Two different types of tandem sequences mediate the overexpression of TinCYP51B in azole-resistant Trichophyton indotineae.

Trichophyton indotineae is an emerging dermatophyte that causes severe tinea corporis and tinea cruris. Numerous cases of terbinafine- and azole-recalcitrant T. indotineae-related dermatophytosis have been observed in India over the past decade, and cases are now being recorded worldwide. Whole genome sequencing of three azole-resistant strains revealed a variable number of repeats of a 2,404 base pair (bp) sequence encoding TinCYP51B in tandem specifically at the CYP51B locus position. However, many other resistant strains (itraconazole MIC ≥0.25 µg/mL; voriconazole MIC ≥0.25 µg/mL) did not contain such duplications. Whole-genome sequencing of three of these strains revealed a variable number of 7,374 bp tandem repeat blocks harboring TinCYP51B. Consequently, two types of T. indotineae azole-resistant strains were found to host TinCYP51B in tandem sequences (type I with 2,404 bp TinCYP51B blocks and type II with 7,374 bp TinCYP51B blocks). Using the CRISPR/Cas9 genome-editing tool, the copy number of TinCYP51B within the genome of types I and II strains was brought back to a single copy. The azole susceptibility of these modified strains was similar to that of strains without TinCYP51B duplication, showing that azole resistance in T. indotineae strains is mediated by one of two types of TinCYP51B amplification. Type II strains were prevalent among 32 resistant strains analyzed using a rapid and reliable PCR test.

Reliable and rapid identification of terbinafine resistance in dermatophytic nail and skin infections.

BACKGROUND: Fungal infections are the most frequent dermatoses. The gold standard treatment for dermatophytosis is the squalene epoxidase (SQLE) inhibitor terbinafine. Pathogenic dermatophytes resistant to terbinafine are an emerging global threat. Here, we determine the proportion of resistant fungal skin infections, analyse the molecular mechanisms of terbinafine resistance, and validate a method for its reliable rapid identification. METHODS: Between 2013 and 2021, we screened 5634 consecutively isolated Trichophyton for antifungal resistance determined by hyphal growth on Sabouraud dextrose agar medium containing 0.2 µg/mL terbinafine. All Trichophyton isolates with preserved growth capacity in the presence of terbinafine underwent SQLE sequencing. Minimum inhibitory concentrations (MICs) were determined by the broth microdilution method. RESULTS: Over an 8-year period, the proportion of fungal skin infections resistant to terbinafine increased from 0.63% in 2013 to 1.3% in 2021. Our routine phenotypic in vitro screening analysis identified 0.83% (n = 47/5634) of Trichophyton strains with in vitro terbinafine resistance. Molecular screening detected a mutation in the SQLE in all cases. Mutations L393F, L393S, F397L, F397I, F397V, Q408K, F415I, F415S, F415V, H440Y, or A398 A399 G400 deletion were detected in Trichophyton rubrum. Mutations L393F and F397L were the most frequent. In contrast, all mutations detected in T. mentagrophytes/T. interdigitale complex strains were F397L, except for one strain with L393S. All 47 strains featured significantly higher MICs than terbinafine-sensitive controls. The mutation-related range of MICs varied between 0.004 and 16.0 µg/mL, with MIC as low as 0.015 µg/mL conferring clinical resistance to standard terbinafine dosing. CONCLUSIONS: Based on our data, we propose MIC of 0.015 µg/mL as a minimum breakpoint for predicting clinically relevant terbinafine treatment failure to standard oral dosing for dermatophyte infections. We further propose growth on Sabouraud dextrose agar medium containing 0.2 µg/mL terbinafine and SQLE sequencing as fungal sporulation-independent methods for rapid and reliable detection of terbinafine resistance.

Survey on Dermatophytes Isolated from Animals in Switzerland in the Context of the Prevention of Zoonotic Dermatophytosis.

