Sorbicillactone A: a structurally unprecedented bioactive novel-type alkaloid from a sponge-derived fungus.

This chapter deals with the discovery of sorbicillactone A, as an illustrative example of the fruitful cooperation within BIOTECmarin--its isolation and chemical characterization, and its biological activities. Sorbicillactone A was isolated from a strain of Penicillium chrysogenum cultured from a sample of the Mediterranean sponge Ircinia fasciculata; it possesses a unique bicyclic lactone structure, seemingly derived from sorbicillin. Among the numerous known sorbicillin-derived structures, it is the first found to contain nitrogen and thus the first representative of a novel type of 'sorbicillin alkaloids', apparently originating from a likewise remarkable biosynthesis. Furthermore, the compound exhibits promising activities in several mammalian and viral test systems, in particular a highly selective cytostatic activity against murine leukemic lymphoblasts (L5178y) and the ability to protect human T cells against the cytopathic effects of HIV-1. These properties qualify sorbicillactone A or one of its derivatives for animal and (hopefully) also future therapeutic human trials.

A fourth gene from the Candida albicans CDR family of ABC transporters.

Using primers derived from a region of the Candida albicans CDR1 (Candida drug resistance) gene that is conserved in other ABC (ATP-binding cassette) transporters, a DNA fragment from a previously unknown CDR gene was obtained by polymerase chain reaction (PCR). After screening a C. albicans genomic library with this fragment as a probe, the complete CDR4 gene was isolated and sequenced. CDR4 codes for a putative ABC transporter of 1490 amino acids with a high degree of homology to Cdr1p, Cdr2p and Cdr3p from C. albicans (62, 59 and 57% amino acid sequence identity, respectively). Cdr4p has a predicted structure typical for cluster I. 1 of yeast ABC transporters, characterized by two homologous halves, each comprising an N-terminal hydrophilic domain with consensus sequences for ATP binding and a C-terminal hydrophobic domain with six transmembrane helices. In contrast to the CDR1/CDR2 genes, the genetic structure of the CDR4 gene was conserved in 59 C. albicans isolates from six different patients. Northern hybridization analysis showed that the CDR4 gene was expressed in most isolates, but no correlation between CDR4 mRNA levels and the degree of fluconazole resistance of the isolates was found. In addition, a C. albicans mutant in which both copies of the CDR4 gene were disrupted by insertional mutagenesis was not hypersusceptible to fluconazole as compared to the parent strain. Unlike CDR1 and CDR2, CDR4 does not, therefore, seem to be involved in fluconazole resistance in C. albicans.

A molecular genetic system for the pathogenic yeast Candida dubliniensis.

Candida dubliniensis is a recently described pathogenic yeast of the genus Candida that is closely related to Candida albicans but differs from it in several phenotypic and genotypic characteristics, including putative virulence traits, which may explain differences in the spectrum of diseases caused by the two species. In contrast to C. albicans, a molecular genetic system to study virulence of C. dubliniensis is lacking. We have developed a system for the genetic transformation of C. dubliniensis that is based on the use of the dominant selection marker MPA(R) from C. albicans that confers resistance to mycophenolic acid (MPA). Using this transformation system, a GFP (green fluorescent protein) reporter gene that was genetically engineered for functional expression in C. albicans and placed under control of the inducible C. albicans SAP2 (secreted aspartic proteinase) promoter was integrated into the C. dubliniensis genome. MPA-resistant transformants containing the SAP2P-GFP fusion fluoresced under SAP2-inducing conditions but not under SAP2-repressing conditions. These results demonstrate that the MPA(R) selection marker is useful for transformation of C. dubliniensis wild-type strains, that the GFP reporter gene is functionally expressed in C. dubliniensis, and that the C. albicans SAP2 promoter can be used for controlled gene expression in C. dubliniensis. These genetic tools will allow the dissection of the differences in virulence characteristics between the two pathogenic yeast species at the molecular level.

Degradation of human subendothelial extracellular matrix by proteinase-secreting Candida albicans.

