Trophozoite fitness dictates the intestinal epithelial cell response to Giardia intestinalis infection.

Giardia intestinalis is a non-invasive, protozoan parasite infecting the upper small intestine of most mammals. Symptomatic infections cause the diarrhoeal disease giardiasis in humans and animals, but at least half of the infections are asymptomatic. However, the molecular underpinnings of these different outcomes of the infection are still poorly defined. Here, we studied the early transcriptional response to G. intestinalis trophozoites, the disease-causing life-cycle stage, in human enteroid-derived, 2-dimensional intestinal epithelial cell (IEC) monolayers. Trophozoites preconditioned in media that maximise parasite fitness triggered only neglectable inflammatory transcription in the IECs during the first hours of co-incubation. By sharp contrast, "non-fit" or lysed trophozoites induced a vigorous IEC transcriptional response, including high up-regulation of many inflammatory cytokines and chemokines. Furthermore, "fit" trophozoites could even suppress the stimulatory effect of lysed trophozoites in mixed infections, suggesting active G. intestinalis suppression of the IEC response. By dual-species RNA-sequencing, we defined the IEC and G. intestinalis gene expression programs associated with these differential outcomes of the infection. Taken together, our results inform on how G. intestinalis infection can lead to such highly variable effects on the host, and pinpoints trophozoite fitness as a key determinant of the IEC response to this common parasite.

Adaptation of the late ISC pathway in the anaerobic mitochondrial organelles of Giardia intestinalis.

Mitochondrial metabolism is entirely dependent on the biosynthesis of the [4Fe-4S] clusters, which are part of the subunits of the respiratory chain. The mitochondrial late ISC pathway mediates the formation of these clusters from simpler [2Fe-2S] molecules and transfers them to client proteins. Here, we characterized the late ISC pathway in one of the simplest mitochondria, mitosomes, of the anaerobic protist Giardia intestinalis that lost the respiratory chain and other hallmarks of mitochondria. In addition to IscA2, Nfu1 and Grx5 we identified a novel BolA1 homologue in G. intestinalis mitosomes. It specifically interacts with Grx5 and according to the high-affinity pulldown also with other core mitosomal components. Using CRISPR/Cas9 we were able to establish full bolA1 knock out, the first cell line lacking a mitosomal protein. Despite the ISC pathway being the only metabolic role of the mitosome no significant changes in the mitosome biology could be observed as neither the number of the mitosomes or their capability to form [2Fe-2S] clusters in vitro was affected. We failed to identify natural client proteins that would require the [2Fe-2S] or [4Fe-4S] cluster within the mitosomes, with the exception of [2Fe-2S] ferredoxin, which is itself part of the ISC pathway. The overall uptake of iron into the cellular proteins remained unchanged as also observed for the grx5 knock out cell line. The pull-downs of all late ISC components were used to build the interactome of the pathway showing specific position of IscA2 due to its interaction with the outer mitosomal membrane proteins. Finally, the comparative analysis across Metamonada species suggested that the adaptation of the late ISC pathway identified in G. intestinalis occurred early in the evolution of this supergroup of eukaryotes.

Insights into translocation mechanism and ribosome evolution from cryo-EM structures of translocation intermediates of Giardia intestinalis.

Giardia intestinalis is a protozoan parasite that causes diarrhea in humans. Using single-particle cryo-electron microscopy, we have determined high-resolution structures of six naturally populated translocation intermediates, from ribosomes isolated directly from actively growing Giardia cells. The highly compact and uniquely GC-rich Giardia ribosomes possess eukaryotic rRNAs and ribosomal proteins, but retain some bacterial features. The translocation intermediates, with naturally bound tRNAs and eukaryotic elongation factor 2 (eEF2), display characteristic ribosomal intersubunit rotation and small subunit's head swiveling-universal for translocation. In addition, we observe the eukaryote-specific 'subunit rolling' dynamics, albeit with limited features. Finally, the eEF2·GDP state features a uniquely positioned 'leaving phosphate (Pi)' that proposes hitherto unknown molecular events of Pi and eEF2 release from the ribosome at the final stage of translocation. In summary, our study elucidates the mechanism of translocation in the protists and illustrates evolution of the translation machinery from bacteria to eukaryotes from both the structural and mechanistic perspectives.

