Variant-specific Surface Proteins Research Articles

Exploration of Giardia small nucleolar RNAs (snoRNAs) and their possible microRNA derivatives.

Small nucleolar RNAs (snoRNAs) are short non-coding RNAs that are abundant in the nucleoli of eukaryotic cells and play a crucial role in various aspects of ribosomal RNA (rRNA) maturation, including modifications such as 2′-O-methylation or pseudouridylation. On the other hand, Giardia duodenalis is a microaerophilic, flagellated, binucleate protozoan responsible for causing giardiasis. Although numerous snoRNAs have been detected in Giardia, their investigation remains limited. Nevertheless, they have been found to play a crucial role in the rRNA precursor processing pathway and influence other cellular functions. In addition, it has been proposed that some microRNAs are generated from these snoRNAs through excision by the Giardia endoribonuclease Dicer. These microRNAs are believed to contribute to the regulation of antigenic variation, which allows the parasite to evade the host immune response. Specifically, they play a role in modulating variant-specific surface proteins (VSPs) and other cysteine-rich surface antigens (CSAs). The main objective of this study was to bring together the available data on snoRNAs in Giardia, uncovering their functions in various processes and their importance on a global scale. In addition, the research delved into potential microRNAs speculated to originate from snoRNAs, exploring their impact on cellular processes.

Giardia VSPAS7 protein attenuates Giardia intestinalis-induced host macrophage pyroptosis

BackgroundThe unicellular protozoan parasite Giardia intestinalis, which primarily infects humans and animals such as cattle and sheep, is having a major negative impact on public health. Giardia is able to evade the recognition and elimination of the host immune system because of the trophozoite surface and extracellular vesicles (EVs) covered by variant-specific surface proteins (VSPs). As key proteins for immune evasion, whether VSPs can regulate Giardia-induced pyroptosis and promote Giardia evasion of host immune responses has not been reported.MethodsTo examine the role of Giardia VSPAS7 on Giardia-induced activation of the signaling pathway, secretion of pro-inflammatory cytokines, pyroptosis and the mechanism involved, we constructed the pcDNA3.1-vspas7 expression plasmid and transfected this plasmid into mouse macrophages. Key proteins for pyroptosis, IL-1β secretion and LDH release were detected in pcDNA3.1-vspas7-transfected wild-type (WT) cells and NLRP3-deficient cells by western blot, ELISA and LDH assays, respectively. The interactions of Giardia VSPAS7 and mouse NLRP3 were examined using immunofluorescence assays (IFA), co-immunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC) assays.ResultsVSPAS7 could decrease the levels of phosphorylated-p65 (P-p65), P-IκBα and P-ERK caused by Giardia and reduce the production levels of Giardia-induced pro-inflammatory cytokine IL-6, IL-12 p40 and TNF-α. The results showed that VSPAS7 inhibited Giardia-mediated activation of NF-κB, ERK/MAPK signaling and secretion of pro-inflammatory cytokines. Furthermore, VSPAS7 suppressed Giardia-induced macrophage pyroptosis by reducing GSDMD cleavage, caspase-1 activation, IL-1β secretion and LDH release. We further found that VSPAS7 could interact with mouse NLRP3 directly, and in NLRP3-deficient cells the suppression of Giardia-induced macrophage pyroptosis by VSPAS7 was significantly attenuated.ConclusionsOverall, VSPAS7 could inhibit Giardia-induced activation of signaling pathways and pyroptosis in host macrophages, allowing Giardia evasion of host immune responses. Studies on Giardia VSP-mediated immune evasion provide an important theoretical basis for in-depth studies on Giardia pathogenicity.Graphical abstract

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.

Comprehensive characterization of Cysteine-rich protein-coding genes of Giardia lamblia and their role during antigenic variation

Giardia lamblia encodes several families of cysteine-rich proteins, including the Variant-specific Surface Proteins (VSPs) involved in the process of antigenic variation. Their characteristics, definition and relationships are still controversial. An exhaustive analysis of the Cys-rich families including organization, features, evolution and levels of expression was performed, by combining pattern searches and predictions with massive sequencing techniques. Thus, a new classification for Cys-rich proteins, genes and pseudogenes that better describes their involvement in Giardia's biology is presented. Moreover, three novel characteristics exclusive to the VSP genes, comprising an Initiator element/Kozak-like sequence, an extended polyadenylation signal and a unique pattern of mutually exclusive transcript accumulation are presented, as well as the finding that High Cysteine Membrane Proteins, upregulated under stress, may protect the parasite during VSP switching. These results allow better interpretation of previous reports providing the basis for further studies of the biology of this early-branching eukaryote.

