Peptide microarrays coupled to machine learning reveal individual epitopes from human antibody responses with neutralizing capabilities against SARS-CoV-2.

The coronavirus SARS-CoV-2 is the causative agent for the disease COVID-19. To capture the IgA, IgG, and IgM antibody response of patients infected with SARS-CoV-2 at individual epitope resolution, we constructed planar microarrays of 648 overlapping peptides that cover the four major structural proteins S(pike), N(ucleocapsid), M(embrane), and E(nvelope). The arrays were incubated with sera of 67 SARS-CoV-2 positive and 22 negative control samples. Specific responses to SARS-CoV-2 were detectable, and nine peptides were associated with a more severe course of the disease. A random forest model disclosed that antibody binding to 21 peptides, mostly localized in the S protein, was associated with higher neutralization values in cellular anti-SARS-CoV-2 assays. For antibodies addressing the N-terminus of M, or peptides close to the fusion region of S, protective effects were proven by antibody depletion and neutralization assays. The study pinpoints unusual viral binding epitopes that might be suited as vaccine candidates.

Identification of Antimotilins, Novel Inhibitors of Helicobacter pylori Flagellar Motility That Inhibit Stomach Colonization in a Mouse Model.

New treatment options against the widespread cancerogenic gastric pathogen Helicobacter pylori are urgently needed. We describe a novel screening procedure for inhibitors of H. pylori flagellar biosynthesis. The assay is based on a flaA flagellin gene-luciferase reporter fusion in H. pylori and was amenable to multi-well screening formats with an excellent Z factor. We screened various compound libraries to identify virulence blockers ("antimotilins") that inhibit H. pylori motility or the flagellar type III secretion apparatus. We identified compounds that either inhibit both motility and the bacterial viability, or the flagellar system only, without negatively affecting bacterial growth. Novel anti-virulence compounds which suppressed flagellar biosynthesis in H. pylori were active on pure H. pylori cultures in vitro and partially suppressed motility directly, reduced flagellin transcript and flagellin protein amounts. We performed a proof-of-principle treatment study in a mouse model of chronic H. pylori infection and demonstrated a significant effect on H. pylori colonization for one antimotilin termed Active2 even as a monotherapy. The diversity of the intestinal microbiota was not significantly affected by Active2. In conclusion, the novel antimotilins active against motility and flagellar assembly bear promise to complement commonly used antibiotic-based combination therapies for treating and eradicating H. pylori infections. IMPORTANCE Helicobacter pylori is one of the most prevalent bacterial pathogens, inflicting hundreds of thousands of peptic ulcers and gastric cancers to patients every year. Antibacterial treatment of H. pylori is complicated due to the need of combining multiple antibiotics, entailing serious side effects and increasing selection for antibiotic resistance. Here, we aimed to explore novel nonantibiotic approaches to H. pylori treatment. We selected an antimotility approach since flagellar motility is essential for H. pylori colonization. We developed a screening system for inhibitors of H. pylori motility and flagellar assembly, and identified numerous novel antibacterial and anti-motility compounds (antimotilins). Selected compounds were further characterized, and one was evaluated in a preclinical therapy study in mice. The antimotilin compound showed a good efficacy to reduce bacterial colonization in the model, such that the antimotilin approach bears promise to be further developed into a therapy against H. pylori infection in humans.

Dual Agents: Fungal Macrocidins and Synthetic Analogues with Herbicidal and Antibiofilm Activities.

Eight analogues of the bioherbicides macrocidin A (1) and Z (2) with structural variance in the size of the macrocycle, its para- or meta-cyclophane character, and its functional groups were synthesized on two modular routes and tested for herbicidal, antibiotic, and antibiofilm activities. Apart from the lead compounds 1 and 2, the structurally simplified dihydromacrocidin Z (3) and normacrocidin Z (4) showed high herbicidal activity in either thistles, dandelions or in both. The derivatives 2, 3, and dibromide 9 also inhibited the growth of Staphylococcus aureus biofilms by ca 70% when applied at subtoxic concentrations as low as ca 20 µM, which are unlikely to induce bacterial resistance. They also led to the dispersion of preformed biofilms of S. aureus, exceeding a similar effect by microporenic acid A, a known biofilm inhibitor. Compounds 3 and 9 showed no noticeable cytotoxicity against human cancer and endothelial cells at concentrations below 50 µM, making them conceivable candidates for application as anti-biofilm agents in a medicinal context.

