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

Cryptococcus neoformans is an opportunistic fungal pathogen that causes life-threatening meningitis primarily in immunocompromised individuals. In order to survive and proliferate during infection, C. neoformans must adapt to a variety of stresses it encounters within the host. Patient outcome depends on the interaction between the pathogen and the host. Understanding the mechanisms that C. neoformans uses to facilitate adaptation to the host and promote pathogenesis is necessary to better predict disease severity and establish proper treatment. Several virulence phenotypes have been characterized in C. neoformans, but the field still lacks a complete understanding of how genotype and phenotype contribute to clinical outcome. Furthermore, while it is known that C. neoformans genotype impacts patient outcome, the mechanisms remain unknown. This lack of understanding may be due to the genetic heterogeneity of C. neoformans and the extensive phenotypic variation observed between and within isolates during infection. In this review, we summarize the current understanding of how the various genotypes and phenotypes observed in C. neoformans correlate with human disease progression in the context of patient outcome and recurrence. We also postulate the mechanisms underlying the genetic and phenotypic changes that occur in vivo to promote rapid adaptation in the host.

Leptospires and other members of the evolutionarily ancient phylum of Spirochaetes are bacteria often characterized by long, highly motile spiral- or wave-shaped cells. Morphology and motility are critical factors in spirochete physiology, contributing to the ability of these bacteria to successfully colonize diverse environments. However, the mechanisms conferring the helical structure of Leptospira spp. have yet to be fully elucidated. We have identified five Leptospira biflexa bactofilin proteins, a recently characterized protein family with cytoskeletal properties. These five bactofilins are conserved in all species of the Leptospiraceae, indicating that these proteins arose early in the evolution of this family. One member of this protein family, LbbD, confers the optimal pitch distance in the helical structure of L. biflexa. Mutants lacking lbbD display a unique compressed helical morphology, a reduced motility and a decreased ability to tolerate cell wall stressors. The change in the helical spacing, combined with the motility and cell wall integrity defects, showcases the intimate relationship and coevolution between shape and motility in these spirochetes.

The mechanisms of latency in the context of C. neoformans infection remain poorly understood. Two reasons for this gap in knowledge are: 1) the lack of standardized criteria for defining latent cryptococcosis in animal models and 2) limited genetic and immunological tools available for studying host parameters against C. neoformans in non-murine models of persistent infection. In this study, we defined criteria required for latency in C. neoformans infection models and used these criteria to develop a murine model of persistent C. neoformans infection using clinical isolates. We analyzed infections with two clinical C. neoformans strains, UgCl223 and UgCl552, isolated from advanced HIV patients with cryptococcal meningitis. Our data show that the majority of C57BL/6 mice infected with the clinical C. neoformans isolates had persistent, stable infections with low fungal burden, survived beyond 90 days-post infection, exhibited weight gain, had no clinical signs of disease, and had yeast cells contained within pulmonary granulomas with no generalized alveolar inflammation. Infected mice exhibited stable relative frequencies of pulmonary immune cells during the course of the infection. Upon CD4+ T-cell depletion, the CD4DTR mice had significantly increased lung and brain fungal burden that resulted in lethal infection, indicating that CD4+ T-cells are important for control of the pulmonary infection and to prevent dissemination. Cells expressing the Tbet transcription factor were the predominant activated CD4 T-cell subset in the lungs during the latent infection. These Tbet-expressing T-cells had decreased IFNγ production, which may have implications in the capacity of the cells to orchestrate the pulmonary immune response. Altogether, these results indicate that clinical C. neoformans isolates can establish a persistent controlled infection that meets most criteria for latency; highlighting the utility of this new mouse model system for studies of host immune responses that control C. neoformans infections.

The human pathogenic fungus Cryptococcus neoformans is a global health concern. Previous research in the field has focused on studies using reference strains to identify virulence factors, generate mutant libraries, define genomic structures, and perform functional studies. In this review, we discuss the benefits and drawbacks of using reference strains to study C. neoformans, describe how the study of clinical isolates has expanded our understanding of pathogenesis, and highlight how studies using clinical isolates can further develop our understanding of the host–pathogen interaction during C. neoformans infection.

