Expression and secretion of foreign proteins by 'Aspergillus'

The feasibility of using the ascomycetes filamentous fungus Aspergillus oryzae as a host organism for foreign protein expression and secretion was assessed. For this purpose, human interleukin-6 (hIL-6) and the Simian virus 5 haemagglutinin-neuraminidase (HN) were used as model foreign proteins. Expression and secretion of the human interleukin 5 gene have been achieved using vector construction containing the entire A.niger glaA coding region linked to the hIL-6 cDNA through a KEX2-motif encoding DNA. hIL-6 containing expression vectors were introduced into A.oryzae nitrate non-utilising mutant strains nlaD14, niaD200, niaD500 (niaD-) and arginine auxotrophic strain 1560-6 (argB-) by means of genetic cotransformation technology. The A.oryzae niaD gene or the A.nidulans argB or amdS genes were used as the transfer and selection system. A.oryzae nitrate and acetamide prototrophic transformants, containing the hIL-6 DNA sequence, failed to secrete free hIL-6 molecules. Recombinant hIL-6 protein was detected in culture filtrates from A.oryzae 1560-6 arginine prototrophic transformants T1560-gla15 and T1560-gpd1. The results suggested that A.oryzae proteases recognized the KEX2-motif yielding free hIL-6 molecules. However, hIL-6 was found to be selective degraded by A.oryzae proteases compared with the heterologous glucoamylase (GLAA). The incubation time for hIL-6 detection has been shown to vary with the host strain and growth conditions. Hybrid GLAA/hIL-6 protein was also found to be secreted. Improvement in hIL-6 yields were obtained by generating 2-deoxy-D-glucose resistant mutants of transformant T1560-gla15 and by manipulating the host growth conditions including the use of microbial solid state culture. The maximum level of secreted hIL-6 was found to be approximately 4 mg/1 as judged from SDS-PAGE polyacrylamide gels and western blots analysis. The Simian virus 5 Haemagglutinin-neuraminidase (HN) glycosylation, folding and oligomerization characteristics have been well studied in mammalian cells. Therefore, HN is an ideal model protein, a priori, for studying secretion / processing in foreign hosts including filamentous fungi. For such studies the entire HN cDNA sequence (designated HN-DNA fragment) was generated by PCR technology. However, as the HN protein is a membrane bound glycoprotein type II, HN might not be secreted due to its anchor-domain, thus a deletion of the first 108 bp of its cDNA sequence, corresponding to the hydrophobic N-terminal region, was carried out in a second PCR reaction (designated ASSHN-DNA fragment). Both DNA fragments, encoding HN or ASSHN protein sequences, were inserted in-frame downstream of the A.niger glaA coding region in plasmid vector pAN56-30, generating pAN56-HN and pAN56-ASSHN. Additionally, both DNA fragments were inserted in-frame downstream of the A.oryzae amy3 gene present in vector pSTA1210, generating pSTA1210-HN and pSTAX210-ASSHN. These expression cassettes were introduced into A.oryzae 1560- 6, an amylase over-producing industrial strain, by means of cotransformation. The A.nidulans argB and amdS genes were used as transfer and selection systems. The HN protein was found in culture filtrates of arginine prototrophic transformants harbouring the glaA/ASSHN and amy3/ASSHN DNA sequences. HN protein was mainly detected by immunoprecipitation of 50 ml of culture filtrate. Such low expression level do not seem to be related to gene copy number, but rather to proteolytic degradation. Another possibility is that HN was not translated efficiently. Improvement in HN yield was achieved by cultivation on solid state culture in which HN protein was detected in 1 ml aliquot of culture filtrate as determine by western blotting. Nevertheless, the yield of HN protein extracted with 50 ml of sterile distilled water from 10 g rice solid state culture is estimated to be less than 1µg per ml as judged by the western blot

Genetic characterization of morphologically variant strains of Paracoccidioides brasiliensis

