<i>cytb</i> as a New Genetic Marker for Differentiation of Prototheca Species

Abstract

Achlorophyllous unicellular microalgae of the genus <i>Prototheca</i> (<i>Trebouxiophyceae</i>, <i>Chlorophyta</i>) are the only known plants that cause infections in both humans and animals, collectively referred to as protothecosis. Human protothecosis, most commonly manifested as cutaneous, articular, and disseminated disease, is primarily caused by <i>Prototheca</i><i>wickerhamii</i>, followed by <i>Prototheca</i><i>zopfii</i> and, sporadically, by <i>Prototheca</i><i>cutis</i> and <i>Prototheca</i><i>miyajii</i> In veterinary medicine, however, <i>P. zopfii</i> is a major pathogen responsible for bovine mastitis, which is a predominant form of protothecal disease in animals. Historically, identification of <i>Prototheca</i> spp. has relied upon phenotypic criteria; these were later replaced by molecular typing schemes, including DNA sequencing. However, the molecular markers interrogated so far, mostly located in the ribosomal DNA (rDNA) cluster, do not provide sufficient discriminatory power to distinguish among all <i>Prototheca</i> spp. currently recognized. Our study is the first attempt to develop a fast, reliable, and specific molecular method allowing identification of all <i>Prototheca</i> spp. We propose the mitochondrial <i>cytb</i> gene as a new and robust marker for diagnostics and phylogenetic studies of the <i>Prototheca</i> algae. The <i>cytb</i> gene displayed important advantages over the rDNA markers. Not only did the <i>cytb</i> gene have the highest discriminatory capacity for resolving all <i>Prototheca</i> species, but it also performed best in terms of technical feasibility, understood as ease of amplification, sequencing, and multiple alignment analysis. Based on the species-specific polymorphisms in the partial <i>cytb</i> gene, we developed a fast and straightforward PCR-restriction fragment length polymorphism (RFLP) assay for identification and differentiation of all <i>Prototheca</i> species described so far. The newly proposed method is advocated to be a new gold standard in diagnostics of protothecal infections in human and animal populations.