Most inflammatory dermatophytoses in humans are caused by zoophilic and geophilic dermatophytes. Knowledge of the epidemiology of these fungi in animals facilitates the prevention of dermatophytosis of animal origin in humans. We studied the prevalence of dermatophyte species in domestic animals in Switzerland and examined the effectiveness of direct mycological examination (DME) for their detection compared to mycological cultures. In total, 3515 hair and skin samples, collected between 2008 and 2022 by practicing veterinarians, were subjected to direct fluorescence microscopy and fungal culture. Overall, 611 dermatophytes were isolated, of which 547 (89.5%) were from DME-positive samples. Cats and dogs were the main reservoirs of Trichophyton mentagrophytes and Microsporum canis, whereas Trichophyton benhamiae was predominantly found in guinea pigs. Cultures with M. canis significantly (p < 0.001) outnumbered those with T. mentagrophytes in DME-negative samples (19.3% versus 6.8%), possibly because M. canis can be asymptomatic in cats and dogs, unlike T. mentagrophytes, which is always infectious. Our data confirm DME as a reliable, quick, and easy method to identify the presence of dermatophytes in animals. A positive DME in an animal hair or skin sample should alert people in contact with the animal to the risk of contracting dermatophytosis.

Identification of Dermatophyte and Non-Dermatophyte Agents in Onychomycosis by PCR and DNA Sequencing-A Retrospective Comparison of Diagnostic Tools.

Rapid and reliable fungal identification is crucial to delineate infectious diseases, and to establish appropriate treatment for onychomycosis. Compared to conventional diagnostic methods, molecular techniques are faster and feature higher accuracy in fungal identification. However, in current clinical practice, molecular mycology is not widely available, and its practical applicability is still under discussion. This study summarizes the results of 16,094 consecutive nail specimens with clinical suspicion of onychomycosis. We performed PCR/sequencing on all primary nail specimens for which conventional mycological diagnostics remained inconclusive. In specimens with a positive direct microscopy but negative or contaminated culture, molecular mycology proved superior and specified a fungal agent in 65% (587/898). In 75% (443/587), the identified pathogen was a dermatophyte. Positive cultures for dermatophytes, yeasts and non-dermatophyte molds (NDMs) were concordant with primary-specimen-DNA PCR/sequencing in 83% (10/12), 34% (22/65) and 45% (76/169), respectively. Among NDMs, agreement was high for Fusarium spp. (32/40; 80%), but low for Penicillium spp. (5/25; 20%) and Alternaria spp. (1/20; 5%). This study underlines the improvement in diagnostic yield by fungal primary-specimen-DNA PCR/sequencing in the event of a negative or contaminated culture, as well as its significance for the diagnosis of dermatophyte and non-dermatophyte onychomycosis. Molecular mycology methods like PCR and DNA sequencing should complement conventional diagnostics in cases of equivocal findings, suspected NDM onychomycosis or treatment-resistant nail pathologies.

Gene Amplification of CYP51B: a New Mechanism of Resistance to Azole Compounds in Trichophyton indotineae.

Trichophyton indotineae causes dermatophytosis that is resistant to terbinafine and azole compounds. The aim of this study was to determine the mechanisms of resistance to itraconazole (ITC) and voriconazole (VRC) in strains of T. indotineae. Two azole-sensitive strains (ITC MIC < 0.125 µg/mL; VRC MIC < 0.06 µg/mL) and four azole-resistant strains (ITC MIC ≥ 0.5 µg/mL; VRC MIC ≥ 0.5 µg/mL) were used for the investigation. The expression of MDR genes encoding multidrug transporters of the ABC family for which orthologs have been identified in Trichophyton rubrum and those of CYP51A and CYP51B encoding the targets of azole antifungal compounds were compared between susceptible and resistant strains. TinMDR3 and TinCYP51B were overexpressed in T. indotineae resistant strains. Only small differences in susceptibility were observed between TinMDR3 disruptants and parental strains overexpressing TinMDR3. Whole-genome sequencing of resistant strains revealed the creation of a variable number of TinCYP51B tandem repeats at the specific position of their genomes in three resistant strains. Downregulation of TinCYP51B by RNA interference (RNAi) restored the susceptibility of azole-resistant strains. In contrast, overexpression of TinCYP51B cDNA conferred resistance to a susceptible strain of T. indotineae. In conclusion, the reduced sensitivity of T. indotineae strains to azoles is mainly due to the overexpression of TinCYP51B resulting from additional copies of this gene.

MFS1, a Pleiotropic Transporter in Dermatophytes That Plays a Key Role in Their Intrinsic Resistance to Chloramphenicol and Fluconazole.

