Project Grant R03DE033304

DEVELOPMENT OF ANTIFUNGAL DRUG RESISTANCE IN THE ORAL ENVIRONMENT OVER THE COURSE OF HIV INFECTION - FUNGAL INFECTIONS ARE AN INCREASING HUMAN HEALTH RISK, AND THE DEVELOPMENT OF FUNGAL RESISTANCE TO THE LIMITED REPERTOIRE OF AVAILABLE DRUGS IS EXACERBATING THIS THREAT. FIVE SPECIES WITHIN THE GENUS CANDIDA PREDOMINANTLY CAUSE DISEASE MANIFESTATIONS IN HUMANS, ESPECIALLY IN IMMUNOCOMPROMISED INDIVIDUALS. INDEED, CANDIDIASIS IS THE MOST COMMON OPPORTUNISTIC ORAL INFECTION AFFECTING PEOPLE WITH AIDS. EARLIER WORK BY OUR GROUP AND OTHERS DEMONSTRATED CHANGES IN ORAL FUNGAL COMPOSITION AND MUTATIONAL CHANGES IN ANTIMICROBIAL RESISTANCE (AMR) GENES THROUGHOUT THE COURSE OF HIV INFECTION AND TREATMENT, SUGGESTING A UNIQUENESS TO THE ORAL ENVIRONMENT. ALTHOUGH THERE ARE SEVERAL KNOWN MOLECULAR MECHANISMS THAT CONFER ANTIFUNGAL RESISTANCE, RECENT STUDIES SUGGEST THE HIGH LIKELIHOOD THAT THERE ARE MANY NOVEL MECHANISMS YET TO BE IDENTIFIED. OUR GOALS ARE TO EMPLOY EVOLUTIONARY GENOMIC APPROACHES TO GAIN BOTH BASIC AND APPLIED INSIGHTS ON THE ORAL ENVIRONMENT THAT WILL INFORM DIAGNOSTICS AND ULTIMATELY TREATMENT. OUR CENTRAL HYPOTHESIS IS THAT FUNGI WILL EVOLVE OVER TIME IN THE ORAL ENVIRONMENT, AND THIS R03 IS DESIGNED TO DEMONSTRATE BOTH SUPPORT FOR THIS HYPOTHESIS AND FEASIBILITY OF THE NOVEL METHODS WE ARE DEVELOPING. OUR PROPOSED WORK LEVERAGES AN EARLIER CASE-CONTROL STUDY ("CROSSTALK") BY ONE OF OUR TEAM MEMBERS AND INCLUDES A SET OF 50 HIV-POSITIVE AND HAART NAIVE INDIVIDUALS, AND 76 HIV-NEGATIVE CONTROLS, OVER A 5-YEAR STUDY PERIOD. DEIDENTIFIED AND CRYOPRESERVED SALIVA, CARIES SWABS, FUNGAL CULTURES, AND BACTERIAL CULTURES FROM THESE PATIENTS ARE IMMEDIATELY AVAILABLE, ALONG WITH ASSOCIATED CLINICAL AND DEMOGRAPHIC DATA. IN AIM 1 WE WILL USE DIRECT LONG-READ SEQUENCING ON PATIENT SALIVA AND CARIES SWABS TO DETERMINE THE FUNGAL MYCOBIOME AT UNPRECEDENTED RESOLUTION. THIS PROFILING WILL COME FROM DIRECT SEQUENCE ANALYSIS OF THE FULL-LENGTH CANDIDA RRNA OPERON FROM PATIENT SAMPLES. IN AIM 2 WE WILL DETERMINE THE FUNGAL RESISTOME OF THE SAME SAMPLES OVER THE COURSE OF TREATMENT. WE WILL PERFORM LONGITUDINAL PROFILING BY HYBRIDIZATION-CAPTURE WITH BIOTINYLATED PROBES TO TARGET KNOWN FUNGAL AMR AND BIOFILM-ASSOCIATED GENES. THESE GENES WILL THEN BE ANALYZED FOR SNPS OVER TIME AND FOR SIGNATURES OF POSITIVE SELECTION PRESSURE. WE WILL NEXT PROPOSE AN R01 WITH A GOAL OF IDENTIFYING NEW FUNGAL GENETIC AMR DETERMINANTS USING PAN-GENOMIC AND MOLECULAR ADAPTATION ANALYSES ON FUNGAL CULTURES FROM CROSSTALK AND MORE RECENT ISOLATES. THIS TAKES ADVANTAGE OF THE RECENT DISCOVERY THAT FUNGI, LIKE BACTERIA, HAVE A CORE AND AN ACCESSORY GENOME; WE PLAN TO CORRELATE THE GENES FROM THE ACCESSORY GENOME, AND SNPS ACROSS THE GENOME, WITH AMR PHENOTYPE DATA, CONCENTRATING ON CANDIDA SPECIES COMMON TO THE HIV ORAL ENVIRONMENT. WE EXPECT THAT THIS PROJECT WILL IDENTIFY PUTATIVE NOVEL CANDIDA ANTIFUNGAL RESISTANCE MECHANISMS AND PROVIDE MUCH NEEDED INFORMATION ON CANDIDA EVOLUTION IN THE ORAL ENVIRONMENT.