Outbreaks of blastomycosis, caused by the fungus Blastomyces dermatitidis, occur in endemic areas of the United States and Canada but the geographic range of blastomycosis is expanding. Previous studies inferred the location of B. dermatitidis through epidemiologic data associated with outbreaks because culture of B. dermatitidis from the environment is often unsuccessful. In this study, we used a culture-independent, PCR-based method to identify B. dermatitidis DNA in environmental samples using the BAD1 promoter region. We tested 250 environmental samples collected in Minnesota, either associated with blastomycosis outbreaks or environmental samples collected from high- and low-endemic regions to determine basal prevalence of B. dermatitidis in the environment. We identified a fifth BAD1 promoter haplotype of B. dermatitidis prevalent in Minnesota. Ecological niche analysis identified latitude, longitude, elevation, and site classification as environmental parameters associated with the presence of B. dermatitidis. Using this analysis, a Random Forest model predicted B. dermatitidis presence in basal environmental samples with 75% accuracy. These data support use of culture-independent, PCR-based environmental sampling to track spread into new regions and to characterize the unknown B. dermatitidis environmental niche.Importance Upon inhalation of spores from the fungus Blastomyces dermatitidis from the environment, humans and animals can develop the disease blastomycosis. Based on disease epidemiology, B. dermatitidis is known to be endemic in the United States and Canada around the Great Lakes and in the Ohio and Mississippi River Valleys but is starting to emerge in other areas. B. dermatitidis is extremely difficult to culture from the environment so little is known about the environmental reservoirs for this pathogen. We used a culture-independent PCR-based assay to identify the presence of B. dermatitidis DNA in soil samples from Minnesota. By combining molecular data with ecological niche modeling, we were able to predict the presence of B. dermatitidis in environmental samples with 75% accuracy and to define characteristics of the B. dermatitidis environmental niche. Importantly, we showed the effectiveness of using a PCR-based assay to identify B. dermatitidis in environmental samples.

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...

S5.3 Cellular pleomorphism and fungal virulence, September 22, 2022, 3:00 PM - 4:30 PM Cryptococcus neoformans is a human pathogenic basidiomycete yeast that can cause cryptococcal meningitis (CM), predominantly in immunocompromised individuals. The patient outcome depends on both host and pathogen-specific factors, including C. neoformans genetics. A groundbreaking 2012 study was the first to show that patient outcome is associated with genetic differences between C. neoformans isolates. Subsequent population-wide sequencing studies have revealed over 100 sequence types (ST) of C. neoformans that are associated with both geographic location and clinical outcome. All these studies have been broad, examining the severity of disease cryptococcal phenotypes in a collection of highly diverse strains. We chose a narrow focus and collected various genotypic and phenotypic data from a single ST: ST93. ST93 is a common sequence type isolated from patients globally and is the most common clinical isolate found in the sub-Saharan African country of Uganda. Previously, we performed whole genome sequencing on 38 ST93 Ugandan clinical isolates. We identified 652 unique SNPs in this ST93 population compared to the H99 reference genome. We also showed that ST93 contained two subpopulations: ST9A and ST93B. In the current study, we further characterized the genotypic, phenotypic, and virulence differences between these 38 clinical isolates. Using Illumina sequence data, we identified a pattern of linkage disequilibrium that suggested that ST93A and ST93B are evolving separately. We performed long-read sequencing on each isolate to investigate chromosomal changes and large structural variations, allowing us to identify a chromosomal translocation event wherein parts of chromosome 11 had recombined with chromosome 3. Additionally, we characterized several in vitro phenotypes for each isolate and identified three distinct phenotypic clusters based on cell wall challenge and growth experiments. Next, we infected mice with 35 isolates and observed eight different disease manifestations, including isolates that caused non-CNS infections. Overall, by working within a single sequence type, we can gain a deeper understanding of how some small genetic changes can impact strain-specific phenotypes while others have no discernable effect. Eventually, these data can be used to provide valuable information about how each clinical isolate impacts patient outcomes.