Genetic Polymorphisms and Susceptibility to Fungal Infections

Invasive fungal infections (IFI) are life-threatening diseases that are of particular concern in specific debilitated or immunosuppressed populations. Invasive candidiasis (IC) is the most frequent of the IFI, being one of the major causes of nosocomial bloodstream infection and a feared complication in patients with recurrent gastrointestinal surgery or prolonged stay in the intensive-care unit [1,2]. Patients with hematological malignancies or prolonged chemotherapy-induced neutropenia, and those with allogeneic hematopoietic stem cell transplantation (allo-HSCT), represent the groups at highest risk for developing invasive aspergillosis (IA), which is associated with a high mortality rate despite the increasing availability of antifungal therapies [3,4]. An increasing incidence of IA has also been reported in non-neutropenic immunosuppressed populations such as solid-organ transplant recipients or steroid-treated patients with chronic pulmonary diseases [5]. Early diagnosis of IFI is crucial for improving chances of survival [6], but is particularly challenging owing to the lack of reliable diagnostic methods [7,8]. Significant efforts during the last few decades have focused on the prevention of these severe complications. Antifungal prophylaxis in high-risk patients has been shown to reduce the incidence of IA in patients with onco-hematological malignancies [9] and that of IC in surgical intensive-care unit patients [10]. However, its widespread use raises concerns about costs, toxicity, and the risk of emergence of resistant fungal species such as non-Aspergillus moulds or non-albicansCandida spp. [4,11,12]. Prophylactic strategies usually rely on the identification of host risk factors resulting from clinical conditions (type and duration of immunosuppression, underlying diseases, and extrinsic interventions) [8,13]. Recent advances in the field of immunogenetics may change our perspective of, and approach to, preventive strategies with the identification of subgroups of patients exhibiting a genetic predisposition to IFI.

Antifungal activity of extracts from Atacama Desert fungi againstParacoccidioides brasiliensis and identification ofAspergillus felis as a promising source of natural bioactive compounds

Fungi of the genus Paracoccidioides are responsible for paracoccidioidomycosis. The occurrence of drug toxicity and relapse in this disease justify the development of new antifungal agents. Compounds extracted from fungal extract have showing antifungal activity. Extracts of 78 fungi isolated from rocks of the Atacama Desert were tested in a microdilution assay against Paracoccidioides brasiliensis Pb18. Approximately 18% (5) of the extracts showed minimum inhibitory concentration (MIC) values≤ 125.0 µg/mL. Among these, extract from the fungus UFMGCB 8030 demonstrated the best results, with an MIC of 15.6 µg/mL. This isolate was identified as Aspergillus felis (by macro and micromorphologies, and internal transcribed spacer, β-tubulin, and ribosomal polymerase II gene analyses) and was grown in five different culture media and extracted with various solvents to optimise its antifungal activity. Potato dextrose agar culture and dichloromethane extraction resulted in an MIC of 1.9 µg/mL against P. brasiliensis and did not show cytotoxicity at the concentrations tested in normal mammalian cell (Vero). This extract was subjected to bioassay-guided fractionation using analytical C18RP-high-performance liquid chromatography (HPLC) and an antifungal assay using P. brasiliensis. Analysis of the active fractions by HPLC-high resolution mass spectrometry allowed us to identify the antifungal agents present in the A. felis extracts cytochalasins. These results reveal the potential of A. felis as a producer of bioactive compounds with antifungal activity.

ESCMID* guideline for the diagnosis and management of Candida diseases 2012: adults with haematological malignancies and after haematopoietic stem cell transplantation (HCT).

