Occurrence and evolutionary conservation analysis of α-helical cationic amphiphilic segments in the human proteome.

The existence of encrypted fragments with antimicrobial activity in human proteins has been thoroughly demonstrated in the literature. Recently, algorithms for the large-scale identification of these segments in whole proteomes were developed, and the pervasiveness of this phenomenon was stated. These algorithms typically mine encrypted cationic and amphiphilic segments of proteins, which, when synthesized as individual polypeptide sequences, exert antimicrobial activity by membrane disruption. In the present report, the human reference proteome was submitted to the software kamal for the uncovering of protein segments that correspond to putative intragenic antimicrobial peptides (IAPs). The assessment of the identity of these segments, frequency, functional classes of parent proteins, structural relevance, and evolutionary conservation of amino acid residues within their corresponding proteins was conducted in silico. Additionally, the antimicrobial and anticancer activity of six selected synthetic peptides was evaluated. Our results indicate that cationic and amphiphilic segments can be found in 2% of all human proteins, but are more common in transmembrane and peripheral membrane proteins. These segments are surface-exposed basic patches whose amino acid residues present similar conservation scores to other residues with similar solvent accessibility. Moreover, the antimicrobial and anticancer activity of the synthetic putative IAP sequences was irrespective to whether these are associated to membranes in the cellular setting. Our study discusses these findings in light of the current understanding of encrypted peptide sequences, offering some insights into the relevance of these segments to the organism in the context of their harboring proteins or as separate polypeptide sequences.

Release of immunomodulatory peptides at bacterial membrane interfaces as a novel strategy to fight microorganisms.

Cationic and amphiphilic peptides can be used as homing devices to accumulate conjugated antibiotics to bacteria-enriched sites and promote efficient microbial killing. However, just as important as tackling bacterial infections, is the modulation of the immune response in this complex microenvironment. In the present report, we designed a peptide chimaera called Chim2, formed by a membrane-active module, an enzyme hydrolysis site and a formyl peptide receptor 2 (FPR2) agonist. This molecule was designed to adsorb onto bacterial membranes, promote their lysis, and upon hydrolysis by local enzymes, release the FPR2 agonist sequence for activation and recruitment of immune cells. We synthesized the isolated peptide modules of Chim2 and characterized their biological activities independently and as a single polypeptide chain. We conducted antimicrobial assays, along with other tests aiming at the analyses of the cellular and immunological responses. In addition, assays using vesicles as models of eukaryotic and prokaryotic membranes were conducted and solution structures of Chim2 were generated by 1H NMR. Chim2 is antimicrobial, adsorbs preferentially to negatively charged vesicles while adopting an α-helix structure and exposes its disorganized tail to the solvent, which facilitates hydrolysis by tryptase-like enzymes, allowing the release of the FPR2 agonist fragment. This fragment was shown to induce accumulation of the cellular activation marker, lipid bodies, in mouse macrophages and the release of immunomodulatory interleukins. In conclusion, these data demonstrate that peptides with antimicrobial and immunomodulatory activities can be considered for further development as drugs.

Probing human proteins for short encrypted antimicrobial peptides reveals Hs10, a peptide with selective activity for gram-negative bacteria.

BACKGROUND: Some cationic and amphiphilic α-helical segments of proteins adsorb to prokaryotic membranes when synthesized as individual polypeptide sequences, resulting in broad and potent antimicrobial activity. However, amphiphilicity, a determinant physicochemical property for peptide-membrane interactions, can also be observed in some ß-sheets. METHODS: The software Kamal was used to scan the human reference proteome for short (7-11 amino acid residues) cationic and amphiphilic protein segments with the characteristic periodicity of ß-sheets. Some of the uncovered peptides were chemically synthesized, and antimicrobial assays were conducted. Biophysical techniques were used to probe the molecular interaction of one peptide with phospholipid vesicles, lipopolysaccharides (LPS) and the bacterium Escherichia coli. RESULTS: Thousands of compatible segments were found in human proteins, five were synthesized, and three presented antimicrobial activity in the micromolar range. Hs10, a nonapeptide fragment of the Complement C3 protein, could inhibit only the growth of tested Gram-negative microorganisms, presenting also little cytotoxicity to human fibroblasts. Hs10 interacted with LPS while transitioning from an unstructured segment to a ß-sheet and increased the hydrodynamic radius of LPS particles. This peptide also promoted morphological alterations in E. coli cells. CONCLUSIONS: Data presented herein introduce yet another molecular template to probe proteins in search for encrypted membrane-active segments and demonstrates that, using this approach, short peptides with low cytotoxicity and high selectivity to prokaryotic cells might be obtained. GENERAL SIGNIFICANCE: This work widens the biotechnological potential of the human proteome as a source of antimicrobial peptides with application in human health.

