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author:("zapa, arnaud")
1.  Unravelling the Transcriptome Profile of the Swine Respiratory Tract Mycoplasmas 
PLoS ONE  2014;9(10):e110327.
The swine respiratory ciliary epithelium is mainly colonized by Mycoplasma hyopneumoniae, Mycoplasma flocculare and Mycoplasma hyorhinis. While colonization by M. flocculare is virtually asymptomatic, M. hyopneumoniae and M. hyorhinis infections may cause respiratory disease. Information regarding transcript structure and gene abundance provides valuable insight into gene function and regulation, which has not yet been analyzed on a genome-wide scale in these Mycoplasma species. In this study, we report the construction of transcriptome maps for M. hyopneumoniae, M. flocculare and M. hyorhinis, which represent data for conducting comparative studies on the transcriptional repertory. For each species, three cDNA libraries were generated, yielding averages of 415,265, 695,313 and 93,578 reads for M. hyopneumoniae, M. flocculare and M. hyorhinis, respectively, with an average read length of 274 bp. The reads mapping showed that 92%, 98% and 96% of the predicted genes were transcribed in the M. hyopneumoniae, M. flocculare and M. hyorhinis genomes, respectively. Moreover, we showed that the majority of the genes are co-expressed, confirming the previously predicted transcription units. Finally, our data defined the RNA populations in detail, with the map transcript boundaries and transcription unit structures on a genome-wide scale.
doi:10.1371/journal.pone.0110327
PMCID: PMC4198240  PMID: 25333523
3.  Genome of the Avirulent Human-Infective Trypanosome—Trypanosoma rangeli 
Background
Trypanosoma rangeli is a hemoflagellate protozoan parasite infecting humans and other wild and domestic mammals across Central and South America. It does not cause human disease, but it can be mistaken for the etiologic agent of Chagas disease, Trypanosoma cruzi. We have sequenced the T. rangeli genome to provide new tools for elucidating the distinct and intriguing biology of this species and the key pathways related to interaction with its arthropod and mammalian hosts.
Methodology/Principal Findings
The T. rangeli haploid genome is ∼24 Mb in length, and is the smallest and least repetitive trypanosomatid genome sequenced thus far. This parasite genome has shorter subtelomeric sequences compared to those of T. cruzi and T. brucei; displays intraspecific karyotype variability and lacks minichromosomes. Of the predicted 7,613 protein coding sequences, functional annotations could be determined for 2,415, while 5,043 are hypothetical proteins, some with evidence of protein expression. 7,101 genes (93%) are shared with other trypanosomatids that infect humans. An ortholog of the dcl2 gene involved in the T. brucei RNAi pathway was found in T. rangeli, but the RNAi machinery is non-functional since the other genes in this pathway are pseudogenized. T. rangeli is highly susceptible to oxidative stress, a phenotype that may be explained by a smaller number of anti-oxidant defense enzymes and heat-shock proteins.
Conclusions/Significance
Phylogenetic comparison of nuclear and mitochondrial genes indicates that T. rangeli and T. cruzi are equidistant from T. brucei. In addition to revealing new aspects of trypanosome co-evolution within the vertebrate and invertebrate hosts, comparative genomic analysis with pathogenic trypanosomatids provides valuable new information that can be further explored with the aim of developing better diagnostic tools and/or therapeutic targets.
Author Summary
Comparative genomics is a powerful tool that affords detailed study of the genetic and evolutionary basis for aspects of lifecycles and pathologies caused by phylogenetically related pathogens. The reference genome sequences of three trypanosomatids, T. brucei, T. cruzi and L. major, and subsequent addition of multiple Leishmania and Trypanosoma genomes has provided data upon which large-scale investigations delineating the complex systems biology of these human parasites has been built. Here, we compare the annotated genome sequence of T. rangeli strain SC-58 to available genomic sequence and annotation data from related species. We provide analysis of gene content, genome architecture and key characteristics associated with the biology of this non-pathogenic trypanosome. Moreover, we report striking new genomic features of T. rangeli compared with its closest relative, T. cruzi, such as (1) considerably less amplification on the gene copy number within multigene virulence factor families such as MASPs, trans-sialidases and mucins; (2) a reduced repertoire of genes encoding anti-oxidant defense enzymes; and (3) the presence of vestigial orthologs of the RNAi machinery, which are insufficient to constitute a functional pathway. Overall, the genome of T. rangeli provides for a much better understanding of the identity, evolution, regulation and function of trypanosome virulence determinants for both mammalian host and insect vector.
doi:10.1371/journal.pntd.0003176
PMCID: PMC4169256  PMID: 25233456
4.  Validation of Suitable Reference Genes for Expression Normalization in Echinococcus spp. Larval Stages 
PLoS ONE  2014;9(7):e102228.
