H3K4 methylation regulates development, DNA repair, and virulence in Mucorales.

Mucorales are basal fungi that opportunistically cause a potentially fatal infection known as mucormycosis (black fungus disease), which poses a significant threat to human health due to its high mortality rate and its recent association with SARS-CoV-2 infections. On the other hand, histone methylation is a regulatory mechanism with pleiotropic effects, including the virulence of several pathogenic fungi. However, the role of epigenetic changes at the histone level never has been studied in Mucorales. Here, we dissected the functional role of Set1, a histone methyltransferase that catalyzes the methylation of H3K4, which is associated with the activation of gene transcription and virulence. A comparative analysis of the Mucor lusitanicus genome (previously known as Mucor circinelloides f. lusitanicus) identified only one homolog of Set1 from Candida albicans and Saccharomyces cerevisiae that contains the typical SET domain. Knockout strains in the gene set1 lacked H3K4 monomethylation, dimethylation, and trimethylation enzymatic activities. These strains also showed a significant reduction in vegetative growth and sporulation. Additionally, set1 null strains were more sensitive to SDS, EMS, and UV light, indicating severe impairment in the repair process of the cell wall and DNA lesions and a correlation between Set1 and these processes. During pathogen-host interactions, strains lacking the set1 gene exhibited shortened polar growth within the phagosome and attenuated virulence both in vitro and in vivo. Our findings suggest that the histone methyltransferase Set1 coordinates several cell processes related to the pathogenesis of M. lusitanicus and may be an important target for future therapeutic strategies against mucormycosis.

Synthesis of novel benzylamine antimycotics and evaluation of their antimycotic potency.

A series of 23 novel benzylamines was synthesized by reductive amination from halogen-substituted 3- and 4-benzyloxybenzaldehyde derivatives and 6-methylhept-2-yl amine or n-octylamine. The antimycotic activity of the resulting amines was evaluated in a microdilution assay against the apathogenic yeast Yarrowia lipolytica as test microorganism. Promising compounds were also tested against human pathogenic Candida species. The influence of halogen substituents at the benzyl ether side chain was studied in this screening, as well as the influence of the branched side chain of (±)-6-methylhept-2-yl amine in comparison with the n-octyl side chain.

Alternative in-vivo models of mucormycosis.

Mucormycosis is still regarded a rare fungal infection, but the high incidences of COVID-associated cases in India and other countries have shown its potential threat to large patient cohorts. In addition, infections by these fast-growing fungi are often fatal and cause disfigurement, badly affecting patients' lives. In advancing our understanding of pathogenicity factors involved in this disease, to enhance the diagnostic toolset and to evaluate novel treatment regimes, animal models are indispensable. As ethical and practical considerations typically favor the use of alternative model systems, this review provides an overview of alternative animal models employed for mucormycosis and discusses advantages and limitations of the respective model.

Blood Serum Stimulates the Virulence Potential of Mucorales through Enhancement in Mitochondrial Oxidative Metabolism and Rhizoferrin Production.

This study analyzed the role of blood serum in enhancing the mitochondrial metabolism and virulence of Mucorales through rhizoferrin secretion. We observed that the spores of clinically relevant Mucorales produced in the presence of serum exhibited higher virulence in a heterologous infection model of Galleria mellonella. Cell-free supernatants of the culture broth obtained from spores produced in serum showed increased toxicity against Caenorhabditis elegans, which was linked with the enhanced secretion of rhizoferrin. Spores from Mucoralean species produced or germinated in serum showed increased respiration rates and reactive oxygen species levels. The addition of non-lethal concentrations of potassium cyanide and N-acetylcysteine during the aerobic or anaerobic growth of Mucorales decreased the toxicity of the cell-free supernatants of the culture broth, suggesting that mitochondrial metabolism is important for serum-induced virulence. In support of this hypothesis, a mutant strain of Mucor lusitanicus that lacks fermentation and solely relies on oxidative metabolism exhibited virulence levels comparable to those of the wild-type strain under serum-induced conditions. Contrary to the lower virulence observed, even in the serum, the ADP-ribosylation factor-like 2 deletion strain exhibited decreased mitochondrial activity. Moreover, spores produced in the serum of M. lusitanicus and Rhizopus arrhizus that grew in the presence of a mitophagy inducer showed low virulence. These results suggest that serum-induced mitochondrial activity increases rhizoferrin levels, making Mucorales more virulent.

