Differences in in vitro interactions between macrophages with pathogenic and environmental strains of Prototheca.

Aim: We investigated the interactions between macrophage and different strains of Prototheca. Materials & method: J774A.1 macrophages were infected with clinical isolates of Prototheca ciferrii 18125 and P. ciferrii 50779 and environmental isolate of P. ciferrii N71. Phagocytosis activities were compared by colony-forming unit assays at 3, 6 and 9 h after infection. Cytokine levels were detected by RT-PCR and ELISA. iNOS protein expression was examined by western blotting. Results: All P. ciferrii strains were phagocytized by macrophages but induced different levels of cytokines in macrophages. Moreover, infected by P. ciferrii N71 upregulated much higher iNOS protein expression in J774A.1 than that infected by the clinical strains. Conclusion: Clinical and environmental P. ciferrii strains show differences in their interactions with macrophages, which may be attributed to their virulence.

Prototheca zopfii genotype II induces mitochondrial apoptosis in models of bovine mastitis.

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 (ΔΨm) 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-α, IL-1ß 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.

Emergence of Fungal-Like Organisms: Prototheca.

The genus Prototheca consists of achlorophyllic algae that are ubiquitous in the environment and animal intestines. However, this organism has forfeited its photosynthetic ability and switched to parasitism. In 1894, Krüger described two microorganisms isolated in Germany from mucous flux of Tilia and Ulmus spp., namely Prototheca moriformis and P. zopfii. Based on their yeast-like colony morphology, Krüger classified these organisms as fungi. The genus is now included within the class Trebouxiophyceae, order Chlorellales, and family Chlorellaceae. Historically, protothecosis and infections caused by green algae have been studied in the field of medical mycology. Prototheca spp. have been found to colonize human skin, fingernails, the respiratory tract, and digestive system. Although human infection by Prototheca is considered rare, an increase in infections has been noted among immunosuppressed patients, those on corticosteroid treatment, or both. Moreover, the first human outbreak of protothecal algaemia and sepsis was recently reported in a tertiary care chemotherapy oncology unit in 2018. Prototheca is also a causative pathogen of bovine disease. Prototheca zopfii and P. blaschkeae are associated with bovine mastitis, which causes a reduction in milk production and secretion of thin, watery milk containing white flakes. Economic losses are incurred either directly via reduced milk production and premature culling of affected animals or indirectly as a result of treatment and veterinary care expenses. Thus, knowledge of this fungal-like pathogen is essential in human and veterinary medicine. In this mini-review, I briefly introduce human and animal protothecoses.

Isobaric tag for relative and absolute quantitation-based comparative proteomic analysis of human pathogenic Prototheca zopfii genotype 2 and environmental genotype 1 strains.

BACKGROUND/PURPOSE: Prototheca species are ubiquitous achlorophyllic microalgae belonging to the family Chlorellaceae, which can cause a wide range of infections in humans and animals. Mainly in individuals with immunologic defects or trauma, Prototheca spp. can cause even lethal diseases. However, the exact pathogenic mechanism of Prototheca in causing disease remains largely unknown. To investigate the differences between pathogenic and nonpathogenic Prototheca spp. genotypes on proteome level, a nonpathogenic Prototheca zopfii genotype 1 strain, isolated from cow manure, and a human pathogenic P. zopfii genotype 2, isolated from human granulomatous lymphadenitis, were studied. METHODS: Differentially expressed proteins between the two genotypes were quantified by isobaric tag for relative and absolute quantitation-based quantitative proteomics, using liquid chromatography-tandem mass spectrometry. RESULTS: A total of 245 proteins were identified from the proteomic analysis after data filtering to eliminate low-scoring spectra. Among these, 35 proteins that displayed a significant (p<0.05) 1.5-fold change were considered as differentially expressed proteins. CONCLUSION: The differentially expressed proteins were associated with suppressed energy production and conversion, carbohydrate transport and metabolism, and enhanced translation in the genotype 2 strain, and are thus potentially relevant in the pathogenic mechanism of P. zopfii genotype 2, but need further investigation.