Production of Oral Vaccines Based on Virus-Like Particles Pseudotyped with Protozoan-Surface Proteins.

Giardia lamblia is the only known parasite that can inhabit the harsh upper gastrointestinal tract, where most of the digestive proteases are secreted. Intestinal and free-living protozoa express surface proteins containing an extraordinarily high percentage of cysteine. These cysteine-rich variant-specific surface proteins (VSPs) form a dense coat on the entire surface of Giardia trophozoites, that coat protects the parasite inside the host intestine. VSPs not only are resistant to proteolytic digestion, extreme pH and temperatures, but also stimulate host immune responses. These properties can be used to protect as well as to increase the immunogenicity of vaccine antigens for oral administration. The incorporation of VSPs onto virus-like particles bearing viral antigens allows oral administration of these vaccines, protecting the antigens from degradation and generating robust and protective immune responses. In this chapter we describe the development of this versatile vaccine platform for the generation of safe, stable, and efficient oral vaccines, including their production and validation, as well as the characterization of immune response to oral immunization.

Differential antibody responses to Giardia lamblia strain variants expressing dissimilar levels of an immunogenic protein.

Giardia lamblia is a protozoan parasite that causes giardiasis, one of the most common worldwide gastrointestinal diseases. For rational development of a Giardia vaccine, increasing our understanding of the host-Giardia interaction is crucial. In this study, we analysed the immunogenicity and antigenicity of two G lamblia strain variants [GS and GS-5G8 (+)], which express different levels of the variant-specific surface protein (VSP) 5G8 and also analysed the intestinal histological changes associated with Giardia infection. We evaluated the antibody responses induced by G lamblia strains in infected, reinfected and immunized C3H/HeJ mice using ELISA, flow cytometry, Western blotting and histological analysis. Our results showed that G lamblia GS-5G8 (+) was more immunogenic and antigenic than the GS strain. The antibody response against the GS-5G8 (+) strain primarily recognized 5G8 protein. Serum antibody from infected and reinfected mice exhibited specific agglutination of trophozoites in vitro. GS-5G8 (+)-infected mice showed higher CD19+ infiltrating cell levels compared to GS-infected animals. G lamblia strains with different expression levels of an immunogenic antigen (VSP 5G8) induce differential antibody responses. A better understanding of the immunogenic proteins of G lamblia will contribute to the rational development of an effective vaccine against this parasitic disease.

Comparative proteomics of three Giardia lamblia strains: investigation of antigenic variation in the post-genomic era.

Giardia lamblia is a causative agent of persistent diarrhoea widespread in regions with low hygienic standards. Laboratory research is based on cloned lines issuing from various patient isolates typed in the late 1980s and 90s using restriction analysis and serology. In the present study, we compared the well-characterized strain WBC6 with another clone of the parent WB isolate termed WBA1 and with a clone from another isolate, GS/M-83-H7, using shotgun mass spectrometry proteomics. We identified 398 proteins differentially expressed between the GS and both WB isolates and 97 proteins differentially expressed between the two WB isolates. We investigated the expression levels of the predominant variant-specific surface proteins (VSPs) in each clone and matched the previously described major VSPs of each strain to the corresponding open reading frame sequences identified by whole-genome sequencing efforts. Furthermore, since the original WB isolate comes from a patient treated with metronidazole, we compared the susceptibilities of the strains to nitro compounds, as well the expression levels of enzymes involved in nitro reduction and on the corresponding enzyme activities and found distinct differences between the three strains.

Benchmarking hybrid assemblies of Giardia and prediction of widespread intra-isolate structural variation

BackgroundCurrently available short read genome assemblies of the tetraploid protozoan parasite Giardia intestinalis are highly fragmented, highlighting the need for improved genome assemblies at a reasonable cost. Long nanopore reads are well suited to resolve repetitive genomic regions resulting in better quality assemblies of eukaryotic genomes. Subsequent addition of highly accurate short reads to long-read assemblies further improves assembly quality. Using this hybrid approach, we assembled genomes for three Giardia isolates, two with published assemblies and one novel, to evaluate the improvement in genome quality gained from long reads. We then used the long reads to predict structural variants to examine this previously unexplored source of genetic variation in Giardia.MethodsWith MinION reads for each isolate, we assembled genomes using several assemblers specializing in long reads. Assembly metrics, gene finding, and whole genome alignments to the reference genomes enabled direct comparison to evaluate the performance of the nanopore reads. Further improvements from adding Illumina reads to the long-read assemblies were evaluated using gene finding. Structural variants were predicted from alignments of the long reads to the best hybrid genome for each isolate and enrichment of key genes was analyzed using random genome sampling and calculation of percentiles to find thresholds of significance.ResultsOur hybrid assembly method generated reference quality genomes for each isolate. Consistent with previous findings based on SNPs, examination of heterozygosity using the structural variants found that Giardia BGS was considerably more heterozygous than the other isolates that are from Assemblage A. Further, each isolate was shown to contain structural variant regions enriched for variant-specific surface proteins, a key class of virulence factor in Giardia.ConclusionsThe ability to generate reference quality genomes from a single MinION run and a multiplexed MiSeq run enables future large-scale comparative genomic studies within the genus Giardia. Further, prediction of structural variants from long reads allows for more in-depth analyses of major sources of genetic variation within and between Giardia isolates that could have effects on both pathogenicity and host range.

