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Genetic engineering is an important tool in helping us to define the molecular basis of pathogenicity and is also useful in helping us to identify new therapeutic targets in pathogenic fungi. Molecular genetic manipulation of micro-organisms requires the development of plasmid-mediated transformation systems that include: (i) infusion of exogenous DNA into recipient cells, (ii) expression of genes present on the incoming DNA, and (iii) stable maintenance and replication of the inserted DNA leading to expression of the desired phenotypic trait. Transformation systems have been developed for only a handful of fungi that are pathogenic to humans including several species of Candida, Cryptococcus neoformans, Histoplasma capsulatum, Blastomyces derrmatitidis, Aspergillus fumigatus, Wangiella dermatitidis (Exophiala dermatitidis) and Coccidioides immitis. Except for Candida species and A. fumigatus, where passage of exogenous DNA into recipient cells has been achieved readily using methods developed for transformation of Saccharomyces cerevisiae and Aspergillus nidulans, respectively, development of transformation systems in other pathogenic fungi has been delayed considerably and has only been possible recently with the introduction of electroporation and biolistic methods. Conventional spheroplasting methods or cell wall permeabilization methods using lithium acetate have not been successful for transformation of C. neoformans and work with only low efficiency in H. capsulatum. The fate of incoming DNA varies greatly in these pathogenic species regardless of their phylogenetic relationships. Understanding the fate of incoming DNA is critical for the construction of transforming vectors and the molecular manipulation of the organisms. In this symposium, recent advances in molecular genetic systems including transformation systems, the fate of incoming DNA and strategies for targeted integration are discussed in relation to four pathogenic fungi.

Recent phylogenetic studies based on ribosomal RNA sequences have confirmed that the organisms traditionally treated as fungi include those that have evolved from several different lines (multiphyletic organisms), as has been suspected. Even organisms causing disease in humans represent at least two evolutional lines. Pythium insidiosum and Prototheca species are both believed to have evolved from one line, while the rest of the pathogens have evolved from another line. P. insidiosum is more closely related to red algae and diatoms than to fungi. Prototheca species, as has been previously postulated, are closer to blue-green algae and plants than to fungi. Pythiosis and protothecosis, however, will still be dealt with by medical mycologists because of the morphological and in vivo staining characteristics of the causative organisms. Molecular genetic studies have revealed that Pneumocystis carinii can best be categorized as a fungus, although questions regarding its fungal status may remain unanswered until additional information becomes available on its life cycle, nuclear division, cell-wall chemistry, nutritional uptake pattern, and lysine biosynthetic pathway as well as the ultrastructural characteristics of its cellular components such as the Golgi complex. The phylogeny of the agents of lobomycosis and rhinosporidiosis, although they are treated as fungi, remains unknown. Although there is no in vitro culture system for Loboa loboi and Rhinosporidium seeberi at present, a molecular approach would allow us to reveal their phylogenetic relationship, and we can hope that such attempts are forthcoming.

Cunninghamella elegans was cultured from autopsy materials of the infected lung of a patient with chronic myelogenous leukemia. The histologic sections revealed extensive growth of nonseptate, broad hyphae in and about the large and small arteries and veins, occluding the vessels. This case is believed to be the second in which human infection by C. elegans has been documented.