Prototheca-ID

Background

Prototheca are unicellular, colourless, nonphotosynthetic microalgae, ubiquitously distributed in nature. They normally live a saprophytic lifestyle, occurring most abundantly in humid and organic-rich environments [1]. However, Prototheca may, under certain conditions, act as opportunistic pathogens, causing a variety of pathologies in both animals and humans, collectively referred to as protothecosis [2-6]. Prototheca are the only known plants which have repeatedly been reported to infect vertebrates, including men. In animals, the disease most commonly affects dairy cattle, resulting in clinical or subclinical mastitis. Bovine mammary protothecosis has recently become an emerging health and economic problem to the veterinary sector worldwide [7-11].

Human protothecosis manifests as cutaneous, articular, and disseminated disease, with the latter being life-threatening and occurring almost exclusively in severely immunocompromised patients [12-14]. Although human protothecosis is a rare condition, with a total of 211 cases reported globally by 2017, the incidence of the disease has been on the rise over the past two decades [13]. This is largely due to the growing population of elderly and otherwise immunocompromised individuals, but also due to improved clinical awareness and technological advancements in the diagnostic approaches.

The Prototheca taxonomy has long been controversial and subject to several revisions. The advent of molecular markers has greatly facilitated elucidating the phylogenetic relationships of the Prototheca algae, leading to considerable improvements in their identification. Until recently, the only target for the molecular taxonomic approaches has been the ribosomal RNA gene cluster. Numerous studies have exploited sequence polymorphisms within the small- (SSU) and large-subunit (LSU) rRNA genes, and the internal transcribed spacer (ITS) region for investigating phylogenetic relatedness among Prototheca species and allied taxa and for developing new typing schemes to achieve fast and accurate identification of the algae [15-22]. However, the rDNA markers do not provide sufficient discriminatory power to effectively separate all Prototheca species currently recognized. What further impedes the use of rDNA markers is a high level of intraspecific and intrastrain sequence variation [23-25].

Recently, we have proposed the mitochondrially encoded CYTB gene as a new and robust marker for diagnostics and phylogenetic studies of the Prototheca algae [23]. The CYTB gene was shown superior to rDNA markers in terms of discriminatory capacity and technical feasibility (i.e. PCR amplification, sequencing, and sequence analysis). Based on the CYTB gene marker, a new taxonomic classification system of the Prototheca algae has been established [26]. Furthermore, a PCR-RFLP assay targeting sequence spolymorphisms within the CYTB gene has been developed as a rapid and reliable means of Prototheca identification at the species level [23,26]. Given, however, that some P. ciferrii strains may produce PCR-RFLP patterns characteristic for P. bovis, to distinguish between the two species, direct sequencing of the partial CYTB gene is required [26]. Consequently, sequencing of the CYTB gene provides the highest accuracy of Prototheca identification. In fact, it is the only approach, currently known, allowing for unambiguous identification of all 15 Prototheca species described so far.

In a clinical setting, we advocate the use of CYTB gene-based typing, and CYTB sequencing in particular, as a new standard in diagnostics of protothecal infections in both human and animal hosts.

Purpose

The success of any sequence-based taxonomic profiling depends critically on high-quality, well-annotated reference sequence databases. However, contamination of publicly-available sequence repositories with incorrectly annotated or otherwise poorly described sequences is quite common, leading to either misidentification or no identification result at all.

Currently, the largest collection of Prototheca-derived DNA sequences provides the GenBank database. As of 1st May 2021, GenBank contained 8948 Prototheca related sequences with 7 assemblies for only 5 different Prototheca species. 1539 out of 8948 sequences (17%) were for the rDNA loci including the ITS region. Only 98 (1%) sequences represented the CYTB gene, of which 87 were submitted by the authors of this website. However, being not actively curated by taxonomic specialists, GenBank contains erroneous, outdated or misleading data, precluding the correct species identification. Moreover, GenBank suffers from the lack of rigorous data collection and structuring, potentially useful for comparative, analytical, and interpretative purposes. These pitfalls have been recognized for many GenBank deposited Prototheca sequences. Given the growing prevalence of Prototheca, and the ongoing discovery of new species, databases such as GenBank are expected to be inflated by incorrectly or insufficiently annotated entries. There is thus a need for an integrative and expert-curated database providing a robust and reliable identification of Prototheca species.

To meet this need, the Prototheca-ID project has been launched, introducing the Prototheca-ID web application, a freely-accessible, easy-to-use toolbox designed for sequence-based, species-level identification of Prototheca isolates.

The construction of Prototheca-ID was essentially based on the CYTB gene marker. However, a number of the LSU rRNA gene sequences were also incorporated, since this locus has been shown the most powerful among all rDNA markers, producing resolution similar to that of CYTB, for most of the Prototheca species. It was the core idea of the project not only to deliver a simple and robust platform for Prototheca identification, but also to establish a comprehensive, dedicated, and fully open database of sequences of Prototheca isolates and isolate-related information, well-annoteted, carefully verified, and rigorously structured in a standardized and easily searchable manner. Prototheca-ID was conceived as a regularly updated and continuously expanding database, to include other taxonomic markers or markers associated with clinically and/or epidemiologically relevant phenotypes, such as drug resistance, virulence, transmissibility etc. The Prototheca-ID aims at integrating sequence data with provenance and phenotypic information on Protothecea, allowing scientists to study the biology of these organisms in the context of their genetic background. An important purpose of such studies is to develop algorithms assessing risk factors predictive for human and animal protothecosis.

Our near-term priority, as the authors of this website, would be to instal within the Prototheca-ID application, a module for accommodating and filtering the whole genome sequence datasets, representing all Prototheca species. The Prototheca WGS project, initiated by our group in 2014, which is still in progress, has already released a complete sequence of the Prototheca wickerhamii genome [27].

Background

Purpose

References