OmpC and OmpF Outer Membrane Proteins of Escherichia coli and Salmonella enterica Form Bona Fide Amyloids.

Outer membrane proteins (Omps) of Gram-negative bacteria represent porins involved in a wide range of virulence- and pathogenesis-related cellular processes, including transport, adhesion, penetration, and the colonization of host tissues. Most outer membrane porins share a specific spatial structure called the ß-barrel that provides their structural integrity within the membrane lipid bilayer. Recent data suggest that outer membrane proteins from several bacterial species are able to adopt the amyloid state alternative to their ß-barrel structure. Amyloids are protein fibrils with a specific spatial structure called the cross-ß that gives them an unusual resistance to different physicochemical influences. Various bacterial amyloids are known to be involved in host-pathogen and host-symbiont interactions and contribute to colonization of host tissues. Such an ability of outer membrane porins to adopt amyloid state might represent an important mechanism of bacterial virulence. In this work, we investigated the amyloid properties of the OmpC and OmpF porins from two species belonging to Enterobacteriaceae family, Escherichia coli, and Salmonella enterica. We demonstrated that OmpC and OmpF of E. coli and S. enterica form toxic fibrillar aggregates in vitro. These aggregates exhibit birefringence upon binding Congo Red dye and show characteristic reflections under X-ray diffraction. Thus, we confirmed amyloid properties for OmpC of E. coli and demonstrated bona fide amyloid properties for three novel proteins: OmpC of S. enterica and OmpF of E. coli and S. enterica in vitro. All four studied porins were shown to form amyloid fibrils at the surface of E. coli cells in the curli-dependent amyloid generator system. Moreover, we found that overexpression of recombinant OmpC and OmpF in the E. coli BL21 strain leads to the formation of detergent- and protease-resistant amyloid-like aggregates and enhances the birefringence of bacterial cultures stained with Congo Red. We also detected detergent- and protease-resistant aggregates comprising OmpC and OmpF in S. enterica culture. These data are important in the context of understanding the structural dualism of Omps and its relation to pathogenesis.

Degradation and decolorization of textile azo dyes by effective fungal-bacterial consortium.

BACKGROUND: Synthetic dyes are one of the main pollutants in the textile industry and bioremediation is considered as an environmentally friendly method to degrade them. Soil microbial consortia (MCs) are reported having the potential of decolorizing most of textile dyes. This study aimed at evaluating dye-degrading ability of MCs developed from fungi and bacteria. METHODS AND RESULTS: Fungi and bacteria were isolated from the soil samples obtained from textile waste dumping site at Horana industrial zone, Sri Lanka and were screened for crystal violet (CV) and congo red (CR) dyes to develop MCs. Decolorization assay was performed for MCs along with individual isolates under variable pH levels. Metabolized products were characterized to confirm the biodegradation. A. tamari (F5) and P. putida (B3) significantly (P < 0.05) decolorized both dyes. All the MCs showed higher decolorization percentages over the individual microorganisms. Neutral pH (pH 7) was the optimum pH for the decolorization of both dyes by individual isolates and the percentages were significantly changed under the acidic and basic pH levels. However, decolorization ability by all MCs was not significantly changed with pH. Consortium with A. tamari - P. putida significantly (P < 0.05) decolourized both dyes under optimum pH 7. CONCLUSION: All MCs showed better pH tolerance in degrading CV and CR. Thus, it can be concluded that the selected MC with A. tamari - P. putida can degrade CV and CR textile dyes efficiently into non-toxic compounds against plants under neutral pH. Degradation and decolorization of textile azo dyes by effective fungal-bacterial consortium.

Recombinant expression and characterization of a new laccase, bioinformatically identified, from the Antarctic thermophilic bacterium Geobacillus sp. ID17.

Geobacillus sp. ID17 is a gram-positive thermophilic bacterium isolated from Deception Island, Antarctica, which has shown to exhibit remarkable laccase activity in crude extract at high temperatures. A bioinformatic search using local databases led to the identification of three putative multicopper oxidase sequences in the genome of this microorganism. Sequence analysis revealed that one of those sequences contains the four-essential copper-binding sites present in other well characterized laccases. The gene encoding this sequence was cloned and overexpressed in Escherichia coli, partially purified and preliminary biochemically characterized. The resulting recombinant enzyme was recovered in active and soluble form, exhibiting optimum copper-dependent laccase activity at 55 °C, pH 6.5 with syringaldazine substrate, retaining over 60% of its activity after 1 h at 55 and 60 °C. In addition, this thermophilic enzyme is not affected by common inhibitors SDS, NaCl and L-cysteine. Furthermore, biodecolorization assays revealed that this laccase is capable of degrading 60% of malachite green, 54% of Congo red, and 52% of Remazol Brilliant Blue R, after 6 h at 55 °C with aid of ABTS as redox mediator. The observed properties of this enzyme and the relatively straightforward overexpression and partial purification of it could be of great interest for future biotechnology applications.

