Cathepsin L of Triatoma brasiliensis (Reduviidae, Triatominae): Sequence characterization, expression pattern and zymography

AbstractTriatoma brasiliensis is considered one of the main vectors of Chagas disease commonly found in semi-arid areas of northeastern Brazil. These insects use proteases, such as carboxypeptidase B, aminopeptidases and different cathepsins for blood digestion. In the present study, two genes encoding cathepsin L from the midgut of T. brasiliensis were identified and characterized. Mature T. brasiliensis cathepsin L-like proteinases (TBCATL-1, TBCATL-2) showed a high level of identity to the cathepsin L-like proteinases of other insects, with highest similarity to Rhodnius prolixus. Both cathepsin L transcripts were highly abundant in the posterior midgut region, the main region of the blood digestion. Determination of the pH in the whole intestine of unfed T. brasiliensis revealed alkaline conditions in the anterior midgut region (stomach) and acidic conditions in the posterior midgut region (small intestine). Gelatine in-gel zymography showed the activity of at least four distinct proteinases in the small intestine and the cysteine proteinase inhibitors transepoxysuccinyl-l-leucylamido-(4-guanidino)butane (E-64) and cathepsin B inhibitor and N-(l-3-trans-propylcarbamoyl-oxirane-2-carbonyl)-l-isoleucyl-l-proline (CA-074) were employed to characterize enzymatic activity. E-64 fully inhibited cysteine proteinase activity, whereas in the samples treated with CA-074 residual proteinase activity was detectable. Thus, proteolytic activity could at least partially be ascribed to cathepsin L. Western blot analysis using specific anti cathepsin L antibodies confirmed the presence of cathepsin L in the lumen of the small intestine of the insects

The Baltic Sea Tracer Release Experiment. Part I: Mixing rates

In this study, results from the Baltic Sea Tracer Release Experiment (BATRE) are described, in which deep water mixing rates and mixing processes in the central Baltic Sea were investigated. In September 2007, an inert tracer gas (CF3SF5) was injected at approximately 200 m depth in the Gotland Basin, and the subsequent spreading of the tracer was observed during six surveys until February 2009. These data describe the diapycnal and lateral mixing during a stagnation period without any significant deep water renewal due to inflow events. As one of the main results, vertical mixing rates were found to dramatically increase after the tracer had reached the lateral boundaries of the basin, suggesting boundary mixing as the key process for basin-scale vertical mixing. Basin-scale vertical diffusivities were of the order of 10−5 m2 s−1 (about 1 order of magnitude larger than interior diffusivities) with evidence for a seasonal and vertical variability. In contrast to tracer experiments in the open ocean, the basin geometry (hypsography) was found to have a crucial impact on the vertical tracer spreading. The e-folding time scale for deep water renewal due to mixing was slightly less than 2 years, the time scale for the lateral homogenization of the tracer patch was of the order of a few months. Key Points: Mixing rates in the Gotland Basin are dominated by boundary mixing processes; The time scale for Gotland Basin deep water renewal is approximately 2 years; Mixing rates determined from the tracer CF3SF

