Cytokines, Antibodies, and Histopathological Profiles during Giardia Infection and Variant-Specific Surface Protein-Based Vaccination.

Giardiasis is one of the most common human intestinal diseases worldwide. Several experimental animal models have been used to evaluate Giardia infections, with gerbils (Meriones unguiculatus) being the most valuable model due to their high susceptibility to Giardia infection, abundant shedding of cysts, and pathophysiological alterations and signs of disease similar to those observed in humans. Here, we report cytokine and antibody profiles both during the course of Giardia infection in gerbils and after immunization with a novel oral vaccine comprising a mixture of purified variant-specific surface proteins (VSPs). Transcript levels of representative cytokines of different immune profiles as well as macro- and microtissue alterations were assessed in Peyer's patches, mesenteric lymph nodes, and spleens. During infection, cytokine responses showed a biphasic profile: an early induction of Th1 (gamma interferon [IFN-γ], interleukin-1ß [IL-1ß], IL-6, and tumor necrosis factor [TNF]), Th17 (IL-17), and Th2 (IL-4) cytokines, together with intestinal alterations typical of inflammation, followed by a shift toward a predominant Th2 (IL-5) response, likely associated with a counterregulatory mechanism. Conversely, immunization with an oral vaccine comprising the entire repertoire of VSPs specifically showed high levels of IL-17, IL-6, IL-4, and IL-5, without obvious signs of inflammation. Both immunized and infected animals developed local (intestinal secretory IgA [S-IgA]) and systemic (serum IgG) humoral immune responses against VSPs; however, only infected animals showed evident signs of giardiasis. This is the first comprehensive report of cytokine expression and anti-Giardia antibody production during infection and VSP vaccination in gerbils, a reliable model of the human disease.

Protein SUMOylation is Involved in Cell-cycle Progression and Cell Morphology in Giardia lamblia.

The unicellular protozoa Giardia lamblia is a food- and waterborne parasite that causes giardiasis. This illness is manifested as acute and self-limited diarrhea and can evolve to long-term complications. Successful establishment of infection by Giardia trophozoites requires adhesion to host cells and colonization of the small intestine, where parasites multiply by mitotic division. The tight binding of trophozoites to host cells occurs by means of the ventral adhesive disc, a spiral array of microtubules and associated proteins such as giardins. In this work we show that knock down of the Small Ubiquitin-like MOdifier (SUMO) results in less adhesive trophzoites, decreased cell proliferation and deep morphological alterations, including at the ventral disc. Consistent with the reduced proliferation, SUMO knocked-down trophozoites were arrested in G1 and in S phases of the cell cycle. Mass spectrometry analysis of anti-SUMO immunoprecipitates was performed to identify SUMO substrates possibly involved in these events. Among the identified SUMOylation targets, α-tubulin was further validated by Western blot and confirmed to be a SUMO target in Giardia trophozoites.

Antibodies to variable surface antigens induce antigenic variation in the intestinal parasite Giardia lamblia.

The genomes of most protozoa encode families of variant surface antigens. In some parasitic microorganisms, it has been demonstrated that mutually exclusive changes in the expression of these antigens allow parasites to evade the host's immune response. It is widely assumed that antigenic variation in protozoan parasites is accomplished by the spontaneous appearance within the population of cells expressing antigenic variants that escape antibody-mediated cytotoxicity. Here we show, both in vitro and in animal infections, that antibodies to Variant-specific Surface Proteins (VSPs) of the intestinal parasite Giardia lamblia are not cytotoxic, inducing instead VSP clustering into liquid-ordered phase membrane microdomains that trigger a massive release of microvesicles carrying the original VSP and switch in expression to different VSPs by a calcium-dependent mechanism. This novel mechanism of surface antigen clearance throughout its release into microvesicles coupled to the stochastic induction of new phenotypic variants not only changes current paradigms of antigenic switching but also provides a new framework for understanding the course of protozoan infections as a host/parasite adaptive process.

Putative SF2 helicases of the early-branching eukaryote Giardia lamblia are involved in antigenic variation and parasite differentiation into cysts.

