Control of protein synthesis through mRNA pseudouridylation by dyskerin.

Posttranscriptional modifications of mRNA have emerged as regulators of gene expression. Although pseudouridylation is the most abundant, its biological role remains poorly understood. Here, we demonstrate that the pseudouridine synthase dyskerin associates with RNA polymerase II, binds to thousands of mRNAs, and is responsible for their pseudouridylation, an action that occurs in chromatin and does not appear to require a guide RNA with full complementarity. In cells lacking dyskerin, mRNA pseudouridylation is reduced, while at the same time, de novo protein synthesis is enhanced, indicating that this modification interferes with translation. Accordingly, mRNAs with fewer pseudouridines due to knockdown of dyskerin are translated more efficiently. Moreover, mRNA pseudouridylation is severely reduced in patients with dyskeratosis congenita caused by inherited mutations in the gene encoding dyskerin (i.e., DKC1). Our findings demonstrate that pseudouridylation by dyskerin modulates mRNA translatability, with important implications for both normal development and disease.

Small Cajal body-associated RNA 2 (scaRNA2) regulates DNA repair pathway choice by inhibiting DNA-PK.

Evidence that long non-coding RNAs (lncRNAs) participate in DNA repair is accumulating, however, whether they can control DNA repair pathway choice is unknown. Here we show that the small Cajal body-specific RNA 2 (scaRNA2) can promote HR by inhibiting DNA-dependent protein kinase (DNA-PK) and, thereby, NHEJ. By binding to the catalytic subunit of DNA-PK (DNA-PKcs), scaRNA2 weakens its interaction with the Ku70/80 subunits, as well as with the LINP1 lncRNA, thereby preventing catalytic activation of the enzyme. Inhibition of DNA-PK by scaRNA2 stimulates DNA end resection by the MRN/CtIP complex, activation of ATM at DNA lesions and subsequent repair by HR. ScaRNA2 is regulated in turn by WRAP53ß, which binds this RNA, sequestering it away from DNA-PKcs and allowing NHEJ to proceed. These findings reveal that RNA-dependent control of DNA-PK catalytic activity is involved in regulating whether the cell utilizes NHEJ or HR.

Serglycin-Deficiency Causes Reduced Weight Gain and Changed Intestinal Cytokine Responses in Mice Infected With Giardia intestinalis.

The proteoglycan serglycin (SG) is expressed by different innate and adaptive immune cells, e.g. mast cells, macrophages, neutrophils, and cytotoxic T lymphocytes, where SG contributes to correct granule storage and extracellular activity of inflammatory mediators. Here the serglycin-deficient (SG-/-) mouse strain was used to investigate the impact of SG on intestinal immune responses during infection with the non-invasive protozoan parasite Giardia intestinalis. Young (≈11 weeks old) oral gavage-infected congenic SG-/- mice showed reduced weight gain as compared with the infected SG+/+ littermate mice and the PBS-challenged SG-/- and SG+/+ littermate mice. The infection caused no major morphological changes in the small intestine. However, a SG-independent increased goblet cell and granulocyte cell count was observed, which did not correlate with an increased myeloperoxidase or neutrophil elastase activity. Furthermore, infected mice showed increased serum IL-6 levels, with significantly reduced serum IL-6 levels in infected SG-deficient mice and decreased intestinal expression levels of IL-6 in the infected SG-deficient mice. In infected mice the qPCR analysis of alarmins, chemokines, cytokines, and nitric oxide synthases (NOS), showed that the SG-deficiency caused reduced intestinal expression levels of TNF-α and CXCL2, and increased IFN-γ, CXCL1, and NOS1 levels as compared with SG-competent mice. This study shows that SG plays a regulatory role in intestinal immune responses, reflected by changes in chemokine and cytokine expression levels and a delayed weight gain in young SG-/- mice infected with G. intestinalis.

High Cysteine Membrane Proteins (HCMPs) Are Up-Regulated During Giardia-Host Cell Interactions.

