William J. Steinbach - US grants

ABSTRACT The need to train scientists to conduct fungal research is greater than ever. Worldwide estimates of human fungal disease include over a billion people with invasive, allergic, or chronic fungal diseases. Plant diseases, caused predominantly by fungi, are estimated to reduce global food yields by 20-40%. These striking statistics showcase the immense human and financial tolls imparted by fungal diseases. Our objective is to develop molecular mycology scientists trained in the latest methods of laboratory, translational, or clinical research who are fully prepared to pursue independent research careers investigating the many aspects of fungal diseases. The Tri-Institutional Molecular Mycology and Pathogenesis Training Program (Tri-I MMPTP) has been funded since 2003 to recruit, support, and train promising postdoctoral scientists and physicians to develop productive research careers. It is the only mycology-focused postdoctoral training program in existence, and it has been highly successful in training the next generation of outstanding scientists. The tremendous productivity of this training program is predicated on its unique design, leveraging the proximity of three prominent research universities: Duke University, the University of North Carolina at Chapel Hill, and North Carolina State University. These institutions offer arguably the highest geographic concentration of researchers who study fungi in the country. The Tri-I MMPTP has trained or is currently training a total of 43 post-doctoral fellows, many of whom have gone on to lead independent NIH-funded laboratories, lead government research efforts, work in industry on cutting-edge projects, and assume positions of leadership in academia, government, and industry. At many institutions, faculty who investigate medical fungi have little contact with geneticists who work on model fungi, biochemists who study cellular mechanisms, or infectious diseases physicians who care for immunocompromised patients. Similarly, those who study phytopathogens are intellectually, and often physically, removed from biomedical researchers. A proven concept underlying our multidisciplinary interaction is that clinical and basic researchers, and plant and animal mycologists, together discover new approaches that are mutually beneficial. With this integrated design, our trainees become broadly knowledgeable, versatile, and more attractive to prospective employers. Several outstanding training themes are responsible for our continued success: careful selection from a large pool of competitive applicants, promotion of diversity (including 3 of 7 current trainees from under-represented backgrounds), and centralized courses on scientific and grant writing. These opportunities are all connected through trainee Individual Research Advisory Committees. At the core is our dedicated mentorship, including a pathway to independence mentality and a formalized continued mentorship program for three years after program completion. Our interconnected strategy has generated highly successful scientists for 15 years, and our constant programmatic updates via trainee and mentor feedback have developed exciting approaches to fill the need for molecular mycologists.

Invasive mold infections (IMI) are a leading infectious cause of death in immunocompromised patients. IMI is most frequently diagnosed in the lungs of children with neutropenia or graft versus host disease (GVHD). Initial clinical suspicion for a pulmonary IMI (PIMI) is often based on lesions identified by chest CT, categorized as ?possible PIMI?. Additional diagnostic testing is usually limited to invasive procedures with concerning risk profiles. The objective of this study is to establish a comprehensive non-invasive diagnostic approach in a prospective multi-center cohort of 300 children with prolonged neutropenia or GVHD that present with possible PIMI. Establishing an accurate non-invasive diagnostic strategy could reduce morbidity from invasive procedures and reduce time to appropriate antifungal therapy, resulting in a reduction in IMI mortality. To accomplish this, we will leverage the International Pediatric Fungal Network, a unique multidisciplinary group of 55 worldwide sites and the only such group dedicated to pediatric invasive fungal disease. This study will benefit from an unparalleled collaboration with the Children's Oncology Group (COG), the world's largest organization devoted to pediatric cancer clinical trials. In Aim 1, we will investigate a comprehensive non-invasive diagnostic testing approach to confirm the presence or absence of proven or probable PIMI in children with newly identified possible PIMI. We hypothesize that a non-invasive diagnostic strategy using available blood-based assays (galactomannan, Aspergillus PCR, and Mucorales PCR) will provide positive and negative post-test probabilities that are clinically informative. In Aim 2, we will leverage this cohort to compare the outcomes for children with possible PIMI managed with empiric antifungal therapy vs. an invasive diagnostic procedure followed by management based on results. Comparison will be using an outcome score measure that incorporates both negative and positive consequences of each exposure, providing a balanced measure of the impact of a clinical decision. We established a minimum clinically important difference (MCID) to dichotomize outcome into `success' or `failure' from surveying experts in clinical mycology. We hypothesize that patients receiving empiric antifungals will have more successful outcomes compared to patients undergoing an invasive diagnostic procedure. This cohort also offers the possibility to study novel non-invasive diagnostic tests not widely available. Therefore, in Aim 3, in a subset of patients we will explore the host response to proven/probable PIMI using RNA-Seq to define transcriptional signatures, and assess plasma cell-free DNA/RNA next-generation sequencing to detect mold pathogens. At select sites, we will also use breath testing to assess volatile metabolite signatures as a marker for IMI. In summary, we will establish the utility of a new comprehensive non-invasive diagnostic approach for possible PIMI using a panel of assays in a unique multi-center prospective pediatric cohort. We will also compare, for the first time, the outcomes of children managed empirically vs. undergoing invasive procedures. These results will provide the foundation for new evidence-based pediatric guidelines.

