Paralogous FgIDO genes with differential roles in tryptophan catabolism, fungal development and virulence in Fusarium graminearum.

Indoleamine 2,3-dioxygenase (Ido) is a tryptophan-degrading enzyme that is widely distributed across species. Ido catalyzes the first step of tryptophan (TRP) degradation and drives the de novo synthesis of nicotinamide adenine dinucleotide (NAD+) coenzymes via the kynurenine (KYN) pathway. The budding yeast Saccharomyces cerevisiae possesses a single IDO gene (BNA2) that is responsible for NAD+ synthesis, whereas a number of fungal species contain multiple IDO genes. However, the biological roles of IDO paralogs in plant pathogens remain unclear. In the current study, we identified three FgIDOs from the wheat head blight fungus Fusarium graminearum. FgIDOA/B/C expression was significantly induced upon TRP treatment. Targeted disruption of FgIDOA and/or FgIDOB caused different levels of NAD+ auxotrophy, thus resulting in pleotropic phenotypic defects. Loss of FgIDOA resulted in abnormal conidial morphology, reduced mycelial growth, decreased virulence in wheat heads and reduced deoxynivalenol accumulation. Exogenous addition of KYN or various intermediates involved in the KYN pathway rescued auxotrophy of the mutants. Metabolomics analysis revealed shifts toward alternative TRP degradation pathways to melatonin and indole derivatives in mutants lacking FgIDOB. Upregulation of partner genes in auxotrophic mutants and the capacity to rescue the auxotroph by overexpressing a partner gene indicated functional complementation among FgIDOA/B/C. Taken together, the results of this study provide insights into differential roles in paralogous FgIDOs and how fungal TRP catabolism modulates fungal development and virulence.

Clinical Utility of Dual Anterior Nares and Oropharynx MRSA Screening PCR for Patients with Suspected Pneumonia.

We reviewed the electronic health records of 1,419 inpatients with anterior nares (AN) and oropharynx (OP) MRSA PCR tests. Concordance was 96.5%. In discordant cases, AN negative-OP positive results increased detection of probable MRSA pneumonia by only 0.3%. A dual testing approach has limited utility in detecting MRSA pneumonia and increases resource utilization.

Aspergillus fumigatus tryptophan metabolic route differently affects host immunity.

Indoleamine 2,3-dioxygenases (IDOs) degrade l-tryptophan to kynurenines and drive the de novo synthesis of nicotinamide adenine dinucleotide. Unsurprisingly, various invertebrates, vertebrates, and even fungi produce IDO. In mammals, IDO1 also serves as a homeostatic regulator, modulating immune response to infection via local tryptophan deprivation, active catabolite production, and non-enzymatic cell signaling. Whether fungal Idos have pleiotropic functions that impact on host-fungal physiology is unclear. Here, we show that Aspergillus fumigatus possesses three ido genes that are expressed under conditions of hypoxia or tryptophan abundance. Loss of these genes results in increased fungal pathogenicity and inflammation in a mouse model of aspergillosis, driven by an alternative tryptophan degradation pathway to indole derivatives and the host aryl hydrocarbon receptor. Fungal tryptophan metabolic pathways thus cooperate with the host xenobiotic response to shape host-microbe interactions in local tissue microenvironments.

Modeling Approaches Reveal New Regulatory Networks in Aspergillus fumigatus Metabolism.

Systems biology approaches are extensively used to model and reverse-engineer gene regulatory networks from experimental data. Indoleamine 2,3-dioxygenases (IDOs)-belonging in the heme dioxygenase family-degrade l-tryptophan to kynurenines. These enzymes are also responsible for the de novo synthesis of nicotinamide adenine dinucleotide (NAD+). As such, they are expressed by a variety of species, including fungi. Interestingly, Aspergillus may degrade l-tryptophan not only via IDO but also via alternative pathways. Deciphering the molecular interactions regulating tryptophan metabolism is particularly critical for novel drug target discovery designed to control pathogen determinants in invasive infections. Using continuous time Bayesian networks over a time-course gene expression dataset, we inferred the global regulatory network controlling l-tryptophan metabolism. The method unravels a possible novel approach to target fungal virulence factors during infection. Furthermore, this study represents the first application of continuous-time Bayesian networks as a gene network reconstruction method in Aspergillus metabolism. The experiment showed that the applied computational approach may improve the understanding of metabolic networks over traditional pathways.