In recent years, a significant amount of sequence data (both genomic and transcriptomic) for Echinococcus spp. has been published, thereby facilitating the analysis of genes expressed during a specific stage or involved in parasite development. To perform a suitable gene expression quantification analysis, the use of validated reference genes is strongly recommended. Thus, the aim of this work was to identify suitable reference genes to allow reliable expression normalization for genes of interest in Echinococcus granulosus sensu stricto (s.s.) (G1) and Echinococcus ortleppi upon induction of the early pre-adult development. Untreated protoscoleces (PS) and pepsin-treated protoscoleces (PSP) from E. granulosus s.s. (G1) and E. ortleppi metacestode were used. The gene expression stability of eleven candidate reference genes (βTUB, NDUFV2, RPL13, TBP, CYP-1, RPII, EF-1α, βACT-1, GAPDH, ETIF4A-III and MAPK3) was assessed using geNorm, Normfinder, and RefFinder. Our qPCR data showed a good correlation with the recently published RNA-seq data. Regarding expression stability, EF-1α and TBP were the most stable genes for both species. Interestingly, βACT-1 (the most commonly used reference gene), and GAPDH and ETIF4A-III (previously identified as housekeeping genes) did not behave stably in our assay conditions. We propose the use of EF-1α as a reference gene for studies involving gene expression analysis in both PS and PSP experimental conditions for E. granulosus s.s. and E. ortleppi. To demonstrate its applicability, EF-1α was used as a normalizer gene in the relative quantification of transcripts from genes coding for antigen B subunits. The same EF-1α reference gene may be used in studies with other Echinococcus sensu lato species. This report validates suitable reference genes for species of class Cestoda, phylum Platyhelminthes, thus providing a foundation for further validation in other epidemiologically important cestode species, such as those from the Taenia genus.
doi:10.1371/journal.pone.0102228
PMCID: PMC4094502  PMID: 25014071
5.  Rapid detection of Echinococcus species by a high-resolution melting (HRM) approach 
Parasites & Vectors  2013;6:327.
Background
High-resolution melting (HRM) provides a low-cost, fast and sensitive scanning method that allows the detection of DNA sequence variations in a single step, which makes it appropriate for application in parasite identification and genotyping. The aim of this work was to implement an HRM-PCR assay targeting part of the mitochondrial cox1 gene to achieve an accurate and fast method for Echinococcus spp. differentiation.
Findings
For melting analysis, a total of 107 samples from seven species were used in this study. The species analyzed included Echinococcus granulosus (n = 41) and Echinococcus ortleppi (n = 50) from bovine, Echinococcus vogeli (n = 2) from paca, Echinococcus oligarthra (n = 3) from agouti, Echinococcus multilocularis (n = 6) from monkey and Echinococcus canadensis (n = 2) and Taenia hydatigena (n = 3) from pig. DNA extraction was performed, and a 444-bp fragment of the cox1 gene was amplified. Two approaches were used, one based on HRM analysis, and a second using SYBR Green Tm-based. In the HRM analysis, a specific profile for each species was observed. Although some species exhibited almost the same melting temperature (Tm) value, the HRM profiles could be clearly discriminated. The SYBR Green Tm-based analysis showed differences between E. granulosus and E. ortleppi and between E. vogeli and E. oligarthra.
Conclusions
In this work, we report the implementation of HRM analysis to differentiate species of the genus Echinococcus using part of the mitochondrial gene cox1. This method may be also potentially applied to identify other species belonging to the Taeniidae family.
doi:10.1186/1756-3305-6-327
PMCID: PMC4029041  PMID: 24517106
Echinococcus species; High-resolution melting (HRM); Genotyping
6.  Performance of the GenoType MTBDRplus Assay Directly on Sputum Specimens from Brazilian Patients with Tuberculosis Treatment Failure or Relapse 
Journal of Clinical Microbiology  2013;51(5):1606-1608.
Rapid identification of drug resistance in clinical isolates of Mycobacterium tuberculosis is important in determining treatment for tuberculosis. The aim of this work was evaluate the performance of the GenoType MDRTBplus assay directly on sputum of patients who had treatment failure or relapse in a routine outpatient setting in southern Brazil.
doi:10.1128/JCM.00364-13
PMCID: PMC3647903  PMID: 23467605
7.  Mycobacterium tuberculosis of the RDRio Genotype Is the Predominant Cause of Tuberculosis and Associated with Multidrug Resistance in Porto Alegre City, South Brazil 
Journal of Clinical Microbiology  2013;51(4):1071-1077.
