The Caspofungin Paradoxical Effect is a Tolerant "Eagle Effect" in the Filamentous Fungal Pathogen Aspergillus fumigatus

Cell responses against antifungals other than resistance have rarely been studied in filamentous fungi, while terms such as tolerance and persistence are well-described for bacteria and increasingly examined in yeast-like organisms. Aspergillus fumigatus is a filamentous fungal pathogen that causes a disease named aspergillosis, for which caspofungin (CAS), a fungistatic drug, is used as a second-line therapy. Some A. fumigatus clinical isolates can survive and grow in CAS concentrations above the minimum effective concentration (MEC), a phenomenon known as "caspofungin paradoxical effect" (CPE). Here, we evaluated the CPE in 67 A. fumigatus clinical isolates by calculating recovery rate (RR) values, where isolates with an RR of ≥0.1 were considered CPE+ while isolates with an RR of <0.1 were classified as CPE-. Conidia produced by three CPE+ clinical isolates, CEA17 (RR = 0.42), Af293 (0.59), and CM7555 (0.38), all showed the ability to grow in high levels of CAS, while all conidia produced by the CPE- isolate IFM61407 (RR = 0.00) showed no evidence of paradoxical growth. Given the importance of the calcium/calcineurin/transcription factor-CrzA pathway in CPE regulation, we also demonstrated that all ΔcrzACEA17 (CPE+) conidia exhibited CPE while 100% of ΔcrzAAf293 (CPE-) did not exhibit CPE. Because all spores derived from an individual strain were phenotypically indistinct with respect to CPE, it is likely that CPE is a genetically encoded adaptive trait that should be considered an antifungal-tolerant phenotype. Because the RR parameter showed that the strength of the CPE was not uniform between strains, we propose that the mechanisms which govern this phenomenon are multifactorial. IMPORTANCE The "Eagle effect," initially described for bacterial species, which reflects the capacity of some strains to growth above the minimum inhibitory concentration (MIC) of specific antimicrobial agents, has been known for more than 70 years. However, its underlying mechanism of action in fungi is not fully understood and its connection with other phenomena such as tolerance or persistence is not clear yet. Here, based on the characterization of the "caspofungin paradoxical effect" in several Aspergillus fumigatus clinical isolates, we demonstrate that all conidia from A. fumigatus CPE+ strains are able to grow in high levels of the drug while all conidia produced by CPE- strains show no evidence of paradoxical growth. This work fills a gap in the understanding of this multifactorial phenomenon by proposing that CPE in A. fumigatus should be considered a tolerant but not persistent phenotype.We thank the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) grants no. 2018/00715-3 (C.V.), 2017/07536-4 (A.C.C.), 2016/12948-7 (P.A.C.), and 2016/07870-9 (G.H.G.) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) grant no. 301058/2019-9 and 404735/2018-5 (G.H.G.), both from Brazil, and the National Institutes of Health/National Institute of Allergy and Infectious Diseases (R01AI153356), from the USA. This work was also supported by the Wellcome Trust grants no. 219551/Z/19/Z and 208396/Z/17/Z to M.B.S