Novel disintegrin-like peptides derived from an amphibian skin cDNA sequence of Hypsiboas punctatus.

Disintegrins comprise a family of small proteins that bind to and alter the physiological function of integrins, especially integrins that mediate platelet aggregation in blood. Here, we report a lysine-glycine-aspartic acid (KGD) disintegrin-like motif present in a 15-amino acid residue peptide identified in a cDNA library of the amphibian Hypsiboas punctatus skin. The original peptide sequence was used as a template from which five new analogs were designed, chemically synthesized by solid phase, and tested for disintegrin activity and tridimensional structural studies using NMR spectroscopy. The original amphibian peptide had no effect on integrin-mediated responses. Nevertheless, derived peptide analogs inhibited integrin-mediated platelet function, including platelet spreading on fibrinogen.

Characterization of the first two toxins isolated from the venom of the ancient scorpion Tityus (Archaeotityus) mattogrossensis (Borelli, 1901).

BACKGROUND: Almost all Tityus characterized toxins are from subgenera Atreus and Tityus, there are only a few data about toxins produced by Archaeotityus, an ancient group in Tityus genus. METHODS: Tityus (Archaeotityus) mattogrossensis crude venom was fractionated by high performance liquid chromatography, the major fractions were tested in a frog sciatic nerve single sucrose-gap technique. Two fractions (Tm1 and Tm2) were isolated, partially sequenced by MALDI-TOF/MS and electrophysiological assayed on HEK293 Nav 1.3, HEK293 Nav 1.6, DUM and DRG cells. RESULTS: The sucrose-gap technique showed neurotoxicity in four fractions. One fraction caused a delay of action potential repolarization and other three caused a reduction in amplitude. An electrophysiological assay showed that Tm1 is active on HEK293 Nav 1.3, HEK293 Nav 1.6, DUM and DRG cells, and Tm2 on HEK293 Nav 1.3 and DRG cells, but not in HEK293 Nav 1.6. In addition, Tm1 and Tm2 did promote a shift to more negative potentials strongly suggesting that both are α-NaScTx. CONCLUSION: Although Tityus (Archaeotityus) mattogrossensis is considered an ancient group in Tityus genus, the primary structure of Tm1 and Tm2 is more related to Tityus subgenus. The patch clamp electrophysiological tests suggest that Tm1 and Tm2 are NaScTx, and also promoted no shift to more negative potentials, strongly suggesting that both are α-NaScTx. This paper aimed to explore and characterize for the first time toxins from the ancient scorpion Tityus (Archaeotityus) mattogrossensis.

Identification of Differential N-Glycan Compositions in the Serum and Tissue of Colon Cancer Patients by Mass Spectrometry.

Colorectal cancer (CRC) ranks second as the leading cause of cancer-related deaths worldwide. N-glycosylation is one of the most common posttranslational protein modifications. Therefore, we studied the total serum N-glycome (TSNG) of 13 colon cancer patients compared to healthy controls using MALDI-TOF/MS and LC-MS. N-glycosylation of cancer tumor samples from the same cohort were further quantified using a similar methodology. In total, 23 N-glycan compositions were down-regulated in the serum of colon cancer patients, mostly galactosylated forms whilst the mannose-rich HexNAc2Hex7, the fucosylated bi-antennary glycan HexNAc4Hex5Fuc1NeuAc2, and the tetra-antennary HexNAc6Hex7NeuAc3 were up-regulated in serum. Hierarchical clustering analysis of TSNG correctly singled out 85% of the patients from controls. Albeit heterogenous, N-glycosylation of tumor samples showed overrepresented oligomannosidic, bi-antennary hypogalactosylated, and branched compositions related to normal colonic tissue, in both MALDI-TOF/MS and LC-MS analysis. Moreover, compositions found upregulated in tumor tissue were mostly uncorrelated to compositions in serum of cancer patients. Mass spectrometry-based N-glycan profiling in serum shows potential in the discrimination of patients from healthy controls. However, the compositions profile in serum showed no parallel with N-glycans in tumor microenvironment, which suggests a different origin of compositions found in serum of cancer patients.