Fungal diseases still play a major role in morbidity and mortality in patients with haematological malignancies, including those undergoing haematopoietic stem cell transplantation. Although Aspergillus and other filamentous fungal diseases remain a major concern, Candida infections are still a major cause of mortality. This part of the ESCMID guidelines focuses on this patient population and reviews pertaining to prophylaxis, empirical/pre-emptive and targeted therapy of Candida diseases. Anti-Candida prophylaxis is only recommended for patients receiving allogeneic stem cell transplantation. The authors recognize that the recommendations would have most likely been different if the purpose would have been prevention of all fungal infections (e.g. aspergillosis). In targeted treatment of candidaemia, recommendations for treatment are available for all echinocandins, that is anidulafungin (AI), caspofungin (AI) and micafungin (AI), although a warning for resistance is expressed. Liposomal amphotericin B received a BI recommendation due to higher number of reported adverse events in the trials. Amphotericin B deoxycholate should not be used (DII); and fluconazole was rated CI because of a change in epidemiology in some areas in Europe. Removal of central venous catheters is recommended during candidaemia but if catheter retention is a clinical necessity, treatment with an echinocandin is an option (CII(t) ). In chronic disseminated candidiasis therapy, recommendations are liposomal amphotericin B for 8 weeks (AIII), fluconazole for >3 months or other azoles (BIII). Granulocyte transfusions are only an option in desperate cases of patients with Candida disease and neutropenia (CIII).

Genetic dissection of azole resistance mechanisms in Candida albicans and their validation in a mouse model of disseminated infection.

Principal mechanisms of resistance to azole antifungals include the upregulation of multidrug transporters and the modification of the target enzyme, a cytochrome P450 (Erg11) involved in the 14alpha-demethylation of ergosterol. These mechanisms are often combined in azole-resistant Candida albicans isolates recovered from patients. However, the precise contributions of individual mechanisms to C. albicans resistance to specific azoles have been difficult to establish because of the technical difficulties in the genetic manipulation of this diploid species. Recent advances have made genetic manipulations easier, and we therefore undertook the genetic dissection of resistance mechanisms in an azole-resistant clinical isolate. This isolate (DSY296) upregulates the multidrug transporter genes CDR1 and CDR2 and has acquired a G464S substitution in both ERG11 alleles. In DSY296, inactivation of TAC1, a transcription factor containing a gain-of-function mutation, followed by sequential replacement of ERG11 mutant alleles with wild-type alleles, restored azole susceptibility to the levels measured for a parent azole-susceptible isolate (DSY294). These sequential genetic manipulations not only demonstrated that these two resistance mechanisms were those responsible for the development of resistance in DSY296 but also indicated that the quantitative level of resistance as measured in vitro by MIC determinations was a function of the number of genetic resistance mechanisms operating in any strain. The engineered strains were also tested for their responses to fluconazole treatment in a novel 3-day model of invasive C. albicans infection of mice. Fifty percent effective doses (ED(50)s) of fluconazole were highest for DSY296 and decreased proportionally with the sequential removal of each resistance mechanism. However, while the fold differences in ED(50) were proportional to the fold differences in MICs, their magnitude was lower than that measured in vitro and depended on the specific resistance mechanism operating.

Defining responses to therapy and study outcomes in clinical trials of invasive fungal diseases: Mycoses Study Group and European Organization for Research and Treatment of Cancer consensus criteria.

Invasive fungal diseases (IFDs) have become major causes of morbidity and mortality among highly immunocompromised patients. Authoritative consensus criteria to diagnose IFD have been useful in establishing eligibility criteria for antifungal trials. There is an important need for generation of consensus definitions of outcomes of IFD that will form a standard for evaluating treatment success and failure in clinical trials. Therefore, an expert international panel consisting of the Mycoses Study Group and the European Organization for Research and Treatment of Cancer was convened to propose guidelines for assessing treatment responses in clinical trials of IFDs and for defining study outcomes. Major fungal diseases that are discussed include invasive disease due to Candida species, Aspergillus species and other molds, Cryptococcus neoformans, Histoplasma capsulatum, and Coccidioides immitis. We also discuss potential pitfalls in assessing outcome, such as conflicting clinical, radiological, and/or mycological data and gaps in knowledge.

