Emerging Fungal Threats in Cystic Fibrosis.

In the past three decades, fungal respiratory colonization and fungal respiratory infections increasingly raised concern in cystic fibrosis (CF). Reasons for this are a better knowledge of the pathogenicity of fungi, whereby detection is sought in more and more CF centers, but also improvement of detection methods. However, differences in fungal detection rates within and between geographical regions exist and indicate the need for standardization of mycological examination of respiratory secretions. The still existing lack of standardization also complicates the assessment of fungal pathogenicity, relevance of fungal detection and risk factors for fungal infections. Nevertheless, numerous studies have now been conducted on differences in detection methods, epidemiology, risk factors, pathogenicity and therapy of fungal diseases in CF. Meanwhile, some research groups now have classified fungal disease entities in CF and developed diagnostic criteria as well as therapeutic guidelines.The following review presents an overview on fungal species relevant in CF. Cultural detection methods with their respective success rates as well as susceptibility testing will be presented, and the problem of increasing azole resistance in Aspergillus fumigatus will be highlighted. Next, current data and conflicting evidence on the epidemiology and risk factors for fungal diseases in patients with CF will be discussed. Finally, an overview of fungal disease entities in CF with their current definitions, diagnostic criteria and therapeutic options will be presented.

Tinea capitis at the University Hospital of Tizi-Ouzou, Algeria, and first isolation of Trichophyton tonsurans.

Tinea capitis, which are fungal infections caused by some dermatophyte species, are common in developing countries, such as Algeria, where they represent a public health concern. In order to define the epidemiological, clinical and diagnostic features of these infections, a prospective study was conducting from September 2018 to May 2019, at the University Hospital of Tizi-Ouzou (Algeria). PATIENTS: All patients addressed to the Laboratory of Parasitology-Mycology of the University Hospital of Tizi-Ouzou for a suspected Tinea capitis, were included in this study. MATERIALS AND METHODS: Before sampling, contact with animals or soil, presence of similar lesions in the family circle, and previous antifungal or corticosteroid treatment were searched. Mycological examination included direct microscopic examination of the samples, and culture on Sabouraud agar slants at 27°C for up to 4weeks. RESULTS: Out of the 87 samples examined, 46 allowed us to confirm the diagnosis of Tinea capitis, representing a positivity rate of 52.9%. The sex ratio was 1.09 (52.2% males vs. 47.8% females among the infected patients), and the infection mainly involved children of 4-6years (43.3%). Thirty-four strains of dermatophytes were isolated. Microsporum canis was the most frequent species identified (44.1%), followed by Trichophyton mentagrophytes (38.2%). During this study, Trichophyton tonsurans, an unusual dermatophyte species in Algeria, was identified for the first time in our hospital. CONCLUSION: Tinea capitis are still common in Algeria, mainly affecting school-aged children and preschool children. Microsporum canis and T. mentagrophytes are the major causative agents, in agreement with previous studies showing a decrease in frequency of anthropophilic species, in favour of zoophilic species.

Antifungal susceptibility testing practices in mycology laboratories in France, 2018.

A survey of mycology laboratories for antifungal susceptibility testing (AFST) was undertaken in France in 2018, to better understand the difference in practices between the participating centers and to identify the difficulties they may encounter as well as eventual gaps with published standards and guidelines. The survey captured information from 45 mycology laboratories in France on how they perform AFST (number of strains tested, preferred method, technical and quality aspects, interpretation of the MIC values, reading and interpretation difficulties). Results indicated that 86% of respondents used Etest as AFST method, with a combination of one to seven antifungal agents tested. Most of the participating laboratories used similar technical parameters to perform their AFST method and a large majority used, as recommended, internal and external quality assessments. Almost all the participating mycology laboratories (98%) reported difficulties to interpret the MIC values, especially when no clinical breakpoints are available. The survey highlighted that the current AFST practices in France need homogenization, particularly for MIC reading and interpretation.

The epidemiology of Candida species in the Middle East and North Africa.