Enzymatic degradation of azo dyes methylene blue and congo red with peroxidase purified from cauliflower using affinity chromatography technique: Kinetic study, optimization and metal binding activity.

The effective results of the enzymatic decolorization of industrial azo dyes found in wastewater, which cause serious health and environmental problems, with peroxidases have recently increased the interest in these enzyme sources. Redox-mediated decolorization of Methylene Blue and Congo Red azo dyes with cauliflower (Brassica oleracea var. botrytis L.) peroxidase (CPOD) purified in one step using 4-amino 3-bromo 2-methyl benzohydrazide molecule was investigated for the first time. The inhibition effect of this molecule, which is used as a ligand in affinity chromatography, on the CPOD enzyme was investigated. The Ki and IC50 values for this enzyme were calculated as 0.113 ± 0.012 mM and 0.196 ± 0.011 mM, respectively. With the affinity gel obtained by binding to the Sepharose-4B-l-tyrosine matrix of this molecule, which shows a reversible inhibition effect, the purification values of CPOD enzyme were determined as 562-fold with a specific activity of 50,250 U mg-1. The purity of the enzyme was checked by the SDS-PAGE technique and its molecular weight was determined. A single band at 44 kDa was observed for the CPOD enzyme. In dye decolorization studies, the effects of dye, enzyme, and hydrogen peroxide concentrations as well as time, pH, and temperature were investigated. The profiles of the optimum conditions for both dyes were similar, and the percentages of decolorization of Methylene Blue and Congo Red under these conditions were 89% and 83%, respectively, at the end of the 40 min reaction time. Again, when examining the effect of metal ions on enzyme activity, it was found that there was no significant negative change in CPOD.

Exploring the use of Congo red agar in improving the serotyping of non-serotypeable Shigella with In-silico evidence.

OBJECTIVES: To assess if congo red could make non-serotypeable Shigella strains serotypeable and to employ molecular docking to determine the basis of the same phenomenon. METHODS: We used 42 bacterial strains of non-serotypeable Shigella collected from 2012 to 2019 for this study. Each bacterial strain was freshly inoculated onto congo red agar and incubated at 37° C for 18-24 h. Bacterial colonies obtained were re-subjected to biochemical tests followed by serotyping and serogrouping. In-silico studies to investigate the interaction between MxiC protein of T3SS and O-antigen LPS with congo red were performed. RESULTS: Of the total 42 non-serotypeable Shigella studied, (26/42)62% were capable of being serotyped following the use of congo red agar, 65% were Shigella flexneri, 19% were Shigella dysenteriae, while 2 strains (7%) each of Shigella boydii and Shigella sonnei were detected. We observed no change in their biochemical properties. The in-silico molecular docking studies revealed high binding affinity between congo red and the B-Chain of Mxi C. Out of the 5 chains of the O-Antigen, congo red showed robust binding with the B-chain with the involvement of a cluster of hydrophobic interactions between them. This may have a crucial role in the conversion of non-serotypeable strains to serotypeable strains on exposure to congo red as observed in our study. CONCLUSION: Congo red agar as a medium converts a sizeable percentage of non-serotypeable Shigella strains to serotypeable Shigella strains.

Eco-friendly detoxification of hazardous Congo red dye using novel fungal strain Trametes flavida WTFP2: Deduced enzymatic biomineralization process through combinatorial in-silico and in-vitro studies.