A recently identified Trichophyton rubrum major facilitator superfamily (MFS)-type transporter (TruMFS1) has been shown to give resistance to azole compounds and cycloheximide (CYH) when overexpressed in Saccharomyces cerevisiae. We investigated the roles of MFS1 in the intrinsic resistance of dermatophytes to CYH and chloramphenicol (CHL), which are commonly used to isolate these fungi, and to what extent MFS1 affects the susceptibility to azole antifungals. Susceptibility to antibiotics and azoles was tested in S. cerevisiae overexpressing MFS1 and ΔMFS1 mutants of Trichophyton benhamiae, a dermatophyte that is closely related to T. rubrum. We found that TruMFS1 functions as an efflux pump for CHL in addition to CYH and azoles in S. cerevisiae. In contrast, the growth of T. benhamiae ΔMFS1 mutants was not reduced in the presence of CYH but was severely impaired in the presence of CHL and thiamphenicol, a CHL analog. The suppression of MFS1 in T. benhamiae also increased the sensitivity of the fungus to fluconazole and miconazole. Our experiments revealed a key role of MFS1 in the resistance of dermatophytes to CHL and their high minimum inhibitory concentration for fluconazole. Suppression of MFS1 did not affect the sensitivity to CYH, suggesting that another mechanism was involved in resistance to CYH in dermatophytes.

Itraconazole resistance of Trichophyton rubrum mediated by the ABC transporter TruMDR2.

BACKGROUND: Dermatophytes showing reduced sensitivity to antifungal agents have emerged in several countries. One terbinafine resistant strain of Trichophyton rubrum, TIMM20092, also showed reduced sensitivity to itraconazole (ITC) and voriconazole (VRC). The expression of two genes (TruMDR2 and TruMDR3) encoding multidrug transporters of the ABC family was found to be highly up-regulated in this strain. Deletion of TruMDR3 in TIMM20092 abolished its resistance to VRC but only slightly reduced its resistance to ITC. OBJECTIVES: We examined the potential of T rubrum to develop resistance to ITC by analysing the mechanism of ITC resistance in TIMM20092. METHODS: The deletion of TruMDR2 by gene replacement was performed in TIMM20092 and one TruMDR3-lacking mutant (∆TruMDR3) previously generated from TIMM20092. TruMDR2 single and TruMDR2/TruMDR3 double mutants (∆TruMDR2 and ∆TruMDR2/3) were successfully obtained, respectively. RESULTS: The suppression of TruMDR2 was shown to abolish resistance to ITC in TIMM20092 and the TruMDR3-lacking mutant, strongly suggesting that TruMDR2 is a major contributor to ITC resistance in TIMM20092. CONCLUSIONS: Our study highlights the possible role of the ABC transporter TruMDR2 in ITC resistance of T. rubrum.

Spread of Terbinafine-Resistant Trichophyton mentagrophytes Type VIII (India) in Germany-"The Tip of the Iceberg?"

Chronic recalcitrant dermatophytoses, due to Trichophyton (T.) mentagrophytes Type VIII are on the rise in India and are noteworthy for their predominance. It would not be wrong to assume that travel and migration would be responsible for the spread of T. mentagrophytes Type VIII from India, with many strains resistant to terbinafine, to other parts of the world. From September 2016 until March 2020, a total of 29 strains of T. mentagrophytes Type VIII (India) were isolated. All patients were residents of Germany: 12 females, 15 males and the gender of the remaining two was not assignable. Patients originated from India (11), Pakistan (two), Bangladesh (one), Iraq (two), Bahrain (one), Libya (one) and other unspecified countries (10). At least two patients were German-born residents. Most samples (21) were collected in 2019 and 2020. All 29 T. mentagrophytes isolates were sequenced (internal transcribed spacer (ITS) and translation elongation factor 1-α gene (TEF1-α)). All were identified as genotype VIII (India) of T. mentagrophytes. In vitro resistance testing revealed 13/29 strains (45%) to be terbinafine-resistant with minimum inhibitory concentration (MIC) breakpoints ≥0.2 µg/mL. The remaining 16 strains (55%) were terbinafine-sensitive. Point mutation analysis revealed that 10/13 resistant strains exhibited Phe397Leu amino acid substitution of squalene epoxidase (SQLE), indicative for in vitro resistance to terbinafine. Two resistant strains showed combined Phe397Leu and Ala448Thr amino acid substitutions, and one strain a single Leu393Phe amino acid substitution. Out of 16 terbinafine-sensitive strains, in eight Ala448Thr, and in one Ala448Thr +, new Val444 Ile amino acid substitutions were detected. Resistance to both itraconazole and voriconazole was observed in three out of 13 analyzed strains. Treatment included topical ciclopirox olamine plus topical miconazole or sertaconazole. Oral itraconazole 200 mg twice daily for four to eight weeks was found to be adequate. Terbinafine-resistant T. mentagrophytes Type VIII are being increasingly isolated. In Germany, transmission of T. mentagrophytes Type VIII from the Indian subcontinent to Europe should be viewed as a significant public health issue.