Head-to-Tail Cyclization after Interaction with Trypsin: A Scorpion Venom Peptide that Resembles Plant Cyclotides.

Peptidase inhibitors (PIs) have been broadly studied due to their wide therapeutic potential for human diseases. A potent trypsin inhibitor from Tityus obscurus scorpion venom was characterized and named ToPI1, with 33 amino acid residues and three disulfide bonds. The X-ray structure of the ToPI1:trypsin complex, in association with the mass spectrometry data, indicate a sequential set of events: the complex formation with the inhibitor Lys32 in the trypsin S1 pocket, the inhibitor C-terminal residue Ser33 cleavage, and the cyclization of ToPI1 via a peptide bond between residues Ile1 and Lys32. Kinetic and thermodynamic characterization of the complex was obtained. ToPI1 shares no sequence similarity with other PIs characterized to date and is the first PI with CS-α/ß motif described from animal venoms. In its cyclic form, it shares structural similarities with plant cyclotides that also inhibit trypsin. These results bring new insights for studies with venom compounds, PIs, and drug design.

Phylloseptin-1 is Leishmanicidal for Amastigotes of Leishmaniaamazonensis Inside Infected Macrophages.

Leishmania protozoans are the causal agents of neglected diseases that represent an important public health issue worldwide. The growing occurrence of drug-resistant strains of Leishmania and severe side effects of available treatments represent an important challenge for the leishmaniases treatment. We have previously reported the leishmanicidal activity of phylloseptin-1 (PSN-1), a peptide found in the skin secretion of Phyllomedusaazurea (=Pithecopus azureus), against Leishmaniaamazonensis promastigotes. However, its impact on the amastigote form of L. amazonensis and its impact on infected macrophages are unknown. In this work, we evaluated the effects of PSN-1 on amastigotes of L. amazonensis inside macrophages infected in vitro. We assessed the production of hydrogen peroxide and nitric oxide, as well as the levels of inflammatory and immunomodulatory markers (TGF-ß, TNF-α and IL-12), in infected and non-infected macrophages treated with PSN-1. Treatment with PSN-1 decreased the number of infected cells and the number of ingested amastigotes per cell when compared with the untreated cells. At 32 µM (64 µg/mL), PSN-1 reduced hydrogen peroxide levels in both infected and uninfected macrophages, whereas it had little effect on NO production or TGF-ß release. The effect of PSN-1 on IL-12 and TNF-α secretion depended on its concentration, but, in general, their levels tended to increase as PSN-1 concentration increased. Further in vitro and in vivo studies are needed to clarify the mechanisms of action of PSN-1 and its interaction with the immune system aiming to develop pharmacological applications.

Intragenic antimicrobial peptides (IAPs) from human proteins with potent antimicrobial and anti-inflammatory activity.