Spoligotyping has shown Mycobacterium tuberculosis strains to be composed of different lineages, and some of them are not just geographically restricted but also affect specific ethnic populations and are associated with outbreaks and drug resistance. We recently described a particular subtype within the Latin American-Mediterranean (LAM) family, called RDRio, widespread in Brazil. Moreover, recent data also indicate that RDRio is present in many countries on all continents and is associated with cavitary disease and multidrug resistance (MDR). To further explore the relationship between RDRio and MDR, we conducted a study in a tuberculosis (TB) reference center responsible for the care of MDR patients in Rio Grande do Sul, the southernmost Brazilian state. From a collection of 237 clinical isolates, RDRio alone was responsible for one-half of all MDR cases, including one large group composed of strains with identical IS6110-restriction fragment length polymorphism (RFLP) and having the LAM5 signature. We additionally had complete data records for 96 patients and could make comparisons between the presence and absence of RDRio. No difference in clinical, radiological or laboratory features was observed, but a significantly greater number of cases with MDR were described in patients infected with an RDRio strain (P = 0.0015). Altogether, RDRio was responsible for 38% of all TB cases. These data support and confirmed previous findings that RDRio is the main agent responsible for TB in Brazil and is associated with drug resistance. Considering that RDRio is a globally distributed genotype, such findings raise concern about the increase in MDR in certain human populations.
doi:10.1128/JCM.01511-12
PMCID: PMC3666761  PMID: 23325819
8.  The Genome of Anopheles darlingi, the main neotropical malaria vector 
Marinotti, Osvaldo | Cerqueira, Gustavo C. | de Almeida, Luiz Gonzaga Paula | Ferro, Maria Inês Tiraboschi | Loreto, Elgion Lucio da Silva | Zaha, Arnaldo | Teixeira, Santuza M. R. | Wespiser, Adam R. | Almeida e Silva, Alexandre | Schlindwein, Aline Daiane | Pacheco, Ana Carolina Landim | da Silva, Artur Luiz da Costa | Graveley, Brenton R. | Walenz, Brian P. | Lima, Bruna de Araujo | Ribeiro, Carlos Alexandre Gomes | Nunes-Silva, Carlos Gustavo | de Carvalho, Carlos Roberto | Soares, Célia Maria de Almeida | de Menezes, Claudia Beatriz Afonso | Matiolli, Cleverson | Caffrey, Daniel | Araújo, Demetrius Antonio M. | de Oliveira, Diana Magalhães | Golenbock, Douglas | Grisard, Edmundo Carlos | Fantinatti-Garboggini, Fabiana | de Carvalho, Fabíola Marques | Barcellos, Fernando Gomes | Prosdocimi, Francisco | May, Gemma | de Azevedo Junior, Gilson Martins | Guimarães, Giselle Moura | Goldman, Gustavo Henrique | Padilha, Itácio Q. M. | Batista, Jacqueline da Silva | Ferro, Jesus Aparecido | Ribeiro, José M. C. | Fietto, Juliana Lopes Rangel | Dabbas, Karina Maia | Cerdeira, Louise | Agnez-Lima, Lucymara Fassarella | Brocchi, Marcelo | de Carvalho, Marcos Oliveira | Teixeira, Marcus de Melo | Diniz Maia, Maria de Mascena | Goldman, Maria Helena S. | Cruz Schneider, Maria Paula | Felipe, Maria Sueli Soares | Hungria, Mariangela | Nicolás, Marisa Fabiana | Pereira, Maristela | Montes, Martín Alejandro | Cantão, Maurício E. | Vincentz, Michel | Rafael, Miriam Silva | Silverman, Neal | Stoco, Patrícia Hermes | Souza, Rangel Celso | Vicentini, Renato | Gazzinelli, Ricardo Tostes | Neves, Rogério de Oliveira | Silva, Rosane | Astolfi-Filho, Spartaco | Maciel, Talles Eduardo Ferreira | Ürményi, Turán P. | Tadei, Wanderli Pedro | Camargo, Erney Plessmann | de Vasconcelos, Ana Tereza Ribeiro
Nucleic Acids Research  2013;41(15):7387-7400.
Anopheles darlingi is the principal neotropical malaria vector, responsible for more than a million cases of malaria per year on the American continent. Anopheles darlingi diverged from the African and Asian malaria vectors ∼100 million years ago (mya) and successfully adapted to the New World environment. Here we present an annotated reference A. darlingi genome, sequenced from a wild population of males and females collected in the Brazilian Amazon. A total of 10 481 predicted protein-coding genes were annotated, 72% of which have their closest counterpart in Anopheles gambiae and 21% have highest similarity with other mosquito species. In spite of a long period of divergent evolution, conserved gene synteny was observed between A. darlingi and A. gambiae. More than 10 million single nucleotide polymorphisms and short indels with potential use as genetic markers were identified. Transposable elements correspond to 2.3% of the A. darlingi genome. Genes associated with hematophagy, immunity and insecticide resistance, directly involved in vector–human and vector–parasite interactions, were identified and discussed. This study represents the first effort to sequence the genome of a neotropical malaria vector, and opens a new window through which we can contemplate the evolutionary history of anopheline mosquitoes. It also provides valuable information that may lead to novel strategies to reduce malaria transmission on the South American continent. The A. darlingi genome is accessible at www.labinfo.lncc.br/index.php/anopheles-darlingi.
doi:10.1093/nar/gkt484
PMCID: PMC3753621  PMID: 23761445
9.  New insights on the biology of swine respiratory tract mycoplasmas from a comparative genome analysis 
BMC Genomics  2013;14:175.