Giardia intestinalis thymidine kinase is a high-affinity enzyme crucial for DNA synthesis and an exploitable target for drug discovery.

Giardiasis is a diarrheal disease caused by the unicellular parasite Giardia intestinalis, for which metronidazole is the main treatment option. The parasite is dependent on exogenous deoxyribonucleosides for DNA replication and thus is also potentially vulnerable to deoxyribonucleoside analogs. Here, we characterized the G. intestinalis thymidine kinase, a divergent member of the thymidine kinase 1 family that consists of two weakly homologous parts within one polypeptide. We found that the recombinantly expressed enzyme is monomeric, with 100-fold higher catalytic efficiency for thymidine compared to its second-best substrate, deoxyuridine, and is furthermore subject to feedback inhibition by dTTP. This efficient substrate discrimination is in line with the lack of thymidylate synthase and dUTPase in the parasite, which makes deoxy-UMP a dead-end product that is potentially harmful if converted to deoxy-UTP. We also found that the antiretroviral drug azidothymidine (AZT) was an equally good substrate as thymidine and was active against WT as well as metronidazole-resistant G. intestinalis trophozoites. This drug inhibited DNA synthesis in the parasite and efficiently decreased cyst production in vitro, which suggests that it could reduce infectivity. AZT also showed a good effect in G. intestinalis-infected gerbils, reducing both the number of trophozoites in the small intestine and the number of viable cysts in the stool. Taken together, these results suggest that the absolute dependency of the parasite on thymidine kinase for its DNA synthesis can be exploited by AZT, which has promise as a future medication effective against metronidazole-refractory giardiasis.

Combined nanometric and phylogenetic analysis of unique endocytic compartments in Giardia lamblia sheds light on the evolution of endocytosis in Metamonada.

BACKGROUND: Giardia lamblia, a parasitic protist of the Metamonada supergroup, has evolved one of the most diverged endocytic compartment systems investigated so far. Peripheral endocytic compartments, currently known as peripheral vesicles or vacuoles (PVs), perform bulk uptake of fluid phase material which is then digested and sorted either to the cell cytosol or back to the extracellular space. RESULTS: Here, we present a quantitative morphological characterization of these organelles using volumetric electron microscopy and super-resolution microscopy (SRM). We defined a morphological classification for the heterogenous population of PVs and performed a comparative analysis of PVs and endosome-like organelles in representatives of phylogenetically related taxa, Spironucleus spp. and Tritrichomonas foetus. To investigate the as-yet insufficiently understood connection between PVs and clathrin assemblies in G. lamblia, we further performed an in-depth search for two key elements of the endocytic machinery, clathrin heavy chain (CHC) and clathrin light chain (CLC), across different lineages in Metamonada. Our data point to the loss of a bona fide CLC in the last Fornicata common ancestor (LFCA) with the emergence of a protein analogous to CLC (GlACLC) in the Giardia genus. Finally, the location of clathrin in the various compartments was quantified. CONCLUSIONS: Taken together, this provides the first comprehensive nanometric view of Giardia's endocytic system architecture and sheds light on the evolution of GlACLC analogues in the Fornicata supergroup and, specific to Giardia, as a possible adaptation to the formation and maintenance of stable clathrin assemblies at PVs.

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.

Dual RNA-seq transcriptome analysis of caecal tissue during primary Eimeria tenella infection in chickens.