BACKGROUND: Regulation of surface antigenic variation in Giardia lamblia is controlled post-transcriptionally by an RNA-interference (RNAi) pathway that includes a Dicer-like bidentate RNase III (gDicer). This enzyme, however, lacks the RNA helicase domain present in Dicer enzymes from higher eukaryotes. The participation of several RNA helicases in practically all organisms in which RNAi was studied suggests that RNA helicases are potentially involved in antigenic variation, as well as during Giardia differentiation into cysts. RESULTS: An extensive in silico analysis of the Giardia genome identified 32 putative Super Family 2 RNA helicases that contain almost all the conserved RNA helicase motifs. Phylogenetic studies and sequence analysis separated them into 22 DEAD-box, 6 DEAH-box and 4 Ski2p-box RNA helicases, some of which are homologs of well-characterized helicases from higher organisms. No Giardia putative helicase was found to have significant homology to the RNA helicase domain of Dicer enzymes. Additionally a series of up- and down-regulated putative RNA helicases were found during encystation and antigenic variation by qPCR experiments. Finally, we were able to recognize 14 additional putative helicases from three different families (RecQ family, Swi2/Snf2 and Rad3 family) that could be considered DNA helicases. CONCLUSIONS: This is the first comprehensive analysis of the Super Family 2 helicases from the human intestinal parasite G. lamblia. The relative and variable expression of particular RNA helicases during both antigenic variation and encystation agrees with the proposed participation of these enzymes during both adaptive processes. The putatives RNA and DNA helicases identified in this early-branching eukaryote provide initial information regarding the biological role of these enzymes in cell adaptation and differentiation.

Efficient oral vaccination by bioengineering virus-like particles with protozoan surface proteins.

Intestinal and free-living protozoa, such as Giardia lamblia, express a dense coat of variant-specific surface proteins (VSPs) on trophozoites that protects the parasite inside the host's intestine. Here we show that VSPs not only are resistant to proteolytic digestion and extreme pH and temperatures but also stimulate host innate immune responses in a TLR-4 dependent manner. We show that these properties can be exploited to both protect and adjuvant vaccine antigens for oral administration. Chimeric Virus-like Particles (VLPs) decorated with VSPs and expressing model surface antigens, such as influenza virus hemagglutinin (HA) and neuraminidase (NA), are protected from degradation and activate antigen presenting cells in vitro. Orally administered VSP-pseudotyped VLPs, but not plain VLPs, generate robust immune responses that protect mice from influenza infection and HA-expressing tumors. This versatile vaccine platform has the attributes to meet the ultimate challenge of generating safe, stable and efficient oral vaccines.

Specific histone modifications play critical roles in the control of encystation and antigenic variation in the early-branching eukaryote Giardia lamblia.

During evolution, parasitic microorganisms have faced the challenges of adapting to different environments to colonize a variety of hosts. Giardia lamblia, a common cause of intestinal disease, has developed fascinating strategies to adapt both outside and inside its host's intestine, such as trophozoite differentiation into cyst and the switching of its major surface antigens. How gene expression is regulated during these adaptive processes remains undefined. Giardia lacks some typical eukaryotic features, like canonical transcription factors, linker histone H1, and complex promoter regions; suggesting that post-transcriptional and translational control of gene expression is essential for parasite survival. However, epigenetic factors may also play critical roles at the transcriptional level. Here, we describe the most common post-translational histone modifications; characterize enzymes involved in these reactions, and analyze their association with the Giardia's differentiation processes. We present evidence that NAD+-dependent and NAD+-independent histone deacetylases regulate encystation; however, a unique NAD+-independent histone deacetylase modulate antigenic switching. The rates of acetylation of H4K8 and H4K16 are critical for encystation, whereas a decrease in acetylation of H4K8 and methylation of H3K9 occur preferentially during antigenic variation. These results show the complexity of the mechanisms regulating gene expression in this minimalistic protozoan parasite.

Vaccination of domestic animals with a novel oral vaccine prevents Giardia infections, alleviates signs of giardiasis and reduces transmission to humans.

