Development of Highly Efficient Universal Pneumocystis Primers and Their Application in Investigating the Prevalence and Genetic Diversity of Pneumocystis in Wild Hares and Rabbits.

Despite its ubiquitous infectivity to mammals with strong host specificity, our current knowledge about Pneumocystis has originated from studies of merely 4% of extant mammalian species. Further studies of Pneumocystis epidemiology across a broader range of animal species require the use of assays with high sensitivity and specificity. To this end, we have developed multiple universal Pneumocystis primers targeting different genetic loci with high amplification efficiency. Application of these primers to PCR investigation of Pneumocystis in free-living hares (Lepus townsendii, n = 130) and rabbits (Oryctolagus cuniculus, n = 8) in Canada revealed a prevalence of 81% (105/130) and 25% (2/8), respectively. Genotyping analysis identified five and two variants of Pneumocystis from hares and rabbits, respectively, with significant sequence divergence between the variants from hares. Based on phylogenetic analysis using nearly full-length sequences of the mitochondrial genome, nuclear rRNA operon and dihydropteroate synthase gene for the two most common variants, Pneumocystis in hares and rabbits are more closely related to each other than either are to Pneumocystis in other mammals. Furthermore, Pneumocystis in both hares and rabbits are more closely related to Pneumocystis in primates and dogs than to Pneumocystis in rodents. The high prevalence of Pneumocystis in hares (P. sp. 'townsendii') suggests its widespread transmissibility in the natural environment, similar to P. oryctolagi in rabbits. The presence of multiple distinct Pneumocystis populations in hares contrasts with the lack of apparent intra-species heterogeneity in P. oryctolagi, implying a unique evolution history of P. sp. 'townsendii' in hares.

A Novel CARMIL2 Immunodeficiency Identified in a Subset of Cavalier King Charles Spaniels with Pneumocystis and Bordetella Pneumonia.

Pet dogs are a valuable natural animal model for studying relationships between primary immunodeficiencies and susceptibility to Pneumocystis and other opportunistic respiratory pathogens. Certain breeds, such as the Cavalier King Charles Spaniel, are over-represented for Pneumocystis pneumonia (PCP), suggesting the presence of a primary immunodeficiency in the breed. Here, we report the discovery of a CARMIL2 nonsense variant in three Cavalier King Charles Spaniel dogs with either PCP (n = 2) or refractory Bordetella pneumonia (n = 1). CARMIL2 encodes a protein that plays critical roles in T-cell activation and other aspects of immune function. Deleterious CARMIL2 variants have recently been reported in human patients with PCP and other recurrent pneumonias. In addition to opportunistic respiratory infection, the affected dogs also exhibited other clinical manifestations of CARMIL2 deficiencies that have been reported in humans, including early-onset gastrointestinal disease, allergic skin disease, mucocutaneous lesions, abscesses, autoimmune disorders, and gastrointestinal parasitism. This discovery highlights the potential utility of a natural canine model in identifying and studying primary immunodeficiencies in patients affected by PCP.

Meta-Analysis and Systematic Literature Review of the Genus Pneumocystis in Pet, Farm, Zoo, and Wild Mammal Species.

A systematic literature search on Pneumocystis in 276 pet, farm, zoo, and wild mammal species resulted in 124 publications originating from 38 countries that were analyzed descriptively and statistically, for which inclusion and exclusion criteria were exactly defined. The range of recorded Pneumocystis prevalence was broad, yet in half of the citations a prevalence of ≤25% was documented. Prevalence was significantly dependent on the method used for Pneumocystis detection, with PCR revealing the highest percentages. Pet animals showed the lowest median Pneumocystis prevalence, followed by farm, wild, and zoo animals. In contrast, pet and farm animals showed higher proportions of high-grade infection levels compared to zoo and wild mammals. Only in individual cases, all of them associated with severe Pneumocystis pneumonia, was an underlying immunosuppression confirmed. Acquired immunosuppression caused by other diseases was frequently discussed, but its significance, especially in highly immunosuppressive cases, needs to be clarified. This meta-analysis supported a potential influence of the social and environmental factors of the host on Pneumocystis transmission in wildlife, which must be further elucidated, as well as the genetic diversity of the fungus.