[A case of cutaneous protothecosis]. / Nablyudenie prototekoza kozhi.

The paper describes a case of a rare opportunistic infection, such as skin lesion caused by achlorophyllic unicellular algae of the genus Prototheca. It provides a detailed pathologic description of the foci of cutaneous protothecosis, such as pandermal inflammatory infiltrate, granulomas, pseudoepitheliomatous hyperplasia, and intraepidermal abscesses. Criteria for pathogen detection in histological sections are given.

Glutathione may have implications in the design of 3-bromopyruvate treatment protocols for both fungal and algal infections as well as multiple myeloma.

In different fungal and algal species, the intracellular concentration of reduced glutathione (GSH) correlates closely with their susceptibility to killing by the small molecule alkylating agent 3-bromopyruvate (3BP). Additionally, in the case of Cryptococcus neoformans cells 3BP exhibits a synergistic effect with buthionine sulfoximine (BSO), a known GSH depletion agent. This effect was observed when 3BP and BSO were used together at concentrations respectively of 4-5 and almost 8 times lower than their Minimal Inhibitory Concentration (MIC). Finally, at different concentrations of 3BP (equal to the half-MIC, MIC and double-MIC in a case of fungi, 1 mM and 2.5 mM for microalgae and 25, 50, 100 µM for human multiple myeloma (MM) cells), a significant decrease in GSH concentration is observed inside microorganisms as well as tumor cells. In contrast to the GSH concentration decrease, the presence of 3BP at concentrations corresponding to sub-MIC values or half maximal inhibitory concentration (IC50) clearly results in increasing the expression of genes encoding enzymes involved in the synthesis of GSH in Cryptococcus neoformans and MM cells. Moreover, as shown for the first time in the MM cell model, the drastic decrease in the ATP level and GSH concentration and the increase in the amount of ROS caused by 3BP ultimately results in cell death.

Biofilm formation by pathogenic Prototheca algae.

UNLABELLED: Prototheca microalgae are the only plants known to cause infections in humans and animals. The mechanisms of Prototheca infections are poorly understood, and no good treatments are available. Biofilms-surface-attached, three-dimensional microbial communities contributing to chronic infections-are formed by many pathogenic bacteria and fungi, but it is not known if Prototheca algae also have this ability. This study shows that various Prototheca species form biofilms composed of surface-attached cells in all growth phases, linked together by matrix containing DNA and polysaccharides. Biofilm formation was modulated by the presence of host plasma or milk. Compared to planktonic cells, Prototheca biofilms caused decreased release of IL-6 by mononuclear immune cells and responded differently to treatment with antimicrobials. Prototheca biofilms possibly contribute to chronic and hard-to-treat character of those algal infections. SIGNIFICANCE AND IMPACT OF THE STUDY: Prototheca algae are the only existing pathogenic plants. Almost nothing is known about mechanisms of Prototheca infections. This study identifies that, similar to pathogenic bacteria and fungi, Prototheca algae can form biofilms. These biofilms induce reduced immune cell activation relative to planktonic cells, and are also less susceptible to antimicrobials. Biofilm formation by Prototheca could be the first in vitro correlate of pathogenicity, opening a new research field for this pathogen.

Experimental infection of bovine mammary gland with prototheca zopfii genotype 1.

Prototheca zopfii is divided into three genotypes, one of which, P. zopfii genotype 2, appears to be the main causative agent of bovine protothecal mastitis. However, the difference in pathogenicity between genotypes 1 and 2 has not been well investigated. In the present study, we experimentally infected normal bovine mammary gland with P. zopfii genotype 1 to investigate its pathogenicity. The mammary gland infected with P. zopfii genotype 1 showed no clinical signs. However, the histopathologic features of the infected mammary gland consisted of interstitial infiltrates of macrophages, plasma cells, lymphocytes, and fibroblasts with neutrophils in acinar lumens. Algae were present in macrophages and free in the alveolar lumens and the interstitium. Histopathology of the resultant tissue samples revealed that genotype 1 also induced a granulomatous lesion in the cow teat, similar to the mastitis lesion due to genotype 2.

