Characterization the rtf A gene in Aspergillus fumigatus

Ref ID: 19543

Author:

R Myers1, T Smith1, AM Calvo1*

Author address:

1Biological Sciences, Northern Illinois University, Dekalb, Illinois, USA

Full conference title:

6th Advances Against Aspergillosis 2014

Abstract:

Purpose
Invasive aspergillosis by Aspergillus fumigatus is a leading cause of infection-related mortality
in immunocompromised patients. This population group includes individuals infected with HIV,
cancer patients undergoing chemotherapy, those individuals with genetic immune deficiencies and
those with hematological malignancies. The number of patients who fall into these categories is
steadily increasing. In order to discover potential genetic targets to control A. fumigatus infections,
we have characterized rtfA, a gene encoding a conserved putative RNA polymerase II elongation
factor. Recently, work from our laboratory has shown that the rtfA ortholog in the model fungus
Aspergillus nidulans influences growth, conidiation, and secondary metabolism. In the present study
we are investigating the regulatory output of rtfA in the opportunistic pathogen A. fumigatus.
Methods
With this purpose, we generated an A. fumigatus rtfA deletion mutant, a complementation strain
where the rtfA wild-type allele was incorporated in the rtfA deletion mutant, and an rtfA overexpression
strain. These strains were compared with the isogenic wild-type control strain, CEA10.
Predicted expression profile of rftA in these strains was confirmed by qRT-PCR. Colony growth was
determined by colony diameter in point-inoculated Czapek-Dox cultures. The strains were allowed
to grow for five days. Quantification of conidial production was performed with an hemacytometer
under a light microscope. Protease activity was measured using an Azo-Casein assay. Oxidative
stress response was evaluated adding increased concentrations of menadione to the culture medium.
All the experiments were carried out with three replicates.
Results
Deletion of A. fumigatus rtfA resulted in a strain with a notable reduction (30%) in colony diameter
compared to the wild-type strain. Complementation of the mtfA deletion strain with the mtfA wilttype
allele was able to restore wild-type phenotype. Deletion of rtfA also resulted in colonies with
delayed but slightly increased condiation levels compared to the controls. Interestingly, the rtfA
deletion strain was also deficient in protease activity. In addition, the rtfA deletion strain presented
higher sensitivity to oxidative stress, being unable to grow in the presence of 20 μM menadione,
condition that allowed normal growth in the wild type strain. The over-expression strain presented
wild-type phenotype.
Conclusion
The notable decrease in colony diameter of the rtfA deletion strain compared to the control strains
indicates that rtfA is necessary for normal growth in A. fumigatus. Furthermore, the delay and increase
in conidiation detected in the absence of rtfA suggests that this gene is a regulator of conidiation. In
addition, the rtfA mutant lacks protease activity, and presents growth inhibition in an oxidative stress
environment. For all these reasons, rtfA is a promising genetic target with high potential against
aspergillosis. Current studies in our laboratory are focused on further characterizing the effect of rtfA
on A. fumigatus secondary metabolism and pathogenicity.

Abstract Number: 70

Conference Year: 2014

Link to conference website: http://www.AAA2014.org

New link: NULL

Conference abstracts, posters & presentations

Showing 10 posts of 17325 posts found.

  • Title

    Author

    Year

    Number

    Poster