Genetic structure and expression of the surface glycoprotein GP82, the main adhesin of Trypanosoma cruzi metacyclic trypomastigotes.

T. cruzi improves the likelihood of invading or adapting to the host through its capacity to present a large repertoire of surface molecules. The metacyclic stage-specific surface glycoprotein GP82 has been implicated in host cell invasion. GP82 is encoded by multiple genes from the trans-sialidase superfamily. GP82 shows a modular organization, with some variation of N-terminal region flanking a conserved central core where the binding sites to the mammalian cell and gastric mucin are located. The function of GP82 as adhesin in host cell invasion process could expose the protein to an intense conservative and selective pressure. GP82 is a GPI-anchored surface protein, synthesized as a 70 kDa precursor devoid of N-linked sugars. GPI-minus variants accumulate in the ER indicating that GPI anchor acts as a forward transport signal for progressing along the secretory pathway as suggested for T. cruzi mucins. It has been demonstrated that the expression of GP82 is constitutive and may be regulated at post-transcriptional level, for instance, at translational level and/or mRNA stabilization. GP82 mRNAs are mobilized to polysomes and consequently translated, but only in metacyclic trypomastigotes. Analysis of transgenic parasites indicates that the mechanism regulating GP82 expression involves multiple elements in the 3'UTR.

Regulatory elements in the 3' untranslated region of the GP82 glycoprotein are responsible for its stage-specific expression in Trypanosoma cruzi metacyclic trypomastigotes.

Gene expression in Trypanosoma cruzi is regulated at the post-transcriptional level and cis-acting elements present in the 3' untranslated region (3'UTR) play an important role by interacting with regulatory proteins. Previous studies demonstrated that the GP82 surface glycoprotein, which is involved in host cell invasion, is up-regulated in the infective metacyclic trypomastigote form, and that GP82 mRNA half-life is longer in this form compared to the non-infective epimastigote form. Here, we demonstrate that the 3'UTR of the GP82 transcript is involved in this developmental regulation, promoting higher expression of the green fluorescent protein (GFP) reporter in metacyclic trypomastigotes than in epimastigotes. A series of stepwise deletions in the 3'UTR was created and results suggest that the mechanism regulating GP82 expression involves multiple elements in the 3'UTR.

Utilização de um meio cromogênico e da técnica de semi-nested PCR para identificação de espécies de Candida / Use of chromogenic medium and semi-nested PCR-based assay to identify Candida species

Nesse trabalho, o meio cromogênico CHROMagar Candida e a técnica de semi-nested PCR (sn-PCR) foram comparados quanto a sua capacidade de identificar a espécie de 52 isolados clínicos de Candida sp. Com o emprego do meio cromogênico, 39 (75%) isolados foram identificados presuntivamente como C. albicans (n = 22), C. glabrata (n = 9), C. tropicalis (n = 5) e C. krusei (n = 3). Treze isolados (25%) não puderam ser identificados. Por meio da técnica de sn-PCR, que se baseia na amplificação de uma região do cluster gênico do RNA ribossomal (5.8S – 28S), 43 (83%) isolados foram identificados como C.albicans (n = 24), C. glabrata (n = 11), C. tropicalis (n = 5), C. parapsilosis (n = 3) e nove isolados não puderam ser identificados em nível de espécie. Entre os 52 isolados analisados, 34 (65,4%) apresentaram resultados concordantes pelos dois métodos, 12 (23,1%) apresentaram resultados discrepantes, e 6 (11,5%) não puderam ser identificadas por nenhuma das duas metodologias. Os resultados indicam que ambas as técnicas apresentam limitações, mas o método de sn-PCR é mais adequado que o meio cromogênico para a identificação de espécies do gênero Candida, devido ao menor número de isolados que não puderam ser identificados.