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The basidiomycete Cryptococcus neoformans causes life-threatening infections in immunocompromised patients, and available chemotherapeutic agents are potentially toxic or have limited efficacy. In vitro, C. neoformans is very sensitive to selected benzimidazole compounds (e.g. albendazole), which act by disrupting microtubules through binding to the beta-tubulin subunit. To understand the basis for this benzimidazole sensitivity, we have characterized C. neoformans beta-tubulin genes and their expression. Analysis of PCR amplification products, genomic and cDNA clones and Southern blots identified two beta-tubulin genes. TUB1 contains seven introns, including one that splits the start codon, and encodes a 447 amino acid protein with &gt; 80% identity to most other beta-tubulins. A partial sequence of TUB2 revealed a higher density of introns and a considerably more divergent beta-tubulin. The relative expression of TUB1 to TUB2 determined by reverse-transcription PCR was about 3:1, consistent with a more limited role for the TUB2 product. Comparisons of beta-tubulin sequences from C. neoformans and from various benzimidazole-sensitive and -resistant organisms strongly suggest that the TUB1 product represents the primary benzimidazole target. This was supported by the identification of a His6 to Gln change in TUB1 from three independently isolated albendazole-resistant mutants.

β-Tubulin genes and the basts for benzimidazole sensitivity of the opportunistic fungus Cryptococcus neoformans