B-cell receptor epitope recognition correlates with the clinical course of chronic lymphocytic leukemia.

BACKGROUND: B-cell receptors (BCRs) and their recognition of specific epitopes may play a pivotal role in the development and progression of chronic lymphocytic leukemia (CLL). In this study, the authors set up a model system to explore epitope reactivity and its clinical relevance in CLL. METHODS: Epitope-mimicking peptides were selected from phage display libraries on 6 CLL BCRs from randomly chosen patients. The binding of the 6 index epitope mimics was evaluated in a set of 100 unrelated CLL samples. Epitope recognition patterns were correlated with the clinical course of the disease. RESULTS: Surprisingly, all CLL samples recognized 1 or several index epitopes, and some revealed marked polyreactivity. Patients with CLL who expressed BCRs that reacted with ≥5 epitope mimics had a significantly worse clinical course than less polyreactive patients (median time to first treatment, 24 months vs 102 months). This effect was independent of otherwise known prognostic markers. CONCLUSIONS: The authors introduced a system with which to model epitope reactivity of CLL BCRs without previous knowledge of potential antigens. The findings indicated that a polyreactive epitope recognition pattern may be a determinant of an aggressive clinical course in this disease. This further emphasizes the functional and prognostic relevance of BCR epitope recognition in CLL.

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.

Stereotypical chronic lymphocytic leukemia B-cell receptors recognize survival promoting antigens on stromal cells.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world. Survival of CLL cells depends on their close contact with stromal cells in lymphatic tissues, bone marrow and blood. This microenvironmental regulation of CLL cell survival involves the stromal secretion of chemo- and cytokines as well as the expression of adhesion molecules. Since CLL survival may also be driven by antigenic stimulation through the B-cell antigen receptor (BCR), we explored the hypothesis that these processes may be linked to each other. We tested if stromal cells could serve as an antigen reservoir for CLL cells, thus promoting CLL cell survival by stimulation through the BCR. As a proof of principle, we found that two CLL BCRs with a common stereotyped heavy chain complementarity-determining region 3 (previously characterized as "subset 1") recognize antigens highly expressed in stromal cells--vimentin and calreticulin. Both antigens are well-documented targets of autoantibodies in autoimmune disorders. We demonstrated that vimentin is displayed on the surface of viable stromal cells and that it is present and bound by the stereotyped CLL BCR in CLL-stroma co-culture supernatant. Blocking the vimentin antigen by recombinant soluble CLL BCR under CLL-stromal cell co-culture conditions reduces stroma-mediated anti-apoptotic effects by 20-45%. We therefore conclude that CLL BCR stimulation by stroma-derived antigens can contribute to the protective effect that the stroma exerts on CLL cells. This finding sheds a new light on the understanding of the pathobiology of this so far mostly incurable disease.

Trichophyton rubrum secreted and membrane-associated carboxypeptidases.

Dermatophytes are the most common agents of superficial mycoses, and exclusively infect stratum corneum, nails or hair. Therefore, secreted proteolytic activity is considered a virulence trait of these fungi. In a medium containing protein as a sole nitrogen and carbon source Trichophyton rubrum secretes a metallocarboxypeptidase (TruMcpA) of the M14 family according to the MEROPS proteolytic enzyme database. TruMcpA is homologous to human pancreatic carboxypeptidase A, and is synthesized as a precursor in a preproprotein form. The propeptide is removed to generate the mature active enzyme alternatively by either one of two subtilisins which are concomitantly secreted by the fungus. In addition, T. rubrum was shown to possess two genes (TruSCPA and TruSCPB) encoding serine carboxypeptidases of the S10 family which are homologues of the previously characterized Aspergillus and Penicillium secreted acid carboxypeptidases. However, in contrast to the Aspergillus and Penicillium homologues, TruScpA and TruScpB enzymes are not secreted into the environment, but are membrane-associated with a glycosylphosphatidylinositol (GPI) anchor. During infection, T. rubrum secreted and GPI-anchored carboxypeptidases may contribute to fungal virulence by cooperating with previously characterized endoproteases and aminopeptidases in the degradation of compact keratinized tissues into assimilable amino acids and short peptides.

Sulphite efflux pumps in Aspergillus fumigatus and dermatophytes.