In recent decades, the epidemiology of invasive candidiasis (IC) has progressively changed worldwide. This notably includes emergence of several Candida species. Although some surveillance programs provided global trends in IC epidemiology, data from countries from the Middle East and North Africa (MENA) remain scarce. In this manuscript, we reviewed the existing available data on the epidemiology of Candida species associated with IC, particularly candidemia, in MENA region regarding species distribution. As witnessed worldwide, an evident shift of Candidaalbicans towards non-albicansCandida (NAC) has been observed in the MENA region. The worrying emergence of multi-drug resistant Candida species in MENA calls for a better understanding of their epidemiology. This represents an essential prerequisite for the implementation of effective infection control strategies and surveillance systems to prevent IC among high-risk patients.

PCR-terminal restriction fragment length polymorphism for direct detection and identification of dermatophytes in veterinary mycology.

The biological diagnosis of dermatophytosis in veterinary medicine usually relies on direct microscopic examination and inoculation of the samples on appropriate culture media. However, identification of dermatophytes needs expertise, and cultures which require from days to weeks to be conclusive, may lack of sensitivity because of the quite common overgrowth of contaminants. Here we developed a polymerase chain reaction (PCR) assay based on terminal restriction fragment length polymorphism (TRFLP), which may improve sensitivity of the biological diagnosis and reduce the delay for initiation of treatment. This study was first conducted on pure cultures of various dermatophytes (27 species), yeasts (14 species) and moulds (45 species). After DNA extraction, the internal transcribed spacer (ITS)-28S region of ribosomal DNA was amplified with primers targeting specifically pathogenic dermatophytes, and species of interest were identified by TRFLP with appropriate restriction enzymes. After validation, this assay was applied to veterinary samples and results were compared to those obtained by direct microscopic examination and cultures. All target species were correctly identified, and none of the yeast or mould species was amplified, demonstrating specificity of the assay. Regarding clinical samples, the causative agent was detected by PCR-TRFLP from 97.1% of the samples with both positive direct microscopic examination and cultures. No dermatophytes were detected when both conventional tests were negative. PCR-TRFLP developed here demonstrated to be highly sensitive and specific, allowing rapid detection and direct identification of dermatophytes in veterinary practice. Therefore, this assay is especially suitable for the biological diagnosis of dermatophytosis in different animal species.

[Yeasts from the CTG clade (Candida clade): Biology, impact in human health, and biotechnological applications]. / Les levures du clade CTG (clade Candida) : biologie, incidence en santé humaine et applications en biotechnologie.

Among the subdivision of Saccharomycotina (ascomycetes budding yeasts), the CTG clade (formerly the Candida clade) includes species that display a particular genetic code. In these yeasts, the CTG codon is predominantly translated as a serine instead of a leucine residue. It is now well-known that some CTG clade species have a major impact on human and its activities. Some of them are recognized as opportunistic agents of fungal infections termed candidiasis. In addition, another series of species belonging to the CTG clade draws the attention of some research groups because they exhibit a strong potential in various areas of biotechnology such as biological control, bioremediation, but also in the production of valuable biocompounds (biofuel, vitamins, sweeteners, industrial enzymes). Here we provide an overview of recent advances concerning the biology, clinical relevance, and currently tested biotechnological applications of species of the CTG clade. Future directions for scientific research on these particular yeasts are also discussed.

Malassezia species in students from universities of Sabzevar, Northeastern Iran.

BACKGROUND: Malassezia species, usually part of normal human skin microbiota, may also cause cutaneous infections, mainly pityriasis versicolor (PV) which may rapidly spread in crowded communities, particularly in students' dormitories and sport leisure centers. OBJECTIVE: Few studies have been conducted on PV in students in the Middle East. The present study was designed to determine prevalence of Malassezia species and related diseases in students from city of Sabzevar, Northeast Iran. METHODS: Specimens were collected from 189 students and analyzed by direct microscopy and cultures. Following PCR amplification of the large subunit of ribosomal DNA, species were identified by restriction fragment length polymorphism analysis (RFL-PCR). RESULTS: PV was suspected for 28 students which was confirmed by direct examination and cultures. Cultures also revealed positive for 13 students with healthy skin. Four Malassezia species were identified, with M. restricta as the most prevalent. A higher rate of PV was observed compared to other regions in Iran. However, despite the lipophilic feature of Malassezia species, no significant association was observed between PV or Malassezia species and fatty skin or gender. CONCLUSION: This study determined the frequencies of Malassezia species in part of Northeast Iran, but further studies are needed to identify risk factors for PV.