Functional characterization of a xylose transporter in Aspergillus nidulans

BACKGROUND: The production of bioethanol from lignocellulosic feedstocks will only become economically feasible when the majority of cellulosic and hemicellulosic biopolymers can be efficiently converted into bioethanol. The main component of cellulose is glucose, whereas hemicelluloses mainly consist of pentose sugars such as D-xylose and L-arabinose. The genomes of filamentous fungi such as A. nidulans encode a multiplicity of sugar transporters with broad affinities for hexose and pentose sugars. Saccharomyces cerevisiae, which has a long history of use in industrial fermentation processes, is not able to efficiently transport or metabolize pentose sugars (e.g. xylose). Subsequently, the aim of this study was to identify xylose-transporters from A. nidulans, as potential candidates for introduction into S. cerevisiae in order to improve xylose utilization. RESULTS: In this study, we identified the A. nidulans xtrD (xylose transporter) gene, which encodes a Major Facilitator Superfamily (MFS) transporter, and which was specifically induced at the transcriptional level by xylose in a XlnR-dependent manner, while being partially repressed by glucose in a CreA-dependent manner. We evaluated the ability of xtrD to functionally complement the S. cerevisiae EBY.VW4000 strain which is unable to grow on glucose, fructose, mannose or galactose as single carbon source. In S. cerevisiae, XtrD was targeted to the plasma membrane and its expression was able to restore growth on xylose, glucose, galactose, and mannose as single carbon sources, indicating that this transporter accepts multiple sugars as a substrate. XtrD has a high affinity for xylose, and may be a high affinity xylose transporter. We were able to select a S. cerevisiae mutant strain that had increased xylose transport when expressing the xtrD gene. CONCLUSIONS: This study characterized the regulation and substrate specificity of an A. nidulans transporter that represents a good candidate for further directed mutagenesis. Investigation into the area of sugar transport in fungi presents a crucial step for improving the S. cerevisiae xylose metabolism. Moreover, we have demonstrated that the introduction of adaptive mutations beyond the introduced xylose utilization genes is able to improve S. cerevisiae xylose metabolism.We would like to thank the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) and the Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) for providing financial support. We also thank Dr Eckardt Boles for providing the EBY.VW4000 yeast strain, Dr Ronald Hector for providing the plasmids pRH274 and pRH195, Dr Michel Flipphi for providing the Delta creA4 strain, and the two anonymous reviewers for their comments and suggestions. We also acknowledge the Program project grant GM068087 (PI Jay Dunlap) for providing the deletion cassettes

Predicting the Proteins of Angomonas deanei, Strigomonas culicis and Their Respective Endosymbionts Reveals New Aspects of the Trypanosomatidae Family

Endosymbiont-bearing trypanosomatids have been considered excellent models for the study of cell evolution because the host protozoan co-evolves with an intracellular bacterium in a mutualistic relationship. Such protozoa inhabit a single invertebrate host during their entire life cycle and exhibit special characteristics that group them in a particular phylogenetic cluster of the Trypanosomatidae family, thus classified as monoxenics. in an effort to better understand such symbiotic association, we used DNA pyrosequencing and a reference-guided assembly to generate reads that predicted 16,960 and 12,162 open reading frames (ORFs) in two symbiont-bearing trypanosomatids, Angomonas deanei (previously named as Crithidia deanei) and Strigomonas culicis (first known as Blastocrithidia culicis), respectively. Identification of each ORF was based primarily on TriTrypDB using tblastn, and each ORF was confirmed by employing getorf from EMBOSS and Newbler 2.6 when necessary. the monoxenic organisms revealed conserved housekeeping functions when compared to other trypanosomatids, especially compared with Leishmania major. However, major differences were found in ORFs corresponding to the cytoskeleton, the kinetoplast, and the paraflagellar structure. the monoxenic organisms also contain a large number of genes for cytosolic calpain-like and surface gp63 metalloproteases and a reduced number of compartmentalized cysteine proteases in comparison to other TriTryp organisms, reflecting adaptations to the presence of the symbiont. the assembled bacterial endosymbiont sequences exhibit a high A+T content with a total of 787 and 769 ORFs for the Angomonas deanei and Strigomonas culicis endosymbionts, respectively, and indicate that these organisms hold a common ancestor related to the Alcaligenaceae family. Importantly, both symbionts contain enzymes that complement essential host cell biosynthetic pathways, such as those for amino acid, lipid and purine/pyrimidine metabolism. These findings increase our understanding of the intricate symbiotic relationship between the bacterium and the trypanosomatid host and provide clues to better understand eukaryotic cell evolution.Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)ERC AdG SISYPHEUniv Fed Rio de Janeiro, Inst Biofis Carlos Chagas Filho, Lab Ultraestrutura Celular Hertha Meyer, BR-21941 Rio de Janeiro, BrazilUniv Fed Rio de Janeiro, Inst Biofis Carlos Chagas Filho, Lab Metab Macromol Firmino Torres de Castro, BR-21941 Rio de Janeiro, BrazilLab Bioinformat, Lab Nacl Computacao Cient, Rio de Janeiro, BrazilINRIA Grenoble Rhone Alpes, BAMBOO Team, Villeurbanne, FranceUniv Lyon 1, CNRS, UMR5558, Lab Biometrie & Biol Evolut, F-69622 Villeurbanne, FranceUniv Estadual Campinas, Inst Biol, Dept Genet Evolucao & Bioagentes, São Paulo, BrazilUniv São Paulo, Fac Ciencias Farmaceut Ribeirao Preto, Dept Ciencias Farmaceut, São Paulo, BrazilLab Nacl Ciencia & Tecnol Bioetano, São Paulo, BrazilUniv Fed Minas Gerais, Inst Ciencias Biol, Dept Bioquim & Imunol, Belo Horizonte, MG, BrazilUniv Fed Goias, Inst Ciencias Biol, Mol Biol Lab, Goiania, Go, BrazilFundacao Oswaldo Cruz, Inst Carlos Chagas, Lab Biol Mol Tripanossomatideos, Curitiba, Parana, BrazilFundacao Oswaldo Cruz, Inst Carlos Chagas, Lab Genom Func, Curitiba, Parana, BrazilUniv Estadual Campinas, Ctr Pluridisciplinar Pesquisas Quim Biol & Agr, São Paulo, BrazilUniv Fed Minas Gerais, Inst Ciencias Biol, Dept Parasitol, Belo Horizonte, MG, BrazilUniv Fed Santa Catarina, Dept Microbiol Imunol & Parasitol, Ctr Ciencias Biol, Lab Protozool & Bioinformat, Florianopolis, SC, BrazilUniv Fed Vicosa, Dept Bioquim & Biol Mol, Ctr Ciencias Biol & Saude, Vicosa, MG, BrazilInst Butantan, Lab Especial Ciclo Celular, São Paulo, BrazilUniv São Paulo, Dept Biol, Fac Filosofia Ciencias & Letras Ribeirao Preto, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilWeb of Scienc