Whole-genome sequencing confirms a persistent candidaemia clonal outbreak due to multidrug-resistant Candida parapsilosis.

OBJECTIVES: Although perceived as a rare clinical entity, recent studies have noted the emergence of MDR C. parapsilosis (MDR-Cp) isolates from single patients (resistant to both azole and echinocandins). We previously reported a case series of MDR-Cp isolates carrying a novel FKS1R658G mutation. Herein, we identified an echinocandin-naive patient infected with MDR-Cp a few months after the previously described isolates. WGS and CRISPR-Cas9 editing were used to explore the origin of the new MDR-Cp isolates, and to determine if the novel mutation confers echinocandin resistance. METHODS: WGS was applied to assess the clonality of these isolates and CRISPR-Cas9 editing and a Galleria mellonella model were used to examine whether FKS1R658G confers echinocandin resistance. RESULTS: Fluconazole treatment failed, and the patient was successfully treated with liposomal amphotericin B (LAMB). WGS proved that all historical and novel MDR-Cp strains were clonal and distant from the fluconazole-resistant outbreak cluster in the same hospital. CRISPR-Cas9 editing and G. mellonella virulence assays confirmed that FKS1R658G confers echinocandin resistance in vitro and in vivo. Interestingly, the FKS1R658G mutant showed a very modest fitness cost compared with the parental WT strain, consistent with the persistence of the MDR-Cp cluster in our hospital. CONCLUSIONS: Our study showcases the emergence of MDR-Cp isolates as a novel threat in clinical settings, which undermines the efficacy of the two most widely used antifungal drugs against candidiasis, leaving only LAMB as a last resort. Additionally, surveillance studies and WGS are warranted to effectively establish infection control and antifungal stewardship strategies.

Candida parapsilosis isolates carrying mutations outside FKS1 hotspot regions confer high echinocandin tolerance and facilitate the development of echinocandin resistance.

Candida parapsilosis is a significant cause of candidemia worldwide. Echinocandin-resistant (ECR) and echinocandin-tolerant (ECT) C. parapsilosis isolates have been reported in various countries but are rare. Resistance and tolerance are predominantly caused by mutations related to the hotspot (HS) regions of the FKS1 gene. A relatively high proportion of clinical C. parapsilosis isolates carrying mutations outside the HS regions has been noted in some studies, but an association with echinocandin (EC) resistance or tolerance was not explored. Herein, CRISPR-Cas9 was used and the association between amino acid substitution in FKS1 outside HS 1/2 (V595I, S745L, M1328I, F1386S, and A1422G) with EC susceptibility profile was delineated. None of the mutations conferred EC resistance, but they resulted in a significantly higher level of EC tolerance than the parental isolate, ATCC 22019. When incubated on agar plates containing ECs, specifically caspofungin and micafungin, ECR colonies were exclusively observed among ECT isolates, particularly mutants carrying V595I, S745L, and F1386S. Additionally, mutants had significantly better growth rates in yeast extract peptone dextrose (YPD) and YPD containing agents inducing membrane and oxidative stresses. The mutants had a trivial fitness cost in the Galleria mellonella model relative to ATCC 22019. Collectively, this study supports epidemiological studies to catalog mutations occurring outside the HS regions of FKS1, even if they do not confer EC resistance. These mutations are important as they potentially confer a higher level of EC tolerance and a higher propensity to develop EC resistance, therefore unveiling a novel mechanism of EC tolerance in C. parapsilosis. The identification of EC tolerance in C. parapsilosis may have direct clinical benefit in patient management.

cotH Genes Are Necessary for Normal Spore Formation and Virulence in Mucor lusitanicus.