Giardia intestinalis colonizes the upper small intestine of humans and animals, causing the diarrheal disease giardiasis. This unicellular eukaryotic parasite is not invasive but it attaches to the surface of small intestinal epithelial cells (IECs), disrupting the epithelial barrier. Here, we used an in vitro model of the parasite's interaction with host IECs (differentiated Caco-2 cells) and RNA sequencing (RNAseq) to identify differentially expressed genes (DEGs) in Giardia, which might relate to the establishment of infection and disease induction. Giardia trophozoites interacted with differentiated Caco-2 cells for 1.5, 3, and 4.5 h and at each time point, 61, 89, and 148 parasite genes were up-regulated more than twofold, whereas 209, 265, and 313 parasite genes were down-regulated more than twofold. The most abundant DEGs encode hypothetical proteins and members of the High Cysteine Membrane Protein (HCMP) family. Among the up-regulated genes we also observed proteins associated with proteolysis, cellular redox balance, as well as lipid and nucleic acid metabolic pathways. In contrast, genes encoding kinases, regulators of the cell cycle and arginine metabolism and cytoskeletal proteins were down-regulated. Immunofluorescence imaging of selected, up-regulated HCMPs, using C-terminal HA-tagging, showed localization to the plasma membrane and peripheral vesicles (PVs). The expression of the HCMPs was affected by histone acetylation and free iron-levels. In fact, the latter was shown to regulate the expression of many putative giardial virulence factors in subsequent RNAseq experiments. We suggest that the plasma membrane localized and differentially expressed HCMPs play important roles during Giardia-host cell interactions.

The compact genome of Giardia muris reveals important steps in the evolution of intestinal protozoan parasites.

Diplomonad parasites of the genus Giardia have adapted to colonizing different hosts, most notably the intestinal tract of mammals. The human-pathogenic Giardia species, Giardia intestinalis, has been extensively studied at the genome and gene expression level, but no such information is available for other Giardia species. Comparative data would be particularly valuable for Giardia muris, which colonizes mice and is commonly used as a prototypic in vivo model for investigating host responses to intestinal parasitic infection. Here we report the draft-genome of G. muris. We discovered a highly streamlined genome, amongst the most densely encoded ever described for a nuclear eukaryotic genome. G. muris and G. intestinalis share many known or predicted virulence factors, including cysteine proteases and a large repertoire of cysteine-rich surface proteins involved in antigenic variation. Different to G. intestinalis, G. muris maintains tandem arrays of pseudogenized surface antigens at the telomeres, whereas intact surface antigens are present centrally in the chromosomes. The two classes of surface antigens engage in genetic exchange. Reconstruction of metabolic pathways from the G. muris genome suggest significant metabolic differences to G. intestinalis. Additionally, G. muris encodes proteins that might be used to modulate the prokaryotic microbiota. The responsible genes have been introduced in the Giardia genus via lateral gene transfer from prokaryotic sources. Our findings point to important evolutionary steps in the Giardia genus as it adapted to different hosts and it provides a powerful foundation for mechanistic exploration of host-pathogen interaction in the G. muris-mouse pathosystem.

The Chymase Mouse Mast Cell Protease-4 Regulates Intestinal Cytokine Expression in Mature Adult Mice Infected with Giardiaintestinalis.

Mast cells have been shown to affect the control of infections with the protozoan parasite Giardia intestinalis. Recently, we demonstrated that Giardia excretory-secretory proteins inhibited the activity of the connective tissue mast cell-specific protease chymase. To study the potential role of the chymase mouse mast cell protease (mMCP)-4 during infections with Giardia, mMCP-4+/+ and mMCP-4-/- littermate mice were gavage-infected with G. intestinalis trophozoites of the human assemblage B isolate GS. No significant changes in weight gain was observed in infected young (≈10 weeks old) mMCP-4-/- and mMCP-4+/+ littermate mice. In contrast, infections of mature adult mice (>18 weeks old) caused significant weight loss as compared to uninfected control mice. We detected a more rapid weight loss in mMCP-4-/- mice as compared to littermate mMCP-4+/+ mice. Submucosal mast cell and granulocyte counts in jejunum increased in the infected adult mMCP-4-/- and mMCP-4+/+ mice. This increase was correlated with an augmented intestinal trypsin-like and chymotrypsin-like activity, but the myeloperoxidase activity was constant. Infected mice showed a significantly lower intestinal neutrophil elastase (NE) activity, and in vitro, soluble Giardia proteins inhibited human recombinant NE. Serum levels of IL-6 were significantly increased eight and 13 days post infection (dpi), while intestinal IL-6 levels showed a trend to significant increase 8 dpi. Strikingly, the lack of mMCP-4 resulted in significantly less intestinal transcriptional upregulation of IL-6, TNF-α, IL-25, CXCL2, IL-2, IL-4, IL-5, and IL-10 in the Giardia-infected mature adult mice, suggesting that chymase may play a regulatory role in intestinal cytokine responses.

