Yun C. Chang

Complementation of a capsule-deficient mutation of Cryptococcus neoformans restores its virulence.

Capsule formation plays a significant role in the pathogenicity of Cryptococcus neoformans. To study the molecular basis of capsule synthesis, the capsule-deficient phenotype of a mutant strain was complemented by transformation. A plasmid rescued from the resulting Cap+ transformant complemented a cap59 mutation which was mapped previously by classical recombination analysis. Gene deletion by homologous integration resulted in an acapsular phenotype, indicating that we have identified the CAP59 gene. The CAP59 gene was assigned to chromosome I by Southern blot analysis of contour-clamped homogeneous electric field gel electrophoresis-resolved chromosomes of C. neoformans var. neoformans. Sequence comparison of genomic and cDNA clones indicated the presence of six introns. CAP59 encoded a 1.9-kb transcript and a deduced protein of 458 amino acids. Analysis of the nucleotide sequence revealed little similarity to existing sequences in the data bank. When the capsule-deficient phenotype was complemented, the originally avirulent C. neoformans strain became virulent for mice. In addition, the acapsular strain created by gene deletion of CAP59 lost its virulence. This work demonstrates the molecular basis for capsule-related virulence and that the CAP59 gene is required for capsule formation.

Cryptococcus neoformans overcomes stress of azole drugs by formation of disomy in specific multiple chromosomes.

Cryptococcus neoformans is a haploid environmental organism and the major cause of fungal meningoencephalitis in AIDS patients. Fluconazole (FLC), a triazole, is widely used for the maintenance therapy of cryptococcosis. Heteroresistance to FLC, an adaptive mode of azole resistance, was associated with FLC therapy failure cases but the mechanism underlying the resistance was unknown. We used comparative genome hybridization and quantitative real-time PCR in order to show that C. neoformans adapts to high concentrations of FLC by duplication of multiple chromosomes. Formation of disomic chromosomes in response to FLC stress was observed in both serotype A and D strains. Strains that adapted to FLC concentrations higher than their minimal inhibitory concentration (MIC) contained disomies of chromosome 1 and stepwise exposure to even higher drug concentrations induced additional duplications of several other specific chromosomes. The number of disomic chromosomes in each resistant strain directly correlated with the concentration of FLC tolerated by each strain. Upon removal of the drug pressure, strains that had adapted to high concentrations of FLC returned to their original level of susceptibility by initially losing the extra copy of chromosome 1 followed by loss of the extra copies of the remaining disomic chromosomes. The duplication of chromosome 1 was closely associated with two of its resident genes: ERG11, the target of FLC and AFR1, the major transporter of azoles in C. neoformans. This adaptive mechanism in C. neoformans may play an important role in FLC therapy failure of cryptococcosis leading to relapse during azole maintenance therapy.

Cryptococcal Yeast Cells Invade the Central Nervous System via Transcellular Penetration of the Blood-Brain Barrier

ABSTRACT Cryptococcal meningoencephalitis develops as a result of hematogenous dissemination of inhaled Cryptococcus neoformans from the lung to the brain. The mechanism(s) by which C. neoformans crosses the blood-brain barrier (BBB) is a key unresolved issue in cryptococcosis. We used both an in vivo mouse model and an in vitro model of the human BBB to investigate the cryptococcal association with and traversal of the BBB. Exposure of human brain microvascular endothelial cells (HBMEC) to C. neoformans triggered the formation of microvillus-like membrane protrusions within 15 to 30 min. Yeast cells of C. neoformans adhered to and were internalized by the HBMEC, and they crossed the HBMEC monolayers via a transcellular pathway without affecting the monolayer integrity. The histopathology of mouse brains obtained after intravenous injection of C. neoformans showed that the yeast cells either were associated with endothelial cells or escaped from the brain capillary vessels into the neuropil by 3 h. C. neoformans was found in the brain parenchyma away from the vessels by 22 h. Association of C. neoformans with the choroid plexus, however, was not detected during up to 10 days of observation. Our findings indicate that C. neoformans cells invade the central nervous system by transcellular crossing of the endothelium of the BBB.

Gliotoxin Is a Virulence Factor of Aspergillus fumigatus: gliP Deletion Attenuates Virulence in Mice Immunosuppressed with Hydrocortisone