TcI/TcII co-infection can enhance Trypanosoma cruzi growth in Rhodnius prolixus

We thank Prof. J. Jurberg (Laboratório Nacional e Internacional de Referência em Taxonomia de Triatomíneos, Fiocruz, Rio de Janeiro) for providing the insects and Prof. Dr. V. Bongertz (Laboratório de AIDS e Imunologia Molecular, Fiocruz, Rio de Janeiro) for the English corrections. This work was supported by Fundação de Amparo à Pesquisa no Estado do Rio de Janeiro (FAPERJ: E-26/ 100.456/2007), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq: Edital Universal 472276/2006-9; PDJ: 152069/2006-2) and Fundação Oswaldo Cruz (FIOCRUZ). CACA was a CNPq Research Fellow (151187/2009-6) and PJW was a FAPERJ Research Fellow (E-26/152.913/2005).Made available in DSpace on 2015-04-22T14:23:07Z (GMT). No. of bitstreams: 2 license.txt: 1914 bytes, checksum: 7d48279ffeed55da8dfe2f8e81f3b81f (MD5) catarina_araujoetal_IOC_2014.pdf: 362757 bytes, checksum: a8aeb5f677f8cc4d71789993e7736fc9 (MD5) Previous issue date: 2014Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia de Tripanossomatídeos. Rio de Janeiro, RJ, BrasilFundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia de Tripanossomatídeos. Rio de Janeiro, RJ, BrasilFundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia de Tripanossomatídeos. Rio de Janeiro, RJ, BrasilBackground: Rhodnius prolixus is an obligate haematophagous insect and one of the most important vectors of Trypanosoma cruzi, the causative agent of Chagas disease in the Americas. T. cruzi is a highly variable parasite which is not transmitted in the same efficiency by the different triatomine vectors. Because different T. cruzi genotypes are aetiopathologically divergent, further elucidation of the transmission abilities of different Chagas disease vectors is extremely important. Findings: In the present study, the growth behaviour of two T. cruzi isolates, MDID/BR/1993/C45 (TcI) and TBRA/BR/ 1999/JCA3 (TcII), sharing the same microhabitat (intestinal tract) in single and mixed infections, was examined. The distribution patterns and parasite population densities were evaluated at 7, 14 and 21 days after feeding (daf) by quantification of parasites using Neubauer haemocytometric measurements and mini-exon PCR to identify TcI and TcII subpopulations. Parasitic colonization in the small intestine was more successful in the mixed infection model than the single infection models at 21 daf. In the rectal lumen and wall, the growth behaviour of the mixed infection was similar to that of the TcI group, although the total parasite number was lower. In the TcII group, no metacyclic trypomastigote forms were found. PCR analysis of the contents of each dissected region showed different genotype fractions in the mixed infection model, in which TcI seemed to be the predominant isolate. Conclusion: The different growth behaviour of the TcI and TcII isolates in single and mixed infection models demonstrated that possibly an intraspecific factor modulates parasitic development in the intestine of R. prolixus

Humoral responses in Rhodnius prolixus: bacterial feeding induces differential patterns of antibacterial activity and enhances mRNA levels of antimicrobial peptides in the midgut

Made available in DSpace on 2015-05-04T17:07:31Z (GMT). No. of bitstreams: 2 license.txt: 1914 bytes, checksum: 7d48279ffeed55da8dfe2f8e81f3b81f (MD5) cecilia_vieiraetal_IOC-2104.pdf: 1062015 bytes, checksum: e8dde228f0bdd6c4004641893450e0cb (MD5) Previous issue date: 2014Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica e Fisiologia de Insetos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica e Fisiologia de Insetos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica e Fisiologia de Insetos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica e Fisiologia de Insetos. Rio de Janeiro, RJ, Brasil.Universidade Federal Fluminense (UFF).Instituto de Biologia. Departamento de Biologia Geral. Laboratório de Biologia de Insetos. Niterói, RJ, Brasil.Universidade Federal Fluminense (UFF).Instituto de Biologia. Departamento de Biologia Geral. Laboratório de Biologia de Insetos. Niterói, RJ, Brasil / Swansea University. College of Science. Swansea, Wales, UK.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica e Fisiologia de Insetos. Rio de Janeiro, RJ, Brasil / Instituto Nacional de Entomologia Molecular (INCT-EM). Departamento de Entomologia Molecular, Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica e Fisiologia de Insetos. Rio de Janeiro, RJ, Brasil / Instituto Nacional de Entomologia Molecular (INCT-EM). Departamento de Entomologia Molecular, Rio de Janeiro, RJ, Brasil.Background The triatomine, Rhodnius prolixus, is a major vector of Trypanosoma cruzi, the causative agent of Chagas disease in Latin America. It has a strictly blood-sucking habit in all life stages, ingesting large amounts of blood from vertebrate hosts from which it can acquire pathogenic microorganisms. In this context, the production of antimicrobial peptides (AMPs) in the midgut of the insect is vital to control possible infection, and to maintain the microbiota already present in the digestive tract. Methods In the present work, we studied the antimicrobial activity of the Rhodnius prolixus midgut in vitro against the Gram-negative and Gram-positive bacteria Escherichia coli and Staphylococcus aureus, respectively. We also analysed the abundance of mRNAs encoding for defensins, prolixicin and lysozymes in the midgut of insects orally infected by these bacteria at 1 and 7 days after feeding. Results Our results showed that the anterior midgut contents contain a higher inducible antibacterial activity than those of the posterior midgut. We observed that the main AMP encoding mRNAs in the anterior midgut, 7 days after a blood meal, were for lysozyme A, B, defensin C and prolixicin while in the posterior midgut lysozyme B and prolixicin transcripts predominated. Conclusion Our findings suggest that R. prolixus modulates AMP gene expression upon ingestion of bacteria with patterns that are distinct and dependent upon the species of bacteria responsible for infection

