Project Grant F31AI203011

CONSEQUENCES OF GENOME CONTENT VARIATION IN THE HUMAN FUNGAL PATHOGEN CRYPTOCOCCUS NEOFORMANS - PROJECT SUMMARY CRYPTOCOCCUS NEOFORMANS IS A HUMAN FUNGAL PATHOGEN ESTIMATED TO CAUSE 147,000 DEATHS ANNUALLY. THERE ARE ONLY THREE CLASSES OF ANTIFUNGALS USED TO TREAT INFECTED PATIENTS, WHO ARE USUALLY IMMUNOCOMPROMISED. WITH SO FEW TREATMENT OPTIONS AVAILABLE FOR PATIENTS, MANY ISOLATES HAVE DEVELOPED RESISTANCE TO CURRENT ANTIFUNGALS. THIS HAS LED TO DECADES OF RESEARCH ON ANTIFUNGAL RESISTANCE AND NEW DRUG TARGETS FOR FUNGAL PATHOGENS. C. NEOFORMANS IS A GENETICALLY COMPLEX PATHOGEN WITH ALMOST 4,000 DIFFERENT ISOLATES COLLECTED AROUND THE WORLD FROM BOTH CLINICAL AND ENVIRONMENTAL SETTINGS. HOWEVER, THIS SPECIES HAS ALMOST EXCLUSIVELY BEEN INVESTIGATED USING THE REFERENCE STRAIN H99, ORIGINALLY ISOLATED FROM A PATIENT. THIS SINGULAR REFERENCE STRAIN HAS FACILITATED THE DEVELOPMENT OF A PLETHORA OF RESOURCES; HOWEVER, IT REPRESENTS JUST ONE ISOLATE. THEREFORE, THIS PROJECT AIMS TO INVESTIGATE POPULATION LEVEL DIVERSITY AMONGST C. NEOFORMANS ISOLATES AND HOW IT CONTRIBUTES TO DRUG RESISTANCE. PREVIOUS RESEARCH HAS FOCUSED ON REFERENCE-BASED TOOLS THAT RELY ON SIMILARITY BETWEEN SEQUENCES TO IDENTIFY SMALL CHANGES, OR SINGLE NUCLEOTIDE POLYMORPHISMS, THAT ARE CAUSATIVE FOR RESISTANCE. IN CONTRAST, PRELIMINARY WORK FOR THIS PROJECT HAS ANNOTATED 384 UNIQUE C. NEOFORMANS GENOMES TO IDENTIFY PATTERNS OF GENE PRESENCE AND ABSENCE ACROSS THE POPULATION. FROM THESE ANNOTATIONS, A PANGENOME WAS CURATED USING THESE 384 ISOLATES THAT CLASSIFIED ORTHOLOGS INTO THREE GROUPS BASED ON THEIR PRESENCE IN THE POPULATION: CORE (CONSERVED IN ALL ISOLATES), ACCESSORY (IN A PORTION OF ISOLATES), OR SINGLETON (PRESENT IN ONLY A SINGLE ISOLATE). ADDITIONALLY, A TRANSPOSON-MUTAGENESIS (TN-SEQ) SYSTEM, WHICH ASSAYS GENE ESSENTIALITY IN THE GENOME, WAS PRELIMINARILY ESTABLISHED IN THE REFERENCE C. NEOFORMANS STRAIN. WITH THESE TWO TOOLS, THIS APPLICANT IS UNIQUELY POISED TO INVESTIGATE HOW GENE CONTENT DIFFERENCES CONTRIBUTE TO DRUG RESISTANCE IN C. NEOFORMANS ISOLATES. THE MAIN HYPOTHESIS OF THIS PROPOSAL IS THAT GENE CONTENT DIFFERENCES, SUCH AS COPY NUMBER VARIATION OR ACCESSORY GENES, CONTRIBUTE TO VARIATION IN ESSENTIALITY ACROSS THE POPULATION AND ARE IMPORTANT FOR DRUG RESISTANCE PHENOTYPES. AIM 1 WILL USE TN-SEQ TO MAP THE VARIATION IN GENE ESSENTIALITY ACROSS THE C. NEOFORMANS POPULATION AND EXPLORE POTENTIAL CONTRIBUTORS TO THESE CHANGES. AIM 2 WILL EXPAND ON THE TN-SEQ LIBRARIES TO IDENTIFY GENES THAT ARE CONDITIONALLY ESSENTIAL FOR GROWTH IN THE ANTIFUNGAL DRUG FLUCONAZOLE. THIS RESEARCH IS INNOVATIVE BOTH TECHNICALLY IN IMPLEMENTING TN-SEQ IN NON-REFERENCE ISOLATES AND CONCEPTUALLY AS IT DEMONSTRATES A NOVEL COMBINATION OF PANGENOME AND TN-SEQ TOOLS. TN-SEQ LIBRARIES WILL ADVANCE THE C. NEOFORMANS COMMUNITY BY PROVIDING A RESOURCE FOR EXPLORATION INTO GENETIC FUNCTIONS IN DIVERSE ISOLATES. FUTURE APPLICATIONS OF THIS RESEARCH COULD INCLUDE USE OF ACCESSORY GENES FOR DIAGNOSTIC PURPOSES OR INVESTIGATION INTO CORE ESSENTIAL GENES AS NOVEL DRUG TARGETS.