A Th17- and Th2-skewed Cytokine Profile in Cystic Fibrosis Lungs Represents a Potential Risk Factor for Pseudomonas aeruginosa Infection.

Rationale: Cystic fibrosis (CF) is characterized by progressive pulmonary inflammation that is infection-triggered. Pseudomonas aeruginosa represents a risk factor for deterioration of lung function and reduced life expectancy. Objectives: To assess T-cell cytokine/chemokine production in clinically stable children with CF and evaluate the association between T-cell subtypes and susceptibility for infection with P. aeruginosa. Methods: T-cell cytokine/chemokine profiles were measured in bronchoalveolar lavage fluid (BALF) from children with CF (n = 57; 6.1 ± 5.9 yr) and non-CF control subjects (n = 18; 5.9 ± 4.3 yr). Memory responses to Aspergillus fumigatus and P. aeruginosa were monitored. High-resolution computed tomography-based Helbich score was assessed. In a prospective observational trial the association between BALF cytokine/chemokine profiles and subsequent infection with P. aeruginosa was studied. Measurements and Main Results: Th1- (INF-γ), Th2- (IL-5, IL-13), Th17- (IL-17A), and Th17-related cytokines (IL-1β, IL-6) were significantly up-regulated in airways of patients with CF. IL-17A, IL-13, and IL-5 were significantly higher in BALF of symptomatic as compared with clinically asymptomatic patients with CF. IL-17A and IL-5 correlated with the percentage of neutrophils in BALF (r = 0.41, P < 0.05 and r = 0.46, P < 0.05, respectively). Th17- (IL-17A, IL-6, IL-1β, IL-8) and Th2-associated cytokines and chemokines (IL-5, IL-13, TARC/CCL17), but not IFN-γ levels, significantly correlated with high-resolution computed tomography changes (Helbich score; P < 0.05). P. aeruginosa- and A. fumigatus-specific T cells from patients with CF displayed significantly higher IL-5 and IL-17A mRNA expression. IL-17A and TARC/CCL17 were significantly augmented in patients that developed P. aeruginosa infection within 24 months. Conclusions: We propose a role for Th17 and Th2 T cells in chronic inflammation in lungs of patients with CF. High concentrations of these cytokines/chemokines in CF airways precede infection with P. aeruginosa.

Toll-like receptor 4 polymorphisms and aspergillosis in stem-cell transplantation.

BACKGROUND: Toll-like receptors (TLRs) are essential components of the immune response to fungal pathogens. We examined the role of TLR polymorphisms in conferring a risk of invasive aspergillosis among recipients of allogeneic hematopoietic-cell transplants. METHODS: We analyzed 20 single-nucleotide polymorphisms (SNPs) in the toll-like receptor 2 gene (TLR2), the toll-like receptor 3 gene (TLR3), the toll-like receptor 4 gene (TLR4), and the toll-like receptor 9 gene (TLR9) in a cohort of 336 recipients of hematopoietic-cell transplants and their unrelated donors. The risk of invasive aspergillosis was assessed with the use of multivariate Cox regression analysis. The analysis was replicated in a validation study involving 103 case patients and 263 matched controls who received hematopoietic-cell transplants from related and unrelated donors. RESULTS: In the discovery study, two donor TLR4 haplotypes (S3 and S4) increased the risk of invasive aspergillosis (adjusted hazard ratio for S3, 2.20; 95% confidence interval [CI], 1.14 to 4.25; P=0.02; adjusted hazard ratio for S4, 6.16; 95% CI, 1.97 to 19.26; P=0.002). The haplotype S4 was present in carriers of two SNPs in strong linkage disequilibrium (1063 A/G [D299G] and 1363 C/T [T399I]) that influence TLR4 function. In the validation study, donor haplotype S4 also increased the risk of invasive aspergillosis (adjusted odds ratio, 2.49; 95% CI, 1.15 to 5.41; P=0.02); the association was present in unrelated recipients of hematopoietic-cell transplants (odds ratio, 5.00; 95% CI, 1.04 to 24.01; P=0.04) but not in related recipients (odds ratio, 2.29; 95% CI, 0.93 to 5.68; P=0.07). In the discovery study, seropositivity for cytomegalovirus (CMV) in donors or recipients, donor positivity for S4, or both, as compared with negative results for CMV and S4, were associated with an increase in the 3-year probability of invasive aspergillosis (12% vs. 1%, P=0.02) and death that was not related to relapse (35% vs. 22%, P=0.02). CONCLUSIONS: This study suggests an association between the donor TLR4 haplotype S4 and the risk of invasive aspergillosis among recipients of hematopoietic-cell transplants from unrelated donors.

