A Custom-Designed Recombinant Multiepitope Protein for Human Cytomegalovirus Diagnosis.

BACKGROUND: The Human Cytomegalovirus (HCMV) has infected more than 90% of the world population and its prevalence can be related to the individuals geographical and socialeconomic status. Serological tests based on ELISA are pivotal for HCMV diagnosis. Due to the lack of standardization in the production/purification of antigens from viral preparations, ELISA tests are based on several recombinant proteins or peptides. As an alternative, multiepitope proteins may be employed. OBJECTIVE: In this work, we developed a recombinant multiepitope protein (rMEHCMV) for HCMV diagnosis based on conserved and immunodominant epitopes derived from tegument (pp150, pp65 and pp28), glycoprotein gB (pp38) and DNA polymerase subunit (pp52) of HCMV. METHODS: The rMEHCMV gene was synthesized de novo and overexpressed in Escherichia coli cells. The recombinant protein was purified to homogeneity using a Ni-NTA column. Biophysical analysis of recombinant protein was performed by circular dichroism. A preliminary biological activity test was performed using 12 positive human sera samples by using an in-house IgG ELISA. The following patents database were consulted: Espacenet, Google Patents and the National Institute of Intellectual Property (INPI, Brazil). RESULTS: The recombinant multiepitope protein was successfully expressed in E. coli. The structural data obtained by circular dichroism spectroscopy showed that rMEHCMV is structurally disordered. An in-house IgG ELISA test with rMEHCMV was successfully used to recognized IgG from human serum samples. CONCLUSION: Together, our results show that rMEHCMV should be considered as a potential antigenic target for HCMV diagnosis.

Comparative Genomics of Sibling Species of Fonsecaea Associated with Human Chromoblastomycosis.

Fonsecaea and Cladophialophora are genera of black yeast-like fungi harboring agents of a mutilating implantation disease in humans, along with strictly environmental species. The current hypothesis suggests that those species reside in somewhat adverse microhabitats, and pathogenic siblings share virulence factors enabling survival in mammal tissue after coincidental inoculation driven by pathogenic adaptation. A comparative genomic analysis of environmental and pathogenic siblings of Fonsecaea and Cladophialophora was undertaken, including de novo assembly of F. erecta from plant material. The genome size of Fonsecaea species varied between 33.39 and 35.23 Mb, and the core genomes of those species comprises almost 70% of the genes. Expansions of protein domains such as glyoxalases and peptidases suggested ability for pathogenicity in clinical agents, while the use of nitrogen and degradation of phenolic compounds was enriched in environmental species. The similarity of carbohydrate-active vs. protein-degrading enzymes associated with the occurrence of virulence factors suggested a general tolerance to extreme conditions, which might explain the opportunistic tendency of Fonsecaea sibling species. Virulence was tested in the Galleria mellonella model and immunological assays were performed in order to support this hypothesis. Larvae infected by environmental F. erecta had a lower survival. Fungal macrophage murine co-culture showed that F. erecta induced high levels of TNF-α contributing to macrophage activation that could increase the ability to control intracellular fungal growth although hyphal death were not observed, suggesting a higher level of extremotolerance of environmental species.

Draft Genome Sequence of Fonsecaea nubica Strain CBS 269.64, Causative Agent of Human Chromoblastomycosis.

On the basis of multilocus phylogenetic data, Fonsecaea nubica was described in 2010 as a molecular sibling of F. monophora, an established agent of the human skin disease chomoblastomycosis in tropical zones. Genome analysis of these pathogens is mandatory to identify genes involved in the interaction with host and virulence.

Draft Genome Sequence of Fonsecaea monophora Strain CBS 269.37, an Agent of Human Chromoblastomycosis.

The black yeast Fonsecaea monophora is one of the main etiologic agents of chromoblastomycosis in humans. Its pathogenicity profile is more invasive than that of related Fonsecaea species, causing brain infection in addition to (sub)cutaneous infections.

Worldwide Phylogenetic Distributions and Population Dynamics of the Genus Histoplasma.

