Limonium brasiliense rhizomes extract against virulence factors of Porphyromonas gingivalis: Inhibition of gingipains, bacterial adhesion, and biofilm formation.

Periodontitis is clinically characterized by destruction of the tooth support system and tooth loss. Porphyromonas gingivalis (Pg) plays a dominant role in periodontitis. Fractions and isolated compounds from an acetone-water extract of the roots of Limonium brasiliense (Lb) were tested in vitro for their anti-adhesive capacity against Pg on human KB buccal cells, influence on gingipains, the main virulence factors of Pg, and biofilm formation. Fractions EAF and FLB7 (50 µg/mL) reduced the bacterial adhesion of Pg to KB cells significantly (63 resp. 70%). The proanthocyanidin samarangenin A inhibited the adhesion (72%, 30 µM), samarangenin B (71%, 20 µM), and the flavan-3-ol epigallocatechin-3-O-gallate (79%, 30 µM). Fraction AQF, representing hydrophilic compounds, reduced the proteolytic activity of Arginin-specific gingipain (IC50 12.78 µg/mL). Fractions EAF and FLB7, characterized by lipohilic constituents, inhibited Arg-gingipain (IC50 3 µg/mL). On Lysine-specific gingipain, AQF has an IC50 15.89, EAF 14.15, and FLB7 6 µg/mL. The reduced bacterial adhesion is due to a strong interaction of proanthocyanidins with gingipains. AQF, EAF, and FLB7 significantly inhibited biofilm formation: IC50 11.34 (AQF), 11.66 (EAF), and 12.09 µg/mL (FLB7). In silico analysis indicated, that the polyphenols act against specific targets of Pg, not affecting mammalian cells. Therefore, Lb might be effective for prevention of periodontal disease by influencing virulence factors of Pg.

Marine-Derived Sulfated Glycans Inhibit the Interaction of Heparin with Adhesion Proteins of Mycoplasma pneumoniae.

Mycoplasma pneumoniae, a notable pathogen behind respiratory infections, employs specialized proteins to adhere to the respiratory epithelium, an essential process for initiating infection. The role of glycosaminoglycans, especially heparan sulfate, is critical in facilitating pathogen-host interactions, presenting a strategic target for therapeutic intervention. In this study, we assembled a glycan library comprising heparin, its oligosaccharide derivatives, and a variety of marine-derived sulfated glycans to screen the potential inhibitors for the pathogen-host interactions. By using Surface Plasmon Resonance spectroscopy, we evaluated the library's efficacy in inhibiting the interaction between M. pneumoniae adhesion proteins and heparin. Our findings offer a promising avenue for developing novel therapeutic strategies against M. pneumoniae infections.

Nicotinamide employs a starvation strategy against Porphyromonas gingivalis virulence by inhibiting the heme uptake system and gingipain activities.

Periodontitis is a common oral bacterial infection characterized by inflammatory responses. Its high prevalence lowers the quality of life for individuals and increases the global economic and disease burden. As microorganisms in dental plaque are responsible for this oral disease, antibacterial drug treatments are effective strategies for preventing and treating periodontitis. In this study, we investigated the inhibitory effect of nicotinamide (NAM), a vitamin B3 derivative, on the growth and virulence of Porphyromonas gingivalis, a key member of the red complex. Our findings revealed that NAM inhibited bacterial growth and gingipain activities, which played a dominant role in protein hydrolysis and heme acquisition. NAM decreased hemagglutination and hemolysis abilities and changed hemin and hemoglobin binding capacities, controlling bacterial infection through a starvation strategy by blocking access to growth-essential nutrients from the outside and reducing bacterial virulence. Several experiments in an animal model showed the effectiveness of NAM in preventing alveolar bone loss and reducing inflammatory cell infiltration, shedding light on its potential therapeutic applicability.

Selective Capture of Anti-N-glucosylated NTHi Adhesin Peptide Antibodies by a Multivalent Dextran Conjugate.

