Purpose of Review Candida auris is a multi-drug-resistant pathogen with many phenotypic variations that contribute to its pathogenicity. This review aims to characterize its phenotypic heterogeneity while highlighting the variants that should be prioritized in future research to advance therapies against C. auris . Recent Findings As the Earth warms, fungi like Candida experience selective pressure to tolerate these higher temperatures and become the few fungal species capable of successfully colonizing the host. The most recent of these is C. auris , which has become an acute concern due to its rapid emergence, high mortality rate, and resistance to all known classes of antifungals. Summary Several studies have contributed rapidly to our general understanding of C. auris , but not enough has been experimentally verified on its morphological variation and its ability to maintain a successful commensal lifestyle on the human skin. Because of its distinct phenotypic variations compared to other fungal species, especially under the selective pressures of its host, C. auris presents a unique opportunity to identify unique targets and strategies to contribute to the antifungal pipeline and control emergent pathogens.

Candida albicans, Candida auris, Candida glabrata, and Cryptococcus neoformans are pathogenic yeasts which can cause systemic infections in immune-compromised as well as immune-competent individuals. These yeasts undergo replicative aging analogous to a process first described in the nonpathogenic yeast Saccharomyces cerevisiae. The hallmark of replicative aging is the asymmetric cell division of mother yeast cells that leads to the production of a phenotypically distinct daughter cell. Several techniques to study aging that have been pioneered in S. cerevisiae have been adapted to study aging in other pathogenic yeasts. The studies indicate that aging is relevant for virulence in pathogenic fungi. As the mother yeast cell progressively ages, every ensuing asymmetric cell division leads to striking phenotypic changes, which results in increased antifungal and antiphagocytic resistance. This review summarizes the various techniques that are used to study replicative aging in pathogenic fungi along with their limitations. Additionally, the review summarizes some key phenotypic variations that have been identified and are associated with changes in virulence or resistance and thus promote persistence of older cells.

Purpose of Review Pervasive fungal infection among the immunocompromised population, in conjunction with a lack of effective treatment options, has demanded further scrutiny. Millions of people are still dying annually from fungal infections. While existing treatment for these fungal infections exists, it is difficult to administer without adverse effects in the immunocompromised and is slowly becoming obsolete due to varying mutation rates and rising resistance in multiple species. Thus, vaccines may be a viable target for preventing and treating fungal infections and addressing the critical challenge of such infections. Recent Findings Candida albicans , along with other non-albicans Candida species, is among the more virulent class of fungal specimens considered for vaccine development. C. albicans is responsible for a large percentage of invasive fungal infections among immunocompromised and immunocompetent populations and carries a relatively high mortality rate. In the last decade, a recent increase in infective capacity among Candida species has shed light on the lack of adequate fungal vaccine choices. While roadblocks still exist in the development of antifungal vaccines, several novel targets have been examined and proposed as candidates. Summary Success in vaccine development has universal appeal; an anti- Candida vaccine formulation could be modified to work against other fungal infections and thus bolster the antifungal pipeline.

Cryptococcus neoformans successfully replicates in low glucose in infected patients. In the serotype A strain, H99, growth in this condition prolongs lifespan regulated by SIR2, and can be modulated with SIR2-specific drugs. Previous studies show that lifespan modulation of a cryptococcal population affects its sensitivity to antifungals, and survival in an infection model. Sirtuins and their role in longevity are conserved among fungi; however, the effect of glucose starvation is not confirmed even in Saccharomyces cerevisiae. Lifespan analysis of C. neoformans strains in low glucose showed that 37.5% exhibited pro-longevity, and lifespan of a serotype D strain, RC2, was shortened. Transcriptome comparison of H99 and RC2 under calorie restriction demonstrated differences, confirmed by real-time PCR showing that SIR2, TOR1, SCH9, and PKA1 expression correlated with lifespan response to calorie restriction. As expected, RC2-sir2Δ cells exhibited a shortened lifespan, which was reconstituted. However, shortened lifespan from calorie restriction was independent of SIR2. In contrast to H99 but consistent with altered SIR2 regulation, SIR2-specific drugs did not affect outcome of RC2 infection. These data suggest that SIR2 regulation and response to calorie restriction varies in C. neoformans, which should be considered when Sirtuins are investigated as potential therapy targets for fungal infections.

