N fowleri enters the host through the nasal cavity and
N. fowleri enters the host through the nasal cavity and travels via the olfactory nerve into the A769662 where it causes PAM (Visvesvara and Stehr-Green, 1990). The ameba most commonly infects healthy young adults and children and is able to evade the immune system but also elicits extensive damaging inflammation, tissue necrosis and hemorrhage of the brain and meninges (Visvesvara and Stehr-Green, 1990). The time from initial exposure to onset of illness is usually 5–7 days but may be as early as 24 h. Initial symptoms include sudden onset of bifrontal or bitemporal headaches, high fever, nuchal rigidity, anorexia, vomiting, irritability and restlessness. Other symptoms such as photophobia, neurological abnormalities, including altered mental status, lethargy, dizziness, ataxia, cranial nerve palsy, hallucinations, delirium, coma may occur late in the clinical course, leading to death in 3–7 days (Visvesvara et al., 2007). Current treatments are inadequate and provide no guarantee for survival. The drug of choice to treat N. fowleri is the antifungal amphotericin B but is not FDA-approved for this indication. Treatment with amphotericin B requires high dosage (1.5 mg/kg/day intravenous in 2 doses × 3 days, then 1 mg/kg/day × 6 days plus 1.5 mg/day intrathecally × 2 days, then 1 mg/day every other day × 8 days) and its use is frequently associated with renal toxicity, manifested as azotemia and hypokalemia (Visvesvara, 2010). Amphotericin B can also cause anemia, and many patients experience chills, fever, nausea, vomiting, and headache. Moreover, no more than a dozen persons with PAM have been treated successfully worldwide with amphotericin B alone or in combination with other drugs (Visvesvara, 2010). Recently, an investigational breast cancer and anti-Leishmania drug, miltefosine, has shown some promise in combination with other drugs and a patient was successfully treated with miltefosine and hypothermia (Centers for Disease, 2013b). But another patient, though treated with miltefosine, suffered permanent brain damage. Therefore, it is imperative to find safe and effective drugs that can treat PAM and increase host survival. Identification of new leads that can be used as a basis to develop drugs to treat Naegleria infection is a critical unmet need to prevent future deaths of children and young adults.
In this study, we first show that the cysteine protease inhibitors inhibited N. fowleri cysteine protease activity present in cell lysates. Because of the inhibitory activity against parasite cysteine proteases, we also tested the effect of the inhibitors on the viability of N. fowleri.
Materials and methods
Discussion The role of cysteine proteases in N. fowleri is not clearly defined. Several studies suggest that cysteine protease may play a role in Naegleria pathogenesis. A 30 kDa secreted N. fowleri cysteine protease was found to have a cytopathic effect on BHK cells (Aldape et al., 1994). A 37 kDa cysteine protease with mucinolytic activity was hypothesized to have a role in mucin degradation and host immune response evasion (Cervantes-Sandoval et al., 2008). Treatment of N. fowleri with the canonical cysteine protease inhibitor, E64, decreased their migration through an extracellular matrix construct, suggesting a role for cysteine protease activity in invasion of the central nervous system (Vyas et al., 2015). In the absence of a complete N. fowleri genome, recent studies reported that N. fowleri lysate and excretory-secretory proteins contain cathepsin B and cathepsin B-like cysteine proteases that are likely virulence factors of N. fowleri and involved in pathogenesis, as they are in E. histolytica infections. The N. fowleri cathepsin B gene was found differentially expressed only in the pathogenic trophozoite stage but not in the cyst stage, compatible with a role in pathogenesis (Lee et al., 2014). Biochemical characterization of N. fowleri protease activity in total crude extract and in conditioned medium was performed and the effect of protease inhibitors, such as E-64 and NEM, on proteolytic activity was also quantified (Serrano-Luna et al., 2007).