Immunodominant proteins ?-1 giardin and ?-giardin are expressed in both assemblages A and B of Giardia lamblia.
ABSTRACT: BACKGROUND: To date, eight assemblages of Giardia lamblia have been described, but only assemblages A and B are known to infect humans. Despite the fact that the genomic, biological, and clinical differences found between these two assemblages has raised the possibility that they may be considered different species, there is relatively limited information on their phenotypic differences. In the present study, we developed monoclonal antibodies against alpha-1 and beta giardin, two immunodominant proteins produced during G. lamblia infection, and studied their expression and localization in WB (assemblage A) and GS trophozoites (assemblage B). RESULTS: The polyclonal antibodies generated against WB trophozoites, particularly those recognizing intracellular proteins as well as the proteins present at the plasma membrane (variable-specific surface proteins), showed cross-reactivity with intracellular proteins in GS trophozoites. The use of monoclonal antibodies against beta giardin indicated ventral disc localization, particularly at the periphery in WB trophozoites. Interestingly, although beta giardin was also restricted to the ventral disc in GS trophozoites, the pattern of localization clearly differed in this assemblage. On the other hand, monoclonal antibodies against alpha-1 giardin showed plasma membrane localization in both assemblages with the bare area of GS trophozoites also being distinguished. Moreover, the same localization at the plasma membrane was observed in Portland-1 (Assemblage A) and in P15 (Assemblage E) trophozoites. CONCLUSIONS: We found differences in localization of the beta giardin protein between assemblages A and B, but the same pattern of localization of alpha-1 giardin in strains from Assemblages A, B and E. These findings reinforce the need for more studies based on phenotypic characteristics in order to disclose how far one assemblage is from the other.
Project description:A Giardia-specific protein family denominated as alpha-giardins, represents the major protein component, besides tubulin, of the cytoskeleton of the human pathogenic parasite Giardia lamblia. One of its members, alpha19-giardin, carries an N-terminal sequence extension of MGCXXS, which in many proteins serves as a target for dual lipid conjugation: myristoylation at the glycine residue after removal of the methionine and palmitoylation at the cysteine residue. As the first experimental evidence of a lipid modification, we found alpha19-giardin to be associated with the membrane fraction of disrupted trophozoites. After heterologous coexpression of alpha19-giardin with giardial N-myristoyltransferase (NMT) in Escherichia coli, we found the protein in a myristoylated form. Additionally, after heterologous expression together with the palmitoyl transferase Pfa3 in Saccharomyces cerevisiae, alpha19-giardin associates with the membrane of the main vacuole. Immunocytochemical colocalization studies on wild-type Giardia trophozoites with tubulin provide evidence that alpha19-giardin exclusively localizes to the ventral pair of the giardial flagella. A mutant in which the putatively myristoylated N-terminal glycine residue was replaced by alanine lost this specific localization. Our findings suggest that the dual lipidation of alpha19-giardin is responsible for its specific flagellar localization.
Project description:Giardia lamblia is a leading protozoan cause of diarrheal disease worldwide, yet preventive medical strategies are not available. A crude veterinary vaccine has been licensed for cats and dogs, but no defined human vaccine is available. We tested the vaccine potential of three conserved antigens previously identified in human and murine giardiasis, ?1-giardin, ?-enolase, and ornithine carbamoyl transferase, in a murine model of G. lamblia infection. Live recombinant attenuated Salmonella enterica Serovar Typhimurium vaccine strains were constructed that stably expressed each antigen, maintained colonization capacity, and sustained total attenuation in the host. Oral administration of the vaccine strains induced antigen-specific serum IgG, particularly IgG(2A), and mucosal IgA for ?1-giardin and ?-enolase, but not for ornithine carbamoyl transferase. Immunization with the ?1-giardin vaccine induced significant protection against subsequent G. lamblia challenge, which was further enhanced by boosting with cholera toxin or sublingual ?1-giardin administration. The ?-enolase vaccine afforded no protection. Analysis of ?1-giardin from divergent assemblage A and B isolates of G. lamblia revealed >97% amino acid sequence conservation and immunological cross-reactivity, further supporting the potential utility of this antigen in vaccine development. Together. These results indicate that ?1-giardin is a suitable candidate antigen for a vaccine against giardiasis.
