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

ABSTRACT: 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.

SUBMITTER: Gruttner J

PROVIDER: S-EPMC10187934 | biostudies-literature | 2023 May

REPOSITORIES: biostudies-literature

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

Grüttner Jana J van Rijn Jorik M JM Geiser Petra P Florbrant Alexandra A Webb Dominic-Luc DL Hellström Per M PM Sundbom Magnus M Sellin Mikael E ME Svärd Staffan G SG

PLoS pathogens 20230504 5

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 ...[more]

PMID: 37141303

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Project description: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.

Project description:In the field of infectious diseases the multifaceted amino acid arginine has reached special attention as substrate for the hosts production of the antimicrobial agent nitric oxide (NO). A variety of infectious organisms interfere with this part of the host immune response by reducing the availability of arginine. This prompted us to further investigate additional roles of arginine during pathogen infections. As a model we used the intestinal parasite Giardia intestinalis that actively consumes arginine as main energy source and secretes an arginine-consuming enzyme, arginine deiminase (ADI). Reduced intestinal epithelial cell (IEC) proliferation is a common theme during bacterial and viral intestinal infections, but it has never been connected to arginine-consumption. Our specific question was thereby, whether the arginine-consumption by Giardia leads to reduced IEC proliferation, in addition to NO reduction. In vitro cultivation of human IEC lines in arginine-free or arginine/citrulline-complemented medium, as well as in interaction with different G. intestinalis isolates, were used to study effects on host cell replication by MTT assay. IEC proliferation was further analyzed by DNA content analysis, polyamine measurements and expressional analysis of cell cycle regulatory genes. IEC proliferation was reduced upon arginine-withdrawal and also in an arginine-dependent manner upon interaction with G. intestinalis or addition of Giardia ADI. We show that arginine-withdrawal by intestinal pathogens leads to a halt in the cell cycle in IECs through reduced polyamine levels and upregulated cell cycle inhibitory genes. This is of importance with regards to intestinal tissue homeostasis that is affected through reduced cell proliferation. Thus, the slower epithelial cell turnover helps the pathogen to maintain a more stable niche for colonization. This study also shows why supplementation therapy of diarrhea patients with arginine/citrulline is helpful and that citrulline especially should gain further attention in future treatment strategies.

Project description:BackgroundGiardia 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.MethodologyTo 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.FindingsThe 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.ConclusionsThis 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.

Project description:Giardia intestinalis is a flagellated protozoan responsible for giardiosis (also called giardiasis in humans), the most prevalent and widespread parasitic infection in humans and mammals worldwide. The intestinal microbiota is highly diverse and any alteration in its composition may impact on the health of the host. While studies on the mouse model of giardiosis described the role of the gut microbiota in host susceptibility to infection by the parasite, little is known about the gut microbiota during natural infections in dogs and particularly in puppies. In this study, we monitored naturally G. intestinalis-infected puppies for 3 months and quantified cyst excretion every 2 weeks. All puppies remained subclinically infected during the sampling period as confirmed by fecal examination. In parallel, we performed 16S Illumina sequencing of fecal samples from the different time points to assess the impact of G. intestinalis infection on gut microbiota development of the puppies, as well as gut health markers of immunity such as fecal IgA and calprotectin. Sequencing results revealed that the canine fecal microbiota of Giardia-infected puppies becomes more complex and less diverse with increasing age. In addition, significant differences in the structure of the microbiota were observed between puppies with high and low Giardia cyst excretion. Chronic subclinical G. intestinalis infection appears to be associated with some detrimental structural changes in the gut microbiota. G. intestinalis-associated dysbiosis is characterized by an enrichment of facultative anaerobic, mucus-degrading, pro-inflammatory species and opportunistic pathogens, as well as a reduction of Lactobacillus johnsonii at specific time points. Calprotectin levels increased with age, suggesting the establishment of chronic low-grade inflammation in puppies. Further work is needed to demonstrate whether these alterations in the canine gut microbiota could lead to a dysbiosis-related disease, such as irritable bowel syndrome (IBS) or inflammatory bowel disease (IBD).

Project description:Enolase, a multifunctional protein expressed by multiple pathogens activates plasminogen to promote proteolysis on components of the extracellular matrix, an important event in early host-pathogen interactions. A secreted form of enolase that is released upon the interaction of trophozoites with epithelial cells has been detected in the secretome of G. duodenalis. However, the role of enolase in the host-pathogen interactions remains largely unknown. In this work, the effects of G. duodenalis enolase (Gd-eno) on the epithelial cell model (IEC-6) were analyzed. Firstly, the coding sequence of Giardia enolase was cloned and the recombinant protein used to raise antibodies that were then used to define the localization and role of enolase in epithelial cell-trophozoite interactions. Gd-eno was detected in small cytoplasmic vesicles as well as at the surface and is enriched in the region of the ventral disk of Giardia trophozoites. Moreover, the blocking of the soluble monomeric form of the enzyme, which is secreted upon interaction with IEC-6 cells by the anti-rGd-eno antibodies, significantly inhibited trophozoite attachment to intestinal IEC-6 cell monolayers. Further, rGd-eno was able to bind human plasminogen (HsPlg) and enhanced plasmin activity in vitro when the trophozoites were incubated with the intrinsic plasminogen activators of epithelial cells. In IEC-6 cells, rGd-eno treatment induced a profuse cell damage characterized by copious vacuolization, intercellular separation and detachment from the substrate; this effect was inhibited by either anti-Gd-eno Abs or the plasmin inhibitor ϵ- aminocaproic acid. Lastly, we established that in epithelial cells rGd-eno treatment induced a necroptotic-like process mediated by tumor necrosis factor α (TNF-α) and the apoptosis inducing factor (AIF), but independent of caspase-3. All together, these results suggest that Giardia enolase is a secreted moonlighting protein that stimulates a necroptotic-like process in IEC-6 epithelial cells via plasminogen activation along to TNFα and AIF activities and must be considered as a virulence factor.

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

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

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