Tn7-Based Genome-Wide Random Insertional Mutagenesis of Candida glabrata

Candida species, primarily Candida albicans and Candida glabrata, are important human pathogens, responsible for 7% of all hospital-acquired blood stream infections (Schaberg et al. 1991). Even with available antifungal therapies, the associated mortality for Candida bloodstream infections is high (up to 30% in cancer patients). We are usingC. glabrata as a model to explore the molecular details of the host–pathogen interaction. In frequency of isolation, C. glabrata is second only to C. albicans and is responsible for 15%–20% of both mucosal (Schuman et al. 1998; Vazquez et al. 1999) and systemic (Pfaller et al. 1999, 2001) candidiasis; in spite of this epidemiological similarity, C. glabrata is phylogenetically distant from C. albicans, more highly related, for example, to Saccharomyces cerevisiae than to C. albicans (Barns et al. 1991). What strategies for host colonization are shared by C. glabrata and C. albicans remain to be determined; no genes essential for virulence have yet been described in C. glabrata. Studies primarily in C. albicans have identified multiple factors important in the pathogenesis of Candida species (for review, see Calderone and Fonzi 2001), including the ability to adhere to host tissue, the ability to grow in hyphal and yeast form (for C. albicans), the capacity to switch between different cellular phenotypes, and the ability to acquire iron in vivo. Like C. albicans, C. glabrata is able to adhere specifically to host tissue, recognizing host carbohydrate (Cormack et al. 1999). On the other hand, C. glabrata does not make hyphae, a feature of prime importance in the pathogenesis of C. albicans; rather, it grows solely in the yeast form, making pseudohyphae under conditions of nitrogen starvation (Csank and Haynes 2000). Because C. glabrata is haploid, the tools of classical genetics can be applied, and mutants defective in various aspects of virulence can be isolated and characterized. An efficient genetic analysis depends on a method of random insertional mutagenesis, and we considered various available options. In other species, numerous approaches have been taken, including the use of bacterial transposons such as Tn3 (Seifert et al. 1986; Ross-Macdonald et al. 1999), Tn7 (Biery et al. 2000), and the Drosophila melanogaster transposon Mariner (Gueiros-Filho and Beverley 1997). Tn3, in particular, has been used to advantage in mutagenesis of S. cerevisiae (Ross-Macdonald et al. 1999). In that efficient and highly random method, fragments of the S. cerevisiae genome are first mutagenized in Escherichia coli; those insertion mutations are then introduced into the S. cerevisiae genome by homologous recombination. For C. glabrata, the options were somewhat limited, in part because there are no known natural transposons in C. glabrata (like the Ty elements of S. cerevisiae). In an earlier study, we exploited nonhomologous recombination in C. glabrata to make insertion mutants and to analyze these for effects on adherence to epithelial cells (Cormack and Falkow 1999; Cormack et al. 1999). We found that the insertions were distributed more or less randomly in many different genes; however, a close analysis of the sites of insertion for 50 mutants showed that the majority (48/50) were in noncoding regions of the genome. If one were able to analyze a very large number of mutants, this bias might not be an important factor. However, for screens in which only modest numbers of mutants (20,000–30,000) are analyzed, the bias against insertions in coding regions would result in a mutational sampling of only a fraction of the genome. As an alternative to the problematic nonhomologous-recombination-based method, we describe in this paper a novel mutagenesis approach similar in principle to the Tn3 method described above (Ross-Macdonald et al. 1999), but which exploits recent studies of in vitro transposition by the bacterial transposon Tn7. This method is of some general interest because the generation of mutants requires only two steps: in vitro mutagenesis by Tn7 followed by homologous recombination into the target genome (here C. glabrata). In theory, therefore, our method can easily be applied to any organism with efficient homologous recombination. We describe modifications to Tn7 to allow its use in C. glabrataand to facilitate the recovery of DNA flanking insertion sites for mutants of interest. We demonstrate that this method can be used in the efficient generation of thousands of randomly distributed insertion mutants, possessing an array of phenotypes.