?1-giardin based live heterologous vaccine protects against Giardia lamblia infection in a murine model.
ABSTRACT: 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:Giardia lamblia, an important cause of diarrheal disease, resides in the small intestinal lumen in close apposition to epithelial cells. Since the disease mechanisms underlying giardiasis are poorly understood, elucidating the specific interactions of the parasite with the host epithelium is likely to provide clues to understanding the pathogenesis. Here we tested the hypothesis that contact of Giardia lamblia with intestinal epithelial cells might lead to release of specific proteins. Using established co-culture models, intestinal ligated loops and a proteomics approach, we identified three G. lamblia proteins (arginine deiminase, ornithine carbamoyl transferase and enolase), previously recognized as immunodominant antigens during acute giardiasis. Release was stimulated by cell-cell interactions, since only small amounts of arginine deiminase and enolase were detected in the medium after culturing of G. lamblia alone. The secreted G. lamblia proteins were localized to the cytoplasm and the inside of the plasma membrane of trophozoites. Furthermore, in vitro studies with recombinant arginine deiminase showed that the secreted Giardia proteins can disable host innate immune factors such as nitric oxide production. These results indicate that contact of Giardia with epithelial cells triggers metabolic enzyme release, which might facilitate effective colonization of the human small intestine.
Project description:BACKGROUND:Giardia lamblia is one of the most common infectious protozoans in human that may cause diarrhea in travelers. Searching for antigens that induced effectively protective immunity has become a key point in the development of vaccine against giardiasis. METHODOLOGY/PRINCIPAL FINDINGS:Mice vaccinated with G. lamblia trophozozite-specific α1-giardin DNA vaccine delivered orally by attenuated Salmonella typhimurium SL7027 elicited 74.2% trophozoite reduction, but only 28% reduction in cyst shedding compared with PBS buffer control. Oral vaccination with Salmonella-delivered cyst-specific CWP2 DNA produced 89% reduction in cysts shedding in feces of vaccinated mice. Significantly, the mice vaccinated with Salmonella-delivered bivalent α1-giardin and CWP2 DNA vaccines produced significant reduction in both trophozoite (79%) and cyst (93%) in feces of vaccinated mice. This parasite reduction is associated with the strong local mucosal IgA secretion and the IgG2a-dominant systemic immune responses in vaccinated mice. CONCLUSIONS:The results demonstrate that bivalent vaccines targeting α1-giardin and CWP2 can protect mice against the colonization of Giardia trophozoite and block the transformation of cyst in host at the same time, and can be used to prevent Giardia infection and block the transmission of giardiasis.
Project description:Alpha-11 Giardin, a protein from the annexin superfamily, is a 35.0 kDa protein from the intestinal protozoan parasite Giardia lamblia which triggers a form of diarrhea called giardiasis. Here, the cloning, expression, purification and the crystallization of alpha-11 giardin under two different conditions and in two different space groups is reported. Crystals from the first condition diffracted to 1.1 A and belong to a primitive orthorhombic space group, while crystals from the second condition, which included calcium in the crystallization solution, diffracted to 2.93 A and belong to a primitive monoclinic space group. Determination of the detailed atomic structure of alpha-11 giardin will provide a better insight into its biological function and might establish whether this class of proteins is a potential drug target against giardiasis.
