Trans-spliced Heat Shock Protein 90 Modulates Encystation in Giardia lamblia

Background: Hsp90 from Giardia lamblia is expressed by splicing of two independently transcribed RNA molecules, coded by genes named HspN and HspC located 777 kb apart. The reasons underlying such unique trans-splicing based generation of GlHsp90 remain unclear. Principle Finding: In this study using mass-spectrometry we identify the sequence of the unique, junctional peptide contributed by the 5' UTR of HspC ORF. This peptide is critical for the catalytic function of Hsp90 as it harbours an essential ``Arg'' in its sequence. We also show that full length GlHsp90 possesses all the functional hall marks of a canonical Hsp90 including its ability to bind and hydrolyze ATP. Using qRT-PCR as well as western blotting approach we find the reconstructed Hsp90 to be induced in response to heat shock. On the contrary we find GlHsp90 to be down regulated during transition from proliferative trophozoites to environmentally resistant cysts. This down regulation of GlHsp90 appears to be mechanistically linked to the encystation process as we find pharmacological inhibition of GlHsp90 function to specifically induce encystation. Significance: Our results implicate the trans-spliced GlHsp90 from Giardia lamblia to regulate an essential stage transition in the life cycle of this important human parasite.

Distribution of rDNA in the nucleus of Giardia lamblia - Detection by Ag-I silver stain

OBJECTIVE: To determine whether rDNA of Giardia lamblia forms a nucleolus organizer region (NOR)-like structure and is in a very primitive state. STUDY DESIGN: G lamblia was used as the experimental animal, with Euglena gracilis as the control. The distribution was demonstrated indirectly by the modified Ag-I silver technique, which can specifically indicate the NOR under both light and electron microscopes. RESULTS: In the ultrathin sections of silver-stained Euglena cells, all the silver grains were concentrated in the fibrosa of the nucleolus, while no grains found in the cytoplasm, nucleoplasm, condensed chromosomes or pars granulosa of the nucleus. In the silver-stained Giardia cells, no nucleolus was found; a few silver grains were scattered in the nucleus but were not concentrated in any specific region. CONCLUSION: The distribution of silver grains in G lamblia showed that the transcription of rDNA occurs inside the nucleus, though no nucleolus is present. It is possible that chromosomes are in a very primitive state in diplomonad cells; as each chromosome has few prRNA genes, the transcription is independent of a nucleolus. These results imply that the rDNA of Giardia does not form a NOR-like structure and seems to represent a very primitive state in the evolution of the nucleolus.

The intraspecific difference of the triose phosphate isomerase ( tim) gene from Giardia lamblia

目的　探讨蓝氏贾第鞭毛虫 (Giardialamblia)磷酸丙糖异构酶基因种内差异。方法　提取虫体总DNA ,对所有虫株磷酸丙糖异构酶 (tim)基因部分片段进行PCR扩增。测定序列后 ,用简约法和NJ法构建系统树进行系统发育分析。结果　共有 12 4个位点存在变异 (占所有测定序列中的 2 3% ) ,且大多数为发生在密码子的同义突变。两种构树方法所得二树的分枝结构相似 ,均将受试的 16株蓝氏贾第虫分为明显的两组。结论　宿主及地理因素对蓝氏贾第虫群体的遗传多样性影响不大。在DNA分子进化水平上 ,自然选择的影响十分显著。可将tim基因作为蓝氏贾第虫群体遗传结构一个十分有效的遗传标记。

DNA sequence analysis of the triose phosphate isomerase gene from isolates of Giardia lamblia

Objective To confirm the genetic relation between Giardia lamblia (G. lamblia) isolates from different geographic regions of China and other countries. Methods Genomic DNA were extracted from the trophozoites or cysts of Giardia lamblia. The triose phosphate isomerase (tim) gene was amplified using polymerase chain reaction (PCR) technique. PCR products were digested with endonuclease and sequenced. The data of sequencing were analyzed with the DNAstar software and compared with that of the isolates acquired from GenBank. Results Of nine isolates of Giardia lamblia from China (C1, C2, CH2 and CH3), Cambodia (CAM), Australia (A1 and A2) and America (BP and CDC), respectively, 3 (A1, A2 and CAM) fit into Group 1 (WB), 2 (CH2 and CH3) into Group 2, and 4 (C1, C2, BP and CDC) into Group 3 (GS). The results confirmed the genetic relatedness of G. lamblia isolates from all over the world. Conclusion Genotyping isolates of G. Lamblia provides important information for establishing the phylogenetic relationship or for the epidemiological evaluation of the spreading of this organism.