BACKGROUND: Histoplasma capsulatum comprises a worldwide complex of saprobiotic fungi mainly found in nitrogen/phosphate (often bird guano) enriched soils. The microconidia of Histoplasma species may be inhaled by mammalian hosts, and is followed by a rapid conversion to yeast that can persist in host tissues causing histoplasmosis, a deep pulmonary/systemic mycosis. Histoplasma capsulatum sensu lato is a complex of at least eight clades geographically distributed as follows: Australia, Netherlands, Eurasia, North American classes 1 and 2 (NAm 1 and NAm 2), Latin American groups A and B (LAm A and LAm B) and Africa. With the exception of the Eurasian cluster, those clades are considered phylogenetic species. METHODOLOGY/PRINCIPAL FINDINGS: Increased Histoplasma sampling (n = 234) resulted in the revision of the phylogenetic distribution and population structure using 1,563 aligned nucleotides from four protein-coding regions. The LAm B clade appears to be divided into at least two highly supported clades, which are geographically restricted to either Colombia/Argentina or Brazil respectively. Moreover, a complex population genetic structure was identified within LAm A clade supporting multiple monophylogenetic species, which could be driven by rapid host or environmental adaptation (~0.5 MYA). We found two divergent clades, which include Latin American isolates (newly named as LAm A1 and LAm A2), harboring a cryptic cluster in association with bats. CONCLUSIONS/SIGNIFICANCE: At least six new phylogenetic species are proposed in the Histoplasma species complex supported by different phylogenetic and population genetics methods, comprising LAm A1, LAm A2, LAm B1, LAm B2, RJ and BAC-1 phylogenetic species. The genetic isolation of Histoplasma could be a result of differential dispersion potential of naturally infected bats and other mammals. In addition, the present study guides isolate selection for future population genomics and genome wide association studies in this important pathogen complex.

A Novel Structurally Stable Multiepitope Protein for Detection of HCV.

Hepatitis C virus (HCV) has emerged as the major pathogen of liver diseases in recent years leading to worldwide blood-transmitted chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Accurate diagnosis for differentiation of hepatitis C from other viruses is thus of pivotal importance for proper treatment. In this work we developed a recombinant multiepitope protein (rMEHCV) for hepatitis C diagnostic purposes based on conserved and immunodominant epitopes from core, NS3, NS4A, NS4B, and NS5 regions of the virus polyprotein of genotypes 1a, 1b, and 3a, the most prevalent genotypes in South America (especially in Brazil). A synthetic gene was designed to encode eight epitopes in tandem separated by a flexible linker and bearing a his-tag at the C-terminal end. The recombinant protein was produced in Escherichia coli and purified in a single affinity chromatographic step with >95% purity. Purified rMEHCV was used to perform an ELISA which showed that the recombinant protein was recognized by IgG and IgM from human serum samples. The structural data obtained by circular dichroism (CD) spectroscopy showed that rMEHCV is a highly thermal stable protein at neutral and alkaline conditions. Together, these results show that rMEHCV should be considered an alternative antigen for hepatitis C diagnosis.

Functional characterization of Sporothrix schenckii glycosidases involved in the N-linked glycosylation pathway.

Protein glycosylation pathways are conserved metabolic processes in eukaryotic organisms and are required for cell fitness. In fungal pathogens, the N-linked glycosylation pathway is indispensable for proper cell wall composition and virulence. In Sporothrix schenckii sensu stricto, the causative agent of sporotrichosis, little is known about this glycosylation pathway. Here, using a genome-wide screening for putative members of the glycosyl hydrolase (CAZy - GH) families 47 and 63, which group enzymes involved in the processing step during N-linked glycan maturation, we found seven homologue genes belonging to family 47 and one to family 63. The eight genes were individually expressed in C. albicans null mutants lacking either MNS1 (for members of family 47) or CWH41 (for the member of family 63). Our results indicate that SsCWH41 is the functional ortholog of CaCWH41, whereas SsMNS1 is the functional ortholog of CaMNS1. The remaining genes of family 47 encode Golgi mannosidases and endoplasmic reticulum degradation-enhancing alpha-mannosidase-like proteins (EDEMs). Since these GH families gather proteins used as target for drugs to control cell growth, identification of these genes could help in the design of antifungals that could be used to treat sporotrichosis and other fungal diseases. In addition, to our knowledge, we are the first to report that Golgi mannosidases and EDEMs are expressed and characterized in yeast cells.

