The genus Prototheca (Trebouxiophyceae, Chlorophyta) revisited: Implications from molecular taxonomic studies
Graphical abstract
Introduction
The pathogenicity of autotrophic green algae towards vertebrates was first recognized in 1973 by Cordy, who described bright green necrotic lesions in the liver and hepatic lymph nodes of a lamb, and attributed them to infection with Chlorella spp., naming the condition as chlorellosis [1]. Since then, Chlorella infections have been reported in seven other sheep, with signs of systemic involvement [[2], [3], [4], [5], [6]], ten slaughtered cows, with lymphadenitis or peritonitis lesions [7,8], a gazelle [9], a dromedary [10], and a dog [11], with cutaneous, intestinal, and disseminated manifestations, respectively. Only twice, have the Chlorella algae been implicated as a causative agent of human (wound) disease [12,13]. Apart from Chlorella spp., the most prominent algal pathogens of animals are members of two heterotrophic genera, namely Prototheca and Helicosporidium. The Helicosporidia (Trebouxiophyceae) were first described by Keilin in 1921, with the original specimen isolated from the ceratopogonid larvae Dasyhelea obscura (Diptera), and named Helicosporidium parasiticum [14]. The organism has since been reported in a wide range of invertebrate hosts, yet with a distinct preference for the insects, representing the only entomopathogenic algae currently known. The Prototheca algae share a close relationship, supported by morphological and molecular data, with the Helicosporidia. They both lost the photosynthetic activity during the transition from autotrophic to heterotrophic growth, yet retained colorless plastids with a varying degree of structural and functional degeneration [15,16]. What differs between the two organisms is that whereas Helicosporidium is an obligate parasite, which cannot survive outside the host unless in the form of a highly characteristic cyst, Prototheca are normally free-living and become pathogenic only under certain conditions, usually related to the impairment of the host's immunity. Another distinctive feature is the range of host species parasitized. Whereas the Helicospordia infect only arthropods, and insect larvae in particular, the protothecans have been exclusively recovered from vertebrates, including humans. Among animals, cattle are the most commonly affected [[17], [18], [19], [20], [21], [22]]. Dogs, cats, and goats are also affected but on much rarer occasions [[23], [24], [25], [26], [27]]. Single cases of Prototheca infections have been reported in a deer [28], a beaver [29], a fruit bat [30], a snake [31], fishes [32,33], and horses [34].
Overall, among all the Viridiplantae, standing at more than a third of a million species [35], only Prototheca and Chlorella microalgae are able to cause disease in humans and other mammals through active invasion and spread within the host tissues. However, while Chlorella infections have been sporadic and have occurred in widely scattered geographical regions, protothecosis is an emerging disease, whose incidence has been on the rise worldwide. This applies particularly to bovine mastitis, the first to be recognized (1952) and the most prevalent form of protothecal disease, which incurs severe economic losses to the global dairy industry, as well as to human infections, whose caseload, calculated cumulatively since the first case reported (1964), has grown by nearly 180% over the last two decades (from 76 cases before 1996 to 211 cases as for mid-2017) [36]. This sharp increase in the incidence of human protothecosis reflects an expanding population of senile and immunocompromised patients on the one hand, and increased clinical awareness and technological advancements in diagnostic procedures on the other.
Since 1894 when the genus Prototheca was erected by Krüger, the taxonomic status and origin of the organisms under this generic name have long remained a source of incessant controversies and speculative theorizing. Noteworthy, Prototheca were originally described as fungi, though they could not be affiliated with Saccharomycetes or any then-recognized Phycomycetes [37].
The Prototheca taxonomy has repeatedly been revised, as more phenotypic, chemotaxonomic, and molecular data have become available. More than a century-old history of the Prototheca genus, summarized in a series of review articles [[37], [38], [39], [40]], clearly show that most of the taxonomic changes introduced were the outcome of the transition from phenotypic to molecular classification tools. Indeed, since the mid-1990s, the taxonomy of Prototheca spp. has reached the molecular level. Until recently, the only target for the molecular taxonomic approaches has been the rRNA 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 inferring phylogenetic relationships among the Prototheca species and allied taxa [41,42], and for developing new typing schemes to achieve fast and accurate identification, comprehensively reviewed in a study by Jagielski et al. [43]. In the same work, the authors have proposed the mitochondrial gene cytb, coding for cytochrome b, a transmembrane protein, forming the core of the mitochondrial cytochrome bc1 complex of the respiratory chain, as a new and efficient marker for diagnostics and phylogenetic studies of the Prototheca algae [43]. The cytb gene offers important advantages over the so-far used rDNA markers owing to its high discriminatory capacity, intra-strain sequence homogeneity, and technical feasibility, including a robust and reproducible amplification assay, and a rapid and unambiguous sequence analysis.
Over the years, a large number of Prototheca strains have been accumulated in public culture collections. Many of these strains still exist under outdated or invalid names identified by more or less experienced scientists, being the heritage of a traditional, phenotype-based taxonomy.
In this study, the cytb gene was used as a marker to revise the taxonomy and nomenclature of a set of Prototheca strains, preserved in major algae culture repositories worldwide.
Section snippets
Algal strains and culture conditions
Eighty-six strains of Prototheca spp. were used in the study. Included in this number were nine authentic Prototheca “type” (T) strains representing eight species and genotypes and 77 additional strains, either isolated in the authors' laboratories, purchased from public culture collections or kindly supplied by collaborating colleagues. The species names, strain numbers, and origins are given in Table 1. Cultures were maintained on Sabouraud Dextrose (SD) agar (Becton Dickinson, Franklin
Results and discussion
A total of eighty-six 599–668-bp sequences of the partial cytb gene, from as many strains of Prototheca spp., were aligned with the equivalent sequences from related genera to assess their phylogenetic relationships shown in Fig. 1. Eighty-one Prototheca spp. strains were distributed into nine (I-IX) clusters (3–17 strains per cluster), each representing distinct species. Cluster I comprised three strains, under different species designations, with their cytb gene sequences being identical
Statement of informed consent, human/animal rights
No conflicts, informed consent, human or animal rights applicable.
Declaration of competing interest
Authors have no competing interests to declare.
Acknowledgements
The authors wish to thank Prof. Uwe Rösler and his team for their contribution to the validation of Prototheca blaschkeae. Consultation by Dr. Maike Lorenz, curator of the Culture Collection of Algae at the University of Göttingen, Göttingen, Germany, as well as technical assistance of Dr. Maja Graniszewska, curator of the Herbarium of the Faculty of Biology at the University of Warsaw is also appreciated.
The authors are greatly indebted to Prof. Uwe Rösler, Institute of Animal Hygiene and
Author contributions
T.J. conceptualized the study, supervised all research procedures, organized and integrated the data, and wrote the manuscript; Z.B. performed all PCR amplifications and restriction enzyme digestions; J.G. and R.G. performed molecular cloning of rDNA amplicons into bacterial plasmids and did all the sequencing procedures; K.M. and A.K. analyzed the sequence data and carried out the molecular phylogenetic analyses; W.-H.K. provided nomenclatural revisions according to the International Code of
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