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Intestinal amebiasis is the disease caused by the extracellular protozoan parasite Entamoeba histolytica (Eh) that induces a dynamic and heterogeneous interaction profile with the host immune system during disease pathogenesis. In 90% of asymptomatic infection, Eh resides with indigenous microbiota in the outer mucus layer of the colon without prompting an immune response. However, for reasons that remain unclear, in a minority of the Eh-infected individuals, this fine tolerated relationship is switched to a pathogenic phenotype and advanced to an increasingly complex host-parasite interaction. Eh disease susceptibility depends on parasite virulence factors and their interactions with indigenous bacteria, disruption of the mucus bilayers, and adherence to the epithelium provoking host immune cells to evoke a robust pro-inflammatory response mediated by inflammatory caspases and inflammasome activation. To understand Eh pathogenicity and innate host immune responses, this review highlights recent advances in our understanding of how Eh induces outside-in signaling via Mϕs to activate inflammatory caspases and inflammasome to regulate pro-inflammatory responses.

The mechanism whereby Entamoeba histolytica (Eh) binding with macrophages at the intercellular junction triggers aggressive pro-inflammatory responses in disease pathogenesis is not well understood. The host intracellular protein degradation process autophagy and its regulatory proteins are involved in maintenance of cellular homeostasis and excessive inflammatory responses. In this study we unraveled how Eh hijacks the autophagy process in macrophages to dysregulate pro-inflammatory responses. Direct contact of live Eh with macrophages activated caspase-6 that induced rapid proteolytic degradation of the autophagy ATG16L1 protein complex independent of NLRP3 inflammasome and caspase-3/8 activation. Crohn's disease susceptible ATG16L1 T300A variant was highly susceptible to Eh-mediated degradation that augmented pro-inflammatory cytokines in mice. Quantitative proteomics revealed downregulation of autophagy and vesicle-mediated transport and upregulation of cysteine-type endopeptidase pathways in response to Eh. We conclude during Eh-macrophage outside-in signaling, ATG16L1 protein complex plays an overlooked regulatory role in shaping the pro-inflammatory landscape in amebiasis.

Even though Entamoeba histolytica (Eh)-induced host pro-inflammatory responses play a critical role in disease, we know very little about the host factors that regulate this response. Direct contact between host cell and Eh signify the highest level of danger, and to eliminate this threat, the host immune system elicits an augmented immune response. To understand the mechanisms of this response, we investigated the induction and release of the endogenous alarmin molecule high-mobility group box 1 (HMGB1) that act as a pro-inflammatory cytokine and chemoattractant during Eh infection. Eh in contact with macrophage induced a dose- and time-dependent secretion of HMGB1 in the absence of cell death. Secretion of HMGB1 was facilitated by Eh surface Gal-lectin-activated phosphoinositide 3-kinase and nuclear factor-κB signaling and up-regulation of histone acetyltransferase activity to trigger acetylated HMGB1 translocation from the nucleus. Unlike lipopolysaccharide, Eh-induced HMGB1 release was independent of caspase-1-mediated inflammasome and gasdermin D pores. In vivo, Eh inoculation in specific pathogen-free but not germ-free mice was associated with high levels of pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin-1β, and keratinocyte-derived chemokine, which was suppressed with HMGB1 neutralization. This study reveals that Eh-induced active secretion of the HMGB1 plays a key role in shaping the pro-inflammatory landscape critical in innate host defense against amebiasis.

Entamoeba histolytica (Eh) is the causative agent of amebiasis, one of the major causes of dysentery-related morbidity worldwide. Recent studies have underlined the importance of the intercellular junction between Eh and host cells as a determinant in the pathogenesis of amebiasis. Despite the fact that direct contact and ligation between Eh surface Gal-lectin and EhCP-A5 with macrophage α5β1 integrin are absolute requirements for NLRP3 inflammasome activation and IL-1β release, many other undefined molecular events and downstream signaling occur at the interface of Eh and macrophage. In this study, we investigated the molecular events at the intercellular junction that lead to recognition of Eh through modulation of the macrophage cytoskeleton. Upon Eh contact with macrophages key cytoskeletal-associated proteins were rapidly post-translationally modified only with live Eh but not with soluble Eh proteins or fragments. Eh ligation with macrophages rapidly activated caspase-6 dependent cleavage of the cytoskeletal proteins talin, Pyk2 and paxillin and caused robust release of the pro-inflammatory cytokine, IL-1β. Macrophage cytoskeletal cleavages were dependent on Eh cysteine proteinases EhCP-A1 and EhCP-A4 but not EhCP-A5 based on pharmacological blockade of Eh enzyme inhibitors and EhCP-A5 deficient parasites. These results unravel a model where the intercellular junction between macrophages and Eh form an area of highly interacting proteins that implicate the macrophage cytoskeleton as a sensor for Eh contact that leads downstream to subsequent inflammatory immune responses., Author summary The protozoan parasite Entamoeba histolytica can establish an enteric infection in human hosts that leads to symptoms ranging from diarrhea to abscesses in the liver and the brain. Host susceptibility to amebic infection is in part determined by the quality and potency of the host immune response that occurs once the parasite overcomes the mucus bilayers and colonic epithelial barriers, and invades underlying tissues. At the cellular level, one of the key events that shape the inflammatory response occurs during direct parasite interaction with host macrophages via surface proteins. The ensuing cascades of intracellular signaling events have only partly been uncovered. Interestingly, only direct interaction between live parasites and macrophages, as opposed to soluble factors or dead parasites, is a prerequisite to the generation of a prompt raging pro-inflammatory response. We have sought to further elucidate the mechanisms by which macrophages distinguish live parasites and found that the macrophage cell skeleton undergoes rapid significant alteration upon Eh contact. Furthermore, we uncovered a previously unknown role for two Eh enzymes in triggering macrophage pro-inflammatory responses. Through this work, we gain a better understanding of the molecular interactions that occur at the macrophage-ameba interface that regulate host inflammatory responses.