ABSTRACT Gliotoxin is an immunosuppressive mycotoxin long suspected to be a potential virulence factor of Aspergillus fumigatus. Recent studies using mutants lacking gliotoxin production, however, suggested that the mycotoxin is not important for pathogenesis of A. fumigatus in neutropenic mice resulting from treatment with cyclophosphomide and hydrocortisone. In this study, we report on the pathobiological role of gliotoxin in two different mouse strains, 129/Sv and BALB/c, that were immunosuppressed by hydrocortisone alone to avoid neutropenia. These strains of mice were infected using the isogenic set of a wild type strain (B-5233) and its mutant strain (gliPΔ) and the the glip reconstituted strain (gliPR). The gliP gene encodes a nonribosomal peptide synthase that catalyzes the first step in gliotoxin biosynthesis. The gliPΔ strain was significantly less virulent than strain B-5233 or gliPR in both mouse models. In vitro assays with culture filtrates (CFs) of B-5233, gliPΔ, and gliPR strains showed the following: (i) deletion of gliP abrogated gliotoxin production, as determined by high-performance liquid chromatography analysis; (ii) unlike the CFs from strains B-5233 and gliPR, gliPΔ CFs failed to induce proapoptotic processes in EL4 thymoma cells, as tested by Bak conformational change, mitochondrial-membrane potential disruption, superoxide production, caspase 3 activation, and phosphatidylserine translocation. Furthermore, superoxide production in human neutrophils was strongly inhibited by CFs from strain B-5233 and the gliPR strain, but not the gliPΔ strain. Our study confirms that gliotoxin is an important virulence determinant of A. fumigatus and that the type of immunosuppression regimen used is important to reveal the pathogenic potential of gliotoxin.

Human Polymorphonuclear Leukocytes Inhibit Aspergillus fumigatus Conidial Growth by Lactoferrin-Mediated Iron Depletion

Aspergillus fumigatus, a common mold, rarely infects humans, except during prolonged neutropenia or in cases of chronic granulomatous disease (CGD), a primary immunodeficiency caused by mutations in the NADPH oxidase that normally produces fungicidal reactive oxygen species. Filamentous hyphae of Aspergillus are killed by normal, but not CGD polymorphonuclear leukocytes (PMN); however, the few studies on PMN-mediated host defenses against infectious conidia (spores) of this organism have yielded conflicting results, some showing that PMN do not inhibit conidial growth, with others showing that they do, most likely using reactive oxygen species. Given that CGD patients are exposed daily to hundreds of viable A. fumigatus conidia, yet considerable numbers of them survive years without infection, we reasoned that PMN use ROS-independent mechanisms to combat Aspergillus. We show that human PMN from both normal controls and CGD patients are equipotent at arresting the growth of Aspergillus conidia in vitro, indicating the presence of a reactive oxygen species-independent factor(s). Cell-free supernatants of degranulated normal and CGD neutrophils both suppressed fungal growth and were found to be rich in lactoferrin, an abundant PMN secondary granule protein. Purified iron-poor lactoferrin at concentrations occurring in PMN supernatants (and reported in human mucosal secretions in vivo) decreased fungal growth, whereas saturation of lactoferrin or PMN supernatants with iron, or testing in the presence of excess iron in the form of ferritin, completely abolished activity against conidia. These results demonstrate that PMN lactoferrin sequestration of iron is important for host defense against Aspergillus.

Sre1p, a regulator of oxygen sensing and sterol homeostasis, is required for virulence in Cryptococcus neoformans

Cryptococcus neoformans is an environmental pathogen requiring atmospheric levels of oxygen for optimal growth. Upon inhalation, C. neoformans disseminates to the brain and causes meningoencephalitis, but the mechanisms by which the pathogen adapts to the low‐oxygen environment in the brain have not been investigated. We found that SRE1, a homologue of the mammalian sterol regulatory element‐binding protein (SREBP), functions in an oxygen‐sensing pathway. Low oxygen decreased sterol synthesis in C. neoformans and triggered activation of membrane‐bound Sre1p by the cleavage‐activating protein, Scp1p. Microarray and Northern blot analysis demonstrated that under low oxygen, Sre1p activates genes required for ergosterol biosynthesis and iron uptake. Consistent with these regulatory functions, sre1Δ cells were hypersensitive to azole drugs and failed to grow under iron‐limiting conditions. Importantly, sre1Δ cells failed to produce fulminating brain infection in mice. Our in vitro data support a model in which Sre1p is activated under low oxygen leading to the upregulation of genes required for sterol biosynthesis and growth in a nutrient‐limiting environment. Animal studies confirm the importance of SRE1 for C. neoformans to adapt to the host environment and to cause fatal meningoencephalitis, thereby identifying the SREBP pathway as a therapeutic target for cryptococcosis.

Agrobacterium tumefaciens-Mediated Transformation of Aspergillus fumigatus: an Efficient Tool for Insertional Mutagenesis and Targeted Gene Disruption

ABSTRACT Agrobacterium tumefaciens was used to transform Aspergillus fumigatus by either random or site-directed integration of transforming DNA (T-DNA). Random mutagenesis via Agrobacterium tumefaciens-mediated transformation (ATMT) was accomplished with T-DNA containing a hygromycin resistance cassette. Cocultivation of A. fumigatus conidia and Agrobacterium (1:10 ratio) for 48 h at 24°C resulted in high frequencies of transformation (>100 transformants/107 conidia). The majority of transformants harbored a randomly integrated single copy of T-DNA and were mitotically stable. We chose alb1, a polyketide synthase gene, as the target gene for homologous integration because of the clear phenotype difference between the white colonies of Δalb1 mutant strains and the bluish-green colonies of wild-type strains. ATMT with a T-DNA-containing alb1 disruption construct resulted in 66% albino transformants. Southern analysis revealed that 19 of the 20 randomly chosen albino transformants (95%) were disrupted by homologous recombination. These results suggest that ATMT is an efficient tool for transformation, random insertional mutagenesis, and gene disruption in A. fumigatus.