Tentacle-like polymers decorated with several copies of peptide antigens can be interesting tools for increasing the ability to capture circulating antibodies in patient sera, using cooperative effects for stronger avidity. We previously showed that antibodies from multiple sclerosis (MS) patient sera preferentially recognize hyperglucosylated adhesin protein HMW1ct of non-typeable Haemophilus influenzae (NTHi). We selected the C-terminal HMW1ct(1347-1354) minimal epitope and prepared the diglucosylated analogue Ac-KAN(Glc)VTLN(Glc)TTG-K(N3 )-NH2 to graft a 40 kDa dextran scaffold modified with glycidyl-propargyl moieties to perform a copper catalyzed alkyne-azide coupling reaction (CuAAC). Quantitative NMR measurements allowed the characterization of the peptide loading (19.5 %) on the multivalent dextran conjugate. This novel polymeric structure displayed optimal capturing properties of both IgG and, more interestingly, IgM antibodies in MS sera. Specific antibodies from a representative MS serum, were successfully depleted using a Sepharose resin bearing the new glucosylated multivalent conjugate, as confirmed by ELISA. These results may offer a promising proof-of-concept for the selective purification of high affinity autoantibodies from sera of autoimmune patients, in general, and of specific high affinity antibodies against a minimally glcosylated epitope Asn(Glc) from sera of multiple sclerosis (MS) patients, in particular.

A tough, adhesive, self-healable, and antibacterial plant-inspired hydrogel based on pyrogallol-borax dynamic cross-linking.

Multifunctional hydrogels that integrate stretchability, adhesion, self-healing, and antibacterial properties may find use in a variety of fields including electronic skin, wound dressings, and wearable devices; however, traditional hydrogels often exhibit short-term adhesiveness, poor mechanical properties, and a lack of antibacterial activity. Herein, a plant-inspired polyacrylamide-soybean protein isolate-pyrogallol/borax (PAM-SPI-P/B) hydrogel has been developed using a facile green method based on dynamic coordination cross-linking between pyrogallol (PG) and borax. The PG-borax dynamic bonds adjusted the network structure of the hydrogels to provide greater structural integrity to the PAM-SPI double network. This hydrogel possessed a high mechanical strength (large elongation up to 760% and compressive strength up to 1.25 MPa at 80% strain), low swelling ratio, and self-healing properties. Inspired by natural polyphenols that contain adhesive molecules, the addition of pyrogallol provided the hydrogel excellent adhesion to various hydrophilic and hydrophobic substrates. And with the inhibition of pyrogallol autoxidation due to the borax protection, the hydrogel showed repeatable and durable adhesion over 20 cycles. The obtained hydrogels also exhibited good antibacterial activities against Escherichia coli and Staphylococcus aureus because they were based on pyrogallol and borax, which have antibacterial properties. Accordingly, we envision that the PAM-SPI-P/B hydrogels have great potential for use in biomimetic tissues and biosensors.

Frequency and expression of genes involved in adhesion and biofilm formation in Staphylococcus aureus strains isolated from periodontal lesions.

BACKGROUND/PURPOSE: The aim of this study was to characterize the Staphylococcus aureus strains isolated from periodontal lesions of patients, to determine the expression of genes involved in cell adhesion upon their infection of human epithelial cells using an in vitro model, its biofilm formation, and its resistance to antibiotics. METHODS: S. aureus was analysed by PCR, Kirby-Bauer, and pulsed-field gel electrophoresis (PFGE), measuring gene expression by real-time PCR after infection of human cells in vitro. RESULTS: S. aureus was identified in 18.6% (50/268) of the samples. All strains (n = 50) possessed the virulence genes spa (Staphylococcal protein A), coa (coagulase), and icaAB (intercellular adhesin); 96% (n = 48) possessed clfB (clumping factor B), and 88% (n = 44) possessed ebps (elastin-binding protein) and sdrD (serine aspartate repeat protein D). All strains were resistant to methicillin, ampicillin, dicloxacillin, cefotaxime, and penicillin, and were multidrug resistant to 6-12 antibiotics. PFGE analysis showed 37 different pulsed-field types and most strains (60.4%) had a unique pulsed-field type. Twenty-four distinct combinations of virulence genes and antibiotic-resistant phenotypes were identified. CONCLUSION: Although S. aureus has been considered a transient member of the oral microbiota, our results indicate a high-level expression of virulence genes and multidrug resistance in the strains isolated from periodontal lesions. These strains might complicate the successful treatment of the disease.

e-Membranome: A Database for Genome-Wide Analysis of Escherichia coli Outer Membrane Proteins.