Background
Mycoplasma hyopneumoniae, Mycoplasma flocculare and Mycoplasma hyorhinis live in swine respiratory tracts. M. flocculare, a commensal bacterium, is genetically closely related to M. hyopneumoniae, the causative agent of enzootic porcine pneumonia. M. hyorhinis is also pathogenic, causing polyserositis and arthritis. In this work, we present the genome sequences of M. flocculare and M. hyopneumoniae strain 7422, and we compare these genomes with the genomes of other M. hyoponeumoniae strain and to the a M. hyorhinis genome. These analyses were performed to identify possible characteristics that may help to explain the different behaviors of these species in swine respiratory tracts.
Results
The overall genome organization of three species was analyzed, revealing that the ORF clusters (OCs) differ considerably and that inversions and rearrangements are common. Although M. flocculare and M. hyopneumoniae display a high degree of similarity with respect to the gene content, only some genomic regions display considerable synteny. Genes encoding proteins that may be involved in host-cell adhesion in M. hyopneumoniae and M. flocculare display differences in genomic structure and organization. Some genes encoding adhesins of the P97 family are absent in M. flocculare and some contain sequence differences or lack of domains that are considered to be important for adhesion to host cells. The phylogenetic relationship of the three species was confirmed by a phylogenomic approach. The set of genes involved in metabolism, especially in the uptake of precursors for nucleic acids synthesis and nucleotide metabolism, display some differences in copy number and the presence/absence in the three species.
Conclusions
The comparative analyses of three mycoplasma species that inhabit the swine respiratory tract facilitated the identification of some characteristics that may be related to their different behaviors. M. hyopneumoniae and M. flocculare display many differences that may help to explain why one species is pathogenic and the other is considered to be commensal. However, it was not possible to identify specific virulence determinant factors that could explain the differences in the pathogenicity of the analyzed species. The M. hyorhinis genome contains differences in some components involved in metabolism and evasion of the host’s immune system that may contribute to its growth aggressiveness. Several horizontal gene transfer events were identified. The phylogenomic analysis places M. hyopneumoniae, M. flocculare and M. hyorhinis in the hyopneumoniae clade.
doi:10.1186/1471-2164-14-175
PMCID: PMC3610235  PMID: 23497205
Mycoplasma; Comparative genomics; Adhesins; Swine respiratory tract
11.  A Transcriptomic Analysis of Echinococcus granulosus Larval Stages: Implications for Parasite Biology and Host Adaptation 
Background
The cestode Echinococcus granulosus - the agent of cystic echinococcosis, a zoonosis affecting humans and domestic animals worldwide - is an excellent model for the study of host-parasite cross-talk that interfaces with two mammalian hosts. To develop the molecular analysis of these interactions, we carried out an EST survey of E. granulosus larval stages. We report the salient features of this study with a focus on genes reflecting physiological adaptations of different parasite stages.
Methodology/Principal Findings
We generated ∼10,000 ESTs from two sets of full-length enriched libraries (derived from oligo-capped and trans-spliced cDNAs) prepared with three parasite materials: hydatid cyst wall, larval worms (protoscoleces), and pepsin/H+-activated protoscoleces. The ESTs were clustered into 2700 distinct gene products. In the context of the biology of E. granulosus, our analyses reveal: (i) a diverse group of abundant long non-protein coding transcripts showing homology to a middle repetitive element (EgBRep) that could either be active molecular species or represent precursors of small RNAs (like piRNAs); (ii) an up-regulation of fermentative pathways in the tissue of the cyst wall; (iii) highly expressed thiol- and selenol-dependent antioxidant enzyme targets of thioredoxin glutathione reductase, the functional hub of redox metabolism in parasitic flatworms; (iv) candidate apomucins for the external layer of the tissue-dwelling hydatid cyst, a mucin-rich structure that is critical for survival in the intermediate host; (v) a set of tetraspanins, a protein family that appears to have expanded in the cestode lineage; and (vi) a set of platyhelminth-specific gene products that may offer targets for novel pan-platyhelminth drug development.
Conclusions/Significance
This survey has greatly increased the quality and the quantity of the molecular information on E. granulosus and constitutes a valuable resource for gene prediction on the parasite genome and for further genomic and proteomic analyses focused on cestodes and platyhelminths.