Growing textile industry is a major global concern, owing to the presence of recalcitrant hazardous pollutants, like synthetic dyes in discharged effluents. To explore new bioresources for mycoremediation, a high laccase-producing novel white-rot fungus (WRF), Trametes flavida WTFP2, was employed. T. flavida is an underexplored member of Polyporales. Using bioinformatic tools, 8 different cis-acting RNA elements were identified in the 5.8 S ITS gene sequence, where CRISPR (CRISPR-DR15), sRNA (RUF1), and snoRNA (ceN111) are uniquely present. Molecular docking was adopted to predict the catalytic interaction of chosen toxic diazo colorant, Congo red (CR), with four dye-degrading enzymes (laccase, lignin peroxidase, azoreductase, and aryl alcohol oxidase). With 376.41 × 103 U/L laccase production, novel WRF exhibited dye-decolorization potential. WTFP2 effectively removed 99.48 ± 0.04% CR (100 mg/L) and demonstrated remarkable recyclability and persistence in consecutive remediation trials. Mycelial dye adsorption was not only substantial driver of colorant elimination; decolorization using active T. flavida was regulated by enzymatic catalysis, as outlined by in-vitro growth, induction of extracellular enzymes, and FESEM. Fifteen metabolites were identified using HRLCMS-QTOF, and novel CR degradation pathway was proposed. Furthermore, microbial and phyto-toxicity tests of metabolites suggested complete detoxification of toxic dye, making the process clean, green, and economically sustainable.

Leveraging molecular docking to understand Congo red degradation by Staphylococcus caprae MB400.

Congo red (CR) is a genotoxic, sulphonated azo dye and poses significant pollution problem. We hereby report its degradation by Staphylococcus caprae MB400. The bacterium initially propagated as a suspected contaminant upon CR dye supplemented nutrient agar plates, forming zones of clearance around its growth area. The bacterium was purified, gram stained and identified as Staphylococcus caprae via 16S rRNA gene sequencing. Dye decolourization was analysed in liquid culture, and Fourier-transform infrared spectroscopy (FTIR) was conducted for analysis of degraded product/metabolites. A decolourization of ~ 96.0% at 100 µg/ml concentration and pH 7 after 24 h of incubation was observed. Structure of the azoreductase enzyme, responsible for breakage of the bond in the dye and ultimately decolourization, was predicted, and molecular docking was harnessed for understanding the mechanism behind the reduction of azo bond (-N=N-) and conversion to metabolites. Our analysis revealed 12 residues critical for structural interaction of the azoreductase enzyme with this dye. Among these, protein backbone region surrounding four residues, i.e. Lys65, Phe122, Ile166 and Phe169, showed major displacement changes, upon binding with the dye. However, overall the conformational changes were not large.

A sustainable approach to the degradation of dyes by fungal species isolated from industrial wastewaters: Performance, parametric optimization, kinetics and degradation mechanism.

Fungal abetted processes are among the finest approaches for the transformation or degradation and decolorization of dyes in effluents. In this piece of research; biodegradation and metabolic pathways of two toxic dyes Congo Red (CR) and Reactive black 5 (RB5) by two strains of Aspergillus sp. fungus in batch experiments has been investigated. Morphological characteristics of the isolates were observed with both light and electron microscopies. Based on molecular characterization the isolates were identified as Aspergillus flavus and Aspergillus niger. The degradation was also optimized via. operational parameters such as pH, temperature, incubation time, inoculums size, dye concentration, carbon sources and nitrogen sources. Degradation measurements revealed that the isolates effectively degraded 90% and 96% of CR and RB5 respectively. Metabolites were identified with Liquid chromatography-mass spectrometry (LCMS) and degradation pathways of the dyes were proposed. Toxicity assay Phaseolus mungo seeds showed that pure CR and RB5 dyes exhibits significant toxicity whereas fungal treated dye solution resulted in an abatement of the toxicity and cell viability was increased. The results stipulated in this article clearly showed the effectiveness of the isolates on detoxification of CR and RB5 dyes.

Evaluation of Congo red dye decolorization and degradation potential of an endophyte Colletotrichum gloeosporioides isolated from Thevetia peruviana (Pers.) K. Schum.

Decolorization and degradation of textile dye by endophytic fungi stand to be a profitable and viable alternative over conventional methods with respect to eco-friendliness, cost-effectiveness, and non-hazardous nature. One of the active fungal endophytes Colletotrichum gloeosporioides isolated from plant Thevetia peruviana (Pers.) K. Schum. was screened for laccase production and Congo red dye decolorization. Various physicochemical parameters like dye concentration, carbon sources, nitrogen sources, temperature, and pH were optimized, and the maximum decolorization (%) was achieved at 100 mg/L of dye concentration (82%), yeast extract (80%), 30 °C temp (80%), glucose (79%), and 7 pH (78%), respectively. SEM image and fungal biomass changes represent that fungus actively participated in the dye decolorization and had less significant effect on biomass. The regenerative ability of fungus C. gloeosporioides after dye decolorization indicated tolerance against the dye and was found to be more advantageous over previous reports of dye decolorization by other endophytic fungi. UV-Vis spectra, TLC, FTIR, and HPLC results confirmed the decolorization and degradation process due to absorption and biodegradation. Phytotoxicity assay depicted that degraded products are less toxic to Phaseolus mungo compared to Congo red. The overall findings showed that C. gloeosporioides possesses a good decolorization and degradation potential against Congo red and this endophyte can be profitably used for dye-containing wastewater treatment.