Ecology of Scedosporium Species: Present Knowledge and Future Research.

The genus Scedosporium, which comprises at least five clinically relevant species, i.e. Scedosporium apiospermum, Scedosporium boydii, Scedosporium aurantiacum, Scedosporium dehoogii and Scedosporium minutisporum, ranks the second among the filamentous fungi colonizing the airways of patients with cystic fibrosis (CF). This colonization of the airways is thought to contribute to the inflammatory reaction leading to a progressive deterioration of the lung function. Additionally, these colonizing fungi may lead to severe disseminated infections in case of lung transplantation. Therefore, considering the low susceptibility of Scedosporium species to all current antifungal drugs, preventive measures should be defined to reduce the risk of exposure to these fungi for non-colonized CF patients. With this in mind, several studies have been conducted to elucidate the ecology of these fungi and to define possible sources of patient contamination. This review will summarize the major outcomes of those studies, including: the clear demonstration that ecological niches of Scedosporium species are strongly impacted by human activities, and the ability of Scedosporium species to degrade aliphatic and aromatic pollutants which supports the high occurrence of these species in contaminated soils and polluted waters and makes them promising candidates for bioremediation purposes. Finally, prospects for future research in this field are proposed.

Enzymatic Mechanisms Involved in Evasion of Fungi to the Oxidative Stress: Focus on Scedosporium apiospermum.

The airways of patients with cystic fibrosis (CF) are frequently colonized by various filamentous fungi, mainly Aspergillus fumigatus and Scedosporium species. To establish within the respiratory tract and cause an infection, these opportunistic fungi express pathogenic factors allowing adherence to the host tissues, uptake of extracellular iron, or evasion to the host immune response. During the colonization process, inhaled conidia and the subsequent hyphae are exposed to reactive oxygen species (ROS) and reactive nitrogen species (RNS) released by phagocytic cells, which cause in the fungal cells an oxidative stress and a nitrosative stress, respectively. To cope with these constraints, fungal pathogens have developed various mechanisms that protect the fungus against ROS and RNS, including enzymatic antioxidant systems. In this review, we summarize the different works performed on ROS- and RNS-detoxifying enzymes in fungi commonly encountered in the airways of CF patients and highlight their role in pathogenesis of the airway colonization or respiratory infections. The potential of these enzymes as serodiagnostic tools is also emphasized. In addition, taking advantage of the recent availability of the whole genome sequence of S. apiospermum, we identified the various genes encoding ROS- and RNS-detoxifying enzymes, which pave the way for future investigations on the role of these enzymes in pathogenesis of these emerging species since they may constitute new therapeutics targets.

Fungal Planet description sheets: 469-557.