Secreted proteases from dermatophytes

Dermatophytes are highly specialized pathogenic fungi that exclusively infect the stratum corneum, nails or hair, and it is evident that secreted proteolytic activity is important for their virulence. Endo- and exoproteases-secreted by dermatophytes are similar to those of species of the genus Aspergillus. However, in contrast to Aspergillus spp., dermatophyte-secreted endoproteases are multiple and are members of two large protein families, the subtilisins (serine proteases) and the fungalysins (metalloproteases). In addition, dermatophytes excrete sulphite as a reducing agent. In the presence of sulphite, disulphide bounds of the keratin substrate are directly cleaved to cysteine and S-sulphocysteine, and reduced proteins become accessible for further digestion by various endo- and exoproteases secreted by the fungi. Sulphitolysis is likely to be an essential step in the digestion of compact keratinized tissues which precedes the action of all proteases.

Neue Nachweise der Rhinopoma hardwickei (Chiroptera) aus Westafrika

From Mali, the first record of the bat Rhinopoma hardwickei is communicated. From Niger, new data of the same bat actualise the problem of subspecific rank of older material from this country

Secreted subtilisin gene family in Trichophyton rubrum.

Secreted proteases constitute potential virulence factors of dermatophytes. A total of seven genes encoding putative serine proteases of the subtilisin family (SUB) were isolated in Trichophyton rubrum. Based on sequence data and intron-exon structure, a phylogenetic analysis of subtilisins from T. rubrum and other fungi revealed a presumed ancestral lineage comprising T. rubrum SUB2 and Aspergillus SUBs. All other SUBs (SUB1, SUB3-7) are dermatophyte-specific and have apparently emerged more recently, through successive gene duplication events. We showed that two subtilisins, Sub3 and Sub4, were detected in culture supernatants of T. rubrum grown in a medium containing soy protein as a sole nitrogen source. Both recombinant enzymes produced in Pichia pastoris are highly active on keratin azure suggesting that these proteases play an important role in invasion of keratinised tissues by the fungus. The set of deduced amino acid sequences of T. rubrum SUB ORFs allowed the identification of orthologous Subs secreted by other dermatophyte species using proteolysis and mass spectrometry.

Les maladies infectieuses ont encore un bel avenir [Infectious diseases have again a good future!].

Invasive fungal diseases (IFDs) have become major causes of morbidity and mortality among highly immunocompromised patients. Authoritative consensus criteria to diagnose IFD have been useful in establishing eligibility criteria for antifungal trials. There is an important need for generation of consensus definitions of outcomes of IFD that will form a standard for evaluating treatment success and failure in clinical trials. Therefore, an expert international panel consisting of the Mycoses Study Group and the European Organization for Research and Treatment of Cancer was convened to propose guidelines for assessing treatment responses in clinical trials of IFDs and for defining study outcomes. Major fungal diseases that are discussed include invasive disease due to Candida species, Aspergillus species and other molds, Cryptococcus neoformans, Histoplasma capsulatum, and Coccidioides immitis. We also discuss potential pitfalls in assessing outcome, such as conflicting clinical, radiological, and/or mycological data and gaps in knowledge.

