Welcome to IMC 2018 International Mycological Congress
RNAi-dependent epimutations evoke transient antifungal drug resistance
- J. Heitman
Microorganisms evolve via sexual/parasexual reproduction, mutators, aneuploidy, Hsp90, or prions. Mechanisms that are detrimental can be repurposed to generate diversity. Microbes are known to evolve resistance to antimicrobial agents via pathways involving both stable and unstable genetic mechanisms, such as aneuploidy underlying azole resistance in Candida albicans and Cryptococcus neoformans. We discovered a new mechanism conferring antifungal drug resistance in the human fungal pathogen Mucor circinelloides. Spontaneous resistance to the antifungal drug FK506 was found to evolve via two distinct mechanisms. One involves Mendelian mutations conferring stable irreversible drug resistance; the other occurs via an epigenetic RNA interference (RNAi)-mediated pathway resulting in unstable, transient drug resistance. The peptidyl-prolyl isomerase FKBP12 interacts with FK506 forming a complex that inhibits the protein phosphatase calcineurin. Calcineurin inhibition by FK506 blocks M. circinelloides dimorphic transition to hyphae and enforces growth as yeasts. In some FK506 resistant isolates, mutations in the fkbA gene encoding FKBP12 or the calcineurin cnbR or cnaA genes confer FK506 resistance and restore hyphal growth. In other resistant isolates, no mutations are found in the known drug targets. Instead, RNAi has been triggered to silence the fkbA gene, yielding drug-resistant epimutants. FK506-resistant epimutants readily reverted to drug-sensitivity in the absence of FK506. The establishment of epimutants is accompanied by generation of abundant fkbA small RNAs and requires some known RNAi pathway components whereas others are dispensable. Surprisingly, epimutants occur at a higher frequency and are more stable in mutants lacking RNA-dependent RNA polymerase 1 (Rdrp1), revealing some RNAi components constrain or reverse epimutation. Silencing of the drug target FKBP12 appears to involve generation of a double-stranded RNA trigger intermediate using the fkbA mature mRNA as template to produce antisense fkbA RNA. Epimutational silencing may be stochastic, similar to Mendelian mutations, but differs as the altered phenotype is reversible in response to fluctuating environmental conditions. Our recent studies reveal novel components required for epimutation, including orthologs of the Neurospora crassa quelling inducing protein (QIP) and Sad-3 helicase (RnhA); interestingly, the rnhA gene is linked to the Mucor sex locus, suggesting sexual reproduction may activate epimutation similar to sex-induced-silencing in Cryptococcus. We found epimutants occur at a higher frequency in mutants lacking RNA-dependent RNA polymerase 3 (Rdrp3) or the RNaseIII-like protein R3B2. Rdrp1, Rdrp3, and R3B2 operate a non-canonical RNA degradation pathway suppressing RNAi-dependent epimutation by competition for targets. We generalized these findings by showing epimutations occur in a second species of Mucor, and identifying epimutations in the pyrF or pyrG genes conferring 5-fluoroorotic acid (5-FOA) resistance. These studies uncover a novel, reversible, transient epigenetic RNAi-based epimutation mechanism controlling phenotypic plasticity, with implications for antimicrobial drug resistance and RNAi-regulatory mechanisms in fungi and other eukaryotes. The full impact of epimutations in this and other genetic systems may have eluded discovery previously given their inherently unstable nature.
Fusarium, the trans-kingdom pathogen
- A. Van Diepeningen
Fusarium head blight, Fusarium ear rot, and vascular wilting are –despite their names- typical diseases caused by Fusarium species in different plant hosts, including many economically important plant species. Fusaria have often specialised on a certain host and in some cases this virulence seems to be organised on supernumerary chromosomes containing the essential information for pathogenicity on one or a limited number of host species. As added bonus, Fusarium is capable of the production of a large variety of mycotoxins, often acting as virulence factors, contaminating harvested products.
However, over the past years we also see Fusarium species as emerging pathogens both in human and animal. There they cause from relatively innocent, but actual quite frequent, nail and skin infections to more rare local, deep and in the growing group of immuno-compromised hosts even disseminated infections. These latter infections are connected with high mortality rates. In Fusarium, it has become customary to cluster closely related sibling species or lineages with little to no morphological differences in so-called species complexes: The opportunists on human and animal group into seven main species complexes: the Fusarium solani, F. oxysporum, F. incarnatum-equiseti, F. fujikuroi, F. clamydosporum, F. dimerum and F. sporotrichioides species complexes. In some cases the human infections can directly be linked to trauma with infected plant materials, making Fusarium a true trans-kingdom pathogen.
