Ribosomal Internal Transcribed Spacer of Prototheca wickerhamii Has Characteristic Structure Useful for Identification and Genotyping

Abstract

Prototheca species are achlorophyllous algae ubiquitous in nature and known to cause localized and systemic infection both in humans and animals. Although identification of the Prototheca species in clinical specimens is a challenge, there are an increasing number of cases in which molecular techniques have successfully been used for diagnosis of protothecosis. In this study, we characterized nuclear ribosomal DNA (rDNA) of a strain of Prototheca (FL11-0001) isolated from a dermatitis patient in Japan for its species identification. When nuclear rDNA of FL11-0001 and that of various other Prototheca strains were compared by polymerase chain reaction (PCR), the results indicated that the sizes of ribosomal internal transcribed spacer (ITS) were different in a species-dependent manner, suggesting that the variation might be useful for differentiation of Prototheca spp. Especially, ITS of P. wickerhamii, the most common cause of human protothecosis, was distinctively larger than that of other Prototheca spp. FL11-0001, whose ITS was comparably large, could easily be identified as P. wickerhamii. The usefulness of the PCR analysis of ITS was also demonstrated by the discovery that one of the clinical isolates that had previously been designated as P. wickerhamii was likely a novel species. Furthermore, our data demonstrated that nucleotide sequences of P. wickerhamii ITS are heterogenous between different rDNA copies in each strain and also polymorphic between strains. Phylogenetic analysis suggested that the ITS sequences could be classified to four clades, based on which P. wickerhamii strains might be grouped into at least two genotypes. Comprehensive characterization of Prototheca rDNA may provide valuable insights into diagnosis and epidemiology of protothecosis, as well as evolution and taxonomy of Prototheca and related organisms.

Prototheca miyajii sp. nov., isolated from a patient with systemic protothecosis

“…S1, ITS, lane 3) was shown to be smaller than that of P. wickerhamii (Fig. S1, ITS, lanes 1 and 2), confirming results of the previous study (Hirose et al, 2013). In addition, the SSU rDNA of IFM 53848 T was demonstrated to be about 3.6 kb long (Fig.…”

“…As it was previously shown that PCR analysis of the rRNA gene is useful for identification of species of the genus Prototheca (Hirose et al, 2013), we amplified the SSU rDNA, the ITS and the D1/D2 region of the LSU rDNA of IFM 53848 T and various other strains of species of the genus Prototheca for comparison (Fig. S1).…”

“…Our previous studies have shown that comprehensive analysis by PCR of the SSU rDNA, internal transcribed spacer (ITS) and the LSU D1/D2 region is also useful for identification of species of the genus Prototheca (Hirose et al, 2013). In addition, an increasing number of studies demonstrate that matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) could be applied to the identification of species and genotypes of Prototheca (von Bergen et al, 2009;Ahrholdt et al, 2012;Murugaiyan et al, 2012;Wirth et al, 2012).…”

Case report of cutaneous protothecosis caused by Prototheca wickerhamii designated as genotype 2 and current status of human protothecosis in Japan

“…The case was recorded in a Japanese female patient, and was discussed as an interesting case during one of the regional meetings of the Japanese Dermatological Association (JDA) in 2011 . In 2013, we characterized the nuclear rDNA of the strain isolated from the patient and identified it to be Prototheca wickerhamii genotype 2 . Also, a review article published in 2014 included this patient as case 7 in its table .…”

“…2 In 2013, we characterized the nuclear rDNA of the strain isolated from the patient and identified it to be Prototheca wickerhamii genotype 2. 3 Also, a review article published in 2014 included this patient as case 7 in its table 1. 4 During the working group symposium of the 19th International Society for Human and Animal Mycology (ISHAM) Congress in 2015, Todd et al 5 listed this case in their presentation.…”