Comparative genomics of the major fungal agents of human and animal Sporotrichosis: Sporothrix schenckii and Sporothrix brasiliensis.

BACKGROUND: The fungal genus Sporothrix includes at least four human pathogenic species. One of these species, S. brasiliensis, is the causal agent of a major ongoing zoonotic outbreak of sporotrichosis in Brazil. Elsewhere, sapronoses are caused by S. schenckii and S. globosa. The major aims on this comparative genomic study are: 1) to explore the presence of virulence factors in S. schenckii and S. brasiliensis; 2) to compare S. brasiliensis, which is cat-transmitted and infects both humans and cats with S. schenckii, mainly a human pathogen; 3) to compare these two species to other human pathogens (Onygenales) with similar thermo-dimorphic behavior and to other plant-associated Sordariomycetes. RESULTS: The genomes of S. schenckii and S. brasiliensis were pyrosequenced to 17x and 20x coverage comprising a total of 32.3 Mb and 33.2 Mb, respectively. Pair-wise genome alignments revealed that the two species are highly syntenic showing 97.5% average sequence identity. Phylogenomic analysis reveals that both species diverged about 3.8-4.9 MYA suggesting a recent event of speciation. Transposable elements comprise respectively 0.34% and 0.62% of the S. schenckii and S. brasiliensis genomes and expansions of Gypsy-like elements was observed reflecting the accumulation of repetitive elements in the S. brasiliensis genome. Mitochondrial genomic comparisons showed the presence of group-I intron encoding homing endonucleases (HE's) exclusively in S. brasiliensis. Analysis of protein family expansions and contractions in the Sporothrix lineage revealed expansion of LysM domain-containing proteins, small GTPases, PKS type1 and leucin-rich proteins. In contrast, a lack of polysaccharide lyase genes that are associated with decay of plants was observed when compared to other Sordariomycetes and dimorphic fungal pathogens, suggesting evolutionary adaptations from a plant pathogenic or saprobic to an animal pathogenic life style. CONCLUSIONS: Comparative genomic data suggest a unique ecological shift in the Sporothrix lineage from plant-association to mammalian parasitism, which contributes to the understanding of how environmental interactions may shape fungal virulence. . Moreover, the striking differences found in comparison with other dimorphic fungi revealed that dimorphism in these close relatives of plant-associated Sordariomycetes is a case of convergent evolution, stressing the importance of this morphogenetic change in fungal pathogenesis.

Differences in cell morphometry, cell wall topography and gp70 expression correlate with the virulence of Sporothrix brasiliensis clinical isolates.

Sporotrichosis is a chronic infectious disease affecting both humans and animals. For many years, this subcutaneous mycosis had been attributed to a single etiological agent; however, it is now known that this taxon consists of a complex of at least four pathogenic species, including Sporothrix schenckii and Sporothrix brasiliensis. Gp70 was previously shown to be an important antigen and adhesin expressed on the fungal cell surface and may have a key role in immunomodulation and host response. The aim of this work was to study the virulence, morphometry, cell surface topology and gp70 expression of clinical isolates of S. brasiliensis compared with two reference strains of S. schenckii. Several clinical isolates related to severe human cases or associated with the Brazilian zoonotic outbreak of sporotrichosis were genotyped and clustered as S. brasiliensis. Interestingly, in a murine subcutaneous model of sporotrichosis, these isolates showed a higher virulence profile compared with S. schenckii. A single S. brasiliensis isolate from an HIV-positive patient not only showed lower virulence but also presented differences in cell morphometry, cell wall topography and abundant gp70 expression compared with the virulent isolates. In contrast, the highly virulent S. brasiliensis isolates showed reduced levels of cell wall gp70. These observations were confirmed by the topographical location of the gp70 antigen using immunoelectromicroscopy in both species. In addition, the gp70 molecule was sequenced and identified using mass spectrometry, and the sequenced peptides were aligned into predicted proteins using Blastp with the S. schenckii and S. brasiliensis genomes.