Eryphus Perty, 1832 e Tacyba, um novo gênero de Heteropsini (Coleoptera, Cerambycidae, Cerambycinae)

Eryphus Perty, 1832 and Tacyba, a new genus of Heteropsini (Coleoptera, Cerambycidae). Some species, up to now, included in Callideriphus Blanchard, 1851 are rearranged in: a) those congeneric with Callideriphus grossipes Blanchard, 1851 and b) not congeneric. The first set of species will be treated in a future paper; the second one, on the other hand, is subdivided into Eryphus Perty, 1832 and Tacyba gen. nov. Eryphus Perty, 1832 (type species: Eryphus bipunctatus Perty, 1832), a valid genus, is redescribed and a key for the species is also provided. The following species are transferred to Eryphus: E. bivittatus (Melzer, 1934) comb. nov., E. carinatus (Zajciw, 1970) comb. nov., E. flavicollis (Fisher, 1938) comb. nov., E. laetus (Blanchard, 1851) comb. nov., E. marginatus (Zajciw, 1970) comb. nov., E. picticollis (Gounelle, 1911) comb. nov., E. transversalis (Fairmaire & Germain, 1864) comb. nov. New synonym proposed: Eryphus bipunctatus Perty, 1832 = Callideriphus atricollis Melzer, 1931. New taxa described: Eryphus tacuarembo sp. nov. (Uruguay, Tacuarembó), E. carioca sp. nov. (Brazil, Rio de Janeiro); Tacyba gen. nov. (type species: Callideriphus maculatus Cerda, 1988). Species transferred to Tacyba and synonyms: T. maculata (Cerda, 1988) comb. nov., T. tenuis (Blanchard, 1851) comb. nov. = Callideriphus testaceicornis Fairmaire & Germain, 1859 syn. nov. = Callideriphus clathratus Fairmaire & Germain, 1860 syn. nov. = Callideriphus niger Philippi & Philippi, 1864 syn. nov. Callideriphus flavicollis m. quadripunctatus Fuchs, 1961 and Callideriphus flavicollis m. reductus Fuchs, 1961, both names of infrasubspecific category (not available under the rules of ICZN), are herein treated as intraspecific variation of Eryphus picticollis (Gounelle, 1911) which occur in southern Brazil and Argentina.

Review of the genus Chalcolepidius Eschscholtz, 1829 (Coleoptera, Elateridae, Agrypninae)

