Systematic discovery of mutation-directed neo-protein-protein interactions in cancer.

Comprehensive sequencing of patient tumors reveals genomic mutations across tumor types that enable tumorigenesis and progression. A subset of oncogenic driver mutations results in neomorphic activity where the mutant protein mediates functions not engaged by the parental molecule. Here, we identify prevalent variant-enabled neomorph-protein-protein interactions (neoPPI) with a quantitative high-throughput differential screening (qHT-dS) platform. The coupling of highly sensitive BRET biosensors with miniaturized coexpression in an ultra-HTS format allows large-scale monitoring of the interactions of wild-type and mutant variant counterparts with a library of cancer-associated proteins in live cells. The screening of 17,792 interactions with 2,172,864 data points revealed a landscape of gain of interactions encompassing both oncogenic and tumor suppressor mutations. For example, the recurrent BRAF V600E lesion mediates KEAP1 neoPPI, rewiring a BRAFV600E/KEAP1 signaling axis and creating collateral vulnerability to NQO1 substrates, offering a combination therapeutic strategy. Thus, cancer genomic alterations can create neo-interactions, informing variant-directed therapeutic approaches for precision medicine.

Interferon-λ Mediates Non-redundant Front-Line Antiviral Protection against Influenza Virus Infection without Compromising Host Fitness.

Lambda interferons (IFNλs) or type III IFNs share homology, expression patterns, signaling cascades, and antiviral functions with type I IFNs. This has complicated the unwinding of their unique non-redundant roles. Through the systematic study of influenza virus infection in mice, we herein show that IFNλs are the first IFNs produced that act at the epithelial barrier to suppress initial viral spread without activating inflammation. If infection progresses, type I IFNs come into play to enhance viral resistance and induce pro-inflammatory responses essential for confronting infection but causing immunopathology. Central to this are neutrophils which respond to both cytokines to upregulate antimicrobial functions but exhibit pro-inflammatory activation only to type I IFNs. Accordingly, Ifnlr1-/- mice display enhanced type I IFN production, neutrophilia, lung injury, and lethality, while therapeutic administration of PEG-IFNλ potently suppresses these effects. IFNλs therefore constitute the front line of antiviral defense in the lung without compromising host fitness.

Proteomic dissection of the role of GliZ in gliotoxin biosynthesis in Aspergillus fumigatus.

Gliotoxin (GT) biosynthesis in fungi is encoded by the gli biosynthetic gene cluster. While GT addition autoinduces biosynthesis, Zn2+ has been shown to attenuate cluster activity, and it was speculated that identification of Zn2Cys6 binuclear transcription factor GliZ binding partners might provide insight into this observation. Using the Tet-ON induction system, doxycycline (DOX) presence induced GliZ fusion protein expression in, and recovery of GT biosynthesis by, A. fumigatus ΔgliZ::HA-gliZ and ΔgliZ::TAP-gliZ strains, respectively. Quantitative RT-PCR confirmed that DOX induces gli cluster gene expression (n = 5) in both A. fumigatus HA-GliZ and TAP-GliZ strains. GT biosynthesis was evident in Czapek-Dox and in Sabouraud media, however tagged GliZ protein expression was more readily detected in Sabouraud media. Unexpectedly, Zn2+ was essential for GliZ fusion protein expression in vivo, following 3 h DOX induction. Moreover, HA-GliZ abundance was significantly higher in either DOX/GT or DOX/Zn2+, compared to DOX-only. This suggests that while GT induction is still intact, Zn2+ inhibition of HA-GliZ production in vivo is lost. Co-immunoprecipitation revealed that GT oxidoreductase GliT associates with GliZ in the presence of GT, suggesting a potential protective role. Additional putative HA-GliZ interacting proteins included cystathionine gamma lyase, ribosomal protein L15 and serine hydroxymethyltransferase (SHMT). Total mycelial quantitative proteomic data revealed that GliT and GtmA, as well as several other gli cluster proteins, are increased in abundance or uniquely expressed with GT addition. Proteins involved in sulphur metabolism are also differentially expressed with GT or Zn2+ presence. Overall, we disclose that under DOX induction GliZ functionality is unexpectedly evident in zinc-replete media, subject to GT induction and that GliT appears to associate with GliZ, potentially to prevent dithiol gliotoxin (DTG)-mediated GliZ inactivation by zinc ejection.