Following the treads of our previous works on the unveiling of bioactive peptides encrypted in plant proteins from diverse species, the present manuscript reports the occurrence of four proof-of-concept intragenic antimicrobial peptides in human proteins, named Hs IAPs. These IAPs were prospected using the software Kamal, synthesized by solid phase chemistry, and had their interactions with model phospholipid vesicles investigated by differential scanning calorimetry and circular dichroism. Their antimicrobial activity against bacteria, yeasts and filamentous fungi was determined, along with their cytotoxicity towards erythrocytes. Our data demonstrates that Hs IAPs are capable to bind model membranes while attaining α-helical structure, and to inhibit the growth of microorganisms at concentrations as low as 1µM. Hs02, a novel sixteen residue long internal peptide (KWAVRIIRKFIKGFIS-NH2) derived from the unconventional myosin 1h protein, was further investigated in its capacity to inhibit lipopolysaccharide-induced release of TNF-α in murine macrophages. Hs02 presented potent anti-inflammatory activity, inhibiting the release of TNF-α in LPS-primed cells at the lowest assayed concentration, 0.1 µM. A three-dimensional solution structure of Hs02 bound to DPC micelles was determined by Nuclear Magnetic Resonance. Our work exemplifies how the human genome can be mined for molecules with biotechnological potential in human health and demonstrates that IAPs are actual alternatives to antimicrobial peptides as pharmaceutical agents or in their many other putative applications.

Identification and characterization of phospholipases A2 from the skin secretion of Pithecopus azureus anuran.

The present work reports the isolation, characterization and the complete sequence of a phospholipase A2 (PLA2) present in the skin secretion of Pithecopus azureus. Among several peptides and small proteins previously described by our group from some species belonging to this amphibian genus (formerly named Phyllomedusa), a 15 kDa N-glycosylated protein showing PLA2 activity was purified, assayed, sequenced and named Pa-PLA2. The Pithecopus azureus skin phospholipase A2 polypeptide chain is composed by 125 amino acid residues linked by seven disulfide bonds and two N-glycosylated sites (N67 and N108). The Pa-PLA2 enzymatic activity was qualitatively evaluated and compared to classical viperid PLA2 showing that both, native and deglycosylated Pa-PLA2 forms, are catalytically functional. The tridimensional molecular model of Pa-PLA2 indicates that the observed glycan moieties are suggestively placed far from the active site of that enzyme and therefore having little or no significant role on the direct interaction of the Pa-PLA2 catalytic pocket and its substrates.

Whey hydrolysate-based ingredient with dual functionality: From production to consumer's evaluation.

The aim of the present study concerns the development, characterization and sensory evaluation of a dual-functional whey hydrolysate. Four concentrations of commercial pepsin (0.48%, 0.95%, 1.43%, 1.91% w/w) were evaluated. The hydrolyses curves and the Reversed-Phase High Performance Liquid Chromatography analyses showed a direct relationship between enzyme concentration and degree of hydrolysis. Through mass spectrometry 21 peptides were identified and 5 of them have never been described in the literature before. The hydrolysate produced (PC3) induced a vascular relaxation of 65.02% in phenylephrine-contracted rat aortic rings. PC3 powder presented a homogeneous aspect with a mean particle size of 86.39 µm, high water solubility (>92%) in a wide pH range (1-12) and an increase of 33% in oil absorption capacity, when compared to the unhydrolyzed product. Sensory analysis showed a high acceptance (7.6 in a 9-point hedonic scale) of the hydrolysate among 100 consumers. The results brought the possibility of developing a whey hydrolysate with high vasorelaxant activity, great technological properties and sensory appeal, as an interesting dual-functional ingredient to be incorporated into food products.

A previously undescribed hexapeptide His-Arg-Phe-Leu-Arg-His-NH2 from amphibian skin secretion shows CO2 and metal biding affinities.

A previously undescribed six residues long peptide His-Arg-Phe-Leu-Arg-His was identified and purified from the skin secretion of the amphibian Phyllomedusa centralis. A synthetic analogue carboxyamidated HRFLRH-NH2 showed structural changes induced by CO2 and metal ions in aqueous solution when analyzed by NMR. The present work reports NMR structures for the carboxyamidated hexapeptide in the presence CO2, Zn2+ and Cd2+, suggesting possible affinity regions on the polypeptide chain for each ligand. The NMR structures were optimized by DFT to identify probable biding sites of these species in the polypeptide structure. To our best knowledge, this is the first time that a putative CO2 binding site is described on a peptide structure obtained in aqueous conditions, at room temperature.

Imaging Mass Spectrometry of Endogenous Polypeptides and Secondary Metabolites from Galls Induced by Root-Knot Nematodes in Tomato Roots.