Project description:Background:Giardia lamblia is a pathogenic intestinal protozoan with high prevalence in developing countries, especially among children. Molecular characterization has revealed the existence of eight assemblages, with A and B being more commonly described in human infections. Despite its importance, to our knowledge this is the first published molecular analysis of G. lamblia assemblages in Angola. Methods:The present study aimed to identify the assemblages of G. lamblia in children with acute diarrhoea presenting at the Bengo General Hospital, Angola. A stool sample was collected and microscopy and immunochromatographic tests were used. DNA was extracted and assemblage determination was performed through amplification of the gene fragment ssu-rRNA (175 bp) and ?-giardin (511 bp) through polymerase chain reaction and DNA sequencing. Results:Of the 16 stool samples screened, 12 were successfully sequenced. Eleven isolates were assigned to assemblage B and one to assemblage A. Subassemblage determination was not possible for assemblage B, while the single isolate assigned to assemblage A was identified as belonging to subassemblage A3. Conclusion:This study provides information about G. lamblia assemblages in Bengo Province, Angola and may contribute as a first step in understanding the molecular epidemiology of this protozoan in the country. GenBank accession numbers for the ssur-RNA gene: MF479750, MF479751, MF479752, MF479753, MF479754, MF479755, MF479756, MF479757, MF479758, MF479759, MF479760, MF479761. GenBank accession numbers for the ?-giardin gene: MF565378, MF565379, MF565380, MF565381.
Project description:BACKGROUND:Giardia lamblia, a protozoan pathogen causing diarrheal outbreaks, has characteristic cytoskeletal structures including eight flagella, a median body and a ventral disc. Gamma-giardin is a unique component protein of the cytoskeleton of this protozoan. RESULTS:Through comparative proteomic analysis between different stages of the cell cycle, G. lamblia γ-giardin (Glγ-giardin) was identified as an upregulated protein in the G2-phase. Increased Glγ-giardin expression in G2 was confirmed by western blot and real-time polymerase chain reaction analyses. Knockdown of this protein using a morpholino affected the formation of ventral discs, especially the microribbons of the discs, but exerted little effect on the binding ability of G. lamblia. The number of cells with four nuclei was increased in Glγ-giardin-knockdown cells. Expression of Glγ-giardin was decreased during encystation, in contrast with the G2-phase. CONCLUSIONS:Knockdown experiments demonstrated that Glγ-giardin is a component of the trilaminar structure of the ventral disc. Expression of Glγ-giardin is induced in the G2-phase prior to active cell division, whereas its expression decreases during encystation, a dormant stage of G. lamblia.
Project description:Two major genotypic assemblages of Giardia lamblia infect humans; the epidemiologic significance of this phenomenon is poorly understood. We developed a single-vessel multiplex real-time PCR (qPCR) assay that genotypes Giardia infections into assemblages A and/or B directly from fecal samples. The assay utilized Scorpion probes that combined genotype-specific primers and probes for the 18S rRNA gene into the same molecule. The protocol was capable of detecting as few as 20 trophozoites per PCR on fecal DNA isolated using a commercial method or 1.25 trophozoites per PCR on fecal DNA isolated using a G. lamblia-specific oligonucleotide capture technique. The assay was specific for fecal specimens, with no amplification of the discordant genotype with the opposite Scorpion probe. When 97 clinical specimens from Bangladesh were used, the multiplex PCR assay detected 95% (21 of 22) of Giardia microscopy-positive specimens and 18% (13 of 74) of microscopy-negative specimens. Microscopy-negative and qPCR-positive specimens had higher average cycle threshold values than microscopy-positive and qPCR-positive specimens, suggesting that they represented true low-burden infections. Most (32 of 35) infections were assemblage B infections. This single-reaction multiplex qPCR assay distinguishes assemblage A Giardia infections from assemblage B infections directly on fecal samples and may aid epidemiologic investigation.
Project description:Giardia intestinalis is a common cause of diarrheal disease and it consists of eight genetically distinct genotypes or assemblages (A-H). Only assemblages A and B infect humans and are suggested to represent two different Giardia species. Correlations exist between assemblage type and host-specificity and to some extent symptoms. Phenotypical differences have been documented between assemblages and genome sequences are available for A, B and E. We have characterized and compared the polyadenylated transcriptomes of assemblages A, B and E. Four genetically different isolates were studied (WB (AI), AS175 (AII), P15 (E) and GS (B)) using paired-end, strand-specific RNA-seq. Most of the genome was transcribed in trophozoites grown in vitro, but at vastly different levels. RNA-seq confirmed many of the present annotations and refined the current genome annotation. Gene expression divergence was found to recapitulate the known phylogeny, and uncovered lineage-specific differences in expression. Polyadenylation sites were mapped for over 70% of the genes and revealed many examples of conserved and unexpectedly long 3' UTRs. 28 open reading frames were found in a non-transcribed gene cluster on chromosome 5 of the WB isolate. Analysis of allele-specific expression revealed a correlation between allele-dosage and allele expression in the GS isolate. Previously reported cis-splicing events were confirmed and global mapping of cis-splicing identified only one novel intron. These observations can possibly explain differences in host-preference and symptoms, and it will be the basis for further studies of Giardia pathogenesis and biology.