Dermatophytes and other filamentous fungi excrete sulphite as a reducing agent during keratin degradation. In the presence of sulphite, cystine in keratin is directly cleaved to cysteine and S-sulphocysteine, and thereby, reduced proteins become accessible to hydrolysis by a variety of secreted endo- and exoproteases. A gene encoding a sulphite transporter in Aspergillus fumigatus (AfuSSU1), and orthologues in the dermatophytes Trichophyton rubrum and Arthroderma benhamiae (TruSSU1 and AbeSSU1, respectively), were identified by functional expression in Saccharomyces cerevisiae. Like the S. cerevisiae sulphite efflux pump Ssu1p, AfuSsu1p, TruSsu1p and AbeSsu1p belong to the tellurite-resistance/dicarboxylate transporter (TDT) family which includes the Escherichia coli tellurite transporter TehAp and the Schizosaccharomyces pombe malate transporter Mae1p. Seven genes in the A. fumigatus genome encode transporters of the TDT family. However, gene disruption of AfuSSU1 and of the two more closely related paralogues revealed that only AfuSSU1 encodes a sulphite efflux pump. TruSsulp and AbeSsulp are believed to be the first members of the TDT family identified in dermatophytes. The relatively high expression of TruSSU1 and AbeSSU1 in dermatophytes compared to that of AfuSSU1 in A. fumigatus likely reflects a property of dermatophytes which renders these fungi pathogenic. Sulphite transporters could be a new target for antifungal drugs in dermatology, since proteolytic digestion of hard keratin would not be possible without prior reduction of disulphide bridges.

Fast and reliable PCR/sequencing/RFLP assay for identification of fungi in onychomycoses.

Fusarium spp. and other non-dermatophyte fungi are repeatedly isolated from abnormal nails. To investigate whether these fungi are the aetiological agents of infection or simply transient contaminants, a PCR/sequencing/RFLP assay was developed for direct and routine identification of the infecting fungi in onychomycosis. Fungal DNA was readily extracted using a commercial kit after dissolving nail fragments in a Na2S solution. Amplification of part of the 28S rDNA by PCR was performed with universal primers and the fungal species were identified by sequencing. The PCR/sequencing results were comparable with microbiological identification from the same nail sample. In addition to dermatophytes, Fusarium spp. and other less frequently isolated non-dermatophyte fungi were identified as single fungal agents in onychomycosis. Moreover, mixed infections were clearly demonstrated in 10% of cases by RFLP analysis of PCR products. Identification of infectious agents could be obtained in 2 days, whilst results from fungal cultures take 1-3 weeks. Rapid and reliable molecular identification of the infectious fungus expedites the choice of appropriate antifungal therapy, thereby improving the cure rate of onychomycosis.

Sedolisins, a new class of secreted proteases from Aspergillus fumigatus with endoprotease or tripeptidyl-peptidase activity at acidic pHs.

The secreted proteolytic activity of Aspergillus fumigatus is of potential importance as a virulence factor and in the industrial hydrolysis of protein sources. The A. fumigatus genome contains sequences that could encode a five-member gene family that produces proteases in the sedolisin family (MEROPS S53). Four putative secreted sedolisins with a predicted 17- to 20-amino-acid signal sequence were identified and termed SedA to SedD. SedA produced heterologously in Pichia pastoris was an acidic endoprotease. Heterologously produced SedB, SedC, and SedD were tripeptidyl-peptidases (TPP) with a common specificity for tripeptide-p-nitroanilide substrates at acidic pHs. Purified SedB hydrolyzed the peptide Ala-Pro-Gly-Asp-Arg-Ile-Tyr-Val-His-Pro-Phe to Arg-Pro-Gly, Asp-Arg-Ile, and Tyr-Val-His-Pro-Phe, thereby confirming TPP activity of the enzyme. SedB, SedC, and SedD were detected by Western blotting in culture supernatants of A. fumigatus grown in a medium containing hemoglobin as the sole nitrogen source. A degradation product of SedA also was observed. A search for genes encoding sedolisin homologues in other fungal genomes indicates that sedolisin gene families are widespread among filamentous ascomycetes.

Deletion of GEL2 encoding for a beta(1-3)glucanosyltransferase affects morphogenesis and virulence in Aspergillus fumigatus.