BACKGROUND: Coccidiosis is an infectious disease with large negative impact on the poultry industry worldwide. It is an enteric infection caused by unicellular Apicomplexan parasites of the genus Eimeria. The present study aimed to gain more knowledge about interactions between parasites and the host immune system during the early asexual replication phase of E. tenella in chicken caeca. For this purpose, chickens were experimentally infected with E. tenella oocysts, sacrificed on days 1-4 and 10 after infection and mRNA from caecal tissues was extracted and sequenced. RESULTS: Dual RNA-seq analysis revealed time-dependent changes in both host and parasite gene expression during the course of the infection. Chicken immune activation was detected from day 3 and onwards with the highest number of differentially expressed immune genes recorded on day 10. Among early (days 3-4) responses up-regulation of genes for matrix metalloproteinases, several chemokines, interferon (IFN)-γ along with IFN-stimulated genes GBP, IRF1 and RSAD2 were noted. Increased expression of genes with immune suppressive/regulatory effects, e.g. IL10, SOCS1, SOCS3, was also observed among early responses. For E. tenella a general up-regulation of genes involved in protein expression and energy metabolism as well as a general down-regulation genes for DNA and RNA processing were observed during the infection. Specific E. tenella genes with altered expression during the experiment include those for proteins in rhoptry and microneme organelles. CONCLUSIONS: The present study provides novel information on both the transcriptional activity of E. tenella during schizogony in ceacal tissue and of the local host responses to parasite invasion during this phase of infection. Results indicate a role for IFN-γ and IFN-stimulated genes in the innate defence against Eimeria replication.

Eukaryote-Conserved Methylarginine Is Absent in Diplomonads and Functionally Compensated in Giardia.

Methylation is a common posttranslational modification of arginine and lysine in eukaryotic proteins. Methylproteomes are best characterized for higher eukaryotes, where they are functionally expanded and evolved complex regulation. However, this is not the case for protist species evolved from the earliest eukaryotic lineages. Here, we integrated bioinformatic, proteomic, and drug-screening data sets to comprehensively explore the methylproteome of Giardia duodenalis-a deeply branching parasitic protist. We demonstrate that Giardia and related diplomonads lack arginine-methyltransferases and have remodeled conserved RGG/RG motifs targeted by these enzymes. We also provide experimental evidence for methylarginine absence in proteomes of Giardia but readily detect methyllysine. We bioinformatically infer 11 lysine-methyltransferases in Giardia, including highly diverged Su(var)3-9, Enhancer-of-zeste and Trithorax proteins with reduced domain architectures, and novel annotations demonstrating conserved methyllysine regulation of eukaryotic elongation factor 1 alpha. Using mass spectrometry, we identify more than 200 methyllysine sites in Giardia, including in species-specific gene families involved in cytoskeletal regulation, enriched in coiled-coil features. Finally, we use known methylation inhibitors to show that methylation plays key roles in replication and cyst formation in this parasite. This study highlights reduced methylation enzymes, sites, and functions early in eukaryote evolution, including absent methylarginine networks in the Diplomonadida. These results challenge the view that arginine methylation is eukaryote conserved and demonstrate that functional compensation of methylarginine was possible preceding expansion and diversification of these key networks in higher eukaryotes.

Dual RNA-Seq transcriptome analysis of chicken macrophage-like cells (HD11) infected in vitro with Eimeria tenella.

The study aimed to monitor parasite and host gene expression during the early stages of Eimeria tenella infection of chicken cells using dual RNA-Seq analysis. For this, we used chicken macrophage-like cell line HD11 cultures infected in vitro with purified E. tenella sporozoites. Cultures were harvested between 2 and 72 h post-infection and mRNA was extracted and sequenced. Dual RNA-Seq analysis showed clear patterns of altered expression for both parasite and host genes during infection. For example, genes in the chicken immune system showed upregulation early (2­4 h), a strong downregulation of genes across the immune system at 24 h and a repetition of early patterns at 72 h, indicating that invasion by a second generation of parasites was occurring. The observed downregulation may be due to immune self-regulation or to immune evasive mechanisms exerted by E. tenella. Results also suggested pathogen recognition receptors involved in E. tenella innate recognition, MRC2, TLR15 and NLRC5 and showed distinct chemokine and cytokine induction patterns. Moreover, the expression of several functional categories of Eimeria genes, such as rhoptry kinase genes and microneme genes, were also examined, showing distinctive differences which were expressed in sporozoites and merozoites.