Project description:Giardia intestinalis is a major cause of diarrheal disease worldwide and two major Giardia genotypes, assemblages A and B, infect humans. The genome of assemblage A parasite WB was recently sequenced, and the structurally compact 11.7 Mbp genome contains simplified basic cellular machineries and metabolism. We here performed 454 sequencing to 16x coverage of the assemblage B isolate GS, the only Giardia isolate successfully used to experimentally infect animals and humans. The two genomes show 77% nucleotide and 78% amino-acid identity in protein coding regions. Comparative analysis identified 28 unique GS and 3 unique WB protein coding genes, and the variable surface protein (VSP) repertoires of the two isolates are completely different. The promoters of several enzymes involved in the synthesis of the cyst-wall lack binding sites for encystation-specific transcription factors in GS. Several synteny-breaks were detected and verified. The tetraploid GS genome shows higher levels of overall allelic sequence polymorphism (0.5 versus <0.01% in WB). The genomic differences between WB and GS may explain some of the observed biological and clinical differences between the two isolates, and it suggests that assemblage A and B Giardia can be two different species.
Project description:Alpha-11 giardin is a member of the multi-gene alpha-giardin family in the intestinal protozoan, Giardia lamblia. This gene family shares an ancestry with the annexin super family, whose common characteristic is calcium-dependent binding to membranes that contain acidic phospholipids. Several alpha giardins are highly expressed during parasite-induced diarrhea in humans. Despite being a member of a large family of proteins, little is known about the function and cellular localization of alpha-11 giardin, although giardins are often associated with the cytoskeleton. It has been shown that Giardia exhibits high levels of alpha-11 giardin mRNA transcript throughout its life cycle; however, constitutive over-expression of this protein is lethal to the parasite. Determining the three-dimensional structure of an alpha-giardin is essential to identifying functional domains shared in the alpha-giardin family. Here we report the crystal structures of the apo and Ca(2+)-bound forms of alpha-11 giardin, the first alpha giardin to be characterized structurally. Crystals of apo and Ca(2+)-bound alpha-11 giardin diffracted to 1.1 A and 2.93 A, respectively. The crystal structure of selenium-substituted apo alpha-11 giardin reveals a planar array of four tandem repeats of predominantly alpha-helical domains, reminiscent of previously determined annexin structures, making this the highest-resolution structure of an annexin to date. The apo alpha-11 giardin structure also reveals a hydrophobic core formed between repeats I/IV and II/III, a region typically hydrophilic in other annexins. Surprisingly, the Ca(2+)-bound structure contains only a single calcium ion, located in the DE loop of repeat I and coordinated differently from the two types of calcium sites observed in previous annexin structures. The apo and Ca(2+)-bound alpha-11 giardin structures assume overall similar conformations; however, Ca(2+)-bound alpha-11 giardin crystallized in a lower-symmetry space group with four molecules in the asymmetric unit. Vesicle-binding studies suggest that alpha-11 giardin, unlike most other annexins, does not bind to vesicles composed of acidic phospholipids in a calcium-dependent manner.
Project description:Real-time PCR, using dual-labeled fluorescent probes targeting the beta-giardin gene, was used to detect Giardia lamblia in human stool specimens and to discriminate between isolates from the two major genetic assemblages of G. lamblia infective to humans, assemblages A and B.
Project description:G. lamblia is a fecal-oral transmitted human enteropathogenic protozoan with extremely high incidence in endemic areas in Africa and Asia particularly in the infant population. It homes to the proximal small intestine and induceses diarrhea and malabsoption. Here we established a novel murine G. lamblia infection model and analyzed the tissue and epithelial response and downstream microbial and metabolic effects in the adult host. Overall design: Neonate mice at the age of 3-4 days were orally infected by gavage with 2x10e5 G. lamblia ATCC 5058 (assemblage B, strain GS, clone H7) trophozoites. The infection was monitored by antigen ELISA and quantitative PCR using weekly fecal samples as well as histology. Transcriptional analysis of total gut tissue and isolated intestinal epithelial cells (Lotz et al., JEM, 2006) as well as metabolic analysis in blood, intestinal content and liver tissue were performed in adult animals.