Giardia excretory-secretory proteins modulate the enzymatic activities of mast cell chymase and tryptase.

BACKGROUND: Mast cells are involved in the host immune response controlling infection with the non-invasive intestinal protozoan parasite Giardia intestinalis. Experimental infections in rodents with G. intestinalis showed increased intestinal expression of mucosal and connective mast cell specific proteases suggesting that both mucosal and connective tissue mast cells are recruited and activated during infection. During infection Giardia excretory-secretory proteins (ESPs) with immunomodulatory capacity are released. However, studies investigating potential interactions between Giardia ESPs and the connective tissue mast cell specific serine proteases, i.e. human chymase and mouse mast cell protease (mMCP)-4 and, human and mouse tryptase (mMCP-6) remain scarce. RESULTS: We first investigated if soluble Giardia proteins (sGPs), which over-lap extensively in protein content with ESP fractions, from the isolates GS, WB and H3, could induce mast cell activation. sGPs induced a minor activation of bone marrow derived mucosal-like mast cells, as indicated by increased IL-6 secretion and no degranulation. Furthermore, sGPs were highly resistant to degradation by human tryptase while human chymase degraded a 65 kDa sGP and, wild-type mouse ear tissue extracts degraded several protein bands in the 10 to 75 kDa range. In striking contrast, sGPs and ESPs were found to increase the enzymatic activity of human and mouse tryptase and to reduce the activity of human and mouse chymase. CONCLUSION: Our finding suggests that Giardia ssp. via enhancement or reduction of mast cell protease activity may modulate mast cell-driven intestinal immune responses. ESP-mediated modulation of the mast cell specific proteases may also increase degradation of tight junctions, which may be beneficial for Giardia ssp. during infection.

Secreted Giardia intestinalis cysteine proteases disrupt intestinal epithelial cell junctional complexes and degrade chemokines.

Giardiasis is a common diarrheal disease caused by the protozoan parasite Giardia intestinalis. Cysteine proteases (CPs) are acknowledged as virulence factors in Giardia but their specific role in the molecular pathogenesis of disease is not known. Herein, we aimed to characterize the three main secreted CPs (CP14019, CP16160 and CP16779), which were identified by mass spectrometry in the medium during interaction with intestinal epithelial cells (IECs) in vitro. First, the CPs were epitope-tagged and localized to the endoplasmic reticulum and cytoplasmic vesicle-like structures. Second, we showed that recombinant CPs, expressed in Pichia pastoris, are more active in acidic environment (pH 5.5-6) and we determined the kinetic parameters using fluorogenic substrates. Third, excretory-secretory proteins (ESPs) from Giardia trophozoites affect the localization of apical junctional complex (AJC) proteins and recombinant CPs cleave or re-localize the AJC proteins (claudin-1 and -4, occludin, JAM-1, ß-catenin and E-cadherin) of IECs. Finally, we showed that the ESPs and recombinant CPs can degrade several chemokines, including CXCL1, CXCL2, CXCL3, IL-8, CCL2, and CCL20, which are up-regulated in IECs during Giardia-host cell interactions. This is the first study that characterizes the role of specific CPs secreted from Giardia and our results collectively indicate their roles in the disruption of the intestinal epithelial barrier and modulating immune responses during Giardia infections.

Characterization of the Giardia intestinalis secretome during interaction with human intestinal epithelial cells: The impact on host cells.