With the control of both plant and human pathogenic Fusarium, we are faced with several major challenges: One foremost being the limited availability of effective treatments as the species are generally very resistant to the available antifungals. Also some species prove to vary in their susceptibility to those few effective drugs available, emphasizing the need for fast identification and suitable diagnostic tools which are at the moment limited available. Additional confusing factors are that especially for some of the clinically important species there has been limited nomenclatural stability over the years, obscuring at times available data, and the recent discovery of many new species.
Exploring possible ecological niches for Coccidioides species endemic in New Mexico
- P. Hamm
- M. Hutchinson
- S. Melman
- D. Natvig
Species in the genus Coccidioides, C. posadasii and C. immitis, are the causative agents for coccidioidomycosis, the disease commonly known as Valley Fever. Coccidioidomycosis is estimated to affect more than 150,000 humans each year and is one of the few fungal diseases to affect otherwise healthy individuals. While progress has been made in the clinical understanding of the disease, little is known of the natural biology. Eighteen clinical isolates derived from 17 individuals diagnosed with coccidioidomycosis collected from New Mexico were used in a multi-locus sequencing analysis to explore genetic variation within the state and between neighboring states. While New Mexico is predicted to have C. posadasii, results of our analysis indicate that both C. immitis and C. posadasii are present among clinical isolates in New Mexico. Five of eight infections for which patient ethnicity was known occurred in Native Americans, suggesting that further studies should be conducted to determine if American Indians represent a risk group for coccidioidomycosis. We are also taking a novel approach to screen small rodents for exposure to Coccidioides by two means: 1) a survey of frozen mammal lung tissue for fungal infections, and 2) an enzyme immunoassay that detects IgG antibodies against Coccidioides in a variety of mammalian species. This will produce critical information regarding animal infection rates, the geographical distribution of infected animals, and relative spore loads in soils. Characterization of clinical and environmental isolates will allow us to understand the genetic variation that affects the virulence of these pathogens.
Emerging pathogen Candida auris evades neutrophil attack
- J. Nett
- C. Johnson
- J. Davis
- A. Huttenlocher
- J. Kernien
Introduction. Candida auris, an emerging fungal pathogen, causes hospital-associated outbreaks of invasive candidiasis with mortality near 60%. Little is known about the pathogenesis of this species that has newly arisen in the last 10 years, and it is unclear why this species is rapidly spreading worldwide. Neutrophils are critical for control of numerous invasive fungal infections, including candidiasis. These leukocytes kill fungi through phagocytosis or the release of neutrophil extracellular traps (NETs), which are structures of DNA, histones, and proteins with antimicrobial activity. Objective. The objective of this study was to delineate the neutrophil response to C. auris. We hypothesized that an ineffective neutrophil response may account for the poor outcomes observed in patients. Methods. We examined interactions of human neutrophils with C. auris and included C. albicans for comparison. Neutrophil-Candida interactions were visualized by time-lapse fluorescent microscopy and scanning electron microscopy (SEM). We utilized oxidative stress indicator CM-H2DCFDA to measure the generation of reactive oxygen species (ROS) in neutrophils. NET formation was quantified by Sytox Green staining and assessed by SEM and immunofluorescent labeling of NET-associated proteins. Fungal viability was evaluated using microbiological counts and viability stains. We utilized a zebrafish larval infection model to evaluate neutrophil-Candida interactions in vivo. Results. Imaging revealed the phagocytosis of C. albicans by human neutrophils at 1 h, followed by the formation of NETs by 4 h. In contrast, neutrophils appeared rounded upon encountering C. auris and rarely engaged in phagocytosis or produced NETs. As shown by Sytox Green staining, C. auris triggered negligible NET release by human neutrophils, with levels 7-fold lower when compared to C. albicans. C. auris did not induce neutrophils to generate ROS, a key signaling mechanism for NET formation. The ineffective neutrophil response to C. auris correlated with diminished fungal killing. Imaging of neutrophils in a zebrafish model of invasive candidiasis revealed the recruitment of approximately 50% fewer neutrophils in response to C. auris as compared to C. albicans. Conclusion. C. auris evades neutrophils by altering multiple aspects of their usual anti-candidal responses. This is linked to improved fungal survival. We propose that this diminished innate immune response may contribute to the unexpected virulence of C. auris.