Detection of Pneumocystis and Morphological Description of Fungal Distribution and Severity of Infection in Thirty-Six Mammal Species.

Pneumocystis spp. are thought to adapt to the lungs of potentially all mammals. However, the full host range, fungal burden and severity of infection are unknown for many species. In this study, lung tissue samples originating from 845 animals of 31 different families of eight mammal orders were screened by in situ hybridization (ISH) using a universal 18S rRNA probe for Pneumocystis, followed by hematoxylin and eosin (H&E) staining for determining histopathological lesions. A total of 216 (26%) samples were positive for Pneumocystis spp., encompassing 36 of 98 investigated mammal species, with 17 of them being described for the first time for the presence of Pneumocystis spp. The prevalence of Pneumocystis spp. as assessed by ISH varied greatly among different mammal species while the organism load was overall low, suggesting a status of colonization or subclinical infection. Severe Pneumocystis pneumonia seemed to be very rare. For most of the Pneumocystis-positive samples, comparative microscopic examination of H&E- and ISH-stained serial sections revealed an association of the fungus with minor lesions, consistent with an interstitial pneumonia. Colonization or subclinical infection of Pneumocystis in the lung might be important in many mammal species because the animals may serve as a reservoir.

Pneumocystis spp. in Pigs: A Longitudinal Quantitative Study and Co-Infection Assessment in Austrian Farms.

While Pneumocystis has been recognized as both a ubiquitous commensal fungus in immunocompetent mammalian hosts and a major opportunistic pathogen in humans responsible for severe pneumonias in immunocompromised patients, in pigs its epidemiology and association with pulmonary diseases have been rarely reported. Nevertheless, the fungus can be quite abundant in porcine populations with up to 51% of prevalence reported so far. The current study was undertaken to longitudinally quantify Pneumocystis carinii f. sp. suis and other pulmonary pathogens in a cohort of 50 pigs from five Austrian farms (i.e., 10 pigs per farm) with a history of respiratory disease at five time points between the first week and the fourth month of life. The fungus was present as early as the suckling period (16% and 26% of the animals in the first and the third week, respectively), yet not in a high amount. Over time, both the organism load (highest 4.4 × 105 copies/mL) and prevalence (up to 88% of positive animals in the third month) increased in each farm. The relative prevalence of various coinfection patterns was significantly different over time. The current study unravelled a complex co-infection history involving Pneumocystis and other pulmonary pathogens in pigs, suggesting a relevant role of the fungus in the respiratory disease scenario of this host.

Genomic and transcriptomic analysis of the toluene degrading black yeast Cladophialophora immunda.

Cladophialophora immunda is an ascomycotal species belonging to the group of the black yeasts. These fungi have a thick and melanized cell wall and other physiological adaptations that allows them to cope with several extreme physical and chemical conditions. Member of the group can colonize some of the most extremophilic environments on Earth. Cladophialophora immunda together with a few other species of the order Chaetothyriales show a special association with hydrocarbon polluted environments. The finding that the fungus is able to completely mineralize toluene makes it an interesting candidate for bioremediation purposes. The present study is the first transcriptomic investigation of a fungus grown in presence of toluene as sole carbon and energy source. We could observe the activation of genes involved in toluene degradatation and several stress response mechanisms which allowed the fungus to survive the toluene exposure. The thorough comparative genomics analysis allowed us to identify several events of horizontal gene transfer between bacteria and Cladophialophora immunda and unveil toluene degradation steps that were previously reported in bacteria. The work presented here aims to give new insights into the ecology of Cladophialophora immunda and its adaptation strategies to hydrocarbon polluted environments.

The Transcriptome of Exophiala dermatitidis during Ex-vivo Skin Model Infection.