Molecular characterization of Paracoccidioides brasiliensis variant strains that had been preserved under mineral oil for decades was carried out by random amplified polymorphic DNA analysis (RAPD). On P. brasiliensis variants in the transitional phase and strains with typical morphology, RAPD produced reproducible polymorphic amplification products that differentiated them. A dendrogram based on the generated RAPD patterns placed the 14 P. brasiliensis strains into five groups with similarity coefficients of 72%. A high correlation between the genotypic and phenotypic characteristics of the strains was observed. A 750 bp-RAPD fragment found only in the wild-type phenotype strains was cloned and sequenced. Genetic similarity analysis using BLASTx suggested that this RAPD marker represents a putative domain of a hypothetical flavin-binding monooxygenase (FMO)-like protein of Neurospora crassa.FiocruzBritish Council Progra

Molecular detection of Mycobacterium bovis in cattle herds of the state of Pernambuco, Brazil

Submitted by Adagilson Silva ([email protected]) on 2017-09-01T12:35:29Z No. of bitstreams: 1 26897524 2016 cez-mol.oa.pdf: 466479 bytes, checksum: 72f2a61e564e72399e0dad54064bf1d4 (MD5)Approved for entry into archive by Adagilson Silva ([email protected]) on 2017-09-01T12:42:28Z (GMT) No. of bitstreams: 1 26897524 2016 cez-mol.oa.pdf: 466479 bytes, checksum: 72f2a61e564e72399e0dad54064bf1d4 (MD5)Made available in DSpace on 2017-09-01T12:42:28Z (GMT). No. of bitstreams: 1 26897524 2016 cez-mol.oa.pdf: 466479 bytes, checksum: 72f2a61e564e72399e0dad54064bf1d4 (MD5) Previous issue date: 2016-02-20Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Recife, PE, BrasilThe present study aimed to direct detect Mycobacterium bovis in milk (n = 401) and blood (n = 401) samples collected from 401 dairy cows of 20 properties located in the state of Pernambuco, Brazil, by real-time quantitative PCR (qPCR) targeting the region of difference 4 (RD4). Risk factors possibly associated with bovine tuberculosis (BTB) were also evaluated

Genetic characterization of morphologically variant strains of Paracoccidioides brasiliensis

Molecular characterization of Paracoccidioides brasiliensis variant strains that had been preserved under mineral oil for decades was carried out by random amplified polymorphic DNA analysis (RAPD). On P. brasiliensis variants in the transitional phase and strains with typical morphology, RAPD produced reproducible polymorphic amplification products that differentiated them. A dendrogram based on the generated RAPD patterns placed the 14 P. brasiliensis strains into five groups with similarity coefficients of 72%. A high correlation between the genotypic and phenotypic characteristics of the strains was observed. A 750 bp-RAPD fragment found only in the wild-type phenotype strains was cloned and sequenced. Genetic similarity analysis using BLASTx suggested that this RAPD marker represents a putative domain of a hypothetical flavin-binding monooxygenase (FMO)-like protein of Neurospora crassa.FiocruzBritish Council Progra

Risk factors for treatment delay in pulmonary tuberculosis in Recife, Brazil

BACKGROUND: Tuberculosis is still a great challenge to public health in Brazil and worldwide. Early detection followed by effective therapy is extremely important in controlling the disease. Recent studies have investigated reasons for delays in treatment, but there is no agreed definition of what constitutes an "acceptable" delay. This study investigates factors associated with total delay in treatment of tuberculosis. METHODS: A cohort of adult cases of pulmonary tuberculosis diagnosed over a two-year period was studied. Patients were interviewed on entry, reporting the duration of symptoms before the start of treatment, and sputum and blood samples were collected. It was decided that sixty days was an acceptable total delay. Associations were investigated using univariable and multivariable analysis and the population attributable fraction was estimated. RESULTS: Of 1105 patients, 62% had a delay of longer than 60 days. Age, sex, alcoholism and difficulty of access were not associated with delays, but associations were found in the case of unemployment, having given up smoking, having lost weight and being treated in two of the six health districts. The proportion attributable to: not being an ex-smoker was 31%; unemployment, 18%; weight loss, 12%, and going to the two worst health districts, 25%. CONCLUSION: In this urban area, delays seem to be related to unemployment and general attitudes towards health. Although they reflect the way health services are organized, delays are not associated with access to care