Epidemiology of Dermatophytoses in Switzerland According to a Survey of Dermatophytes Isolated in Lausanne between 2001 and 2018.

Dermatophytes are the most common pathogenic agents of superficial mycoses in humans and animals. Knowledge of their epidemiology can facilitate the prevention of dermatophytosis and improve prophylactic measures. We sought to determine the incidence of the different dermatophyte species diagnosed in Lausanne (Switzerland) from 2001 to 2018. In total, 10,958 dermatophytes were isolated from patients and 459 from pets. Overall, 99% of tinea unguium and tinea pedis were caused by Trichophyton rubrum and Trichophyton interdigitale with a prevalence ratio of 3:1. Trichophyton violaceum and Trichophyton soudanense were mainly found in tinea capitis in patients of African and Mediterranean origin. Interestingly, while Epidermophyton floccosum and Trichophyton verrucosum were prevalent 50 years ago in an epidemiological analysis carried out in the same laboratory from 1967 to 1970, these two species were rarely detected from 2001 to 2018. Trichophyton mentagrophytes, Trichophyton benhamiae and Microsporum canis were the prevalent zoophilic pathogenic species in children and young adults. Our investigation of animal samples revealed the main reservoirs of these zoophilic species to be cats and dogs for T. mentagrophytes and M. canis, and Guinea pigs for T. benhamiae. This study provides an epidemiological overview of dermatophytoses in Switzerland to improve their surveillance.

Alarming India-wide phenomenon of antifungal resistance in dermatophytes: A multicentre study.

BACKGROUND: An alarming increase in recalcitrant dermatophytosis has been witnessed in India over the past decade. Drug resistance may play a major role in this scenario. OBJECTIVES: The aim of the present study was to determine the prevalence of in vitro resistance to terbinafine, itraconazole and voriconazole in dermatophytes, and to identify underlying mutations in the fungal squalene epoxidase (SQLE) gene. PATIENTS/METHODS: We analysed skin samples from 402 patients originating from eight locations in India. Fungi were identified by microbiological and molecular methods, tested for antifungal susceptibility (terbinafine, itraconazole, voriconazole), and investigated for missense mutations in SQLE. RESULTS: Trichophyton (T.) mentagrophytes internal transcribed spacer (ITS) Type VIII was found in 314 (78%) samples. Eighteen (5%) samples harboured species identified up to the T interdigitale/mentagrophytes complex, and T rubrum was detected in 19 (5%) samples. 71% of isolates were resistant to terbinafine. The amino acid substitution Phe397Leu in the squalene epoxidase of resistant T mentagrophytes was highly prevalent (91%). Two novel substitutions in resistant Trichophyton strains, Ser395Pro and Ser443Pro, were discovered. The substitution Ala448Thr was found in terbinafine-sensitive and terbinafine-resistant isolates but was associated with increased MICs of itraconazole and voriconazole. CONCLUSIONS: The high frequencies of terbinafine resistance in dermatophytes are worrisome and demand monitoring and further research. Squalene epoxidase substitutions between Leu393 and Ser443 could serve as markers of resistance in the future.

Trichophyton rubrum Azole Resistance Mediated by a New ABC Transporter, TruMDR3.

