Date Published: October 20, 2016
Publisher: Public Library of Science
Author(s): Fumika Mi-ichi, Hiroki Yoshida, Shinjiro Hamano, Marc-Jan Gubbels.
Amebiasis is caused by Entamoeba histolytica infection and can produce a broad range of clinical signs, from asymptomatic cases to patients with obvious symptoms. The current epidemiological and clinical statuses of amebiasis make it a serious public health problem worldwide. The Entamoeba life cycle consists of the trophozoite, the causative agent for amebiasis, and the cyst, the form responsible for transmission. These two stages are connected by “encystation” and “excystation.” Hence, developing novel strategies to control encystation and excystation will potentially lead to new measures to block the transmission of amebiasis by interrupting the life cycle of the causative agent. Here, we highlight studies investigating encystation using inhibitory chemicals and categorize them based on the molecules inhibited. We also present a perspective on new strategies to prevent the transmission of amebiasis.
Amebiasis is caused by Entamoeba histolytica infection, a protozoan parasite belonging to the phylum Amoebozoa. Infected individuals show a wide range of clinical signs and can be asymptomatic or have obvious symptoms, such as diarrhea, dysentery, fever, and abdominal pains owing to invasive infection. As a consequence of the invasive infection, various extra-intestinal manifestations may also arise: for example, amebic liver, lung, or brain abscesses. Worldwide, 35–50 million symptomatic cases occur annually, leading to approximately 55,000 deaths . However, only a few drugs are available, and no effective vaccines exist [2,3]. Furthermore, because of the high occurrence of asymptomatic infections, amebiasis morbidity is thought to be much higher than estimates that are only based on the number of reported symptomatic cases. These clinical and epidemiological statuses make amebiasis a serious public health problem worldwide .
All the studies described in this review have investigated E. invadens, a reptilian parasite, and not E. histolytica. This is because the in vitro culture of E. invadens has been adopted as a model system for encystation studies (Box 1) [8,10,11].
Distinct lines of evidence demonstrate that galactose (Gal)-terminated ligands and their receptors are involved in encystation:
Catecholamine is a vertebrate hormone synthesized from tyrosine and exerts crucial roles via adrenergic receptor (AR)-mediated signal transduction [19,20]. Coppi and colleagues demonstrated several lines of evidence that an autocrine catecholamine system is involved in Entamoeba encystation .
Cholesteryl sulfate (CS) is a common sulfate metabolite in mammals. Recently, CS was shown to play an important role in Entamoeba encystation .
In the process of encystation, negative as well as positive factors are involved. Heat shock protein 90 (Hsp90) is such a negative regulator of encystation . Hsp90 is a chaperone that is highly conserved from bacteria to mammals and plays crucial roles via interaction with co-chaperones [31–34]. Protozoan parasites are, however, an exception; they maintain the conserved Hsp90 but lack some co-chaperones. Indeed, E. histolytica has a highly conserved Hsp90 but lacks the co-chaperones p23, cyclophilin 40 (Cyp40), cell division cycle 37 (Cdc37), and full-length Activator of the Hsp90 ATPase-1 (Aha1), although it does possess a novel truncated Aha1 [32,35]. Singh et al. demonstrated that the ATPase activity of purified recombinant E. histolytica Hsp90 (EhHsp90) was inhibited by 17-allylamino-17-demethoxygeldanamycin (17-AAG), a well-known inhibitor of Hsp90 with an IC50 value of 30.9 μM [35,36]. Furthermore, 17-AAG inhibited the growth of E. histolytica trophozoites with an IC50 value of 546 nM. These results suggest that 17-AAG targets native EhHsp90 in vivo and that EhHsp90 plays an indispensable role in E. histolytica trophozoites .
Chitin metabolism may provide potential targets for developing strategies to block the transmission of amebiasis because chitin is present in the cyst wall [5,8,9,15,18] and is not a component of mammalian cells [37,38]. Two independent groups published inconsistent results; one study showed that polyoxin D and Nikkomycin (structural analogs of uridine diphosphate-N-acetylglucosamine, a substrate for chitin synthase) inhibited the cyst formation in a dose-dependent manner when added to the in vitro culture (2–500 and 10–50 μg/mL, respectively) . The other study showed that chitin synthase activity in E. invadens cyst lysates was not inhibited by polyoxin D or Nikkomycin even at 100 μg/mL . This inconsistency raises two questions: Is the Entamoeba chitin synthase a target of polyoxin D and Nikkomycin? Is the Entamoeba chitin synthase a redundant enzyme? The Entamoeba chitin synthase may indeed be redundant because two chitin synthases are encoded in the genome (AmoebaDB); however, the critical question of what is the target of polyoxin D and Nikkomycin remains unanswered.
In eukaryotes, two proteolytic systems—i.e., the lysosome system and the ubiquitin (Ub) proteasome system (UPS)—control intracellular protein levels within optimal ranges [43,44]. In Entamoeba, these two systems play important roles in encystation as well as in proliferation of trophozoites and pathogenicity [1,45].
Segovia-Gamboa et al. reported the isolation of a monoclonal antibody, B4F2, by screening monoclonal antibodies raised against E. invadens intact cysts that could inhibit encystation in the in vitro system . The movement of B4F2’s target molecule during encystation was also shown. Moreover, enolase was demonstrated to be a potential target for B4F2. These findings indicate a role of enolase, a glycolytic enzyme, in encystation; however, further study is needed to confirm whether the B4F2 antigen is indeed enolase.
Amebiasis is a serious public health problem; therefore, the development of novel strategies to manipulate E. histolytica encystation is important for realizing new preventive measures. Impairing the ability to form cysts can block the spread of the disease because dormancy of the E. histolytica cyst is crucial for disease transmission. This type of approach cannot directly eliminate the causative agent, the trophozoite, from infected patients, so it would not cure amebiasis. However, it could be an effective approach to mitigate the disease because high numbers of asymptomatic patients who do not require clinical treatment are unconsciously spreading the disease (see Fig 1). A combination approach of eliminating the causative agent itself and interrupting its life cycle would be effective against infectious diseases such as amebiasis and would accelerate a reduction in endemicity.