Identification of pathogen genomic differences that impact human immune response and disease duringCryptococcus neoformansinfection

Abstract

1 2Patient outcomes during infection are due to a complex interplay between the quality of medical 3 care, host immunity factors, and the infecting pathogen's characteristics. To probe the influence 4 of pathogen genotype on human immune response and disease, we examined Cryptococcus 5 neoformans isolates collected during the Cryptococcal Optimal ART Timing (COAT) trial in 6 Uganda. We measured human participants' immunologic phenotypes, meningitis disease 7 parameters, and survival. We compared this clinical data to whole genome sequences from 38 C. 8 neoformans isolates of the most frequently observed sequence type (ST) ST93 in our Ugandan 9 participant population, and an additional 18 strains from 9 other sequence types representing the 10 known genetic diversity within the Ugandan Cryptococcus clinical isolates. We focused our 11 analyses on 652 polymorphisms that: were variable among the ST93 genomes, were not in 12 centromeres or extreme telomeres, and were predicted to have a fitness effect. Logistic 13 regression and principal component analyses identified 40 candidate Cryptococcus genes and 3 14 hypothetical RNAs associated with human immunologic response or clinical parameters. We 15 infected mice with 17 available KN99α gene deletion strains for these candidate genes and found 16 that 35% (6/17) directly influenced murine survival. Four of the six gene deletions that impacted 17 murine survival were novel. Such bedside-to-bench translational research provides important 18 candidate genes for future studies on virulence-associated traits in human Cryptococcus 19 infections.Even with the best available care, mortality rates in cryptococcal meningitis range from 23 20-60%. Disease is often due to infection by the fungus Cryptococcus neoformans and involves a 24 complex interaction between the human host and the fungal pathogen. Although previous studies 25 have suggested genetic differences in the pathogen impact human disease, it has proven quite 26 difficult to identify the specific C. neoformans genes that impact the outcome of the human 27 infection. Here, we take advantage of a Ugandan patient cohort infected with closely related C. 28 neoformans strains to examine to role of pathogen genetic variants on several human disease 29 characteristics. Using a pathogen whole genome sequencing approach, we showed that 40 C. 30neoformans genes are associated with human disease. Surprisingly, many of these genes are 31 specific to Cryptococcus and have unknown functions. We also show deletion of these genes 32 alters disease in a mouse model of infection, confirming their role in disease. These findings are 33 particularly important because they are the first to identify C. neoformans genes associated with 34 human cryptococcal meningitis and lay the foundation for future studies that may lead to new 35 treatment strategies aimed at reducing patient mortality. 36 37 38Cryptococcus neoformans is the etiological agent of cryptococcal meningitis, the most 39 common brain infection in Sub-Saharan Africa, which enc...