Novel species of fungi described in this study include those from various countries as follows: Australia: Apiognomonia lasiopetali on Lasiopetalum sp., Blastacervulus eucalyptorum on Eucalyptus adesmophloia, Bullanockia australis (incl. Bullanockia gen. nov.) on Kingia australis, Caliciopsis eucalypti on Eucalyptus marginata, Celerioriella petrophiles on Petrophile teretifolia, Coleophoma xanthosiae on Xanthosia rotundifolia, Coniothyrium hakeae on Hakea sp., Diatrypella banksiae on Banksia formosa, Disculoides corymbiae on Corymbia calophylla, Elsinoë eelemani on Melaleuca alternifolia, Elsinoë eucalyptigena on Eucalyptus kingsmillii, Elsinoë preissianae on Eucalyptus preissiana, Eucasphaeria rustici on Eucalyptus creta, Hyweljonesia queenslandica (incl. Hyweljonesia gen. nov.) on the cocoon of an unidentified microlepidoptera, Mycodiella eucalypti (incl. Mycodiella gen. nov.) on Eucalyptus diversicolor, Myrtapenidiella sporadicae on Eucalyptus sporadica, Neocrinula xanthorrhoeae (incl. Neocrinula gen. nov.) on Xanthorrhoea sp., Ophiocordyceps nooreniae on dead ant, Phaeosphaeriopsis agavacearum on Agave sp., Phlogicylindrium mokarei on Eucalyptus sp., Phyllosticta acaciigena on Acacia suaveolens, Pleurophoma acaciae on Acacia glaucoptera, Pyrenochaeta hakeae on Hakea sp., Readeriella lehmannii on Eucalyptus lehmannii, Saccharata banksiae on Banksia grandis, Saccharata daviesiae on Daviesia pachyphylla, Saccharata eucalyptorum on Eucalyptus bigalerita, Saccharata hakeae on Hakea baxteri, Saccharata hakeicola on Hakea victoria, Saccharata lambertiae on Lambertia ericifolia, Saccharata petrophiles on Petrophile sp., Saccharata petrophilicola on Petrophile fastigiata, Sphaerellopsis hakeae on Hakea sp., and Teichospora kingiae on Kingia australis.Brazil: Adautomilanezia caesalpiniae (incl. Adautomilanezia gen. nov.) on Caesalpina echinata, Arthrophiala arthrospora (incl. Arthrophiala gen. nov.) on Sagittaria montevidensis, Diaporthe caatingaensis (endophyte from Tacinga inamoena), Geastrum ishikawae on sandy soil, Geastrum pusillipilosum on soil, Gymnopus pygmaeus on dead leaves and sticks, Inonotus hymenonitens on decayed angiosperm trunk, Pyricularia urashimae on Urochloa brizantha, and Synnemellisia aurantia on Passiflora edulis. Chile: Tubulicrinis australis on Lophosoria quadripinnata.France: Cercophora squamulosa from submerged wood, and Scedosporium cereisporum from fluids of a wastewater treatment plant. Hawaii: Beltraniella acaciae, Dactylaria acaciae, Rhexodenticula acaciae, Rubikia evansii and Torula acaciae (all on Acacia koa).India: Lepidoderma echinosporum on dead semi-woody stems, and Rhodocybe rubrobrunnea from soil. Iran: Talaromyces kabodanensis from hypersaline soil. La Réunion: Neocordana musarum from leaves of Musa sp. Malaysia: Anungitea eucalyptigena on Eucalyptus grandis × pellita, Camptomeriphila leucaenae (incl. Camptomeriphila gen. nov.) on Leucaena leucocephala, Castanediella communis on Eucalyptus pellita, Eucalyptostroma eucalypti (incl. Eucalyptostroma gen. nov.) on Eucalyptus pellita, Melanconiella syzygii on Syzygium sp., Mycophilomyces periconiae (incl. Mycophilomyces gen. nov.) as hyperparasite on Periconia on leaves of Albizia falcataria, Synnemadiella eucalypti (incl. Synnemadiella gen. nov.) on Eucalyptus pellita, and Teichospora nephelii on Nephelium lappaceum.Mexico: Aspergillus bicephalus from soil. New Zealand: Aplosporella sophorae on Sophora microphylla, Libertasomyces platani on Platanus sp., Neothyronectria sophorae (incl. Neothyronectria gen. nov.) on Sophora microphylla, Parastagonospora phoenicicola on Phoenix canariensis, Phaeoacremonium pseudopanacis on Pseudopanax crassifolius, Phlyctema phoenicis on Phoenix canariensis, and Pseudoascochyta novae-zelandiae on Cordyline australis.Panama: Chalara panamensis from needle litter of Pinus cf. caribaea. South Africa: Exophiala eucalypti on leaves of Eucalyptus sp., Fantasmomyces hyalinus (incl. Fantasmomyces gen. nov.) on Acacia exuvialis, Paracladophialophora carceris (incl. Paracladophialophora gen. nov.) on Aloe sp., and Umthunziomyces hagahagensis (incl. Umthunziomyces gen. nov.) on Mimusops caffra.Spain: Clavaria griseobrunnea on bare ground in Pteridium aquilinum field, Cyathus ibericus on small fallen branches of Pinus halepensis, Gyroporus pseudolacteus in humus of Pinus pinaster, and Pseudoascochyta pratensis (incl. Pseudoascochyta gen. nov.) from soil. Thailand: Neoascochyta adenii on Adenium obesum, and Ochroconis capsici on Capsicum annuum. UK: Fusicolla melogrammae from dead stromata of Melogramma campylosporum on bark of Carpinus betulus. Uruguay: Myrmecridium pulvericola from house dust. USA: Neoscolecobasidium agapanthi (incl. Neoscolecobasidium gen. nov.) on Agapanthus sp., Polyscytalum purgamentum on leaf litter, Pseudopithomyces diversisporus from human toenail, Saksenaea trapezispora from knee wound of a soldier, and Sirococcus quercus from Quercus sp. Morphological and culture characteristics along with DNA barcodes are provided.