OBJECTIVE: Lectin-like adhesins of enteric bacterial pathogens such as Escherichia coli are an attractive target for vaccine or drug development. Here, we have developed e-Membranome as a database of genome-wide putative adhesins in Escherichia coli (E. coli). METHODS: The outer membrane adhesins were predicted from the annotated genes of Escherichia coli strains using the PSORTb program. Further analysis was performed using Interproscan and the String database. The candidate proteins can be investigated for homology modeling of the Three-Dimensional (3D) structure (I-TASSER version 5.1), epitope region (ABCpred), and the glycan array. RESULTS: e-Membranome is implemented using the Django (version 2.2.5) framework. The Web Application Server Apache Tomcat 6.0 is integrated into the platform on Ubuntu Linux (version 16.04). MySQL database (version 5.7) is used as a database engine. The information on homology model of the 3D structure, epitope region, and affinity information from the glycan array will be stored in the e- Membranome database. As a case study, we performed a genome-wide screening of outer membraneembedded proteins from the annotated genes of E. coli using the e-Membranome pipeline. CONCLUSION: This platform is expected to be a valuable resource for advancing research of outer membrane proteins for the construction of lectin-glycan interaction network of E. coli. In addition, the e- Membranome pipeline can be extended to other similar biological systems that need to address hostpathogen interactions.

Wheat Germ-Derived Peptides Exert Antiadhesive Activity against Helicobacter pylori: Insights into Structural Characteristics of Identified Peptides.

Approximately 50-80% of the world population are infected with H. pylori, which is categorized as a class I carcinogen. Antiadhesive therapy is emerging as a promising alternative to antibiotics against bacterial infection. This study demonstrated that defatted wheat germ protein hydrolysates (DWGPH) effectively inhibited H. pylori adhesion to gastric epithelial cells. DWGPH prepared by pronase possessed the best activity where its inhibitory percentage at 10 mg/mL was 51.7 ± 6.8% and the minimum antiadhesive concentration was 0.31 mg/mL. The antiadhesive activity is attributable to peptides acting as receptor analogs in binding to H. pylori. Peptides with potential H. pylori-binding ability (n = 267) were identified, and their structural characteristics were comprehensively analyzed, including net charge, Boman index, instability index, aliphatic index, molecular weight, isoelectric point, hydrophobicity, and Hmoment (α-helix and ß-sheet). This work provided an array of peptide sequences for further exploration as putative ligands of H. pylori adhesins and for elucidating molecular mechanisms.

Analyzing the adhesion mechanism of FnBPA, a surface adhesin from Staphylococcus aureus on its interaction with nanoparticle.

Staphylococcus aureus expresses many Microbial Surface Recognizing Adhesive Matrix Molecules (MSCRAMM's) to recognize host extracellular matrix (ECM) molecules to initiate colonization. The MSCRAMM, fibronectin binding protein A (FnBPA), is an important adhesin for S. aureus infection. FnBPA also binds with fibrinogen (Fg) by using a unique ligand binding mechanism called dock, lock and latch. Nanoparticles, especially nanosilver particles have been widely used in a variety of biomedical applications which includes disease diagnosis and treatment, drug delivery and implanted medical device coating. In a biological system, when protein molecules encounter nanoparticle, they can be absorbed onto its surface which results in the formation of protein corona. In the present study, we have analysed the fibrinogen binding ability of rFnBPA(189-512) in the presence of silver nanoparticles by employing techniques like gel shift assay, Western blot, size exclusion chromatography, enzyme-linked immunosorbent assay, bio-layer interferometry and circular dichroism spectroscopy. The results indicate that rFnBPA(189-512) is unable to bind to Fg in the presence of a nanoparticle. This could be due to the inaccessibility of the Fg binding site and conformational change in rFnBPA(189-512). With nanoparticles, rFnBPA(189-512) undergoes significant structural changes as the ß-sheet content has drastically reduced to 10% from the initial 60% at higher concentration of the nanoparticle. Pathogenic bacteria interact with its surrounding environment through their surface molecules which includes MSCRAMMs. Therefore MSCRAMMs play an important role when bacteria encounter nanoparticles. The results of the present study suggest that the orientation of the protein during the absorption on the surface of a nanoparticle as well as the concentration of the nanoparticle, will dictate the function of the absorbed protein and in this case the Fg binding property of rFnBPA(189-512).