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.

Molecular genotyping, diversity studies and high-resolution molecular markers unveiled by microsatellites in Giardia duodenalis.

BackgroundGiardia duodenalis (synonyms G. lamblia and G. intestinalis) is an enteric protozoan parasite of a wide range of mammalian hosts, including humans and various domestic and wild animals. There is considerable genetic variability in G. duodenalis and isolates of this parasite have been divided into eight genetic assemblages. Microsatellites markers can be used to discriminate isolates with a high level of sensitivity. This study was conducted to identify and characterize genomic microsatellites (simple sequence repeats—SSRs), sequences of one- to six-nucleotide motifs repeated in tandem, present in the available genomes of G. duodenalis and to develop new markers that can serve as a tool for detection and for characterizing the genetic diversity of this parasite.Methodology/ Principal findingsFor each genetic assemblage, polymorphism levels for the microsatellite markers were evaluated. After performing the analysis using the MISA and SciRoKo software, 1,853 simple sequence repeats (SSRs) were identified. In all the genomes, trinucleotide repeats were the most common class followed by tetranucleotide. Many of the SSR loci are assemblage-specific, and 36 SSR loci shared among all the genomes were identified. Together with hypothetical proteins, variant-specific surface proteins represented nearly half of the annotated SSR loci. The results regarding the most common repeat among the SSRs led us to infer that positive selection occurred to avoid frameshift mutations. Additionally, based on inter- and intra-genetic assemblages polymorphism analyses, we unveiled previously undetected genetic variation, indicating that the microsatellite markers we developed are useful molecular tools for epidemiological inferences based on population genetics patterns and processes.ConclusionsThere is increasing demand for the development of new molecular markers and for the characterization of pathogens at a higher resolution level. In this study, we present 60 G. duodenalis microsatellites markers that exhibited high polymerase chain reaction (PCR) amplification efficiency among the different genetic assemblages. Twenty of these markers presented nucleotide sequence polymorphisms and may be used as a genotyping tool. The monomorphic markers can be used for the detection of the parasite at the species and genetic assemblage level. These polymorphic markers revealed a genetic diversity that was previously undetectable, thus they can be considered valuable molecular tools for high resolution markers in future studies investigating Giardia and may also be used for epidemiological inferences based on populations genetics patterns and processes.

Longitudinal cohort study of serum antibody responses towards Giardia lamblia variant-specific surface proteins in a non-endemic area

Introduction/AimsThe long-term humoral immune response after a natural giardiasis infection is not well understood. The aim of this study was to evaluate longitudinal serum IgA and IgG/M responses towards conserved regions of two Giardia variant-specific surface proteins (VSP) and whether these responses differ between Giardia assemblages and durations of infection. MethodsWe recruited thirty Giardia-positive patients, mainly returning travellers, and eighteen healthy adults presumed to be Giardia unexposed. Blood samples were collected before treatment, and at 6 weeks, 6 months and 12 months after the infection cleared. We used a multiplex bead-based flow cytometry immunoassay to measure Giardia specific IgA and IgG/M responses targeting two recombinant antigens from G. lamblia VSP proteins 3 and 5 (VSP3 and VSP5). ResultsSerum levels of anti-VSP5 and anti-VSP3 IgA decreased rapidly to low levels after treatment but continued to be substantially higher than that of presumed unexposed controls even after 6 and 12 months. The IgG/M response decreased more gradually but remained significantly higher than presumed unexposed controls at all time points, except for anti-VSP3 at 12 months. There were no significant difference in responses for infections with assemblage A and assemblage B Giardia lamblia. Chronic infections (>8 weeks) were associated with a significantly lower anti-VSP5 IgG/M response. ConclusionThis study describes the kinetics of the humoral immune response against two Giardia VSP proteins over one year, and the considerable cross reactivity between the two human infective Giardia assemblages. Persons with chronic Giardia infection seem to have lower levels of VSP antibodies.