Nematodes are devastating pests that infect most cultivated plant species and cause considerable agricultural losses worldwide. The understanding of metabolic adjustments induced during plant-nematode interaction is crucial to generate resistant plants or to select more efficient molecules to fight against this pest. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has been used herein for in situ detection and mapping endogenous polypeptides and secondary metabolites from nematode-induced gall tissue. One of the major critical features of this technique is sample preparation; mainly, the generation of intact sections of plant cells with their rigid cell walls and vacuolated cytoplasm. Our experimental settings allowed us to obtain sections without contamination of exogenous ions or diffusion of molecules and to map the differential presence of low and high molecular weight ions in uninfected roots compared with nematode-induced galls. We predict the presence of lipids in both uninfected roots and galls, which was validated by MALDI time-of-flight tandem mass spectrometry and high-resolution mass spectrometry analysis of lipid extracts. Based on the isotopic ion distribution profile, both esters and glycerophospholipids were predicted compounds and may be playing an important role in gall development. Our results indicate that the MALDI-MSI technology is a promising tool to identify secondary metabolites as well as peptides and proteins in complex plant tissues like galls to decipher molecular processes responsible for infection and maintenance of these feeding sites during nematode parasitism.

The Antifungal Plant Defensin HsAFP1 Is a Phosphatidic Acid-Interacting Peptide Inducing Membrane Permeabilization.

HsAFP1, a plant defensin isolated from coral bells (Heuchera sanguinea), is characterized by broad-spectrum antifungal activity. Previous studies indicated that HsAFP1 binds to specific fungal membrane components, which had hitherto not been identified, and induces mitochondrial dysfunction and cell membrane permeabilization. In this study, we show that HsAFP1 reversibly interacts with the membrane phospholipid phosphatidic acid (PA), which is a precursor for the biosynthesis of other phospholipids, and to a lesser extent with various phosphatidyl inositol phosphates (PtdInsP's). Moreover, via reverse ELISA assays we identified two basic amino acids in HsAFP1, namely histidine at position 32 and arginine at position 52, as well as the phosphate group in PA as important features enabling this interaction. Using a HsAFP1 variant, lacking both amino acids (HsAFP1[H32A][R52A]), we showed that, as compared to the native peptide, the ability of this variant to bind to PA and PtdInsP's is reduced (≥74%) and the antifungal activity of the variant is reduced (≥2-fold), highlighting the link between PA/PtdInsP binding and antifungal activity. Using fluorescently labelled HsAFP1 in confocal microscopy and flow cytometry assays, we showed that HsAFP1 accumulates at the cell surface of yeast cells with intact membranes, most notably at the buds and septa. The resulting HsAFP1-induced membrane permeabilization is likely to occur after HsAFP1's internalization. These data provide novel mechanistic insights in the mode of action of the HsAFP1 plant defensin.

A Linear 19-Mer Plant Defensin-Derived Peptide Acts Synergistically with Caspofungin against Candida albicans Biofilms.

Public health problems are associated with device-associated biofilm infections, with Candida albicans being the major fungal pathogen. We previously identified potent antibiofilm combination treatment in which the antifungal plant defensin HsAFP1 is co-administered with caspofungin, the preferred antimycotic to treat such infections. In this study, we identified the smallest linear HsAFP1-derived peptide that acts synergistically with caspofungin or anidulafungin against C. albicans as HsLin06_18, a 19-mer peptide derived from the C-terminal part of HsAFP1. The [caspofungin + HsLin06_18] combination significantly reduced in vitro biofilm formation of Candida glabrata and C. albicans on catheters, as well as biofilm formation of a caspofungin-resistant C. albicans strain. The [caspofungin + HsLin06_18] combination was not cytotoxic and reduced biofilm formation of C. albicans in vivo using a subcutaneous rat catheter model, as compared to control treatment. Mode of action research on the [caspofungin + HsLin06_18] combination pointed to caspofungin-facilitated HsLin06_18 internalization and immediate membrane permeabilization. All these findings point to broad-spectrum antibiofilm activity of a combination of HsLin06_18 and caspofungin.

Ruthenium(II) complexes of 1,3-thiazolidine-2-thione: Cytotoxicity against tumor cells and anti-Trypanosoma cruzi activity enhanced upon combination with benznidazole.

