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The role of the Aspergillus fumigatus rheb homologue, rhbA, in nitrogen sensing and the pathogenesis of invasive pulmonary aspergillosis
| Title: | The role of the Aspergillus fumigatus rheb homologue, rhbA, in nitrogen sensing and the pathogenesis of invasive pulmonary aspergillosis |
| Author: | PANEPINTO, JOHN CARLO |
| Description: | A gene encoding a ras protein with homology to the rheb family was cloned from Aspergillus fumigatus. The rhbA gene contains four exons separated by three introns, and is predicted to encode a protein of 187 amino acids. Although conserved ras domains are present, the RhbA protein sequence deviates from the ras consensus in a manner that is characteristic of rheb proteins. The invariant Gly-Gly in the first GTP-binding domain of ras proteins is replaced by Arg-Ser in RhbA, and a conserved Asp in the effector region of ras proteins is replaced by Asn in RhbA. Although the levels of rhbA mRNA remained similar throughout the A. fumigatus asexual developmental cycle, they accumulated over five-fold in response to nitrogen starvation. The rhbA gene was able to complement the canavanine hypersensitivity of Saccharomyces cerevisiae Δrhb1 mutants, suggesting that the two proteins share overlapping function. To determine the function of the A. fumigatus rheb homologue, rhbA, in growth and pathogenesis, a strain of A. fumigatus lacking rhbA was constructed. Three of the four coding exons of the rhbA gene were replaced with a hygromycin resistance cassette. In vitro analysis of the growth rate of the rhbA mutant compared with that of the wild type and complemented strain revealed a significant reduction in growth of the mutant on medium containing poor nitrogen sources (p<0.05), while maintaining wild type growth rate on ammonium containing medium. The ΔrhbA strain exhbited hypersensitivity to the TOR kinase inhibitor rapamycin, but was hyposensitive to the histidine analog 3-amino triazole. Survival of mice inoculated with the ΔrhbA strain was significantly increased when compared to those groups inoculated with the wild type or reconstituted strain (p<0.05). Image analysis of lung sections from mice infected with the ΔrhbA strain revealed a smaller average fungal lesion area compared to the lesions from mice infected with the wild type or reconstituted strain at 4 and 7 days post-inoculation (p<0.05). These data suggest that rhbA is involved in nitrogen discrimination and contributes to the virulence of A. fumigatus. |
| Permanent Link: |
http://rave.ohiolink.edu/etdc/view?acc_num=ucin1008598595
http://hdl.handle.net/2374.OX/9024 |
| Date: | 2001 |
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