The black yeast Exophiala dermatitidis is a widespread polyextremophile and human pathogen, that is found in extreme natural habitats and man-made environments such as dishwashers. It can cause various diseases ranging from phaeohyphomycosis and systemic infections, with fatality rates reaching 40%. While the number of cases in immunocompromised patients are increasing, knowledge of the infections, virulence factors and host response is still scarce. In this study, for the first time, an artificial infection of an ex-vivo skin model with Exophiala dermatitidis was monitored microscopically and transcriptomically. Results show that Exophiala dermatitidis is able to actively grow and penetrate the skin. The analysis of the genomic and RNA-sequencing data delivers a rich and complex transcriptome where circular RNAs, fusion transcripts, long non-coding RNAs and antisense transcripts are found. Changes in transcription strongly affect pathways related to nutrients acquisition, energy metabolism, cell wall, morphological switch, and known virulence factors. The L-Tyrosine melanin pathway is specifically upregulated during infection. Moreover the production of secondary metabolites, especially alkaloids, is increased. Our study is the first that gives an insight into the complexity of the transcriptome of Exophiala dermatitidis during artificial skin infections and reveals new virulence factors.

Pathogenic Yet Environmentally Friendly? Black Fungal Candidates for Bioremediation of Pollutants.

A collection of 163 strains of black yeast-like fungi from the CBS Fungal Biodiversity Center (Utrecht, The Netherlands), has been screened for the ability to grow on hexadecane, toluene and polychlorinated biphenyl 126 (PCB126) as the sole carbon and energy source. These compounds were chosen as representatives of relevant environmental pollutants. A microtiter plate-based culture assay was set up in order to screen the fungal strains for growth on the selected xenobiotics versus glucose, as a positive control. Growth was observed in 25 strains on at least two of the tested substrates. Confirmation of substrate assimilation was performed by cultivation on closed vials and analysis of the headspace composition with regard to the added volatile substrates and the generated carbon dioxide. Exophiala mesophila (CBS 120910) and Cladophialophora immunda (CBS 110551), both of the order Chaetothyriales and isolated from a patient with chronic sinusitis and a polluted soil sample, respectively, showed the ability to grow on toluene as the sole carbon and energy source. Toluene assimilation has previously been described for C. immunda but this is the first account for E. mesophila. Also, this is the first time that the capacity to grow on alkylbenzenes has been demonstrated for a clinical isolate. Assimilation of toluene could not be demonstrated for the human opportunistic pathogen Pseudoallescheria boydii (CBS 115.59, Microascales), but the results from microtiter plate assays suggest that strains of this species are promising candidates for further studies. The outstanding abilities of black yeast-like fungi to thrive in extreme environments makes them ideal agents for the bioremediation of polluted soils, and for the treatment of contaminated gas streams in biofilters. However, interrelations between hydrocarbonoclastic and potentially pathogenic strains need to be elucidated in order to avoid the possibility of biohazards occurring.

From Glacier to Sauna: RNA-Seq of the Human Pathogen Black Fungus Exophiala dermatitidis under Varying Temperature Conditions Exhibits Common and Novel Fungal Response.

Exophiala dermatitidis (Wangiella dermatitidis) belongs to the group of the so-called black yeasts. Thanks in part to its thick and strongly melanized cell walls, E. dermatitidis is extremely tolerant to various kinds of stress, including extreme pH, temperature and desiccation. E. dermatitidis is also the agent responsible for various severe illnesses in humans, such as pneumonia and keratitis, and might lead to fatal brain infections. Due to its association with the human environment, its poly-extremophilic lifestyle and its pathogenicity in humans, E. dermatitidis has become an important model organism. In this study we present the functional analysis of the transcriptional response of the fungus at 1°C and 45°C, in comparison with that at 37°C, for two different exposition times, i.e. 1 hour and 1 week. At 1°C, E. dermatitidis uses a large repertoire of tools to acclimatize, such as lipid membrane fluidization, trehalose production or cytoskeleton rearrangement, which allows the fungus to remain metabolically active. At 45°C, the fungus drifts into a replicative state and increases the activity of the Golgi apparatus. As a novel finding, our study provides evidence that, apart from the protein coding genes, non-coding RNAs, circular RNAs as well as fusion-transcripts are differentially regulated and that the function of the fusion-transcripts can be related to the corresponding temperature condition. This work establishes that E. dermatitidis adapts to its environment by modulating coding and non-coding gene transcription levels and through the regulation of chimeric and circular RNAs.