Mutational analysis of the Hsp70 substrate-binding domain: Correlating molecular-level changes with in vivo function.

Hsp70 is an evolutionarily conserved chaperone involved in maintaining protein homeostasis during normal growth and upon exposure to stresses. Mutations in the ß6/ß7 region of the substrate-binding domain (SBD) disrupt the SBD hydrophobic core resulting in impairment of the heat-shock response and prion propagation in yeast. To elucidate the mechanisms behind Hsp70 loss of function due to disruption of the SBD, we undertook targeted mutational analysis of key residues in the ß6/ß7 region. We demonstrate the critical functional role of the F475 residue across yeast cytosolic Hsp70-Ssa family. We identify the size of the hydrophobic side chain at 475 as the key factor in maintaining SBD stability and functionality. The introduction of amino acid variants to either residue 475, or close neighbor 483, caused instability and cleavage of the Hsp70 SBD and subsequent degradation. Interestingly, we found that Hsp70-Ssa cleavage may occur through a vacuolar carboxypeptidase (Pep4)-dependent mechanism rather than proteasomal. Mutations at 475 and 483 result in compromised ATPase function, which reduces protein re-folding activity and contributes to depletion of cytosolic Hsp70 in vivo. The combination of reduced functionality and stability of Hsp70-Ssa results in yeast cells that are compromised in their stress response and cannot propagate the [PSI+ ] prion.

The Aspergillus fumigatus transcription factor RglT is important for gliotoxin biosynthesis and self-protection, and virulence.

Aspergillus fumigatus is an opportunistic fungal pathogen that secretes an array of immune-modulatory molecules, including secondary metabolites (SMs), which contribute to enhancing fungal fitness and growth within the mammalian host. Gliotoxin (GT) is a SM that interferes with the function and recruitment of innate immune cells, which are essential for eliminating A. fumigatus during invasive infections. We identified a C6 Zn cluster-type transcription factor (TF), subsequently named RglT, important for A. fumigatus oxidative stress resistance, GT biosynthesis and self-protection. RglT regulates the expression of several gli genes of the GT biosynthetic gene cluster, including the oxidoreductase-encoding gene gliT, by directly binding to their respective promoter regions. Subsequently, RglT was shown to be important for virulence in a chemotherapeutic murine model of invasive pulmonary aspergillosis (IPA). Homologues of RglT and GliT are present in eurotiomycete and sordariomycete fungi, including the non-GT-producing fungus A. nidulans, where a conservation of function was described. Phylogenetically informed model testing led to an evolutionary scenario in which the GliT-based resistance mechanism is ancestral and RglT-mediated regulation of GliT occurred subsequently. In conclusion, this work describes the function of a previously uncharacterised TF in oxidative stress resistance, GT biosynthesis and self-protection in both GT-producing and non-producing Aspergillus species.

Production of a functionally active recombinant SARS-CoV-2 (COVID-19) 3C-like protease and a soluble inactive 3C-like protease-RBD chimeric in a prokaryotic expression system.