Candida albicans infections in severely immunocompromized patients are not confined to mucosal surfaces; instead the fungus can invade through epithelial and endothelial layers into the bloodstream and spread to other organs, causing disseminated infections with often fatal outcome. We investigated whether secretion of the C. albicans acid proteinase facilitates invasion into deeper tissues by degrading the subendothelial basement membrane. After cultivation under conditions that induce the secretion of the acid proteinase, C. albicans degraded radioactively metabolically labeled extracellular matrix proteins from a human endothelial cell line. The degradation was inhibited in the presence of pepstatin A, an inhibitor of acid proteinases. Pepstatin A-sensitive degradation of the soluble and immobilized extracellular matrix proteins fibronectin and laminin by proteinase-producing C. albicans was also detected, whereas no degradation was observed when the expression of the acid proteinase was repressed. Our results demonstrate that the C. albicans acid proteinase degrades human subendothelial extracellular matrix; this may be of importance in the penetration of C. albicans into circulation and deep organs.

Chlamydospore formation on Staib agar as a species-specific characteristic of Candida dubliniensis.

Staib agar (Syn. Guizotia abyssinica creatinine agar) was evaluated for differentiation between the highly related yeast species Candida albicans and Candida dubliniensis. On these agar plates C. dubliniensis formed rough colonies due to mycelial growth and produced abundant chlamydospores whereas C. albicans grew only in smooth colonies and without chlamydospore formation. The rough colonies of C. dubliniensis could be readily distinguished from the smooth C. albicans colonies. These results demonstrate that, under certain growth conditions, mycelial growth with chlamydospore formation is a species-specific marker that can be used for the identification of C. dubliniensis.

Analysis of phase-specific gene expression at the single-cell level in the white-opaque switching system of Candida albicans.

The opportunistic fungal pathogen Candida albicans can switch spontaneously and reversibly between different cell forms, a capacity that may enhance adaptation to different host niches and evasion of host defense mechanisms. Phenotypic switching has been studied intensively for the white-opaque switching system of strain WO-1. To facilitate the molecular analysis of phenotypic switching, we have constructed homozygous ura3 mutants from strain WO-1 by targeted gene deletion. The two URA3 alleles were sequentially inactivated using the MPA(R)-flipping strategy, which is based on the selection of integrative transformants carrying a mycophenolic acid (MPA) resistance marker that is subsequently deleted again by site-specific, FLP-mediated recombination. To investigate a possible cell type-independent switching in the expression of individual phase-specific genes, two different reporter genes that allowed the analysis of gene expression at the single-cell level were integrated into the genome, using URA3 as a selection marker. Fluorescence microscopic analysis of cells in which a GFP reporter gene was placed under the control of phase-specific promoters demonstrated that the opaque-phase-specific SAP1 gene was detectably expressed only in opaque cells and that the white-phase-specific WH11 gene was detectably expressed only in white cells. When MPA(R) was used as a reporter gene, it conferred an MPA-resistant phenotype on opaque but not white cells in strains expressing it from the SAP1 promoter, which was monitored at the level of single cells by a significantly enlarged size of the corresponding colonies on MPA-containing indicator plates. Similarly, white but not opaque cells became MPA resistant when MPA(R) was placed under the control of the WH11 promoter. The analysis of these reporter strains showed that cell type-independent phase variation in the expression of the SAP1 and WH11 genes did not occur at a detectable frequency. The expression of these phase-specific genes of C. albicans in vitro, therefore, is tightly linked to the cell type.

Expression of a chromosomally integrated, single-copy GFP gene in Candida albicans, and its use as a reporter of gene regulation.