Functional Identification of Arthrinium phaeospermum Effectors Related to Bambusa pervariabilis × Dendrocalamopsis grandis Shoot Blight.

The shoot blight of Bambusa pervariabilis × Dendrocalamopsis grandis caused by Arthrinium phaeospermum made bamboo die in a large area, resulting in serious ecological and economic losses. Dual RNA-seq was used to sequence and analyze the transcriptome data of A. phaeospermum and B. pervariabilis × D. grandis in the four periods after the pathogen infected the host and to screen the candidate effectors of the pathogen related to the infection. After the identification of the effectors by the tobacco transient expression system, the functions of these effectors were verified by gene knockout. Fifty-three differentially expressed candidate effectors were obtained by differential gene expression analysis and effector prediction. Among them, the effectors ApCE12 and ApCE22 can cause programmed cell death in tobacco. The disease index of B. pervariabilis × D. grandis inoculated with mutant ΔApCE12 and mutant ΔApCE22 strains were 52.5% and 47.5%, respectively, which was significantly lower than that of the wild-type strains (80%), the ApCE12 complementary strain (77.5%), and the ApCE22 complementary strain (75%). The tolerance of the mutant ΔApCE12 and mutant ΔApCE22 strains to H2O2 and NaCl stress was significantly lower than that of the wild-type strain and the ApCE12 complementary and ApCE22 complementary strains, but there was no difference in their tolerance to Congo red. Therefore, this study shows that the effectors ApCE12 and ApCE22 play an important role in A. phaeospermum virulence and response to H2O2 and NaCl stress.

Construction of CdS-Tetrahymena thermophila hybrid system by efficient cadmium adsorption for dye removal under light irradiation.

The water pollution caused by heavy metals and dyes emitted by industries has become a worldwide problem. These pollutants are difficult to be biodegraded. Even at low concentrations, they are toxic and at last threaten human health. Herein, while using Tetrahymena thermophila, a single-celled ciliate protozoa, to enrich and remove the heavy metal Cd2+ from water, CdS nanoparticle-Tetrahymena thermophila hybrid system (CdS-T. thermophila) for dye pollution remediation under light irradiation was developed. The conditions of Cd2+ enrichment and removal by T. thermophila, construction of efficient CdS-T. thermophila, and decolorization of Congo red using CdS-T. thermophila were investigated. In the presence of cysteine ethyl ester, the removal rate of Cd2+ by T. thermophila was 94% at low Cd2+ concentration of 1 mg L-1. The adsorption capacity of T. thermophila to Cd2+ reached 43 mg g-1 at Cd2+ concentration of 80 mg L-1. Using 0.1 g L-1 constructed CdS-T. thermophila, the decolorization rate of 50 mg L-1 Congo red solution reached 95% in 60 min under light irradiation. This study provides a new insight to effective removing Cd2+ from water by T. thermophila to construct the CdS-T. thermophila and using it to remediate dye pollution in the environment.

Isolation and characterization of amyloid-like protein aggregates from soya beans and the effect of low pH and heat treatment on their stability.