During the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) intracellular life-cycle, two large polyproteins, pp1a and pp1ab, are produced. Processing of these by viral cysteine proteases, the papain-like protease (PLpro) and the chymotrypsin-like 3C-like protease (3CL-pro) release non-structural proteins necessary for the establishment of the viral replication and transcription complex (RTC), crucial for viral replication. Hence, these proteases are considered prime targets against which anti-coronavirus disease 2019 (COVID-19) drugs could be developed. Here, we describe the expression of a highly soluble and functionally active recombinant 3CL-pro using Escherichia coli BL21 cells. We show that the enzyme functions in a dimeric form and exhibits an unexpected inhibitory profile because its activity is potently blocked by serine rather than cysteine protease inhibitors. In addition, we assessed the ability of our 3CL-pro to function as a carrier for the receptor binding domain (RBD) of the Spike protein. The co-expressed chimeric protein, 3CLpro-RBD, did not exhibit 3CL-pro activity, but its enhanced solubility made purification easier and improved RBD antigenicity when tested against serum from vaccinated individuals in ELISAs. Chimeric proteins containing the 3CL-pro could represent an innovative approach to developing new COVID-19 vaccines.

Acquisition of taxane resistance by p53 inactivation in ovarian cancer cells.

Ovarian cancer is one of the most common gynecologic malignancies in women and has a poor prognosis. Taxanes are a class of standard first-line chemotherapeutic agents for the treatment of ovarian cancer. However, tumor-intrinsic and acquired resistance to taxanes poses major challenges to improving clinical outcomes. Hence, there is an urgent clinical need to understand the mechanisms of resistance in order to discover potential biomarkers and therapeutic strategies to increase taxane sensitivity in ovarian cancer. Here, we report the identification of an association between the TP53 status and taxane sensitivity in ovarian cancer cells through complementary experimental and informatics approaches. We found that TP53 inactivation is associated with taxane resistance in ovarian cancer cells, supported by the evidence from (i) drug sensitivity profiling with bioinformatic analysis of large-scale cancer therapeutic response and genomic datasets and (ii) gene signature identification based on experimental isogenic cell line models. Further, our studies revealed TP53-dependent gene expression patterns, such as overexpression of ACSM3, as potential predictive biomarkers of taxane resistance in ovarian cancer. The TP53-dependent hyperactivation of the WNT/ß-catenin pathway discovered herein revealed a potential vulnerability to exploit in developing combination therapeutic strategies. Identification of this genotype-phenotype relationship between the TP53 status and taxane sensitivity sheds light on TP53-directed patient stratification and therapeutic discoveries for ovarian cancer treatment.

Highdicom: a Python Library for Standardized Encoding of Image Annotations and Machine Learning Model Outputs in Pathology and Radiology.

Machine learning (ML) is revolutionizing image-based diagnostics in pathology and radiology. ML models have shown promising results in research settings, but the lack of interoperability between ML systems and enterprise medical imaging systems has been a major barrier for clinical integration and evaluation. The DICOM® standard specifies information object definitions (IODs) and services for the representation and communication of digital images and related information, including image-derived annotations and analysis results. However, the complexity of the standard represents an obstacle for its adoption in the ML community and creates a need for software libraries and tools that simplify working with datasets in DICOM format. Here we present the highdicom library, which provides a high-level application programming interface (API) for the Python programming language that abstracts low-level details of the standard and enables encoding and decoding of image-derived information in DICOM format in a few lines of Python code. The highdicom library leverages NumPy arrays for efficient data representation and ties into the extensive Python ecosystem for image processing and machine learning. Simultaneously, by simplifying creation and parsing of DICOM-compliant files, highdicom achieves interoperability with the medical imaging systems that hold the data used to train and run ML models, and ultimately communicate and store model outputs for clinical use. We demonstrate through experiments with slide microscopy and computed tomography imaging, that, by bridging these two ecosystems, highdicom enables developers and researchers to train and evaluate state-of-the-art ML models in pathology and radiology while remaining compliant with the DICOM standard and interoperable with clinical systems at all stages. To promote standardization of ML research and streamline the ML model development and deployment process, we made the library available free and open-source at https://github.com/herrmannlab/highdicom .

Quantitative Proteomic Analysis Reveals Yeast Cell Wall Products Influence the Serum Proteome Composition of Broiler Chickens.