Isolation and molecular characterization of a major hemolymph serpin from the triatomine, Panstrongylus megistus

Made available in DSpace on 2015-06-08T14:01:47Z (GMT). No. of bitstreams: 2 license.txt: 1914 bytes, checksum: 7d48279ffeed55da8dfe2f8e81f3b81f (MD5) richard_valenteetal_IOC_2014.pdf: 1457087 bytes, checksum: fae84319b3b51565ca75569b60621e0c (MD5) Previous issue date: 2014Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Doenças Parasitárias. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica e Fisiologia de Insetos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Toxinologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Proteômica e Engenharia de Proteínas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Toxinologia. Rio de Janeiro, RJ, Brasil.Universidade Federal Fluminense. Laboratório de Biologia de Insetos. Niterói, Brasil.Universidade Federal Fluminense. Laboratório LABIEMol. Niterói, RJ, Brasil.Universidade Federal Fluminense. Laboratório LABIEMol. Niterói, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica e Fisiologia de Insetos. Rio de Janeiro, RJ, Brasil.Universidade Federal Fluminense. Laboratório de Biologia de Insetos. Niterói, Brasil / Swansea University. College of Science. Department od Biosciences. Wales, UK.Universidade Federal Fluminense. Laboratório de Biologia de Insetos. Niterói, Brasil.Background: Chagas disease kills 2.5 thousand people per year of 15 million persons infected in Latin America. The disease is caused by the protozoan, Trypanosome cruzi, and vectored by triatomine insects, including Panstrongylus megistus, an important vector in Brazil. Medicines treating Chagas disease have unpleasant side effects and may be ineffective, therefore, alternative control techniques are required. Knowledge of the T. cruzi interactions with the triatomine host needs extending and new targets/strategies for control identified. Serine and cysteine peptidases play vital roles in protozoan life cycles including invasion and entry of T. cruzi into host cells. Peptidase inhibitors are, therefore, promising targets for disease control. Methods: SDS PAGE and chromatograpy detected and isolated a P. megistus serpin which was peptide sequenced by mass spectrometry. A full amino acid sequence was obtained from the cDNA and compared with other insect serpins. Reverse transcription PCR analysis measured serpin transcripts of P. megistus tissues with and without T. cruzi infection. Serpin homology modeling used the Swiss Model and Swiss-PDB viewer programmes. Results: The P. megistus serpin (PMSRP1) has a ca. 40 kDa molecular mass with 404 amino acid residues. A reactive site loop contains a highly conserved hinge region but, based on sequence alignment, the normal cleavage site for serine proteases at P1-P1′ was translocated to the putative position P4′-P5′. A small peptide obtained corresponded to the C-terminal 40 amino acid region. The secondary structure of PMSRP1 indicated nine α-helices and three β-sheets, similar to other serpins. PMSRP1 transcripts occurred in all tested tissues but were highest in the fat body and hemocytes. Levels of mRNA encoding PMSRP1 were significantly modulated in the hemocytes and stomach by T. cruzi infection indicating a role for PMSRP1 in the parasite interactions with P. megistus. Conclusions: For the first time, a constitutively expressed serpin has been characterized from the hemolymph of a triatomine. This opens up new research avenues into the roles of serine peptidases in the T. cruzi/P. megistus association. Initial experiments indicate a role for PMSRP1 in T. cruzi interactions with P. megistus and will lead to further functional studies of this molecule

Rhodnius prolixus interaction with Trypanosoma rangeli: modulation of the immune system and microbiota population