Identification of glucose transporters in Aspergillus nidulans

o characterize the mechanisms involved in glucose transport, in the filamentous fungus Aspergillus nidulans, we have identified four glucose transporter encoding genes hxtB-E. We evaluated the ability of hxtB-E to functionally complement the Saccharomyces cerevisiae EBY.VW4000 strain that is unable to grow on glucose, fructose, mannose or galactose as single carbon source. In S. cerevisiae HxtB-E were targeted to the plasma membrane. The expression of HxtB, HxtC and HxtE was able to restore growth on glucose, fructose, mannose or galactose, indicating that these transporters accept multiple sugars as a substrate through an energy dependent process. A tenfold excess of unlabeled maltose, galactose, fructose, and mannose were able to inhibit glucose uptake to different levels (50 to 80 %) in these s. cerevisiae complemented strains. Moreover, experiments with cyanide-m-chlorophenylhydrazone (CCCP), strongly suggest that hxtB, -C, and –E mediate glucose transport via active proton symport. The A. nidulans ΔhxtB, ΔhxtC or ΔhxtE null mutants showed ~2.5-fold reduction in the affinity for glucose, while ΔhxtB and -C also showed a 2-fold reduction in the capacity for glucose uptake. The ΔhxtD mutant had a 7.8-fold reduction in affinity, but a 3-fold increase in the capacity for glucose uptake. However, only the ΔhxtB mutant strain showed a detectable decreased rate of glucose consumption at low concentrations and an increased resistance to 2-deoxyglucose.The authors would like to thank the Fundacao de Amparo a Pesquisa do Estado de Sao Paulo and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, Brazil for financial support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

The <i>A. nidulans</i> YpkA interacts with BarA.