With an ever-growing market and continual financial pressures associated with the prohibition of antibiotic growth promoters, the poultry industry has had to rapidly develop non-antibiotic alternatives to increase production yields. A possible alternative is yeast and its derivatives, such as the yeast cell wall (YCW), which have been proposed to confer selected beneficial effects on the host animal. Here, the effect of YCW supplementation on the broiler chicken was investigated using a quantitative proteomic strategy, whereby serum was obtained from three groups of broilers fed with distinct YCW-based Gut Health Products (GHP) or a control basal diet. Development of a novel reagent enabled application of ProteoMiner™ technology for sample preparation and subsequent comparative quantitative proteomic analysis revealed proteins which showed a significant change in abundance (n = 167 individual proteins; p < 0.05); as well as proteins which were uniquely identified (n = 52) in, or absent (n = 37) from, GHP-fed treatment groups versus controls. An average of 7.1% of proteins showed changes in abundance with GHP supplementation. Several effects of these GHPs including immunostimulation (via elevated complement protein detection), potential alterations in the oxidative status of the animal (e.g., glutathione peroxidase and catalase), stimulation of metabolic processes (e.g., differential abundance of glyceraldehyde-3-phosphate dehydrogenase), as well as evidence of a possible hepatoprotective effect (attenuated levels of serum α-glutathione s-transferase) by one GHP feed supplement, were observed. It is proposed that specific protein detection may be indicative of GHP efficacy to stimulate broiler immune status, i.e., may be biomarkers of GHP efficacy. In summary, this work has developed a novel technology for the preparation of high dynamic range proteomic samples for LC-MS/MS analysis, is part of the growing area of livestock proteomics and, importantly, provides evidential support for beneficial effects that GHP supplementation has on the broiler chicken.

A Single Aspergillus fumigatus Gene Enables Ergothioneine Biosynthesis and Secretion by Saccharomyces cerevisiae.

The naturally occurring sulphur-containing histidine derivative, ergothioneine (EGT), exhibits potent antioxidant properties and has been proposed to confer human health benefits. Although it is only produced by select fungi and prokaryotes, likely to protect against environmental stress, the GRAS organism Saccharomyces cerevisiae does not produce EGT naturally. Herein, it is demonstrated that the recombinant expression of a single gene, Aspergillus fumigatus egtA, in S. cerevisiae results in EgtA protein presence which unexpectedly confers complete EGT biosynthetic capacity. Both High Performance Liquid Chromatography (HPLC) and LC−mass spectrometry (MS) analysis were deployed to detect and confirm EGT production in S. cerevisiae. The localisation and quantification of the resultant EGT revealed a significantly (p < 0.0001) larger quantity of EGT was extracellularly present in culture supernatants than intracellularly accumulated in 96 h yeast cultures. Methionine addition to cultures improved EGT production. The additional expression of two candidate cysteine desulfurases from A. fumigatus was thought to be required to complete EGT biosynthesis, namely AFUA_2G13295 and AFUA_3G14240, termed egt2a and egt2b in this study. However, the co-expression of egtA and egt2a in S. cerevisiae resulted in a significant decrease in the observed EGT levels (p < 0.05). The AlphaFold prediction of A. fumigatus EgtA 3-Dimensional structure illuminates the bidomain structure of the enzyme and the opposing locations of both active sites. Overall, we clearly show that recombinant S. cerevisiae can biosynthesise and secrete EGT in an EgtA-dependent manner which presents a facile means of producing EGT for biotechnological and biomedical use.

At the metal-metabolite interface in Aspergillus fumigatus: towards untangling the intersecting roles of zinc and gliotoxin.