Mucormycosis is an invasive fungal infection caused by certain members of the fungal order of Mucorales. The species most frequently identified as the etiological agents of mucormycosis belong to the genera Rhizopus, Lichtheimia, and Mucor. The frequency of systemic mucormycosis has been increasing, mainly because of increasing numbers of susceptible patients. Furthermore, Mucorales display intrinsic resistance to the majority of routinely used antifungal agents (e.g., echinocandins and short-tailed azoles), which limits the number of possible therapeutic options. All the above-mentioned issues urge the improvement of molecular identification methods and the discovery of new antifungal targets and strategies. Spore coat proteins (CotH) constitute a kinase family present in many pathogenic bacteria and fungi and participate in the spore formation in these organisms. Moreover, some of them can act as virulence factors being receptors of the human GRP78 protein during Rhizopus delemar-induced mucormycosis. We identified 17 cotH-like genes in the Mucor lusitanicus genome database. Successful disruption of five cotH genes in Mucor was performed using the CRISPR-Cas9 system. The CotH3 and CotH4 proteins play a role in adaptation to different temperatures as well as in developing the cell wall structure. We also show CotH4 protein is involved in spore wall formation by affecting the total chitin content and, thus, the composition of the spore wall. The role of CotH3 and CotH4 proteins in virulence was confirmed in two invertebrate models and a diabetic ketoacidosis (DKA) mouse model. IMPORTANCE Current treatment options for mucormycosis are inadequate, resulting in high mortality rates, especially among immunosuppressed patients. The development of novel therapies for mucormycosis has been hampered by lack of understanding of the pathogenetic mechanisms. The importance of the cell surface CotH proteins in the pathogenesis of Rhizopus-mediated mucormycosis has been recently described. However, the contribution of this family of proteins to the virulence of other mucoralean fungi and their functionality in vital processes remain undefined. Through the use of the CRISPR-Case9 gene disruption system, we demonstrate the importance of several of the CotH proteins to the virulence of Mucor lusitanicus by using three infection models. We also report on the importance of one of these proteins, CotH4, to spore wall formation by affecting chitin content. Therefore, our studies extend the importance of CotH proteins to Mucor and identify the mechanism by which one of the CotH proteins contributes to the development of a normal fungal cell wall, thereby indicating that this family of proteins can be targeted for future development of novel therapeutic strategies of mucormycosis.

Aspergillus terreus and the Interplay with Amphotericin B: from Resistance to Tolerance?

Aspergillus terreus is an opportunistic causative agent of invasive aspergillosis and, in most cases, it is refractory to amphotericin B (AMB) therapy. Notably, AMB-susceptible Aspergillus terreus sensu stricto (s.s.) representatives exist which are also associated with poor clinical outcomes. Such findings may be attributable to drug tolerance, which is not detectable by antifungal susceptibility testing. Here, we tested in vitro antifungal susceptibility (AFST) and the fungicidal activity of AMB against 100 clinical isolates of A. terreus species complex in RPMI 1640 and antibiotic medium 3 (AM3). MICs ranged from 0.5 to 16 µg/mL for RPMI 1640 and from 1 to >16 mg/L for AM3. AMB showed medium-dependent activity, with fungicidal effects only in antibiotic medium 3, not in RPMI 1640. Furthermore, the presence of AMB-tolerant phenotypes of A. terreus has been examined by assessing the minimum duration for killing 99% of the population (MDK99) and evaluating the data obtained in a Galleria mellonella infection model. A time-kill curve analysis revealed that A. terreus with AMB MICs of ≤1 mg/L (susceptible range) displayed AMB-tolerant phenotypes, exhibiting MDK99s at 18 and 36 h, respectively. Survival rates of infected G. mellonella highlighted that AMB was effective against susceptible A. terreus isolates, but not against tolerant or resistant isolates. Our analysis reveals that A. terreus isolates which are defined as susceptible based on MIC may comprise tolerant phenotypes, which may, in turn, explain the worse outcome of AMB therapy for phenotypically susceptible isolates.

Galleria mellonella Larvae as a Model for Investigating Fungal-Host Interactions.