Identifying fungal determinants of keratitis pathogenesis through a reverse-translational approach
- K. Fuller
- R. Cramer
- J. Dunlap
- T. Lietman
- K. Dharmalingam
- M. Zegans
AbstractFungal infections of the cornea (mycotic keratitis) are a significant cause of ocular morbidity and blindness worldwide. These infections are difficult to manage and many cases, despite treatment, result in corneal perforation or require corneal transplantation. The aim of our work, therefore, is to better understand fungal genes and pathways that drive pathogenesis in the eye, and could consequently inform novel therapeutic strategies. To this end, our multi-site collaboration is analyzing clinical data and fungal isolates from patients enrolled in the Mycotic Ulcer Treatment Trial (MUTT), a large-prospective study completed in India to compare the efficacy of natamycin vs voriconazole monotherapies. We hypothesize that (1) the fungal isolates will be heterogenous with respect to various in vitro phenotypes, and (2) some of those phenotypes will correlate with patient outcome. Such phenotypes will then serve as putative virulence determinants and will be the focus of downstream molecular analysis. In this way, our search for fungal virulence determinants can be informed by context-specific clinical data, as opposed to candidate gene analysis based on work in disparate fungal species and/or disease models (e.g. the lung).
Fusarium is the most common mold associated with keratitis both in the MUTT (50% of culture-positive cases) and in other studies, and so these isolates are the current focus our analyses. We first employed multi-locus sequencing to determine the species-level distribution across the 128 Fusarium isolates. The majority were F. solani (80%), followed by F. delphinoides (10%) and varying others. In addition to its prevalence, F. solani was also statistically associated with larger ulcer size across the patients. Initial assessment of the F. solani isolates has already revealed marked heterogeneity with respect to colonial morphology (e.g. pigmentation) and growth rates across physiological temperatures (30-37C). Ongoing efforts are aimed at screening metabolic and stress-related phenotypes, secreted protein profiles, as well as immune cell interactions. We predict that that these parameters will also vary widely across the isolates, and that correlation analyses against the clinical data will provide novel insights into Fusarium pathogenesis in the eye.
Surface proteins and the interaction of Lichtheimia corymbifera and phagocytes
- M. Hassan
- T. Krueger
- H. Dahse
- Z. Cseresnyés
- O. Kniemeyer
- T. Figge
- K. Voigt
Mucoralean fungi can cause mucormycosis, a life-threatening disease in immunocompromised patients. In our study, we analysed the influence of different enzymatic treatments of the spore surface alterations on the phagocytosis by murine alveolar macrophages. Two strains which were shown to be virulent and attenuated in avian, invertebrate and murine infection models were used in this study. The spore surface was treated with different cell wall-degrading cell wall enzymes targeting carbohydrate and protein cell wall components. The highest phagocytosis index was achieved with the proteolytic treatments which encouraged us to do focus our research on the protein surface of spores. Proteomic analysis of the spore surface was conducted for both strains. About four-teen candidate proteins were found which were differentially abundant in either the virulent strain or in the attenuated strain leading to the hypothesis that these proteins may play a role in virulence. One of these candidate proteins is the hydrophobic surface binding protein A (HsbA) which was first found in higher abundance in the virulent strain of L. corymbifera. HsbA was first described as an adhesin in Aspergillus oryzae. Additionally, HsbA was purified from the insect-killing ascomycete Beauveria bassiana and identified to play a role in immunogenicity. The HsbA protein from the virulent strain of L. corymbifera was heterologously overexpressed in Pichia pastoris. After pretreatment of murine alveolar macrophages and spores with purified fractions of the HsbA protein, the phagocytic index was found to be enhanced in comparison with unstimulated host cells. The findings presented in this study will open the door for the role of surface protein in the recognition of L. corymbifera by phagocytes of the innate immune system which raise important measures to mammalian infection models. Our prospect for the future research will focus on the identification of potential stimulatory effects of L. corymbifera surface proteins and their putative receptors on the surface of macrophages which possibly contribute to virulence.