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-1β [IL-1β], 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.

Giardia and Cryptosporidium antibody prevalence and correlates of exposure among Alaska residents, 2007-2008.

Giardia duodenalis and Cryptosporidium spp. are common intestinal protozoa that can cause diarrhoeal disease. Although cases of infection with Giardia and Cryptosporidium have been reported in Alaska, the seroprevalence and correlates of exposure to these parasites have not been characterised. We conducted a seroprevalence survey among 887 residents of Alaska, including sport hunters, wildlife biologists, subsistence bird hunters and their families and non-exposed persons. We tested serum using a multiplex bead assay to evaluate antibodies to the Giardia duodenalis variant-specific surface protein conserved structural regions and to the Cryptosporidium parvum 17- and 27-kDa antigens. Approximately one third of participants in each group had evidence of exposure to Cryptosporidium. Prevalence of Giardia antibody was highest among subsistence hunters and their families (30%), among whom positivity was associated with lack of community access to in-home running water (adjusted prevalence ratio [aPR] 1.15, 95% confidence interval (CI) 1.02-1.28) or collecting rain, ice, or snow to use as drinking water (aPR 1.09, 95% CI 1.01-1.18). Improving in-home water access for entire communities could decrease the risk of exposure to Giardia.

Transcriptomics Indicates Active and Passive Metronidazole Resistance Mechanisms in Three Seminal Giardia Lines.

Giardia duodenalis is an intestinal parasite that causes 200–300 million episodes of diarrhoea annually. Metronidazole (Mtz) is a front-line anti-giardial, but treatment failure is common and clinical resistance has been demonstrated. Mtz is thought to be activated within the parasite by oxidoreductase enzymes, and to kill by causing oxidative damage. In G. duodenalis, Mtz resistance involves active and passive mechanisms. Relatively low activity of iron-sulfur binding proteins, namely pyruvate:ferredoxin oxidoreductase (PFOR), ferredoxins, and nitroreductase-1, enable resistant cells to passively avoid Mtz activation. Additionally, low expression of oxygen-detoxification enzymes can allow passive (non-enzymatic) Mtz detoxification via futile redox cycling. In contrast, active resistance mechanisms include complete enzymatic detoxification of the pro-drug by nitroreductase-2 and enhanced repair of oxidized biomolecules via thioredoxin-dependent antioxidant enzymes. Molecular resistance mechanisms may be largely founded on reversible transcriptional changes, as some resistant lines revert to drug sensitivity during drug-free culture in vitro, or passage through the life cycle. To comprehensively characterize these changes, we undertook strand-specific RNA sequencing of three laboratory-derived Mtz-resistant lines, 106-2ID10, 713-M3, and WB-M3, and compared transcription relative to their susceptible parents. Common up-regulated genes encoded variant-specific surface proteins (VSPs), a high cysteine membrane protein, calcium and zinc channels, a Mad-2 cell cycle regulator and a putative fatty acid α-oxidase. Down-regulated genes included nitroreductase-1, putative chromate and quinone reductases, and numerous genes that act proximal to PFOR. Transcriptional changes in 106-2ID10 diverged from those in 713-r and WB-r (r ≤ 0.2), which were more similar to each other (r = 0.47). In 106-2ID10, a nonsense mutation in nitroreductase-1 transcripts could enhance passive resistance whereas increased transcription of nitroreductase-2, and a MATE transmembrane pump system, suggest active drug detoxification and efflux, respectively. By contrast, transcriptional changes in 713-M3 and WB-M3 indicated a higher oxidative stress load, attributed to Mtz- and oxygen-derived radicals, respectively. Quantitative comparisons of orthologous gene transcription between Mtz-resistant G. duodenalis and Trichomonas vaginalis, a closely related parasite, revealed changes in transcripts encoding peroxidases, heat shock proteins, and FMN-binding oxidoreductases, as prominent correlates of resistance. This work provides deep insight into Mtz-resistant G. duodenalis, and illuminates resistance-associated features across parasitic species.

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.

Coordinated Changes in Gene Expression Throughout Encystation of Giardia intestinalis.