Purified soya bean proteins (glycinin and conglycinin) are known to form amyloid-like aggregates in vitro at a higher temperature. Soya beans (chunks) are textured proteinaceous vegetables made from defatted soya flour by heating it above 100°C and extruding under high pressure. Therefore, it was assumed that subjecting the soya bean proteins to high temperatures raises the possibility of forming amyloids or amyloid-like protein aggregates. Hence, the present study aimed to examine the presence of amyloid-like protein aggregates in soya beans. The isolated protein aggregates from hydrated soya beans displayed typical characteristics of amyloids, such as the red shift in the absorption maximum (λmax ) of Congo red (CR), high Thioflavin T (ThT), and 8-Anilinonapthalene-1-sulfonate (ANS) binding, and fibrilar morphology. Furthermore, these aggregates were found to be stable against proteolytic hydrolysis, confirming the specific property of amyloids. The presence of amyloid-like structures in soya beans raises concerns about their implications for human nutrition and health. PRACTICAL APPLICATIONS: Protein aggregation has usually been considered detrimental. The traditional food-processing conditions, such as thermal processing, are associated with protein denaturation and aggregation. The formation of ordered protein aggregates with extensive ß-sheet are progressively evident in various protein-rich foods known as amyloid, which expands food safety concerns. Instead, it is also associated with poor nutritional characteristics. The present study concerns the presence of amyloid-like protein aggregates in widely consumed native soya beans, which are manufactured by extensive heat treatment of defatted soy flour. Although there is no indication of their toxicity, these aggregates are found to be proteolytically resistant. The seminal findings in this manuscript suggest that it is time to adapt innovative food processing and supplementation of bioactive molecules that can prevent the formation of such protein aggregates and help maximize the utilization of protein-based nutritional values.

Antimicrobial, Polarizing Light, and Paired Helical Filament Properties of Fragmented Tau Peptides of Selected Putative Gingipains.

BACKGROUND: Tau is an established substrate for gingipains secreted by Porphyromonas gingivalis. Hyperphosphorylation of tau and neurofibrillary tangle (NFT) formation is a defining lesion of Alzheimer's disease (AD) where NFT distribution is related to Braak stage and disease severity. OBJECTIVE: To assess gingipains'-fragmented tau peptides for their antimicrobial properties and for the likelihood of paired helical/straight filament (PHF/SF) formation with implications for the NFT lesion. METHODS: Seven non-phosphorylated (A-G) and three phosphorylated (A-C) tau peptides, were tested for antimicrobial properties against P. gingivalis. Polarizing light properties were determined using Congo Red staining. Secondary and tertiary structures of peptides B-F were determined using transmission electron microscopy (TEM) and circular dichroism (CD) was undertaken for the soluble peptides A in phosphorylated and non-phosphorylated states. RESULTS: Phosphorylated tau peptide A displayed a significant effect against planktonic P. gingivalis. The CD results demonstrated that both peptides A, in phosphorylated and non-phosphorylated states, in aqueous solution, adopted mainly ß-type structures. Non-phosphorylated peptides B-F and phosphorylated peptides B-C were insoluble and fibrillar under the TEM. The secondary and tertiary structures of the non-phosphorylated peptide B demonstrated fewer helical twists, whereas peptide C displayed significantly more helical twists along the whole fiber(s) length following its phosphorylation. CONCLUSION: Phosphorylated peptide A reduced P. gingivalis viability. CD spectroscopy demonstrated the phosphorylated and the non-phosphorylated peptide A predominantly formed from ß-sheet structures in aqueous solution with potential antimicrobial activity. Phosphorylation of tau peptides physically changed their tertiary structure into PHFs with potential for self-aggregation and binding to the NFT lesion.

Islet amyloid polypeptide aggregation exerts cytotoxic and proinflammatory effects on the islet vasculature in mice.

AIMS/HYPOTHESIS: The islet vasculature, including its constituent islet endothelial cells, is a key contributor to the microenvironment necessary for normal beta cell health and function. In type 2 diabetes, islet amyloid polypeptide (IAPP) aggregates, forming amyloid deposits that accumulate between beta cells and islet capillaries. This process is known to be toxic to beta cells but its impact on the islet vasculature has not previously been studied. Here, we report the first characterisation of the effects of IAPP aggregation on islet endothelial cells/capillaries using cell-based and animal models. METHODS: Primary and immortalised islet endothelial cells were treated with amyloidogenic human IAPP (hIAPP) alone or in the presence of the amyloid blocker Congo Red or the Toll-like receptor (TLR) 2/4 antagonist OxPAPc. Cell viability was determined0 along with mRNA and protein levels of inflammatory markers. Islet capillary abundance, morphology and pericyte coverage were determined in pancreases from transgenic mice with beta cell expression of hIAPP using conventional and confocal microscopy. RESULTS: Aggregated hIAPP decreased endothelial cell viability in immortalised and primary islet endothelial cells (by 78% and 60%, respectively) and significantly increased expression of inflammatory markers Il6, Vcam1 and Edn1 mRNA relative to vehicle treatment in both cell types (p<0.05; n=4). Both cytotoxicity and the proinflammatory response were ameliorated by Congo Red (p<0.05; n=4); whereas TLR2/4-inhibition blocked inflammatory gene expression (p<0.05; n=6) without improving viability. Islets from high-fat-diet-fed amyloid-laden hIAPP transgenic mice also exhibited significantly increased expression of most markers of endothelial inflammation (p<0.05; n=5) along with decreased capillary density compared with non-transgenic littermates fed the same diet (p<0.01). Moreover, a 16% increase in capillary diameter was observed in amyloid-adjacent capillaries (p<0.01), accompanied by a doubling in pericyte structures positive for neuron-glial antigen 2 (p<0.001). CONCLUSIONS/INTERPRETATION: Islet endothelial cells are susceptible to hIAPP-induced cytotoxicity and exhibit a TLR2/4-dependent proinflammatory response to aggregated hIAPP. Additionally, we observed amyloid-selective effects that decreased islet capillary density, accompanied by increased capillary diameter and increased pericyte number. Together, these data demonstrate that the islet vasculature is a target of the cytotoxic and proinflammatory effects of aggregated hIAPP that likely contribute to the detrimental effects of hIAPP aggregation on beta cell function and survival in type 2 diabetes.