Predicting antimicrobial peptides from eukaryotic genomes: in silico strategies to develop antibiotics.

A remarkable and intriguing challenge for the modern medicine consists in the development of alternative therapies to avoid the problem of microbial resistance. The cationic antimicrobial peptides present a promise to be used to develop more efficient drugs applied to human health. The in silico analysis of genomic databases is a strategy utilized to predict peptides of therapeutic interest. Once the main antimicrobial peptides' physical-chemical properties are already known, the correlation of those features to search on these databases is a tool to shorten identifying new antibiotics. This study reports the identification of antimicrobial peptides by theoretical analyses by scanning the Paracoccidioides brasiliensis transcriptome and the human genome databases. The identified sequences were synthesized and investigated for hemocompatibility and also antimicrobial activity. Two peptides presented antifungal activity against Candida albicans. Furthermore, three peptides exhibited antibacterial effects against Staphylococcus aureus and Escherichia coli; finally one of them presented high potential to kill both pathogens with superior activity in comparison to chloramphenicol. None of them showed toxicity to mammalian cells. In silico structural analyses were performed in order to better understand function-structure relation, clearly demonstrating the necessity of cationic peptide surfaces and the exposition of hydrophobic amino acid residues. In summary, our results suggest that the use of computational programs in order to identify and evaluate antimicrobial peptides from genomic databases is a remarkable tool that could be used to abbreviate the search of peptides with biotechnological potential from natural resources.

Identification and characterization of Tc1/mariner-like DNA transposons in genomes of the pathogenic fungi of the Paracoccidioides species complex.

BACKGROUND: Paracoccidioides brasiliensis (Eukaryota, Fungi, Ascomycota) is a thermodimorphic fungus, the etiological agent of paracoccidioidomycosis, the most important systemic mycoses in Latin America. Three isolates corresponding to distinct phylogenetic lineages of the Paracoccidioides species complex had their genomes sequenced. In this study the identification and characterization of class II transposable elements in the genomes of these fungi was carried out. RESULTS: A genomic survey for DNA transposons in the sequence assemblies of Paracoccidioides, a genus recently proposed to encompass species P. brasiliensis (harboring phylogenetic lineages S1, PS2, PS3) and P. lutzii (Pb01-like isolates), has been completed. Eight new Tc1/mariner families, referred to as Trem (Transposable element mariner), labeled A through H were identified. Elements from each family have 65-80% sequence similarity with other Tc1/mariner elements. They are flanked by 2-bp TA target site duplications and different termini. Encoded DDD-transposases, some of which have complete ORFs, indicated that they could be functionally active. The distribution of Trem elements varied between the genomic sequences characterized as belonging to P. brasiliensis (S1 and PS2) and P. lutzii. TremC and H elements would have been present in a hypothetical ancestor common to P. brasiliensis and P. lutzii, while TremA, B and F elements were either acquired by P. brasiliensis or lost by P. lutzii after speciation. Although TremD and TremE share about 70% similarity, they are specific to P. brasiliensis and P. lutzii, respectively. This suggests that these elements could either have been present in a hypothetical common ancestor and have evolved divergently after the split between P. brasiliensis and P. Lutzii, or have been independently acquired by horizontal transfer. CONCLUSIONS: New families of Tc1/mariner DNA transposons in the genomic assemblies of the Paracoccidioides species complex are described. Families were distinguished based on significant BLAST identities between transposases and/or TIRs. The expansion of Trem in a putative ancestor common to the species P. brasiliensis and P. lutzii would have given origin to TremC and TremH, while other elements could have been acquired or lost after speciation had occurred. The results may contribute to our understanding of the organization and architecture of genomes in the genus Paracoccidioides.

Transcriptome profiling of Paracoccidioides brasiliensis yeast-phase cells recovered from infected mice brings new insights into fungal response upon host interaction.