Contributions of Spore Secondary Metabolites to UV-C Protection and Virulence Vary in Different Aspergillus fumigatus Strains.

Fungi are versatile organisms which thrive in hostile environments, including the International Space Station (ISS). Several isolates of the human pathogen Aspergillus fumigatus have been found contaminating the ISS, an environment with increased exposure to UV radiation. Secondary metabolites (SMs) in spores, such as melanins, have been shown to protect spores from UV radiation in other fungi. To test the hypothesis that melanin and other known spore SMs provide UV protection to A. fumigatus isolates, we subjected SM spore mutants to UV-C radiation. We found that 1,8-dihydroxynaphthalene (DHN)-melanin mutants of two clinical A. fumigatus strains (Af293 and CEA17) but not an ISS-isolated strain (IF1SW-F4) were more sensitive to UV-C than their respective wild-type (WT) strains. Because DHN-melanin has been shown to shield A. fumigatus from the host immune system, we examined all DHN mutants for virulence in the zebrafish model of invasive aspergillosis. Following recent studies highlighting the pathogenic variability of different A. fumigatus isolates, we found DHN-melanin to be a virulence factor in CEA17 and IF1SW-F4 but not Af293. Three additional spore metabolites were examined in Af293, where fumiquinazoline also showed UV-C-protective properties, but two other spore metabolites, monomethylsulochrin and fumigaclavine, provided no UV-C-protective properties. Virulence tests of these three SM spore mutants indicated a slight increase in virulence of the monomethylsulochrin deletion strain. Taken together, this work suggests differential roles of specific spore metabolites across Aspergillus isolates and by types of environmental stress.IMPORTANCE Fungal spores contain secondary metabolites that can protect them from a multitude of abiotic and biotic stresses. Conidia (asexual spores) of the human pathogen Aspergillus fumigatus synthesize several metabolites, including melanin, which has been reported to be important for virulence in this species and to be protective against UV radiation in other fungi. Here, we investigate the role of melanin in diverse isolates of A. fumigatus and find variability in its ability to protect spores from UV-C radiation or impact virulence in a zebrafish model of invasive aspergillosis in two clinical strains and one ISS strain. Further, we assess the role of other spore metabolites in a clinical strain of A. fumigatus and identify fumiquinazoline as an additional UV-C-protective molecule but not a virulence determinant. The results show differential roles of secondary metabolites in spore protection dependent on the environmental stress and strain of A. fumigatus As protection from elevated levels of radiation is of paramount importance for future human outer space explorations, the discovery of small molecules with radiation-protective potential may result in developing novel safety measures for astronauts.

Comparative study of Revogene GBS LB assay and GeneXpert GBS LB assay for the detection of group B Streptococcus in prenatal screening samples.

BACKGROUND: Group B Streptococcal (GBS) infections in the United States are a leading cause of meningitis and sepsis in newborns. The CDC therefore recommends GBS screening for all pregnant women at 35-37 weeks of gestation and administration of intrapartum prophylaxis (in those that tested positive) as an effective means of controlling disease transmission. Several FDA approved molecular diagnostic tests are available for rapid and accurate detection of GBS in antepartum women. METHOD: In this study, we report a clinical comparison of the Xpert GBS LB assay and a novel FDA-cleared test, Revogene GBS LB assay. A total of 250 vaginal-rectal swabs from women undergoing prenatal screening were submitted to the University of Wisconsin's clinical microbiology laboratory for GBS testing. RESULTS: We found 96.8% of samples were concordant between the two tests, while 3.2% were discordant with a positive percent agreement of 98.0% and a negative percent agreement of 96.5% between the Revogene GBS LB assay and the GeneXpert GBS LB assay. CONCLUSION: Overall, we report that both assays perform well for the detection of GBS colonization in pregnant women.

