Molecular Docking of Phytochemicals against Streptococcus mutans Virulence Targets: A Proteomic Insight into Drug Planning

Streptococcus mutans (S. mutans) is the most prevalent and most associated with dental caries. Here we aim to identify, through an in silico study, potential bioactive molecules against S. mutans. Twenty-four bioactive molecules with proven action against S. mutans were selected: 1-methoxyficifolinol; 5,7,2′,4′-tetrahydroxy-8-lavandulylflavanone (sophoraflavanone G); 6,8-diprenylgenistein; apigenin; artocarpesin; artocarpin; darbergioidin; dihydrobiochanin A; dihydrocajanin (5,2′,4′-trihydroxy-7-methoxyisoflavanone); erycristagallin; Erystagallin; ferreirin; fisetin; kaempferol; licoricidin; licorisoflavan A; licorisoflavan C; licorisoflavan E; luteolin (3′,4′,5,7-tetrahydroxyflavone); malvidin-3,5-diglucoside; myricetin; orientanol B; quercetin; and quercitrin. Moreover, we selected nine important target proteins for the virulence of this microorganism to perform as drug targets: antigen I/II (region V) (PDB: 1JMM); Antigen I/II (carbox-terminal region) (PDB: 3QE5); Spap (PDB: 3OPU); UA159sp signaling peptide (PDB: 2I2J); TCP3 signaling peptide (PDB: 2I2H); ATP-binding protein ComA (PDB: 3VX4); glucanosucrase (PDB: 3AIC); dextranase (PDB: 3VMO), and Hemolysin (PDB: 2RK5). Five molecules were revealed to be the best ligands for at least three target proteins, highlighting the following compounds: 11 (erystagallin), 10 (erycristagallin), 1 (methoxyficifonilol), 20 (malvidin-3,5-diglucoside), and 2 (sophoraflavanone G), which indicates a possible multi-target action of these compounds. Therefore, based on these findings, in vitro and in vivo tests should be performed to validate the effectiveness of these compounds in inhibiting S. mutans virulence factors. Furthermore, the promising results of these assays will allow the incorporation of these phytoconstituents in products for oral use for the control of tooth decay.

Mapping of Genetic Locus for Leaf Trichome Formation in Chinese Cabbage Based on Bulked Segregant Analysis

Chinese cabbage is a leafy vegetable, and its leaves are the main edible organs. The formation of trichomes on the leaves can significantly affect its taste, so studying this phenomenon is of great significance for improving the quality of Chinese cabbage. In this study, two varieties of Chinese cabbage, W30 with trichome leaves and 082 with glabrous leaves, were crossed to generate F1 and F1 plants, which were self-fertilized to develop segregating populations with trichome or glabrous morphotypes. The two bulks of the different segregating populations were used to conduct bulked segregant analysis (BSA). A total of 293.4 M clean reads were generated from the samples, and plants from the trichome leaves (AL) bulk and glabrous leaves (GL) bulk were identified. Between the two DNA pools generated from the trichome and glabrous plants, 55,048 SNPs and 272 indels were generated. In this study, three regions (on chromosomes 6, 10 and scaffold000100) were identified, and the annotation revealed three candidate genes that may participate in the formation of leaf trichomes. These findings suggest that the three genes—Bra025087 encoding a cyclin family protein, Bra035000 encoding an ATP-binding protein/kinase/protein kinase/protein serine/threonine kinase and Bra033370 encoding a WD-40 repeat family protein–influence the formation of trichomes by participating in trichome morphogenesis (GO: 0010090). These results demonstrate that BSA can be used to map genes associated with traits and provide new insights into the molecular mechanism of leafy trichome formation in Chinese cabbage.

Autophosphorylation of the KaiC-like protein ArlH inhibits oligomerisation and interaction with ArlI, the motor ATPase of the archaellum

Motile archaea are propelled by the archaellum, whose motor complex consists of the membrane protein ArlJ, the ATPase ArlI, and the ATP-binding protein ArlH. Despite its essential function and the existence of structural and biochemical data on ArlH, the role of ArlH in archaellum assembly and function remains elusive. ArlH is a structural homolog of KaiC, the central component of the cyanobacterial circadian clock. Similar to KaiC, ArlH exhibits autophosphorylation activity, which was observed for both ArlH of the euryarchaeon Pyrococcus furiosus (PfArlH) and the crenarchaeon Sulfolobus acidocaldarius (SaArlH). Using a combination of single molecule fluorescence measurements and biochemical assays, it is shown that autophosphorylation of ArlH is closely linked to the oligomeric state of ArlH bound to ArlI. These experiments also strongly suggest that ArlH is a hexamer in its functional ArlI bound state. Mutagenesis of the putative catalytic residue Glu-57 in SaArlH results in a reduced autophosphorylation activity and abolished archaellation and motility, suggesting that optimum phosphorylation activity of ArlH is essential for both archaellation and motility.

