The Staphylococcus aureus exfoliative toxins (ETs) are the main toxins that produce staphylococcal scalded skin syndrome (SSSS), an abscess skin disorder. The victims of the disease are usually newborns and kids, as well as grown-up people. Five ETs namely, exfoliative toxins A, B, C, D, and E have been identified in S. aureus. The three-dimensional (3D) structure of exfoliative toxins A, B, C and E is known, while that of exfoliative toxin D (ETD) is still unknown. In this work, we have predicted the 3D structure of ETD using protein modeling techniques (software used for 3D structure modeling comprising the MODELLER 9v19 program, SWISS-Model, and I-TESSER). The validation of the build model was done using PROCHECK (Ramachandran plot), ERRAT2, and Verify 3D programs. The results from 3D modeling show that the build model was of good quality as indicated by a GMQE score of 0.88 and by 91.1% amino acid residues in the most favored region of the Ramachandran plot, the ERRAT2 quality factor of 90.1%, and a verify3D score of >0.2 for 99.59% of amino acid residues. The 3D structure analysis indicates that the overall structure of ETD is similar to the chymotrypsin-like serine protease fold. The structure is composed of 13 β-strands and seven α-helices that fold into two well-defined six-strand β-barrels whose axes are roughly perpendicular to each other. The active site residues include histidine-97, aspartic acid-147, and serine-221. This represents the first structure report of ETD. Structural comparison with the other ETs shows some differences, particularly in the loop region, which also change the overall surface charge of these toxins. This may convey variable substrate specificity to these toxins. The inhibition of these toxins by natural (2S albumin and flocculating proteins from Moringa oleifera seeds) and synthetic inhibitors (suramin) was also carried out in this study. The results from docking indicate that the inhibitors bind near the C-terminal domain which may restrict the movement of this domain and may halt the access of the substrate to the active site of this enzyme. Molecular dynamic simulation was performed to see the effect of inhibitor binding to the enzyme. This work will further elucidate the structure–function relationship of this enzyme. The inhibition of this enzyme will lead to a new treatment for SSSS.

Background:The emerging antimicrobial resistance will be responsible for more casualty by 2050 (AMR review, UK). In this context, a group of antimicrobial resistant ESKAPE pathogen is of prime importance. Methicillin-resistant Staphylococcus aureus is one of the members of ESKAPE pathogens, is clinically important and responsible for various major life-threatening nosocomial infections. Its inherent antibiotic resistance and biofilm-forming capability make it more persistent and difficult to encounter. This scenario will demand to search for new molecules with better antimicrobial and anti-virulence properties against MRSA. Polyalthia longifolia is an ornamental plant containing clerodane diterpenoids (i.e. 16-oxo-cleroda-3, 13( 14) E-diene-15 oic acid (1) and kolavenic acid (2) which posses important pharmacological properties was isolated from leaves of this plant further derivatized to synthetic lactones and studied for their antibacterial and biofilm inhibitory potential.Methods and materials: Compound 1 (16-oxo-cleroda-3, 13(14) E-diene-15 oic acid) and Compound 2 (Kolavanic acid) were isolated from the leaves of Polyalthia longifolia; Compound 1 was further derivatized to synthetic lactones as compound 3, 4, 5 and 6. The isolated compounds and synthetic lactones were screened for their antimicrobial and biofilm inhibition and eradication was evaluated by crystal violet based assay, MTT and calculating CFU/ml. Fluorescence and electron microscopy was conducted to confirm the biofilm inhibitory activities. Molecular mechanism of biofilm inhibition was done by evaluating gene expression analysis by using PCR.Results: Among the tested compounds 1 and 3 were found to have better antistaphylococcal properties with better biofilm inhibiting potential. At 10-30 g/ml 1 and 3 were found to be antivirulence, antibacterial and antibiofilm. Compound 1 was also found to have better biofilm eradicating potential as compared to compound 3. Biofilm inhibitory potential of both the compounds was further confirmed by fluorescence and scanning electron microscopy at MIC and sub MIC levels. Parent compound (1) and its synthetic ␥-lactone (3) significantly delayed the bacterial growth after their short exposure of 10 min. These compounds also altered the gene expression of MRSA biofilm-related genes (icaA). Conclusion:The observed anti-virulence properties and delayed bacterial growth suggested these compounds can be a drug lead to overcome MRSA related infections.

Staphylococcus aureus is deliberated as one of the most challenging bacteria owing to its ability to develop resistance against antibacterial drugs. In an attempt to explore new approaches for enhancing the activity of antibiotics, here in this work, ampicillin is conjugated to Ag and Au nanoparticles (NPs) and its antibacterial potential was investigated against S. aureus. The antibacterial activity was assessed and the associated changes in the bacterial cell morphology were analyzed using atomic force microscopy (AFM) as well as other characterization techniques. Results showed that the antibacterial activity of ampicillin conjugated to gold and silver NPs was enhanced up to 10 and 5 times respectively, when compared with the non-conjugated antibiotic. The kinetics of the conjugated ampicillin were improved. Bacterial membrane destruction was scarcely evident after treating a cell culture with pure ampicillin for four hours. However, Ag conjugates have severely disrupted the cell membranes and Au conjugates have completely destroyed the cell morphology. The study gave an insight of the enhanced antimicrobial action of ampicillin and can be exploited for the devising nanoparticle’s based antimicrobial agents. More sophisticated approaches such as faster and more efficient diagnostics, non-antimicrobial methodologies to prevent and treat infections and a better understanding of staphylococcal pathogenesis will also be required to forestall the future of the bacterial resistance.