Oxygen induces the expression of invasion and stress response genes in the anaerobic salmon parasite Spironucleus salmonicida.

BACKGROUND: Spironucleus salmonicida is an anaerobic parasite that can cause systemic infections in Atlantic salmon. Unlike other diplomonad parasites, such as the human pathogen Giardia intestinalis, Spironucleus species can infiltrate the blood stream of their hosts eventually colonizing organs, skin and gills. How this presumed anaerobe can persist and invade oxygenated tissues, despite having a strictly anaerobic metabolism, remains elusive. RESULTS: To investigate how S. salmonicida response to oxygen stress, we performed RNAseq transcriptomic analyses of cells grown in the presence of oxygen or antioxidant-free medium. We found that over 20% of the transcriptome is differentially regulated in oxygen (1705 genes) and antioxidant-depleted (2280 genes) conditions. These differentially regulated transcripts encode proteins related to anaerobic metabolism, cysteine and Fe-S cluster biosynthesis, as well as a large number of proteins of unknown function. S. salmonicida does not encode genes involved in the classical elements of oxygen metabolism (e.g., catalases, superoxide dismutase, glutathione biosynthesis, oxidative phosphorylation). Instead, we found that genes encoding bacterial-like oxidoreductases were upregulated in response to oxygen stress. Phylogenetic analysis revealed some of these oxygen-responsive genes (e.g., nadh oxidase, rubrerythrin, superoxide reductase) are rare in eukaryotes and likely derived from lateral gene transfer (LGT) events into diplomonads from prokaryotes. Unexpectedly, we observed that many host evasion- and invasion-related genes were also upregulated under oxidative stress suggesting that oxygen might be an important signal for pathogenesis. CONCLUSION: While oxygen is toxic for related organisms, such as G. intestinalis, we find that oxygen is likely a gene induction signal for host invasion- and evasion-related pathways in S. salmonicida. These data provide the first molecular evidence for how S. salmonicida could tolerate oxic host environments and demonstrate how LGT can have a profound impact on the biology of anaerobic parasites.

Endoplasmic reticulum is the sorting core facility in the Golgi-lacking protozoan Giardia lamblia.

Our understanding of protein and lipid trafficking in eukaryotic cells has been challenged by the finding of different forms of compartmentalization and cargo processing in protozoan parasites. Here, we show that, in the absence of a Golgi compartment in Giardia, proteins destined for secretion are directly sorted and packaged at specialized ER regions enriched in COPII coatomer complexes and ceramide. We also demonstrated that ER-resident proteins are retained at the ER by the action of a KDEL receptor, which, in contrast to other eukaryotic KDEL receptors, showed no interorganellar dynamic but instead acts specifically at the limit of the ER membrane. Our study suggests that the ER-exit sites and the perinuclear ER-membranes are capable of performing protein-sorting functions. In our view, the description presented here suggests that Giardia adaptation represents an extreme example of reductive evolution without loss of function.

Identification of Conserved Candidate Vaccine Antigens in the Surface Proteome of Giardia lamblia.

