State of the Art and Future Directions of <em>Cryptosporidium</em> spp.

Cryptosporidium species are protozoan parasites that infect epithelium surfaces in gastrointestinal and respiratory tracts of humans and a range of animals worldwide. Cryptosporidiosis has been associated with considerable morbidity and, under certain circumstances, mortality. Humans can acquire it by consuming food and drink containing oocysts, which have been recognised as a major cause for diarrhoeal disease. The ubiquitousness of the infective oocyst, its resilience to environmental pressures, and the low dose of oocyst exposure needed for infection amplify to outbreaks of Cryptosporidium traced to drinking and recreational water. Unlike in developing countries where lack of sustained access to safe water creates tremendous burdens of Cryptosporidium diarrhoea, this scenario is aggravated due to limited diagnosis and therapeutics. However, over the past few decades, growing information on Cryptosporidium genomes have allowed novel insight into the host-parasite relationship. Future field research on potential tools will focus on biology-derived parasite products applicable to drugs and diagnosis. This chapter reviews available data on biology, transmission, life cycle, diagnosis, genome, and a few but important progresses in the field of cryptosporidiosis

Identification of novel genes and proteoforms in Angiostrongylus costaricensis through a proteogenomic approach

RNA sequencing (RNA-Seq) and mass-spectrometry-based proteomics data are often integrated in proteogenomic studies to assist in the prediction of eukaryote genome features, such as genes, splicing, single-nucleotide (SNVs), and single-amino-acid variants (SAAVs). Most genomes of parasite nematodes are draft versions that lack transcript- and protein-level information and whose gene annotations rely only on computational predictions

Cisteína-proteinases em promastigotas de Leishmania (Viannia) braziliensis

Made available in DSpace on 2016-04-04T12:35:28Z (GMT). No. of bitstreams: 2 karina_rebello_ioc_mest_2008.pdf: 6872283 bytes, checksum: a0cb112c96e6862c0ff341afeb28dbf5 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2008Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, BrasilNo presente trabalho foram detectadas cisteína-proteinases (CPs) em promastigotas infectivas de Leishmania (Viannia). braziliensis. A estratégia de purificação consistiu na associação do método de extração por Triton X-114 com cromatografia em coluna de Concanavalina A-Sepharose, seguida por outra de DEAE-Sephacel. No ensaio das cromatografias, observamos um pico majoritário de atividade enzimática na presença do substrato pEFLpNan (165 x 10³² \03BCM de pNan/minuto) para cerca de 10¹0 parasitas, coincidentes com o pico majoritário da proteína eluído da coluna de troca iônica. A análise por SDS-PAGE do material eluído da coluna de troca iônica mostrou quatro principais bandas de proteínas com massas moleculares relativas de 63, 43, 30 e 27 kDa. Os ensaios da atividade enzimática após eletroforese mostraram que as bandas de 63 kDa e 43 kDa, hidrolisam substratos como gelatina em pH 7,0 e são sensíveis à presença de E-64. Além disso, as duas enzimas são capazes de hidrolisar o substrato pEFLpNan: 63 kDa (2,2 ± 0,3 \03BCM de pNan/minuto) e 43 kDa (0,05 ± 0,2 \03BCM de pNan/minuto), e são inibidas por 10\03BCM E-64 (47 % e 36 % respectivamente). Os ensaios de reconhecimento imunológico utilizando um anti-soro policlonal específico contra cisteína-proteinase B [anti-CPB de L. (L.) mexicana] revelaram que as enzimas de 63 kDa são reconhecidas por este anti-soro. Os experimentos de aglutinação, citometria de fluxo e imunocitoquímica utilizando esse mesmo antisoro revelaram que homólogos de CPBs estão localizados na superfície da membrana de promastigotas. Além disso, a incubação dos promastigotas com fosfolipase C (PLC) reduziu o número de células positivas pra os homólogos de CPB. Os anti-soros anti-CRD (cross reactive determinat) e anti-CPB reconhecem bandas de 63kDa e 43 kDa do sobrenadante das células tratadas com PLC em ensaios de immunoblotting sugerindo que isoformas destas proteínas são ancoradas por glicosilfostatidilinositol (GP (GPI) à membrana plasmática. Também observamos que os homólogos de CPBs são presos a membrana por âncora GPI e se concentram em plataformas lipídicas. Nós observamos que as proteínas homólogas a CPB não permanecem estáveis na superfície da membrana quando os parasitas são mantidos em culturas sucessivas, assim como a atividade enzimática total sobre o substrato pEFLpNan é alterada. Mostramos ainda através da técnica RT-PCR em tempo real um aumento da transcrição de cpb de L. (V.) braziliensis quando os parasitas foram submetidos a culturas sucessivas. De uma forma geral os dados apresentados suportam a hipótese de que o parasita estudado apresentam CPs de membrana ativas, além de homólogos de CPB intracelulares.It was detected cysteine-pro teinases (CPs) in infective Leishmania (Viannia) braziliensis promastigotes . The purification strategy c onsisted of an association of Triton X-114 extraction method with chromatography in Concanavalin A-Sepharose column, followed by chromatography in DEAE-Sephacell column. In the assay pf chromatographic fractions, we observed a peak of enzymatic activity against the pEFLpNan substrate (165 x 10 -32 μM of pNan/minute) in over 10 10 parasites, coincident with the ma jor protein peak eluted from the column. SDS-PAGE analysis of the material eluted from the ionic exchange column showed four main protein bands with relative molecular mass of 63 , 43, 30 and 27 kDa. Gelatin-SDS-PAGE assays indicated that the 43kDa and the 63kDa bands can hydrolyze gelatin at neutral pH and are sensitive to E-64. Also, both enzymes can hydrolyze pEFLpNan substrate: 63 kDa (2,2 ± 0,3 μM of pNan/minute) and 43 kDa (0,05 ± 0,2 μM of pNan/minute) bei ng both inhibited by E-64 (47 % and 36 % inhibition, respectively) . Immunological recognition assays , with a specific polyclonal antibody against CPB from L. (L.) mexicana , showed that bands of 63 kDa and 43 kDa are recognized in the fractions of the ionic exchange column. Aggl utination, flow cytometry and immunocytochemistry assays performed with an ti-CPB antiserum revealed that homologous of CPB are located on the promastigote membrane surface. Moreover, the incubation of promastigotes with phospholipase C reduced th e number of CPBs homologues-positive cells. Both anti-cross-reacting determinant (CRD) a nd anti-CPB antisera recognized 63kDa and 43kDa bands in the supernatant of phospholipase C-treat ed cells, suggesting that isoforms of these proteins are attached to the plasma membra ne by glycosylphosphatidylinositol (GPI)-anchors. Also, our data suggest that GPI-anchored CPBs are present in the detergent - resistant lipid rafts. We observed that the CPB homologues do not remain stable on the membrane surface when the parasites are maintained under successive cultur es; also, the total enzymatic activity over the substrate pEFLpNan is altered. We add itionally showed by real-time RT-PCR that L. (V). braziliensis cpb genes are active and their relative expression is increased throughout the successive cultures. Thus, the presented data support the hypothesis of that studied parasite presents CPs of membrane active, beyond homologous of intracellular CPB