Author Summary
Cestodes are a neglected group of platyhelminth parasites, despite causing chronic infections to humans and domestic animals worldwide. We used Echinococcus granulosus as a model to study the molecular basis of the host-parasite cross-talk during cestode infections. For this purpose, we carried out a survey of the genes expressed by parasite larval stages interfacing with definitive and intermediate hosts. Sequencing from several high quality cDNA libraries provided numerous insights into the expression of genes involved in important aspects of E. granulosus biology, e.g. its metabolism (energy production and antioxidant defences) and the synthesis of key parasite structures (notably, the one exposed to humans and livestock intermediate hosts). Our results also uncovered the existence of an intriguing set of abundant repeat-associated non-protein coding transcripts that may participate in the regulation of gene expression in all surveyed stages. The dataset now generated constitutes a valuable resource for gene prediction on the parasite genome and for further genomic and proteomic studies focused on cestodes and platyhelminths. In particular, the detailed characterization of a range of newly discovered genes will contribute to a better understanding of the biology of cestode infections and, therefore, to the development of products allowing their efficient control.
doi:10.1371/journal.pntd.0001897
PMCID: PMC3510090  PMID: 23209850
12.  Echinococcus granulosus Antigen B Structure: Subunit Composition and Oligomeric States 
Background
Antigen B (AgB) is the major protein secreted by the Echinococcus granulosus metacestode and is involved in key host-parasite interactions during infection. The full comprehension of AgB functions depends on the elucidation of several structural aspects that remain unknown, such as its subunit composition and oligomeric states.
Methodology/Principal Findings
The subunit composition of E. granulosus AgB oligomers from individual bovine and human cysts was assessed by mass spectrometry associated with electrophoretic analysis. AgB8/1, AgB8/2, AgB8/3 and AgB8/4 subunits were identified in all samples analyzed, and an AgB8/2 variant (AgB8/2v8) was found in one bovine sample. The exponentially modified protein abundance index (emPAI) was used to estimate the relative abundance of the AgB subunits, revealing that AgB8/1 subunit was relatively overrepresented in all samples. The abundance of AgB8/3 subunit varied between bovine and human cysts. The oligomeric states formed by E. granulosus AgB and recombinant subunits available, rAgB8/1, rAgB8/2 and rAgB8/3, were characterized by native PAGE, light scattering and microscopy. Recombinant subunits showed markedly distinct oligomerization behaviors, forming oligomers with a maximum size relation of rAgB8/3>rAgB8/2>rAgB8/1. Moreover, the oligomeric states formed by rAgB8/3 subunit were more similar to those observed for AgB purified from hydatid fluid. Pressure-induced dissociation experiments demonstrated that the molecular assemblies formed by the more aggregative subunits, rAgB8/2 and rAgB8/3, also display higher structural stability.
Conclusions/Significance
For the first time, AgB subunit composition was analyzed in samples from single hydatid cysts, revealing qualitative and quantitative differences between samples. We showed that AgB oligomers are formed by different subunits, which have distinct abundances and oligomerization properties. Overall, our findings have significantly contributed to increase the current knowledge on AgB expression and structure, highlighting issues that may help to understand the parasite adaptive response during chronic infection.
Author Summary
Antigen B (AgB) is the major secretory protein of the Echinococcus granulosus hydatid cyst, the causative agent of cystic hydatid disease. Structurally, AgB is a multisubunit protein formed by 8-kDa subunits, but it is not known which subunits are secreted by a single parasite (cyst) and how they interact in the formation of distinct AgB oligomeric states. Here, we investigated AgB subunit composition and oligomeric states in individual samples from bovine and human cysts. We identified AgB8/1, AgB8/2, AgB8/3 and AgB8/4 subunits in AgB oligomers of all samples analyzed. Quantitative and qualitative differences in the expression of AgB subunits were observed within and between samples. Using recombinant subunits as models, we showed that AgB subunits form distinct oligomeric states, with a rAgB8/3>rAgB8/2>rAgB8/1 maximum size relation. We also demonstrated by different experimental approaches that rAgB8/3 oligomers are more similar, both in size and morphology, to those observed for E. granulosus AgB. Overall, we provided experimental evidences that AgB is composed of different subunits within a single cyst, and that subunits have different abundances and oligomerization properties. These issues are important for the understanding of AgB expression and structure variations, and their impact for the host-parasite cross-talk.
doi:10.1371/journal.pntd.0001551
PMCID: PMC3295803  PMID: 22413028
13.  Streptomycin Resistance and Lineage-Specific Polymorphisms in Mycobacterium tuberculosis gidB Gene ▿ 
Journal of Clinical Microbiology  2011;49(7):2625-2630.
