Genome structure of pathogenic fungi.
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Animals↗
Biomedical subjects
J Morschhäuser
Publications and source records attributed to J Morschhäuser.
Gene regulation and host adaptation mechanisms in Candida albicans.
The yeast Candida albicans is a harmless member of the normal microflora on the mucosal surfaces of most healthy persons, but it can cause severe opportunistic infections in immunosuppressed patients. To become a successful human commensal and pathogen, C. albicans has evolved host adaptation mechanisms on different levels. The regulated expression of virulence and other genes in response to environmental signals allows an optimal adaptation to new host niches during the course of an infection. In addition, C. albicans is able to switch between different cell types in a reversible and apparently random fashion. Phenotypic switching involves the coordinated regulation of phase-specific genes, and the resulting generation of selected, pre-programmed cell types may represent an additional strategy to adapt to certain host environments. Finally, C. albicans produces genetically altered variants at a high rate. This microevolution ensures survival when the pathogen encounters new adverse conditions, as exemplified by the development of stable drug-resistant variants under the selection pressure caused by antimycotic therapy. Thus, rather than the possession of single dominant virulence factors, it is its remarkable versatility that makes C. albicans the most important fungal pathogen of humans.
Adaptation, Physiological↗
MDR1-mediated drug resistance in Candida dubliniensis.
Candida dubliniensis is a recently described opportunistic fungal pathogen that is closely related to Candida albicans. Candida dubliniensis readily develops resistance to the azole antifungal agent fluconazole, both in vitro and in infected patients, and this resistance is usually associated with upregulation of the CdMDR1 gene, encoding a multidrug efflux pump of the major facilitator superfamily. To determine the role of CdMDR1 in drug resistance in C. dubliniensis, we constructed an mdr1 null mutant from the fluconazole-resistant clinical isolate CM2, which overexpressed the CdMDR1 gene. Sequential deletion of both CdMDR1 alleles was performed by the MPA(R)-flipping method, which is based on the repeated use of a dominant mycophenolic acid resistance marker for selection of integrative transformants and its subsequent deletion from the genome by FLP-mediated, site-specific recombination. In comparison with its parental strain, the mdr1 mutant showed decreased resistance to fluconazole but not to the related drug ketoconazole. In addition, we found that CdMDR1 confers resistance to the structurally unrelated drugs 4-nitroquinoline-N-oxide, cerulenin, and brefeldin A, since the enhanced resistance to these compounds of the parent strain CM2 compared with the matched susceptible isolate CM1 was abolished in the mdr1 mutant. In contrast, CdMDR1 inactivation did not cause increased susceptibility to amorolfine, terbinafine, fluphenazine, and benomyl, although overexpression of CdMDR1 in a hypersusceptible Saccharomyces cerevisiae strain had previously been shown to confer resistance to these compounds. The effect of CdMDR1 inactivation was identical to that seen in two similarly constructed C. albicans mdr1 mutants. Therefore, despite species-specific differences in the amino acid sequences of the Mdr1 proteins, overexpression of CaMDR1 and CdMDR1 in clinical C. albicans and C. dubliniensis strains seems to confer the same drug resistance profile in both species.
Antifungal Agents↗
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.
Aspartic Acid Endopeptidases↗
Isogenic strain construction and gene targeting in Candida dubliniensis.
Candida dubliniensis is a recently described opportunistic fungal pathogen that is closely related to Candida albicans but differs from it with respect to epidemiology, certain virulence characteristics, and the ability to develop fluconazole resistance in vitro. A comparison of C. albicans and C. dubliniensis at the molecular level should therefore provide clues about the mechanisms used by these two species to adapt to their human host. In contrast to C. albicans, no auxotrophic C. dubliniensis strains are available for genetic manipulations. Therefore, we constructed homozygous ura3 mutants from a C. dubliniensis wild-type isolate 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 resistance marker that is subsequently deleted again by site-specific, FLP-mediated recombination. The URA3 gene from C. albicans (CaURA3) was then used as a selection marker for targeted integration of a fusion between the C. dubliniensis MDR1 (CdMDR1) promoter and a C. albicans-adapted GFP reporter gene. Uridine-prototrophic transformants were obtained with high frequency, and all transformants of two independent ura3-negative parent strains had correctly integrated the reporter gene fusion into the CdMDR1 locus, demonstrating that the CaURA3 gene can be used for efficient and specific targeting of recombinant DNA into the C. dubliniensis genome. Transformants carrying the reporter gene fusion did not exhibit detectable fluorescence during growth in yeast extract-peptone-dextrose medium in vitro, suggesting that CdMDR1 is not significantly expressed under these conditions. Fluconazole had no effect on MDR1 expression, but the addition of the drug benomyl strongly activated the reporter gene fusion in a dose-dependent fashion, demonstrating that the CdMDR1 gene, which encodes an efflux pump mediating resistance to toxic compounds, is induced by the presence of certain drugs.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Evolution of microbial pathogens.
Various genetic mechanisms including point mutations, genetic rearrangements and lateral gene transfer processes contribute to the evolution of microbes. Long-term processes leading to the development of new species or subspecies are termed macroevolution, and short-term developments, which occur during days or weeks, are considered as microevolution. Both processes, macro- and microevolution need horizontal gene transfer, which is particularly important for the development of pathogenic microorganisms. Plasmids, bacteriophages and so-called pathogenicity islands (PAIs) play a crucial role in the evolution of pathogens. During microevolution, genome variability of pathogenic microbes leads to new phenotypes, which play an important role in the acute development of an infectious disease. Infections due to Staphylococcus epidermidis, Candida albicans and Escherichia coli will be described with special emphasis on processes of microevolution. In contrast, the development of PAIs is a process involved in macroevolution. PAIs are especially important in processes leading to new pathotypes or even species. In this review, particular attention will be given to the fact that the evolution of pathogenic microbes can be considered as a specific example for microbial evolution in general.