Genetically engineered versions of the GFP gene, which encodes the green fluorescent protein of Aequorea victoria, were placed under the control of the constitutively active Candida albicans ACT1 promoter and integrated in single copy into the genome of this pathogenic yeast. Integrative transformants in which one of the two ACT1 alleles had been replaced by a GFP gene exhibited a homogeneous, constitutive fluorescent phenotype. Cells expressing GFP with the wild-type chromophore exhibited very weak fluorescence compared to those GFP proteins with the S65T or S65A, V68L, S72A (GFPmut2) chromophore mutations. Substitution of the CTG codon, which specifies serine instead of leucine in C. albicans, by TTG was absolutely necessary for GFP expression. Although GFP mRNA levels in cells containing a GFP gene with the CTG codon were comparable to those of transformants containing GFP with the TTG substitution, only the latter produced GFP protein, as detected by Western blotting, suggesting that the frequent failure to express heterologous genes in C. albicans is principally due to the noncanonical codon usage. Transformants expressing the modified GFP gene from the promoter of the SAP2 gene, which encodes one of the secreted acid proteinases of C. albicans, showed fluorescence only under conditions which promote proteinase expression, thereby demonstrating the utility of stable, chromosomally integrated GFP reporter genes for the study of gene activation in C. albicans.

[Are there pathogenicity factors in fungi?]. / Gibt es Pathogenitätsfaktoren bei Pilzen?

Since many years so-called pathogenicity or virulence factors of pathogenic bacteria have been studied intensively. Whereas these factors are produced especially by obligate but also facultative pathogenic bacteria, their presence and function in pathogenic fungi is still a matter of debate. In this minireview we summarize some data about possible pathogenicity factors of Cryptococcus neoformans, Aspergillus fumigatus and Candida albicans. The production of adhesins, proteases and capsules contributes to the virulence of these fungi. As fungal infections usually affect immunocompromised patients, the immune status of the host plays a decisive role in addition to the virulence factors produced by fungi.

Targeted gene disruption in Candida albicans wild-type strains: the role of the MDR1 gene in fluconazole resistance of clinical Candida albicans isolates.

Resistance of the pathogenic yeast Candida albicans to the antifungal agent fluconazole is often caused by active drug efflux out of the cells. In clinical C. albicans strains, fluconazole resistance frequently correlates with constitutive activation of the MDR1 gene, encoding a membrane transport protein of the major facilitator superfamily that is not expressed detectably in fluconazole-susceptible isolates. However, the molecular changes causing MDR1 activation have not yet been elucidated, and direct proof for MDR1 expression being the cause of drug resistance in clinical C. albicans strains is lacking as a result of difficulties in the genetic manipulation of C. albicans wild-type strains. We have developed a new strategy for sequential gene disruption in C. albicans wild-type strains that is based on the repeated use of a dominant selection marker conferring resistance against mycophenolic acid upon transformants and its subsequent excision from the genome by FLP-mediated, site-specific recombination (MPAR-flipping). This mutagenesis strategy was used to generate homozygous mdr1/mdr1 mutants from two fluconazole-resistant clinical C. albicans isolates in which drug resistance correlated with stable, constitutive MDR1 activation. In both cases, disruption of the MDR1 gene resulted in enhanced susceptibility of the mutants against fluconazole, providing the first direct genetic proof that MDR1 mediates fluconazole resistance in clinical C. albicans strains. The new gene disruption strategy allows the generation of specific knock-out mutations in any C. albicans wild-type strain and therefore opens completely novel approaches for studying this most important human pathogenic fungus at the molecular level.

Molecular aspects of fluconazole resistance development in Candida albicans.

Serial Candida albicans isolates from recurrent episodes of oropharyngeal candidosis (OPC) in four AIDS patients which became fluconazole-resistant during therapy were analysed by molecular methods. The CARE-2 fingerprint patterns of the isolates demonstrated that in all four patients fluconazole resistance developed in a previously more susceptible strain. In two cases resistance correlated with enhanced expression of genes encoding multiple drug resistance proteins that mediate active drug efflux. Enhanced mRNA levels of the CDR1/CDR2 genes encoding ABC transporters were observed in fluconazole-resistant isolates from one patient compared with the corresponding susceptible isolates. The fluconazole-resistant isolates from another patient exhibited high mRNA levels of the MDR1 gene encoding a membrane transport protein of the major facilitator superfamily that was not detectably expressed in any of the fluconazole-susceptible isolates. These results demonstrate that in AIDS patients with recurrent OPC the development of fluconazole resistance is usually caused by molecular changes in a previously susceptible C. albicans strain from the same patient.