Cryptic links between apparently unrelated metabolic systems represent potential new drug targets in fungi. Evidence of such a link between zinc and gliotoxin (GT) biosynthesis in Aspergillus fumigatus is emerging. Expression of some genes of the GT biosynthetic gene cluster gli is influenced by the zinc-dependent transcription activator ZafA, zinc may relieve GT-mediated fungal growth inhibition and, surprisingly, GT biosynthesis is influenced by zinc availability. In A. fumigatus, dithiol gliotoxin (DTG), which has zinc-chelating properties, is converted to either GT or bis-dethiobis(methylthio)gliotoxin (BmGT) by oxidoreductase GliT and methyltransferase GtmA, respectively. A double deletion mutant lacking both GliT and GtmA was previously observed to be hypersensitive to exogenous GT exposure. Here we show that compared to wild-type exposure, exogenous GT and the zinc chelator N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN) inhibit A. fumigatus ΔgliTΔgtmA growth, specifically under zinc-limiting conditions, which can be reversed by zinc addition. While GT biosynthesis is evident in zinc-depleted medium, addition of zinc (1 µM) suppressed GT and activated BmGT production. In addition, secretion of the unferrated siderophore, triacetylfusarinine C (TAFC), was evident by A. fumigatus wild-type (at >5 µM zinc) and ΔgtmA (at >1 µM zinc) in a low-iron medium. TAFC secretion suggests that differential zinc-sensing between both strains may influence fungal Fe3+ requirement. Label-free quantitative proteomic analysis of both strains under equivalent differential zinc conditions revealed protein abundance alterations in accordance with altered metabolomic observations, in addition to increased GliT abundance in ΔgtmA at 5 µM zinc, compared to wild-type, supporting a zinc-sensing deficiency in the mutant strain. The relative abundance of a range of oxidoreductase- and secondary metabolism-related enzymes was also evident in a zinc- and strain-dependent manner. Overall, we elaborate new linkages between zinc availability, natural product biosynthesis and oxidative stress homeostasis in A. fumigatus.

Effects of antifungal agents on the fungal proteome: informing on mechanisms of sensitivity and resistance.

INTRODUCTION: Antifungal agents are essential in the fight against serious fungal disease, however emerging resistance is threatening an already limited collection of therapeutics. Proteomic analyses of effects of antifungal agents can expand our understanding of multifactorial mechanisms of action and have also proven valuable to elucidate proteomic changes associated with antifungal resistance. AREAS COVERED: This review covers the application of proteomic techniques to examine sensitivity and resistance to antifungals including commonly used therapeutics, amphotericin B, echinocandins and the azoles, based predominantly on studies involving Aspergillus fumigatus, Candida albicans and Candida glabrata from the last 10 years. In addition, non-clinical antimicrobial agents are also discussed, which highlight the potential of proteomics to identify new antifungal targets. EXPERT COMMENTARY: Fungal proteomics has evolved in the last decade with increased genome availability and developments in mass spectrometry. Collectively, these have led to the advancement of proteomic techniques, allowing increased coverage of the proteome. Gel-based proteomics laid the foundation for these types of studies, which has now shifted to the more powerful gel-free proteomics. This has resulted in the identification of key mediators and potential biomarkers of antifungal resistance, as well as elucidating the mechanisms of action of novel and established antifungal agents.

Generation and characterisation of a semi-synthetic siderophore-immunogen conjugate and a derivative recombinant triacetylfusarinine C-specific monoclonal antibody with fungal diagnostic application.

Invasive pulmonary aspergillosis (IPA) is a severe life-threatening condition. Diagnosis of fungal disease in general, and especially that caused by Aspergillus fumigatus is problematic. A. fumigatus secretes siderophores to acquire iron during infection, which are also essential for virulence. We describe the chemoacetylation of ferrated fusarinine C to diacetylated fusarinine C (DAFC), followed by protein conjugation, which facilitated triacetylfusarinine C (TAFC)-specific monoclonal antibody production with specific recognition of the ferrated form of TAFC. A single monoclonal antibody sequence was ultimately elucidated by a combinatorial strategy involving protein LC-MS/MS, cDNA sequencing and RNAseq. The resultant murine IgG2a monoclonal antibody was secreted in, and purified from, mammalian cell culture (5 mg) and demonstrated to be highly specific for TAFC detection by competitive ELISA (detection limit: 15 nM) and in a lateral flow test system (detection limit: 3 ng), using gold nanoparticle conjugated- DAFC-bovine serum albumin for competition. Overall, this work reveals for the first time a recombinant TAFC-specific monoclonal antibody with diagnostic potential for IPA diagnosis in traditional and emerging patient groups (e.g., COVID-19) and presents a useful strategy for murine Ig sequence determination, and expression in HEK293 cells, to overcome unexpected limitations associated with aberrant or deficient murine monoclonal antibody production.

