Microbial Pathogenesis: Fungi
José Vargas-Muñiz is a 5th year Ph.D. candidate in the Steinbach lab. The Steinbach lab specialize in the etiological agent of invasive aspergillosis, the filamentous fungus Aspergillus fumigatus. Invasive aspergillosis is a leading cause of death in immunocompromised patients and carries a mortality rate of 40-60%. For clinical disease and invasive growth into host tissue, A. fumigatus requires hyphal growth and these hyphae are compartmentalized by the septum. Although the full function of the septum is unknown, it has been suggested that it is involved in increasing hyphal rigidity, limiting mechanical damage, and facilitating differentiation. Despite the probable role of septation in hyphal growth, the regulation of septum formation in Aspergillus fumigatus is unknown. Understanding septum formation would allow a better comprehension of fundamental disease pathogenesis and facilitate development of new, targeted fungal-specific therapeutics. In order to provide a critical insight into septum formation we decided to study a conserved family of GTP-binding proteins called septins. Septins are involved in a myriad of cellular processes, including septation, cell wall organization, and cytokinesis. A. fumigatus contains five septin genes: aspA, aspB, aspC, aspD and aspE. The first four, known as core septins, have orthologs in the model yeast Saccharomyces cerevisiae, but aspE is only found in filamentous ascomycetes. Septins organize in higher-order structures, yet it remains unclear how these complexes are regulated. Previous work in S. cerevisiae has shown that mutation of phosphorylation sites affects septin ring organization. However, the role of septin post-translational modification in the Aspergillus proteins has not been studied before my dissertation. Our overall goal is to delineate the role of septins and their post-translational modification in A. fumigatus growth and pathogenesis.
We have deleted all five septins genes in A. fumigatus, and found that AspA, AspB, AspC and AspE are required for regular septation. Only the deletion of core septin genes significantly reduced conidiation. the ΔaspB strain was also sensitive to anti-cell wall agents. Moreover, using a strain expressing AspB-GFP we were able to observe alteration of AspB localization patterns as well as a 7-fold increase in the number of structures found in the apical compartment after 2 hours of caspofungin, a b-glucan synthase inhibitor, treatment. While infection with the ΔaspB strain in a Galleria mellonella model of invasive aspergillosis showed hypervirulence, no virulence difference was noted when compared to the wild-type strain in a murine model of invasive aspergillosis. Due to the pleotropic role of AspB in A. fumigatus growth, we decided to study how AspB is regulated. We have deleted two non-essential kinases (Gin4 and Cla4) and one protein phosphatase 2A subunit (ParA). This approach revealed that deletion of these genes in a strains expressing AspB-GFP fusion protein resulted in altered AspB. LC-MS/MS phosphoproteomics show that AspB is phosphorylated in vivo at 7 residues. Additionally, deletion of the parA gene resulted in AspB being phosphorylated at two additional sites: T68 and S447. Phosphomimetics and non-phosphorylatable alleles of AspB were created. Mutation of T68 or S447 altered the localization of AspB but only the mutation of T68 resulted in an increased apical compartment. Taken together, these results point out the importance of AspB phosphorylation/dephosphorylation in the regulation of septation.