Galleria mellonella larvae have become a widely accepted and utilised infection model due to the functional homology displayed between their immune response to infection and that observed in the mammalian innate immune response. Due to these similarities, comparable results to murine studies can be obtained using G. mellonella larvae in assessing the virulence of fungal pathogens and the in vivo toxicity or efficacy of anti-fungal agents. This coupled with their low cost, rapid generation of results, and lack of ethical/legal considerations make this model very attractive for analysis of host-pathogen interactions. The larvae of G. mellonella have successfully been utilised to analyse various fungal virulence factors including toxin and enzyme production in vivo providing in depth analysis of the processes involved in the establishment and progression of fungal pathogens (e.g., Candida spps, Aspergillus spp., Madurella mycetomatis, Mucormycetes, and Cryptococcus neoformans). A variety of experimental endpoints can be employed including analysis of fungal burdens, alterations in haemocyte density or sub-populations, melanisation, and characterisation of infection progression using proteomic, histological or imaging techniques. Proteomic analysis can provide insights into both sides of the host-pathogen interaction with each respective proteome being analysed independently following infection and extraction of haemolymph from the larvae. G. mellonella can also be employed for assessing the efficacy and toxicity of antifungal strategies at concentrations comparable to those used in mammals allowing for early stage investigation of novel compounds and combinations of established therapeutic agents. These numerous applications validate the model for examination of fungal infection and development of therapeutic approaches in vivo in compliance with the need to reduce animal models in biological research.

Synthesis, Biological Evaluation, and Structure-Activity Relationships of 4-Aminopiperidines as Novel Antifungal Agents Targeting Ergosterol Biosynthesis.

The aliphatic heterocycles piperidine and morpholine are core structures of well-known antifungals such as fenpropidin and fenpropimorph, commonly used as agrofungicides, and the related morpholine amorolfine is approved for the treatment of dermal mycoses in humans. Inspired by these lead structures, we describe here the synthesis and biological evaluation of 4-aminopiperidines as a novel chemotype of antifungals with remarkable antifungal activity. A library of more than 30 4-aminopiperidines was synthesized, starting from N-substituted 4-piperidone derivatives by reductive amination with appropriate amines using sodium triacetoxyborohydride. Antifungal activity was determined on the model strain Yarrowia lipolytica, and some compounds showed interesting growth-inhibiting activity. These compounds were tested on 20 clinically relevant fungal isolates (Aspergillus spp., Candida spp., Mucormycetes) by standardized microbroth dilution assays. Two of the six compounds, 1-benzyl-N-dodecylpiperidin-4-amine and N-dodecyl-1-phenethylpiperidin-4-amine, were identified as promising candidates for further development based on their in vitro antifungal activity against Candida spp. and Aspergillus spp. Antifungal activity was determined for 18 Aspergillus spp. and 19 Candida spp., and their impact on ergosterol and cholesterol biosynthesis was determined. Toxicity was determined on HL-60, HUVEC, and MCF10A cells, and in the alternative in vivo model Galleria mellonella. Analysis of sterol patterns after incubation gave valuable insights into the putative molecular mechanism of action, indicating inhibition of the enzymes sterol C14-reductase and sterol C8-isomerase in fungal ergosterol biosynthesis.

Antifungal Siderophore Conjugates for Theranostic Applications in Invasive Pulmonary Aspergillosis Using Low-Molecular TAFC Scaffolds.

Invasive pulmonary aspergillosis (IPA) is a life-threatening form of fungal infection, primarily in immunocompromised patients and associated with significant mortality. Diagnostic procedures are often invasive and/or time consuming and existing antifungals can be constrained by dose-limiting toxicity and drug interaction. In this study, we modified triacetylfusarinine C (TAFC), the main siderophore produced by the opportunistic pathogen Aspergillus fumigatus (A. fumigatus), with antifungal molecules to perform antifungal susceptibility tests and molecular imaging. A variation of small organic molecules (eflornithine, fludioxonil, thiomersal, fluoroorotic acid (FOA), cyanine 5 (Cy5) with antifungal activity were coupled to diacetylfusarinine C (DAFC), resulting in a "Trojan horse" to deliver antifungal compounds specifically into A. fumigatus hyphae by the major facilitator transporter MirB. Radioactive labeling with gallium-68 allowed us to perform in vitro characterization (distribution coefficient, stability, uptake assay) as well as biodistribution experiments and PET/CT imaging in an IPA rat infection model. Compounds chelated with stable gallium were used for antifungal susceptibility tests. [Ga]DAFC-fludioxonil, -FOA, and -Cy5 revealed a MirB-dependent active uptake with fungal growth inhibition at 16 µg/mL after 24 h. Visualization of an A. fumigatus infection in lungs of a rat was possible with gallium-68-labeled compounds using PET/CT. Heterogeneous biodistribution patterns revealed the immense influence of the antifungal moiety conjugated to DAFC. Overall, novel antifungal siderophore conjugates with promising fungal growth inhibition and the possibility to perform PET imaging combine both therapeutic and diagnostic potential in a theranostic compound for IPA caused by A. fumigatus.