Validation of a novel real-time PCR for detecting Rasamsonia argillacea species complex in respiratory secretions from cystic fibrosis patients.

Members of the recently introduced fungal genus Rasamsonia (formerly included in the Geosmithia genus) have been described as emerging pathogens in immunosuppressed hosts or patients with cystic fibrosis (CF). Rasamsonia species have often been misidentified as Penicillium or Paecilomyces because of similar morphological characteristics. We validated a commercially available real-time PCR assay (Primerdesign™, UK) for accurate detection of species from the Rasamsonia argillacea complex. First, we tested this assay with a collection of 74 reference strains and clinical isolates and then compared the PCR with cultures of 234 respiratory samples from 152 patients with CF from two University Hospitals in Germany and France. The assay reliably detected the three main species within the Rasamsonia argillacea species complex (R. argillacea, R. piperina, R. aegroticola), which are typically encountered in CF patients. The limit of DNA detection was between 0.01 and 1 pg/µL. Analysis of the DNA extracts from respiratory specimens of CF patients revealed that four out of the 153 patients studied (2.6%) were colonized with R. argillacea species complex. Two species from the R. argillacea complex grew in the parallel cultures from the same patients. In one patient the PCR was positive 5 months before culture. The real-time PCR assay is a sensitive and specific method for detecting the three most important species of the R. argillacea species complex encountered in the CF context. Detection of these emerging pathogens in respiratory secretions from CF patients by this novel assay may increase our understanding of the occurrence and epidemiology of the R. argillacea species complex.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry for fast and accurate identification of Pseudallescheria/Scedosporium species.

An increasing number of infections due to Pseudallescheria/Scedosporium species has been reported during the past decades, both in immunocompromised and immunocompetent patients. Additionally, these fungi are now recognized worldwide as common agents of fungal colonization of the airways in cystic fibrosis patients, which represents a risk factor for disseminated infections after lung transplantation. Currently six species are described within the Pseudallescheria/Scedosporium genus, including Scedosporium prolificans and species of the Pseudallescheria/Scedosporium apiospermum complex (i.e. S. apiospermum sensu stricto, Pseudallescheria boydii, Scedosporium aurantiacum, Pseudallescheria minutispora and Scedosporium dehoogii). Precise identification of clinical isolates at the species level is required because these species differ in their antifungal drug susceptibility patterns. Matrix-assisted laser desorption ionization (MALDI)-time of flight (TOF)/mass spectrometry (MS) is a powerful tool to rapidly identify moulds at the species level. We investigated the potential of this technology to discriminate Pseudallescheria/Scedosporium species. Forty-seven reference strains were used to build a reference database library. Profiles from 3-, 5- and 7-day-old cultures of each reference strain were analysed to identify species-specific discriminating profiles. The database was tested for accuracy using a set of 64 clinical or environmental isolates previously identified by multilocus sequencing. All isolates were unequivocally identified at the species level by MALDI-TOF/MS. Our results, obtained using a simple protocol, without prior protein extraction or standardization of the culture, demonstrate that MALDI-TOF/MS is a powerful tool for rapid identification of Pseudallescheria/Scedosporium species that cannot be currently identified by morphological examination in the clinical setting.