Differentiation into infectious cysts through the process of encystation is crucial for transmission and survival of the intestinal protozoan parasite Giardia intestinalis. Hitherto the majority of studies have focused on the early events, leaving late encystation poorly defined. In order to further study encystation, focusing on the later events, we developed a new encystation protocol that generates a higher yield of mature cysts compared to standard methods. Transcriptome changes during the entire differentiation from trophozoites to cysts were thereafter studied using RNA sequencing (RNA-seq). A high level of periodicity was observed for up- and down-regulated genes, both at the level of the entire transcriptome and putative regulators. This suggests the trajectory of differentiation to be coordinated through developmentally linked gene regulatory activities. Our study identifies a core of 13 genes that are consistently up-regulated during initial encystation. Of these, two constitute previously uncharacterized proteins that we were able to localize to a new type of encystation-specific vesicles. Interestingly, the largest transcriptional changes were seen in the late phase of encystation with the majority of the highly up-regulated genes encoding hypothetical proteins. Several of these were epitope-tagged and localized to further characterize these previously unknown genetic components of encystation and possibly excystation. Finally, we also detected a switch of variant specific surface proteins (VSPs) in the late phase of encystation. This occurred at the same time as nuclear division and DNA replication, suggesting a potential link between the processes.

Induction of virulence factors in Giardia duodenalis independent of host attachment.

Giardia duodenalis is responsible for the majority of parasitic gastroenteritis in humans worldwide. Host-parasite interaction models in vitro provide insights into disease and virulence and help us to understand pathogenesis. Using HT-29 intestinal epithelial cells (IEC) as a model we have demonstrated that initial sensitisation by host secretions reduces proclivity for trophozoite attachment, while inducing virulence factors. Host soluble factors triggered up-regulation of membrane and secreted proteins, including Tenascins, Cathepsin-B precursor, cystatin, and numerous Variant-specific Surface Proteins (VSPs). By comparison, host-cell attached trophozoites up-regulated intracellular pathways for ubiquitination, reactive oxygen species (ROS) detoxification and production of pyridoxal phosphate (PLP). We reason that these results demonstrate early pathogenesis in Giardia involves two independent host-parasite interactions. Motile trophozoites respond to soluble secreted signals, which deter attachment and induce expression of virulence factors. Trophozoites attached to host cells, in contrast, respond by up-regulating intracellular pathways involved in clearance of ROS, thus anticipating the host defence response.

Comparative genomic analyses of freshly isolated Giardia intestinalis assemblage A isolates.

BackgroundThe diarrhea-causing protozoan Giardia intestinalis makes up a species complex of eight different assemblages (A-H), where assemblage A and B infect humans. Comparative whole-genome analyses of three of these assemblages have shown that there is significant divergence at the inter-assemblage level, however little is currently known regarding variation at the intra-assemblage level. We have performed whole genome sequencing of two sub-assemblage AII isolates, recently axenized from symptomatic human patients, to study the biological and genetic diversity within assemblage A isolates.ResultsSeveral biological differences between the new and earlier characterized assemblage A isolates were identified, including a difference in growth medium preference. The two AII isolates were of different sub-assemblage types (AII-1 [AS175] and AII-2 [AS98]) and showed size differences in the smallest chromosomes. The amount of genetic diversity was characterized in relation to the genome of the Giardia reference isolate WB, an assemblage AI isolate. Our analyses indicate that the divergence between AI and AII is approximately 1 %, represented by ~100,000 single nucleotide polymorphisms (SNP) distributed over the chromosomes with enrichment in variable genomic regions containing surface antigens. The level of allelic sequence heterozygosity (ASH) in the two AII isolates was found to be 0.25–0.35 %, which is 25–30 fold higher than in the WB isolate and 10 fold higher than the assemblage AII isolate DH (0.037 %). 35 protein-encoding genes, not found in the WB genome, were identified in the two AII genomes. The large gene families of variant-specific surface proteins (VSPs) and high cysteine membrane proteins (HCMPs) showed isolate-specific divergences of the gene repertoires. Certain genes, often in small gene families with 2 to 8 members, localize to the variable regions of the genomes and show high sequence diversity between the assemblage A isolates. One of the families, Bactericidal/Permeability Increasing-like protein (BPIL), with eight members was characterized further and the proteins were shown to localize to the ER in trophozoites.ConclusionsGiardia genomes are modular with highly conserved core regions mixed up by variable regions containing high levels of ASH, SNPs and variable surface antigens. There are significant genomic variations in assemblage A isolates, in terms of chromosome size, gene content, surface protein repertoire and gene polymorphisms and these differences mainly localize to the variable regions of the genomes. The large genetic differences within one assemblage of G. intestinalis strengthen the argument that the assemblages represent different Giardia species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1893-6) contains supplementary material, which is available to authorized users.

Giardia and Vilém Dušan Lambl.

Giardia and Vilém Dušan Lambl.

Structural Basis for Inactivation of Giardia lamblia Carbamate Kinase by Disulfiram

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.