Giardia lamblia, one of the most common protozoal infections of the human intestine, is an important worldwide cause of diarrheal disease, malabsorption, malnutrition, delayed cognitive development in children, and protracted postinfectious syndromes. Despite its medical importance, no human vaccine is available against giardiasis. A crude veterinary vaccine has been developed, and experimental vaccines based on expression of multiple variant-specific surface proteins have been reported, but poorly defined vaccine components and excessive antigen variability are problematic for pharmaceutical vaccine production. To expand the repertoire of antigen candidates for vaccines, we reasoned that surface proteins may provide an enriched source of such antigens since key host effectors, such as secretory IgA, can directly bind to such antigens in the intestinal lumen and interfere with epithelial attachment. Here, we have applied a proteomics approach to identify 23 novel surface antigens of G. lamblia that show >90% amino acid sequence identity between the two human-pathogenic genetic assemblages (A and B) of the parasite. Surface localization of a representative subset of these proteins was confirmed by immunostaining. Four selected proteins, uridine phosphorylase-like protein-1, protein 21.1 (GL50803_27925), α1-giardin, and α11-giardin, were subsequently produced in recombinant form and shown to be immunogenic in mice and G. lamblia-infected humans and confer protection against G. lamblia infection upon intranasal immunization in rodent models of giardiasis. These results demonstrate that identification of conserved surface antigens provides a powerful approach for overcoming a key rate-limiting step in the design and construction of an effective vaccine against giardiasis.

Giardia intestinalis mitosomes undergo synchronized fission but not fusion and are constitutively associated with the endoplasmic reticulum.

BACKGROUND: Mitochondria of opisthokonts undergo permanent fission and fusion throughout the cell cycle. Here, we investigated the dynamics of the mitosomes, the simplest forms of mitochondria, in the anaerobic protist parasite Giardia intestinalis, a member of the Excavata supergroup of eukaryotes. The mitosomes have abandoned typical mitochondrial traits such as the mitochondrial genome and aerobic respiration and their single role known to date is the formation of iron-sulfur clusters. RESULTS: In live experiments, no fusion events were observed between the mitosomes in G. intestinalis. Moreover, the organelles were highly prone to becoming heterogeneous. This suggests that fusion is either much less frequent or even absent in mitosome dynamics. Unlike in mitochondria, division of the mitosomes was absolutely synchronized and limited to mitosis. The association of the nuclear and the mitosomal division persisted during the encystation of the parasite. During the segregation of the divided mitosomes, the subset of the organelles between two G. intestinalis nuclei had a prominent role. Surprisingly, the sole dynamin-related protein of the parasite seemed not to be involved in mitosomal division. However, throughout the cell cycle, mitosomes associated with the endoplasmic reticulum (ER), although none of the known ER-tethering complexes was present. Instead, the ER-mitosome interface was occupied by the lipid metabolism enzyme long-chain acyl-CoA synthetase 4. CONCLUSIONS: This study provides the first report on the dynamics of mitosomes. We show that together with the loss of metabolic complexity of mitochondria, mitosomes of G. intestinalis have uniquely streamlined their dynamics by harmonizing their division with mitosis. We propose that this might be a strategy of G. intestinalis to maintain a stable number of organelles during cell propagation. The lack of mitosomal fusion may also be related to the secondary reduction of the organelles. However, as there are currently no reports on mitochondrial fusion in the whole Excavata supergroup, it is possible that the absence of mitochondrial fusion is an ancestral trait common to all excavates.

Giardia-specific cellular immune responses in post-giardiasis chronic fatigue syndrome.

BACKGROUND: The role of pathogen specific cellular immune responses against the eliciting pathogen in development of post-infectious chronic fatigue syndrome (PI-CFS) is not known and such studies are difficult to perform. The aim of this study was to evaluate specific anti-Giardia cellular immunity in cases that developed CFS after Giardia infection compared to cases that recovered well. Patients reporting chronic fatigue in a questionnaire study three years after a Giardia outbreak were clinically evaluated five years after the outbreak and grouped according to Fukuda criteria for CFS and idiopathic chronic fatigue. Giardia specific immune responses were evaluated in 39 of these patients by proliferation assay, T cell activation and cytokine release analysis. 20 Giardia exposed non-fatigued individuals and 10 healthy unexposed individuals were recruited as controls. RESULTS: Patients were clinically classified into CFS (n = 15), idiopathic chronic fatigue (n = 5), fatigue from other causes (n = 9) and recovered from fatigue (n = 10). There were statistically significant antigen specific differences between these Giardia exposed groups and unexposed controls. However, we did not find differences between the Giardia exposed fatigue classification groups with regard to CD4 T cell activation, proliferation or cytokine levels in 6 days cultured PBMCs. Interestingly, sCD40L was increased in patients with PI-CFS and other persons with fatigue after Giardia infection compared to the non-fatigued group, and correlated well with fatigue levels at the time of sampling. CONCLUSION: Our data show antigen specific cellular immune responses in the groups previously exposed to Giardia and increased sCD40L in fatigued patients.