Improved diagnosis of SARS-CoV-2 by using nucleoprotein and spike protein fragment 2 in quantitative dual ELISA tests.

The novel coronavirus, severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), is the causative agent of the 2020 worldwide coronavirus pandemic. Antibody testing is useful for diagnosing historic infections of a disease in a population. These tests are also a helpful epidemiological tool for predicting how the virus spreads in a community, relating antibody levels to immunity and for assessing herd immunity. In the present study, SARS-CoV-2 viral proteins were recombinantly produced and used to analyse serum from individuals previously exposed, or not, to SARS-CoV-2. The nucleocapsid (Npro) and spike subunit 2 (S2Frag) proteins were identified as highly immunogenic, although responses to the former were generally greater. These two proteins were used to develop two quantitative enzyme-linked immunosorbent assays (ELISAs) that when used in combination resulted in a highly reliable diagnostic test. Npro and S2Frag-ELISAs could detect at least 10% more true positive coronavirus disease-2019 (COVID-19) cases than the commercially available ARCHITECT test (Abbott). Moreover, our quantitative ELISAs also show that specific antibodies to SARS-CoV-2 proteins tend to wane rapidly even in patients who had developed severe disease. As antibody tests complement COVID-19 diagnosis and determine population-level surveillance during this pandemic, the alternative diagnostic we present in this study could play a role in controlling the spread of the virus.

The effect of surface inclination and limb on knee loading measures in transtibial prosthesis users.

BACKGROUND: Osteoarthritis (OA) is a degenerative disease caused by the wearing of joint cartilage and bone. Literature has established that a prosthesis user's intact limb is at greater risk of developing OA. This study analyzed the effect of commonly encountered surface inclinations on knee joint loading measures in able-bodied and transtibial prosthesis users. METHODS: 12 transtibial prosthesis users and 12 able-bodied participants walked across level ground, up slope, down slope, and cross slope (further divided into top and bottom slope depending on the location of the limb being analyzed). First and second peak external knee adduction moment (KAM), external knee adduction moment rate, and external knee adduction moment impulse were extracted from the stance phase of gait. Mixed ANOVA statistics with Bonferonni post hoc analyses were performed. RESULTS: Significant limb differences were only found for KAM rate and first peak KAM. When compared to all other surfaces up slope had the significantly lowest KAM rate and was not significantly lower for all other tested variables. Down slope had significantly greater KAM rate than all surfaces except bottom slope. KAM second peak and KAM impulse analysis resulted in no significant differences. CONCLUSIONS: Individuals at risk for developing, or currently dealing with, knee OA could avoid walking for extended periods on down slope. Walking up moderate slopes may be considered as a complementary activity to level walking for rehabilitation and delaying OA progression. The lack of significant limb differences suggests that second peak KAM and KAM impulse may not be appropriate load-related indicators of OA initiation among prosthesis users without OA. KAM rate was the most sensitive joint loading variable and therefore should be investigated further as an appropriate variable for identifying OA risk in individuals with transtibial amputations.

Activation of Epithelial Signal Transducer and Activator of Transcription 1 by Interleukin 28 Controls Mucosal Healing in Mice With Colitis and Is Increased in Mucosa of Patients With Inflammatory Bowel Disease.