The first case of feline Prototheca wickerhamii infection in Japan.

A 3-year-old castrated male domestic short-haired cat was presented with nodules on the left nasal wing and the left earflap. Prototheca cells were found after excision biopsy of one of the nodules located on the left earflap. The patient cat was generally in good condition without skin problems. Prototheca wickerhamii was isolated from all 6 masses after they were surgically nucleated. The cat was recovered two months after intervention with no recurrence of skin nodules. This report deals with the first case of feline protothecosis in Japan.

An unusual case of granulomatous lymphadenitis due to Prototheca zopfii var. portoricensis in an immunocompetent man in China.

BACKGROUND: Protothecosis is an uncommon human infection caused by Prototheca. Prototheca spp can be considered as saprophytes, and in spite of their frequency in the environment, they are of low virulence and may cause chronic infection with low-grade inflammation in humans. At present, only three species are recognized: Prototheca wickerhamii, Prototheca zopfii and Prototheca stagnora. Of these, the former two have been associated with human disease. This study was an investigation of the clinical and microbiological features of a case of granulomatous lymphadenitis due to P. zopfii var. portoricensis in an immunocompetent man in China. METHODS: We report the case of a 39-year-old male, who presented with swollen lymph nodes, from which the organism was isolated and identified by the RapidID Yeast Plus test (Remel, Santa Fe, NM, USA) and PCR molecular analysis. The pathogenicity of the isolate was confirmed in a mouse model and antifungal drug susceptibility testing was carried out. RESULTS: The pathogen was identified as Prototheca zopfii. The DNA sequence of the 18S SSU rDNA regions of the isolate strain were 100% (1205/1205) identical with Prototheca zopfii var. portoricensis. Antifungal susceptibility tests revealed that it was sensitive to amphotericin B, but resistant to 5-flucytosine, fluconazole, ketoconazole, and itraconazole. The patient responded to treatment with intravenous itraconazole and amphotericin B. CONCLUSIONS: Based on the patient's symptoms and microscopic evaluation, cultures, and molecular analyses of the isolate, granulomatous lymphadenitis due to P. zopfii var. portoricensis was diagnosed. P. zopfii var. portoricensis as a causative agent of human lymphadenitis in an immunocompetent case has not been reported, though a few cases of protothecosis have been reported in China. The real number of protothecosis cases may be greater than that reported in the literature. Thus, clinicians should be vigilant for any unknown cause of granulomatous lymphadenitis and should undertake an intensive histopathology, mycology examination, and even molecular analysis to rule out or confirm a potential Prototheca infection.

Disseminated protothecosis following traumatic Hickman line removal in a patient with leukaemia.

Human protothecosis is a rare infection caused by a member of the genus Prototheca, an ubiquitous alga. Traumatic inoculation can cause infections in the immunocompetent host while disseminated infection is mainly seen in patients with compromised immunity. We report here an unusual case of disseminated infection in a cancer patient. Traumatic removal of a Hickman catheter in this patient, led to the development of a severe skin and subcutaneous tissue infection with algaemia. These infections are often indolent and difficult to treat, with paucity of information available as to guidelines for diagnosis and effective therapeutic options.

Prototheca cutis sp. nov., a newly discovered pathogen of protothecosis isolated from inflamed human skin.

A strain of a novel pathogenic, achlorophyllic alga belonging to the genus Prototheca was isolated from the inflamed skin of a patient with protothecosis in a Japanese hospital. The pathogen was detected and isolated in biopsy specimens by histopathology and culture-based examination. Analyses of the nuclear 18S rDNA gene and 26S rDNA gene D1/D2 domain sequences and chemotaxonomic studies indicated that this strain represents a novel species with a close phylogenetic relationship to Prototheca wickerhamii and Auxenochlorella protothecoides. This strain grew well at 28-30 degrees C, showed slow and weak growth at 37 degrees C, and no growth at 40 degrees C. This strain grew in vitamin-free medium and assimilated acetate (pH 5.1), l-arabinose and soluble starch as a carbon source. The taxonomic description of Prototheca cutis sp. nov. is proposed (type strain JCM 15793(T) =CBS 11262(T) =DSM 22084(T)) as a pathogen of dermatitis.