Sequential gene disruption in Candida albicans by FLP-mediated site-specific recombination.

The genetic manipulation of the human fungal pathogen Candida albicans is difficult because of its diploid genome, the lack of a known sexual phase and its unusual codon usage. We devised a new method for sequential gene disruption in C. albicans that is based on the repeated use of the URA3 marker for selection of transformants and its subsequent deletion by FLP-mediated, site-specific recombination. A cassette was constructed that, in addition to the URA3 selection marker, contained an inducible SAP2P-FLP fusion and was flanked by direct repeats of the minimal FLP recognition site (FRT). This URA3 flipper cassette was used to generate homozygous C. albicans mutants disrupted for both alleles of either the CDR4 gene, encoding an ABC transporter, or the MDR1 gene, encoding a membrane transport protein of the major facilitator superfamily. After insertion of the URA3 flipper into the first copy of the target gene, the whole cassette could be efficiently excised by induced FLP-mediated recombination, leaving one FRT site in the disrupted allele of the target gene. The URA3 flipper was then used for another round of mutagenesis to disrupt the second allele. Deletion of the cassette from primary and secondary transformants occurred exclusively by intrachromosomal recombination of the FRT sites flanking the URA3 flipper, whereas interchromosomal recombination between FRT sites on the homologous chromosomes was never observed. This new gene disruption strategy facilitates the generation of specific, homozygous C. albicans mutants as it eliminates the need for a negative selection scheme for marker deletion and minimizes the risk of mitotic recombination in sequential disruption experiments.

Activation of the multiple drug resistance gene MDR1 in fluconazole-resistant, clinical Candida albicans strains is caused by mutations in a trans-regulatory factor.

Resistance of Candida albicans against the widely used antifungal agent fluconazole is often due to active drug efflux from the cells. In many fluconazole-resistant C. albicans isolates the reduced intracellular drug accumulation correlates with constitutive strong expression of the MDR1 gene, encoding a membrane transport protein of the major facilitator superfamily that is not detectably expressed in vitro in fluconazole-susceptible isolates. To elucidate the molecular changes responsible for MDR1 activation, two pairs of matched fluconazole-susceptible and resistant isolates in which drug resistance coincided with stable MDR1 activation were analyzed. Sequence analysis of the MDR1 regulatory region did not reveal any promoter mutations in the resistant isolates that might account for the altered expression of the gene. To test for a possible involvement of trans-regulatory factors, a GFP reporter gene was placed under the control of the MDR1 promoter from the fluconazole-susceptible C. albicans strain CAI4, which does not express the MDR1 gene in vitro. This MDR1P-GFP fusion was integrated into the genome of the clinical C. albicans isolates with the help of the dominant selection marker MPA(R) developed for the transformation of C. albicans wild-type strains. Integration was targeted to an ectopic locus such that no recombination between the heterologous and resident MDR1 promoters occurred. The transformants of the two resistant isolates exhibited a fluorescent phenotype, whereas transformants of the corresponding susceptible isolates did not express the GFP gene. These results demonstrate that the MDR1 promoter was activated by a trans-regulatory factor that was mutated in fluconazole-resistant isolates, resulting in deregulated, constitutive MDR1 expression.

Standardized molecular typing of Candida albicans strains.

A method is presented for the standardization of Candida albicans DNA fingerprinting, which is based on Southern hybridization of EcoRI-digested chromosomal DNA with the moderately repetitive DNA element CARE-2 and the subsequent rehybridization of the blots with a molecular size marker also included in each DNA sample. This method resulted in extremely precise alignment of all strain-specific CARE-2 hybridization patterns, even when analysed on different gels, and will enhance the accuracy of genetic relationship determinations in epidemiological studies including large numbers of strains.