Invasive aspergillosis due to Aspergillus fumigatus is a leading infectious killer of immunocompromised patients. A significant barrier to developing effective antifungal therapeutics is the lack of knowledge regarding the regulation of A. fumigatus growth and pathogenesis. We established calcineurin (CN) as a critical phosphatase required for A. fumigatus hyphal growth, septation, and invasive disease. We were the first to crystallize the fungal CN complex, and structure-guided inhibitor strategies showed targeting CN effective in animal models. However, exactly how CN regulates growth and pathogenesis is largely undefined. We demonstrated that the CN complex, comprised of catalytic (CnaA) and regulatory (CnaB) subunits, is dynamically localized at active points of growth, the hyphal tip and septum, likely interacting with key effectors at these active points to regulate septation and hyphal extension. We confirmed that defects in septation and hyphal extension are not mediated via the CN-dependent transcription factor CrzA or the major CN-binding protein CbpA. Using mutational approaches, we showed that CN binds to these unknown effectors at the septum via short linear substrate binding motifs (PxIxIT/LxVP) and CN mislocalization from the septum leads to aberrant septation and stunted growth, indicating CN?s direct regulatory role. Our recent CN proteomic and phosphoproteomic approaches uncovered several hyphal growth-related and septum-associated proteins (SAPs) as potential CN effectors. As a logical next step, we will now define CN?s main function as a phosphatase to orchestrate growth and virulence via its interaction with these effectors. Our overall objective is to leverage our strong genetic, structural and new robust proteomic data to define CN specific control. Our central hypothesis is that CN orchestrates invasive hyphal growth by binding and regulating key effector proteins through phosphorylation-dephosphorylation at the septum, thereby governing invasive disease. In Aim 1, we will identify key CN signaling effectors at the hyphal septum by bimolecular fluorescence complementation and affinity assays. Localization studies of the effectors in CN inhibited/conditional expression/deletion backgrounds will confirm their CN-dependency for function. Molecular modeling of CN-effector binding and molecular dynamics simulations will guide our targeted mutations of predicted CN-binding motifs in the effectors to confirm CN-effector interactions. Effector mutants will be screened for hyphal growth and septation defects to correlate CN-dependent effector regulation. In Aim 2, we will perform in vivo phosphorylation analyses of the prioritized SAPs by utilizing orthogonal approaches and LC- MS/MS analysis to gain in-depth insight into specific residues phosphorylated in the respective SAPs in CN inhibited/deletion backgrounds. Finally, contributions of CN-dependent septal effectors to septation, septal pore sealing, hyphal extension, and virulence will be validated using an iterative approach of genetic deletion, targeted mutations, in vitro growth screening, and murine model validation. This study will, for the first time, identify and define the CN signaling network of novel septal effectors controlling fungal growth and virulence.