Identification, characteristic and phylogenetic analysis of type II DNA topoisomerase gene in Giardia lamblia

The genes encoding type II DNA topoisomerases were investigated in Giardia lamblia genome, and a type IIA gene, GlTop 2 was identified. It is a single copy gene with a 4476 by long ORF without intron. The deduced amino acid sequence shows strong homology to eukaryotic DNA Top 2. However, some distortions were found, such as six insertions in the ATPase domain and the central domain, a similar to 100 as longer central domain; a similar to 200 as shorter C-terminal domain containing rich charged residues. These features revealed by comparing with Top 2 of the host, human, might be helpful in exploiting drug selectivity for antigiardial therapy. Phylogenetic analysis of eukaryotic enzymes showed that kinetoplastids, plants, fungi, and animals were monophyletic groups, and the animal and fungi lineages shared a more recent common ancestor than either did with the plant lineage; microsporidia grouped with fungi. However, unlike many previous phylogenetic analyses, the "amitochondriate" G. lamblia was not the earliest branch but diverged after mitochondriate kinetoplastids in our trees. Both the finding of typical eukaryotic type IIA topoisomerase and the phylogenetic analysis suggest G. lamblia is not possibly as primitive as was regarded before and might diverge after the acquisition of mitochondria. This is consistent with the recent discovery of mitochondrial remnant organelles in G. lamblia.

Genome-wide computational identification of microRNAs and their targets in the deep-branching eukaryote Giardia lamblia

Using a combined computational program. we identified 50 potential microRNAs (miRNAs) in Giardia lamblia. one of the most primitive unicellular eukaryotes. These miRNAs are unique to G. lamblia and no homologues have been found in other organisms; miRNAs.

Gene duplication in the genome of parasitic Giardia lamblia

Background: Giardia are a group of widespread intestinal protozoan parasites in a number of vertebrates. Much evidence from G. lamblia indicated they might be the most primitive extant eukaryotes. When and how such a group of the earliest branching unicellular eukaryotes developed the ability to successfully parasitize the latest branching higher eukaryotes (vertebrates) is an intriguing question. Gene duplication has long been thought to be the most common mechanism in the production of primary resources for the origin of evolutionary novelties. In order to parse the evolutionary trajectory of Giardia parasitic lifestyle, here we carried out a genome-wide analysis about gene duplication patterns in G. lamblia. Results: Although genomic comparison showed that in G. lamblia the contents of many fundamental biologic pathways are simplified and the whole genome is very compact, in our study 40% of its genes were identified as duplicated genes. Evolutionary distance analyses of these duplicated genes indicated two rounds of large scale duplication events had occurred in G. lamblia genome. Functional annotation of them further showed that the majority of recent duplicated genes are VSPs (Variant-specific Surface Proteins), which are essential for the successful parasitic life of Giardia in hosts. Based on evolutionary comparison with their hosts, it was found that the rapid expansion of VSPs in G. lamblia is consistent with the evolutionary radiation of placental mammals. Conclusions: Based on the genome-wide analysis of duplicated genes in G. lamblia, we found that gene duplication was essential for the origin and evolution of Giardia parasitic lifestyle. The recent expansion of VSPs uniquely occurring in G. lamblia is consistent with the increment of its hosts. Therefore we proposed a hypothesis that the increment of Giradia hosts might be the driving force for the rapid expansion of VSPs.

最原始的现存真核生物Giardia lamblia核基质的检测

国家自然科学基金

Actividad biológica de factores difusibles de probióticos (fdp) sobre Giardia Lamblia y Trichomonas vaginalis bajo condiciones axénicas in vitro.

Tesis (Maestría en Ciencias con Acentuación en Microbiología) UANL, 2011.

Efecto de los extractos de syzygium aromaticum sobre el crecimiento de entamoeba histolytica, giardia lamblia y trichonomas vaginalis.

Tesis (Doctor en Ciencias con acentuación en Química de Productos Naturales) UANL, 2014.

Infecção por Giardia lamblia (Kunstler, 1882) em cães (Canis familiaris) determinada através do método de Faust e Cols. (1939) e da técnica de coloração da auramina, no município de Canoas, RS, Brasil.

Giardia lamblia é um protozoário que acomete mais comumente animais jovens e que convivem em grupos. Apesar da alta prevalência, nem todos animais apresentam a doença clínica. Mesmo assim, a giardíase tem importância epidemiológica por possuir um elevado potencial zoonótico. O presente estudo teve como objetivo determinar a freqüência de Giardia lamblia em cães no município de Canoas, RS, Brasil, através do Método de Faust e cols. (1939) e da Técnica de Coloração da Auramina. Os grupos experimentais foram divididos de acordo com a procedência e o sexo. Das 332 amostras analisadas com o Método de Faust e cols, a estimativa em ponto da freqüência obtida foi de 34,04%, podendo variar de 28,95 a 39,13%, dentro de um intervalo de confiança de 95%. Destas amostras, 40,96% foram positivas em animais de canil e 27,11% de rua. O Teste Exato de Fisher aplicado a esses dados revelou existir uma diferença significativa (p = 0,0107) entre as variáveis resultado e procedência. A variável sexo, neste método não apresentou diferença significativa em relação ao resultado (p = 0,8162) totalizando 33,11% de machos positivos e 34,08% de fêmeas infectadas com o parasita. Das 147 amostras realizadas com a Técnica de Coloração da Auramina, 23 foram positivas, totalizando 15,65%. A análise estatística através do Teste McNemar revelou existir diferença significativa entre as duas técnicas (p = 0,0004). O valor Kappa foi igual a 0,07, considerado como um grau de concordância fraco. Os resultados encontrados neste estudo nos permitem afirmar que o Método de Faust e cols. foi o mais adequado para o diagnóstico na infecção por Giardia lamblia, entre os métodos analisados.