Three new mixed and mononuclear Ru(II) complexes containing 1,3-thiazolidine-2-thione (tzdtH) were synthesized and characterized by spectroscopic analysis, molar conductivity, cyclic voltammetry, high-resolution electrospray ionization mass spectra and X-ray diffraction. The complexes presented unique stereochemistry and the proposed formulae are: [Ru(tzdt)(bipy)(dppb)]PF6 (1), cis-[Ru(tzdt)2(PPh3)2] (2) and trans-[Ru(tzdt)(PPh3)2(bipy)]PF6 (3), where dppb=1,4-bis(diphenylphosphino)butane and bipy=2,2'-bipyridine. These complexes demonstrated strong cytotoxicity against cancer cell lines when compared to cisplatin. Specifically, complex 2 was the most potent cytotoxic agent against MCF-7 breast cells, while complexes 1 and 3 were more active in DU-145 prostate cells. Binding of complexes to ctDNA was determined by UV-vis titration and viscosity measurements and revealed binding constant (Kb) values in range of 1.0-4.9×10(3)M(-1), which are characteristic of compounds possessing weak affinity to ctDNA. In addition, these complexes presented antiparasitic activity against Trypanosoma cruzi. Specifically, complex 3 demonstrated strong potency, moderate selectivity index and acted in synergism with the approved antiparasitic drug, benznidazole. Additionally, complex 3 caused parasite cell death through a necrotic process. In conclusion, we demonstrated that Ru(II) complexes have powerful pharmacological activity, while the metal-free tzdtH does not provoke the same outcome.

Secretome analysis of the mycoparasitic fungus Trichoderma harzianum ALL 42 cultivated in different media supplemented with Fusarium solani cell wall or glucose.

Trichoderma harzianum is a fungus well known for its potential as a biocontrol agent against many fungal phytopathogens. The aim of this study was to characterize the proteins secreted by T. harzianum ALL42 when its spores were inoculated and incubated for 48 h in culture media supplemented with glucose (GLU) or with cell walls from Fusarium solani (FSCW), a phytopathogen that causes severe losses in common bean and soy crops in Brazil, as well as other crop diseases around the world. Trichoderma harzianum was able to grow in Trichoderma Liquid Enzyme Production medium (TLE) and Minimal medium (MM) supplemented with FSCW and in TLE+GLU, but was unable to grow in MM+GLU medium. Protein quantification showed that TLE+FSCW and MM+FSCW had 45- and 30- fold, respectively, higher protein concentration on supernatant when compared to TLE+GLU, and this difference was observable on 2D gel electrophoresis (2DE). A total of 94 out of 105 proteins excised from 2DE maps were identified. The only protein observed in all three conditions was epl1. In the media supplemented with FSCW, different hydrolases such as chitinases, ß-1,3-glucanases, glucoamylases, α-1,3-glucanases and proteases were identified, along with other proteins with no known functions in mycoparasitism, such as npp1 and cys. Trichoderma harzianum showed a complex and diverse arsenal of proteins that are secreted in response to the presence of FSCW, with novel proteins not previously described in mycoparasitic-related studies.

Skin secretion peptides: the molecular facet of the deimatic behavior of the four-eyed frog, Physalaemus nattereri (Anura, Leptodactylidae).

RATIONALE: Amphibians can produce a large amount of bioactive peptides over the skin. In order to map the precise tissue localization of these compounds and evaluate their functions, mass spectrometry imaging (MSI) and gene expression studies were used to investigate a possible correlation between molecules involved in the antimicrobial defense mechanisms and anti-predatory behavior by Physalaemus nattereri. METHODS: Total skin secretion of P. nattereri was analyzed by classical Protein Chemistry and proteomic techniques. Intact inguinal macroglands were dissected from the rest of the skin and both tissues were analyzed by MSI and real-time polymerase chain reaction (RT-PCR) experiments. Peptides were primarily identified by de novo sequencing, automatic Edman degradation and cDNA data. RESULTS: Fifteen bradykinin (BK)-related peptides and two antimicrobial peptides were sequenced and mapped by MSI on the inguinal macrogland and the rest of P. nattereri skin. RT-PCR results revealed that BK-related peptide levels of expression were about 30,000 times higher on the inguinal macroglands than on the any other region of the skin, whilst antimicrobial peptide ions appear to be evenly distributed in both investigated regions. CONCLUSIONS: The presence of antimicrobial peptides in all investigated tissue regions is in accordance with the defensive role against microorganisms thoroughly demonstrated in the literature, whereas BK-related molecules are largely found on the inguinal macroglands suggesting an intriguing link between their noxious activities against potential predators of P. nattereri and the frog's deimatic behavior.

