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Displaying One Session

01/01/1970

02. Parasites of humans
Session Type
02. Parasites of humans
Date
08/22/2022
Session Time
17:00 - 18:30
Room
Auditorium 11
02. Parasites of humans

ANTI-INFLAMMATORY ACTIVITIES OF GIARDIA INTESTINALIS- ON OR OFF MAKES A DIFFERENCE (ID 129)

Session Type
02. Parasites of humans
Date
08/22/2022
Session Time
17:00 - 18:30
Room
Auditorium 11
Lecture Time
17:00 - 17:30
Onsite or Pre-Recorded
Onsite

Abstract

Abstract Body

Background and aims

Giardia intestinalis is a major contributor to the enormous burden of diarrheal diseases with 180 million symptomatic human infections per year and more than 0.5 billion humans are currently asymptomatically colonized by the parasite. Most infectious agents causing symptomatic gastrointestinal infections cause a severe inflammation in the intestine but this is usually not seen during a Giardia infection. Our aim in this study was to try to find out why Giardia is not inducing a high level of inflammation during infections.

Methods

We used an in vitro model of the parasite’s interaction with host IECs (differentiated Caco-2 cells and human enteroids) and Giardia WB trophozoites, early encysting cells (7 h), and cysts. Dual RNA sequencing, RT-PCR, Western blotting, immuno-localization, transfections with gene fusions, tester cell lines and specific inhibitors of signaling pathways were used characterize the host-parasite interactions.

Results

Many inflammatory chemokines (e.g. CXCL1 to 3 and CCL20) are first induced in the intestinal epithelial cells by binding of parasite-specific factors to pattern recognition receptors. Both trophozoites and cysts induce these chemokines but there are also chemokines specifically induced by cysts (CCL4L2, CCL5 and CXCL5). The transcription factors AP-1 and NFκB are activated early during the interaction and initiate transcription of chemokines. However, this initial induction of inflammatory signals is quickly reversed by several different anti-inflammatory pathways to induce an anti-inflammatory state and trophozoites have a better ability to suppress the inflammatory responses than cysts. The RNA binding protein Tristetraprolin (TTP) is highly and constantly expressed in the presence of attached parasites and the chemokine mRNAs are degraded in P bodies. NFκB subunits are down-regulated and blocked from the nucleus after the initial induction. Secreted parasite cysteine proteases (CPs) degrade released chemokines and the amount of free-arginine is reduced in the local environment, inhibiting immune cell replication and NO production.

Conclusions

Giardia can actively reduce inflammation in the intestine using several different complementary mechanisms, even if it initially induces inflammatory chemokines. This anti-inflammatory activity is important during Giardia infections but also during co-infections by other intestinal pathogens.

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02. Parasites of humans

BIOLOGICAL AND IMMUNOLOGICAL CHARACTERIZATION OF ENTAMOEBA HISTOLYTICA-DERIVED EXTRACELLULAR VESICLES (ID 1281)

Session Type
02. Parasites of humans
Date
08/22/2022
Session Time
17:00 - 18:30
Room
Auditorium 11
Lecture Time
17:30 - 17:45
Onsite or Pre-Recorded
Onsite

Abstract

Introduction

Entamoeba histolytica is the causative agent of amebiasis. While 90 % of intestinal infections remain asymptomatic, 10 % result in invasive disease. In some cases, parasites can invade the mucosa and migrate to the liver via the bloodstream, where amebic liver abscesses (ALAs) are formed. Interestingly, ALA formation is more prevalent in men compared with women. Onset of invasive disease is the result of parasite pathogenicity factors on the one hand and an overshooting immune response mainly involving infiltrating monocytes and neutrophils on the other. In this context, extracellular vesicles (EVs) are investigated to gain a better understanding of the interaction between parasite and host.

Methods

EVs were isolated from E. histolytica culture supernatant using an ultracentrifugation-based approach and characterized using transmission electron microscopy, nanoparticle tracking analysis, proteomics and miRNA sequencing. Two clonal cell lines differing in their pathogenicity were used for EV isolation. Analysis of putative immunostimulatory properties of these EVs was performed in vitro on primary murine monocytes from both male and female mice. RNAseq, flow cytometry and cytokine profiling were implemented for the characterization of immune cell response.

Results

More than 1000 E. histolytica proteins and over 7000 miRNAs were detected in EVs. Vesicles range in size from approximately 50 – 400 nm. Stimulation of primary murine monocytes with EVs resulted in increased expression of activation markers on the cell surface and an increase in the secretion of pro-inflammatory cytokines. Gene regulation upon EV stimulation was observed using RNAseq.

Conclusions

Our data suggest an involvement of EVs in the immune response to E. histolytica infection.

