The invisibility cloak: Chitin binding protein of Verticillium nonalfalfae disguises fungus from plant chitinases

2018 ◽  
Author(s):  
Helena Volk ◽  
Kristina Marton ◽  
Marko Flajšman ◽  
Sebastjan Radišek ◽  
Ingo Hein ◽  
...  
Keyword(s):  
Fungal Infections ◽  
Binding Protein ◽  
Homology Modelling ◽  
Binding Motif ◽  
Carbohydrate Binding ◽  
Fungal Cell ◽  
Verticillium Nonalfalfae ◽  
Chitin Binding Protein ◽  
Plant Chitinases

AbstractDuring fungal infections, plant cells secrete chitinases that digest chitin in the fungal cell walls. The recognition of released chitin oligomers via lysin motif (LysM)-containing immune receptors results in the activation of defence signalling pathways. We report here that Verticillium nonalfalfae, a hemibiotrophic xylem-invading fungus, prevents this recognition process by secreting a CBM18 (carbohydrate binding motif 18)-chitin binding protein, VnaChtBP, which is transcriptionally activated specifically during the parasitic life stages. VnaChtBP is encoded by the Vna8.213 gene which is highly conserved within the species, suggesting high evolutionary stability and importance for the fungal lifestyle. In a pathogenicity assay, however, Vna8.213 knockout mutants exhibit wilting symptoms similar to the wild type fungus, suggesting that Vna8.213 activity is functionally redundant during fungal infection of hop. In binding assay, recombinant VnaChtBP binds chitin and chitin oligomers in vitro with submicromolar affinity and protects fungal hyphae from degradation by plant chitinases. Using a yeast-two-hybrid assay, homology modelling and molecular docking, we demonstrated that VnaChtBP forms dimers in the absence of ligands and that this interaction is stabilized by the binding of chitin hexamers with a similar preference in the two binding sites. Our data suggest that, in addition to chitin binding LysM (CBM50) and Avr4 (CBM14) fungal effectors, structurally unrelated CBM18 effectors have convergently evolved to prevent hydrolysis of the fungal cell wall against plant chitinases.

scholarly journals Chitin-Binding Protein of Verticillium nonalfalfae Disguises Fungus from Plant Chitinases and Suppresses Chitin-Triggered Host Immunity

2019 ◽  
Vol 32 (10) ◽  
pp. 1378-1390 ◽  
Author(s):  
Helena Volk ◽  
Kristina Marton ◽  
Marko Flajšman ◽  
Sebastjan Radišek ◽  
Hui Tian ◽  
...  
Keyword(s):  
Fungal Infections ◽  
Binding Protein ◽  
Host Immunity ◽  
Binding Motif ◽  
Carbohydrate Binding ◽  
Suspension Cells ◽  
Fungal Cell ◽  
Verticillium Nonalfalfae ◽  
Chitin Binding Protein ◽  
Plant Chitinases

During fungal infections, plant cells secrete chitinases, which digest chitin in the fungal cell walls. The recognition of released chitin oligomers via lysin motif (LysM)-containing immune host receptors results in the activation of defense signaling pathways. We report here that Verticillium nonalfalfae, a hemibiotrophic xylem-invading fungus, prevents these digestion and recognition processes by secreting a carbohydrate-binding motif 18 (CBM18)-chitin-binding protein, VnaChtBP, which is transcriptionally activated specifically during the parasitic life stages. VnaChtBP is encoded by the Vna8.213 gene, which is highly conserved within the species, suggesting high evolutionary stability and importance for the fungal lifestyle. In a pathogenicity assay, however, Vna8.213 knockout mutants exhibited wilting symptoms similar to the wild-type fungus, suggesting that Vna8.213 activity is functionally redundant during fungal infection of hop. In a binding assay, recombinant VnaChtBP bound chitin and chitin oligomers in vitro with submicromolar affinity and protected fungal hyphae from degradation by plant chitinases. Moreover, the chitin-triggered production of reactive oxygen species from hop suspension cells was abolished in the presence of VnaChtBP, indicating that VnaChtBP also acts as a suppressor of chitin-triggered immunity. Using a yeast-two-hybrid assay, circular dichroism, homology modeling, and molecular docking, we demonstrated that VnaChtBP forms dimers in the absence of ligands and that this interaction is stabilized by the binding of chitin hexamers with a similar preference in the two binding sites. Our data suggest that, in addition to chitin-binding LysM (CBM50) and Avr4 (CBM14) fungal effectors, structurally unrelated CBM18 effectors have convergently evolved to prevent hydrolysis of the fungal cell wall against plant chitinases and to interfere with chitin-triggered host immunity.