Mutations related to streptomycin resistance in the rpsL and rrs genes are well known and can explain about 70% of this phenotypic resistance. Recently, the gidB gene was found to be associated with low-level streptomycin resistance in Mycobacterium tuberculosis. Mutations in gidB have been reported with high frequency, and this gene appears to be very polymorphic, with frameshift and point mutations occurring in streptomycin-susceptible and streptomycin-resistant strains. In this study, mutations in gidB appeared in 27% of streptomycin-resistant strains that contained no mutations in the rpsL or rrs genes, and they were associated with low-level streptomycin resistance. However, the association of certain mutations in gidB with streptomycin resistance needs to be further investigated, as we also found mutations in gidB in streptomycin-susceptible strains. This occurred only when the strain was resistant to rifampin and isoniazid. Two specific mutations appeared very frequently in this and other studies of streptomycin-susceptible and -resistant strains; these mutations were not considered related to streptomycin resistance, but as a polymorphism. We stratified the strains according to the different phylogenetic lineages and showed that the gidB16 polymorphism (16G allele) was exclusively present in the Latin American-Mediterranean (LAM) genotype, while the gidB92 polymorphism (92C allele) was associated with the Beijing lineage in another population. In the sample studied, the two characterized single-nucleotide polymorphisms could distinguish LAM and Beijing lineages from the other lineages.
doi:10.1128/JCM.00168-11
PMCID: PMC3147840  PMID: 21593257
14.  Structure-based functional inference of hypothetical proteins from Mycoplasma hyopneumoniae 
Journal of Molecular Modeling  2011;18(5):1917-1925.
Enzootic pneumonia caused by Mycoplasma hyopneumoniae is a major constraint to efficient pork production throughout the world. This pathogen has a small genome with 716 coding sequences, of which 418 are homologous to proteins with known functions. However, almost 42% of the 716 coding sequences are annotated as hypothetical proteins. Alternative methodologies such as threading and comparative modeling can be used to predict structures and functions of such hypothetical proteins. Often, these alternative methods can answer questions about the properties of a model system faster than experiments. In this study, we predicted the structures of seven proteins annotated as hypothetical in M. hyopneumoniae, using the structure-based approaches mentioned above. Three proteins were predicted to be involved in metabolic processes, two proteins in transcription and two proteins where no function could be assigned. However, the modeled structures of the last two proteins suggested experimental designs to identify their functions. Our findings are important in diminishing the gap between the lack of annotation of important metabolic pathways and the great number of hypothetical proteins in the M. hyopneumoniae genome.
doi:10.1007/s00894-011-1212-3
PMCID: PMC3340535  PMID: 21870198
Comparative modeling; Known function; Modeller; Mollicutes; Threading; Chemistry; Health Informatics; Life Sciences, general; Computer Appl. in Life Sciences; Molecular Medicine; Biomedicine general; Computer Applications in Chemistry
15.  Evaluation of real-time PCR of patient pleural effusion for diagnosis of tuberculosis 
BMC Research Notes  2011;4:279.
Background
Pleural tuberculosis (TB) diagnosis often requires invasive procedures such as pleural biopsy. The aim of this study was to evaluate the role of real-time polymerase chain reaction (PCR) for the IS6110 sequence of M. tuberculosis in pleural fluid specimens as a rapid and non-invasive test for pleural TB diagnosis.
Findings
For this cross-sectional study, 150 consecutive patients with pleural effusion diagnosed by chest radiography, who were referred for diagnostic thoracocentesis and pleural biopsy and met eligibility criteria, had a pleural fluid specimen submitted for real-time PCR testing. Overall, 98 patients had pleural TB and 52 had pleural effusion secondary to other disease. TB diagnosis was obtained using acid-fast bacilli (AFB) smear or culture for mycobacteria and/or histopathologic examination in 94 cases and by clinical findings in 4 cases. Sensitivity, specificity, positive and negative predictive values of PCR testing for pleural TB diagnosis were 42.8% (95% CI 38.4 - 44.8), 94.2% (95% CI 85.8 - 98.0), 93.3% (95% CI 83.6 - 97.7), and 48.5% (95% CI 44.2 - 50.4), respectively. The real-time PCR test improved TB detection from 30.6% to 42.9% when compared to AFB smear and culture methods performed on pleural fluid specimens, although the best sensitivity was achieved by combining the results of culture and histopathology of pleural tissue specimens.
Conclusion
The real-time PCR test of pleural fluid specimens is a useful and non-invasive additional assay for fast diagnosis of pleural TB.
doi:10.1186/1756-0500-4-279
PMCID: PMC3224496  PMID: 21819571
16.  Survey of transcripts expressed by the invasive juvenile stage of the liver fluke Fasciola hepatica 
BMC Genomics  2010;11:227.
Background
The common liver fluke Fasciola hepatica is the agent of a zoonosis with significant economic consequences in livestock production worldwide, and increasing relevance to human health in developing countries. Although flukicidal drugs are available, re-infection and emerging resistance are demanding new efficient and inexpensive control strategies. Understanding the molecular mechanisms underlying the host-parasite interaction provide relevant clues in this search, while enlightening the physiological adaptations to parasitism. Genomics and transcriptomics are still in their infancy in F. hepatica, with very scarce information available from the invasive newly excysted juveniles (NEJ). Here we provide an initial glimpse to the transcriptomics of the NEJ, the first stage to interact with the mammalian host.
