Label-Free Quantitative Proteomic Analysis of Harmless and Pathogenic Strains of Infectious Microalgae, Prototheca spp.
ABSTRACT: Microalgae of the genus Prototheca (P.) spp are associated with rare algal infections of invertebrates termed protothecosis. Among the seven generally accepted species, P. zopfii genotype 2 (GT2) is associated with a severe form of bovine mastitis while P. blaschkeae causes the mild and sub-clinical form of mastitis. The reason behind the infectious nature of P. zopfii GT2, while genotype 1 (GT1) remains non-infectious, is not known. Therefore, in the present study we investigated the protein expression level difference between the genotypes of P. zopfii and P. blaschkeae. Cells were cultured to the mid-exponential phase, harvested, and processed for LC-MS analysis. Peptide data was acquired on an LTQ Orbitrap Velos, raw spectra were quantitatively analyzed with MaxQuant software and matching with the reference database of Chlorella variabilis and Auxenochlorella protothecoides resulted in the identification of 226 proteins. Comparison of an environmental strain with infectious strains resulted in the identification of 51 differentially expressed proteins related to carbohydrate metabolism, energy production and protein translation. The expression level of Hsp70 proteins and their role in the infectious process is worth further investigation. All mass spectrometry data are available via ProteomeXchange with identifier PXD005305.
Project description:Prototheca zopfii associated with bovine mastitis and human protothecosis exists as two genotypes, of which genotype 1 is considered as non-infectious and genotype 2 as infectious. The mechanism of infection has not yet been described. The present study was aimed to identify genotype 2-specific immunodominant proteins. Prototheca proteins were separated using two-dimensional gel electrophoresis. Subsequent western blotting with rabbit hyperimmune serum revealed 28 protein spots. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis resulted in the identification of 15 proteins including malate dehydrogenase, elongation factor 1-alpha, heat shock protein 70, and 14-3-3 protein, which were previously described as immunogenic proteins of other eukaryotic pathogens.
Project description:Microalgae of the genus Prototheca (P.) are associated with rare but severe infections (protothecosis) and represent a potential zoonotic risk. Genotype (GT) 2 of P. zopfii has been established as pathogenic agent for humans, dogs, and cattle, whereas GT1 is considered to be non-pathogenic. Since pathogenesis is poorly understood, the aim of this study was to determine immunogenic proteins and potential virulence factors of P. zopfii GT2. Therefore, 2D western blot analyses with sera and isolates of two dogs naturally infected with P. zopfii GT2 have been performed. Cross-reactivity was determined by including the type strains of P. zopfii GT2, P. zopfii GT1, and P. blaschkeae, a close relative of P. zopfii, which is known to cause subclinical forms of bovine mastitis. The sera showed a high strain-, genotype-, and species-cross-reactivity. A total of 198 immunogenic proteins have been analyzed via MALDI-TOF MS. The majority of the 86 identified proteins are intracellularly located (e.g., malate dehydrogenase, oxidoreductase, 3-dehydroquinate synthase) but some antigens and potential virulence factors, known from other pathogens, have been found (e.g., phosphomannomutase, triosephosphate isomerase). One genotype-specific antigen could be identified as heat shock protein 70 (Hsp70), a well-known antigen of eukaryotic pathogens with immunological importance when located extracellularly. Both sera were reactive to glyceraldehyde-3-phosphate-dehydrogenase of all investigated strains. This house-keeping enzyme is found to be located on the surface of several pathogens as virulence factor. Flow-cytometric analysis revealed its presence on the surface of P. blaschkeae.
Project description:We report a reactive Aspergillus galactomannan enzymatic immunoassay against the serum of a patient with invasive Prototheca zopfii infection. Analysis of the supernatants of suspensions of P. zopfii and other Prototheca isolates revealed positive results as well. These data suggest cross-reactivity with the serum Aspergillus galactomannan assay in invasive protothecosis.
Project description:Bovine mastitis is an important and complex disease responsible for economic losses in the dairy industry. Biotype II strains of the green alga Prototheca zopfii can be involved, most often resulting in chronic mastitis of difficult treatment associated with reduced milk production. This type of infection is rare, but the number of reported cases is increasing worldwide. In order to determine the kind of species involved in mastitis by Prototheca in northwest Portugal, 41 Prototheca isolates were genetically characterized. The algae are part of Prototheca isolates that were collected during a 6-year period, isolated from the milk of 41 dairy cows in a total of 22 herds with a history of increasing somatic cell counts, mild clinical signs of udder infection, and unsuccessful response to the usual therapy. PCR amplification of the 18S ribosomal DNA (rDNA), amplified rDNA restriction analysis, and phylogenetic analyses of the 18S rDNA sequences were performed. Thirty-seven isolates were identified as P. zopfii var. hydrocarbonea and four as Prototheca blaschkeae. These data suggest a high incidence of P. zopfii var. hydrocarbonea mastitis in the region and demonstrate for the first time the involvement of P. blaschkeae with bovine mammary gland infections.