Real-time in vivo imaging of extracellular ATP in the brain with a hybrid-type fluorescent sensor

Adenosine 5’ triphosphate (ATP) is a ubiquitous extracellular signaling messenger. Here, we describe a method for in-vivo imaging of extracellular ATP with high spatiotemporal resolution. We prepared a comprehensive set of cysteine-substitution mutants of ATP-binding protein, Bacillus FoF1-ATP synthase ε subunit, labeled with small-molecule fluorophores at the introduced cysteine residue. Screening revealed that the Cy3-labeled glutamine-105 mutant (Q105C-Cy3; designated ATPOS) shows a large fluorescence change in the presence of ATP, with submicromolar affinity, pH-independence, and high selectivity for ATP over ATP metabolites and other nucleotides. To enable in-vivo validation, we introduced BoNT/C-Hc for binding to neuronal plasma membrane and Alexa Fluor 488 for ratiometric measurement. The resulting ATPOS complex binds to neurons in cerebral cortex of living mice, and clearly visualized a concentrically propagating wave of extracellular ATP release in response to electrical stimulation. ATPOS should be useful to probe the extracellular ATP dynamics of diverse biological processes in vivo.

Characterization and Functional Annotation of Uncharacterized ABC Transporter ATP-Binding Protein Rv0986 of Mycobacterium tuberculosis (Strain ATCC 25618 / H37Rv)

The most significant ancient infectious disease tuberculosis is causes by a human pathogen, Mycobacterium tuberculosis (MTB). Amazingly, tuberculosis (TB) has become one of the major causes of human death worldwide. The protein Rv0986 is associated with the ATP-binding cassette domain of the transporters involved in the export of lipoprotein and macrolide, and cell division protein, therefore, related to mycobacterial infection. But the protein Rv0986 is not yet explored. As a result, identification, characterization, and functional annotation of uncharacterized protein Rv0986 were predicted where the structure modeling was generated by using Modeller, Phyre2, and Swiss Model with the structural quality assessment by Ramachandran Plot (PROCHECK), Verify 3d, and Swiss-Model Interactive Workplace as well. Z-scores obtained from Prosa-web were also applied for overall 3D model quality. This in-silico method will uncover the significance of undiscovered uncharacterized protein Rv0986 present in MTB, and indeed it can accelerate the way to enrich our knowledge in the pathogenesis and drug-targeting opportunity against infection by MTB.

Differential protein profiling of soil diazotroph Rhodococcus qingshengii S10107 towards low-temperature and nitrogen deficiency

Protein-based biomarkers can be a promising approach for identification and real-time monitoring of the bio-inoculants employed under sustainable agricultural plans. In this perspective, differential proteomics of psychrophilic diazotroph Rhodococcus qingshengii S10107 (JX173283) was performed to unravel its adaptive responses towards low-temperature nitrogen deficiency and identification of a biomarker for respective physiological conditions. LC-MS/MS-based proteome analysis mapped more than 4830 proteins including 77 up-regulated and 47 down-regulated proteins (p ≤ 0.05). Differential expression of the structural genes of nif regulon viz. nifH, nifD, and nifK along with their response regulators i.e. nifA, nifL, and nifB indicated that the nitrogenase complex was activated successfully. Besides up-regulating the biosynthesis of certain amino acids viz. Leucine, Lysine, and Alanine; the expression of the peptidoglycan synthesis proteins were also increased; while, the enzymes involved in Lipid biosynthesis were found to decrease. Furthermore, two important enzymes of the pentose phosphate pathway viz. Transketolase and Transaldolase along with Ribose import ATP-binding protein RbsA were also found to induce significantly under low temperature a nitrogen deficient condition, which suggests the cellular need for ample ribose sugar instantly. Additionally, comparative protein profiling of S10107 strain with our previous studies revealed that CowN protein was significantly up-regulated in all the cases under low-temperature nitrogen deficient conditions and therefore, can be developed as a biomarker. Conclusively, present study for the first time provides an in-depth proteome profiling of R. qingshengii S10107 and proclaims CowN as a potential protein biomarker for monitoring BNF under cold niches.

Characterization of bacteriocin ABC transporter ATP-binding protein produced by a newly isolated Enterococcus casseliflavus MI001 strain

Background ATP-binding cassette (ABC) transporters constitute one of the largest transporter protein families and play a role in diverse biological processes. Results In the present study, bacteriocin isolated from the Enterococcus casseliflavus MI001 strain was identified as an ABC transporter ATP-binding protein. The optimal conditions for the production of bacteriocin were found to be at 35 °C, a pH 5.5, and an incubation time of 24 h. Purification was performed using ammonium sulphate precipitation, gel filtration, and DEAE ion exchange chromatography. The bacteriocin was purified with an eightfold purification scheme resulting with a specific activity of 15,000 AU/mg. The NMR spectrum of purified bacteriocin revealed the presence of amino acids, namely lysine, methionine, cysteine, proline, threonine, tryptophan, and histidine. Further, the bacteriocin ABC transporter showed antimicrobial activity against food spoilage microorganisms. Conclusions The ABC transporter ATP-binding protein could be used as a potential alternative for food preservation, and it may be considered as a bio-preservative agent in food processing industries.