K.J. Kwon-Chung

Taxonomic studies on Filobasidiella species and their anamorphs

The taxonomy of Filobasidiella neoformans Kwon-Chung and F. bacillispora Kwon-Chung and their anamorphs were reinvestigated. Although the cross between the type cultures of the two species failed to produce viable basidio-spores, another pair of isolates did yield viable basidiospores. The segregation of phenotypic markers among the tetrads isolated from this interspecific cross proved that meiosis had occurred. On the basis of other previously known differences and the present genetic study, the two species are now considered to be two varieties of the species, F. neoformans. The anamorph of F. neoformans var. neoformans grew well at 37°C in vitro and produced fatal infection in mice while that of F. neoformans var. bacillispora grew poorly at 37°C and failed to produce fatal infection in mice. Cryptococcus bacillisporus Kwon-Chung et Bennett is regarded as a synonym of C. neoformans var. gattii Vanbreuseghem et Takashio.

Concentrations of airborne Aspergillus compared to the incidence of invasive aspergillosis: lack of correlation

Air sampling of the rooms and corridors of the oncology wards of the hospital was carried out over a 54-week period to assess the concentration of viable Aspergillus conidia. A. fumigatus and A. flavus were recovered at a mean of 1.83 cfu m-3 air sampled. Individual samplings yielded concentrations of up to 11.6 cfu m-3. Other Aspergillus spp. were recovered at a mean of 2.38 cfu m-3 (maximum 32.6 cfu m-3). Concentration was not correlated with season or hospital ward. Review of autopsy results showed an average of 6.6 cases of aspergillosis annually over a 22-year period. No seasonal variation in case incidence was found. Six cases of invasive aspergillosis were diagnosed on the three cancer wards during the air-sampling period, but no association was seen linking these cases with changes in recovery of airborne Aspergillus. A seasonal pattern was not observed in the overall incidence of aspergillosis cases nor concentrations of airborne conidia.

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.

Nutritional physiology and taxonomy of human- pathogenic Cladosporium-Xylohypha species

Physiological profiles of type, authentic and some additional isolates of Cladosporium-Xylohypha species of purported herpotrichiellaceous relationship are established. This group comprises melanized catenate hyphomycetes which are prevalently found on the human host. The species are excluded from the genus Cladosporium and are classified in the genus Cladophialophora. Taeniolella boppii is also transferred to this genus. Cladosporium bantianum (= Xylohypha emmonsii) and C. trichoides are considered conspecific and are now referred to as Cladophialophora bantiana. Meso-erythritol, L-arabinitol, ethanol and growth at 40 degrees C are found to be the most useful criteria for species distinction. The species Cladosporium carrionii is found to be heterogeneous. The anamorph of the saprophytic ascomycete Capronia pilosella is morphologically similar to an authentic strain of Cladosporium carrionii, but physiologically distinct. A diagnostic key for the recognized Cladophialophora species and to morphologically similar taxa is provided.

Biochemical differences between serotypes of Cryptococcus neoformans.

Prior studies have shown that C. neoformans isolates belonging to serotypes B and C differed from serotype A and D isolates in respect to the morphology of the perfect state, geographic distribution within the U.S.A. and frequency of isolation from avian droppings. The current study found that uptake of radiolabeled l-malic acid was approximately tenfold greater in serotype B and C than in A and D isolates. Assimilation of l-malic, fumaric and succinic acids also distinguished serotypes B and C from A and D. Greening of Guizotia seed agar occurred with 18 of 32 serotype B and C but in none of 91 serotype A or D isolates. The one property not following the same line of division was relatively slow creatinine assimilation, which distinguished type A alone.

Haploid fruiting in Cryptococcus neoformans is not mating type α-specific

Under appropriate conditions, haploid Cryptococcus neoformans cells can undergo a morphological switch from a budding yeast form to develop hyphae and viable basidiospores, which resemble those produced by mating. This process, known as haploid fruiting, was previously thought to occur only in MATα strains. We identified two new strains of C. neoformans var. neoformans serotype D that are MATa type and are able to haploid fruit. Further, a MATa reference strain, B-3502, also produced hyphae and fruited after prolonged incubation on filament agar. Over-expression of STE12a dramatically enhanced the ability of all MATa strains tested to filament. Segregation analysis of haploid fruiting ability confirmed that haploid fruiting is not MATα-specific. Our results indicate that MATa cells are intrinsically able to haploid fruit and previous observations that they do not were probably biased by the examination of a small number of genetically related isolates that have been maintained in the laboratory for many years.