Draft Genome Sequence of Exophiala mesophila, a Black Yeast with High Bioremediation Potential.

The fungal genus Exophiala comprises both pathogen species, which cause severe infections in humans, and environmental species, which are able to degrade alkylbenzene compounds. The draft genome sequence of Exophiala mesophila presented here is the first genome assembly of an alkylbenzene-degrading organism belonging to the genus Exophiala.

Draft Genome of Cladophialophora immunda, a Black Yeast and Efficient Degrader of Polyaromatic Hydrocarbons.

The fungal genus Cladophialophora comprises many species which cause severe and even fatal infections in humans as well as environmental strains able to degrade polyaromatic hydrocarbons. The draft genome of Cladophialophora immunda presented here is the first whole-genome sequence within this important genus.

Nothing special in the specialist? Draft genome sequence of Cryomyces antarcticus, the most extremophilic fungus from Antarctica.

The draft genome of the Antarctic endemic fungus Cryomyces antarcticus is presented. This rock inhabiting, microcolonial fungus is extremely stress tolerant and it is a model organism for exobiology and studies on stress resistance in Eukaryots. Since this fungus is a specialist in the most extreme environment of the Earth, the analysis of its genome is of important value for the understanding of fungal genome evolution and stress adaptation. A comparison with Neurospora crassa as well as with other microcolonial fungi shows that the fungus has a genome size of 24 Mbp, which is the average in the fungal kingdom. Although sexual reproduction was never observed in this fungus, 34 mating genes are present with protein homologs in the classes Eurotiomycetes, Sordariomycetes and Dothideomycetes. The first analysis of the draft genome did not reveal any significant deviations of this genome from comparative species and mesophilic hyphomycetes.

Red blood cell subpopulations in freshly drawn blood: application of proteomics and metabolomics to a decades-long biological issue.

BACKGROUND: It has long been known that red blood cells comprise various subpopulations, which can be separated through Percoll density gradients. MATERIALS AND METHODS: In this study, we performed integrated flow cytometry, proteomic and metabolomic analyses on five distinct red blood cell subpopulations obtained by Percoll density gradient separation of freshly drawn leucocyte-depleted erythrocyte concentrates. The relation of density gradient fractions to cell age was confirmed through band 4.1a/4.1b assays. RESULTS: We observed a decrease in size and increase in cell rugosity in older (denser) populations. Metabolomic analysis of fraction 5 (the oldest population) showed a decrease of glycolytic metabolism and of anti-oxidant defence-related mechanisms, resulting in decreased activation of the pentose phosphate pathway, less accumulation of NADPH and reduced glutathione and increased levels of oxidized glutathione. These observations strengthen conclusions about the role of oxidative stress in erythrocyte ageing in vivo, in analogy with results of recent in vitro studies. On the other hand, no substantial proteomic differences were observed among fractions. This result was partly explained by intrinsic technical limitations of the two-dimensional gel electrophoresis approach and the probable clearance from the bloodstream of erythrocytes with membrane protein alterations. Since this clearance effect is not present in vitro (in blood bank conditions), proteomic studies have shown substantial membrane lesions in ageing red blood cells in vitro. CONCLUSION: This analysis shows that the three main red blood cell subpopulations, accounting for over 92% of the total RBC, are rather homogeneous soon after withdrawal. Major age-related alterations in vivo probably affect enzyme activities through post-translational mechanisms rather than through changes in the overall proteomic profile of RBC.

Oxidative stress and caspase-mediated fragmentation of cytoplasmic domain of erythrocyte band 3 during blood storage.

