set of genes that allows its survival in the host. The identification of genes that are actually expressed by the bacterium is of high significance in the context of mycobacterial pathogenesis. In this current study, a proteomic approach was undertaken to identify the proteins present in the soluble/cytosol and membrane subcellular fractions obtained from armadillo derived M. leprae. Proteins from each fraction were separated by two-dimensional gel electrophoresis (2-DE) and identified by mass spectrometry. A total of 147 protein spots were identified from 2-DE patterns and shown to comprise products of 44 different genes, twenty eight of them corresponding to new proteins. Additionally, two highly basic proteins (with pI >10.0) were isolated by heparin affinity chromatography and identified by N-terminal sequencing. This study constitutes the first application of proteomics to a host-derived Mycobacterium.

Spoilt for choice: protein target selection in a time of plenty.

Experiences in the application of Boolean logic to the clusters of orthologous groups of proteins (COGs) database for target selection in the Mycobacterium tuberculosis genome are described.