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.

Abstract Body

Metronidazole (MTZ) is a clinically important antimicrobial agent that is active against both bacterial and protozoan anaerobic and microaerophilic organisms. A recent and dramatic increase in the number of MTZ resistant bacteria and protozoa is of great concern since there are few alternative drugs with a similarly broad activity spectrum. To identify key factors and mechanisms underlying MTZ resistance in the protozoan parasite Giardia intestinalis we characterized two in vitro selected, metronidazole resistant parasite lines, as well as one revertant, by analyzing fitness aspects associated with increased drug resistance, as well as changes in their genomes, transcriptomes and proteomes. We also conducted meta-analyses using already existing omicsdata from additional MTZ resistant G. intestinalis isolates. The combined data suggest that in vitro generated MTZ resistance has a substantial fitness cost to the parasite, which may partly explain why resistance is not widespread despite decades of heavy use. Mechanistically, MTZ resistance in Giardia is multifactorial and associated with complex changes, yet a core set of pathways involving reduced expression of drug-activating oxidoreductases, changed plasma membrane composition and increased intracellular oxygen levels are central to MTZ resistance in both Giardia and bacteria.