Cryptococcus neoformans (C. neoformans) is estimated to cause about 220,000 new cases every year in patients with AIDS, despite advances in antifungal treatments. C. neoformans possesses a remarkable ability to disseminate through an immunocompromised host, making treatment difficult. Here, we examine the mechanism of survival of C. neoformans under varying host conditions and find a role for ceramide synthase in C. neoformans virulence. This study also provides a detailed lipidomics resource for the fungal lipid research community in addition to discovering a potential target for antifungal therapy.

2The Jackson Laboratory, Bar Harbor, ME 04609, USA 3Department of Biochemistry, University of Lucknow, Lucknow, Uttar Pradesh 226007, India 4Department of Physiology and Biophysics, Stony Brook University, Stony Brook, NY 11794, USA 5Proteomics Center, Stony Brook University, Stony Brook, NY 11794, USA 6Department of Biomedical Sciences, School of Health Professions and Nursing, Long Island University, Brookville, NY 11548, USA

Strains: C. auris strains from the Centers for Disease Control, USA, were maintained on YPD agar and 37 o C. In vitro studies: Minimum Inhibitory Concentration (MIC) was performed per Clinical and Laboratory Standards Institute guidelines.Growth curves were measured by spectrophotometry on a logarithmic culture.In vivo studies: 10 5 CFU of C. auris strains or PBS was injected into Galleria mellonella as described previously (Cotter et al., 2000).10 6 CFU was injected intravenously into C57BL/6 mice.Mice were euthanized at various days post infection, and fungal burden was calculated on various organs.Macrophage killing assay was performed as previously described (Bouklas et.al. 2013). Infection StudiesStrain MIC (μg/mL) CAU-01 4 CAU-02 16 CAU-03

The fungal pathogen, Cryptococcus neoformans, has been shown to undergo replicative aging. Old cells are characterized by advanced generational age and phenotypic changes that appear to mediate enhanced resistance to host and antifungal-based killing. As a consequence of this age-associated resilience, old cells accumulate during chronic infection. Based on these findings, we hypothesized that shifting the generational age of a pathogenic yeast population would alter its vulnerability to the host and affect its virulence. SIR2 is a well-conserved histone deacetylase, and a pivotal target for the development of anti-aging drugs. We tested its effect on C. neoformans’ replicative lifespan (RLS). First, a mutant C. neoformans strain (sir2Δ) was generated, and confirmed a predicted shortened RLS in sir2Δ cells consistent with its known role in aging. Next, RLS analysis showed that treatment of C. neoformans with Sir2p-agonists resulted in a significantly prolonged RLS, whereas treatment with a Sir2p-antagonist shortened RLS. RLS modulating effects were dependent on SIR2 and not observed in sir2Δ cells. Because SIR2 loss resulted in a slightly impaired fitness, effects of genetic RLS modulation on virulence could not be compared with wild type cells. Instead we chose to chemically modulate RLS, and investigated the effect of Sir2p modulating drugs on C. neoformans cells in a Galleria mellonella infection model. Consistent with our hypothesis that shifts in the generational age of the infecting yeast population alters its vulnerability to host cells, we observed decreased virulence of C. neoformans in the Galleria host when RLS was prolonged by treatment with Sir2p agonists. In contrast, treatment with a Sir2p antagonist, which shortens RLS enhanced virulence in Galleria. In addition, combination of Sir2p agonists with antifungal therapy enhanced the antifungal’s effect. Importantly, no difference in virulence was observed with drug treatment when sir2Δ cells were used for infection, which confirmed target specificity and ruled out non-specific effects of the drugs on the Galleria host. Thus, this study suggests that RLS modulating drugs, such as Sir2p agonists, shift lifespan and vulnerability of the fungal population, and should be further investigated as a potential class of novel antifungal drug targets that can enhance antifungal efficacy.

