Restriction-modification systems in Mycoplasma spp

Restriction and Modification (R-M) systems are present in all Mycoplasma species sequenced so far. The presence of these genes poses barriers to gene transfer and could protect the cell against phage infections. The number and types of R-M genes between different Mycoplasma species are variable, which is characteristic of a polymorphism. The majority of the CDSs code for Type III R-M systems and particularly for methyltransferase enzymes, which suggests that functions other than the protection against the invasion of heterologous DNA may exist. A possible function of these enzymes could be the protection against the invasion of other but similar R-M systems. In Mycoplasma hyopneumoniae strain J, three of the putative methyltransferase genes were clustered in a region forming a genomic island. Many R-M CDSs were mapped in the vicinity of transposable elements suggesting an association between these genes and reinforcing the idea of R-M systems as mobile selfish DNA. Also, many R-M genes present repeats within their coding sequences, indicating that their expression is under the control of phase variation mechanisms. Altogether, these data suggest that R-M systems are a remarkable characteristic of Mycoplasma species and are probably involved in the adaptation of these bacteria to different environmental conditions.236244Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Evolutionary fates of universal stress protein paralogs in Platyhelminthes

Background Universal stress proteins (USPs) are present in all domains of life. Their expression is upregulated in response to a large variety of stress conditions. The functional diversity found in this protein family, paired with the sequence degeneration of the characteristic ATP-binding motif, suggests a complex evolutionary pattern for the paralogous USP-encoding genes. In this work, we investigated the origin, genomic organization, expression patterns and evolutionary history of the USP gene family in species of the phylum Platyhelminthes. Results Our data showed a cluster organization, a lineage-specific distribution, and the presence of several pseudogenes among the USP gene copies identified. The absence of a well conserved -CCAATCA- motif in the promoter region was positively correlated with low or null levels of gene expression, and with amino acid changes within the ligand binding motifs. Despite evidence of the pseudogenization of various USP genes, we detected an important functional divergence at several residues, mostly located near sites that are critical for ligand interaction. Conclusions Our results provide a broad framework for the evolution of the USP gene family, based on the emergence of new paralogs that face very contrasting fates, including pseudogenization, subfunctionalization or neofunctionalization. This framework aims to explain the sequence and functional diversity of this gene family, providing a foundation for future studies in other taxa in which USPs occur

Diagnóstico da angiostrongilíase abdominal utilizando PCR em soro de pacientes

Abdominal angiostrongyliasis is a zoonotic infection caused by an intra-vascular nematode parasitic of wild rodents, Angiostrongylus costaricensis. No parasitological diagnosis is currently available and immunodiagnosis presents several drawbacks. Primers constructed based on a congeneric species, A. cantonensis, were able to amplify a 232 bp fragment from serum samples of 3 patients with histopathological diagnosis. Extraction was better performed with DNAzol and the specificity of the primers was confirmed by Southern blot. This disease has been diagnosed with frequency in south of Brazil, thus, this method appears like the important and unpublished alternative to improve diagnostic of disease.Angiostrongilíase abdominal é uma infecção zoonótica causada por um parasito nematódeo intravascular de roedores silvestres, Angiostrongylus costaricensis. Nenhum diagnóstico parasitológico é atualmente disponível e o imunodiagnóstico apresenta alguns obstáculos. Oligonucleotídeos foram construídos baseados em um gênero específico, Angiostrongylus cantonensis, e foi capaz de amplificar um fragmento de 232 bp de amostras de soro de 3 pacientes com diagnóstico histopatológico. O melhor método de extração foi com DNAzol e a especificidade dos oligonucleotídeos foi confirmada por Southern Blot. A doença tem sido diagnosticada com freqüência no sul do Brasil, assim, este método surge como uma importante e inédita alternativa no auxílio do diagnóstico desta doença

Comparative proteomic analysis of pathogenic and non-pathogenic strains from the swine pathogen Mycoplasma hyopneumoniae

<p>Abstract</p> <p>Background</p> <p><it>Mycoplasma hyopneumoniae </it>is a highly infectious swine pathogen and is the causative agent of enzootic pneumonia (EP). Following the previous report of a proteomic survey of the pathogenic 7448 strain of swine pathogen, <it>Mycoplasma hyopneumoniae</it>, we performed comparative protein profiling of three <it>M. hyopneumoniae </it>strains, namely the non-pathogenic J strain and the two pathogenic strains 7448 and 7422.</p> <p>Results</p> <p>In 2DE comparisons, we were able to identify differences in expression levels for 67 proteins, including the overexpression of some cytoadherence-related proteins only in the pathogenic strains. 2DE immunoblot analyses allowed the identification of differential proteolytic cleavage patterns of the P97 adhesin in the three strains. For more comprehensive protein profiling, an LC-MS/MS strategy was used. Overall, 35% of the <it>M. hyopneumoniae </it>genome coding capacity was covered. Partially overlapping profiles of identified proteins were observed in the strains with 81 proteins identified only in one strain and 54 proteins identified in two strains. Abundance analysis of proteins detected in more than one strain demonstrates the relative overexpression of 64 proteins, including the P97 adhesin in the pathogenic strains.</p> <p>Conclusions</p> <p>Our results indicate the physiological differences between the non-pathogenic strain, with its non-infective proliferate lifestyle, and the pathogenic strains, with its constitutive expression of adhesins, which would render the bacterium competent for adhesion and infection prior to host contact.</p

