|Skory, Christopher - Chris|
|EDWARDS, JR, J|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 9/20/2007
Publication Date: 9/20/2007
Citation: Lin, L., Spellberg, B.J., Fu, Y., Skory, C.D., Gebremariam, T., Husseiny, M.I., Edwards, Jr, J.E., Ibrahim, A.S. 2007. High affinity iron permease is required for virulence of Rhizopus oryzae [abstract]. The American Society for Microbiology's 47th Annual Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC). Presentation #B-1444.
Technical Abstract: Rhizopus oryzae is the most common cause of mucormycosis. Clinical and animal model data clearly demonstrate that the presence of elevated available serum iron predisposes the host to develop mucormycosis. The high affinity iron permease gene (rFTR1) is required for R. oryzae iron transport in iron-limited environments, such as those found in the host during infection. To define its role in R. oryzae virulence, we used RNA interference (RNAi) to abrogate the function of rFTR1 because gene disruption is problematic in R. oryzae. Methods: We cloned a 400 bp invert repeat of rFTR1 into an RNAi expression vector under the control of the pdc promoter of R. oryzae. Transformants were selected on minimal medium lacking uracil. RT-PCR was used to confirm inhibition of rFTR1 expression in iron limiting conditions. Virulence of rFTR1 expression-inhibited transformants was compared to the control strain (transformed with empty plasmid) using our standard diabetic ketoacidosis (DKA) mouse model of hematogenously disseminated mucormycosis. Results: Analysis of transformants revealed that the RNAi plasmid was maintained episomally. RT-PCR confirmed RNAi-plasmid mediated inhibition of rFTR1 expression by >95% compared to the control strain. Mice infected with the RNAi-transformant strain of R. oryzae demonstrated reduced virulence compared to the control strain. However, over time virulence was restored in the RNAi-transformed strain, and analysis of the organism recovered from moribund mice demonstrated that the strain had lost the RNAi plasmid prior to restoration of virulence. Conclusions: RNAi is a viable alternative for gene silencing in R. oryzae. However methods of transformation that ensure stable integration of the construct are desirable to prevent plasmid loss from affecting pathogenicity studies. Finally, we confirm that rFTR1 is required for full virulence of R. oryzae in DKA mice.