Presence of biofilm and adhesin genes in Staphylococcus aureus strains taken from chronic wound infections and their genotypic and phenotypic antimicrobial sensitivity patterns.

The purpose of this research was to examine biofilm (icaA, icaD and bap) and adhesin (clfA, fnbA, cna) genes, and also assess the genotypic and phenotypic antimicrobial resistance patterns of Staphylococcus aureus strains taken from wound specimens in Mardin, Turkey. A total of 220 wound specimens were investigated. The biofilm forming ability and resistance pattern for eleven antimicrobial agents were investigated by conventional and multiplex PCR methods. S. aureus were taken from 112 (50.9%) of 220 wound specimens. Moreover, biofilm production was found in 79 (70.5%) of the 112 S. aureus isolates. 97 (86.6%) strains of all isolates were positive for icaA and icaD, and 15 (13.4%) for bap. The adhesin genes, cna, fnbA and clfA were detected in 98 (87.5%), 87 (77.7%), and 75 (66.9%) strains, respectively. The numbers of MSSA and MRSA bearing antimicrobial resistance genes were 19 (16.96%) and 32 (28.57%) for blaZ, 9 (8.04%) and 17 (15.18%) for tetK, 6 (5.36%) and 14 (12.5%) for ermC, 2 (1.79%) and 7 (6.25%) for tetM, 0 (0%) and 5 (4.46%) for mecA, 2 (1.79%) and 4 (3.57%) for ermA, 1 (0.89%) and 2 (1.79%) for both tetK and tetM, respectively. Our findings indicate that multiplex PCR is a suitable way for identifying biofilm and adhesin producing S. aureus. Our data also provided a country-wide oversight of the S. aureus antimicrobial resistance gene profiles for the properly therapy of patients and to control the spreading of the resistance genes.

Antimicrobial activities of peptide Cbf-K16 against drug-resistant Helicobacter pylori infection in vitro and in vivo.

Helicobacter pylori (H. pylori) infection is highly prevalent, and has developed antimicrobial resistance to virtually all existing antibiotics. Currently, treatment of H. pylori infection (involving proton pump inhibitors and broad-spectrum antibiotics) is suboptimal, with high failure rates. Thus, there is a pressing need to develop new anti-H. pylori therapies. Cbf-K16, a cathelicidin-like antimicrobial peptide, presented broad antimicrobial activity during our previous research. This study further evaluated the therapeutic potential and the mode of action underlying Cbf-K16 against clarithromycin- and amoxicillin-resistant H. pylori SS1. The MIC and MBC of Cbf-K16 against the tested H. pylori were 16 and 32 µg/ml, respectively, and its killing kinetics was time-dependent, reflecting the thorough elimination of drug-resistant bacteria within 24 h. This peptide also protected H. pylori-infected gastric epithelial cells (GES-1) from death by reducing the cell supernatant and intracellular bacterial counts by 1.9 and 2.9-log10 units, respectively. These data indicated the powerful antimicrobial effects of Cbf-K16in vitro. Meanwhile, notable antimicrobial activity in the mouse gastritis model was observed, with decreasing bacterial counts by 3.9-log10 units in stomach tissues and Cbf-K16 could effectively suppress the secretion of inflammatory cytokine IL-8. For its mode of action, Cbf-K16 not only neutralized the negative potential and increased the membrane uptake of NPN and PI by 78.5% and 85.1%, respectively, but also bound to genomic DNA, which in turn downregulated the expression of adhesion genes (alpA and alpB) and virulence gene (cagA), indicating its effective activities on membrane disruption, DNA-binding and gene expression. The data above demonstrated that Cbf-K16 possessed effective antimicrobial and anti-inflammatory activities and downregulated the expression of adhesion- and cytotoxin-associated genes of drug-resistant H. pylori SS1, making it a potential candidate for anti-infective therapy.

Dual-targeting potential of active constituents of Nigella sativa against FimH and CTX-M-15: A plausible therapeutic strategy against drug-resistant uropathogenic strains.