Results
We catalogued more than 500 clusters generated from the analysis of F. hepatica juvenile expressed sequence tags (EST), several of them not detected in the adult stage. A set of putative F. hepatica specific transcripts, and a group of sequences conserved exclusively in flatworms were identified. These novel sequences along with a set of parasite transcripts absent in the host genomes are putative new targets for future anti-parasitic drugs or vaccine development.
Comparisons of the F. hepatica sequences with other metazoans genomes or EST databases were consistent with the basal positioning of flatworms in the bilaterian phylogeny. Notably, GC content, codon usage and amino acid frequencies are remarkably different in Schistosomes to F. hepatica and other trematodes.
Functional annotation of predicted proteins showed a general representation of diverse biological functions. Besides proteases and antioxidant enzymes expected to participate in the early interaction with the host, various proteins involved in gene expression, protein synthesis, cell signaling and mitochondrial enzymes were identified. Differential expression of secreted protease gene family members between juvenile and adult stages may respond to different needs during host colonization.
Conclusion
The knowledge of the genes expressed by the invasive stage of Fasciola hepatica is a starting point to unravel key aspects of this parasite's biology. The integration of the emerging transcriptomics, and proteomics data and the advent of functional genomics tools in this organism are positioning F. hepatica as an interesting model for trematode biology.
doi:10.1186/1471-2164-11-227
PMCID: PMC2867827  PMID: 20374642
17.  Comparative proteomic analysis of pathogenic and non-pathogenic strains from the swine pathogen Mycoplasma hyopneumoniae 
Proteome Science  2009;7:45.
Background
Mycoplasma hyopneumoniae 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, Mycoplasma hyopneumoniae, we performed comparative protein profiling of three M. hyopneumoniae strains, namely the non-pathogenic J strain and the two pathogenic strains 7448 and 7422.
Results
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 M. hyopneumoniae 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.
Conclusions
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.
doi:10.1186/1477-5956-7-45
PMCID: PMC2804596  PMID: 20025764
18.  Transcriptome analysis of Taenia solium cysticerci using Open Reading Frame ESTs (ORESTES) 
Parasites & Vectors  2009;2:35.
Background
Human infection by the pork tapeworm Taenia solium affects more than 50 million people worldwide, particularly in underdeveloped and developing countries. Cysticercosis which arises from larval encystation can be life threatening and difficult to treat. Here, we investigate for the first time the transcriptome of the clinically relevant cysticerci larval form.
Results
Using Expressed Sequence Tags (ESTs) produced by the ORESTES method, a total of 1,520 high quality ESTs were generated from 20 ORESTES cDNA mini-libraries and its analysis revealed fragments of genes with promising applications including 51 ESTs matching antigens previously described in other species, as well as 113 sequences representing proteins with potential extracellular localization, with obvious applications for immune-diagnosis or vaccine development.
Conclusion
The set of sequences described here will contribute to deciphering the expression profile of this important parasite and will be informative for the genome assembly and annotation, as well as for studies of intra- and inter-specific sequence variability. Genes of interest for developing new diagnostic and therapeutic tools are described and discussed.
doi:10.1186/1756-3305-2-35
PMCID: PMC2731055  PMID: 19646239
19.  Identification of Mutations Related to Streptomycin Resistance in Clinical Isolates of Mycobacterium tuberculosis and Possible Involvement of Efflux Mechanism▿ † 
The MIC for streptomycin in the presence of efflux pump (EP) inhibitors and the sequencing of rpsL, rrs, and gidB genes provided evidence for the possible participation of EP in low-level streptomycin (STR) resistance of some isolates without mutations. Mutation in the gidB gene and an EP could act synergistically to confer low STR resistance.