Detection of anti-Giardia lamblia serum antibody among children of day care centers

OBJETIVOS: Detectar anticorpos séricos anti-Giardia lamblia entre crianças atendidas em creches e estimar a freqüência de infecção por Giardia lamblia em área endêmica. MÉTODOS: Foram coletadas três amostras de fezes de cada uma das 147 crianças de três creches da rede municipal de Botucatu, SP, com idade variando de 0 a 6 anos, e as amostras foram processadas pelos métodos de sedimentação espontânea e flutuação pelo sulfato de zinco. Amostras de sangue foram obtidas da polpa digital, coletadas em papel de filtro e testadas pelos métodos de imunofluorescência indireta (IFI) e de reação imunoenzimática (Elisa) para pesquisa de IgG anti-Giardia. RESULTADOS E CONCLUSÕES: de um total de 147 crianças, 93 (63,3%) apresentaram cistos de Giardia nas fezes. Dos 147 eluatos testados, 93 (63,3%) e 100 (68%) foram positivos para Giardia em IFI e em Elisa, respectivamente. A sensibilidade de IFI foi de 82% e de Elisa, 72%. Contudo, Elisa foi menos específica (39%) do que IFI (70%). A imunofluorescência indireta apresentou maior concordância com o exame de fezes do que Elisa.

INTRAGASTRIC INFECTION OF CONVENTIONAL AND GERM-FREE MICE WITH GIARDIA-LAMBLIA

The effects of experimental infection with Giardia lamblia were studied in 30-day old conventional and germfree CFW mice (7 animals in each group) of both sexes. Cysts were observed in the feces of both groups 6 to 7 days after intragastric infection of each animal with about 2.5 x 10(5) G. lamblia trophozoites. Fecal cyst level was statistically higher in germfree mice (about 10(5) cysts/g feces) when compared with the conventional group (about 10(4) cysts/g feces). The peak of infection in the conventional group apparently occurred on the 10th day after infection as indicated by an increase of fecal weight and by histopathological examination. Intense infiltration of the lamina propria and high reactional hyperplasia of the lymphoid component were observed in the conventional group. There was no infiltration or hyperplasia in germfree infected mice and fecal weight was relatively constant throughout the experiment. These results suggest that, as is the case for other intestinal pathogenic protozoa, the intestinal microflora is indispensable for the expression of the pathogenicity but not for the multiplication of G. lamblia.

EFFECTS OF PROTEIN-DEFICIENCY AND NATURAL INTESTINAL INFECTION WITH GIARDIA-LAMBLIA ON JEJUNAL INTRAEPITHELIAL LYMPHOCYTES IN RATS OF DIFFERENT AGES

1. The interaction between experimental protein deprivation and natural intestinal infection by Giardia lamblia was studied in terms of its effects on the intraepithelial lymphocyte (IEL) population and morphology of the jejunal mucosa of rats of different ages.2. Young, adult and old male Wistar rats received a protein-deficient diet (2% casein) or a control diet (20% casein) for 42 days. Mucosal height and the number of lymphocytes located among 500 consecutive epithelial cells (EC) along the villi or crossing the basement membrane were determined in PAS-stained jejunal fragments.3. The number of IEL increased progressively with animal age, from 14 to 25 per 100 epithelial cells, with significant differences between age ranges. However, the number of IEL did not differ between control and protein-deficient rats in any of the age groups. The proportion of lymphocytes crossing the basement membrane was approximately two-fold greater in young (2.8/100 EC) and adult (5.8/100 EC) protein-deficient animals than in their respective controls (1.6 and 2.8/100 EC). The intensity of parasite colonization was moderate, from 3 to 5/100 EC and did not differ between groups. The pattern of morphologic changes of jejunal mucosa in protozoal infection did not differ between control and protein-deficient animals in any of the three age groups.4. We conclude that intestinal infection with Giardia lamblia probably stimulated the local immune response, masking the reduction of the IEL population induced by protein deficiency. The increase in lymphocyte numbers with age may be related to prolonged antigenic stimulation promoted by infection.