Different colonization patterns of Aspergillus terreus in patients with cystic fibrosis.

Aspergillus terreus is a common soil saprophyte. After Aspergillus fumigatus and Scedosporium apiospermum it ranks third amongst the filamentous fungi colonizing the airways of patients with cystic fibrosis. In this context, the clinical presentation of A. terreus infection mainly corresponds to allergic broncho-pulmonary aspergillosis. In the work presented here, we studied colonization patterns of A. terreus in CF patients by genotyping using nine short tandem repeat markers. A total of 115 clinical isolates from respiratory secretions collected from five French CF patients were studied. The number of isolates varied from 15 to 39 per patient, and the duration of the follow-up period ranged from 2 months to 7.5 years. Seventeen genotypes were identified, corresponding to three distinct colonization patterns. The first colonization pattern consisted of a chronic colonization by one dominant genotype associated with few other genotypes found only incidentally. The second colonization pattern consisted of a prolonged colonization by two distinct genotypes detected simultaneously. The last pattern was characterized by multiple different genotypes that were present only transiently. These results demonstrate the importance of genotyping clinical isolates before making conclusions about chronic colonization of the airways in CF patients in the case of repeated isolation of the fungus.

Taxonomy and antifungal susceptibility of clinically important Rasamsonia species.

In recent years, Geosmithia argillacea has been increasingly reported in humans and animals and can be considered an emerging pathogen. The taxonomy of Geosmithia was recently studied, and Geosmithia argillacea and related species were transferred to the new genus Rasamsonia. The diversity among a set of Rasamsonia argillacea strains, including 28 clinical strains, was studied, and antifungal susceptibility profiles were generated. Data obtained from morphological studies and from phylogenetic analyses of internal transcribed spacer (ITS) and partial ß-tubulin and calmodulin sequences revealed the presence of four species in the Rasamsonia argillacea complex, two of which are newly described here: R. piperina sp. nov. and R. aegroticola sp. nov. In contrast to other related genera, all Rasamsonia species can be identified with ITS sequences. A retrospective identification was performed on recently reported clinical isolates from animal or human patients. Susceptibility tests showed that the antifungal susceptibility profiles of the four members of the R. argillacea complex are similar, and caspofungin showed significant activity in vitro, followed by amphotericin B and posaconazole. Voriconazole was the least active of the antifungals tested. The phenotypically similar species R. brevistipitata and R. cylindrospora had different antifungal susceptibility profiles, and this indicates that correct species identification is important to help guide appropriate antifungal therapy.

Reverse line blot hybridisation screening of Pseudallescheria/Scedosporium species in patients with cystic fibrosis.

The PCR-RLB (reverse line blot hybridisation) was applied as a molecular technique for the detection of members of Pseudallescheria and Scedosporium from sputum of patients with cystic fibrosis (CF). Fifty-nine sputum samples were collected from 52 CF patients, which were analysed by culture and PCR-RLB. Conventional and semi-selective culture yielded five positive samples, but the PCR-RLB hybridisation assay permitted the detection of members of Pseudallescheria/Scedosporium in 32 out of 52 patients (61.5%). In total, PCR-RLB yielded 47 positives. Pseudallescheria apiosperma was detected in 20 samples, while Pseudallescheria boydii and Pseudallescheria aurantiacum were detected in 17 and eight samples, respectively. Six samples gave a positive reaction with two distinct species-specific probes and one sample with three probes. In conclusion, the PCR-RLB assay described in this study allows the detection of Scedosporium spp. in CF sputum samples and the identification of Pseudallescheria apiosperma, P. boydii, S. aurantiacum, Scedosporium prolificans and Pseudallescheria minutispora.