Changing taxonomic concepts and their impact on nomenclatural stability

Experimental techniques, which are routinely applied in yeast systematics, are currently finding recognition in a growing number of filamentous taxa. Some other biochemical markers have recently been developed in hyphomycete taxonomy. The spectrum of potential criteria now comprises characters of coenzyme-Q systems, secondary metabolites, protein electrophoresis, serology, nuclear (n) DNA/DNA reassociation, mole% G+C of DNA, protein electrophoresis, nutritional physiology and ultrastructural and karyological data. In addition, a wide range of molecular techniques is gaining rapid acceptance in evolutionary, systematic, ecological and epidemiological studies. Depending on the aim of the study, partial DNA sequencing (mainly of SS and LS ribosomal genes and their spacers, but also of other genes), PCR-ribotyping and mitochondrial (mt) DNA restriction analyses are particularly powerful; for the establishment of the taxonomic position of taxa, 5.8S ribosomal (r) rRNA sequencing and Southern blotting with conserved genes are useful. Together with renewed in-depth morphological studies and the elucidation of teleomorph connections and (syn)anamorph life cycles, these techniques provide tools for an improved understanding of the phylogeny and ecological role of the distinguished taxa. Taxa are increasingly being classified along natural lines. The resulting changes in the taxonomic system, and thereby its impact on nomenclature, may be considerable. This does not help in making taxonomy any more popular among medical mycologists. However, the significance to medical mycology of an improved taxonomic system should not be underestimated. Taxonomy is now becoming enabled to describe fungi as living entities in their natural ecological niche and thereby provides a better insight into their behaviour on the human body. We are now beginning to realize that several fungi have a narrow ecological amplitude and hence their role as aetiological agents of human disease may be well defined and characteristic for the species. The clinical significance of found taxa can thus be evaluated with more precision. Below some examples of fungal groups, in which the taxonomic concepts are subject to rapid change, are presented.

Taxonomic clarification of Cladosporium trichoides Emmons and its subsequent synonyms

Cladosporium trichoides Emmons has been treated by some mycologists as a synonym of Cladosporium bantianum (Sacc.) Borelli and has been transferred to the genus Xylohypha (Fr.) Mason. In the present study, a herbarium specimen of C. bantianum (Torula bantiana Sacc.) Borelli, prepared by Saccardo, was compared with a herbarium specimen and a living type culture of C. triochoides by light and scanning electron microscopy (SEM) and was found to be dissimilar. Herbarium specimens and living cultures of Xylohypha nigrescens, the type species of the genus Xylohypha, were also compared with those of C. trichoides and other pathogenic Cladosporium species. Fundamental differences were found between X. nigrescens and Cladosporium species, in colony morphology, manner of sporulation and conidial morphology. All Cladosporium isolates produced olive-black colonies regardless of environmental conditions, bore brown pigment on the walls of the vegetative hyphae as well as on the walls of the fruiting structures and produced branched chains of conidia either from well differentiated or poorly differentiated conidiophores, or directly from the hyphae. By SEM, conidia showed strong to moderately protruded hila, and the basal contour of the conidia was always truncated. On germination, hyphal tubes were produced randomly from the surface of the conidia. In contrast, X. nigrescens produced white colonies with or without brown centres, depending on the culture medium, bore pigment on the conidial walls and on conidiogenous cells but not on the vegetative hyphae and produced infrequently branched conidial chains, usually from intercalary conidiogenous cells which were globose to hat-shaped. Conidial hila were nonprotruding but, instead, were deeply concave and pore-like. The basal contour of the conidia was round and germ tubes were produced only from the pore-like hila. These results indicate that C. triochoides Emmons is different from C. bantianum (Sacc.) Borelli and that the reclassification of C. trichoides into the genus Xylohypha was not warranted.

Production and regeneration of protoplasts from Cryptococcus

Protoplasts were quickly and efficiently produced from both varieties of Cryptococcus neoformans and from C. laurentii by use of the multi-enzyme product Novozym 234. Conditions for regeneration of protoplasts are described. DNA yield from the Novozym-produced protoplasts was superior to that from snail gut enzyme-derived protoplasts.

Unilateral involvement of kidneys in mice infected with Candida albicans

Mice injected with 10(3) cells of a virulent isolate of Candida albicans via the lateral tail vein developed frequent unilateral abnormalities of the right but not the left kidneys. Initially the number of colony forming units in the right and left kidneys were similar but the number of colonies became consistently higher in the right kidneys as the infection progressed. The frequency of unilateral involvement decreased when the inoculum size was increased to 5 X 10(3) cells. These observations indicate that when growth of C. albicans in vivo is monitored over a period of time starting with a low inoculum, it is critical to be consistent in culturing kidneys from the same side.