The first fungal glycosylphosphatidylinositol anchored beta(1-3)glucanosyltranferase (Gel1p) has been described in Aspergillus fumigatus and its encoding gene GEL1 identified. Glycosylphosphatidylinositol-anchored glucanosyltransferases play an active role in the biosynthesis of the fungal cell wall. We characterize here GEL2, a homologue of GEL1. Both homologues share common characteristics: (i) GEL1 and GEL2 are constitutively expressed during over a range of growth conditions; (ii) Gel2p is also a putative GPI-anchored protein and shares the same beta(1-3)glucanosyltransferase activity as Gel1p and (iii) GEL2, like GEL1, is able to complement the Deltagas1 deletion in Saccharomyces cerevisiae confirming that Gelp and Gasp have the same enzymatic activity. However, disruption of GEL1 did not result in a phenotype whereas a Deltagel2 mutant and the double mutant Deltagel1Deltagel2 exhibit slower growth, abnormal conidiogenesis, and an altered cell wall composition. In addition, the Deltagel2 and the Deltagel1Deltagel2 mutant have reduced virulence in a murine model of invasive aspergillosis. These data suggest for the first time that beta(1-3)glucanosyltransferase activity is required for both morphogenesis and virulence in A. fumigatus.

Aminopeptidases and dipeptidyl-peptidases secreted by the dermatophyte Trichophyton rubrum.

The nature of secreted aminopeptidases in Trichophyton rubrum was investigated by using a reverse genetic approach. T. rubrum genomic and cDNA libraries were screened with Aspergillus spp. and Saccharomyces cerevisiae aminopeptidase genes as the probes. Two leucine aminopeptidases, ruLap1 and ruLap2, and two dipeptidyl-peptidases, ruDppIV and ruDppV, were characterized and compared to orthologues secreted by Aspergillus fumigatus using a recombinant protein from Pichia pastoris. RuLap1 is a 33 kDa nonglycosylated protein, while ruLap2 is a 58-65 kDa glycoprotein. The hydrolytic activity of ruLap1, ruLap2 and A. fumigatus orthologues showed various preferences for different aminoacyl-7-amido-4-methylcoumarin substrates, and various sensitivities to inhibitors and cations. ruDppIV and ruDppV showed similar activities to A. fumigatus orthologues. In addition to endopeptidases, the four aminopeptidases ruLap1, ruLap2, ruDppIV and ruDppV were produced by T. rubrum in a medium containing keratin as the sole nitrogen source. Synergism between endo- and exopeptidases is likely to be essential for dermatophyte virulence, since these fungi grow only in keratinized tissues.

Molecular identification of Fusarium species in onychomycoses.

BACKGROUND: Fusarium species are isolated from about 3% of onychomycoses in the Swiss native population. On the basis of macroscopic characters and microscopic examination of the cultures, identification of Fusarium often remains difficult or uncertain because of variations from one isolate to another and overlapping characteristics between species. OBJECTIVE: To obtain information about the prevailing species of Fusarium collected from onychomycoses. METHODS: An analysis of the Fusarium specimens isolated in the Department of Dermatology at the University Hospital of Lausanne was conducted during a 2-year period (71 isolates). A 311-bp fragment of the gene encoding 28S rDNA was amplified by PCR and sequenced. DNA sequences were compared to those available for reference strains. RESULTS: Fusarium oxysporum was the most frequently isolated species, accounting for 54% of the isolates. F. proliferatum and 4 taxons belonging to the F. solani species complex were identified with an appreciable frequency ranging from 4 to 14%. CONCLUSION: The Fusarium species identified were the same as those known to cause disseminated fusariosis in immunocompromised patients. The presence of these Fusarium species in onychomycoses warrants that careful attention should be paid to abnormal nails before beginning immunosuppressive treatments in patients.

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.

First report of Arthroderma benhamiae in Switzerland.

BACKGROUND: Dermatophytes are usually identified on the basis of macroscopic characteristics and microscopic examination of the cultures. Identification of dermatophytes often remains difficult or uncertain because there are variations from one isolate to another and overlapping characteristics between species. OBJECTIVE: To identify dermatophyte species producing numerous microconidia and resembling Trichophyton mentagrophytes by DNA sequence analysis. METHODS: The complete ITS1 + 5.6s + ITS2 rDNA region of various dermatophytes isolated in culture was amplified by PCR and sequenced. RESULTS: Nine isolates of a fast-growing dermatophyte species were identified as Arthroderma benhamiae by DNA sequencing. Retrospective investigations revealed that the isolates were from 8 children and 1 adult suffering from inflammatory dermatophytosis. Eight of the 9 patients had had previous contact with rodents, mostly guinea pigs. CONCLUSION: It is the first time that A. benhamiae is reported in Switzerland. In cases of dermatophytosis attributed to A. benhamiae, a rodent is the most likely cause of infection.