Proteomic analysis of free-living Bradyrhizobium diazoefficiens: highlighting potential determinants of a successful symbiosis.

BACKGROUND: Strain CPAC 7 (=SEMIA 5080) was recently reclassified into the new species Bradyrhizobium diazoefficiens; due to its outstanding efficiency in fixing nitrogen, it has been used in commercial inoculants for application to crops of soybean [Glycine max (L.) Merr.] in Brazil and other South American countries. Although the efficiency of B. diazoefficiens inoculant strains is well recognized, few data on their protein expression are available. RESULTS: We provided a two-dimensional proteomic reference map of CPAC 7 obtained under free-living conditions, with the successful identification of 115 spots, representing 95 different proteins. The results highlighted the expression of molecular determinants potentially related to symbiosis establishment (e.g. inositol monophosphatase, IMPase), fixation of atmospheric nitrogen (N2) (e.g. NifH) and defenses against stresses (e.g. chaperones). By using bioinformatic tools, it was possible to attribute probable functions to ten hypothetical proteins. For another ten proteins classified as "NO related COG" group, we analyzed by RT-qPCR the relative expression of their coding-genes in response to the nodulation-gene inducer genistein. Six of these genes were up-regulated, including blr0227, which may be related to polyhydroxybutyrate (PHB) biosynthesis and competitiveness for nodulation. CONCLUSIONS: The proteomic map contributed to the identification of several proteins of B. diazoefficiens under free-living conditions and our approach-combining bioinformatics and gene-expression assays-resulted in new information about unknown genes that might play important roles in the establishment of the symbiosis with soybean.

MALDI-TOF mass spectrometry applied to identifying species of insect-pathogenic fungi from the Metarhizium anisopliae complex.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has proven to be a powerful tool for taxonomic resolution of microorganisms. In this proof-of-concept study, we assessed the effectiveness of this technique to track the current gene sequence-based phylogenetic classification of species in the Metarhizium anisopliae complex. Initially the phylogenetic analysis of 5' strains by sequencing of the 59' end of the TEF-1α gene region revealed seven species within M. anisopliae sensu lato and two varieties outside this complex. Because initial studies on MS profiles from different cell types showed that mycelial fragments or conidia produced on nutrient-poor medium may yield too much background noise, all subsequent spectrometric analyses were performed with acidhydrolyzed conidia from 10-12 d old PDA cultures. The initial MALDI-TOF reference library included protein spectral profiles from nine taxonomically distinct, molecularly identified isolates sharing high genetic homology with the ex-type or ex-epitype isolates of these taxa in Metarhizium. A second reference library added one isolate each for M. anisopliae sensu stricto and M. robertsii. The second, larger reference library (including 11 taxa) allowed nearly perfect MALDI-TOF matching of DNA-based species identification for the 40 remaining isolates molecularly recognized as M. anisopliae sensu stricto (n = 19), M. robertsii (n = 6), M. majus (n = 3), M. lepidiotae (n = 1), M. acridum (n = 3), M. flavoviride var. pemphigi (n = 1), plus seven unidentified strains (six of them phylogenetically close to M. anisopliae sensu stricto and one outside the Metarhizium pingshaense-anisopliae-robertsii-brunneum clade). Due to the increasing frequency of phylogenetically (genomically) based taxonomic revisions of fungi, this approach is especially useful for culture collections, because once the protein profiles of Metarhizium isolates are obtained taxonomic updating of MALDI-TOF library data is easily accomplished by comparing stored profiles with those of newly proposed taxa.