c-Fos-induced activation of a TATA-box-containing promoter involves direct contact with TATA-box-binding protein

1994 ◽  
Vol 14 (9) ◽  
pp. 6021-6029
Author(s):  
R Metz ◽  
A J Bannister ◽  
J A Sutherland ◽  
C Hagemeier ◽  
E C O'Rourke ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Transcriptional Activation ◽  
Binding Protein ◽  
Tata Box ◽  
Binding Motif ◽  
Protein Protein Interactions ◽  
High Concentrations ◽  
Tata Box Binding Protein

Transcriptional activation in eukaryotes involves protein-protein interactions between regulatory transcription factors and components of the basal transcription machinery. Here we show that c-Fos, but not a related protein, Fra-1, can bind the TATA-box-binding protein (TBP) both in vitro and in vivo and that c-Fos can also interact with the transcription factor IID complex. High-affinity binding to TBP requires c-Fos activation modules which cooperate to activate transcription. One of these activation modules contains a TBP-binding motif (TBM) which was identified through its homology to TBP-binding viral activators. This motif is required for transcriptional activation, as well as TBP binding. Domain swap experiments indicate that a domain containing the TBM can confer TBP binding on Fra-1 both in vitro and in vivo. In vivo activation experiments indicate that a GAL4-Fos fusion can activate a promoter bearing a GAL4 site linked to a TATA box but that this activity does not occur at high concentrations of GAL4-Fos. This inhibition (squelching) of c-Fos activity is relieved by the presence of excess TBP, indicating that TBP is a direct functional target of c-Fos. Removing the TBM from c-Fos severely abrogates activation of a promoter containing a TATA box but does not affect activation of a promoter driven only by an initiator element. Collectively, these results suggest that c-Fos is able to activate via two distinct mechanisms, only one of which requires contact with TBP. Since TBP binding is not exhibited by Fra-1, TBP-mediated activation may be one characteristic that discriminates the function of Fos-related proteins.

Structural characterization of amorphous calcium carbonate-binding protein: an insight into the mechanism of amorphous calcium carbonate formation

2013 ◽  
Vol 453 (2) ◽  
pp. 179-186 ◽  
Author(s):  
Jingtan Su ◽  
Xiao Liang ◽  
Qiang Zhou ◽  
Guiyou Zhang ◽  
Hongzhong Wang ◽  
...  
Keyword(s):  
Calcium Carbonate ◽  
Binding Sites ◽  
Binding Protein ◽  
Homology Modelling ◽  
Size Exclusion ◽  
Structural Basis ◽  
Amorphous Calcium Carbonate ◽  
Carbonate Formation

ACC (amorphous calcium carbonate) plays an important role in biomineralization process for its function as a precursor for calcium carbonate biominerals. However, it is unclear how biomacromolecules regulate the formation of ACC precursor in vivo. In the present study, we used biochemical experiments coupled with bioinformatics approaches to explore the mechanisms of ACC formation controlled by ACCBP (ACC-binding protein). Size-exclusion chromatography, chemical cross-linking experiments and negative staining electron microscopy reveal that ACCBP is a decamer composed of two adjacent pentamers. Sequence analyses and fluorescence quenching results indicate that ACCBP contains two Ca2+-binding sites. The results of in vitro crystallization experiments suggest that one Ca2+-binding site is critical for ACC formation and the other site affects the ACC induction efficiency. Homology modelling demonstrates that the Ca2+-binding sites of pentameric ACCBP are arranged in a 5-fold symmetry, which is the structural basis for ACC formation. To the best of our knowledge, this is the first report on the structural basis for protein-induced ACC formation and it will significantly improve our understanding of the amorphous precursor pathway.

scholarly journals Identification of a novel two-component system SenS/SenR modulating the production of the catalase-peroxidase CpeB and the haem-binding protein HbpS in Streptomyces reticuli