Albumin Binds Doxorubicin via Self-Assembling Dyes as Specific Polymolecular Ligands.

Congo red (CR) type self-assembled ribbon-like structures (SRLS) were previously shown to interact with some proteins, including albumin. SRLS also complex with some drugs with a flat, ring-shaped structure with aromatic characteristics, intercalating them into their ribbon structure. The combination of interaction with proteins and drug binding by SRLS enables the use of such systems for immunotargeting. It is especially interesting in the case of chemotherapeutic agents. The present experiments aimed to show that the model carrier system composed of supramolecular albumin and Congo red efficiently binds doxorubicin (Dox) and that the drug can be released at reduced pH. The presented results come from the studies on such complexes differing in the molar ratio of CR to Dox. The following methods were used for the analysis: electrophoresis, dialysis, gel filtration, spectral analysis, and analysis of the size of the hydrodynamic radius using the dynamic light scattering method (DLS). The applied methods confirmed the formation of the CR-Dox complex, with large dimensions and changed properties compared with free CR. The presented results show that albumin binds both CR and its complex with Dox. Various CR-Dox molar ratios, 5:1, 2:1, and 1:1, were analyzed. The confirmation of the possibility of releasing the drug from the carriers thus formed was also obtained. The presented research is important due to the search for optimal solutions for the use of SRLS in drug immunotargeting, with particular emphasis on chemotherapeutic agents.

Analysis of CYP450 gene expression and function in white-rot fungus, Lenzites gibbosa, treated with Congo red.

We investigated the function of cytochrome P450 (CYP450) genes in degradation of the diazo dye, Congo red, by white-rot fungus Lenzites gibbosa. Hyphae treated with Congo red at different times were sequenced to obtain transcription data. CYP450 genes in transcriptomes were identified using a gene-encoding protein functional search and analyzed in Cluster of Orthologous Genes, Gene Ontology, Kyoto Encyclopedia of Genes and Genomes Databases. Differentially expressed genes (DEGs) in different groups were analyzed using EdgeR. We present the relation between transcription factors (TFs) and CYP450 regulation by analysis of the co-expression network. One hundred sixty CYP450 genes obtained by a functional annotation search were related to the oxido-reduction reaction of secondary metabolism, defense mechanism and aromatic compound degradation. The fastest decolorization and the greatest expression of CYP450 genes, which were related to the decolorization effect, occurred at 0-3 h. Seven CYP450 genes (7522, 6568, 4482, 9118, 10935, 7521 and 10926) were identified. The key TFs that regulate these genes belong to the zinc finger family. CYP450 genes and their products in L. gibbosa participated in degradation of Congo red and stress resistance. We provide a reference value for degradation and decolorization of Congo red.

Experimental Evidence of Intrinsic Disorder and Amyloid Formation by the Henipavirus W Proteins.