Antigen B from Echinococcus granulosus enters mammalian cells by endocytic pathways

Background Cystic hydatid disease is a zoonosis caused by the larval stage (hydatid) of Echinococcus granulosus (Cestoda, Taeniidae). The hydatid develops in the viscera of intermediate host as a unilocular structure filled by the hydatid fluid, which contains parasitic excretory/secretory products. The lipoprotein Antigen B (AgB) is the major component of E. granulosus metacestode hydatid fluid. Functionally, AgB has been implicated in immunomodulation and lipid transport. However, the mechanisms underlying AgB functions are not completely known. Methodology/Principal findings In this study, we investigated AgB interactions with different mammalian cell types and the pathways involved in its internalization. AgB uptake was observed in four different cell lines, NIH-3T3, A549, J774 and RH. Inhibition of caveolae/raft-mediated endocytosis causes about 50 and 69% decrease in AgB internalization by RH and A549 cells, respectively. Interestingly, AgB colocalized with the raft endocytic marker, but also showed a partial colocalization with the clathrin endocytic marker. Finally, AgB colocalized with an endolysosomal tracker, providing evidence for a possible AgB destination after endocytosis. Conclusions/Significance The results indicate that caveolae/raft-mediated endocytosis is the main route to AgB internalization, and that a clathrin-mediated entry may also occur at a lower frequency. A possible fate for AgB after endocytosis seems to be the endolysosomal system. Cellular internalization and further access to subcellular compartments could be a requirement for AgB functions as a lipid carrier and/or immunomodulatory molecule, contributing to create a more permissive microenvironment to metacestode development and survival

Variant predictions in congenital adrenal hyperplasia caused by mutations in CYP21A2

CYP21A2 deficiency represents 95% of congenital adrenal hyperplasia (CAH) cases, a group of genetic disorders that affect steroid biosynthesis. The genetic and functional analysis provide critical tools to elucidate complex CAH cases. One of the most accessible tools to infer the pathogenicity of new variants is in silico prediction. Here, we analyzed the performance of in silico prediction tools to categorize missense single nucleotide variants (SNVs) of CYP21A2. SNVs of CYP21A2 characterized in vitro by functional assays were selected to assess the performance of online single and meta predictors. SNVs were tested separately or in combination with the related phenotype (severe or mild CAH form). In total, 103 SNVs of CYP21A2 (90 pathogenic and 13 neutral) were used to test the performance of 13 single-predictors and four meta-predictors. All SNVs associated with the severe phenotypes were well categorized by all tools, with an accuracy of between 0.69 (PredictSNP2) and 0.97 (CADD), and Matthews’ correlation coefficient (MCC) between 0.49 (PoredicSNP2) and 0.90 (CADD). However, SNVs related to the mild phenotype had more variation, with the accuracy between 0.47 (S3Ds&GO and MAPP) and 0.88 (CADD), and MCC between 0.18 (MAPP) and 0.71 (CADD). From our analysis, we identified four predictors of CYP21A2 variant pathogenicity with good performance, CADD, ConSurf, DANN, and PolyPhen2. These results can be used for future analysis to infer the impact of uncharacterized SNVs in CYP21A2

Unraveling oxidative stress response in the cestode parasite Echinococcus granulosus

Cystic hydatid disease (CHD) is a worldwide neglected zoonotic disease caused by Echinococcus granulosus. The parasite is well adapted to its host by producing protective molecules that modulate host immune response. An unexplored issue associated with the parasite’s persistence in its host is how the organism can survive the oxidative stress resulting from parasite endogenous metabolism and host defenses. Here, we used hydrogen peroxide (H₂O₂) to induce oxidative stress in E. granulosus protoescoleces (PSCs) to identify molecular pathways and antioxidant responses during H₂O₂ exposure. Using proteomics, we identified 550 unique proteins; including 474 in H₂O₂-exposed PSCs (H-PSCs) samples and 515 in non-exposed PSCs (C-PSCs) samples. Larger amounts of antioxidant proteins, including GSTs and novel carbonyl detoxifying enzymes, such as aldo-keto reductase and carbonyl reductase, were detected after H₂O₂ exposure. Increased concentrations of caspase-3 and cathepsin-D proteases and components of the 26S proteasome were also detected in H-PSCs. Reduction of lamin-B and other caspase-substrate, such as filamin, in H-PSCs suggested that molecular events related to early apoptosis were also induced. We present data that describe proteins expressed in response to oxidative stress in a metazoan parasite, including novel antioxidant enzymes and targets with potential application to treatment and prevention of CHD