The genome of Spironucleus salmonicida highlights a fish pathogen adapted to fluctuating environments.

Spironucleus salmonicida causes systemic infections in salmonid fish. It belongs to the group diplomonads, binucleated heterotrophic flagellates adapted to micro-aerobic environments. Recently we identified energy-producing hydrogenosomes in S. salmonicida. Here we present a genome analysis of the fish parasite with a focus on the comparison to the more studied diplomonad Giardia intestinalis. We annotated 8067 protein coding genes in the ∼12.9 Mbp S. salmonicida genome. Unlike G. intestinalis, promoter-like motifs were found upstream of genes which are correlated with gene expression, suggesting a more elaborate transcriptional regulation. S. salmonicida can utilise more carbohydrates as energy sources, has an extended amino acid and sulfur metabolism, and more enzymes involved in scavenging of reactive oxygen species compared to G. intestinalis. Both genomes have large families of cysteine-rich membrane proteins. A cluster analysis indicated large divergence of these families in the two diplomonads. Nevertheless, one of S. salmonicida cysteine-rich proteins was localised to the plasma membrane similar to G. intestinalis variant-surface proteins. We identified S. salmonicida homologs to cyst wall proteins and showed that one of these is functional when expressed in Giardia. This suggests that the fish parasite is transmitted as a cyst between hosts. The extended metabolic repertoire and more extensive gene regulation compared to G. intestinalis suggest that the fish parasite is more adapted to cope with environmental fluctuations. Our genome analyses indicate that S. salmonicida is a well-adapted pathogen that can colonize different sites in the host.

On the reversibility of parasitism: adaptation to a free-living lifestyle via gene acquisitions in the diplomonad Trepomonas sp. PC1.

BACKGROUND: It is generally thought that the evolutionary transition to parasitism is irreversible because it is associated with the loss of functions needed for a free-living lifestyle. Nevertheless, free-living taxa are sometimes nested within parasite clades in phylogenetic trees, which could indicate that they are secondarily free-living. Herein, we test this hypothesis by studying the genomic basis for evolutionary transitions between lifestyles in diplomonads, a group of anaerobic eukaryotes. Most described diplomonads are intestinal parasites or commensals of various animals, but there are also free-living diplomonads found in oxygen-poor environments such as marine and freshwater sediments. All these nest well within groups of parasitic diplomonads in phylogenetic trees, suggesting that they could be secondarily free-living. RESULTS: We present a transcriptome study of Trepomonas sp. PC1, a diplomonad isolated from marine sediment. Analysis of the metabolic genes revealed a number of proteins involved in degradation of the bacterial membrane and cell wall, as well as an extended set of enzymes involved in carbohydrate degradation and nucleotide metabolism. Phylogenetic analyses showed that most of the differences in metabolic capacity between free-living Trepomonas and the parasitic diplomonads are due to recent acquisitions of bacterial genes via gene transfer. Interestingly, one of the acquired genes encodes a ribonucleotide reductase, which frees Trepomonas from the need to scavenge deoxyribonucleosides. The transcriptome included a gene encoding squalene-tetrahymanol cyclase. This enzyme synthesizes the sterol substitute tetrahymanol in the absence of oxygen, potentially allowing Trepomonas to thrive under anaerobic conditions as a free-living bacterivore, without depending on sterols from other eukaryotes. CONCLUSIONS: Our findings are consistent with the phylogenetic evidence that the last common ancestor of diplomonads was dependent on a host and that Trepomonas has adapted secondarily to a free-living lifestyle. We believe that similar studies of other groups where free-living taxa are nested within parasites could reveal more examples of secondarily free-living eukaryotes.