ASPERGILLUS FUMIGATUS ARP1 MODULATES CONIDIAL PIGMENTATION AND COMPLEMENT DEPOSITION

Aspergillus fumigatus is an important pathogen causing invasive pulmonary aspergillosis in immunocompromised patients. The fungus propagates by conidia, which are the infectious structures inhaled by the human host. Opsonophagocytosis is thought to contribute to clearance of the inhaled conidia, a process that is facilitated by complement deposition on conidial surfaces. We now show that conidial colour mutants exhibit significant increases in C3 binding capacity compared with wild type. A reddish‐pink mutation that led to enhanced C3 binding was complemented by a cosmid clone. A 3.3 kb DNA fragment from the subsequently rescued cosmid was sufficient to restore the bluish‐green conidial pigment. The bluish‐green transformant exhibited a level of C3 binding similar to that of the parental strain. A gene, designated arp1, was responsible for the complementation. Comparison of the genomic and cDNA sequences of arp1 revealed that it has two introns and encodes a putative protein of 168 amino acids. Arp1 is very similar to scytalone dehydratase, an enzyme involved in 1,8‐dihydroxynaphthalene‐melanin synthesis in Colletotrichum lagenarium and Magnaporthe grisea. Northern hybridization analysis revealed that arp1 is developmentally regulated, being expressed during conidiation. Disruption of arp1 resulted in reddish‐pink conidia and increased C3 binding. Our studies suggest that arp1 modulates the bluish‐green pigmentation of conidia as well as complement deposition.

Cryptococcus neoformans CAP59 (or Cap59p) Is Involved in the Extracellular Trafficking of Capsular Glucuronoxylomannan

ABSTRACT Several genes are essential for Cryptococcus neoformans capsule synthesis, but their functions are unknown. We examined the localization of glucuronoxylomannan (GXM) in strain B-3501 and in cap59 mutants B-4131 and C536. Wild-type strain B-3501 showed a visible capsule by India ink staining and immunofluorescence with anticapsular monoclonal antibodies (MAbs) 12A1 and 18B7. B-4131, a mutant containing a missense mutation in CAP59, showed no capsule by India ink staining but revealed the presence of capsular polysaccharide on the cell surface by immunofluorescence. The cap59 gene deletion mutant (C536), however, did not show a capsule by either India ink staining or immunofluorescence. Analysis of cell lysates for GXM by enzyme-linked immunosorbent assay revealed GXM in C536 samples. Furthermore, the epitopes recognized by MAbs 12A1, 2D10, 13F1, and 18B7 were each detected in the cytoplasm of all strains by immunogold electron microscopy, although there were differences in location consistent with differences in epitope synthesis and/or transport. In addition, the cells of B-3501 and B-4131, but not those of the cap59 deletant, assimilated raffinose or urea. Hence, the missense mutation of CAP59 in B-4131 partially hampered the trafficking of GXM but allowed the secretion of enzymes involved in hydrolysis of raffinose or urea. Furthermore, the cell diameter and volume for strain C536 are higher than those for strain B-3501 or B-4131 and may suggest the accumulation of cellular material in the cytoplasm. Our results suggest that CAP59 is involved in capsule synthesis by participating in the process of GXM (polysaccharide) export.

Heteroresistance to Fluconazole in Cryptococcus neoformans Is Intrinsic and Associated with Virulence

ABSTRACT In 1999, heteroresistance to triazoles was reported in Cryptococcus neoformans strains isolated from an azole therapy failure case of cryptococcosis in an AIDS patient and in a diagnostic strain from a non-AIDS patient. In this study, we analyzed 130 strains of C. neoformans isolated from clinical and environmental sources before 1979, prior to the advent of triazoles, and 16 fluconazole (FLC)-resistant strains isolated from AIDS patients undergoing FLC maintenance therapy during 1990 to 2000. All strains isolated prior to 1979 manifested heteroresistance (subset of a population that grows in the presence of FLC) at concentrations between 4 and 64 μg/ml, and all 16 FLC-resistant AIDS isolates manifested heteroresistance at concentrations between 16 and 128 μg/ml. Upon exposure to stepwise increases in the concentration of FLC, subpopulations that could grow at higher concentrations emerged. Repeated transfer on drug-free media caused the highly resistant subpopulations to revert to the original level of heteroresistance. The reversion pattern fell into four categories based on the number of transfers required. The strains heteroresistant at ≥32 μg/ml were significantly more resistant to other xenobiotics and were also more virulent in mice than were those heteroresistant at ≤8 μg/ml. During FLC treatment of mice infected by strains with low levels of heteroresistance, subpopulations exhibiting higher levels of heteroresistance emerged after a certain period of time. The ABC transporter AFR1, known to efflux FLC, was unrelated to the heteroresistance mechanism. Our study showed that heteroresistance to azole is universal and suggests that heteroresistance contributes to relapse of cryptococcosis during azole maintenance therapy.