BACKGROUND & AIMS: We investigated the roles of interleukin 28A (also called IL28A or interferon λ2) in intestinal epithelial cell (IEC) activation, studying its effects in mouse models of inflammatory bowel diseases (IBD) and intestinal mucosal healing. METHODS: Colitis was induced in C57BL/6JCrl mice (controls), mice with IEC-specific disruption of Stat1 (Stat1IEC-KO), mice with disruption of the interferon λ receptor 1 gene (Il28ra-/-), and mice with disruption of the interferon regulatory factor 3 gene (Irf3-/-), with or without disruption of Irf7 (Irf7-/-). We used high-resolution mini-endoscopy and in vivo imaging methods to assess colitis progression. We used 3-dimensional small intestine and colon organoids, along with RNA-Seq and gene ontology methods, to characterize the effects of IL28 on primary IECs. We studied the effects of IL28 on the human intestinal cancer cell line Caco-2 in a wound-healing assay, and in mice colon wounds. Colonic biopsies and resected tissue from patients with IBD (n = 62) and patients without colon inflammation (controls, n = 23) were analyzed by quantitative polymerase chain rection to measure expression of IL28A, IL28RA, and other related cytokines; biopsy samples were also analyzed by immunofluorescence to identify sources of IL28 production. IECs were isolated from patient tissues and incubated with IL28; signal transducer and activator of transcription 1 (STAT1) phosphorylation was measured by immunoblots and confocal imaging. RESULTS: Lamina propria cells in colon tissues of patients with IBD, and mice with colitis, had increased expression of IL28 compared with controls; levels of IL28R were increased in the colonic epithelium of patients with IBD and mice with colitis. Administration of IL28 induced phosphorylation of STAT1 in primary human and mouse IECs, increasing with dose. Il28ra-/-, Irf3-/-, Irf3-/-Irf7-/-, as well as Stat1IEC-KO mice, developed more severe colitis after administration of dextran sulfate sodium than control mice, with reduced epithelial restitution. Il28ra-/- and Stat1IEC-KO mice also developed more severe colitis in response to oxazolone than control mice. We found IL28 to induce phosphorylation (activation) of STAT1 in epithelial cells, leading to their proliferation in organoid culture. Administration of IL28 to mice with induced colonic wounds promoted mucosal healing. CONCLUSIONS: IL28 controls proliferation of IECs in mice with colitis and accelerates mucosal healing by activating STAT1. IL28 might be developed as a therapeutic agent for patients with IBD.

Aspergillus fumigatus protein phosphatase PpzA is involved in iron assimilation, secondary metabolite production, and virulence.

Metal restriction imposed by mammalian hosts during an infection is a common mechanism of defence to reduce or avoid the pathogen infection. Metals are essential for organism survival due to its involvement in several biological processes. Aspergillus fumigatus causes invasive aspergillosis, a disease that typically manifests in immunocompromised patients. A. fumigatus PpzA, the catalytic subunit of protein phosphatase Z (PPZ), has been recently identified as associated with iron assimilation. A. fumigatus has 2 high-affinity mechanisms of iron acquisition during infection: reductive iron assimilation and siderophore-mediated iron uptake. It has been shown that siderophore production is important for A. fumigatus virulence, differently to the reductive iron uptake system. Transcriptomic and proteomic comparisons between ∆ppzA and wild-type strains under iron starvation showed that PpzA has a broad influence on genes involved in secondary metabolism. Liquid chromatography-mass spectrometry under standard and iron starvation conditions confirmed that the ΔppzA mutant had reduced production of pyripyropene A, fumagillin, fumiquinazoline A, triacetyl-fusarinine C, and helvolic acid. The ΔppzA was shown to be avirulent in a neutropenic murine model of invasive pulmonary aspergillosis. PpzA plays an important role at the interface between iron starvation, regulation of SM production, and pathogenicity in A. fumigatus.

The Aspergillus fumigatus SchASCH9 kinase modulates SakAHOG1 MAP kinase activity and it is essential for virulence.