Retrospective analysis of microorganisms isolated from cystic fibrosis patients in Southern Italy, 2002-2010.

OBJECTIVE: This study aim was to determine the prevalence of microorganisms in the respiratory tract of patients with cystic fibrosis (CF) admitted to the CF Reference Centre in Southern Italy between 2002-2010. METHODS: Microbiology assessment of samples (sputum and tracheal aspirates) collected from patients with pulmonary exacerbation admitted to hospital was carried out. All patients were registered in a database and clinical and microbiological data were retrospectively analysed. RESULTS: Overall, 188 patients were included and a total of 1217 samples were analysed. The most common microorganisms were Staphylococcus aureus (78.7% of the patients) and Pseudomonas aeruginosa (58%), followed by Candida albicans (19.1%), Haemophilus influenzae (13.3%) and Aspergillus fumigatus (9.6%). CONCLUSION: Compared to similar studies performed in other European countries, our microbiological data, especially the low occurrence of filamentous fungi, suggest a specific local epidemiology, probably related to some uncommon CFTR mutations, which are specific to Southern Italy.

Proteomic analysis of cytosolic proteins associated with petite mutations in Candida glabrata

The incidence of superficial or deep-seated infections due to Candida glabrata has increased markedly, probably because of the low intrinsic susceptibility of this microorganism to azole antifungals and its relatively high propensity to acquire azole resistance. To determine changes in the C. glabrata proteome associated with petite mutations, cytosolic extracts from an azole-resistant petite mutant of C. glabrata induced by exposure to ethidium bromide, and from its azole-susceptible parent isolate were compared by two-dimensional polyacrylamide gel electrophoresis. Proteins of interest were identified by peptide mass fingerprinting or sequence tagging using a matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometer. Tryptic peptides from a total of 160 Coomassie-positive spots were analyzed for each strain. Sixty-five different proteins were identified in the cytosolic extracts of the parent strain and 58 in the petite mutant. Among the proteins identified, 10 were higher in the mutant strain, whereas 23 were lower compared to the parent strain. The results revealed a significant decrease in the enzymes associated with the metabolic rate of mutant cells such as aconitase, transaldolase, and pyruvate kinase, and changes in the levels of specific heat shock proteins. Moreover, transketolase, aconitase and catalase activity measurements decreased significantly in the ethidium bromide-induced petite mutant. These data may be useful for designing experiments to obtain a better understanding of the nuclear response to impairment of mitochondrial function associated with this mutation in C. glabrata.

Proteomic analysis of cytosolic proteins associated with petite mutations in Candida glabrata.

The incidence of superficial or deep-seated infections due to Candida glabrata has increased markedly, probably because of the low intrinsic susceptibility of this microorganism to azole antifungals and its relatively high propensity to acquire azole resistance. To determine changes in the C. glabrata proteome associated with petite mutations, cytosolic extracts from an azole-resistant petite mutant of C. glabrata induced by exposure to ethidium bromide, and from its azole-susceptible parent isolate were compared by two-dimensional polyacrylamide gel electrophoresis. Proteins of interest were identified by peptide mass fingerprinting or sequence tagging using a matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometer. Tryptic peptides from a total of 160 Coomassie-positive spots were analyzed for each strain. Sixty-five different proteins were identified in the cytosolic extracts of the parent strain and 58 in the petite mutant. Among the proteins identified, 10 were higher in the mutant strain, whereas 23 were lower compared to the parent strain. The results revealed a significant decrease in the enzymes associated with the metabolic rate of mutant cells such as aconitase, transaldolase, and pyruvate kinase, and changes in the levels of specific heat shock proteins. Moreover, transketolase, aconitase and catalase activity measurements decreased significantly in the ethidium bromide-induced petite mutant. These data may be useful for designing experiments to obtain a better understanding of the nuclear response to impairment of mitochondrial function associated with this mutation in C. glabrata.