Paracoccidioides brasiliensis is a fungal human pathogen with a wide distribution in Latin America. It causes paracoccidioidomycosis, the most widespread systemic mycosis in Latin America. Although gene expression in P. brasiliensis had been studied, little is known about the genome sequences expressed by this species during the infection process. To better understand the infection process, 4934 expressed sequence tags (ESTs) derived from a non-normalized cDNA library from P. brasiliensis (isolate Pb01) yeast-phase cells recovered from the livers of infected mice were annotated and clustered to a UniGene (clusters containing sequences that represent a unique gene) set with 1602 members. A large-scale comparative analysis was performed between the UniGene sequences of P. brasiliensis yeast-phase cells recovered from infected mice and a database constructed with sequences of the yeast-phase and mycelium transcriptome (isolate Pb01) (https://dna.biomol.unb.br/Pb/), as well as with all public ESTs available at GenBank, including sequences of the P. brasiliensis yeast-phase transcriptome (isolate Pb18) (http://www.ncbi.nlm.nih.gov/). The focus was on the overexpressed and novel genes. From the total, 3184 ESTs (64.53%) were also present in the previously described transcriptome of yeast-form and mycelium cells obtained from in vitro cultures (https://dna.biomol.unb.br/Pb/) and of those, 1172 ESTs (23.75% of the described sequences) represented transcripts overexpressed during the infection process. Comparative analysis identified 1750 ESTs (35.47% of the total), comprising 649 UniGene sequences representing novel transcripts of P. brasiliensis, not previously described for this isolate or for other isolates in public databases. KEGG pathway mapping showed that the novel and overexpressed transcripts represented standard metabolic pathways, including glycolysis, amino acid biosynthesis, lipid and sterol metabolism. The unique and divergent representation of transcripts in the cDNA library of yeast cells recovered from infected mice suggests differential gene expression in response to the host milieu.

The transcriptome analysis of early morphogenesis in Paracoccidioides brasiliensis mycelium reveals novel and induced genes potentially associated to the dimorphic process.

BACKGROUND: Paracoccidioides brasiliensis is a human pathogen with a broad distribution in Latin America. The fungus is thermally dimorphic with two distinct forms corresponding to completely different lifestyles. Upon elevation of the temperature to that of the mammalian body, the fungus adopts a yeast-like form that is exclusively associated with its pathogenic lifestyle. We describe expressed sequence tags (ESTs) analysis to assess the expression profile of the mycelium to yeast transition. To identify P. brasiliensis differentially expressed sequences during conversion we performed a large-scale comparative analysis between P. brasiliensis ESTs identified in the transition transcriptome and databases. RESULTS: Our analysis was based on 1107 ESTs from a transition cDNA library of P. brasiliensis. A total of 639 consensus sequences were assembled. Genes of primary metabolism, energy, protein synthesis and fate, cellular transport, biogenesis of cellular components were represented in the transition cDNA library. A considerable number of genes (7.51%) had not been previously reported for P. brasiliensis in public databases. Gene expression analysis using in silico EST subtraction revealed that numerous genes were more expressed during the transition phase when compared to the mycelial ESTs 1. Classes of differentially expressed sequences were selected for further analysis including: genes related to the synthesis/remodeling of the cell wall/membrane. Thirty four genes from this family were induced. Ten genes related to signal transduction were increased. Twelve genes encoding putative virulence factors manifested increased expression. The in silico approach was validated by northern blot and semi-quantitative RT-PCR. CONCLUSION: The developmental program of P. brasiliensis is characterized by significant differential positive modulation of the cell wall/membrane related transcripts, and signal transduction proteins, suggesting the related processes important contributors to dimorphism. Also, putative virulence factors are more expressed in the transition process suggesting adaptation to the host of the yeast incoming parasitic phase. Those genes provide ideal candidates for further studies directed at understanding fungal morphogenesis and its regulation.

Swine and poultry pathogens: the complete genome sequences of two strains of Mycoplasma hyopneumoniae and a strain of Mycoplasma synoviae.