doi:10.1128/AAC.01570-07
PMCID: PMC2493096  PMID: 18541729
21.  Swine and Poultry Pathogens: the Complete Genome Sequences of Two Strains of Mycoplasma hyopneumoniae and a Strain of Mycoplasma synoviae†  
Vasconcelos, Ana Tereza R. | Ferreira, Henrique B. | Bizarro, Cristiano V. | Bonatto, Sandro L. | Carvalho, Marcos O. | Pinto, Paulo M. | Almeida, Darcy F. | Almeida, Luiz G. P. | Almeida, Rosana | Alves-Filho, Leonardo | Assunção, Enedina N. | Azevedo, Vasco A. C. | Bogo, Maurício R. | Brigido, Marcelo M. | Brocchi, Marcelo | Burity, Helio A. | Camargo, Anamaria A. | Camargo, Sandro S. | Carepo, Marta S. | Carraro, Dirce M. | de Mattos Cascardo, Júlio C. | Castro, Luiza A. | Cavalcanti, Gisele | Chemale, Gustavo | Collevatti, Rosane G. | Cunha, Cristina W. | Dallagiovanna, Bruno | Dambrós, Bibiana P. | Dellagostin, Odir A. | Falcão, Clarissa | Fantinatti-Garboggini, Fabiana | Felipe, Maria S. S. | Fiorentin, Laurimar | Franco, Gloria R. | Freitas, Nara S. A. | Frías, Diego | Grangeiro, Thalles B. | Grisard, Edmundo C. | Guimarães, Claudia T. | Hungria, Mariangela | Jardim, Sílvia N. | Krieger, Marco A. | Laurino, Jomar P. | Lima, Lucymara F. A. | Lopes, Maryellen I. | Loreto, Élgion L. S. | Madeira, Humberto M. F. | Manfio, Gilson P. | Maranhão, Andrea Q. | Martinkovics, Christyanne T. | Medeiros, Sílvia R. B. | Moreira, Miguel A. M. | Neiva, Márcia | Ramalho-Neto, Cicero E. | Nicolás, Marisa F. | Oliveira, Sergio C. | Paixão, Roger F. C. | Pedrosa, Fábio O. | Pena, Sérgio D. J. | Pereira, Maristela | Pereira-Ferrari, Lilian | Piffer, Itamar | Pinto, Luciano S. | Potrich, Deise P. | Salim, Anna C. M. | Santos, Fabrício R. | Schmitt, Renata | Schneider, Maria P. C. | Schrank, Augusto | Schrank, Irene S. | Schuck, Adriana F. | Seuanez, Hector N. | Silva, Denise W. | Silva, Rosane | Silva, Sérgio C. | Soares, Célia M. A. | Souza, Kelly R. L. | Souza, Rangel C. | Staats, Charley C. | Steffens, Maria B. R. | Teixeira, Santuza M. R. | Urmenyi, Turan P. | Vainstein, Marilene H. | Zuccherato, Luciana W. | Simpson, Andrew J. G. | Zaha, Arnaldo
Journal of Bacteriology  2005;187(16):5568-5577.
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.
doi:10.1128/JB.187.16.5568-5577.2005
PMCID: PMC1196056  PMID: 16077101
22.  Characterization of pncA Mutations in Pyrazinamide-Resistant Mycobacterium tuberculosis in Brazil 
In this study the nucleotide sequence of the pncA gene from 59 Mycobacterium tuberculosis clinical isolates was analyzed. Mutations in the pncA gene were identified in 29 of 40 pyrazinamide-resistant isolates, and no pyrazinamidase activity was detected in 39 of them. Twelve mutations found in this work have not been described previously.
doi:10.1128/AAC.49.1.444-446.2005
PMCID: PMC538919  PMID: 15616332
23.  Mutations in katG, inhA, and ahpC Genes of Brazilian Isoniazid-Resistant Isolates of Mycobacterium tuberculosis 
Journal of Clinical Microbiology  2003;41(9):4471-4474.
The presence of mutations in specific regions of the katG, inhA, and ahpC genes was analyzed with 69 Mycobacterium tuberculosis isoniazid-resistant isolates from three Brazilian states. Point mutations in codon 315 of the katG gene were observed in 87.1, 60.9, and 60% of the isolates from Rio Grande do Sul, Rio de Janeiro, and São Paulo, respectively. Mutations in the inhA gene were identified only in one isolate from RJ State, and the ahpC promoter region revealed mutations in distinct positions in 12.9, 21.7, and 6.7% of the isolates from RS, RJ and SP, respectively.
doi:10.1128/JCM.41.9.4471-4474.2003
PMCID: PMC193791  PMID: 12958298
24.  Detection of Mycobacterium avium in Blood Samples of Patients with AIDS by Using PCR 
Journal of Clinical Microbiology  2002;40(6):2297-2299.
Sixty-nine blood samples from 47 patients infected with human immunodeficiency virus were analyzed by using PCR to detect Mycobacterium avium. The sensitivity can be up to 95.7%, depending on the detection method used and the number of blood samples analyzed from each patient. The procedure can be helpful in the diagnosis of mycobacterial disease.
doi:10.1128/JCM.40.6.2297-2299.2002
PMCID: PMC130774  PMID: 12037115
25.  Mutations in the rpoB Gene of Multidrug-Resistant Mycobacterium tuberculosis Isolates from Brazil 
Journal of Clinical Microbiology  2000;38(8):3119-3122.
Mutations in a 69-bp region of the rpoB gene associated with rifampin resistance (Rifr) in 100 isolates (82 Rifr) from three states of Brazil were studied. Twenty-one different kinds of mutations were identified in the Rifr isolates, and six new alleles are described.
PMCID: PMC87207  PMID: 10921994

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