Entamoeba histolytica (Eh) is a protozoan parasite that infects 10% of the world’s population and results in 100,000 deaths/year from amebic dysentery and/or liver abscess. In most cases, this extracellular parasite colonizes the colon by high affinity binding to MUC2 mucin without disease symptoms, whereas in some cases, Eh triggers an aggressive inflammatory response upon invasion of the colonic mucosa. The specific host-parasite factors critical for disease pathogenesis are still not well characterized. From the parasite, the signature events that lead to disease progression are cysteine protease cleavage of the C-terminus of MUC2 that dissolves the mucus layer followed by Eh binding and cytotoxicity of the mucosal epithelium. The host mounts an ineffective excessive host pro-inflammatory response following contact with host cells that causes tissue damage and participates in disease pathogenesis as Eh escapes host immune clearance by mechanisms that are not completely understood. Ameba can modulate or destroy effector immune cells by inducing neutrophil apoptosis and suppressing respiratory burst or nitric oxide (NO) production from macrophages. Eh adherence to the host cells also induce multiple cytotoxic effects that can promote cell death through phagocytosis, apoptosis or by trogocytosis (ingestion of living cells) that might play critical roles in immune evasion. This review focuses on the immune evasion mechanisms that Eh uses to survive and induce disease manifestation in the host.

Children in developing countries are continually exposed to enteric pathogens [1–4]. This results in high rates of morbidity and mortality, especially in infants [5]. The outcome of an Entamoeba histolytica exposure can be diarrhea, dysentery, or amebic liver abscess, with the latter occurring mainly in men [6]. Disease, however, occurs in a minority of E. histolytica infections (approximately 20%) [6]. While both host and parasite factors contribute to outcome, they do not entirely explain susceptibility [7–9]. A prospective longitudinal study of enteric pathogens in a slum community in Dhaka, Bangladesh, has provided a rich data set to identify parameters important for protection and disease. It has been previously shown that children in this study cohort who are malnourished at birth are more susceptible to amebiasis and in general experience more-severe diarrhea [2]. There is evidence in support of both passive and active acquired immunity. Infants with mothers with high levels of breast milk anti-galactose/N-acetylgalactosamine (Gal/GalNAc) lectin immunoglobulin A (IgA) had fewer E. histolytica infections, and children with fecal IgA anti-Gal/GalNAc lectin IgA also had a lower incidence of new infection [10, 11]. Weaning and the introduction of supplementary food into the diet is known to trigger changes in the bacterial composition of the microbiome that include increases in Prevotellaceae and Bacteroides species [12, 13]. The first 2 years of life in general is a period of rapid immune and microbiome maturation, which, in this population, may be disrupted by malnutrition and environmental insult [13–15]. Here we describe the natural history of amebiasis in the first 2 years of life in infants from an urban slum in Dhaka. The cumulative incidence of amebic infection and diarrhea, the association of protection with anti-Gal/GalNAc lectin IgA, the impact of parasite burden on symptoms, and potential role of Prevotella copri in E. histolytica diarrhea are described.

(See the editorial commentary by Ryan on pages 1732–3.) Diarrhea accounts for 26.1% of childhood deaths in South Asia [1], with a peak incidence in the first year of life [2–4]. Beyond this immediate mortality burden, diarrheal episodes contribute to intestinal barrier dysfunction and malnutrition, which underlie additional mortality [5] and disability-adjusted life-years lost [6]. This large burden of disease continues despite improvements from measures such as oral rehydration solution, antibiotics, cleaner water, sanitation, breast-feeding, and rotavirus vaccination [7–10]. The etiology of diarrhea must be understood to accelerate additional preventive measures. Unfortunately, diarrhea is a nonspecific syndrome defined as ≥3 loose stools in a day and can be caused by a diversity of viruses, bacteria, protozoa, helminths, fungi, as well as non infectious triggers [11]. Rotavirus is widely accepted as the major diarrheal pathogen in the first year of life [12–14], but the relative importance of enteropathogens thereafter is less clear. Several methods are needed to detect these enteropathogens including culture, immunoassay, microscopy, and polymerase chain reaction (PCR), yet these are generally applied selectively and vary in their sensitivity [9, 10, 15, 16]. Certain bacteria, such as Campylobacter and Shigella, are difficult to grow, particularly in the global context of widespread antibiotic use. Mixed infections are common but difficult to interpret. For these reasons we developed a series of quantitative multiplex PCR assays for 32 of the main enteropathogen targets, encompassing the major viruses, bacteria, protozoa, helminths, and fungi [17–21]. In this work we applied these assays to infants in Dhaka, Bangladesh, starting from birth, testing all assays with both monthly surveillance and diarrheal specimens . This detection strategy and knowledge of pathogen history preceding diarrhea allowed for a temporal examination of the etiology of diarrhea not possible with most study designs.