The mechanisms of terbinafine resistance in a set of clinical isolates of Trichophyton rubrum have been studied recently. Of these isolates, TIMM20092 also showed reduced sensitivity to azoles. The azole resistance of TIMM20092 could be inhibited by milbemycin oxime, prompting us to examine the potential of T. rubrum to develop resistance through multidrug efflux transporters. The introduction of a T. rubrum cDNA library into Saccharomyces cerevisiae allowed the isolation of one transporter of the major facilitator superfamily (MFS) conferring resistance to azoles (TruMFS1). To identify more azole efflux pumps among 39 ABC and 170 MFS transporters present within the T. rubrum genome, we performed a BLASTp analysis of Aspergillus fumigatus, Candida albicans, and Candida glabrata on transporters that were previously shown to confer azole resistance. The identified candidates were further tested by heterologous gene expression in S. cerevisiae Four ABC transporters (TruMDR1, TruMDR2, TruMDR3, and TruMDR5) and a second MFS transporter (TruMFS2) proved to be able to operate as azole efflux pumps. Milbemycin oxime inhibited only TruMDR3. Expression analysis showed that both TruMDR3 and TruMDR2 were significantly upregulated in TIMM20092. TruMDR3 transports voriconazole (VRC) and itraconazole (ITC), while TruMDR2 transports only ITC. Disruption of TruMDR3 in TIMM20092 abolished its resistance to VRC and reduced its resistance to ITC. Our study highlights TruMDR3, a newly identified transporter of the ABC family in T. rubrum, which can confer azole resistance if overexpressed. Finally, inhibition of TruMDR3 by milbemycin suggests that milbemycin analogs could be interesting compounds to treat dermatophyte infections in cases of azole resistance.

Terbinafine Resistance of Trichophyton Clinical Isolates Caused by Specific Point Mutations in the Squalene Epoxidase Gene.

Terbinafine is one of the allylamine antifungal agents whose target is squalene epoxidase (SQLE). This agent has been extensively used in the therapy of dermatophyte infections. The incidence of patients with tinea pedis or unguium tolerant to terbinafine treatment prompted us to screen the terbinafine resistance of all Trichophyton clinical isolates from the laboratory of the Centre Hospitalier Universitaire Vaudois collected over a 3-year period and to identify their mechanism of resistance. Among 2,056 tested isolates, 17 (≈1%) showed reduced terbinafine susceptibility, and all of these were found to harbor SQLE gene alleles with different single point mutations, leading to single amino acid substitutions at one of four positions (Leu393, Phe397, Phe415, and His440) of the SQLE protein. Point mutations leading to the corresponding amino acid substitutions were introduced into the endogenous SQLE gene of a terbinafine-sensitive Arthroderma vanbreuseghemii (formerly Trichophyton mentagrophytes) strain. All of the generated A. vanbreuseghemii transformants expressing mutated SQLE proteins exhibited obvious terbinafine-resistant phenotypes compared to the phenotypes of the parent strain and of transformants expressing wild-type SQLE proteins. Nearly identical phenotypes were also observed in A. vanbreuseghemii transformants expressing mutant forms of Trichophyton rubrum SQLE proteins. Considering that the genome size of dermatophytes is about 22 Mb, the frequency of terbinafine-resistant clinical isolates was strikingly high. Increased exposure to antifungal drugs could favor the generation of resistant strains.

AoS28D, a proline-Xaa carboxypeptidase secreted by Aspergillus oryzae.

Prolyl peptidases of the MEROPS S28 family are of particular interest because they are key enzymes in the digestion of proline-rich peptides. A BLAST analysis of the Aspergillus oryzae genome revealed sequences coding for four proteases of the S28 family. Three of these proteases, AoS28A, AoS28B, and AoS28C, were previously characterized as acidic prolyl endopeptidases. The fourth protease, AoS28D, showed high sequence divergence with other S28 proteases and belongs to a phylogenetically distinct cluster together with orthologous proteases from other Aspergillus species. The objective of the present paper was to characterize AoS28D protease in terms of substrate specificity and activity. AoS28D produced by gene overexpression in A. oryzae and in Pichia pastoris was a 70-kDa glycoprotein with a 10-kDa sugar moiety. In contrast with other S28 proteases, AoS28D did not hydrolyze internal Pro-Xaa bonds of several tested peptides. Similarly, to human lysosomal Pro-Xaa carboxypeptidase, AoS28D demonstrated selectivity for cleaving C-terminal Pro-Xaa bonds which are resistant to carboxypeptidases of the S10 family concomitantly secreted by A. oryzae. Therefore, AoS28D could act in synergy with these enzymes during sequential degradation of a peptide from its C-terminus.