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02. Parasites of humans

GIARDIA DUODENALIS MRNA BOUND PROTEOME REVEALED THE EMERGENCE OF EUKARYOTIC SPECIFIC POST-TRANSCRIPTIONAL REGULATORY NETWORKS (ID 1094)

Session Type
02. Parasites of humans
Date
08/22/2022
Session Time
17:00 - 18:30
Room
Auditorium 11
Lecture Time
17:45 - 18:00
Onsite or Pre-Recorded
Onsite

Abstract

Introduction

RNA binding proteins (RBPs) are major post-transcriptional regulators (PTR). In higher eukaryotes RBPs control transcription, RNA transport, splicing and degradation, translation and translational repression and play key roles in cell fating, pluripotency and differentiation. Surprisingly, the eukaryotic RBPome is fundamentally unchanged from yeast to humans. This suggests many novel RBPs emerged in basal eukaryotes. Yet this is largely unstudied. Here, we characterised the RBPome of G. duodenalis, which belongs to one of the earliest branching eukaryotic lineages, and used this to understand the evolution of a conserved and essential layer of eukaryotic regulation.

Methods

We undertook in silico curation, RNA interactome capture (RIC) and high-resolution mass spectrometry to characterise Giardia’s RBPome. We then explored the function of key RBPs including the earliest known Pumilio homologs (PUF, PUM) and two significant helicases, DDX3X and EIF4A in Giardia, CRISPRi mediated knockdowns, RBP-crosslinking immunoprecipitation (CLiP), quantitative proteomics and in vitro protein expression.

Results

Despite Giardia’s basal evolutionary origins and minimalistic regulatory systems, its RBPome has most novel functions found in higher eukaryotes including the capacity to form membraneless organelles, translationally repressing complex mRNA networks and use this to control major changes in its life-cycle and stress responses.

Conclusions

Giardia provides the earliest eukaryotic record of many of the RBP families acquired by higher eukaryotes and highlights the central role these have played in eukaryotic evolution.

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02. Parasites of humans

ANALYSIS OF INTESTINAL INVASION PROCESS BY E. HISTOLYTICA USING ORGANOID-DERIVED 2D MONOLAYERS (ID 328)

Session Type
02. Parasites of humans
Date
08/22/2022
Session Time
17:00 - 18:30
Room
Auditorium 11
Lecture Time
18:00 - 18:05
Onsite or Pre-Recorded
Onsite

Abstract

Introduction

Entamoeba histolytica is the causative agent of amoebiasis. E. histolytica can live in the human intestine for months or years without causing symptoms in the host. For reasons that are still unclear, the amoebae can suddenly destroy the intestinal mucosa and become invasive. This can lead to amoebic colitis or extraintestinal amoebic liver abscesses. To analyze the invasion process, only very artificial animal models exist so far.

Methods

We are trying to better understand the invasion process using human intestinal organoid-derived 2D monolayers and clones as well as transfectants of E. histolytica that differ in pathogenicity.

Results

Preliminary results indicate that pathogenic and non-pathogenic amoebae can invade a human organoid-derived 2D monolayer at the same rate. Inhibition of cysteine peptidases, which are postulated to be an important factor in the invasion process, significantly reduce monolayer destruction only in pathogenic amoebae. In contrast, inhibition of metallopeptidases leads to significant reduction of monolayer destruction only by the non-pathogenic amoebae.

After 4-hour co-incubation of non-pathogenic amoebae with the 2D monolayer, 260 human genes were identified as significantly differentially expressed. In contrast, co-incubation with pathogenic amoebae significantly changed the expression of only 48 genes. The genes most affected by regulation are those encoding proteins of the immune system.

Conclusions

Through this result, it can be postulated that the non-pathogenic amoebae stimulate the immune system more and are therefore better eliminated than the pathogenic amoebae.

This work is supported by the Deutsche Forschungsgemeinschaft (BR 1744).

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02. Parasites of humans

COMPARATIVE PROTEOMIC PROFILING OF EXTRACELLULAR SECRETED VESICLES BETWEEN GIARDIA DUODENALIS ASSEMBLAGE A AND B (ID 786)

Session Type
02. Parasites of humans
Date
08/22/2022
Session Time
17:00 - 18:30
Room
Auditorium 11
Lecture Time
18:05 - 18:10
Onsite or Pre-Recorded
Onsite

Abstract

Introduction

The parasitic protozoan Giardia duodenalis is responsible for human and animal giardiasis, a diarrheal disease with a worldwide distribution. Human disease ranges from asymptomatic, to acute and chronic diarrhea, eventually causing malnutrition and stunting in children, and long term post-infectious sequelae. Two Giardia genetic groups (Assemblages A and B) cause human infection. Differences at genomic level between and within Assemblages can account for variations in growth rate, infectivity, and pathogenicity. Extracellular vesicles (EVs) operate as cargoes from cell to cell, carrying proteins and nucleic acids, being implicated in physiological and pathological processes. Giardia also releases EVs. Aim of this work was to isolate EVs from assemblage A and B reference isolates to define and compare EVs proteomes in the attempt to find a link with differences in assemblage pathogenicity.

Methods

Released EVs and freely secreted protein were purified from trophozoites, grown in well-defined culture condition, by differential ultracentrifugation. EVs were morphologically and biochemically characterized by electron microscopy and dynamic light scatter (DLS). EVs and secretome was characterized by proteomic analysis.