Henipaviruses are severe human pathogens within the Paramyxoviridae family. Beyond the P protein, the Henipavirus P gene also encodes the V and W proteins which share with P their N-terminal, intrinsically disordered domain (NTD) and possess a unique C-terminal domain. Henipavirus W proteins antagonize interferon (IFN) signaling through NTD-mediated binding to STAT1 and STAT4, and prevent type I IFN expression and production of chemokines. Structural and molecular information on Henipavirus W proteins is lacking. By combining various bioinformatic approaches, we herein show that the Henipaviruses W proteins are predicted to be prevalently disordered and yet to contain short order-prone segments. Using limited proteolysis, differential scanning fluorimetry, analytical size exclusion chromatography, far-UV circular dichroism and small-angle X-ray scattering, we experimentally confirmed their overall disordered nature. In addition, using Congo red and Thioflavin T binding assays and negative-staining transmission electron microscopy, we show that the W proteins phase separate to form amyloid-like fibrils. The present study provides an additional example, among the few reported so far, of a viral protein forming amyloid-like fibrils, therefore significantly contributing to enlarge our currently limited knowledge of viral amyloids. In light of the critical role of the Henipavirus W proteins in evading the host innate immune response and of the functional role of phase separation in biology, these studies provide a conceptual asset to further investigate the functional impact of the phase separation abilities of the W proteins.

Biosorptive and Biodegradative Mechanistic Approach for the Decolorization of Congo Red Dye by Aspergillus Species.

In this work, Aspergillus terreus GS28 and Aspergillus flavus CR500 isolated from industrial waste sludge examined for the decolorization of Congo red (CR) dye. The rate of CR decolorization raised due to optimum pH, temperature, carbon, nitrogen, and heavy metals. In the comparative study, A. terreus has the maximum ability (95%) to decolorize CR (≈ 100 mg L-1) as compared with A. flavus (92.96%) under optimized condition after 120 h. GC-MS and FTIR analysis of the fungal-metabolite and fungal-biomass shows bio-degradation and biosorption processes respectively. The degraded products were benzenepropanic (Rt-26.147), 3, 4-diaminonapthelene-1-sulfonic acid, and benzenedicarboxylic acid (Rt-26.660) by A. terreus, and benzenedicarboxylic acid (Rt-41.467) by A. flavus. The phytotoxicity assay revealed that a decrease in toxicity of the degraded product towards the growth and germination rate of two plant seeds compared to CR. Thus, the finding suggests that both the fungi act promising CR remediation candidates, induces restoration of CR polluted wastewater and save soil-land.

Identification of a Region in the Common Amino-terminal Domain of Hendra Virus P, V, and W Proteins Responsible for Phase Transition and Amyloid Formation.

Henipaviruses are BSL-4 zoonotic pathogens responsible in humans for severe encephalitis. Their V protein is a key player in the evasion of the host innate immune response. We previously showed that the Henipavirus V proteins consist of a long intrinsically disordered N-terminal domain (NTD) and a ß-enriched C-terminal domain (CTD). These terminals are critical for V binding to DDB1, which is a cellular protein that is a component of the ubiquitin ligase E3 complex, as well as binding to MDA5 and LGP2, which are two host sensors of viral RNA. Here, we serendipitously discovered that the Hendra virus V protein undergoes a liquid-to-hydrogel phase transition and identified the V region responsible for this phenomenon. This region, referred to as PNT3 and encompassing residues 200-310, was further investigated using a combination of biophysical and structural approaches. Congo red binding assays, together with negative-staining transmisison electron microscopy (TEM) studies, show that PNT3 forms amyloid-like fibrils. Fibrillation abilities are dramatically reduced in a rationally designed PNT3 variant in which a stretch of three contiguous tyrosines, falling within an amyloidogenic motif, were replaced by three alanines. Worthy to note, Congo red staining experiments provided hints that these amyloid-like fibrils form not only in vitro but also in cellula after transfection or infection. The present results set the stage for further investigations aimed at assessing the functional role of phase separation and fibrillation by the Henipavirus V proteins.

Old dog, new tricks: Influenza A virus NS1 and in vitro fibrillogenesis.

The influenza NS1 protein is involved in suppression of the host immune response. Recently, there is growing evidence that prion-like protein aggregation plays an important role in cellular signaling and immune responses. In this work, we obtained a recombinant, influenza A NS1 protein and showed that it is able to form amyloid-like fibrils in vitro. Using proteolysis and subsequent mass spectrometry, we showed that regions resistant to protease hydrolysis highly differ between the native NS1 form (NS1-N) and fibrillar form (NS1-F); this indicates that significant structural changes occur during fibril formation. We also found a protein fragment that is capable of inducing the process of fibrillogenesis at 37 °C. The discovery of the ability of NS1 to form amyloid-like fibrils may be relevant to uncovering relationships between influenza A infection and modulation of the immune response.