Unveiling potential inhibitors targeting the nucleocapsid protein of SARS-CoV-2: Structural insights into their binding sites.

The Nucleocapsid (N) protein of SARS-CoV-2 plays a crucial role in viral replication and pathogenesis, making it an attractive target for developing antiviral therapeutics. In this study, we used differential scanning fluorimetry to establish a high-throughput screening method for identifying high-affinity ligands of N-terminal domain of the N protein (N-NTD). We screened an FDA-approved drug library of 1813 compounds and identified 102 compounds interacting with N-NTD. The screened compounds were further investigated for their ability to inhibit the nucleic-acid binding activity of the N protein using electrophoretic mobility-shift assays. We have identified three inhibitors, Ceftazidime, Sennoside A, and Tannic acid, that disrupt the N protein's interaction with RNA probe. Ceftazidime and Sennoside A exhibited nano-molar range binding affinities with N protein, determined through surface plasmon resonance. The binding sites of Ceftazidime and Sennoside A were investigated using [1H, 15N]-heteronuclear single quantum coherence (HSQC) NMR spectroscopy. Ceftazidime and Sennoside A bind to the putative RNA binding site of the N protein, thus providing insights into the inhibitory mechanism of these compounds. These findings will contribute to the development of novel antiviral agents targeting the N protein of SARS-CoV-2.

Structural characterization of transcription-coupled repair protein UVSSA and its interaction with TFIIH protein.

UV-stimulated scaffold protein A (UVSSA) is a key protein in the Transcription-Coupled Nucleotide Excision Repair (TC-NER) pathway. UVSSA, an intrinsically disordered protein, interacts with multiple members of the pathway, tethering them into the complex. Several studies have reported that UVSSA recruits Transcription Factor IIH (TFIIH) via direct interaction, following which CSB is degraded and the lesion recognition TC-NER complex dissociates from the damage site to facilitate the DNA repair. Structural insights into these events remain largely unknown. Herein, we have investigated the interaction of human UVSSA with the Pleckstrin-Homology-domain of p62 subunit of TFIIH (p62-PHD) using biophysical techniques. We observed that UVSSA forms a stable complex with the p62-PHD in vitro. Small-angle scattering measurements using X-rays and neutrons revealed a significant change in pair-distance distribution function for UVSSA662/p62-PHD complex compared to UVSSA alone. Additionally, a significant decrease was observed in the radius of gyration of the complex. Our findings suggest that TFIIH binding to UVSSA causes significant conformational changes in UVSSA. We hypothesize that these conformational changes play an important role in the dissociation of the lesion recognition TC-NER complex.

Transcription coupled DNA repair protein UVSSA binds to DNA and RNA: Mapping of nucleic acid interaction sites on human UVSSA.

Transcription-coupled repair (TCR) is a dedicated pathway for the preferential repair of bulky transcription-blocking DNA lesions. These lesions stall the elongating RNA-polymerase II (RNAPII) triggering the recruitment of TCR proteins at the damaged site. UV-stimulated scaffold protein A (UVSSA) is a recently identified cofactor which is involved in stabilization of the TCR complex, recruitment of DNA-repair machinery and removal/restoration of RNAPII from the lesion site. Mutations in UVSSA render the cells TCR-deficient and have been linked to UV-sensitive syndrome. Human UVSSA is a 709-residue long protein with two short conserved domains; an N-terminal (residues 1-150) and a C-terminal (residues 495-605) domain, while the rest of the protein is predicted to be intrinsically disordered. The protein is well conserved in eukaryotes, however; none of its homologs have been characterized yet. Here, we have purified the recombinant human UVSSA and have characterized it using bioinformatics, biophysical and biochemical techniques. Using EMSA, SPR and fluorescence-based methods, we have shown that human UVSSA interacts with DNA and RNA. Furthermore, we have mapped the nucleic acid binding regions using several recombinant protein fragments containing either the N-terminal or the C-terminal domains. Our data indicate that UVSSA possesses at least two nucleic acid binding regions; the N-terminal domain and a C-terminal tail region (residues 606-662). These regions, far apart in sequence space, are predicted to be in close proximity in structure-space suggesting a coherent interaction with target DNA/RNA. The study may provide functional clues about the novel family of UVSSA proteins.

Gallic Acid: A Dietary Polyphenol that Exhibits Anti-neoplastic Activities by Modulating Multiple Oncogenic Targets.