Inhibition of Respiration of Candida albicans by Small Molecules Increases Phagocytosis Efficacy by Macrophages.

Candida albicans adapts to various conditions in different body niches by regulating gene expression, protein synthesis, and metabolic pathways. These adaptive reactions not only allow survival but also influence the interaction with host cells, which is governed by the composition and structure of the fungal cell wall. Numerous studies had shown linkages between mitochondrial functionality, cell wall integrity and structure, and pathogenicity. Thus, we decided to inhibit single complexes of the respiratory chain of C. albicans and to analyze the resultant interaction with macrophages via their phagocytic activity. Remarkably, inhibition of the fungal bc1 complex by antimycin A increased phagocytosis, which correlated with an increased accessibility of ß-glucans. To contribute to mechanistic insights, we performed metabolic studies, which highlighted significant changes in the abundance of constituents of the plasma membrane. Collectively, our results reinforce the strong linkage between fungal energy metabolism and other components of fungal physiology, which also determine the vulnerability to immune defense reactions.IMPORTANCE The yeast Candida albicans is one of the major fungal human pathogens, for which new therapeutic approaches are required. We aimed at enhancements of the phagocytosis efficacy of macrophages by targeting the cell wall structure of C. albicans, as the coverage of the ß-glucan layer by mannans is one of the immune escape mechanisms of the fungus. We unambiguously show that inhibition of the fungal bc1 complex correlates with increased accessibilities of ß-glucans and improved phagocytosis efficiency. Metabolic studies proved not only the known direct effects on reactive oxygen species (ROS) production and fermentative pathways but also the clear downregulation of the ergosterol pathway and upregulation of unsaturated fatty acids. The changed composition of the plasma membrane could also influence the interaction with the overlying cell wall. Thus, our work highlights the far-reaching relevance of energy metabolism, indirectly also for host-pathogen interactions, without affecting viability.

Inhibition of Type IV Secretion Activity and Growth of Helicobacter pylori by Cisplatin and Other Platinum Complexes.

Type IV secretion systems are protein secretion machineries that are frequently used by pathogenic bacteria to inject their virulence factors into target cells of their respective hosts. In the case of the human gastric pathogen Helicobacter pylori, the cytotoxin-associated gene (Cag) type IV secretion system is considered a major cause for severe disease, such as gastric cancer, and thus constitutes an attractive target for specific treatment options against H. pylori infections. Here, we have used a Cag type IV secretion reporter assay for screening a repurposing compound library for inhibitors targeting this system. We found that the antitumor agent cisplatin, a platinum coordination complex that kills target cells by formation of DNA crosslinks, is a potent inhibitor of the Cag type IV secretion system. Strikingly, we found that this inhibitory activity of cisplatin depends on a ligand exchange reaction which incorporates a solvent molecule (dimethylsulfoxide) into the complex, a modification which is known to be deleterious for DNA crosslinking, and for its anticancer activity. We extended our analysis to several analogous platinum complexes containing N-heterocyclic carbene, as well as DMSO or other ligands, and found varying inhibitory activities toward the Cag system which were not congruent with their DNA-binding properties, suggesting that protein interactions may cause the inhibitory effect. Inhibition experiments under varying conditions revealed effects on adherence and bacterial viability as well, and showed that the type IV secretion-inhibitory capacity of platinum complexes can be inactivated by sulfur-containing reagents and in complex bacterial growth media. Taken together, our results demonstrate DNA binding-independent inhibitory effects of cisplatin and other platinum complexes against different H. pylori processes including type IV secretion.

