cytb as a New Genetic Marker for Differentiation of Prototheca Species

Abstract

Achlorophyllous unicellular microalgae of the genus Prototheca (Trebouxiophyceae, Chlorophyta) 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 Protothecawickerhamii, followed by Protothecazopfii and, sporadically, by Protothecacutis and Protothecamiyajii In veterinary medicine, however, P. zopfii is a major pathogen responsible for bovine mastitis, which is a predominant form of protothecal disease in animals. Historically, identification of Prototheca 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 Prototheca spp. currently recognized. Our study is the first attempt to develop a fast, reliable, and specific molecular method allowing identification of all Prototheca spp. We propose the mitochondrial cytb gene as a new and robust marker for diagnostics and phylogenetic studies of the Prototheca algae. The cytb gene displayed important advantages over the rDNA markers. Not only did the cytb gene have the highest discriminatory capacity for resolving all Prototheca 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 cytb gene, we developed a fast and straightforward PCR-restriction fragment length polymorphism (RFLP) assay for identification and differentiation of all Prototheca 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.

Keywords: Prototheca; algae; cytb; cytochrome b; genotypic identification.

FIG 1

PCR products of the partial cytb gene (above) and restriction patterns of these products (below) produced upon RsaI/TaiI double digestion for nine Prototheca species (genotypes) type strains. Lane 1, P. zopfii genotype 1; lane 2, P. zopfii genotype 2; lane 3, P. blaschkeae; lane 4, P. cutis; lane 5, P. ulmea; lane 6, P. stagnora; lane 7, P. wickerhamii; lane 8, P. miyajii; lane 9, P. tumulicola; lanes M, size marker (Gene Ruler Low Range; Thermo Fisher Scientific, Waltham, MA, USA).

FIG 2

Restriction patterns of the partial cytb gene produced upon MboI digestion for two Prototheca species indistinguishable by RsaI/TaiI double digestion. Lane 1, P. miyajii; lane 2, P. tumulicola; lane M, size marker (Gene Ruler Low Range; Thermo Fisher Scientific, Waltham, MA, USA).

FIG 3

Evaluation of the cytb PCR restriction enzyme analysis profiling for selected Prototheca sp. strains. Lanes 1 to 3, P. zopfii genotype 1; lanes 4 to 6, P. zopfii genotype 2; lanes 7 to 9, P. blaschkeae; lanes 10 to 12 – P. wickerhamii; lanes M, size marker (Gene Ruler Low Range; Thermo Fisher Scientific, Waltham, MA, USA).

FIG 4

Phylogenetic tree constructed through maximum likelihood analysis based on cytb sequences. The bootstrap values obtained by the analysis are marked at the nodes.

FIG 5

Phylogenetic tree constructed through maximum likelihood analysis based on SSU rDNA sequences. The bootstrap values obtained by the analysis are marked at the nodes.

FIG 6

Phylogenetic tree constructed through maximum likelihood analysis based on ITS sequences. The bootstrap values obtained by the analysis are marked at the nodes.

FIG 7

Phylogenetic tree constructed through maximum likelihood analysis based on D1/D2 LSU sequences. The bootstrap values obtained by the analysis are marked at the nodes.

FIG 8

Multiple alignment of the SSU sequences of the Prototheca sp. type strains. Nucleotides identical across all displayed species are shaded in red, and those present in at least six sequences are boxed in blue. Blue and orange arrows indicate forward and reverse primers, respectively. Alignment positions boxed in gray are nucleotide coordinates for the adjacent alignment block. Additional black lines indicate sequences based on which species-specific primers were designed. Primer names are given above the arrows, and numbers in brackets correspond to numbers provided in Table S7 in the supplemental material. Dots in the alignment represent the intersequence gaps, while an ellipsis beneath the proto18S-4r-1 (#8) primer in the P. miyajii IFM 53848 SSU sequence indicates a large insert in that region.

FIG 9

Multiple alignment of the ITS sequences of the Prototheca sp. type strains. Nucleotides identical across all displayed species are shaded in red, and those present in at least six sequences are boxed in blue. Blue and orange arrows indicate forward and reverse primers, respectively. Alignment positions boxed in gray are nucleotide coordinates for the adjacent alignment block. Additional black lines indicate specific strains for which identification primers were designed. Primer names are given above the arrows, and numbers in brackets correspond to numbers provided in Table S7 in the supplemental material. Dots in the alignment represent the intersequence gaps.

FIG 10

Multiple alignment of the D1/D2 LSU sequences of the Prototheca spp. type strains. Nucleotides identical across all displayed species are shaded in red, and those present in at least six sequences are boxed in blue. Blue and orange arrows indicate forward and reverse primers, respectively. Alignment positions boxed in gray are nucleotide coordinates for the adjacent alignment block. Primer names are given above the arrows, and numbers in brackets correspond to numbers provided in Table S7 in the supplemental material. Dots in the alignment represent the intersequence gaps.

FIG 11

Multiple alignment of the partial sequences of the cytb gene of the Prototheca sp. type strains. Nucleotides identical across all displayed species are shaded in red, and positions with a maximum of two different nucleotides are boxed in blue. The blue-, green-, and black-shaded nucleotides indicate recognition sites for RsaI (GT∧AC), TaiI (ACGT∧), and MboI (∧GATC) restriction enzymes, respectively. The caret represents where the enzyme cuts the sequence.