The genus Chalcolepidius is revised. Type specimens of 65 nominal species, except C. costatus Pjatakowa, 1941, C. fleutiauxi Pjatakowa, 1941 and C. viriditarsus Schwarz, 1906, are examined. Eighty five species are studied, of which 34 are synonymyzed and 12 new species described; three species, C. alicii Pjatakowa, 1941, C. haroldi Candèze, 1878 and C. unicus Fleutiaux, 1910, formely included in this genus, are not congeneric and are removed; C. validus Candèze, 1857 is revalidated. The genus is now formed by 63 species. Redescriptions, illustrations and a key for the examined species, and a cladistic analysis for groups of species are also included. New synonyms established: C. apacheanus Casey, 1891 = C. simulans Casey, 1907 syn. nov. = C. acuminatus Casey, 1907 syn. nov. = C. nobilis Casey, 1907 syn. nov.; C. approximatus Erichson, 1841 = C. aztecus Casey, 1907 syn. nov. = C. niger Pjatakowa, 1941 syn. nov.; C. attenuatus Erichson, 1841 = C. cuneatus Champion, 1894 syn. nov. = C. tenuis Champion, 1894 syn. nov.; C. aurulentus Candèze, 1874 = C. candezei Dohrn, 1881 syn. nov. = C. grossheimi Pjatakowa, 1941 syn. nov.; C. bomplandii Guérin, 1844 = C. humboldti Candèze, 1881 syn. nov.; C. chalcantheus Candèze, 1857 = C. violaceous Pjatakowa, 1941 syn. nov.; C. cyaneus Candèze, 1881 = C. scitus Candèze, 1889 syn. nov. = C. abbreviatovittatus Pjatakowa, 1941 syn. nov.; C. desmarestii Chevrolat, 1835 = C. brevicollis Casey, 1907 syn. nov.; C. gossipiatus Guérin, 1844 = C. erichsonii Guérin-Méneville, 1844 syn. nov. = C. lemoinii Candèze, 1857 syn. nov.; C. inops Candèze, 1886 = C. murinus Champion, 1894 syn. nov.; C. jansoni Candèze, 1874 = C. mucronatus Candèze, 1889 syn. nov.; C. lacordairii Candèze, 1857 = C. exquisitus Candèze, 1886 syn. nov. = C. monachus Candèze, 1893 syn. nov.; C. lenzi Candèze, 1886 = C. behrensi Candèze, 1886 syn. nov.; C. oxydatus Candèze, 1857 = C. jekeli Candèze, 1874 syn. nov.; C. porcatus (Linnaeus, 1767) = C. peruanus Candèze, 1886 syn. nov. = C. flavostriatus Pjatakowa, 1941 syn. nov. = C. herbstii multistriatus Golbach, 1977 syn. nov.; C. rugatus Candèze, 1857 = C. amictus Casey, 1907 syn. nov.; C. smaragdinus LeConte, 1854 = C. ostentus Casey, 1907 syn. nov. = C. rectus Casey, 1907 syn. nov.; C. sulcatus (Fabricius, 1777) = C. herbstii Erichson, 1841 syn. nov; C. virens (Fabricius, 1787) = C. perrisi Candèze, 1857 syn. nov.; C. virginalis Candèze, 1857 = C. championi Casey, 1907 syn. nov.; C. viridipilis (Say, 1825) = C. debilis Casey, 1907 syn. nov.; C. webbi LeConte, 1854 = C. sonoricus Casey, 1907 syn. nov.; C. zonatus Eschscholtz, 1829 = C. longicollis Candèze, 1857 syn. nov. New species described: C. albisetosus sp. nov. (Ecuador), C. albiventris sp. nov. (Mexico: Veracruz), C. copulatuvittatus sp. nov. (Venezuela), C. extenuatuvittatus sp. nov. (Venezuela), C. fasciatus sp. nov. (Mexico: Durango), C. ferratuvittatus sp. nov. (Ecuador), C. proximus sp. nov. (Mexico: Sinaloa), C. serricornis sp. nov. (Mexico: Veracruz), C. spinipennis sp. nov. (Mexico: Veracruz), C. supremus sp. nov. (Venezuela), C. truncuvittatus sp. nov. (Mexico: Tamaulipas) and C. virgatipennis sp. nov. (Mexico: Durango). Redescribed species: C. angustatus Candèze, 1857, C. apacheanus Casey, 1891, C. approximatus Erichson, 1841, C. attenuatus Erichson, 1841, C. aurulentus Candèze, 1874, C. bomplandii Guérin-Méneville, 1844, C. boucardi Candèze, 1874, C. chalcantheus Candèze, 1857, C. corpulentus Candèze, 1874, C. cyaneus Candèze, 1881, C. desmarestii Chevrolat, 1835, C. dugesi Candèze, 1886, C. erythroloma Candèze, 1857, C. eschscholtzi Chevrolat, 1833, C. exulatus Candèze, 1874, C. fabricii Erichson, 1841, C. forreri Candèze, 1886, C. fryi Candèze, 1874, C. gossipiatus Guérin-Méneville, 1844, C. inops Candèze, 1886, C. jansoni Candèze, 1874, C. lacordairii Candèze, 1857, C. lafargi Chevrolat, 1835, C. lenzi Candèze, 1886, C. limbatus (Fabricius, 1777), C. mexicanus Castelnau, 1836, C. mniszechi Candèze, 1881, C. mocquerysii Candèze, 1857, C. morio Candèze, 1857, C. obscurus Castelnau, 1836, C. oxydatus Candèze, 1857, C. porcatus (Linnaeus, 1767), C. pruinosus Erichson, 1841, C. rodriguezi Candèze, 1886, C. rostainei Candèze, 1889, C. rubripennis LeConte, 1861, C. rugatus Candèze, 1857, C. silbermanni Chevrolat, 1835, C. smaragdinus LeConte, 1854, C. sulcatus (Fabricius, 1777), C. tartarus Fall, 1898, C. validus Candèze, 1857, reval., C. villei Candèze, 1878, C. virens (Fabricius, 1787), C. virginalis Candèze, 1857, C. viridipilis (Say, 1825), C. webbi LeConte, 1854, C. zonatus Eschscholtz, 1829.