This work reports the results of analyses of three complete mycoplasma genomes, a pathogenic (7448) and a nonpathogenic (J) strain of the swine pathogen Mycoplasma hyopneumoniae and a strain of the avian pathogen Mycoplasma synoviae; the genome sizes of the three strains were 920,079 bp, 897,405 bp, and 799,476 bp, respectively. These genomes were compared with other sequenced mycoplasma genomes reported in the literature to examine several aspects of mycoplasma evolution. Strain-specific regions, including integrative and conjugal elements, and genome rearrangements and alterations in adhesin sequences were observed in the M. hyopneumoniae strains, and all of these were potentially related to pathogenicity. Genomic comparisons revealed that reduction in genome size implied loss of redundant metabolic pathways, with maintenance of alternative routes in different species. Horizontal gene transfer was consistently observed between M. synoviae and Mycoplasma gallisepticum. Our analyses indicated a likely transfer event of hemagglutinin-coding DNA sequences from M. gallisepticum to M. synoviae.

The antigenic and catalytically active formamidase of Paracoccidioides brasiliensis: protein characterization, cDNA and gene cloning, heterologous expression and functional analysis of the recombinant protein.

Paracoccidioides brasiliensis is a well-characterized pathogen of humans. To identify proteins involved in the fungus-host interaction, P. brasiliensis yeast proteins were separated by liquid isoelectric focusing, and fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Immunoreactive bands were detected with pooled sera of patients with P. brasiliensis infection. A protein species with a molecular mass of 45 kDa was subsequently purified to homogeneity by preparative gel electrophoresis. The amino acid sequence of four endoproteinase Lys-C-digested peptides indicated that the protein was a formamidase (FMD) (E.C. 3.5.1.49) of P. brasiliensis. The complete cDNA and a genomic clone (Pbfmd) encoding the isolated FMD were isolated. An open reading frame predicted a 415-amino acid protein. The sequence contained each of the peptide sequences obtained from amino acid sequencing. The Pbfmd gene contained five exons interrupted by four introns. Northern and Southern blot analysis suggested that there is one copy of the gene in P. brasiliensis and that it is preferentially expressed in mycelium. The complete coding cDNA was expressed in Escherichia coli to produce a recombinant fusion protein with glutathione S-transferase (GST). The purified recombinant protein was recognized by sera of patients with proven paracoccidioidomycosis and not by sera of healthy individuals. The recombinant 45-kDa protein was shown to be catalytically active; FMD activity was detected in P. brasiliensis yeast and mycelium.

Proteomic identification, nucleotide sequence, heterologous expression and immunological reactivity of the triosephosphate isomerase of Paracoccidioides brasiliensis.

An antigen of Paracoccidioides brasiliensis (Pb) was gel isolated and characterized. Endoproteinase Lys-C-digested peptides of the purified protein, which presented a molecular mass of 29 kDa and pI of 5.8, were subjected to sequence analysis of their amino acids. Searches at databases comparing the sequence of amino acids from the three peptides of the native protein revealed strong homology to triosephosphate isomerase (TPI: E.C. 5.3.1.1) from several sources. The complete cDNA and gene encoding PbTPI were obtained and both contained an open reading frame predicted to encode a 249-amino acid protein that presented all the peptides characterized in the native PbTPI. The Pbtpi gene contained six exons interrupted by five introns. Analysis performed with the deduced PbTPI suggested its usefulness in providing phylogenetic relatedness, as well as evidencing the correlation between the phylogeny provided by the deduced protein and intron positions in the cognate genes. The immunological reactivity of PbTPI was examined. The complete coding cDNA of PbTPI was overexpressed in an Escherichia coli host to produce high levels of recombinant fusion protein with glutathione S-transferase (GST) that had been purified by affinity chromatography. The purified recombinant TPI was recognized by sera of patients with confirmed paracoccidioidomycosis and not by sera of healthy individuals. Thus, recombinant PbTPI can be a valuable addition to the still small arsenal of P. brasiliensis immunoreactive proteins, which could be tested for incorporation into assays for serodiagnosis of the disease.