Cruz Maria Cristina, Edlind Thomas
Department of Microbiology and Immunology, MCP Hahnemann School of Medicine, Allegheny University of the Health Sciences, 2900 Queen Lane, Philadelphia, PA 19129, USA.
Microbiology (Reading). 1997 Jun;143 ( Pt 6):2003-2008. doi: 10.1099/00221287-143-6-2003.
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 > 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.
担子菌新生隐球菌可在免疫功能低下的患者中引发危及生命的感染，而现有的化疗药物可能具有毒性或疗效有限。在体外，新生隐球菌对某些苯并咪唑化合物（如阿苯达唑）非常敏感，这些化合物通过与β-微管蛋白亚基结合来破坏微管发挥作用。为了解这种对苯并咪唑敏感性的基础，我们对新生隐球菌的β-微管蛋白基因及其表达进行了表征。对聚合酶链反应扩增产物、基因组和互补脱氧核糖核酸克隆以及Southern印迹的分析鉴定出两个β-微管蛋白基因。TUB1包含七个内含子，其中一个将起始密码子分开，编码一种447个氨基酸的蛋白质，与大多数其他β-微管蛋白具有>80%的同一性。TUB2的部分序列显示内含子密度更高，且β-微管蛋白差异更大。通过逆转录聚合酶链反应确定的TUB1与TUB2的相对表达约为3:1，这与TUB2产物的作用更有限一致。对新生隐球菌以及各种对苯并咪唑敏感和耐药生物体的β-微管蛋白序列比较强烈表明，TUB1产物是主要的苯并咪唑靶点。从三个独立分离的阿苯达唑耐药突变体中鉴定出TUB1中His6到Gln的变化，这支持了这一观点。
