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.

Corrigendum: Responses of the Differentiated Intestinal Epithelial Cell Line Caco-2 to Infection With the Giardia intestinalis GS Isolate.

[This corrects the article DOI: 10.3389/fcimb.2018.00244.].

Responses of the Differentiated Intestinal Epithelial Cell Line Caco-2 to Infection With the Giardia intestinalis GS Isolate.

Giardia intestinalis is a parasitic protist that causes diarrhea in humans, affecting mainly children of the developing world, elderly and immunocompromised individuals. Humans are infected by two major Giardia assemblages (i.e. genetic subtypes), A and B, with the latter being the most common. So far, there is little information on molecular or cellular changes during infections with assemblage B. Here, we used RNA sequencing to study transcriptional changes in Caco-2 intestinal epithelial cells (IECs) co-incubated with assemblage B (GS isolate) trophozoites for 1.5, 3, and 4.5 h. We aimed to identify early molecular events associated with the establishment of infection and followed cellular protein changes up to 10 h. IEC transcriptomes showed a dominance of immediate early response genes which was sustained across all time points. Transcription of inflammatory cytokines (e.g., cxcl1-3, ccl2, 1l1a, and il1b) peaked at 1.5 and 3 h of infection. Compared to co-incubation with assemblage A Giardia, we identified the induction of novel cytokines (cxcl8, cxcl10, csf1, cx3cl1, il12a, il11) and showed that inflammatory signaling is mediated by Erk1/2 phosphorylation (mitogen activated protein kinase, MAPK), nuclear factor kappa B (NFκB) and adaptor protein-1 (AP-1). We also showed that GS trophozoites attenuate P38 (MAPK) phosphorylation in IECs. Low amounts of IL-8, CXCL1 and CCL20 proteins were measured in the interaction medium, which was attributed to cytokine degradation by trophozoite secreted proteases. Based on the transcriptome, the decay of cytokines mRNA mediated by zinc finger protein 36 might be another mechanism controlling cytokine levels at later time points. IEC transcriptomes suggested homeostatic responses to counter oxidative stress, glucose starvation, and disturbances in amino acid and lipid metabolism. A large group of differentially transcribed genes were associated with cell cycle arrest and induction of apoptosis, which was validated at protein level. IEC transcriptomes also suggested changes in tight junction's integrity, microvilli structure and the extracellular mucin layer. This is the first study to illuminate transcriptional and protein regulatory events underlying IECs responses and pathogenesis during Giardia assemblage B infection. It highlights differences compared to assemblage A infections which might account for the differences observed in human infections with the two assemblages.

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.

Transcriptional profiling of Giardia intestinalis in response to oxidative stress.

Giardia intestinalis is a microaerophilic parasite that infects the human upper small intestine, an environment that is fairly aerobic with reactive oxygen species being produced to fight off the parasite. It is quite perplexing how Giardia, lacking conventional eukaryotic antioxidant machinery (e.g. catalase, superoxide dismutase and glutathione peroxidase), can cope with the oxidative stress in this environment. We used transcriptomics (RNA sequencing and quantitative PCR) to study giardial gene expression changes in response to oxygen (O2; 1h) and hydrogen peroxide (H2O2; 150 µM, 500 µM and 1mM for 1h). The results showed phenotypic and transcriptional differences between Giardia isolates of different genotypes (WB, assemblage A and GS, assemblage B), with GS being more tolerant to H2O2 and exhibiting higher basic transcript levels of antioxidant genes (e.g. NADH oxidase lateral transfer candidate, peroxiredoxin 1 (Prx1) and thioredoxin (Trx)-like proteins). Cysteine is a major antioxidant in Giardia and its role in oxidative defense could be highlighted here by the up-regulation of gene transcripts encoding the cysteine-rich variable surface proteins (VSPs) and high cysteine membrane proteins (HCMPs). Genes in the thioredoxin system (Prx1, Trx and Trx reductase) occupied a central role in the gene expression response to oxidative stress, together with genes encoding metabolic (NADPH-producing enzymes, glutathione and glycerol biosynthetic enzymes) and O2-consuming nitric oxide detoxification enzymes (e.g. nitroreductase, flavohemoprotein and a flavodiiron protein). This study reveals the intricate network of genes associated with the oxidative stress response in Giardia, and provides a stepping-stone towards future studies at the protein level.

Drug resistance in Giardia duodenalis.

Giardia duodenalis is a microaerophilic parasite of the human gastrointestinal tract and a major contributor to diarrheal and post-infectious chronic gastrointestinal disease world-wide. Treatment of G. duodenalis infection currently relies on a small number of drug classes. Nitroheterocyclics, in particular metronidazole, have represented the front line treatment for the last 40 years. Nitroheterocyclic-resistant G. duodenalis have been isolated from patients and created in vitro, prompting considerable research into the biomolecular mechanisms of resistance. These compounds are redox-active and are believed to damage proteins and DNA after being activated by oxidoreductase enzymes in metabolically active cells. In this review, we explore the molecular phenotypes of nitroheterocyclic-resistant G. duodenalis described to date in the context of the protist's unusual glycolytic and antioxidant systems. We propose that resistance mechanisms are likely to extend well beyond currently described resistance-associated enzymes (i.e., pyruvate ferredoxin oxidoreductases and nitroreductases), to include NAD(P)H- and flavin-generating pathways, and possibly redox-sensitive epigenetic regulation. Mechanisms that allow G. duodenalis to tolerate oxidative stress may lead to resistance against both oxygen and nitroheterocyclics, with implications for clinical control. The present review highlights the potential for systems biology tools and advanced bioinformatics to further investigate the multifaceted mechanisms of nitroheterocyclic resistance in this important pathogen.