Sensitivity of Enzymatic Toxins from Corneal Isolate of Acanthamoeba Protozoan to Physicochemical Parameters

Abstract

Acanthamoeba is a free-living amoeba that causes severe corneal infection (Acanthamoeba keratitis) and produces a variety of extracellular enzymes, called exoproteome. Since physicochemical characters are suggested being associated with therapeutic profile and clinical severity of the infection, we investigated the physicochemical properties of proteolysis mediated by amoebic exoproteome. Corneal scraping was collected from a patient who showed typical symptoms of acute Acanthamoeba keratitis. Axenic amoeba was phylogenetically identified by 18S rDNA sequencing analysis. Effects of pH, temperature and diamidines on proteolysis mediated by exoproteome were assessed using zymography assays. Proteolytic enzymes were most active at pH 7.0 and 37 °C. Calcium ions decreased enzymatic activity. The main components of amoebic exoproteome were characterized as serine proteases. We demonstrated for the first time that commercial antimicrobial diamidines used for Acanthamoeba keratitis therapy inhibit enzymatic activity of amoebic exoproteome. Results showed the thermostability of Acanthamoeba proteases, which suggest a long-term effect of these virulence factors at the central and peripheral cornea with possible role in degradation of extracellular matrix components. Our findings open new perspectives about the complementary and unreported properties of antimicrobial compounds of the diamidine class on the inhibition of enzymatic activity and presumptive control of amoebic infection in the cornea.

A history of over 40 years of potentially pathogenic free-living amoeba studies in Brazil - a systematic review

“…for human health in Brazil -As shown in Fig. 3, 29 out of 178 references were classified in the clinical category, including mainly Acanthamoeba keratitis, (99)(100)(101)(102)(103)(104) PAM, (105) and GAE, (106) reports (Table I).…”

“…Between 2010 and 2020, 23 out of 34 FLA environmental and clinical investigations included molecular-based tools for FLA detection, in contrast with five "morphological" 3 Encephalitis / Human -Death Leptomyxid amoeba M, I (152) 4 Encephalitis / Human -Death Balamuthia mandrillaris M, I (154) 5 Encephalitis / Human -Death Balamuthia mandrillaris M, I, D (116) 6 Encephalitis / Cattle -Death N. fowleri M, I (105) 7 Encephalitis / Cow -Death N. fowleri M, I (107) 8 Keratitis / Human A. astronyxis M, D (39) 9 Keratitis / Human Acanthamoeba spp. M 16 Keratitis / Human Acanthamoeba T4 M, D (74) 17 Keratitis / Human Acanthamoeba T4, T11, T2 and T4/T2; T4/T13; T4/T16 M, D (103) 18 Keratitis / Human A. castellanii, A. rhysodes M, I, D (61) 19 Keratitis / Human A. castellanii T4 M, D (104) 20 Keratitis / Human Acanthamoeba spp. M, D (21) 21 Keratitis / Human Acanthamoeba spp.…”

“… M 101 14 2009 Keratitis / Human Acanthamoeba spp. M 102 15 2011 Keratitis / Human A. castellanii M 155 16 2011 Keratitis / Human Acanthamoeba T4 M, D 74 17 2013 Keratitis / Human Acanthamoeba T4, T11, T2 and T4/T2; T4/T13; T4/T16 M, D 103 18 2013 Keratitis / Human A. castellanii , A. rhysodes M, I, D 61 19 2017 Keratitis / Human A. castellanii T4 M, D 104 20 2017 Keratitis / Human Acanthamoeba spp. M, D 21 ...…”