The impact of episporic modification of Lichtheimia corymbifera on virulence and interaction with phagocytes.

Fungal infections caused by the ancient lineage Mucorales are emerging and increasingly reported in humans. Comprehensive surveys on promising attributes from a multitude of possible virulence factors are limited and so far, focused on Mucor and Rhizopus. This study addresses a systematic approach to monitor phagocytosis after physical and enzymatic modification of the outer spore wall of Lichtheimia corymbifera, one of the major causative agents of mucormycosis. Episporic modifications were performed and their consequences on phagocytosis, intracellular survival and virulence by murine alveolar macrophages and in an invertebrate infection model were elucidated. While depletion of lipids did not affect the phagocytosis of both strains, delipidation led to attenuation of LCA strain but appears to be dispensable for infection with LCV strain in the settings used in this study. Combined glucano-proteolytic treatment was necessary to achieve a significant decrease of virulence of the LCV strain in Galleria mellonella during maintenance of the full potential for spore germination as shown by a novel automated germination assay. Proteolytic and glucanolytic treatments largely increased phagocytosis compared to alive resting and swollen spores. Whilst resting spores barely (1-2%) fuse to lysosomes after invagination in to phagosomes, spore trypsinization led to a 10-fold increase of phagolysosomal fusion as measured by intracellular acidification. This is the first report of a polyphasic measurement of the consequences of episporic modification of a mucormycotic pathogen in spore germination, spore surface ultrastructure, phagocytosis, stimulation of Toll-like receptors (TLRs), phagolysosomal fusion and intracellular acidification, apoptosis, generation of reactive oxygen species (ROS) and virulence.

Efficacy of LAMB against Emerging Azole- and Multidrug-Resistant Candida parapsilosis Isolates in the Galleria mellonella Model.

While being the third leading cause of candidemia worldwide, numerous studies have shown severe clonal outbreaks due to fluconazole-resistant (FLCR) Candida parapsilosis isolates associated with fluconazole therapeutic failure (FTF) with enhanced mortality. More recently, multidrug resistant (MDR) C. parapsilosis blood isolates have also been identified that are resistant to both azole and echinocandin drugs. Amphotericin B (AMB) resistance is rarely reported among C. parapsilosis isolates and proper management of bloodstream infections due to FLZR and MDR isolates requires prompt action at the time of outbreak. Therefore, using a well-established Galleria mellonella model, we assessed whether (a) laboratory-based findings on azole or echinocandin (micafungin) resistance in C. parapsilosis lead to therapeutic failure, (b) LAMB could serve as an efficient salvage treatment option, and (c) distinct mutations in ERG11 impact mortality. Our in vivo data confirm fluconazole inefficacy against FLCR C. parapsilosis isolates carrying Y132F, Y132F + K143R, Y132F + G307A, and G307A + G458S in Erg11p, while LAMB proved to be an efficacious accessible option against both FLCR and MDR C. parapsilosis isolates. Moreover, positive correlation of in vitro and in vivo data further highlights the utility of G. melonella as a reliable model to investigate azole and polyene drug efficacy.

Arginine Auxotrophy Affects Siderophore Biosynthesis and Attenuates Virulence of Aspergillus fumigatus.

Aspergillus fumigatus is an opportunistic human pathogen mainly infecting immunocompromised patients. The aim of this study was to characterize the role of arginine biosynthesis in virulence of A. fumigatus via genetic inactivation of two key arginine biosynthetic enzymes, the bifunctional acetylglutamate synthase/ornithine acetyltransferase (argJ/AFUA_5G08120) and the ornithine carbamoyltransferase (argB/AFUA_4G07190). Arginine biosynthesis is intimately linked to the biosynthesis of ornithine, a precursor for siderophore production that has previously been shown to be essential for virulence in A. fumigatus. ArgJ is of particular interest as it is the only arginine biosynthetic enzyme lacking mammalian homologs. Inactivation of either ArgJ or ArgB resulted in arginine auxotrophy. Lack of ArgJ, which is essential for mitochondrial ornithine biosynthesis, significantly decreased siderophore production during limited arginine supply with glutamine as nitrogen source, but not with arginine as sole nitrogen source. In contrast, siderophore production reached wild-type levels under both growth conditions in ArgB null strains. These data indicate that siderophore biosynthesis is mainly fueled by mitochondrial ornithine production during limited arginine availability, but by cytosolic ornithine production during high arginine availability via cytosolic arginine hydrolysis. Lack of ArgJ or ArgB attenuated virulence of A. fumigatus in the insect model Galleria mellonella and in murine models for invasive aspergillosis, indicating limited arginine availability in the investigated host niches.