Phytochemicals are being used for thousands of years to prevent dreadful malignancy. Side effects of existing allopathic treatment have also initiated intense research in the field of bioactive phytochemicals. Gallic acid, a natural polyphenolic compound, exists freely as well as in polymeric forms. The anti-cancer properties of gallic acid are indomitable by a variety of cellular pathways such as induction of programmed cell death, cell cycle apprehension, reticence of vasculature and tumor migration, and inflammation. Furthermore, gallic acid is found to show synergism with other existing chemotherapeutic drugs. Therefore, the antineoplastic role of gallic acid suggests its promising therapeutic candidature in the near future. The present review describes all these aspects of gallic acid at a single platform. In addition nanotechnology-mediated approaches are also discussed to enhance bioavailability and therapeutic efficacy.

Structure-function analysis reveals Trichoderma virens Tsp1 to be a novel fungal effector protein modulating plant defence.

Trichoderma virens colonizes roots and develops a symbiotic relationship with plants where the fungal partner derives nutrients from plants and offers defence, in return. Tsp1, a small secreted cysteine-rich protein, was earlier found to be upregulated in co-cultivation of T. virens with maize roots. Tsp1 is well conserved in Ascomycota division of fungi, but none of its homologs have been studied yet. We have expressed and purified recombinant Tsp1, and resolved its structure to 1.25 Å resolutions, from two crystal forms, using Se-SAD methods. The Tsp1 adopts a ß barrel fold and forms dimer in structure as well as in solution form. DALI based structure analysis revealed the structure similarity with two known fungal effector proteins: Alt a1 and PevD1. Structure and evolutionary analysis suggested that Tsp1 belongs to a novel effector protein family. Tsp1 acted as an inducer of salicylic acid mediated susceptibility in plants, rendering maize plants more susceptible to a necrotrophic pathogen Cochliobolus heterostrophus, as observed using plant defence assay and RT-qPCR analysis.

Structural insights into SARS-CoV-2 proteins.

The unprecedented scale of the ongoing COVID-19 pandemic has catalyzed an intense effort of the global scientific community to unravel different aspects of the disease in a short time. One of the crucial aspects of these developments is the determination of more than three hundred experimental structures of SARS-CoV-2 proteins in the last few months. These include structures of viral non-structural, structural, and accessory proteins and their complexes determined by either X-ray diffraction or cryo-electron microscopy. These structures elucidate the intricate working of different components of the viral machinery at the atomic level during different steps of the viral life cycle, including attachment to the host cell, viral genome replication and transcription, and genome packaging and assembly of the virion. Some of these proteins are also potential targets for drug development against the disease. In this review, we discuss important structural features of different SARS-CoV-2 proteins with their function, and their potential as a target for therapeutic interventions.

Expression, purification, crystallization and X-ray diffraction studies of a novel root-induced secreted protein from Trichoderma virens.

Small secreted cysteine-rich proteins (SSCPs) from fungi play an important role in fungi-host interactions. The plant-beneficial fungi Trichoderma spp. are in use worldwide as biocontrol agents and protect the host plant from soil-borne as well as foliar pathogens. Recently, a novel SSCP, Tsp1, has been identified in the secreted protein pool of T. virens and is overinduced upon its interaction with the roots of the maize plant. The protein was observed to be well conserved in the Ascomycota division of fungi, and its homologs are present in many plant-pathogenic fungi such as Fusarium oxysporum and Magnaporthe oryzae. However, none of these homologs have yet been characterized. Recombinant Tsp1 protein has been expressed and purified using an Escherichia coli expression system. The protein, with four conserved cysteines, forms a dimer in solution as observed by size-exclusion chromatography. The dimerization, however, does not involve disulfide bonds. Circular-dichroism data suggested that the protein has a ß-strand-rich secondary structure that matched well with the secondary structure predicted using bioinformatics methods. The protein was crystallized using sodium malonate as a precipitant. The crystals diffracted X-rays to 1.7 Šresolution and belonged to the orthorhombic space group P212121 (Rmeas = 5.4%), with unit-cell parameters a = 46.3, b = 67.0, c = 173.2 Å. The Matthews coefficient (VM) of the crystal is 2.32 Å3 Da-1, which corresponds to nearly 47% solvent content with four subunits of Tsp1 protein in the asymmetric unit. This is the first report of the structural study of any homolog of the novel Tsp1 protein. These structural studies will help in understanding the classification and function of the protein.

Correction: Prevalence and Characterization of Oxacillin Susceptible mecA-Positive Clinical Isolates of Staphylococcus aureus Causing Bovine Mastitis in India.

[This corrects the article DOI: 10.1371/journal.pone.0162256.].

Potential Sabotage of Host Cell Physiology by Apicomplexan Parasites for Their Survival Benefits.