Uropathogenic strains belonging to the Enterobacteriaceae family are considered one of factors for urinary tract infections, and type 1 pilus fimbrial adhesin (FimH) and beta lactamase CTX-M-15 play crucial roles in their pathogenesis and resistance. Thus, a promising approach is to explore dual-targeting therapeutic agents that act against both FimH and CTX-M-15. In the present study, active constituents of Nigella sativa were selected on the basis of significant activity against UTIs. Molecular docking was used to target active constituents of Nigella sativa to the active sites of FimH and CTX-M-15; these included thymoquinone, dithymoquinone, carvacrol, p-cymene, thymol, thymohydroquinone and longifolene. Dithymoquinone was found to be the most potent dual inhibitor, with binding energy of -7.01 and -5.38kcal/mol against CTX-M-15 and FimH, respectively; In addition, Dithymoquinone exhibited superior activity compared to positive controls avibactam and heptyl α-D-mannopyranoside. Further molecular dynamic simulation studies were carried out to assess the stability of dithymoquinone-target protein complexes via RMSD, Rg, SASA, hydrogen bond number, and RMSF analysis. Both protein-ligand complexes were conserved and attained equilibrium at around 2.0 to 2.5 ns during 10 ns runs. These results suggest that active constituents of Nigella sativa, particularly dithymoquinone, might represent a plausible therapeutic strategy against resistant uropathogenic bacteria.

Virulence Factor Genes Incidence among Enterococci from Sewage Sludge in Eastern Slovakia following Safety Aspect.

The sewage sludges represent a potential health hazard because of the quantity of different microbiota detected in sewages. Among microbiota detected in sewages, also belong representatives of the phylum Firmicutes. In the past, environmental enterococci in addition to coliforms were widely used as indicators of faecal contamination. Regarding the enterococcal strains as potential pathogenic bacteria, their pathogenicity is mainly caused by production of virulence factors. Therefore, the aim of the study was to analyse incidence of virulence factors in enterococci from cows' dung water. Species identification of 24 enterococci using MALDI-TOF MS system allotted 23 strains to the species Enterococcus faecium with highly probable species identification and E. faecalis EEV20 with a score value meaning secure genus identification/probable species identification. Enterococci were absent of cytolysin A gene, hyaluronidase gene, and element IS gene. It can be concluded that they are not invasive which is very important from safety aspect. The most frequently detected gene was adhesin E. faecium (efaAfm, in 22 E. faecium strains and in one E. faecalis). Adhesin efaAfs gene was detected in E. faecalis EEV20 and in two E. faecium. GelE gene was present in three strains. E. faecium EF/EC31 was absent of virulence factor genes.

Bacterial Anti-adhesives: Inhibition of Staphylococcus aureus Nasal Colonization.

Bacterial adhesion to the skin and mucosa is often a fundamental and early step in host colonization, the establishment of bacterial infections, and pathology. This process is facilitated by adhesins on the surface of the bacterial cell that recognize host cell molecules. Interfering with bacterial host cell adhesion, so-called anti-adhesive therapeutics, offers promise for the development of novel approaches to control bacterial infections. In this review, we focus on the discovery of anti-adhesives targeting the high priority pathogen Staphylococcus aureus. This organism remains a major clinical burden, and S. aureus nasal colonization is associated with poor clinical outcomes. We describe the molecular basis of nasal colonization and highlight potentially efficacious targets for the development of novel nasal decolonization strategies.

Glycocluster Tetrahydroxamic Acids Exhibiting Unprecedented Inhibition of Pseudomonas aeruginosa Biofilms.

Opportunistic Gram-negative Pseudomonas aeruginosa uses adhesins (e.g., LecA and LecB lectins, type VI pili and flagella) and iron to invade host cells with the formation of a biofilm, a thick barrier that protects bacteria from drugs and host immune system. Hindering iron uptake and disrupting adhesins' function could be a relevant antipseudomonal strategy. To test this hypothesis, we designed an iron-chelating glycocluster incorporating a tetrahydroxamic acid and α-l-fucose bearing linker to interfere with both iron uptake and the glycan recognition process involving the LecB lectin. Iron depletion led to increased production of the siderophore pyoverdine by P. aeruginosa to counteract the loss of iron uptake, and strong biofilm inhibition was observed not only with the α-l-fucocluster (72%), but also with its α-d-manno (84%), and α-d-gluco (92%) counterparts used as negative controls. This unprecedented finding suggests that both LecB and biofilm inhibition are closely related to the presence of hydroxamic acid groups.