Comprehensive proteomic profiling of adult Angiostrongylus costaricensis, a human parasitic nematode

This research was supported by Brazilian grants from Fiocruz (PAPES V and PDTIS), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). We thank the staff of the Toxinology and Pathology Laboratories-IOC/Fiocruz for excellent technical assistance. We gratefully acknowledge Dr. André Teixeira S. Ferreira and Dr. Richard H. Valente (Toxinology Laboratory-IOC/Fiocruz) for their assistance with the mass spectrometric analysis. We also thank Monique R. O. Trugilho (Toxinology Laboratory-IOC/Fiocruz) for helping with 2-DE gel image analysis. We are grateful to Heloisa M. N. Diniz and Cristina S. Ferreira (Image Production and Treatment Service - IOC/Fiocruz) for processing the figures and creating the life cycle cartoon. KMR thanks CAPES for her PhD fellowship. PCC thanks CAPES/Fiocruz 30–2006 for his fellowship.Submitted by Gentil Jeorgina ([email protected]) on 2011-12-13T15:57:33Z No. of bitstreams: 1 Comprehensive proteomic profiling of adult Angiostrongylus.pdf: 1766325 bytes, checksum: b9dd765b435673e904949dbfcb4721fa (MD5)Made available in DSpace on 2011-12-13T15:57:33Z (GMT). No. of bitstreams: 1 Comprehensive proteomic profiling of adult Angiostrongylus.pdf: 1766325 bytes, checksum: b9dd765b435673e904949dbfcb4721fa (MD5) Previous issue date: 2011Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Toxinologia. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Patologia. Rio de Janeiro, RJ, Brasil / Proteomic Network, Rio de Janeiro, RJ, BrasilFundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Toxinologia. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Patologia. Rio de Janeiro, RJ, BrasilFundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Patologia. Rio de Janeiro, (RJ), BrasilFundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Toxinologia. Rio de Janeiro, (RJ), Brasil / Fundação Oswaldo Cruz. Centro de Desenvolvimento Tecnológico em Saúde, Rio de Janeiro, RJ, Brasil / Proteomic Network, Rio de Janeiro, RJ, BrasilFundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Toxinologia. Rio de Janeiro, RJ, Brasil / Proteomic Network, Rio de Janeiro, RJ, BrasilFundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Patologia. Rio de Janeiro, RJ, BrasilFundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Toxinologia. Rio de Janeiro, RJ, Brasil / Proteomic Network, Rio de Janeiro, RJ, BrasilAngiostrongylus costaricensis is a nematode helminth that causes an intestinal acute inflammatory process known as abdominal angiostrongyliasis, which is a poorly understood human disease occurring in Latin America. Our aim was to study the proteomic profiles of adult parasites focusing on immunogenic proteins. Total cellular extracts from both genders showed similar 2-DE profiles, with 60% of all protein spots focused between pH 5–7 and presenting molecular masses from 20.1 to 66 kDa. A total of 53 different dominant proteins were identified in our dataset and were mainly associated with the following over-represented Gene Ontology Biological Process terms: “macromolecule metabolic process”, “developmental process”, “response to stress”, and “biological regulation”. Female and male immunoblots showed similar patterns of reactive proteins. Immunoreactive spots identified by MALDI-PSD were found to represent heat shock proteins, a putative abnormal DAuer Formation family member, and galectins. To date, very few biochemical analyses have focused on the nematode Angiostrongylus costaricensis. As such, our results contribute to a better understanding of its biology and the mechanisms underlying the host–parasite relationship associated with this species. Moreover, our findings represent a first step in the search for candidate proteins for diagnostic assays and the treatment of this parasitic infection