Candida albicans↗
Differential activation of a Candida albicans virulence gene family during infection.
The yeast Candida albicans is a harmless commensal in most healthy people, but it causes superficial as well as life-threatening systemic infections in immunocompromised patients. C. albicans can colonize or infect virtually all body sites because of its high adaptability to different host niches, which involves the activation of appropriate sets of genes in response to complex environmental signals. We have used an in vivo expression technology that is based on genetic recombination as a reporter of gene expression to monitor the differential activation of individual members of a gene family encoding secreted aspartic proteinases (Saps), which have been implicated in C. albicans virulence, at various stages of the infection process. Our results demonstrate that SAP expression depends on the type of infection, with different SAP isogenes being activated during systemic disease as compared with mucosal infection. In addition, the activation of individual SAP genes depends on the progress of the infection, some members of the gene family being induced immediately after contact with the host, whereas others are expressed only after dissemination into deep organs. In the latter case, the number of invading organisms determines whether induction of a virulence gene is necessary for successful infection. The in vivo expression technology allows the elucidation of gene expression patterns at different stages of the fungus-host interaction, thereby revealing regulatory adaptation mechanisms that make C. albicans the most successful fungal pathogen of humans and, at the same time, identifying the stage of an infection at which certain virulence genes may play a role.
Animals↗
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.
Aspartic Acid Endopeptidases↗
Transcriptional analysis of the sfa and pap determinants of uropathogenic Escherichia coli strains.
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Bacterial Proteins↗
Expression of virulence genes in Candida albicans.
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Candida albicans↗
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.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
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.
Acquired Immunodeficiency Syndrome↗
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.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Host-induced, stage-specific virulence gene activation in Candida albicans during infection.
An understanding of the complex interactions between pathogenic microbes and their host must include the identification of gene expression patterns during infection. To detect the activation of virulence genes in the opportunistic fungal pathogen Candida albicans in vivo by host signals, we devised a reporter system that is based on FLP-mediated genetic recombination. The FLP gene, encoding the site-specific recombinase FLP, was genetically modified for expression in C. albicans and fused to the promoter of the SAP2 gene that codes for one of the secreted aspartic proteinases, which are putative virulence factors of C. albicans. The SAP2P-FLP fusion was integrated into one of the SAP2 alleles in a strain that contained a deletable marker that conferred resistance to mycophenolic acid and was flanked by direct repeats of the FLP recognition target (FRT). Using this reporter system, a transient gene induction could be monitored at the level of single cells by the mycophenolic acid-sensitive phenotype of the colonies generated from such cells after FLP-mediated marker excision. In two mouse models of disseminated candidiasis, SAP2 expression was not observed in the initial phase of infection, but the SAP2 gene was strongly induced after dissemination into deep organs. In contrast, in a mouse model of oesophageal candidiasis in which dissemination into internal organs did not occur, no SAP2 expression was detected at any time. Our results support a role of the SAP2 gene in the late stages of an infection, after fungal spread into deep tissue. This new in vivo expression technology (IVET) for a human fungal pathogen allows the detection of virulence gene induction at different stages of an infection, and therefore provides clues about the role of these genes in the disease process.
Animals↗
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.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Identification of CARE-2-negative Candida albicans isolates as Candida dubliniensis.
Among 302 clinical yeast isolates originally identified as Candida albicans, we found 16 isolates that did not hybridize with the C. albicans-specific repetitive DNA element CARE-2. These 16 isolates produced abundant chlamydospores, did not grow at 43 degrees C, and exhibited a distinct randomly amplified polymorphic DNA profile. Sequence analysis of part of the 28S rDNA demonstrated that the CARE-2-negative isolates are not an atypical subgroup of C. albicans but belong to the recently described new species C. dubliniensis.
Candida↗
Genotypic relatedness of yeast isolates from women infected with human immunodeficiency virus and their children.
The objective of this study was to compare polymerase chain reaction (PCR) fingerprinting with other molecular typing methods as an epidemiologic tool to investigate the transmission of Candida strains between HIV-positive mothers and their children. Forty-nine yeast strains (including Candida albicans, Candida glabrata, Rhodotorula rubra, Candida tropicalis, Candida famata, Candida dubliniensis, Saccharomyces cerevisiae) from 30 individuals (15 children and 15 HIV-infected mothers or accompanying person) were isolated. Colonization/infection with yeast was observed in 80% of all individuals in the oral cavity, and in 33% from hand cultures, respectively. Thirteen out of 15 children (86%) and 12 out of 15 adults (80%) were colonized/infected with yeasts. Candida dubliniensis strains were found in four HIV-infected women but not in children. The results with an arbitrarily primed (AP)-PCR mediated genotyping assay using phage M13 core sequence were compared with the hybridization patterns using the species-specific DNA probe CARE-2 for the C. albicans isolates. Typing of non-C. albicans strains was done using AP-PCR in comparison with pulsed-field gel electrophoresis (PFGE). Twenty-six C. albicans strains gave two different genotypes by AP-PCR but 16 genotypes by CARE-2 hybridization. The CARE-2 probe appeared to have a higher discriminatory power compared with the primer 'M13'-mediated AP-PCR in typing C. albicans isolates.
Adult↗
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.
AIDS-Related Opportunistic Infections↗