Plasmodium, Toxoplasma, Cryptosporidium, Babesia, and Theileria are the major apicomplexan parasites affecting humans or animals worldwide. These pathogens represent an excellent example of host manipulators who can overturn host signaling pathways for their survival. They infect different types of host cells and take charge of the host machinery to gain nutrients and prevent itself from host attack. The mechanisms by which these pathogens modulate the host signaling pathways are well studied for Plasmodium, Toxoplasma, Cryptosporidium, and Theileria, except for limited studies on Babesia. Theileria is a unique pathogen taking into account the way it modulates host cell transformation, resulting in its clonal expansion. These parasites majorly modulate similar host signaling pathways, however, the disease outcome and effect is different among them. In this review, we discuss the approaches of these apicomplexan to manipulate the host-parasite clearance pathways during infection, invasion, survival, and egress.

Identification of Variable Traits among the Methicillin Resistant and Sensitive Coagulase Negative Staphylococci in Milk Samples from Mastitic Cows in India.

Methicillin resistant Staphylococcus aureus causing bovine mastitis has been very well investigated worldwide. However, there are only limited reports on the characterization of methicillin resistant and sensitive coagulase negative staphylococci (CoNS) across the globe. Hence, in the present study, we aim to determine the phenotypic traits based on antimicrobial susceptibility profile and genotypic characterization by verifying the presence of resistance determinants, virulence and toxin genes present in the CoNS causing clinical mastitis. We obtained 62 CoNS isolates from 167 mastitic milk samples collected from three different states of India. The 62 isolates comprises of 10 different CoNS species S. sciuri, S. haemolyticus, S. chromogenes, S. saprophyticus, S. xylosus, S. simulans, S. agnetis, S. epidermidis, S. gallinarum, and S. cohinii. Susceptibility screening against 11 antibiotics determined 45.16% isolates as multidrug resistant (resistant to more than two class of antibiotic), 46.74% resistant (one or two antibiotic class) and 8.06% isolates were pan-sensitive (sensitive to all drugs). High resistance was observed against oxacillin and cefoxitin, whereas all isolates were susceptible toward vancomycin and linezolid. Fifty three isolates were methicillin resistant and 9 isolates were sensitive as determined by oxacillin susceptibility assay. The methicillin resistance gene, mecA was found in 95.16% isolates and staphylococcal cassette chromosome mec (SCCmec) typing predominantly revealed Type III (n = 34) and Type V (n = 18). Interestingly, 11.9% of mecA positive isolates were oxacillin susceptible and referred as oxacillin susceptible mecA positive staphylococci (OS-MRS). Additionally, genes encoding for enterotoxin, (sea, seb, seh, see) toxic shock syndrome (tsst), exfoliatin (eta, etb, etd) and virulence (pvl, Y-hlg) were also screened. Of all the genes examined, 67.74% of isolate were positive for the Y-hlg gene, followed by the sea gene in 25.8% whereas in none of the isolates the eta and the etb gene was amplified. The study also highlights the incidence of clinical isolates of CoNS, which are harboring the toxin and the virulence genes rendering them as a more potential threat. This is the first report of animal origin OS-MRS from India, which emphasizes on the inclusion of both the genetic and phenotypic test for proper characterization of CoNS and preventing resistant strain misidentification.

Prevalence and Characterization of Oxacillin Susceptible mecA-Positive Clinical Isolates of Staphylococcus aureus Causing Bovine Mastitis in India.

Bovine mastitis caused by multidrug resistant Staphylococcus aureus is a huge problem reported worldwide, resulting in prolonged antibiotic treatment and death of livestock. The current study is focused on surveillance of antibiotic susceptibility along with genotypic and phenotypic characterization of the pathogenic S. aureus strains causing mastitis in India. One hundred and sixty seven milk samples were collected from mastitis-affected cows from different farms in India resulting in thirty nine isolated S. aureus strains. Antibiotic sensitivity profiling revealed the majority of the strains (n = 24) to be multidrug resistant and eleven strains showed reduced susceptibility to vancomycin (MICs = 2µg/ml). All strains were oxacillin sensitive, but 19 strains were positive for the mecA gene, which revealed the occurrence of oxacillin susceptible mecA positive strains (OS-MRSA) for the first time from India. Additionally, 32 strains were positive for the pvl gene, a virulence determinant; of these 17 were also OS-MRSA strains. Molecular characterization based on multilocus sequence typing (MLST), spa typing, agr typing and SCCmec classification revealed strains belonging to different groups. Moreover, strains showed spa types (t2526, t9602) and MLST sequence types, ST-72, ST-88 and ST-239 which have been earlier reported in human infections. The prevalence of OS-MRSA strains indicates the importance of including both the genetic and phenotypic tests in characterizing S. aureus strains. Increased genotypic variability with strain related to human infections and pvl positive isolates indicates a worrisome situation with the possibility of bilateral transfer.