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: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: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:Carbamate kinase from Giardia lamblia is an essential enzyme for the survival of the organism. The enzyme catalyzes the final step in the arginine dihydrolase pathway converting ADP and carbamoyl phosphate to ATP and carbamate. We previously reported that disulfiram, a drug used to treat chronic alcoholism, inhibits G. lamblia CK and kills G. lamblia trophozoites in vitro at submicromolar IC50 values. Here, we examine the structural basis for G. lamblia CK inhibition of disulfiram and its analog, thiram, their activities against both metronidazole-susceptible and metronidazole-resistant G. lamblia isolates, and their efficacy in a mouse model of giardiasis. The crystal structure of G. lamblia CK soaked with disulfiram revealed that the compound thiocarbamoylated Cys-242, a residue located at the edge of the active site. The modified Cys-242 prevents a conformational transition of a loop adjacent to the ADP/ATP binding site, which is required for the stacking of Tyr-245 side chain against the adenine moiety, an interaction seen in the structure of G. lamblia CK in complex with AMP-PNP. Mass spectrometry coupled with trypsin digestion confirmed the selective covalent thiocarbamoylation of Cys-242 in solution. The Giardia viability studies in the metronidazole-resistant strain and the G. lamblia CK irreversible inactivation mechanism show that the thiuram compounds can circumvent the resistance mechanism that renders metronidazole ineffectiveness in drug resistance cases of giardiasis. Together, the studies suggest that G. lamblia CK is an attractive drug target for development of novel antigiardial therapies and that disulfiram, an FDA-approved drug, is a promising candidate for drug repurposing.
Project description:Giardia lamblia is one of most common agents causing persistent abdominal symptoms in developed and developing countries. There are several diagnostic methods for Giardia infection, but none are optimal. In this study our aim was to find a new method based on Giardia microRNA (miRNA) that would contribute to the currently available diagnostic methods of giardiasis. Profiling Giardia small RNAs by deep sequencing revealed that the previously reported putative miR5 and miR6 are expressed in several G. lamblia isolates. These miRNAs were later tested by PCR in duodenal biopsies from 8 patients with positive pathology for giardiasis, while gastric biopsies served as matched negative controls. Additionally, these miRNAs were evaluated in stool samples of patients with proven giardiasis. All 8 duodenal samples of patients with histologically proven G. lamblia infection were positive for Giardia miR5 with a mean Ct of 23.7. These results were superior to Ct levels of G. lamblia DNA, which were 26.3 (p=0.004). The miR6 results were close to negative. All 10 gastric biopsies were negative for miR5. Stool studies showed 90% specificity but only 50% sensitivity in diagnosing giardiasis using miR6. The results of miR5 in stool were even less accurate. In conclusion, miR5 testing for Giardia infection in duodenal biopsies, may be a breakthrough method for diagnosis of giardiasis. It seems to be superior to G. lamblia DNA in duodenal biopsies. It would be important to investigate the contribution of routine Giardia miRNA testing in duodenal biopsies and duodenal aspirates from patients with persistent abdominal symptoms. Overall design: For this part of the project, we profiled small RNA from Giardia lamblia trophozoites of 5 strains obtained from the BEI Resources
Project description:For over 50 years, metronidazole and other nitro compounds such as nitazoxanide have been used as a therapy of choice against giardiasis and more and more frequently, resistance formation has been observed. Model systems allowing studies on biochemical aspects of resistance formation to nitro drugs are, however, scarce since resistant strains are often unstable in culture. In order to fill this gap, we have generated a stable metronidazole- and nitazoxanide-resistant Giardia lamblia WBC6 clone, the strain C4. Previous studies on strain C4 and the corresponding wild-type strain WBC6 revealed marked differences in the transcriptomes of both strains. Here, we present a physiological comparison between trophozoites of both strains with respect to their ultrastructure, whole cell activities such as oxygen consumption and resazurin reduction assays, key enzyme activities, and several metabolic key parameters such as NAD(P)+/NAD(P)H and ADP/ATP ratios and FAD contents. We show that nitro compound-resistant C4 trophozoites exhibit lower nitroreductase activities, lower oxygen consumption and resazurin reduction rates, lower ornithine-carbamyl-transferase activity, reduced FAD and NADP(H) pool sizes and higher ADP/ATP ratios than wildtype trophozoites. The present results suggest that resistance formation against nitro compounds is correlated with metabolic adaptations resulting in a reduction of the activities of FAD-dependent oxidoreductases.