EU-OPENSCREEN: A Novel Collaborative Approach to Facilitate Chemical Biology.

Compound screening in biological assays and subsequent optimization of hits is indispensable for the development of new molecular research tools and drug candidates. To facilitate such discoveries, the European Research Infrastructure EU-OPENSCREEN was founded recently with the support of its member countries and the European Commission. Its distributed character harnesses complementary knowledge, expertise, and instrumentation in the discipline of chemical biology from 20 European partners, and its open working model ensures that academia and industry can readily access EU-OPENSCREEN's compound collection, equipment, and generated data. To demonstrate the power of this collaborative approach, this perspective article highlights recent projects from EU-OPENSCREEN partner institutions. These studies yielded (1) 2-aminoquinazolin-4(3 H)-ones as potential lead structures for new antimalarial drugs, (2) a novel lipodepsipeptide specifically inducing apoptosis in cells deficient for the pVHL tumor suppressor, (3) small-molecule-based ROCK inhibitors that induce definitive endoderm formation and can potentially be used for regenerative medicine, (4) potential pharmacological chaperones for inborn errors of metabolism and a familiar form of acute myeloid leukemia (AML), and (5) novel tankyrase inhibitors that entered a lead-to-candidate program. Collectively, these findings highlight the benefits of small-molecule screening, the plethora of assay designs, and the close connection between screening and medicinal chemistry within EU-OPENSCREEN.

Chemical and Biological Aspects of Nutritional Immunity-Perspectives for New Anti-Infectives that Target Iron Uptake Systems.

Upon bacterial infection, one of the defense mechanisms of the host is the withdrawal of essential metal ions, in particular iron, which leads to "nutritional immunity". However, bacteria have evolved strategies to overcome iron starvation, for example, by stealing iron from the host or other bacteria through specific iron chelators with high binding affinity. Fortunately, these complex interactions between the host and pathogen that lead to metal homeostasis provide several opportunities for interception and, thus, allow the development of novel antibacterial compounds. This Review focuses on iron, discusses recent highlights, and gives some future perspectives which are relevant in the fight against antibiotic resistance.

Regulation of Candida albicans Interaction with Macrophages through the Activation of HOG Pathway by Genistein.

The severity of infections caused by Candida albicans, the most common opportunistic human fungal pathogen, needs rapid and effective antifungal treatments. One of the effective ways is to control the virulence factors of the pathogen. Therefore, the current study examined the effects of genistein, a natural isoflavone present in soybeans, on C. albicans. The genistein-treated C. albicans cells were then exposed to macrophages. Although no inhibition effect on the growth rates of C. albicans was noted an enhancement of the immune response to macrophages has been observed, indicated by phagocytosis and release of cytokines TNF-α and IL-10. The effect of genistein on the enhanced phagocytosis can be mimicked by the fungicides fludioxonil or iprodione, which inhibit the histidine kinase Cos1p and lead to activation of HOG pathway. The western blot results showed a clear phosphorylation of Hog1p in the wild type strain of C. albicans after incubation with genistein. In addition, effects of genistein on the phosphorylation of Hog1p in the histidine kinase mutants Δcos1 and Δsln1 were also observed. Our results thus indicate a new bio-activity of genistein on C. albicans by activation of the HOG pathway of the human pathogen C. albicans.

Short peptides allowing preferential detection of Candida albicans hyphae.

Whereas the detection of pathogens via recognition of surface structures by specific antibodies and various types of antibody mimics is frequently described, the applicability of short linear peptides as sensor molecules or diagnostic tools is less well-known. We selected peptides which were previously reported to bind to recombinant S. cerevisiae cells, expressing members of the C. albicans Agglutinin-Like-Sequence (ALS) cell wall protein family. We slightly modified amino acid sequences to evaluate peptide sequence properties influencing binding to C. albicans cells. Among the selected peptides, decamer peptides with an "AP"-N-terminus were superior to shorter peptides. The new decamer peptide FBP4 stained viable C. albicans cells more efficiently in their mature hyphal form than in their yeast form. Moreover, it allowed distinction of C. albicans from other related Candida spp. and could thus be the basis for the development of a useful tool for the diagnosis of invasive candidiasis.

