180Validation of a new smartphone application for the diagnosis of atrial fibrillation in primary care

Abstract

Secreted cysteine proteases are relevant actors in parasite biology,taking part in critical host colonization roles such as traversingtissue barriers, immune evasion and nutrient digestion. In the trematodeFasciola hepatica, the initial step to successful infection of themammalian host is the excystment of metacercariae and the invasionthrough the intestinal wall by the newly excysted juveniles (NEJ).While the cathepsin L-like cysteine proteinases secreted by the adultfluke have been extensively characterized, the cataloguing and descriptionof the cathepsins B and L reported in the invasive stages is onlysketchy. To identify the cathepsins expressed during excystment andearly invasion we constructed cDNA libraries encoding NEJ cathepsinsB and L. We found two cathepsin L-like cysteine proteinases (CL3,CL4) and three cathepsins B (CB1, CB2, CB3) which are predominantlyexpressed in NEJ. Phylogenetic analysis showed that NEJ-expressedcathepsins L constitute a well-defined clade separate from the adultenzymes. Excystment induction resulted in a significant incrementin activity towards cathepsin-specific fluorogenic substrates inmetacercariae homogenates, consistent with the detection of precursorand mature forms of cathepsins B and L before and after induction.In NEJ culture supernatants, protein and relative activity profilesshow subtle changes during the first 48 h, with prevalence of cathepsinL-like activity, although cathepsins CB3 and CL3 were detected bymass spectrometry. Noticeably, the hydrolysis of a substrate withproline in the P2 position was predominant, a property only sharedwith adult CL2 and vertebrate cathepsin K among the C1A subfamilyof cysteine proteases. Collectively these mRNA, protein and enzymaticdata demonstrate the existence of a NEJ-specific repertoire of cathepsinsexpressed early in invasion, distinct to those used by other trematodes,potentially relevant for specific vaccine and chemotherapy design.The diversity of proteases employed by trematodes in the invasionprocess is discussed.