Cutaneous protothecosis: a case report and review of the literature.

Protothecosis is a rare cause of systemic and/or cutaneous infection. Because approximately 100 cases of human infection with Prototheca wickerhamii have been reported, little is known about the pathogenesis of this infection. Cases include both immunocompetent and immunocompromised patients. However, most cases contain an element of immune compromise, either local or systemic. We present a case of an 81-year-old white woman with a history of multiple nonmelanoma skin cancers, including squamous and basal cell carcinomas and actinic keratoses, throughout her legs and thighs bilaterally. She presented to a central Florida dermatology clinic with complaints of persistent bumps on her right lower leg despite therapy with diclofenac sodium gel 3% and 5-fluorouracil cream 5%. Biopsies of skin specimens from the right lower leg revealed a dermal abscess that contained spherical funguslike organisms, with periodic acid-Schiff staining revealing 6- to 10-microm organisms with internal septations, which are characteristic of P wickerhamii.

Prevalência da mastite bovina causada por Prototheca zopfii em rebanhos leiteiros, na regiäo norte do Paraná / Prevalence of bovine mastitis due to Prototheca zopfii in dairy cattle in north Paraná, Brazil

Algas do gênero Prototheca têm sido relacionadas como agente etiológico na ocorrência de mastite bovina, entretanto, nenhum caso foi relatado no estado do Paraná. O presente trabalho objetivou determinar a prevalência desta alga, enquanto agente etiológico nas mastites bovinas, em rebanhos leiteiros, na regiäo norte do Paraná. Foram visitadas 20 propriedades produtoras de leite e realizados exames clínicos e teste do CMT (California Mastitis Test) em todos os animais em lactaçäo. A alga Prototheca zopfii foi isolada de dois animais de uma das propriedades. Todas as cepas isoladas de Prototheca zopfii mostraram-se resistentes aos antimicrobianos testados.

Actividad inhibitoria "in vitro" de algunos antisépticos frente a cultivos de Prototheca wickerhamii / \"In vitro\" inhibitory activity of some antiseptic drugs against Prototheca wickerhamii strains

Se estudió la acción inhibidora "in vitro" de los siguientes antisépticos: etil mercurio tiosalicilato de sodio, azida sódica, borato de sodio, yoduro de potasio, fenol y cloro, frente a cultivos de 5 cepas de Prototheca wickerhamii. La acción "in vitro" de estas drogas fue medida mediante el halo de inhibición que produjeron en los cultivos de Prototheca en medio de agar-miel de Sabouraud incubados a 28ºC. Los resultados obtenidos permitieron establecer que todas las cepas eran uniformemente susceptibles al etilmercurio tiosalicilato de sodio y a la azida sódica, la mayor parte fue inhibida por el yoduro de potasio, en tanto que, el borato de sodio, el fenol y el cloro resultaron inactivos en las diluciones utilizadas

Algaemia due to Prototheca wickerhamii in a patient with myasthenia gravis.

Prototheca wickerhamii is a rare cause of systemic infection in humans. While some cases occur in previously healthy individuals, others are associated with a variety of preexisting diseases. Here we present, for the first time, a case of P. wickerhamii algaemia in a patient with myasthenia gravis. The patient was successfully treated with amphotericin B.

Canine cutaneous protothecosis: an immunohistochemical analysis of the inflammatory cellular infiltrate.

In this immunohistochemical study, the distribution of the cellular inflammatory infiltrate associated with cutaneous canine protothecosis (Prototheca wickerhamii) was evaluated by consecutive biopsies taken before, during and after treatment. Antibodies specific to canine immunoglobulins (IgG, IgM, IgA), human CD3 antigen (pan T-lymphocyte marker) and human myeloid/ histiocyte antigen (macrophage/neutrophil marker) were used. Before treatment, cellular infiltrate was very scanty in the inflamed areas, but it increased during the treatment, whereas the number of protothecal organisms decreased. Statistical analysis revealed an inverse relation between the number of protothecal organisms and the number of infiltrating macrophage/neutrophils (P < 0.004), T lymphocytes (P < 0.001), and cells containing immunoglobulin G (P < 0.001), M (P < 0.001) and A (P < 0.001) at different stages of the disease. These findings suggest either that protothecal organisms inhibit the migration or proliferation of cellular inflammatory infiltrate or that only dead protothecal organisms induce an effective local immune response.