RcLS2F - A Novel Fungal Class 1 KDAC Co-repressor Complex in Aspergillus nidulans.

The fungal class 1 lysine deacetylase (KDAC) RpdA is a promising target for prevention and treatment of invasive fungal infection. RpdA is essential for survival of the most common air-borne mold pathogen Aspergillus fumigatus and the model organism Aspergillus nidulans. In A. nidulans, RpdA depletion induced production of previously unknown small bioactive substances. As known from yeasts and mammals, class 1 KDACs act as components of multimeric protein complexes, which previously was indicated also for A. nidulans. Composition of these complexes, however, remained obscure. In this study, we used tandem affinity purification to characterize different RpdA complexes and their composition in A. nidulans. In addition to known class 1 KDAC interactors, we identified a novel RpdA complex, which was termed RcLS2F. It contains ScrC, previously described as suppressor of the transcription factor CrzA, as well as the uncharacterized protein FscA. We show that recruitment of FscA depends on ScrC and we provide clear evidence that ΔcrzA suppression by ScrC depletion is due to a lack of transcriptional repression caused by loss of the novel RcLS2F complex. Moreover, RcLS2F is essential for sexual development and engaged in an autoregulatory feed-back loop.

The leucine biosynthetic pathway is crucial for adaptation to iron starvation and virulence in Aspergillus fumigatus.

In contrast to mammalia, fungi are able to synthesize the branched-chain amino acid leucine de novo. Recently, the transcription factor LeuB has been shown to cross-regulate leucine biosynthesis, nitrogen metabolism and iron homeostasis in Aspergillus fumigatus, the most common human mold pathogen. Moreover, the leucine biosynthetic pathway intermediate α-isopropylmalate (α-IPM) has previously been shown to posttranslationally activate LeuB homologs in S. cerevisiae and A. nidulans. Here, we demonstrate that in A. fumigatus inactivation of both leucine biosynthetic enzymes α-IPM synthase (LeuC), which disrupts α-IPM synthesis, and α-IPM isomerase (LeuA), which causes cellular α-IPM accumulation, results in leucine auxotrophy. However, compared to lack of LeuA, lack of LeuC resulted in increased leucine dependence, a growth defect during iron starvation and decreased expression of LeuB-regulated genes including genes involved in iron acquisition. Lack of either LeuA or LeuC decreased virulence in an insect infection model, and inactivation of LeuC rendered A. fumigatus avirulent in a pulmonary aspergillosis mouse model. Taken together, we demonstrate that the lack of two leucine biosynthetic enzymes, LeuA and LeuC, results in significant phenotypic consequences indicating that the regulator LeuB is activated by α-IPM in A. fumigatus and that the leucine biosynthetic pathway is an attractive target for the development of antifungal drugs.

Minimal Inhibitory Concentration (MIC)-Phenomena in Candida albicans and Their Impact on the Diagnosis of Antifungal Resistance.

Antifungal susceptibility testing (AFST) of clinical isolates is a tool in routine diagnostics to facilitate decision making on optimal antifungal therapy. The minimal inhibitory concentration (MIC)-phenomena (trailing and paradoxical effects (PXE)) observed in AFST complicate the unambiguous and reproducible determination of MICs and the impact of these phenomena on in vivo outcome are not fully understood. We aimed to link the MIC-phenomena with in vivo treatment response using the alternative infection model Galleria mellonella. We found that Candida albicans strains exhibiting PXE for caspofungin (CAS) had variable treatment outcomes in the Galleria model. In contrast, C. albicans strains showing trailing for voriconazole failed to respond in vivo. Caspofungin- and voriconazole-susceptible C. albicans strains responded to the respective antifungal therapy in vivo. In conclusion, MIC data and subsequent susceptibility interpretation of strains exhibiting PXE and/or trailing should be carried out with caution, as both effects are linked to drug adaptation and treatment response is uncertain to predict.