Project description:Protothecosis is a severe form of mastitis in cattle that is caused by colorless algae of the genus Prototheca. So far, no suitable serological test for the identification of infected animals is available for routine diagnosis. In this study an indirect enzyme-linked immunosorbent assay (ELISA) for the identification of infected cows and for discriminating among infected cows at various clinical stages was developed. Immunoglobulin G (IgG) in serum and IgA and IgG1 in whey were used as antibody isotypes. The ELISA was evaluated using serum and whey from animals at different clinical stages of infection. A total of 12 cows with acute clinical manifestation of protothecal mastitis, 22 cows with clinical signs of chronic mastitis, 40 Prototheca zopfii-negative cows, and 18 cows with chronic clinical signs and earlier cultures positive for P. zopfii but with presently negative culturing results were investigated. A sensitivity of 96% and a specificity of 94% were calculated for the ELISA based on IgA levels. Intra-assay and interassay variations were calculated to be 6.08 and 6.32%, respectively. Based on these data, this ELISA was found to be suitable for discrimination between infected and uninfected animals and might therefore be useful for screening affected herds.
Project description: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.
Project description:Human protothecosis is a rare microalgae infection, and its dissemination typically occurs in immunocompromised individuals, but no specific immune defect has been reported. Here, we describe an 8-year-old daughter of a consanguineous union with abdominal pain and bloody diarrhea for 3 months who was found to have pancolitis with numerous microalgae identified as Prototheca zopfii. In the absence of a known immunodeficiency, exome sequencing was performed, which uncovered a novel recessive frameshift mutation in CARD9 (p.V261fs). This report highlights that CARD9 deficiency should be investigated in patients with unexplained systemic/visceral protothecosis and suggests a new mechanistic insight into anti-Prototheca immunity.
Project description:Prototheca zopfii is an alga increasingly isolated from bovine mastitis. Of the two genotypes of P. zopfii (genotype I and II (GT-I and -II)), P. zopfii GT-II is the genotype associated with acute mastitis and decreased milk production, although its pathogenesis is not well known. The objective was to determine inflammatory and apoptotic roles of P. zopfii GT-II in cultured mammary epithelial cells (from cattle and mice) and murine macrophages and using a murine model of mastitis. Prototheca zopfii GT-II (but not GT-I) invaded bovine and murine mammary epithelial cells (MECs) and induced apoptosis, as determined by the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling assay. This P. zopfii GT-II driven apoptosis corresponded to mitochondrial pathways; mitochondrial transmembrane resistance (??m) was altered and modulation of mitochondrion-mediated apoptosis regulating genes changed (increased transcriptional Bax, cytochrome-c and Apaf-1 and downregulated Bcl-2), whereas caspase-9 and -3 expression increased. Apoptotic effects by P. zopfii GT-II were more pronounced in macrophages compared to MECs. In a murine mammary infection model, P. zopfii GT-II replicated in the mammary gland and caused severe inflammation with infiltration of macrophages and neutrophils and upregulation of pro-inflammatory genes (TNF-?, IL-1? and Cxcl-1) and also apoptosis of epithelial cells. Thus, we concluded P. zopfii GT-II is a mastitis-causing pathogen that triggers severe inflammation and also mitochondrial apoptosis.
Project description:Of the Prototheca genus, Prototheca wickerhamii has the highest clinical significance in humans. However, neither nuclear nor organellar genomes of this species were sequenced until now. The hitherto determined and analyzed mitochondrial and plastid genomes of the alleged P. wickerhamii species belong in fact to another species, recently named Prototheca xanthoriae. This study provides a first insight into the organellar genomes of a true P. wickerhamii (type strain ATCC 16529). The P. wickerhamii mitochondrion had a 53.8-kb genome, which was considerably larger than that of Prototheca ciferrii (formerly Prototheca zopfii gen. 1) and Prototheca bovis (formerly Prototheca zopfii gen. 2), yet similarly functional, with the differences in size attributable to a higher number of introns and the presence of extra unique putative genes. The 48-kb plastid genome of P. wickerhamii, compared to autotrophic Trebouxiophyceae, was highly reduced due to the elimination of the photosynthesis-related genes. The gene content of the plastid genome of P. wickerhamii was, however, very similar to other colorless Prototheca species. Plastid genome-based phylogeny reinforced the polyphyly of the genus Prototheca, with Helicosporidium and Auxenochlorella branching within clades of Prototheca species. Phylogenetic reconstruction also confirmed the close relationship of P. wickerhamii and P. xanthoriae, which is reflected in the synteny of their organellar genomes. Interestingly, the entire set of atp genes was lost in P. wickerhamii plastid genome while being preserved in P. xanthoriae.
Project description:Prototheca bovis (formerly P. zopfii genotype-II) is an opportunistic, achlorophyllous alga that causes mastitis in cows and skin disease in cats and dogs, as well as cutaneous lesions in both immunocompetent and immunosuppressed humans. Antifungal medications are commonly ineffective. This study aimed to investigate innate immune responses contributed by cathelicidins to P. bovis in the mammary gland using a mastitis model in mice deficient in the sole murine cathelicidin (Camp). We determined P. bovis caused acute mastitis in mice and induced Camp gene transcription. Whereas, Camp -/- and Camp +/+ littermates had similar local algae burden, Camp +/+ mice produced more pro-inflammatory cytokines, TNF-?, and Cxcl-1. Likewise, Camp +/+ bone marrow-derived macrophages were more responsive to P. bovis, producing more TNF-? and Cxcl-1. Human cathelicidin (LL-37) exhibited a different effect against P. bovis; it had direct algicidal activity against P. bovis and lowered TNF-?, Cxcl-1, and IL-1? production in both cultured murine macrophages and mammary epithelial cells exposed to the pathogenic algae. In conclusion, cathelicidins were involved in protothecosis pathogenesis, with unique roles among the diverse peptide family. Whereas, endogenous cathelicidin (Camp) was key in mammary gland innate defense against P. bovis, human LL-37 had algicidal and immunomodulatory functions.