Candida auris can colonize patients for a prolonged time causing life-threatening systemic infections. Replicative aging is the result of asymmetric division, which cause phenotypic changes between mother and daughter cells. We have previously published that older Cryptococcus neoformans and C. glabrata cells exhibit enhanced resistance to antifungals including fluconazole (FLC), five flucytosine (5FC), and sub-therapeutic amphotericin B (AMB). Additionally, they are more resistant to macrophage and neutrophil killing. This is relevant because older fungal cells accumulate in chronic infections. We hypothesized that older C. auris cells would also exhibit enhanced resistance to phagocytic cells and can alter drug resistance. Magnetically labeled cells were grown for 10 generations (Gen) and then were isolated from daughter cells by magnetic column separation. Transmission electron microscopy (TEM) was performed to measure their cell wall thickness. Standard in vitro killing assays were performed with human neutrophils. In vivo virulence was compared in a Galleria injection model. Minimum inhibitory concentration to the antifungals was performed by CSLI methods. Rhodamine 6G (R6G), which is a fluorescent substrate for ABC-transporters, was used to measure efflux. In C. auris, 10 Gen old cells are larger than younger cells (0–3 Gen) and exhibit a thicker cell wall on TEM. The older cells are significantly more resistant to neutrophil killing (24.5% vs. 51.6%, P < 0.05 by paired t-test)). Eleven distinct isolates manifest variable virulence in the Galleria infection model (range 1–8 days). In C. auris, 10 Gen cells were significantly more virulent than 0 Gen cells (2 days vs. 4 days). Most (8 of 11) isolates were highly resistant to FLC and showed high efflux of R6G, whereas the three FLC sensitive isolates showed low efflux of R6G. Using these methods, we observed significant (1.5 to 2.0 fold, P < 0.05) increase in efflux activity of older cells in both sensitive and resistant isolates. In C. auris replicative aging is associated with enhanced resistance to phagocytic killing, increased cell body size, and cell wall thickness. Aging also enhances efflux activity and thus may contribute even further to antifungal resistance. All authors: No reported disclosures.

Similar to other yeasts, the human pathogen Candida glabrata ages when it undergoes asymmetric, finite cell divisions, which determines its replicative lifespan. We sought to investigate if and how aging changes resilience of C. glabrata populations in the host environment. Our data demonstrate that old C. glabrata are more resistant to hydrogen peroxide and neutrophil killing, whereas young cells adhere better to epithelial cell layers. Consequently, virulence of old compared to younger C. glabrata cells is enhanced in the Galleria mellonella infection model. Electron microscopy images of old C. glabrata cells indicate a marked increase in cell wall thickness. Comparison of transcriptomes of old and young C. glabrata cells reveals differential regulation of ergosterol and Hog pathway associated genes as well as adhesion proteins, and suggests that aging is accompanied by remodeling of the fungal cell wall. Biochemical analysis supports this conclusion as older cells exhibit a qualitatively different lipid composition, leading to the observed increased emergence of fluconazole resistance when grown in the presence of fluconazole selection pressure. Older C. glabrata cells accumulate during murine and human infection, which is statistically unlikely without very strong selection. Therefore, we tested the hypothesis that neutrophils constitute the predominant selection pressure in vivo. When we altered experimentally the selection pressure by antibody-mediated removal of neutrophils, we observed a significantly younger pathogen population in mice. Mathematical modeling confirmed that differential selection of older cells is sufficient to cause the observed demographic shift in the fungal population. Hence our data support the concept that pathogenesis is affected by the generational age distribution of the infecting C. glabrata population in a host. We conclude that replicative aging constitutes an emerging trait, which is selected by the host and may even play an unanticipated role in the transition from a commensal to a pathogen state.