The serine-threonine kinase TOR, the Target of Rapamycin, is an important regulator of nutrient, energy and stress signaling in eukaryotes. Sch9, a Ser/Thr kinase of AGC family (the cAMP-dependent PKA, cGMP- dependent protein kinase G and phospholipid-dependent protein kinase C family), is a substrate of TOR. Here, we characterized the fungal opportunistic pathogen Aspergillus fumigatus Sch9 homologue (SchA). The schA null mutant was sensitive to rapamycin, high concentrations of calcium, hyperosmotic stress and SchA was involved in iron metabolism. The ΔschA null mutant showed increased phosphorylation of SakA, the A. fumigatus Hog1 homologue. The schA null mutant has increased and decreased trehalose and glycerol accumulation, respectively, suggesting SchA performs different roles for glycerol and trehalose accumulation during osmotic stress. The schA was transcriptionally regulated by osmotic stress and this response was dependent on SakA and MpkC. The double ΔschA ΔsakA and ΔschA ΔmpkC mutants were more sensitive to osmotic stress than the corresponding parental strains. Transcriptomics and proteomics identified direct and indirect targets of SchA post-exposure to hyperosmotic stress. Finally, ΔschA was avirulent in a low dose murine infection model. Our results suggest there is a complex network of interactions amongst the A. fumigatus TOR, SakA and SchA pathways.

Quantitative proteomics reveals new insights into calcium-mediated resistance mechanisms in Aspergillus flavus against the antifungal protein PgAFP in cheese.

The ability of Aspergillus flavus to produce aflatoxins in dairy products presents a potential hazard. The antifungal protein PgAFP from Penicillium chrysogenum inhibits various foodborne toxigenic fungi, including Aspergillus flavus. However, PgAFP did not inhibit A. flavus growth in cheese, which was related to the associated cation content. CaCl2 increased A. flavus permeability and prevented PgAFP-mediated inhibition in potato dextrose broth (PDB). PgAFP did not elicit any additional increase in permeability of CaCl2-incubated A. flavus. Furthermore, PgAFP did not alter metabolic capability, chitin deposition, or hyphal viability of A. flavus grown with CaCl2. Comparative proteomic analysis after PgAFP treatment of A. flavus in calcium-enriched PDB revealed increased abundance of 125 proteins, including oxidative stress-related proteins, as determined by label-free mass spectrometry (MS)-based proteomics. Seventy proteins were found at lower abundance, with most involved in metabolic pathways and biosynthesis of secondary metabolites. These changes do not support the blockage of potential PgAFP receptors in A. flavus by calcium as the main cause of the protective role. A. flavus resistance appears to be mediated by calcineurin, G-protein, and γ-glutamyltranspeptidase that combat oxidative stress and impede apoptosis. These findings could serve to design strategies to improve PgAFP activity against aflatoxigenic moulds in dairy products.

Exploration of Sulfur Assimilation of Aspergillus fumigatus Reveals Biosynthesis of Sulfur-Containing Amino Acids as a Virulence Determinant.

Fungal infections are of major relevance due to the increased numbers of immunocompromised patients, frequently delayed diagnosis, and limited therapeutics. To date, the growth and nutritional requirements of fungi during infection, which are relevant for invasion of the host, are poorly understood. This is particularly true for invasive pulmonary aspergillosis, as so far, sources of (macro)elements that are exploited during infection have been identified to only a limited extent. Here, we have investigated sulfur (S) utilization by the human-pathogenic mold Aspergillus fumigatus during invasive growth. Our data reveal that inorganic S compounds or taurine is unlikely to serve as an S source during invasive pulmonary aspergillosis since a sulfate transporter mutant strain and a sulfite reductase mutant strain are fully virulent. In contrast, the S-containing amino acid cysteine is limiting for fungal growth, as proven by the reduced virulence of a cysteine auxotroph. Moreover, phenotypic characterization of this strain further revealed the robustness of the subordinate glutathione redox system. Interestingly, we demonstrate that methionine synthase is essential for A. fumigatus virulence, defining the biosynthetic route of this proteinogenic amino acid as a potential antifungal target. In conclusion, we provide novel insights into the nutritional requirements ofA. fumigatus during pathogenesis, a prerequisite to understanding and fighting infection.