Discovery and structural characterization of chicoric acid as a SARS-CoV-2 nucleocapsid protein ligand and RNA binding disruptor.

The nucleocapsid (N) protein plays critical roles in coronavirus genome transcription and packaging, representing a key target for the development of novel antivirals, and for which structural information on ligand binding is scarce. We used a novel fluorescence polarization assay to identify small molecules that disrupt the binding of the N protein to a target RNA derived from the SARS-CoV-2 genome packaging signal. Several phenolic compounds, including L-chicoric acid (CA), were identified as high-affinity N-protein ligands. The binding of CA to the N protein was confirmed by isothermal titration calorimetry, 1H-STD and 15N-HSQC NMR, and by the crystal structure of CA bound to the N protein C-terminal domain (CTD), further revealing a new modulatory site in the SARS-CoV-2 N protein. Moreover, CA reduced SARS-CoV-2 replication in cell cultures. These data thus open venues for the development of new antivirals targeting the N protein, an essential and yet underexplored coronavirus target.

Structural dynamics of SARS-CoV-2 nucleocapsid protein induced by RNA binding.

The nucleocapsid (N) protein of the SARS-CoV-2 virus, the causal agent of COVID-19, is a multifunction phosphoprotein that plays critical roles in the virus life cycle, including transcription and packaging of the viral RNA. To play such diverse roles, the N protein has two globular RNA-binding modules, the N- (NTD) and C-terminal (CTD) domains, which are connected by an intrinsically disordered region. Despite the wealth of structural data available for the isolated NTD and CTD, how these domains are arranged in the full-length protein and how the oligomerization of N influences its RNA-binding activity remains largely unclear. Herein, using experimental data from electron microscopy and biochemical/biophysical techniques combined with molecular modeling and molecular dynamics simulations, we show that, in the absence of RNA, the N protein formed structurally dynamic dimers, with the NTD and CTD arranged in extended conformations. However, in the presence of RNA, the N protein assumed a more compact conformation where the NTD and CTD are packed together. We also provided an octameric model for the full-length N bound to RNA that is consistent with electron microscopy images of the N protein in the presence of RNA. Together, our results shed new light on the dynamics and higher-order oligomeric structure of this versatile protein.

Draft genome sequence of Psychrobacter nivimaris LAMA 639 and its biotechnological potential.

Bacteria of the genus Psychrobacter are known for their psychrophilic characteristics, being extremophilic organisms capable of surviving and reproducing in hostile environments of low temperature and high pressure. Among many of the genus characteristics, there is the ability to produce enzymes and molecules of industrial biotechnology importance, such as pigments and proteins related to heavy metal bioremediation. The bacterium strain Psychrobacter nivimaris LAMA 639 was isolated from sediments from the Walvis Ridge ocean crest at a depth of 4.400 m (33.40 S 2.35 E). It is a nonmotile, halotolerant, cream-colored gram-negative aerobic bacterium. Its cultivation was performed in marine agar plates and inoculated into test tubes with NaCl at an optimal temperature of 30 °C and with shaking at 100 rpm. Genome extraction was performed with the DNeasy Blood & Tissue Kit (QIAGEN®). Sequencing was performed by Macrogen using the NovaSeq® 6000 platform (Illumina) applying the whole genome shotgun (WGS) method. Thereafter, 14.712.526 reads of 151 bp were generated, totaling 2.2 G bp with a GC content of 42.9%. Assembly and mapping were performed with a CLC Genomics Workbench. The best assembly considered was the one with the lowest number of contigs and the highest base length pair. The assemblies were evaluated using QUAST, and the best resulting variant was selected for annotation. Genome annotation was performed with RAST and PATRIC; the antiSMASH tool was used for secondary metabolites; NaPDoS was used for domains; and three-dimensional structural prediction of relevant proteins was performed using Phyre2. Annotation with ClassicRAST generated 2,891 coding sequences (CDSs) distributed in 402 subsystems. Annotation with PATRIC generated 2,896 coding sequences, among them 776 hypothetical proteins. The antiSMASH tool visualized a beta-lactone cluster in contig 06. In the search for natural products with NaPDoS, two ketosynthase domains were identified. The search for relevant proteins was performed using the AMFEP list as a criterion. From these data, 34 possible enzymes with biotechnological potential were found. Finally, the organism is presented as a new reference regarding the potential of deep-sea marine bacteria, demonstrating that, from the annotated and cured genome, it is possible to find in its genetic repertory products of interest for biotechnological applications.