Giardia lamblia is a human intestinal parasite and one of the most frequent enteric pathogen of companion animals. Clinical manifestations of giardiasis, such as diarrhoea, anorexia, weight loss and lethargy, have been associated with Giardia infections in both domestic and farm animals. A few anti-parasitic drugs are routinely used to treat giardiasis, but re-infections are common and drug-resistant strains have already been reported. Unfortunately, efficient vaccines against Giardia are not available. Giardia undergoes antigenic variation; through this mechanism, parasites can avoid the host's immune defenses, causing chronic infections and/or re-infections. Antigenic variation is characterised by a continuous switch in the expression of members of a homologous family of genes encoding surface antigens. In a previous report, we indicated that in Giardia, the mechanism responsible for the exchange of variant-specific surface proteins (VSPs) involves the RNA interference (RNAi) pathway. From a repertoire of ~200 VSP genes, only one is expressed on the surface of single trophozoites; however, RNAi machinery disruption generates trophozoites that express the complete VSP repertoire. We also demonstrated that gerbils orally immunised with VSPs isolated from these altered parasites showed high levels of protection. Here we tested this vaccine in cats and dogs, and found that it is highly efficient in preventing new infections and reducing chronic giardiasis in domestic animals both in experimental and natural infections. Remarkably, immunisation of dogs in a highly endemic area strongly decreased the percentage of infected children in the community, suggesting that this vaccine would block the zoonotic transmission of the disease.

In silico analysis of trypanosomatids' helicases.

Trypanosomatids are unicellular protozoan parasites that cause many diseases in animals, including humans, and plants. These early divergent eukaryotes have many singular structures and processes, including the hyper-modified 'base J', a mitochondrial DNA network, RNA editing, and trans-splicing; all of these unique features involve a wide variety of specific DNA/RNA helicases. In this work, the genomes of trypanosomatids were analyzed by data mining, searching for genes coding for DNA/RNA helicases. Specific motifs and full-length sequences from all families present in the helicase's superfamilies (SFs) 1 and 2 were used as baits for genome analyses. A total of 328 putative helicases were identified; 204 genes were assigned to the SF2, 42 genes to the SF1, and 76 genes remain unclassified. Eight species-specific SF2 helicases were also found; Trypanosoma cruzi has three DEAD-box and one DEAH/RHA-specific helicases, while Leishmania major has three Swi2/Snf2 and Trypanosoma brucei has only one RigI helicase. Finally, to identify helicases that could be used as future therapeutic targets, all obtained genes were compared with those present in the human genome. Forty-two helicases underrepresented in the human genome were identified; constituting 16 orthologs groups from L. major, T. brucei, and T. cruzi.

A CDPK isoform participates in the regulation of nodule number in Medicago truncatula.

Medicago spp. are able to develop root nodules via symbiotic interaction with Sinorhizobium meliloti. Calcium-dependent protein kinases (CDPKs) are involved in various signalling pathways in plants, and we found that expression of MtCPK3, a CDPK isoform present in roots of the model legume Medicago truncatula, is regulated during the nodulation process. Early inductions were detected 15 min and 3-4 days post-inoculation (dpi). The very early induction of CPK3 messengers was also present in inoculated M. truncatula dmi mutants and in wild-type roots subjected to salt stress, indicating that this rapid response is probably stress-related. In contrast, the later response was concomitant with cortical cell division and the formation of nodule primordia, and was not observed in wild-type roots inoculated with nod (-) strains. This late induction correlated with a change in the subcellular distribution of CDPK activities. Accordingly, an anti-MtCPK3 antibody detected two bands in soluble root extracts and one in the particulate fraction. CPK3::GFP fusions are targeted to the plasma membrane in epidermal onion cells, a localization that depends on myristoylation and palmitoylation sites of the protein, suggesting a dual subcellular localization. MtCPK3 mRNA and protein were also up-regulated by cytokinin treatment, a hormone linked to the regulation of cortical cell division and other nodulation-related responses. An RNAi-CDPK construction was used to silence CPK3 in Agrobacterium rhizogenes-transformed roots. Although no major phenotype was detected in these roots, when infected with rhizobia, the total number of nodules was, on average, twofold higher than in controls. This correlates with the lack of MtCPK3 induction in the inoculated super-nodulator sunn mutant. Our results suggest that CPK3 participates in the regulation of the symbiotic interaction.