BACKGROUND: Giardia intestinalis is a non-invasive protozoan parasite that causes giardiasis in humans, the most common form of parasite-induced diarrhea. Disease mechanisms are not completely defined and very few virulence factors are known. METHODOLOGY: To identify putative virulence factors and elucidate mechanistic pathways leading to disease, we have used proteomics to identify the major excretory-secretory products (ESPs) when Giardia trophozoites of WB and GS isolates (assemblages A and B, respectively) interact with intestinal epithelial cells (IECs) in vitro. FINDINGS: The main parts of the IEC and parasite secretomes are constitutively released proteins, the majority of which are associated with metabolism but several proteins are released in response to their interaction (87 and 41 WB and GS proteins, respectively, 76 and 45 human proteins in response to the respective isolates). In parasitized IECs, the secretome profile indicated effects on the cell actin cytoskeleton and the induction of immune responses whereas that of Giardia showed anti-oxidation, proteolysis (protease-associated) and induction of encystation responses. The Giardia secretome also contained immunodominant and glycosylated proteins as well as new candidate virulence factors and assemblage-specific differences were identified. A minor part of Giardia ESPs had signal peptides (29% for both isolates) and extracellular vesicles were detected in the ESPs fractions, suggesting alternative secretory pathways. Microscopic analyses showed ESPs binding to IECs and partial internalization. Parasite ESPs reduced ERK1/2 and P38 phosphorylation and NF-κB nuclear translocation. Giardia ESPs altered gene expression in IECs, with a transcriptional profile indicating recruitment of immune cells via chemokines, disturbances in glucose homeostasis, cholesterol and lipid metabolism, cell cycle and induction of apoptosis. CONCLUSIONS: This is the first study identifying Giardia ESPs and evaluating their effects on IECs. It highlights the importance of host and parasite ESPs during interactions and reveals the intricate cellular responses that can explain disease mechanisms and attenuated inflammatory responses during giardiasis.

Human Memory CD4+ T Cell Immune Responses against Giardia lamblia.

The intestinal protozoan parasite Giardia lamblia may cause severe prolonged diarrheal disease or pass unnoticed as an asymptomatic infection. T cells seem to play an important role in the immune response to Giardia infection, and memory responses may last years. Recently, TH17 responses have been found in three animal studies of Giardia infection. The aim of this study was to characterize the human CD4(+) T cell responses to Giardia. Peripheral blood mononuclear cells (PBMCs) were obtained from 21 returning travelers with recent or ongoing giardiasis and 12 low-risk healthy controls and stimulated in vitro with Giardia lamblia proteins. Production of tumor necrosis factor alpha (TNF-α), gamma interferon, interleukin-17A (IL-17A), IL-10, and IL-4 was measured in CD4(+) effector memory (EM) T cells after 24 h by flow cytometry. After 6 days of culture, activation and proliferation were measured by flow cytometry, while an array of inflammatory cytokine levels in supernatants were measured with multiplex assays. We found the number of IL-17A-producing CD4(+) EM T cells, as well as that of cells simultaneously producing both IL-17A and TNF-α, to be significantly elevated in the Giardia-exposed individuals after 24 h of antigen stimulation. In supernatants of PBMCs stimulated with Giardia antigens for 6 days, we found inflammation-associated cytokines, including 1L-17A, as well as CD4(+) T cell activation and proliferation, to be significantly elevated in the Giardia-exposed individuals. We conclude that symptomatic Giardia infection in humans induces a CD4(+) EM T cell response of which IL-17A production seems to be an important component.

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.

High level of heterogeneity among Listeria monocytogenes isolates from clinical and food origin specimens in Greece.

In order to examine the genetic variation of clinical and food isolates of Listeria monocytogenes in Greece, a total of 61 L. monocytogenes non-duplicate isolates, recovered from clinical specimens (n=19) and food (n=42), were serotyped and genotyped using two different Random Amplification of Polymorphic DNA (RAPD) protocols and Multiple Locus Variable Number Tandem Repeat Analysis (MLVA). Serotype group 4b, 4d, 4e prevailed (39.4%), among both clinical and food isolates, followed by serotype group 1/2a, 3a (23.0%), which nevertheless was detected only among food isolates. The most discriminatory typing protocol was MLVA, which grouped four isolates into two pairs, while the remaining isolates produced unique fingerprints. Similar results were obtained when taking into account the combination of the two RAPD protocols (Simpson index 0.999); six isolates were grouped into three pairs, two of which were the pairs that were identified also by MLVA. Single use of each RAPD protocol resulted in inferior discrimination (Simpson index 0.978 and 0.997, respectively). In conclusion, the two molecular procedures, MLVA, and the combined RAPD protocols, produced similar results, showing that L. monocytogenes isolates from clinical and food specimens were highly heterogenous and that clustering was very uncommon.