Identification of whole pathogenic cells by monoclonal antibodies generated against a specific peptide from an immunogenic cell wall protein.

We selected the immunogenic cell wall ß-(1,3)-glucosyltransferase Bgl2p from Candida albicans as a target protein for the production of antibodies. We identified a unique peptide sequence in the protein and generated monoclonal anti- C. albicans Bgl2p antibodies, which bound in particular to whole C. albicans cells.

[Effect of genistein on the TLR and MAPK transduction cascades in lipopolysaccharide -stimulated macrophages].

OBJECTIVE: To delineate and confirm the signaling processes involving mitogen-activated protein kinase (MAPK) and Toll-like receptor (TLR) in the regulation of lipopolysaccharide (LPS)-induced activity of macrophages by genistein at the protein and transcriptional levels. METHODS: RAW264.7 macrophages were treated with genistein and then stimulated with LPS (100 ng/mL) for different time duration. We evaluated the induction of MAPK phosphorylation by Western blot analysis; RT2 Profiler(TM); PCR array was used to investigate the expressions of TLR pathway-related genes after different treatments. RESULTS: LPS led to the phosphorylation of Raf, MEK1/2 and ERK1/2 at 15 minutes post-stimulation and it lasted till 30 minutes. Phosphorylation of p38 was also observed, but it was not as obvious as p-ERK1/2. Addition of genistein further increased the phosphorylation of ERK1/2, its downstream protein p90rsk and p38. Cells treated with LPS and LPS together with genistein demonstrated significant number of genes to be differentially regulated as compared with control cells. Genistein alone could up-regulate the gene CD80, MEKK1, c-fos, Rela, and Ticam2. LPS could up-regulate 20 genes including cytokines IFN-ß, IL-10, IL-1α, IL-1ß, IL-6, TNF-α, colony stimulating factor 2 (CSF-2) and CSF-3, chemokines CCL2 and CXCL10, transcription factor NF-κB1, IκB-α and cyclooxygenase 2 (COX-2). The presence of genistein led to a strong inhibition of the expressions of these genes and up-regulated the transcription factors IκB-ß and c-Rel, a subunit of NF-κB. CONCLUSION: Genistein strongly enhances the LPS-induced activities of MAPK transduction cascades and inhibits TLR pathway.

Genistein induces morphology change and G2/M cell cycle arrest by inducing p38 MAPK activation in macrophages.

Genistein is a well known natural compound which is present in soy foods and exerts many beneficial functions such as anticancer, anti-inflammatory and antioxidant. However, until now little is known about the effects of genistein on the function of macrophages. The murine macrophage cell line RAW264.7 was used as target cell line. The results show that at concentrations of 50-100µM, genistein reduced cell viability to 70%-80% (after 24h) and 50%-60% (after 48h), which was due to G2/M phase cell cycle arrest. Treatment of the macrophages with genistein for 24 or 48h also led to significant morphological changes, such as elongation of the cells and development of long pseudopodia-like protrusions. By staining the F-actin cytoskeleton, we observed accumulation of actin-filaments at the edges of the cells. The morphology change and G2/M phase arrest after genistein treatment is due to the activation of the phosphorylation of MAP kinase p38. The morphology change and cell cycle arrest can be significantly reverted when treatment is combined with p38 inhibitor SB203580. Moreover, after treatment of the macrophages with genistein for 24 and 48h, the phagocytotic efficiency for Candida albicans was decreased in a time- and dose-dependent manner which correlates to the morphology change. The production of cytokines (TNF-α) stimulated by C. albicans was strongly inhibited by genistein. In conclusion, genistein showed a strong immune modulatory effect on the macrophages.