<p>The radial growth of the wild-type, <i>niiA::ypkA</i>, <i>barA1</i>, and <i>niiA::ypkA barA1</i> mutant strains were grown for 72 hours at 37°C on MM+sodium nitrate 10 mM or MM+ammonium tartrate 50 mM (Wt = Wild-type).</p

Functional Characterization of <em>Aspergillus nidulans ypkA,</em> a Homologue of the Mammalian Kinase SGK

<div><p>The serum- and glucocorticoid-regulated protein kinase (SGK) is an AGC kinase involved in signal cascades regulated by glucocorticoid hormones and serum in mammals. The <i>Saccharomyces cerevisiae ypk1</i> and <i>ypk2</i> genes were identified as SGK homologues and Ypk1 was shown to regulate the balance of sphingolipids between the inner and outer plasma membrane. This investigation characterized the <i>Aspergillus nidulans YPK1</i> homologue, YpkA, representing the first filamentous fungal <i>YPK1</i> homologue. Two conditional mutant strains were constructed by replacing the endogenous <i>ypk1</i> promoter with two different regulatable promoters, <i>alcA</i> (from the alcohol dehydrogenase gene) and <i>niiA</i> (from the nitrate reductase gene). Both constructs confirmed that <i>ypkA</i> was an essential gene in <i>A. nidulans</i>. Repression of <i>ypkA</i> caused decreased radial growth, a delay in conidial germination, deficient polar axis establishment, intense branching during late stages of growth, a lack of asexual spores, and a terminal phenotype. Membrane lipid polarization, endocytosis, eisosomes and vacuolar distribution were also affected by <i>ypkA</i> repression, suggesting that YpkA plays a role in hyphal morphogenesis via coordinating the delivery of cell membrane and wall constituents to the hyphal apex. The <i>A. nidulans</i> Pkh1 homologue <i>pkhA</i> was also shown to be an essential gene, and preliminary genetic analysis suggested that the ypkA gene is not directly downstream of <i>pkhA</i> or epistatic to <i>pkhA</i>, rather, <i>ypkA</i> and <i>pkhA</i> are genetically independent or in parallel. <i>BarA</i> is a homologue of the yeast <i>Lag1</i> acyl-CoA-dependent ceramide synthase, which catalyzes the condensation of phytosphingosine with a fatty acyl-CoA to form phytoceramide. When <i>barA</i> was absent, <i>ypkA</i> repression was lethal to the cell. Therefore, there appears to be a genetic interaction between <i>ypkA</i>, <i>barA</i>, and the sphingolipid synthesis. Transcriptional profiling of <i>ypkA</i> overexpression and down-regulation revealed several putative YpkA targets associated with the observed phenotypes.</p> </div

The <i>pkhA</i> gene is essential to <i>A. nidulans</i> and interacts with <i>ypkA</i>.

<p>(A) The wild-type and <i>niiA::pkhA</i> mutant strains were grown for 72 hours at 37°C on MM+sodium nitrate 10 mM or MM+ammonium tartrate 50 mM (Wt = Wild-type). (B) The <i>alcA::ypkA</i>, <i>niiA::pkhA</i>, and <i>alcA::ypkA niiA::pkhA</i> strains were grown for 72 hours at 37°C on different combinations of MM+glucose 2% or glycerol 2% plus threonine 100 mM plus sodium nitrate 10 mM or ammonium tartrate 50 mM (Wt = Wild-type).</p

polarized delivery of membrane lipids and cell wall deposition was not confined to the hyphal apex in the <i>niiA::ypkA</i> mutant upon repression.

<p>In all experiments, germlings were grown for 16 hours at 37°C on inducing (sodium nitrate) and repressing conditions (ammonium tartrate). Stains utilized: (A) Hoescht, (B) Filipin, (C) FITC-conjugated wheat germ, and (D) CFW. Bars, 5 and 10 µm.</p

<i>A. nidulans</i> YpkA is involved in polarized growth.

<p>The percentage of wild-type and <i>niiA::ypkA</i> mutant germlings that exhibited polar growth (defined here as the emergence of the germ tube) (A) and the number of nuclear per germling (B). Conidia were grown for 2 to 8 hours at 37°C. Averages (± standard deviation) represent 100 germlings from three independent experiments (Wt = Wild-type). (C) The germination pattern of the wild-type and <i>niiA::ypkA</i> conidiospores. Conidia were allowed to germinate on MM media for 8 to 16 hours. Conidia possessing germ tubes were classified as displaying (left to right) unipolar (1), bipolar (2), unipolar plus lateral branches (3) or bipolar plus lateral branches (4).</p