ABSTRACT Cryptococcus neoformans is a facultative intracellular fungal pathogen that has a polysaccharide capsule and causes life-threatening meningoencephalitis. Its capsule, as well as its ability to survive in the acidic environment of the phagolysosome, contributes to the pathogen's resilience in the host environment. Previously, we reported that downregulation of allergen 1 ( ALL1 ) results in the secretion of a shorter, more viscous exopolysaccharide with less branching and structural complexity, as well as altered iron homeostasis. Now, we report on a homologous coregulated gene, allergen 2 ( ALL2 ). ALL2 's function was characterized by generating null mutants in C. neoformans . In contrast to ALL1 , loss of ALL2 attenuated virulence in the pulmonary infection model. The all2 Δ mutant shed a less viscous exopolysaccharide and exhibited higher sensitivity to hydrogen peroxide than the wild type, and as a result, the all2 Δ mutant was more resistant to macrophage-mediated killing. Transcriptome analysis further supported the distinct function of these two genes. Unlike ALL1 's involvement in iron homeostasis, we now present data on ALL2 's unique function in maintaining intracellular pH in low-pH conditions. Thus, our data highlight that C. neoformans , a human-pathogenic basidiomycete, has evolved a unique set of virulence-associated genes that contributes to its resilience in the human niche.

The pathogenic fungus, Cryptococcus neoformans, is known to undergo phenotypic variation, which affects its virulence in the host. Recent investigations on C. neoformans cells in humans have validated the concept that phenotypic variation is present and relevant for the outcome of chronic cryptococcosis. The C. neoformans capsule is not the only trait that varies among strains. An emerging variant is the "old cell phenotype" generated when C. neoformans undergoes replicative aging. This phenotype, which other than larger size also exhibits a thickened cell wall, inhibits phagocytosis and killing by antifungals in vitro. In concert with the finding that old cells accumulate in vivo, this emergent trait could have significant impact on cryptococcal virulence and infection, and contribute to treatment failure.

Cryptococcus neoformans, similar to other eukaryotes, undergoes replicative aging. Replicative life spans have been determined for clinical C. neoformans strains, and although they are a reproducible trait, life spans vary considerably among strains. C. neoformans has been proposed as an ideal model organism to investigate the contribution of replicative aging in a fungal pathogen population to emerging phenotypic variation during chronic cryptococcal infections. C. neoformans cells of advanced generational age manifest a distinct phenotype; specifically, a larger cell size, a thicker cell wall, drug resistance, as well as resistance to hydrogen peroxide-mediated killing. Consequently, old cells are selected in the host environment during chronic infection and aging could be an unanticipated mechanism of pathogen adaptation that contributes to persistent disease. Aging as a natural process of phenotypic variation should be further studied as it likely is also relevant for other eukaryotic pathogen populations that undergo asymmetric replicative aging.

Aging affects all organisms, from unicellular yeasts to multicellular humans. Studies in model organisms demonstrate that the pathways that mediate the two forms of aging, replicative and chronological, are highly conserved. Most studies are focused on the effect of aging on an individual cell rather than a whole population. Complex longevity regulation, however, makes aging a highly adaptive trait that is subject to natural selection. Recent studies have shed light on the potential relevance of aging in fungal pathogens, which undergo replicative aging when they expand in the host environment. Hence, pathogens causing chronic infections can constitute ideal model organisms in unraveling the contribution of selection to aging within a population and help elucidate the contribution of aging itself to the virulence of infections.

ABSTRACT Does cell age matter in virulence? The emergence of persister cells during chronic infections is critical for persistence of infection, but little is known how this occurs. Here, we demonstrate for the first time that the replicative age of the fungal pathogen Cryptococcus neoformans contributes to persistence during chronic meningoencephalitis. Generationally older C. neoformans cells are more resistant to hydrogen peroxide stress, macrophage intracellular killing, and antifungal agents. Older cells accumulate in both experimental rat infection and in human cryptococcosis. Mathematical modeling supports the concept that the presence of older C. neoformans cells emerges from in vivo selection pressures. We propose that advanced replicative aging is a new unanticipated virulence trait that emerges during chronic fungal infection and facilitates persistence. Therapeutic interventions that target old cells could help in the clearance of chronic infections. IMPORTANCE Our findings that the generational age of Cryptococcus neoformans cells matters in pathogenesis introduces a novel concept to eukaryotic pathogenesis research. We propose that emerging properties of aging C. neoformans cells and possibly also other fungal pathogens contribute to persistence and virulence. Whereas the replicative life span of strains may not matter for virulence per se, age-related resilience and thus the generational age of individual C. neoformans cells within a pathogen population could greatly affect persistence of the pathogen population and therefore impact outcome.