Microbiology ◽  
2005 ◽  
Vol 151 (11) ◽  
pp. 3603-3614 ◽  
Author(s):  
Darío Ortiz de Orué Lucana ◽  
Peijian Zou ◽  
Marc Nierhaus ◽  
Hildgund Schrempf
Keyword(s):  
Peroxidase Activity ◽  
Response Regulator ◽  
Binding Protein ◽  
Streptomyces Avermitilis ◽  
Binding Motif ◽  
Two Component System ◽  
Component System ◽  
Regulatory Cascade ◽  
Two Component

The Gram-positive soil bacterium and cellulose degrader Streptomyces reticuli synthesizes the mycelium-associated enzyme CpeB, which displays haem-dependent catalase and peroxidase activity, as well as haem-independent manganese-peroxidase activity. The expression of the furS–cpeB operon depends on the redox regulator FurS and the presence of the haem-binding protein HbpS. Upstream of hbpS, the neighbouring senS and senR genes were identified. SenS is a sensor histidine kinase with five predicted N-terminally located transmembrane domains. SenR is the corresponding response regulator with a C-terminal DNA-binding motif. Comparative transcriptional and biochemical studies with a designed S. reticuli senS/senR chromosomal disruption mutant and a set of constructed Streptomyces lividans transformants showed that the presence of the novel two-component system SenS/SenR negatively modulates the expression of the furS–cpeB operon and the hbpS gene. The presence of SenS/SenR enhances considerably the resistance of S. reticuli to haemin and the redox-cycling compound plumbagin, suggesting that this system could participate directly or indirectly in the sensing of redox changes. Epitope-tagged HbpS (obtained from an Escherichia coli transformant) as well as the native S. reticuli HbpS interact in vitro specifically with the purified SenS fusion protein. On the basis of these findings, together with data deduced from the S. reticuli hbpS mutant strain, HbpS is suggested to act as an accessory protein that communicates with the sensor protein to modulate the corresponding regulatory cascade. Interestingly, close and distant homologues, respectively, of the SenS/SenR system are encoded within the Streptomyces coelicolor A3(2) and Streptomyces avermitilis genomes, but not within other known bacterial genomes. Hence the SenS/SenR system appears to be confined to streptomycetes.

scholarly journals Identification of a New Class of Antifungals Targeting the Synthesis of Fungal Sphingolipids

mBio ◽  
2015 ◽  
Vol 6 (3) ◽  
Author(s):  
Visesato Mor ◽  
Antonella Rella ◽  
Amir M. Farnoud ◽  
Ashutosh Singh ◽  
Mansa Munshi ◽  
...  
Keyword(s):  
Cell Cycle Progression ◽  
Fungal Infections ◽  
Pathogenic Fungi ◽  
Antifungal Drugs ◽  
Sequencing Analysis ◽  
Fungal Cell ◽  
New Class ◽  
Narrow Spectrum

ABSTRACT Recent estimates suggest that >300 million people are afflicted by serious fungal infections worldwide. Current antifungal drugs are static and toxic and/or have a narrow spectrum of activity. Thus, there is an urgent need for the development of new antifungal drugs. The fungal sphingolipid glucosylceramide (GlcCer) is critical in promoting virulence of a variety of human-pathogenic fungi. In this study, we screened a synthetic drug library for compounds that target the synthesis of fungal, but not mammalian, GlcCer and found two compounds [N′-(3-bromo-4-hydroxybenzylidene)-2-methylbenzohydrazide (BHBM) and its derivative, 3-bromo-N′-(3-bromo-4-hydroxybenzylidene) benzohydrazide (D0)] that were highly effective in vitro and in vivo against several pathogenic fungi. BHBM and D0 were well tolerated in animals and are highly synergistic or additive to current antifungals. BHBM and D0 significantly affected fungal cell morphology and resulted in the accumulation of intracellular vesicles. Deep-sequencing analysis of drug-resistant mutants revealed that four protein products, encoded by genes APL5, COS111, MKK1, and STE2, which are involved in vesicular transport and cell cycle progression, are targeted by BHBM. IMPORTANCE Fungal infections are a significant cause of morbidity and mortality worldwide. Current antifungal drugs suffer from various drawbacks, including toxicity, drug resistance, and narrow spectrum of activity. In this study, we have demonstrated that pharmaceutical inhibition of fungal glucosylceramide presents a new opportunity to treat cryptococcosis and various other fungal infections. In addition to being effective against pathogenic fungi, the compounds discovered in this study were well tolerated by animals and additive to current antifungals. These findings suggest that these drugs might pave the way for the development of a new class of antifungals.

