Evaluation of Acanthamoeba Myosin-IC as a Potential Therapeutic Target

Abstract

bMembers of the genus Acanthamoeba are facultative pathogens of humans, causing a sight-threatening keratitis and a fatal encephalitis. We have targeted myosin-IC by using small interfering RNA (siRNA) silencing as a therapeutic approach, since it is known that the function of this protein is vital for the amoeba. In this work, specific siRNAs against the Acanthamoeba myosin-IC gene were developed. Treated and control amoebae were cultured in growth and encystment media to evaluate the induced effects after myosin-IC gene knockdown, as we have anticipated that cyst formation may be impaired. The effects of myosin-IC gene silencing were inhibition of cyst formation, inhibition of completion of cytokinesis, inhibition of osmoregulation under osmotic stress conditions, and death of the amoebae. The finding that myosin-IC silencing caused incompletion of cytokinesis is in agreement with earlier suggestions that the protein plays a role in cell locomotion, which is necessary to pull daughter cells apart after mitosis in a process known as "traction-mediated cytokinesis". We conclude that myosin-IC is a very promising potential drug target for the development of much-needed antiamoebal drugs and that it should be further exploited for Acanthamoeba therapy.A canthamoeba is a genus of small amoebae that are ubiquitous in the human environment. They alternate between feeding moving trophozoites and, when conditions are unfavorable, a resting cyst stage. Members of this genus are facultative human pathogens that cause the serious eye infection Acanthamoeba keratitis and a rare but usually fatal infection known as granulomatous Acanthamoeba encephalitis (1). The fact that this organism is able to form a highly resistant cyst stage is a significant clinical problem since it is extremely resilient when exposed to many disinfectants, antibiotics, and even acids (2). The persistence of the cyst stage after cessation of therapies that are effective against the growing amoebae can cause reinfection (3). Specific drugs that are capable of eliminating the amoebae rapidly enough to prevent cyst formation are urgently required. The search for therapeutic targets is currently focused on processes that are unique to the amoebic biological processes. To this end, we have selected the motor protein myosin-IC, as it performs a function in amoebae that human myosin-IC lacks. The myosin-IC family proteins are single-headed myosins that were first identified and characterized in a strain of Acanthamoeba (4). Following their discovery in Acanthamoeba, this family of proteins was also found in other organisms, including humans (5). Acanthamoeba myosin-IC has been shown to be essential for the contraction of the contractile vacuole, an organelle unique to these protozoa that functions as a water pump to maintain osmotic balance. Furthermore, inhibition of this motor protein by antibodies prevents the vacuole from excreting excess water so the amoeba explodes (6). Therefore, Acanthamoeba myosin-IC is a promising therapeutic target, as it is ver...

An update onAcanthamoebakeratitis: diagnosis, pathogenesis and treatment

“…Other drug targets that have been validated using the same approach, but without the elucidation of an available chemical alternative (active principle/drug) include glycogen phosphorylase and other cellulose synthesis related enzymes, serine proteases and myosin IC [ 4 , 27 , 53 , 55 , 57 , 65 ]. The recently published Acanthamoeba castellani genome data will further assist in the development of novel therapeutics in the near future [ 19 ].…”

Acanthamoeba spp. monoclonal antibody against a CPA2 transporter: a promising molecular tool for acanthamoebiasis diagnosis and encystment study

“…The greatest challenges concerning amoebic keratitis are finding a fast specific diagnostic method and effective treatment. Some groups are studying Acanthamoeba pathophysiology and searching for potential new candidates for pharmaceutical targets (Martín-Navarro et al ., 2013, 2014; Lakhundi et al ., 2015; Rice et al ., 2018; Siddiqui et al ., 2019). The findings presented in this study suggest that the monoclonal antibody mAb3 recognizes an extracellular target present only in pathogenic Acanthamoeba , demonstrating its potential application in AK diagnosis.…”

Drug Discovery against Acanthamoeba Infections: Present Knowledge and Unmet Needs

“…Myosin-IA functions in cytoplasmic vesicle transport, myosin-IB functions in pseudopod extension and phagocytosis, and myosin-IC is the only subtype that functions in contractile vacuole [25]. Acanthamoeba myosin-IC was of particular interest as it performs functions that human myosin-IC lacks and it is only 44% homologous to human myosin-IC [111]. The contractile vacuole is highly important in Acanthamoeba because it maintains homeostasis by regulating the amount of water within amoeba.…”