[Anti-inflammatory effect of Syk inhibitor in LPS stimulated macrophages].

OBJECTIVE: To investigate the role of spleen tyrosine kinase (Syk)on the Toll-like receptor 4 (TLR4) signal transduction cascade. METHODS: RAW264.7 macrophages were treated with different concentrations of Syk inhibitor and then stimulated with 100 ng/mL lipopolysaccharide (LPS). The cell viability was tested by the Alarmarblue assay. The NO production was detected by Griess reagents and cytokines in culture supernatants were quantified by ELISA. The expression of iNOS was detected by Western blotting. Moreover, the activation of the transcription factors NF-κB and activator protein 1 (AP-1) was investigated using the reporter cell line RAW-Blue(TM);. RESULTS: The Syk inhibitor had no effect on the cell proliferation even at concentrations of 14 µmol/L. However, the NO production (P<0.01) and iNOS expression resulting from LPS stimulation were inhibited, when the inhibitor concentration exceeded 1.5 µmol/L, and the TNF-α and IL-6 production were inhibited even at lower concentrations (P<0.01). Moreover, inhibition of the activation of both the transcription factors NF-κB and AP-1 was observed. CONCLUSION: The Syk inhibitor shows a significant anti-inflammatory property due to its inhibitory effect on NO and cytokine production by LPS stimulated macrophages. This effect correlated with the reduced activation of both transcription factors NF-κB and AP-1, which places Syk downstream of the TLR4 signal pathway but upstream of the point of divergence of both transcription factors.

Involvement of the mitogen activated protein kinase Hog1p in the response of Candida albicans to iron availability.

BACKGROUND: Iron is an essential nutrient for almost all organisms, and generating iron limiting conditions for pathogens is one of the host defense strategies against microbial infections. Excess of iron can be toxic; therefore, iron uptake is tightly controlled. The high affinity iron uptake system of the opportunistic pathogenic yeast Candida albicans has been shown to be essential for virulence. Several transcription factors and regulators of iron uptake genes were identified, but the knowledge of signaling pathways is still limited. Gene expression profiling of the Δhog1 deletion mutant indicated an involvement of the mitogen activated protein (MAP) kinase Hog1p. However, the function of Hog1p in the response of C. albicans to iron availability was not studied in detail. Thus, we analyzed phenotypic and molecular responses of C. albicans to different iron concentrations particularly with respect to the activity of the Hog1p MAP kinase module. RESULTS: We observed flocculation of yeast cells, when the iron ion concentration was equal to or higher than 5 µM. This phenotype was dependent on the MAP kinase Hog1p and the corresponding MAP kinase kinase Pbs2p. Moreover, high extracellular iron ion concentrations led to hyper-phosphorylation of Hog1p. We determined lower amounts of multicopper ferroxidase (MCFO) proteins and lower ferric reductase activity, when the iron ion concentration in the medium was increased. This effect was also observed for the Δhog1 mutant. However, the amounts of MCFO proteins and the cell surface ferric reductase activity were increased in the Δhog1 in comparison to wild type cells. This effect was independent of iron availability in growth media. CONCLUSIONS: In C. albicans, the MAP kinase Hog1p is part of the network regulating the response of the organism to iron availability. Hog1p was transiently phosphorylated under high iron concentrations and was essential for a flocculent phenotype. Furthermore, deletion of HOG1 led to increased levels of components of the reductive iron uptake system in comparison to the wild-type, independent of iron concentrations in the media. However, the additional induction of this system by low iron concentrations was independent of HOG1.

Identification of inhibitors of yeast-to-hyphae transition in Candida albicans by a reporter screening assay.