Adhesin regulatory genes within large, unstable DNA regions of pathogenic Escherichia coli: cross-talk between different adhesin gene clusters.

The uropathogenic Escherichia coli strain 536 possesses two large, unstable DNA regions on its chromosome, which were termed pathogenicity islands (pais). Deletions of pais, which occur with relatively high frequency in vitro and in vivo, lead to avirulent mutants. The genetic determinants for production of haemolysin (Hly) and P-related fimbriae (Prf) are located in one of these islands. Deletion of this pathogenicity island (paill) not only removes the hly- and prf-specific genes, but also represses S fimbriae (Sfa), although the sfa genes of this virulence factor are not located on paill. We have identified two regulatory genes, prfB and prfl, of the prf gene cluster that are homologous to the sfa regulatory genes sfaB and sfaC, respectively. Mutations in sfaB and sfaC that inhibit transcription of the major fimbrial subunit gene sfaA were complemented by the homologous prf genes, suggesting communication between the two fimbrial gene clusters in the wild-type strain. Chromosomal mutagenesis of the two prf regulators in strain 536 repressed transcription of sfaA, detected by Northern hybridization and a chromosomal sfaA-lacZ fusion. In addition, haemagglutination assays measured a lower level of S fimbriae in these mutants. Expression of the cloned prf regulators in trans reversed the effect of the mutations; furthermore, constitutive expression of prfB or prfl could also over-come the repression of S fimbriae in a strain that had lost the pathogenicity islands. Virulence assays in mice established that the prf mutants were less virulent than the wild-type strain. The results demonstrate that cross-regulation of two unlinked virulence gene clusters together with the co-ordinate loss of large DNA regions significantly influences the virulence of an extraintestinal E. coli wild-type isolate.

Functional analysis of the sialic acid-binding adhesin SfaS of pathogenic Escherichia coli by site-specific mutagenesis.

The gene coding for the sialic acid-specific adhesin SfaS produced by the S fimbrial adhesin (sfa) determinant of Escherichia coli has been modified by oligonucleotide-directed, site-specific mutagenesis. Lysine 116, arginine 118, and lysine 122 were replaced by threonine, serine, and threonine, respectively. The mutagenized gene clusters were able to produce S fimbrial adhesin complexes consisting of the S-specific subunit proteins including the adhesin SfaS. The mutant clones were further characterized by hemagglutination and by enzyme-linked immunoassay tests with antifimbria- and anti-adhesin-specific monoclonal antibodies, one of which is able to block S-specific binding (Moch et al., Proc. Natl. Acad. Sci. USA 84:3462-3466, 1987). The lysine-122 mutant clone was indistinguishable from the wild-type clone in these assays. Replacement of lysine 116 and arginine 118, however, abolished hemagglutination and resulted in clones which showed a weak (lysine 116) or a negative (arginine 118) reaction with the antiadhesin-specific antibody A1. We therefore suggest that lysine 116 and arginine 118 have an influence on binding of SfaS to the sialic acid residue of the receptor molecule. Substitution of arginine 118 by serine also had a negative effect on the amount of SfaS adhesin proteins isolated from the S fimbrial adhesin complex.

Development of simultaneous resistance to fluconazole in Candida albicans and Candida dubliniensis in a patient with AIDS.

In this report, we describe a patient with recurrent episodes of oral candidosis who finally suffered from fluconazole-refractory oral and oesophageal candidosis. The patient was monitored for 4 years until his death from AIDS. During the observation period, persistent colonization with both Candida albicans and Candida dubliniensis was observed. From the appearance of the first episode of oral candidosis, the patient was treated with fluconazole for 18 months. The infection became unresponsive to fluconazole 400 mg/day. In vitro susceptibility testing revealed the development of resistance to fluconazole in C. albicans and C. dubliniensis. Molecular typing confirmed the persistence of the same C. albicans and C. dubliniensis strains which developed resistance after up to 3 years of asymptomatic colonization. This observation demonstrates that Candida spp. other than C. albicans may develop resistance to fluconazole in a patient who is repeatedly exposed to the drug.