Hypoxia Decreases Diagnostic Biomarkers for Aspergillosis In Vitro.

The aim of the study was to evaluate the influence of hypoxia on galactomannan and (1,3)-ß-d-glucan release of clinically relevant Aspergilli in vitro. Hypoxia decreased biomass and consequently led to lower biomarker release. However, when normalized to biomass, hypoxia led to increased levels of biomarkers at early growth stages (24 h). Antifungals (amphotericin B and voriconazole) decreased the galactomannan amount of A. fumigatus, even more prominently in hypoxia.

Cryptic species of Aspergillus section Terrei display essential physiological features to cause infection and are similar in their virulence potential in Galleria mellonella.

Aspergillus species account for the majority of invasive mold infections in immunocompromised patients. Most commonly, members of the Aspergillus section Fumigati are isolated from clinical material, followed by isolates belonging to section Terrei. The section Terrei contains 16 accepted species. Six species were found to be of clinical relevance and studied for differences in growth adaptability and virulence potential. Therefore, a set of 73 isolates (22 A. terreus s.s., 8 A. alabamensis, 27 A. citrinoterreus, 2 A. floccosus, 13 A. hortai, and 1 A. neoafricanus) was studied to determine differences in (a) germination kinetics, (b) temperature tolerance, (c) oxygen stress tolerance (1% O2), and (d) a combination of the latter two. Virulence potential of phialidic (PC) and accessory conidia (AC) was studied in G. mellonella larvae, using survival as read out. Further, the formation of AC was evaluated in larval tissue. All isolates were able to grow at elevated temperature and hypoxia, with highest growth and germination rates at 37°C. A. terreus s.s., A. citrinoterreus, and A. hortai exhibited highest growth rates. Virulence potential in larvae was inoculum and temperature dependent. All species except A. floccosus formed AC and germination kinetics of AC was variable. Significantly higher virulence potential of AC was found for one A. hortai isolate. AC could be detected in larval tissue 96 h post infection. Based on these findings, cryptic species of section Terrei are well adapted to the host environment and have similar potential to cause infections.

Precise genome editing using a CRISPR-Cas9 method highlights the role of CoERG11 amino acid substitutions in azole resistance in Candida orthopsilosis.

BACKGROUND: Azoles are one of the main antifungal classes for the treatment of candidiasis. In the current context of emerging drug resistance, most studies have focused on Candida albicans, Candida glabrata or Candida auris but, so far, less is known about the underlying mechanisms of resistance in other species, including Candida orthopsilosis. OBJECTIVES: We investigated azole resistance in a C. orthopsilosis clinical isolate recovered from a patient with haematological malignancy receiving fluconazole prophylaxis. METHODS: Antifungal susceptibility to fluconazole was determined in vitro (CLSI M27-A3) and in vivo (in a Galleria mellonella model of invasive candidiasis). The CoERG11 gene was then sequenced and amino acid substitutions identified were mapped on the predicted 3D structure of CoErg11p. A clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) genome-editing strategy was used to introduce relevant mutations into a fluconazole-susceptible C. orthopsilosis isolate. RESULTS: Compared with unrelated C. orthopsilosis isolates, the clinical isolate exhibited both in vitro and in vivo fluconazole resistance. Sequencing of the CoERG11 gene identified several amino acid substitutions, including two possibly involved in fluconazole resistance (L376I and G458S). Both mutations mapped close to the active site of CoErg11p. Engineering these mutations in a different genetic background using CRISPR-Cas9 demonstrated that G458S, but not L376I, confers resistance to fluconazole and voriconazole. CONCLUSIONS: Our data show that the G458S amino acid substitution in CoERG11p, but not L376I, contributes to azole resistance in C. orthopsilosis. In addition to highlighting the potential of CRISPR-Cas9 technology for precise genome editing in the field of antifungal resistance, we discuss some points that are critical to improving its efficiency.