BACKGROUND: During blood bank storage, red blood cells (RBCs) undergo a number of biological and biochemical alterations collectively referred to as "storage lesions". These injuries include loss and oxidative cross-linking of band 3, the major integral protein of RBC membranes. Denaturation of hemoglobin (Hb) and damage to the amino-terminal of band 3 are recognised as the starting events for immunological recognition mechanisms and phagocytic removal of senescent or impaired RBCs from circulation. Consequently, studies focusing on the Hb-association and oxidative status of the cytoskeleton of stored RBCs intended for transfusion are of extreme interest. In this work, two storage-related fragments of band 3 were documented and biochemically characterised. METHODS: Four RBC units were collected from normal volunteers and stored for 21 days under (i) standard blood bank conditions, (ii) anaerobic conditions, or (iii) in the presence of caspase 3-inhibitor. Degradation products of band 3 were followed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis coupled with western blot and mass spectrometry analyses. RESULTS: Two different degradation products of the cytoplasmic domain of the erythrocyte band 3 (CDB3) were detected in RBC membranes during storage in saline-adenine-glucosemannitol (SAGM) preservation medium. One of these fragments showed an apparent molecular weight of 34 kDa and was demonstrated to be the product of a free-radical attack on the protein main chain, whereas another fragment of 24 kDa was the result of a caspase 3-mediated cleavage. DISCUSSION: Although to different extent, anaerobic conditions reduced the formation of both truncated products indicating an enhanced activity of the pro-apoptotic caspase 3 enzyme following oxidative stress. Interestingly, both CDB3 fragments were tightly associated to the erythrocyte membrane supporting the involvement of Cys-201 and/or Cys-317 in clustering different band 3 monomers.

Native analysis of plasma-derived clotting factor VIII concentrates: "sponge effect" and contaminants.

In the present study, we analysed two commercially available plasma-derived FVIII preparations, Beriate and Emoclot, through native gel-based approaches (CN-PAGE). The rationale behind this study was to assess whether protein complexes from plasma resisted the aggressive manufacturing processes. A preliminary analysis of plasma complexes was performed focusing on the molecular weight range between 45 kDa and 1 MDa. As a result, we could evidence in both preparations the presence of complexes, which resisted the cryoprecipitation, chromatographic and heat treatment processing steps. These complexes behaved in a "sponge-effect"-like fashion through the enrichment of otherwise less abundant contaminants. The use of an alternative electrophoretic approach such as CN-PAGE allowed us to confirm 2DE-based observations and expand the list of non-FVIII proteins which contaminate the preparations.

Characterization of stress responses of heavy metal and metalloid inducible promoters in synechocystis PCC6803.

In several biotechnological applications of living bacterial cells with inducible gene expression systems, the extent of overexpression and the specificity to the inducer are key elements. In the present study, we established the concentration ranges of Zn(2+), Ni(2+), Co(2+), AsO(2)(-), and Cd(2+) ions that caused significant activation of the respective promoters of Synechocystis sp. without concomitant unspecific stress responses. The low expression levels can be increased up to 10-100-fold upon treatments with Cd(2+), AsO(2)(-), Zn(2+), and Co(2+) ions and up to 800-fold upon Ni(2+) treatment. These results facilitate the development of conditional gene expression systems in cyanobacteria.

Plasma-derived clotting factor VIII: heterogeneity evaluation in the quest for potential inhibitory-antibody stimulating factors.

In this study, we investigated the heterogeneity and the purity grade of three commercially available plasma-derived clotting factor VIII (FVIII) concentrates, which highly differ with regard to purification strategies, relative concentrations of stabilizers (von Willebrand factor, with or without albumin) and virus inactivation strategies (solvent/detergent and/or heat/pasteurization treatments). Western blot analyses were used to evaluate product-specific variations from Emoclot(®) , Alphanate(®) and Haemate(®) both in the presence and absence of reducing agents (dithiotreithol). All the plasma-derived concentrates showed a strong heterogeneity, as they all included a significant amount of truncated forms of the full-length (FL) clotting FVIII protein. The intact protein accounted for the 38% of the total FVIII proteins in Haemate(®) and 29 and 23% in Alphanate(®) and Emoclot(®) , respectively. Lower intact FVIII amounts in Emoclot might be mainly due to the low von Willebrand factor dosage and the absence of albumin. Upon addition of thrombin, both the FL and truncated forms of the FVIII protein were almost completely digested. Indeed, after thrombin activation, we could still observe a mixture of B-domain truncated forms of the FL protein along with biologically active digested-A1 forms. Batch-to-batch variation was tested with no evident changes appearing among different batches. Despite the variables in manufacturing processes, inter-product comparisons yielded similar results for all the plasma-derived FVIII considered in this study. However, we could individuate in Emoclot a band that was not digested by thrombin, which we could characterize as the 200 kDa FVIII heavy chain. This investigation prompts new concerns about the strong heterogeneity observed upon thrombin digestion of plasma-derived FVIII, which might contribute to the development of inhibitory antibodies at an early stage of therapy, and to which extent these untoward phenomena could be avoided through direct intervention on routine manufacturing processes.