Epithelioid cell granulomas experimentally induced by prototheca in the skin of mice: a light microscopic study.

Prototheca wickerhamii, an achlorophyllous algae, was previously found to induce massive epithelioid cell granulomas in the skin of mice. By means of light microscopy, examination was made of the histological reactions involved in epithelioid cell granulomas induced by intradermal and/or subcutaneous inoculation of Prototheca wickerhamii in BALB/c and ICR mice. Six BALB/c mice showed granuloma nodules while only three of six ICR mice did so. Based on the results of the present and previous studies, BALB/c mice may be considered a strain particularly vulnerable to contracting epithelioid cell granuloma and ICR mice, a resistant strain. In very early lesions at one week following initial prototheca inoculation, cellular infiltration with varying numbers of polymorphonuclear leukocytes, lymphocytes and some macrophages was observed throughout the dermis and subcutaneous fat tissue. In early lesions at one to two months after inoculation, focal granulomas composed of histiocytic cells and/or macrophages were observed. Mast cells were occasionally present among the histiocytic cell infiltrates. In the granulomatous lesions at two to three months, scattered eosinophils and some lymphocytes were seen. Central necrosis, with numerous neutrophils and many endospores surrounded by the granuloma, was often observed. In late stage lesions at six months, massive lymphocyte and plasma cell infiltration surrounding and/or intervening between vacuolated epithelioid cell clusters was evident. Histological reactions in epithelioid cell granuloma and the ultimate course of this disease can be staged from the histological point of view as follows: 1) diffuse inflammation, 2) cell proliferation leading to epithelioid cell formation, 3) hypertrophy of epithelioid cells with consequent formation of cell aggregates and/or organized granuloma and 4) degeneration of granuloma.(ABSTRACT TRUNCATED AT 250 WORDS)

[Human protothecosis and environment]. / Protothécoses humaines et environnement.

Protothecosis are uncommon infections caused by Prototheca, considered to be achlorophylous algae. Nearly 80 human cases are reported in the literature since the first case described by Davies and Wakelin in 1964 in Sierra Leone (11). The disease have been identified in Europe, Asia (Japan, Thailand, China), Oceania and in the United States with 40 cases, particularly in the Southeast. Clinically, three clinical manifestations can be observed: 1) cutaneous lesions: papules, plaques or eczematoid, papulo-nodular areas of the extremities, 2) olecranon bursitis which occurred in 25% of cases, 3) systemic protothecosis. An immunosuppressive factor local or general can be found in half of the cases and the first description of algal meningitis was reported in a patient with AIDS in association with Cryptococcus neoformans. Because the disease is clinically not suspected, the diagnosis is often provided by histology showing a dermic granuloma with endospores. The characteristic feature of protothecosis in tissues is the presence of specific mature sporangia of Prototheca wickerhamii with the pattern of morula. The organism was PAS, Grocott and mucicarmin positive. The ecology was studied by Clark (7), Pidoux (23), Pore (25) and Sudman (27). Prototheca are ubiquitous inhabitants of sewages and are found in slime flux and animal wastes contaminating different aquatic systems. The transmission generally occurred by traumatic inoculation. Prototheca are also found in the digestive system of man and animals without never invasion of the epithelium and mucosae in experimental models. The pathogenicity and virulence are moderate and they are considered as rare opportunistic agents.

Prototecosis: Informe de tres Casos / Prothotecosis: Report of 3 cases

Se presentan tres casos clinicos de prototecosis vistos y estudiados en el Instituto Nacional de Salud. Uno corresponde a la forma tegumentaria y los otros dos a formas de bursitis del olecranon. Se discuten los aspectos biologicos, clinicos y de laboratorio de esta rara entidad causada por algas