scholarly journals Interaction of the Antiactivator FleN with the Transcriptional Activator FleQ Regulates Flagellar Number inPseudomonas aeruginosa

2001 ◽  
Vol 183 (22) ◽  
pp. 6636-6644 ◽  
Author(s):  
Nandini Dasgupta ◽  
Reuben Ramphal
Keyword(s):  
Binding Protein ◽  
Direct Interaction ◽  
Transcriptional Activator ◽  
Binding Motif ◽  
Yeast Two Hybrid System ◽  
Gtp Binding ◽  
C Terminus ◽  
Enhancer Binding Protein ◽  
Two Hybrid

ABSTRACT Flagellar number in Pseudomonas aeruginosa is controlled by FleN, a putative ATP/GTP binding protein. Disruption offleN results in multiflagellation of the otherwise monoflagellate strains PAK and PAO1 and is associated with a chemotactic defect. We propose that flagellar number is maintained by the antiactivator FleN, which downregulates flagellar genes by binding to their transcriptional activator, FleQ, an enhancer binding protein belonging to the NifA subfamily. In this report we demonstrate direct interaction of FleN and FleQ in the yeast two-hybrid system. Mutagenesis of the putative ATP/GTP binding motif in FleN24K→Q and truncation of FleN at either the N or C terminus abrogates this interaction. FleN does not inhibit the DNA binding ability of FleQ in vitro, thus indicating that it probably utilizes another mechanism(s) to serve as a FleQ antiactivator.

scholarly journals A Bacillus thuringiensis Chitin-Binding Protein is Involved in Insect Peritrophic Matrix Adhesion and Takes Part in the Infection Process

Toxins ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 252
Author(s):  
Jiaxin Qin ◽  
Zongxing Tong ◽  
Yiling Zhan ◽  
Christophe Buisson ◽  
Fuping Song ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Galleria Mellonella ◽  
Binding Protein ◽  
Insect Pest ◽  
Infection Process ◽  
Peritrophic Matrix ◽  
Limited Information ◽  
Chitin Binding Protein

Bacillus thuringiensis (Bt) is used for insect pest control, and its larvicidal activity is primarily attributed to Cry toxins. Other factors participate in infection, and limited information is available regarding factors acting on the peritrophic matrix (PM). This study aimed to investigate the role of a Bt chitin-binding protein (CBPA) that had been previously shown to be expressed at pH 9 in vitro and could therefore be expressed in the alkaline gut of lepidopteron larvae. A ∆cbpA mutant was generated that was 10-fold less virulent than wild-type Bt HD73 towards Ostrinia furnacalis neonate larvae, indicating its important role in infection. Purified recombinant Escherichia coli CBPA was shown to have a chitin affinity, thus indicating a possible interaction with the chitin-rich PM. A translational GFP–CBPA fusion elucidated the localization of CBPA on the bacterial surface, and the transcriptional activity of the promoter PcbpA was immediately induced and confirmed at pH 9. Next, in order to connect surface expression and possible in vivo gut activity, last instar Galleria mellonella (Gm) larvae (not susceptible to Bt HD-73) were used as a model to follow CBPA in gut expression, bacterial transit, and PM adhesion. CBPA-GFP was quickly expressed in the Gm gut lumen, and more Bt HD73 strain bacteria adhered to the PM than those of the ∆cbpA mutant strain. Therefore, CBPA may help to retain the bacteria, via the PM binding, close to the gut surface and thus takes part in the early steps of Bt gut interactions.

scholarly journals Galectin-3 is an important mediator of VEGF- and bFGF-mediated angiogenic response

2010 ◽  
Vol 207 (9) ◽  
pp. 1981-1993 ◽  
Author(s):  
Anna I. Markowska ◽  
Fu-Tong Liu ◽  
Noorjahan Panjwani
Keyword(s):  
Growth Factor ◽  
Binding Protein ◽  
Integrin Αvβ3 ◽  
Dominant Negative ◽  
Carbohydrate Binding ◽  
Αvβ3 Integrin ◽  
Angiogenic Response ◽  
Inhibition Of Angiogenesis ◽  
Galectin 3