Revitalization of a Forward Genetic Screen Identifies Three New Regulators of Fungal Secondary Metabolism in the Genus Aspergillus.

The study of aflatoxin in Aspergillus spp. has garnered the attention of many researchers due to aflatoxin's carcinogenic properties and frequency as a food and feed contaminant. Significant progress has been made by utilizing the model organism Aspergillus nidulans to characterize the regulation of sterigmatocystin (ST), the penultimate precursor of aflatoxin. A previous forward genetic screen identified 23 A. nidulans mutants involved in regulating ST production. Six mutants were characterized from this screen using classical mapping (five mutations in mcsA) and complementation with a cosmid library (one mutation in laeA). The remaining mutants were backcrossed and sequenced using Illumina and Ion Torrent sequencing platforms. All but one mutant contained one or more sequence variants in predicted open reading frames. Deletion of these genes resulted in identification of mutant alleles responsible for the loss of ST production in 12 of the 17 remaining mutants. Eight of these mutations were in genes already known to affect ST synthesis (laeA, mcsA, fluG, and stcA), while the remaining four mutations (in laeB, sntB, and hamI) were in previously uncharacterized genes not known to be involved in ST production. Deletion of laeB, sntB, and hamI in A. flavus results in loss of aflatoxin production, confirming that these regulators are conserved in the aflatoxigenic aspergilli. This report highlights the multifaceted regulatory mechanisms governing secondary metabolism in Aspergillus Additionally, these data contribute to the increasing number of studies showing that forward genetic screens of fungi coupled with whole-genome resequencing is a robust and cost-effective technique.IMPORTANCE In a postgenomic world, reverse genetic approaches have displaced their forward genetic counterparts. The techniques used in forward genetics to identify loci of interest were typically very cumbersome and time-consuming, relying on Mendelian traits in model organisms. The current work was pursued not only to identify alleles involved in regulation of secondary metabolism but also to demonstrate a return to forward genetics to track phenotypes and to discover genetic pathways that could not be predicted through a reverse genetics approach. While identification of mutant alleles from whole-genome sequencing has been done before, here we illustrate the possibility of coupling this strategy with a genetic screen to identify multiple alleles of interest. Sequencing of classically derived mutants revealed several uncharacterized genes, which represent novel pathways to regulate and control the biosynthesis of sterigmatocystin and of aflatoxin, a societally and medically important mycotoxin.

Aspergillus fumigatus Copper Export Machinery and Reactive Oxygen Intermediate Defense Counter Host Copper-Mediated Oxidative Antimicrobial Offense.

The Fenton-chemistry-generating properties of copper ions are considered a potent phagolysosome defense against pathogenic microbes, yet our understanding of underlying host/microbe dynamics remains unclear. We address this issue in invasive aspergillosis and demonstrate that host and fungal responses inextricably connect copper and reactive oxygen intermediate (ROI) mechanisms. Loss of the copper-binding transcription factor AceA yields an Aspergillus fumigatus strain displaying increased sensitivity to copper and ROI in vitro, increased intracellular copper concentrations, decreased survival in challenge with murine alveolar macrophages (AMΦs), and reduced virulence in a non-neutropenic murine model. ΔaceA survival is remediated by dampening of host ROI (chemically or genetically) or enhancement of copper-exporting activity (CrpA) in A. fumigatus. Our study exposes a complex host/microbe multifactorial interplay that highlights the importance of host immune status and reveals key targetable A. fumigatus counter-defenses.

A Multifaceted Role of Tryptophan Metabolism and Indoleamine 2,3-Dioxygenase Activity in Aspergillus fumigatus-Host Interactions.