Production and characterization of two major Aspergillus oryzae secreted prolyl endopeptidases able to efficiently digest proline-rich peptides of gliadin.

Prolyl endopeptidases are key enzymes in the digestion of proline-rich proteins. Fungal extracts rich in prolyl endopeptidases produced by a species such as Aspergillus oryzae used in food fermentation would be of particular interest for the development of an oral enzyme therapy product in patients affected by intolerance to gluten. Two major A. oryzae secreted prolyl endopeptidases of the MEROPS S28 peptidase family, AoS28A and AoS28B, were identified when this fungus was grown at acidic pH in a medium containing soy meal protein or wheat gliadin as the sole source of nitrogen. AoS28B was produced by 12 reference A. oryzae strains used in food fermentation. AoS28A was secreted by six of these 12 strains. This protease is the orthologue of the previously characterized Aspergillus fumigatus (AfuS28) and Aspergillus niger (AN-PEP) prolyl endopeptidases which are encoded by genes with a similar intron-exon structure. Large amounts of secreted AoS28A and AoS28B were obtained by gene overexpression in A. oryzae. AoS28A and AoS28B are endoproteases able to cleave N-terminally blocked proline substrates. Both enzymes very efficiently digested the proline-rich 33-mer of gliadin, the most representative immunotoxic peptide deriving from gliadin, with some differences in terms of specificity and optimal pH. Digestion of the gliadin peptide in short peptides with both enzymes was found to occur from its N terminus.

Identification of infectious agents in onychomycoses by PCR-terminal restriction fragment length polymorphism.

A fast and reliable assay for the identification of dermatophyte fungi and nondermatophyte fungi (NDF) in onychomycosis is essential, since NDF are especially difficult to cure using standard treatment. Diagnosis is usually based on both direct microscopic examination of nail scrapings and macroscopic and microscopic identification of the infectious fungus in culture assays. In the last decade, PCR assays have been developed for the direct detection of fungi in nail samples. In this study, we describe a PCR-terminal restriction fragment length polymorphism (TRFLP) assay to directly and routinely identify the infecting fungi in nails. Fungal DNA was easily extracted using a commercial kit after dissolving nail fragments in an Na(2)S solution. Trichophyton spp., as well as 12 NDF, could be unambiguously identified by the specific restriction fragment size of 5'-end-labeled amplified 28S DNA. This assay enables the distinction of different fungal infectious agents and their identification in mixed infections. Infectious agents could be identified in 74% (162/219) of cases in which the culture results were negative. The PCR-TRFLP assay described here is simple and reliable. Furthermore, it has the possibility to be automated and thus routinely applied to the rapid diagnosis of a large number of clinical specimens in dermatology laboratories.

Identification of novel secreted proteases during extracellular proteolysis by dermatophytes at acidic pH.

The dermatophytes are a group of closely related fungi which are responsible for the great majority of superficial mycoses in humans and animals. Among various potential virulence factors, their secreted proteolytic activity attracts a lot of attention. Most dermatophyte-secreted proteases which have so far been isolated in vitro are neutral or alkaline enzymes. However, inspection of the recently decoded dermatophyte genomes revealed many other hypothetical secreted proteases, in particular acidic proteases similar to those characterized in Aspergillus spp. The validation of such genome predictions instigated the present study on two dermatophyte species, Microsporum canis and Arthroderma benhamiae. Both fungi were found to grow well in a protein medium at acidic pH, accompanied by extracellular proteolysis. Shotgun MS analysis of secreted protein revealed fundamentally different protease profiles during fungal growth in acidic versus neutral pH conditions. Most notably, novel dermatophyte-secreted proteases were identified at acidic pH such as pepsins, sedolisins and acidic carboxypeptidases. Therefore, our results not only support genome predictions, but demonstrate for the first time the secretion of acidic proteases by dermatophytes. Our findings also suggest the existence of different pathways of protein degradation into amino acids and short peptides in these highly specialized pathogenic fungi.

Secreted glutamic protease rescues aspartic protease Pep deficiency in Aspergillus fumigatus during growth in acidic protein medium.