Chemical Elicitors Induce Rare Bioactive Secondary Metabolites in Deep-Sea Bacteria under Laboratory Conditions.

Bacterial genome sequencing has revealed a vast number of novel biosynthetic gene clusters (BGC) with potential to produce bioactive natural products. However, the biosynthesis of secondary metabolites by bacteria is often silenced under laboratory conditions, limiting the controlled expression of natural products. Here we describe an integrated methodology for the construction and screening of an elicited and pre-fractionated library of marine bacteria. In this pilot study, chemical elicitors were evaluated to mimic the natural environment and to induce the expression of cryptic BGCs in deep-sea bacteria. By integrating high-resolution untargeted metabolomics with cheminformatics analyses, it was possible to visualize, mine, identify and map the chemical and biological space of the elicited bacterial metabolites. The results show that elicited bacterial metabolites correspond to ~45% of the compounds produced under laboratory conditions. In addition, the elicited chemical space is novel (~70% of the elicited compounds) or concentrated in the chemical space of drugs. Fractionation of the crude extracts further evidenced minor compounds (~90% of the collection) and the detection of biological activity. This pilot work pinpoints strategies for constructing and evaluating chemically diverse bacterial natural product libraries towards the identification of novel bacterial metabolites in natural product-based drug discovery pipelines.

Carotenoids produced by the deep-sea bacterium Erythrobacter citreus LAMA 915: detection and proposal of their biosynthetic pathway.

Technologies based on synthetic biology to produce bacterial natural carotenoids depend on information regarding their biosynthesis. Although the biosynthetic pathway of common carotenoids is known, there are carotenoids whose pathways are not completely described. This work aimed to mine the genome of the deep-sea bacterium Erythrobacter citreus LAMA 915, an uncommon bacterium that forms yellow colonies under cultivation. This work further explores the potential application of the carotenoids found and low-cost substrates for bacterial growth. A combined approach of genome mining and untargeted metabolomics analysis was applied. The carotenoid erythroxanthin sulfate was detected in E. citreus LAMA 915 cell extract. A proposal for carotenoid biosynthesis by this bacterium is provided, involving the genes crtBIYZWG. These are responsible for the biosynthesis of carotenoids from the zeaxanthin pathway and their 2,2'-hydroxylated derivatives. E. citreus LAMA 915 extracts showed antioxidant and sun protection effects. Based on the high content of proteases and lipases, it was possible to rationally select substrates for bacterial growth, with residual oil from fish processing the best low-cost substrate selected. This work advances in the understanding of carotenoid biosynthesis and provides a genetic basis that can be further explored as a biotechnological route for carotenoid production.

Merulinic acid C overcomes gentamicin resistance in Enterococcus faecium.

Enterococci are gram-positive, widespread nosocomial pathogens that in recent years have developed resistance to various commonly employed antibiotics. Since finding new infection-control agents based on secondary metabolites from organisms has proved successful for decades, natural products are potentially useful sources of compounds with activity against enterococci. Herein are reported the results of a natural product library screening based on a whole-cell assay against a gram-positive model organism, which led to the isolation of a series of anacardic acids identified by analysis of their spectroscopic data and by chemical derivatizations. Merulinic acid C was identified as the most active anacardic acid derivative obtained against antibiotic-resistant enterococci. Fluorescence microscopy analyses showed that merulinic acid C targets the bacterial membrane without affecting the peptidoglycan and causes rapid cellular ATP leakage from cells. Merulinic acid C was shown to be synergistic with gentamicin against Enterococcus faecium, indicating that this compound could inspire the development of new antibiotic combinations effective against drug-resistant pathogens.

Structure and function of interleukin-22 and other members of the interleukin-10 family.

The IL-10 family of cytokines is comprised of IL-10, IL-19, IL-20, IL-22, IL-24, IL-26, and IFN-lambdas (IL-28A, IL-28B, and IL-29). The IL-10 family members bind to shared class II cytokine receptor chains that associate in various combinations in heterodimeric complexes. Upon interleukin/receptor complex formation, these proteins switch on the Jak/STAT pathway and elicit pleiotropic biological responses whose variety sharply contrasts with their structural similarities. IL-10 family members are involved in several human diseases and health conditions and hence their structural analyses may provide valuable information to design specific therapeutic strategies. In this review, we describe the human interleukin-10 family of cytokines, focusing on their structures and functions, with particular attention given to IL-22 and IL-10. We report on the recently published structures of IL-10 cytokine family members and their complexes with cognate transmembrane and soluble receptors as well as on interleukin physiology and physiopathology.