Submitted by sandra infurna ([email protected]) on 2016-05-11T11:06:19Z No. of bitstreams: 1 cecilia_vieira_etal_IOC_2015.pdf: 1447403 bytes, checksum: 9b50113b9471461d6d9b11f81121be77 (MD5)Approved for entry into archive by sandra infurna ([email protected]) on 2016-05-11T13:05:36Z (GMT) No. of bitstreams: 1 cecilia_vieira_etal_IOC_2015.pdf: 1447403 bytes, checksum: 9b50113b9471461d6d9b11f81121be77 (MD5)Made available in DSpace on 2016-05-11T13:05:36Z (GMT). No. of bitstreams: 1 cecilia_vieira_etal_IOC_2015.pdf: 1447403 bytes, checksum: 9b50113b9471461d6d9b11f81121be77 (MD5) Previous issue date: 2015Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica e Fisiologia de Insetos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica e Fisiologia de Insetos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica e Fisiologia de Insetos. Rio de Janeiro, RJ, Brasil.Universidade Federal Rural do Rio de Janeiro (UFRRJ). Instituto de Florestas. Departamento de Ciências Ambientais. Seropédica, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica e Fisiologia de Insetos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica e Fisiologia de Insetos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica e Fisiologia de Insetos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica e Fisiologia de Insetos. Rio de Janeiro, RJ, Brasil / Instituto Nacional de Entomologia Molecular (INCT-EM). Departamento de Entomologia Molecular. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica e Fisiologia de Insetos. Rio de Janeiro, RJ, Brasil / Instituto Nacional de Entomologia Molecular (INCT-EM). Departamento de Entomologia Molecular. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica e Fisiologia de Insetos. Rio de Janeiro, RJ, Brasil / Instituto Nacional de Entomologia Molecular (INCT-EM). Departamento de Entomologia Molecular. Rio de Janeiro, RJ. Brasil.Background: Trypanosoma rangeli is a protozoan that infects a variety of mammalian hosts, including humans. Its main insect vector is Rhodnius prolixus and is found in several Latin American countries. The R. prolixus vector competence depends on the T. rangeli strain and the molecular interactions, as well as the insect’s immune responses in the gut and haemocoel. This work focuses on the modulation of the humoral immune responses of the midgut of R. prolixus infected with T. rangeli Macias strain, considering the influence of the parasite on the intestinal microbiota. Methods: The population density of T. rangeli Macias strain was analysed in different R. prolixus midgut compartments in long and short-term experiments. Cultivable and non-cultivable midgut bacteria were investigated by colony forming unit (CFU) assays and by 454 pyrosequencing of the 16S rRNA gene, respectively. The modulation of R. prolixus immune responses was studied by analysis of the antimicrobial activity in vitro against different bacteria using turbidimetric tests, the abundance of mRNAs encoding antimicrobial peptides (AMPs) defensin (DefA, DefB, DefC), prolixicin (Prol) and lysozymes (LysA, LysB) by RT-PCR and analysis of the phenoloxidase (PO) activity. Results: Our results showed that T. rangeli successfully colonized R. prolixus midgut altering the microbiota population and the immune responses as follows: 1 - reduced cultivable midgut bacteria; 2 - decreased the number of sequences of the Enterococcaceae but increased those of the Burkholderiaceae family; the families Nocardiaceae, Enterobacteriaceae and Mycobacteriaceae encountered in control and infected insects remained the same; 3 - enhanced midgut antibacterial activities against Serratia marcescens and Staphylococcus aureus; 4 - downregulated LysB and Prol mRNA levels; altered DefB, DefC and LysA depending on the infection (short and longterm); 5 - decreased PO activity. Conclusion: Our findings suggest that T. rangeli Macias strain modulates R. prolixus immune system and modifies the natural microbiota composition

Selbst berichtete Infektionen in der NAKO Gesundheitsstudie: Einordnung in die gegenwärtige Forschungslandschaft

Infectious diseases continue to play an important role for disease perception, health-economic considerations and public health in Germany. In recent years, infectious diseases have been linked to the development of non-communicable diseases. Analyses of the German National Cohort (GNC) may provide deeper insights into this issue and pave the way for new targeted approaches in disease prevention. Objectives The aim was to describe the tools used to assess infectious diseases and to present initial data on infectious disease frequencies, as well as to relate the GNC assessment tools to data collection methods in other studies in Germany. Methods As part of the baseline examination, questions regarding infectious diseases were administered using both an interview and a self-administered touchscreen questionnaire. Data from the initial 101,787 GNC participants were analysed. Results In the interview, 0.2% (HIV/AIDS) to 8.6% (shingles) of respondents reported ever having a medical diagnosis of shingles, postherpetic neuralgia (in cases where shingles was reported), hepatitis B/C, HIV/AIDS, tuberculosis or sepsis if treated in hospital. In the questionnaire, 12% (cystitis) to 81% (upper respiratory tract infections) of respondents reported having experienced at least one occurrence of upper or lower respiratory tract infections, gastrointestinal infections, cystitis or fever within the past 12 months. Outlook The cross-sectional analyses of data and tools presented here – for example on determinants of susceptibility to self-reported infections – can be anticipated from the year 2021 onward. Beyond that, more extensive research into infectious disease epidemiology will follow, particularly once analyses of GNC biological materials have been performed