|IBRAHIM, A - University Of California|
|GEBREMARIAM, T - University Of California|
|Skory, Christopher - Chris|
|FU, Y - University Of California|
|EDWARDS, JR., J - University Of California|
|SPELLBERG, B - University Of California|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 9/15/2009
Publication Date: 9/15/2009
Citation: Ibrahim, A.S., Gebremariam, T., Skory, C.D., Fu, Y., Edwards, Jr., J.E., Spellberg, B.J. 2009. rFTR1 is required for pathogenesis, and appears to be an essential gene, of Rhizopus oryzae [abstract]. Interscience Conference on Antimicrobial Agents and Chemotherapy. M-361.
Technical Abstract: BACKGROUND: Rhizopus oryzae is a multinucleated fungus responsible for the majority of cases of mucormycosis. The high affinity iron permease gene (rFTR1) is required for R. oryzae iron transport in iron-limited environments. We sought to disrupt the gene to define its role in virulence. METHODS: A disruption cassette was constructed by flanking the pyrF with ~ 600 bp of the rFTR1- 5’ and rFTR1- 3’ UTR. Transformants were obtained by biolistics on minimal medium lacking uracil and purified with 1 mM FeCl3 (iron rich media). Integration of the disruption cassette and rFTR1 disruption were confirmed by PCR. Iron uptake was assayed for by 0.1 uM 59Fe. Virulence was determined using our standard diabetic ketoacidosis mouse models of disseminated and intranasal mucormycosis. RESULTS: After 14 passages on iron rich media, PCR confirmed integration of the disruption cassette in the rFTR1 locus, and absence of rFTR1 ORF from several strains. Two such strains demonstrated lagging growth on iron limited (50 'M ferroxamine) medium. Furthermore, the strains had 23-38% reductions in 59Fe uptake compared to a PYRF corrected control strain (p<0.01). However, after the first 72 h of growth on iron-limited media, growth of the transformants increased and became similar to the PYRF corrected strain. PCR analysis confirmed that the rFTR1 ORF was once again detectable in these strains. Transformants showed reduced virulence compared to the PYRF corrected strain in mice (38% vs. 0% and 56% vs. 25% survival for transformants vs. control strain in disseminated and intranasal models, respectively, p<0.001). CONCLUSION: PCR detected apparent disruption of rftr1 in multinucleated R. oryzae when cells were grown on iron rich media. However, the cells reverted to rFTR1 after growth on iron-limited media. Hence, the rFTR1 appears to be an essential gene, which can be reduced but not eliminated from multinucleated R. oryzae. Nevertheless, even reduction in rFTR1 content within the fungus without complete disruption resulted in diminished ability to obtain iron in vitro and decreased virulence of R. oryzae in mouse models of infections.