Comparative and functional genomics provide insights into the pathogenicity of dermatophytic fungi.

ABSTRACT: BACKGROUND: Millions of humans and animals suffer from superficial infections caused by a group of highly specialized filamentous fungi, the dermatophytes, which exclusively infect keratinized host structures. To provide broad insights into the molecular basis of the pathogenicity-associated traits, we report the first genome sequences of two closely phylogenetically related dermatophytes, Arthroderma benhamiae and Trichophyton verrucosum, both of which induce highly inflammatory infections in humans. RESULTS: 97% of the 22.5 megabase genome sequences of A. benhamiae and T. verrucosum are unambiguously alignable and collinear. To unravel dermatophyte-specific virulence-associated traits, we compared sets of potentially pathogenicity-associated proteins, such as secreted proteases and enzymes involved in secondary metabolite production, with those of closely related onygenales (Coccidioides species) and the mould Aspergillus fumigatus. The comparisons revealed expansion of several gene families in dermatophytes and disclosed the peculiarities of the dermatophyte secondary metabolite gene sets. Secretion of proteases and other hydrolytic enzymes by A. benhamiae was proven experimentally by a global secretome analysis during keratin degradation. Molecular insights into the interaction of A. benhamiae with human keratinocytes were obtained for the first time by global transcriptome profiling. Given that A. benhamiae is able to undergo mating, a detailed comparison of the genomes further unraveled the genetic basis of sexual reproduction in this species. CONCLUSIONS: Our results enlighten the genetic basis of fundamental and putatively virulence-related traits of dermatophytes, advancing future research on these medically important pathogens.

Secretion of an endogenous subtilisin by Pichia pastoris strains GS115 and KM71.

The methylotrophic yeast Pichia pastoris is widely used for the expression of heterologous enzymes. While the purity of the desired expression product is of major importance for many applications, we found that recombinant enzymes produced in methanol medium were contaminated by a 37-kDa endogenous yeast protease. This enzyme was completely inhibited by phenylmethanesulfonyl fluoride (PMSF) but not by 1,10-phenanthroline, EDTA, and pepstatin A, suggesting the nature of a serine protease. Its secretion was abolished in P. pastoris strains GS115 and KM71 by specific mutagenesis of a subtilisin gene (SUB2) but not by inactivation of the gene encoding vacuolar proteinase B (PRB). Bioinformatic comparisons of Sub2 protein with subtilisins from other fungal genomes and phylogenetic analyses indicated that this enzyme is not an orthologue of the vacuolar protease cerevisin generally present in yeasts but is more closely related to another putative subtilisin found in a small number of yeast genomes. During growth of P. pastoris, Sub2 was produced as a secreted enzyme at a concentration of 10 microg/ml of culture supernatant after overexpression of the full-length SUB2 gene. During fermentative production of recombinant enzymes in methanol medium, 1 ml of P. pastoris culture supernatant was found to contain approximately 3 ng of Sub2, while the enzyme was not detected during growth in a medium containing glycerol as a carbon source. The mutant strain GS115-sub2 was subsequently used as a host for the production of recombinant proteases without endogenous subtilisin contamination.