Lack of accurate diagnosis and the use of formulations designed to address the poor aqueous solubility of antifungal agents, but not optimized for delivery, contribute to unsatisfactory outcomes for ...topical treatment of cutaneous mycoses. The objective of this study was to develop a micelle-based antifungal formulation containing econazole (ECZ), terbinafine (TBF) and amorolfine (AMF) using D-α-tocopheryl polyethylene glycol succinate (TPGS) for simultaneous cutaneous delivery of three agents with complementary mechanisms of action. The antifungal "tri-therapy" micelle-based formulation containing 0.1% ECZ, 0.1% TBF and 0.025% AMF had a drug loading 10-fold lower than the "reference" marketed formulations (Pevaryl
, 1% ECZ; Lamisil
, 1% TBF; Loceryl
, 0.25% AMF). Finite dose application of the micelle-based formulation for 6 h resulted in a statistically equivalent deposition of ECZ (
> 0.05) and TBF (
> 0.05) from the 2 systems, and a 2-fold higher accumulation of AMF (
= 0.017). Antifungal concentrations above MIC
against
were achieved in each skin layer with the "tri-therapy", which also exhibited a preferential deposition of each antifungal agent in pilosebaceous unit (PSU)-containing biopsies as compared with PSU-free biopsies (
< 0.05). A planned clinical study will test whether these promising results translate to improved therapeutic outcomes in vivo.
Summary
Colletotrichum graminicola causes maize anthracnose, an agronomically important disease with a worldwide distribution. We have identified a fungalysin metalloprotease (Cgfl) with a role in ...virulence. Transcriptional profiling experiments and live cell imaging show that Cgfl is specifically expressed during the biotrophic stage of infection. To determine whether Cgfl has a role in virulence, we obtained null mutants lacking Cgfl and performed pathogenicity and live microscopy assays. The appressorium morphology of the null mutants is normal, but they exhibit delayed development during the infection process on maize leaves and roots, showing that Cgfl has a role in virulence. In vitro chitinase activity assays of leaves infected with wild‐type and null mutant strains show that, in the absence of Cgfl, maize leaves exhibit increased chitinase activity. Phylogenetic analyses show that Cgfl is highly conserved in fungi. Similarity searches, phylogenetic analysis and transcriptional profiling show that C. graminicola encodes two LysM domain‐containing homologues of Ecp6, suggesting that this fungus employs both Cgfl‐mediated and LysM protein‐mediated strategies to control chitin signalling.
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.
Several dermatophytes producing numerous pyriform or round microconidia were called
Trichophyton mentagrophytes
. Among these dermatophytes are the teleomorph species
Arthroderma benhamiae
,
...Arthroderma vanbreuseghemii
and
Arthroderma simii
, and other species such as
Trichophyton interdigitale
,
Trichophyton erinacei
and
Trichophyton quinckeanum
for which only the anamorph is known. Confusion exists about which fungus should be really called
T. mentagrophytes
and about the rational use of this name in practice. We report a case of beard ringworm (tinea barbae) with
A. vanbreuseghemii
. According to both clinical signs and the type of hair parasitism, this case was exactly compatible to the first description of a non-favic dermatophytosis by Gruby under the name of “mentagrophyte” from which was derived the dermatophyte epithet
mentagrophytes
. In addition, the phenotypic characters of the isolated fungus in cultures perfectly matched with those of the first description of a dermatophyte under
T. mentagrophytes
by Blanchard (Parasites animaux et parasites végétaux à l’exclusion des Bactéries, Masson, Paris,
1896
). In conclusion,
T. mentagrophytes
corresponds to the fungus later named
A. vanbreuseghemii
. However, because the neotype of
T. mentagrophytes
was not adequately designated in regard to the ancient literature, we would privilege the use of
A. vanbreuseghemii
and abandon the name of
T. mentagrophytes
.
The ability of fungi isolated from nails of patients suffering from onychomycosis to induce de novo production of bioactive compounds in co-culture was examined. Comparison between the metabolite ...profiles produced by Sarocladium strictum, by Fusarium oxysporum, and by these two species in co-culture revealed de novo induction of fusaric acid based on HRMS. Structure confirmation of this toxin, using sensitive microflow NMR, required only three 9-cm Petri dishes of fungal culture. A targeted metabolomics study based on UHPLC-HRMS confirmed that the production of fusaric acid was strain-dependent. Furthermore, the detected toxin levels suggested that onychomycosis-associated fungal strains of the F. oxysporum and F. fujikuroi species complexes are much more frequently producing fusaric acid, and in higher amount, than strains of the F. solani species complex. Fusarium strains producing no significant amounts of this compound in pure culture, were shown to de novo produce that compound when grown in co-culture. The role of fusaric acid in fungal virulence and defense is discussed.