Oxidative stress-dependent oligomeric status of erythrocyte peroxiredoxin II (PrxII) during storage under standard blood banking conditions.

Although biochemical properties of 2-Cys peroxiredoxins have been extensively studied in various cell lines and organisms, redox-induced structural transitions of peroxiredoxin II (PrxII) in human erythrocytes certainly warrant further investigation. In this work, cytosol and membrane ghosts of both fresh erythrocytes (cells obtained just after blood collection) and 28-day stored erythrocytes were analyzed by proteomics tools. We demonstrated that in fresh red blood cells PrxII exhibits four different oligomeric states in cytosol, whereas no PrxII complexes are in the membrane. The highest molecular weight PrxII protein complex (440 kDa) was proven to derive from the association between tetrameric catalase (CAT, 232 kDa) and decameric PrxII, whereas oligomers at 140, 100 and 67 kDa resulted to be homo-polymeric complexes composed of variable copies of PrxII monomeric subunits. Interestingly, the 440 kDa complex contained both reduced and oxidized (disulphide-linked dimers) PrxII decamers. Upon oxidative stress (28-day storage), the PrxII oligomers at 100 kDa in the cytosol disappeared and the CAT-PrxII hetero-oligomeric complex at 440 kDa is converted to a higher molecular weight structure (480 kDa) due to the presence therein of cross-linked species of PrxII and hemoglobin. More interestingly, oxidized red cell membranes contained the CAT-PrxII complex detected in 0-day cytosol as a consequence of protein recruitments induced by oxidative stress, however it showed a greater percentage of PrxII dimers. Finally, since the adoption of distinct PrxII structures is known to be closely related to different functions, peroxidase activity assays were performed demonstrating a positive reaction for oligomers at 440 kDa (both in cytosol and membrane compartment) and at 140 kDa. Our results contribute to clarify structural and functional switching of peroxiredoxin II in erythrocytes, thus possibly opening new scenarios in the biological roles played by this protein in defense mechanisms against oxidative stress, especially with the reference to red cell storage lesions.

Peroxiredoxin-2 as a candidate biomarker to test oxidative stress levels of stored red blood cells under blood bank conditions.

BACKGROUND: Several researches on aging red blood cells (RBCs)--performed both in vivo and under blood bank conditions--revealed that RBC membrane proteins undergo a number of irreversible alterations, mainly due to oxidative stress. The individuation of proteins to be used as indicators of irreversible RBC injury and to be proposed as candidate biomarkers of oxidative damage or aging status during blood storage is therefore of great interest. STUDY DESIGN AND METHODS: Based on this purpose we performed proteomic analysis of the membranes of RBCs during various storage periods under blood bank conditions. Changes in protein composition of RBC membranes were monitored as a function of the storage period by means of polyacrylamide gel electrophoresis coupled with immunoblotting and mass spectrometry analyses. RESULTS: During storage, a progressive linkage of typical cytosolic proteins to the membrane was detected, including both antioxidant and metabolic enzymes (such as catalase, peroxiredoxin-2 [Prx2], and 2,3-bisphosphoglycerate-mutase), as well as nonreducible cross-linkings of probably oxidized or denatured hemoglobin. This phenomenon was unequivocally related to oxidative stress, since storage of RBCs under anaerobic conditions showed a suppression of these protein recruitments to the membrane. CONCLUSION: The detailed analysis of these protein associations to the membrane of aged RBCs allowed Prx2 to be suggested as a potential RBC oxidative stress marker for the sake of developing new approaches in quality assurance of blood components.