Results

EVs with different sizes and shapes were released by both Giardia isolates, in different amount. EVs, but not the secretome, showed the presence of typical EVs markers. Unique Giardia proteins, such as virulence factor candidates and assemblage-specific proteins were also present.

Conclusions

Our results provide evidence that differences in amount and content of EVs occur between Assemblage A and B pointing on their role in determining difference in Giardia isolates pathogenicity and virulence.

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02. Parasites of humans

EXPERIMENTAL INFECTIONS WITH TWO DIFFERENT BLASTOCYSTIS SUBTYPES ARE ASSOCIATED WITH STRIKINGLY DIFFERENT MICROBIOME FEATURES AND PATHOBIOLOGICAL OUTCOMES (ID 688)

Session Type
02. Parasites of humans
Date
08/22/2022
Session Time
17:00 - 18:30
Room
Auditorium 11
Lecture Time
18:10 - 18:15
Onsite or Pre-Recorded
Onsite

Abstract

Introduction

The associations between Blastocystis and gut microbiota have been extensively studied, but no study so far has examined the influence of different subtypes (STs) of Blastocystis on gut microbiota under experimentally controlled conditions. Furthermore, whether Blastocystis-altered gut microbiota affects the development of intestinal inflammatory diseases remains to be determined.

Methods

In this study, we first investigated the effect of ST4, a common subtype in Europe, and ST7, a rare pathogenic subtype in humans, on the intestinal microbiota in normal healthy mice, and then explored the role of Blastocystis-altered gut microbiome in the development of dextran sulfate sodium (DSS)-induced colitis in antibiotic treated wild type and Rag1-/- mice.

Results

We showed that ST4 infection increased the bacterial diversity and abundance of Clostridia, whereas ST7 infection showed opposite effects. Transplantation of ST4-altered microbiota was able to prevent DSS-induced colitis by enhancing the Treg response (increased Foxp3 and IL-10) in the colon lamina propria of recipient mice, while ST7-altered microbiota exacerbated the severity of the colitis by inducing Th1 cell differentiation (increased IFN-γ, and TNF-α). Furthermore, the protective or exacerbating effects of Blastocystis-altered gut microbiota on colitis are adaptive immune cell dependent.

Conclusions

Our data showed that ST4 and ST7 infections are associated with strikingly different microbiome features, and these alterations have significantly different effects on the severity of DSS-induced colitis. This study supports accumulating evidence that clinical outcomes of Blastocystis infection is subtype dependent.

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02. Parasites of humans

GIARDIA DUODENALIS EXTRACELLULAR VESICLES MODULATE THE GROWTH AND BEHAVIOR OF COMMENSAL BACTERIA VIA SMALL RNAS ACTIVITY (ID 1003)

Session Type
02. Parasites of humans
Date
08/22/2022
Session Time
17:00 - 18:30
Room
Auditorium 11
Lecture Time
18:15 - 18:20
Onsite or Pre-Recorded
Onsite

Abstract

Introduction

Extracellular vesicles (EVs) play important roles in the pathophysiology of parasites. While the protozoan parasite Giardia duodenalis can produces EVs, their exact role in the pathogenesis of giardiasis remains to be fully understood. In this study, we examined whether Giardia EVs could mediate interactions with commensal bacteria.

Methods

G. duodenalis NF EVs were isolated using Exo-Easy Kit. EVs were characterized using Nanoparticle track analysis and transmission electron microscopy. EVs Small-RNAs were characterized by RNA-Sequencing analysis. Kinetic experiments were conducted on EVs-treated E. coli HB101 and Enterobacter cloacae (human isolate). Swimming motility of EVs-treated E. coli HB101 and E. cloacae was assessed on agar. Biofilm biomass of EVs treated bacteria was investigated using the Calgary Biofilm Device. Adhesion of EVs-treated bacteria was assessed on SCBN intestinal epithelial cells.

Results

Giardia EVs exerted bacteriostatic effects on E. coli HB101 and E. cloacae (p<0.05). EVs decreased the ability of E. coli HB101 to form biofilms. Giardia EVs significantly increased the swimming motility of both E. coli HB101 and E. cloacae as well as their adhesion to the intestinal epithelial cells (p<0.05). RNase assays showed that EVs total RNAs but not proteins modulate bacterial swimming motility and biofilm formation. Small RNAs analysis revealed the presence of heat stable rRNA, miRNA, tRNA, snRNA and snoRNA.

Conclusions

This study indicates that Giardia EVs can mediate interactions with the commensal bacteria by increasing their virulence. G. duodenalis EVs, via their nucleic acid content, can trigger the formation of pathobionts, depicting a novel trans-kingdom cross-talk in the gut.

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02. Parasites of humans

Q&A (ID 2335)

Session Type
02. Parasites of humans
Date
08/22/2022
Session Time
17:00 - 18:30
Room
Auditorium 11
Lecture Time
18:20 - 18:25
Onsite or Pre-Recorded
Onsite