Aspergillus fumigatus is the most prevalent filamentous fungal pathogen of humans, causing either severe allergic bronchopulmonary aspergillosis or often fatal invasive pulmonary aspergillosis (IPA) in individuals with hyper- or hypo-immune deficiencies, respectively. Disease is primarily initiated upon the inhalation of the ubiquitous airborne conidia-the initial inoculum produced by A. fumigatus-which are complete developmental units with an ability to exploit diverse environments, ranging from agricultural composts to animal lungs. Upon infection, conidia initially rely on their own metabolic processes for survival in the host's lungs, a nutritionally limiting environment. One such nutritional limitation is the availability of aromatic amino acids (AAAs) as animals lack the enzymes to synthesize tryptophan (Trp) and phenylalanine and only produce tyrosine from dietary phenylalanine. However, A. fumigatus produces all three AAAs through the shikimate-chorismate pathway, where they play a critical role in fungal growth and development and in yielding many downstream metabolites. The downstream metabolites of Trp in A. fumigatus include the immunomodulatory kynurenine derived from indoleamine 2,3-dioxygenase (IDO) and toxins such as fumiquinazolines, gliotoxin, and fumitremorgins. Host IDO activity and/or host/microbe-derived kynurenines are increasingly correlated with many Aspergillus diseases including IPA and infections of chronic granulomatous disease patients. In this review, we will describe the potential metabolic cross talk between the host and the pathogen, specifically focusing on Trp metabolism, the implications for therapeutics, and the recent studies on the coevolution of host and microbe IDO activation in regulating inflammation, while controlling infection.

FleA Expression in Aspergillus fumigatus Is Recognized by Fucosylated Structures on Mucins and Macrophages to Prevent Lung Infection.

The immune mechanisms that recognize inhaled Aspergillus fumigatus conidia to promote their elimination from the lungs are incompletely understood. FleA is a lectin expressed by Aspergillus fumigatus that has twelve binding sites for fucosylated structures that are abundant in the glycan coats of multiple plant and animal proteins. The role of FleA is unknown: it could bind fucose in decomposed plant matter to allow Aspergillus fumigatus to thrive in soil, or it may be a virulence factor that binds fucose in lung glycoproteins to cause Aspergillus fumigatus pneumonia. Our studies show that FleA protein and Aspergillus fumigatus conidia bind avidly to purified lung mucin glycoproteins in a fucose-dependent manner. In addition, FleA binds strongly to macrophage cell surface proteins, and macrophages bind and phagocytose fleA-deficient (∆fleA) conidia much less efficiently than wild type (WT) conidia. Furthermore, a potent fucopyranoside glycomimetic inhibitor of FleA inhibits binding and phagocytosis of WT conidia by macrophages, confirming the specific role of fucose binding in macrophage recognition of WT conidia. Finally, mice infected with ΔfleA conidia had more severe pneumonia and invasive aspergillosis than mice infected with WT conidia. These findings demonstrate that FleA is not a virulence factor for Aspergillus fumigatus. Instead, host recognition of FleA is a critical step in mechanisms of mucin binding, mucociliary clearance, and macrophage killing that prevent Aspergillus fumigatus pneumonia.

TrpE feedback mutants reveal roadblocks and conduits toward increasing secondary metabolism in Aspergillus fumigatus.

Small peptides formed from non-ribosomal peptide synthetases (NRPS) are bioactive molecules produced by many fungi including the genus Aspergillus. A subset of NRPS utilizes tryptophan and its precursor, the non-proteinogenic amino acid anthranilate, in synthesis of various metabolites such as Aspergillus fumigatus fumiquinazolines (Fqs) produced by the fmq gene cluster. The A. fumigatus genome contains two putative anthranilate synthases - a key enzyme in conversion of anthranilic acid to tryptophan - one beside the fmq cluster and one in a region of co-linearity with other Aspergillus spp. Only the gene found in the co-linear region, trpE, was involved in tryptophan biosynthesis. We found that site-specific mutations of the TrpE feedback domain resulted in significantly increased production of anthranilate, tryptophan, p-aminobenzoate and fumiquinazolines FqF and FqC. Supplementation with tryptophan restored metabolism to near wild type levels in the feedback mutants and suggested that synthesis of the tryptophan degradation product kynurenine could negatively impact Fq synthesis. The second putative anthranilate synthase gene next to the fmq cluster was termed icsA for its considerable identity to isochorismate synthases in bacteria. Although icsA had no impact on A. fumigatus Fq production, deletion and over-expression of icsA increased and decreased respectively aromatic amino acid levels suggesting that IcsA can draw from the cellular chorismate pool.