Identification of fungi in dermatological samples using PCR is reliable and provides significantly improved results in comparison with cultures. It is possible to identify the infectious agent when ...negative results are obtained from cultures. In addition, identification of the infectious agent can be obtained in 1 day. Conventional and real-time PCR methods used for direct fungus identification in collected samples vary by DNA extraction methods, targeted DNA and primers, and the way of analysing the PCR products. The choice of a unique method in a laboratory is complicated because the results expected from skin and hair sample analysis are different from those expected in cases of onychomycosis. In skin and hair samples, one dermatophyte among about a dozen possible species has to be identified. In onychomycosis, the infectious agents are mainly
Trichophyton rubrum
and, to a lesser extent,
Trichophyton interdigitale
, but also moulds insensitive to oral treatments used for dermatophytes, which renders fungal identification mandatory. The benefits obtained with the use of PCR methods for routine analysis of dermatological samples have to be put in balance with the relative importance of getting a result in a short time, the price of molecular biology reagents and equipment, and especially the time spent conducting laboratory manipulations.
Intracellular and secreted proteases fulfill multiple functions in microorganisms. In pathogenic microorganisms extracellular proteases may be adapted to interactions with host cells. Here we ...describe two cell surface-associated aspartic proteases, Sap9 and Sap10, which have structural similarities to yapsins of Saccharomyces cerevisiae and are produced by the human pathogenic yeast Candida albicans. Sap9 and Sap10 are glycosylphosphatidylinositol-anchored and located in the cell membrane or the cell wall. Both proteases are glycosylated, cleave at dibasic or basic processing sites similar to yapsins and Kex2-like proteases, and have functions in cell surface integrity and cell separation during budding. Overexpression of SAP9 in mutants lacking KEX2 or SAP10, or of SAP10 in mutants lacking KEX2 or SAP9, only partially restored these phenotypes, suggesting distinct target proteins of fungal origin for each of the three proteases. In addition, deletion of SAP9 and SAP10 modified the adhesion properties of C. albicans to epithelial cells and caused attenuated epithelial cell damage during experimental oral infection suggesting a unique role for these proteases in both cellular processes and host-pathogen interactions.
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.
Biological processes can be elucidated by investigating complex networks of relevant factors and genes. However, this is not possible in species for which dominant selectable markers for genetic ...studies are unavailable. To overcome the limitation in selectable markers for the dermatophyte Arthroderma vanbreuseghemii (anamorph: Trichophyton mentagrophytes), we adapted the flippase (FLP) recombinase-recombination target (FRT) site-specific recombination system from the yeast Saccharomyces cerevisiae as a selectable marker recycling system for this fungus. Taking into account practical applicability, we designed FLP/FRT modules carrying two FRT sequences as well as the flp gene adapted to the pathogenic yeast Candida albicans (caflp) or a synthetic codon-optimized flp (avflp) gene with neomycin resistance (nptII) cassette for one-step marker excision. Both flp genes were under control of the Trichophyton rubrum copper-repressible promoter (PCTR4). Molecular analyses of resultant transformants showed that only the avflp-harbouring module was functional in A. vanbreuseghemii. Applying this system, we successfully produced the Ku80 recessive mutant strain devoid of any selectable markers. This strain was subsequently used as the recipient for sequential multiple disruptions of secreted metalloprotease (fungalysin) (MEP) or serine protease (SUB) genes, producing mutant strains with double MEP or triple SUB gene deletions. These results confirmed the feasibility of this system for broad-scale genetic manipulation of dermatophytes, advancing our understanding of functions and networks of individual genes in these fungi.
The use of recombinant antigens has been shown to improve both the sensitivity and the standardization of the serological diagnosis of Farmer's lung disease (FLD). The aim of this study was to ...complete the panel of recombinant antigens available for FLD serodiagnosis with antigens of Lichtheimia corymbifera, known to be involved in FLD. L. corymbifera proteins were thus separated by 2D electrophoresis and subjected to western blotting with sera from 7 patients with FLD and 9 healthy exposed controls (HEC). FLD-associated immunoreactive proteins were identified by mass spectrometry based on a protein database specifically created for this study and subsequently produced as recombinant antigens. The ability of recombinant antigens to discriminate patients with FLD from controls was assessed by ELISA performed with sera from FLD patients (n = 41) and controls (n = 43) recruited from five university hospital pneumology departments of France and Switzerland. Forty-one FLD-associated immunoreactive proteins from L. corymbifera were identified. Six of them were produced as recombinant antigens. With a sensitivity and specificity of 81.4 and 77.3% respectively, dihydrolipoyl dehydrogenase was the most effective antigen for discriminating FLD patients from HEC. ELISA performed with the putative proteasome subunit alpha type as an antigen was especially specific (88.6%) and could thus be used for FLD confirmation. The production of recombinant antigens from L. corymbifera represents an additional step towards the development of a standardized ELISA kit for FLD diagnosis.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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