Recent studies have shown that a carbohydrate-binding protein, galectin-3, is a novel pro-angiogenic molecule. The mechanism by which galectin-3 promotes angiogenesis remains unknown. We demonstrate here that galectin-3 is a mediator of vascular endothelial growth factor (VEGF)- and basic fibroblast growth factor (bFGF)-mediated angiogenic response. Angiogenesis assays revealed that galectin-3 inhibitors, β-lactose and dominant-negative galectin-3, reduce VEGF- and bFGF-mediated angiogenesis in vitro and that VEGF- and bFGF-mediated angiogenic response is reduced in galectin-3 knockdown cells and Gal3−/− animals. Integrin αvβ3 was identified as the major galectin-3–binding protein and anti-αv, -β3, and -αvβ3 integrin function-blocking antibodies significantly inhibited the galectin-3–induced angiogenesis. Furthermore, galectin-3 promoted the clustering of integrin αvβ3 and activated focal adhesion kinase. Knockdown of GnTV, an enzyme that synthesizes high-affinity glycan ligands for galectin-3, substantially reduced: (a) complex N-glycans on αvβ3 integrins and (b) VEGF- and bFGF-mediated angiogenesis. Collectively, these data suggest that galectin-3 modulates VEGF- and bFGF-mediated angiogenesis by binding via its carbohydrate recognition domain, to the GnTV synthesized N-glycans of integrin αvβ3, and subsequently activating the signaling pathways that promote the growth of new blood vessels. These findings have broad implications for developing novel, carbohydrate-based therapeutic agents for inhibition of angiogenesis.

scholarly journals Mo-CBP4, a purified chitin-binding protein from Moringa oleifera seeds, is a potent antidermatophytic protein: In vitro mechanisms of action, in vivo effect against infection, and clinical application as a hydrogel for skin infection

2020 ◽  
Vol 149 ◽  
pp. 432-442 ◽  
Author(s):  
Tiago Deiveson Pereira Lopes ◽  
Pedro Filho Noronha Souza ◽  
Helen Paula Silva da Costa ◽  
Mirella Leite Pereira ◽  
João Xavier da Silva Neto ◽  
...  
Keyword(s):  
Clinical Application ◽  
Moringa Oleifera ◽  
Skin Infection ◽  
Binding Protein ◽  
Mechanisms Of Action ◽  
Chitin Binding Protein

AmBisome: liposomal formulation, structure, mechanism of action and pre-clinical experience

2002 ◽  
Vol 49 (suppl_1) ◽  
pp. 21-30 ◽  
Author(s):  
Jill Adler-Moore ◽  
Richard T. Proffitt
Keyword(s):  
Animal Models ◽  
Amphotericin B ◽  
Mechanism Of Action ◽  
Mammalian Cells ◽  
Fungal Infections ◽  
Liposomal Formulation ◽  
Fungal Cell ◽  
High Transition ◽  
High Doses

Abstract Amphotericin B is the treatment of choice for life-threatening systemic fungal infections such as candidosis and aspergillosis. To improve this drug's efficacy and reduce its acute and chronic toxicities, several lipid formulations of the drug have been developed, including AmBisome, a liposomal formulation of amphotericin B. The liposome is composed of high transition temperature phospholipids and cholesterol, designed to incorporate amphotericin B securely into the liposomal bilayer. AmBisome can bind to fungal cell walls, where the liposome is disrupted. The amphotericin B, after being released from the liposomes, is thought to transfer through the cell wall and bind to ergosterol in the fungal cell membrane. This mechanism of action of AmBisome results in its potent in vitro fungicidal activity while the integrity of the liposome is maintained in the presence of mammalian cells, for which it has minimal toxicity. In animal models, AmBisome is effective in treating both intracellular (leishmaniasis and histoplasmosis) and extracellular (candidosis and aspergillosis) systemic infections. Because of its low toxicity at the organ level, intravenous AmBisome can be safely delivered at markedly high doses of amphotericin B (1–30 mg/kg) for the treatment of systemic fungal infections. AmBisome has a circulating half-life of 5–24 h in animals, and in animal models appears to localize at sites of infection in the brain (cryptococcosis, aspergillosis, coccidioidomycosis), lungs (blastomycosis, paracoccidioidomycosis, aspergillosis) and kidneys (candidosis), delivering amphotericin B that remains bioavailable in tissues for several weeks following treatment.

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