Candida albicans is one of the most common opportunistic fungal pathogens, causing life-threatening disease in immunocompromised patients. As it is not primarily a pathogen, but can exist in a commensal state, we aimed at the identification of new anti-infective compounds which do not eradicate the fungus, but primarily disable a virulence determinant. The yeast­hyphae-dimorphism of C. albicans is considered a major contributor to fungal disease, as mutants locked into either yeast or hyphal state have been shown to be less virulent in the mouse-model. We devised a high-throughput screening procedure which allows us to find inhibitors of the induction of hyphae. Hyphae-formation was induced by nitrogen starvation at 37 °C and neutral pH in a reporter strain, which couples promoter activity of the hyphae-specific HWP1 to ß-galactosidase expression. In a pilot screening of 720 novel synthetic compounds, we identified substances which inhibited the outgrowth of germ tubes. They belonged to chemical classes not yet known for antimycotic properties, namely methyl aryl-oxazoline carboxylates, dihydrobenzo[d]isoxazolones and thiazolo[4,5-e]benzoisoxazoles. In conclusion we developed a novel screening assay, which addresses the morphological switch from the yeast form of C. albicans to its hyphal form and identified novel chemical structures with activity against C. albicans.

Deletion of the Candida albicans histidine kinase gene CHK1 improves recognition by phagocytes through an increased exposure of cell wall beta-1,3-glucans.

The pathogenic fungus Candida albicans is able to cover its most potent proinflammatory cell wall molecules, the ß-glucans, underneath a dense mannan layer, so that the pathogen becomes partly invisible for immune cells such as phagocytes. As the C. albicans histidine kinases Chk1p, Cos1p and CaSln1p had been reported to be involved in virulence and cell wall biosynthesis, we investigated whether deletion of the respective genes influences the activity of phagocytes against C. albicans. We found that among all histidine kinase genes, CHK1 plays a prominent role in phagocyte activation. Uptake of the deletion mutant Δchk1 as well as the acidification of Δchk1-carrying phagosomes was significantly increased compared with the parental strain. These improved activities could be correlated with an enhanced accessibility of the mutant ß-1,3-glucans for immunolabelling. In addition, any inhibition of ß-1,3-glucan-mediated phagocytosis resulted in a reduced uptake of Δchk1, while ingestion of the parental strain was hardly affected. Moreover, deletion of CHK1 caused an enhanced release of interleukins 6 and 10, indicating a stronger activation of the ß-1,3-glucan receptor dectin-1. In conclusion, the Chk1p protein is likely to be involved in masking ß-1,3-glucans from immune recognition. As there are no homologues of fungal histidine kinases in mammals, Chk1p has to be considered as a promising target for new antifungal agents.

Characterization of the vascular endothelial growth factor-receptor interaction and determination of the recombinant protein by an optical receptor sensor.

Vascular endothelial growth factor (VEGF) is one of the most important factors controlling angiogenesis. It is a homodimeric glycoprotein belonging to the family of cysteine-knot proteins. The biological activity is transduced via membrane-spanning receptors of the tyrosine kinase receptor family. Each biologically active VEGF has two receptor binding sites leading to receptor dimerization as first step following ligand binding. The ligand-binding site of the receptor is localized on extracellular Ig-like domains. The extracellular part of the receptor Flt-1 (VEGFR-1) was expressed as soluble protein and was used as receptor in an optical affinity sensor system (BIAcore). Suitable conditions allowed the determination of the association and dissociation rate constants as k(a)=4+/-1.2 x 10(6) M(-1) s(-1) and k(d)=3+/-0.8 x 10(-5) s(-1), respectively, leading to an affinity constant of K(D)=7.5+/-3 pM, which is within the range published already from other investigations and methods. Increasing receptor loadings of the sensor surface decreased the binding efficiency, as the ratio of bound VEGF-molecules to theoretically available binding sites increased from 1:1.5 to 1:2.6. Increasing the surface loading further, allowed the establishment of a quantitative assay with the analytical performance being influenced by the receptor loading and the contact time between sample and immobilized receptor, i.e. sample volume. This assay was used for VEGF determination during the cultivation of a recombinant Pichia pastoris strain.