Purpose of Review Entamoeba histolytica is a protozoan parasite that causes amebiasis, which remains a significant cause of morbidity and mortality worldwide. E. histolytica causes tissue destruction which leads to clinical disease. This review outlines some of the recent advances that have furthered our understanding of the processes that lead to the tissue damage caused by E. histolytica. Recent Findings Recent studies have identified new mechanisms involved in E. histolytica –induced tissue damage. These include (i) new form of contact-dependent killing called trogocytosis; (ii) parasite-produced cytokine, macrophage migration inhibitory factor, that contributes to inflammation; (iii) exploitation of host immune response to promote invasion; and (iv) the contribution of the gut microbiome to clinical disease. Summary Targeting these mechanisms that result in tissue injury should be a focus of future research for the development of improved preventive and therapeutic strategies for amebiasis.

Understanding how the protozoan protein degradation pathway is regulated could uncover new parasite biology for drug discovery. We found the COP9 signalosome (CSN) conserved in multiple pathogens such as Leishmania, Trypanosoma, Toxoplasma, and used the severe diarrhea-causing Entamoeba histolytica to study its function in medically significant protozoa. We show that CSN is an essential upstream regulator of parasite protein degradation. Genetic disruption of E. histolytica CSN by two distinct approaches inhibited cell proliferation and viability. Both CSN5 knockdown and dominant negative mutation trapped cullin in a neddylated state, disrupting UPS activity and protein degradation. In addition, zinc ditiocarb (ZnDTC), a main metabolite of the inexpensive FDA-approved globally-available drug disulfiram, was active against parasites acting in a COP9-dependent manner. ZnDTC, given as disulfiram-zinc, had oral efficacy in clearing parasites in vivo. Our findings provide insights into the regulation of parasite protein degradation, and supports the significant therapeutic potential of COP9 inhibition.

Targeting virulence factors represents a promising alternative approach to antimicrobial therapy, through the inhibition of pathogenic pathways that result in host tissue damage. Yet, virulence inhibition remains an understudied area in parasitology. Several medically important protozoan parasites such as Plasmodium, Entamoeba, Toxoplasma, and Leishmania secrete an inflammatory macrophage migration inhibitory factor (MIF) cytokine homolog, a virulence factor linked to severe disease. The aim of this study was to investigate the effectiveness of targeting parasite-produced MIF as combination therapy with standard antibiotics to reduce disease severity. Here, we used Entamoeba histolytica as the model MIF-secreting protozoan, and a mouse model that mirrors severe human infection. We found that intestinal inflammation and tissue damage were significantly reduced in mice treated with metronidazole when combined with anti–E. histolytica MIF antibodies, compared to metronidazole alone. Thus, this preclinical study provides proof-of-concept that combining antiparasite MIF-blocking antibodies with current standard-of-care antibiotics might improve outcomes in severe protozoan infections.

Understanding how the protozoan protein degradation pathway is regulated could uncover new parasite biology for drug discovery. We found the COP9 signalosome (CSN) conserved in multiple pathogens such as Leishmania, Trypanosoma, Toxoplasma, and used the severe diarrhea-causing Entamoeba histolytica to study its function in medically significant protozoa. We show that CSN is an essential upstream regulator of parasite protein degradation. Genetic disruption of E. histolytica CSN by two distinct approaches inhibited cell proliferation and viability. Both CSN5 knockdown and dominant negative mutation trapped cullin in a neddylated state, disrupting UPS activity and protein degradation. In addition, zinc ditiocarb (ZnDTC), a main metabolite of the inexpensive FDA-approved alcohol-abuse drug disulfiram, was active against parasites acting in a COP9-dependent manner. ZnDTC, given as disulfiram-zinc, had oral efficacy in clearing parasites in vivo. Our findings provide insights into the regulation of parasite protein degradation, and supports the significant therapeutic potential of COP9 inhibition.Summary sentenceParasite-encoded COP9 signalosome is an essential upstream regulator of ubiquitin-proteasome mediated protein degradation, and shows significant potential as a therapeutic target.

The Ubiquitin‐Proteasomal System (UPS) is essential for cell survival because it is responsible for the large majority of protein degradation within the cell. Thus, targeting the UPS has emerged as an attractive approach for combating protozoan parasites. Yet, the regulation of UPS‐mediated protein degradation remains poorly understood in clinically important protozoans. We identified COP9 signalosome to be conserved in multiple pathogenic parasites including Leishmania, Trypanosoma, Toxoplasma and Entamoeba. Here, we show that COP9 signalosome is an essential and druggable parasite target that regulates protein degradation. Using Entamoeba histolytica as a model parasite, we found that genetic disruption of COP9 subunit 5 (CSN5) by two independent but complementary approaches, inhibited parasite cell proliferation and viability. Both CSN5 knockdown and dominant negative mutation trapped cullin1 in a neddylated state disrupting UPS activity and protein degradation. Using virtual screening by molecular docking, we identified zinc dithiocarbamate (ZnDTC), a metabolite of the FDA approved alcohol drug disulfiram, as a potential COP9 inhibitor. We found that ZnDTC acts in a COP9‐dependent manner, phenocopy CSN5 gene disruption, and is active against E. histolytica parasites at nanomolar concentrations. In addition, in‐vivo studies revealed that ZnDTC had oral efficacy in clearing E. histolytica parasites in a mouse model that mirrors human amebic colitis. Parasite clearance was assessed by live bioluminescent imaging, amebic culture and immunohistochemistry. Also, ZnDTC treatment promoted resolution of inflammation and tissue damage. Our findings provide insights into how COP9 signalosome regulates parasite protein degradation, and supports COP9 inhibition as a potential anti‐parasite therapy.