Multiplication of an ancestral gene encoding secreted fungalysin preceded species differentiation in the dermatophytes Trichophyton and Microsporum.

Dermatophytes are human and animal pathogenic fungi which cause cutaneous infections and grow exclusively in the stratum corneum, nails and hair. In a culture medium containing soy proteins as sole nitrogen source a substantial proteolytic activity was secreted by Trichophyton rubrum, Trichophyton mentagrophytes and Microsporum canis. This proteolytic activity was 55-75 % inhibited by o-phenanthroline, attesting that metalloproteases were secreted by all three species. Using a consensus probe constructed on previously characterized genes encoding metalloproteases (MEP) of the M36 fungalysin family in Aspergillus fumigatus, Aspergillus oryzae and M. canis, a five-member MEP family was isolated from genomic libraries of T. rubrum, T. mentagrophytes and M. canis. A phylogenetic analysis of genomic and protein sequences revealed a robust tree consisting of five main clades, each of them including a MEP sequence type from each dermatophyte species. Each MEP type was remarkably conserved across species (72-97 % amino acid sequence identity). The tree topology clearly indicated that the multiplication of MEP genes in dermatophytes occurred prior to species divergence. In culture medium containing soy proteins as a sole nitrogen source secreted Meps accounted for 19-36 % of total secreted protein extracts; characterization of protein bands by proteolysis and mass spectrometry revealed that the three dermatophyte species secreted two Meps (Mep3 and Mep4) encoded by orthologous genes.

Identification of dermatophyte species by 28S ribosomal DNA sequencing with a commercial kit.

We have shown that dermatophyte species can be easily identified on the basis of a DNA sequence encoding a part of the large-subunit (LSU) rRNA (28S rRNA) by using the MicroSeq D2 LSU rRNA Fungal Sequencing Kit. Two taxa causing distinct dermatophytoses were clearly distinguished among isolates of the Trichophyton mentagrophytes species complex.

Secreted proteases from pathogenic fungi.

Many species of human pathogenic fungi secrete proteases in vitro or during the infection process. Secreted endoproteases belong to the aspartic proteases of the pepsin family, serine proteases of the subtilisin family, and metalloproteases of two different families. To these proteases has to be added the non-pepsin-type aspartic protease from Aspergillus niger and a unique chymotrypsin-like protease from Coccidioides immitis. Pathogenic fungi also secrete aminopeptidases, carboxypeptidases and dipeptidyl-peptidases. The function of fungal secreted proteases and their importance in infections vary. It is evident that secreted proteases are important for the virulence of dermatophytes since these fungi grow exclusively in the stratum corneum, nails or hair, which constitutes their sole nitrogen and carbon sources. The aspartic proteases secreted by Candida albicans are involved in the adherence process and penetration of tissues, and in interactions with the immune system of the infected host. For Aspergillus fumigatus, the role of proteolytic activity has not yet been proved. Although the secreted proteases have been intensively investigated as potential virulence factors, knowledge on protease substrate specificities is rather poor and few studies have focused on the research of inhibitors. Knowledge of substrate specificities will increase our understanding about the action of each protease secreted by pathogenic fungi and will help to determine their contribution to virulence.

Survey of dermatophyte infections in the Lausanne area Switzerland.

BACKGROUND: The dermatophytes are important in the Swiss medical environment since 5-10% of consultations in dermatology concern mycotic infections. OBJECTIVE: To obtain information about the prevailing species of dermatophytes in the south-west of Switzerland and their pattern of infection. METHODS: An analysis was made of the dermatophytes isolated in the Department of Dermatology at the University Hospital of Lausanne and from samples collected in private practices of Switzerland during an 8-year period (1993-2000). The total number of samples sent for mycological analysis was 33,725. RESULTS: 4,193 cultures revealed a dermatophyte. Trichophyton rubrum was the most frequently isolated species accounting for 62.5% of the strains followed by T. mentagrophytes (24.5%) and Microsporum canis (5.0%). Less frequent isolates included Epidermophyton floccosum, M. langeroni, M. gypseum, T. soudanense, T. violaceum, T. verrucosum, T. gourvili and T. tonsurans. Analysis of the localisation of the isolated fungi confirms that the dermatophyte species have a predilection for certain body areas. CONCLUSIONS: The relative frequencies of isolation of the dermatophyte species partially depending of the record of the different tinea vary from one country to another. Our study reveals the importance of T. rubrum and the appreciable frequency of M. canis in the Swiss autochthonous population and the apparition of new species with immigrants.