In an acidic protein medium Aspergillus fumigatus secretes an aspartic endoprotease (Pep) as well as tripeptidyl-peptidases, a prolyl-peptidase and carboxypeptidases. In addition, LC-MS/MS revealed a novel glutamic protease, AfuGprA, homologous to Aspergillus niger aspergillopepsin II. The importance of AfuGprA in protein digestion was evaluated by deletion of its encoding gene in A. fumigatus wild-type D141 and in a pepΔ mutant. Either A. fumigatus Pep or AfuGprA was shown to be necessary for fungal growth in protein medium at low pH. Exoproteolytic activity is therefore not sufficient for complete protein hydrolysis and fungal growth in a medium containing proteins as the sole nitrogen source. Pep and AfuGprA constitute a pair of endoproteases active at low pH, in analogy to A. fumigatus alkaline protease (Alp) and metalloprotease I (Mep), where at least one of these enzymes is necessary for fungal growth in protein medium at neutral pH. Heterologous expression of AfuGprA in Pichia pastoris showed that the enzyme is synthesized as a preproprotein and that the propeptide is removed through an autoproteolytic reaction at low pH to generate the mature protease. In contrast to A. niger aspergillopepsin II, AfuGprA is a single-chain protein and is structurally more similar to G1 proteases characterized in other non-Aspergillus fungi.

Immuno-reactive proteins from Mycobacterium immunogenum useful for serodiagnosis of metalworking fluid hypersensitivity pneumonitis.

Metalworking fluid-associated hypersensitivity pneumonitis (MWF-HP) is a pulmonary disease caused by inhaling microorganisms present in the metalworking fluids used in the industrial sector. Mycobacterium immunogenum is the main etiological agent. Among the clinical, radiological and biological tools used for diagnosis, serological tests are important. The aim of this study was to identify immunogenic proteins in M. immunogenum and to use recombinant antigens for serological diagnosis of MWF-HP. Immunogenic proteins were detected by two-dimensional Western blot and candidate proteins were identified by mass spectrometry. Recombinant antigens were expressed in Escherichia coli and tested by enzyme-linked immunosorbent assay (ELISA) with the sera of 14 subjects with MWF-HP and 12 asymptomatic controls exposed to M. immunogenum. From the 350 spots visualized by two-dimensional gel electrophoresis with M. immunogenum extract, 6 immunogenic proteins were selected to be expressed as recombinant antigens. Acyl-CoA dehydrogenase antigen allowed for the best discrimination of MWF-HP cases against controls with an area under the receiver operating characteristics (ROC) curve of 0.930 (95% CI=0.820-1), a sensitivity of 100% and a specificity of 83% for the optimum threshold. Other recombinant antigens correspond to acyl-CoA dehydrogenase FadE, cytosol aminopeptidase, dihydrolipoyl dehydrogenase, serine hydroxymethyltransferase and superoxide dismutase. This is the first time that recombinant antigens have been used for the serodiagnosis of hypersensitivity pneumonitis. The availability of recombinant antigens makes it possible to develop standardized serological tests which in turn could simplify diagnosis, thus making it less invasive.

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.

Aspergillus protein degradation pathways with different secreted protease sets at neutral and acidic pH.

Aspergillus fumigatus grows well at neutral and acidic pH in a medium containing protein as the sole nitrogen source by secreting two different sets of proteases. Neutral pH favors the secretion of neutral and alkaline endoproteases, leucine aminopeptidases (Laps) which are nonspecific monoaminopeptidases, and an X-prolyl dipeptidase (DppIV). Acidic pH environment promotes the secretion of an aspartic endoprotease of pepsin family (Pep1) and tripeptidyl-peptidases of the sedolisin family (SedB and SedD). A novel prolyl peptidase, AfuS28, was found to be secreted in both alkaline and acidic conditions. In previous studies, Laps were shown to degrade peptides from their N-terminus until an X-Pro sequence acts as a stop signal. X-Pro sequences can be then removed by DppIV, which allows Laps access to the following residues. We have shown that at acidic pH Seds degrade large peptides from their N-terminus into tripeptides until Pro in P1 or P'1 position acts as a stop for these exopeptidases. However, X-X-Pro and X-X-X-Pro sequences can be removed by AfuS28 thus allowing Seds further sequential proteolysis. In conclusion, both alkaline and acidic sets of proteases contain exoprotease activity capable of cleaving after proline residues that cannot be removed during sequential digestion by nonspecific exopeptidases.