Identification of Novel Genes and Proteoforms in Angiostrongylus costaricensis through a Proteogenomic Approach

RNA sequencing (RNA-Seq) and mass-spectrometry-based proteomics data are often integrated in proteogenomic studies to assist in the prediction of eukaryote genome features, such as genes, splicing, single-nucleotide (SNVs), and single-amino-acid variants (SAAVs). Most genomes of parasite nematodes are draft versions that lack transcript- and protein-level information and whose gene annotations rely only on computational predictions. Angiostrongylus costaricensis is a roundworm species that causes an intestinal inflammatory disease, known as abdominal angiostrongyliasis (AA). Currently, there is no drug available that acts directly on this parasite, mostly due to the sparse understanding of its molecular characteristics. The available genome of A. costaricensis, specific to the Costa Rica strain, is a draft version that is not supported by transcript- or protein-level evidence. This study used RNA-Seq and MS/MS data to perform an in-depth annotation of the A. costaricensis genome. Our prediction improved the reference annotation with (a) novel coding and non-coding genes; (b) pieces of evidence of alternative splicing generating new proteoforms; and (c) a list of SNVs between the Brazilian (Crissiumal) and the Costa Rica strain. To the best of our knowledge, this is the first time that a multi-omics approach has been used to improve the genome annotation of A. costaricensis. We hope this improved genome annotation can assist in the future development of drugs, kits, and vaccines to treat, diagnose, and prevent AA caused by either the Brazil strain (Crissiumal) or the Costa Rica strain

Leishmania (Viannia) braziliensis: Influence of successive in vitro cultivation on the expression of promastigote proteinases

Cysteine proteinases are an important virulence factor in Leishmania parasites. In this study we analyzed the cysteine proteinase expression of infective Leishmania (Viannia) braziliensis promastigotes, examining the expression induced by successive in vitro passages in culture. We observed that this parasite presents a decrease in its virulence over BALB/c macrophages, after successive passages in culture, but still they present proteinase activity, being capable of hydrolyzing the substrate pGlu-Phe-Leu-p Nitroanilide at pH 7.0. This proteinase activity also decreases in the course of the successive passages. Additionally, the decrease in the amount of CPB proteins following successive passages of promastigotes was verified by immunoblotting assays, using an anti-CPB antiserum. Real-time PCR assays were performed to assess the relative cpb expression when compared to a housekeeping gene in promastigote cDNA preparations from the first, fourth and seventh passages. Interestingly, the data indicate a relative increase in cpb gene transcripts as the promastigotes were maintained under in vitro culture: 2.2 times higher for fourth and 2.7 times higher for seventh passages when compared to the first passage. Thus, the information gathered here shows that the expression of cysteine proteinases is modified during in vitro cultivation of L. (V.) braziliensis promastigotes

Proteolytic activity in the adult and larval stages of the human roundworm parasite Angiostrongylus costaricensis

Angiostrongylus costaricensis is a nematode that causes abdominal angiostrongyliasis, a widespread human parasitism in Latin America. This study aimed to characterize the protease profiles of different developmental stages of this helminth. First-stage larvae (L1) were obtained from the faeces of infected Sigmodon hispidus rodents and third-stage larvae (L3) were collected from mollusks Biomphalaria glabrata previously infected with L1. Adult worms were recovered from rodent mesenteric arteries. Protein extraction was performed after repeated freeze-thaw cycles followed by maceration of the nematodes in 40 mM Tris base. Proteolysis of gelatin was observed by zymography and found only in the larval stages. In L3, the gelatinolytic activity was effectively inhibited by orthophenanthroline, indicating the involvement of metalloproteases. The mechanistic class of the gelatinases from L1 could not be precisely determined using traditional class-specific inhibitors. Adult worm extracts were able to hydrolyze haemoglobin in solution, although no activity was observed by zymography. This haemoglobinolytic activity was ascribed to aspartic proteases following its effective inhibition by pepstatin, which also inhibited the haemoglobinolytic activity of L1 and L3 extracts. The characterization of protease expression throughout the A. costaricensis life cycle may reveal key factors influencing the process of parasitic infection and thus foster our understanding of the disease pathogenesis