Divergent Transcriptional Responses to Physiological and Xenobiotic Stress in Giardia duodenalis.

Understanding how parasites respond to stress can help to identify essential biological processes. Giardia duodenalis is a parasitic protist that infects the human gastrointestinal tract and causes 200 to 300 million cases of diarrhea annually. Metronidazole, a major antigiardial drug, is thought to cause oxidative damage within the infective trophozoite form. However, treatment efficacy is suboptimal, due partly to metronidazole-resistant infections. To elucidate conserved and stress-specific responses, we calibrated sublethal metronidazole, hydrogen peroxide, and thermal stresses to exert approximately equal pressure on trophozoite growth and compared transcriptional responses after 24 h of exposure. We identified 252 genes that were differentially transcribed in response to all three stressors, including glycolytic and DNA repair enzymes, a mitogen-activated protein (MAP) kinase, high-cysteine membrane proteins, flavin adenine dinucleotide (FAD) synthetase, and histone modification enzymes. Transcriptional responses appeared to diverge according to physiological or xenobiotic stress. Downregulation of the antioxidant system and α-giardins was observed only under metronidazole-induced stress, whereas upregulation of GARP-like transcription factors and their subordinate genes was observed in response to hydrogen peroxide and thermal stressors. Limited evidence was found in support of stress-specific response elements upstream of differentially transcribed genes; however, antisense derepression and differential regulation of RNA interference machinery suggest multiple epigenetic mechanisms of transcriptional control.

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

BACKGROUND: The 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. RESULTS: Several 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. CONCLUSIONS: Giardia 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.

Rolf Bernander (1956-2014): pioneer of the archaeal cell cycle.

On 19 January 2014 Rolf ('Roffe') Bernander passed away unexpectedly. Rolf was a dedicated scientist; his research aimed at unravelling the cell biology of the archaeal domain of life, especially cell cycle-related questions, but he also made important contributions in other areas of microbiology. Rolf had a professor position in the Molecular Evolution programme at Uppsala University, Sweden for about 8 years, and in January 2013 he became chair professor at the Department of Molecular Biosciences, The Wenner-Gren Institute at Stockholm University in Sweden. Rolf was an exceptional colleague and will be deeply missed by his family and friends, and the colleagues and co-workers that he leaves behind in the scientific community. He will be remembered for his endless enthusiasm for science, his analytical mind, and his quirky sense of humour.

UV irradiation responses in Giardia intestinalis.

The response to ultraviolet light (UV) radiation, a natural stressor to the intestinal protozoan parasite Giardia intestinalis, was studied to deepen the understanding of how the surrounding environment affects the parasite during transmission. UV radiation at 10 mJ/cm(2) kills Giardia cysts effectively whereas trophozoites and encysting parasites can recover from UV treatment at 100 mJ/cm(2) and 50 mJ/cm(2) respectively. Staining for phosphorylated histone H2A showed that UV treatment induces double-stranded DNA breaks and flow cytometry analyses revealed that UV treatment of trophozoites induces DNA replication arrest. Active DNA replication coupled to DNA repair could be an explanation to why UV light does not kill trophozoites and encysting cells as efficiently as the non-replicating cysts. We also examined UV-induced gene expression responses in both trophozoites and cysts using RNA sequencing (RNA seq). UV radiation induces small overall changes in gene expression in Giardia but cysts show a stronger response than trophozoites. Heat shock proteins, kinesins and Nek kinases are up-regulated, whereas alpha-giardins and histones are down-regulated in UV treated trophozoites. Expression of variable surface proteins (VSPs) is changed in both trophozoites and cysts. Our data show that Giardia cysts have limited ability to repair UV-induced damage and this may have implications for drinking- and waste-water treatment when setting criteria for the use of UV disinfection to ensure safe water.