Comparison of adhesin genes expression among Klebsiella oxytoca ESBL-non-producers in planktonic and biofilm mode of growth, and imipenem sublethal exposure.

BACKGROUND: Bacterial adhesins play an important role in the bacterial attachment and colonization. The aim of this study was comparison of adhesin genes expression in the planktonic and biofilm mode of growth among ESBL-non-producers isolates of K. oxytoca and effect of imipenem. MATERIALS AND METHODS: A total of eight extended-spectrum beta-lactamase (ESBL) non-producer K. oxytoca isolates were included from patients with hemorrhagic colitis. The adhesin genes including fimA (type 1 fimbria), mrkA (type 3 fimbria), pilQ and the capsular matB genes were adopted. Phenotypic biofilm production was assessed by microtiter tissue plate assay. Expression of adhesin genes in the planktonic and biofilm growth conditions was calculated using quantitative Real-time PCR (RT-qPCR) technique and sub-MIC (0.25 µg/ml) levels of imipenem were also added to broth culture of isolates to evaluate the gene expression. RESULTS: The isolates produced biofilm in moderate level. The expression of pilQ, mrkA and matB but not fimA genes was significantly higher in biofilm conditions compared to the planktonic mode of growth (p = 0.002, p = 0.011 and p = 001, respectively). In addition, imipenem sub-MIC treatment led to a significant overexpression of matB (p = 0.002) and mrkA (p = 0.003) genes compared to the control group. CONCLUSION: Although none of isolates produced strong biofilm, biofilm conditions led to the increase in the expression of adhesin encoding genes in non-ESBL-producing K. oxytoca. Furthermore, ß-lactams; and especially carbapenems possibly increase the colonization of K. oxytoca and increase the biofilm formation. Hence, accurate consumption of antibiotics must be considered.

Immunomodulatory molecules regulate adhesin gene expression in Staphylococcus aureus: Effect on bacterial internalization into bovine mammary epithelial cells.

Staphylococcus aureus is a major pathogen of subclinical bovine mastitis that usually is chronic and recurrent, which has been related to its ability to internalize into bovine mammary epithelial cells (bMECs). Previously, we reported that short and medium fatty acids and cholecalciferol reduce S. aureus internalization into pretreated-bMECs with these molecules suggesting a role as immunomodulatory agents. Hence, we assessed the role of sodium butyrate (NaB), sodium octanoate (NaO) and cholecalciferol on S. aureus adhesin expression and its internalization into bMECs. S. aureus pre-treated 2 h with 0.5 mM or 2 mM NaB showed a reduction in internalization into bMECs (∼35% and ∼55%; respectively), which coincided with a down-regulated expression of clumping factor B (ClfB). Also, the S. aureus internalization reduction by 2 mM NaB (2 h) agreed with a down-regulated expression of sdrC. Moreover, the 2 mM NaB (24 h) pre-treatment induced bacterial internalization (∼3-fold), which was related with an up-regulation of spa, clfB and sdrC genes. Also, NaO (0.25 mM and 1 mM) only reduced S. aureus internalization when bacteria were grown 2 h with this molecule but there was no relationship with adhesin expression. In addition, cholecalciferol (50 nM) reduced bacteria internalization at similar levels (∼50%) when bacteria were grown 2 and 24 h in broth supplemented with this compound, which correlated with spa and sdrC mRNA expression down-regulated at 2 h, and fnba and clfB mRNA expression decreased at 24 h. In conclusion, our data support the fact that fatty acids and cholecalciferol regulate adhesin gene expression as well as bacteria internalization in nonprofessional phagocytic cells, which may lead to development of anti-virulence agents for control of pathogens.

Baicalin Weakens Staphylococcus aureus Pathogenicity by Targeting Sortase B.

Staphylococcus aureus (S. aureus) is a human and other animal pathogen that contributes to the primary etiology of nosocomial pneumonia, a disease with high mortality rates and costs. Treatment of multidrug-resistant S. aureus infection is extremely challenging, and new therapeutic strategies beyond antibiotic treatment are needed. Anti-virulence agents that specifically target the molecular determinants of virulence may be a novel method for treating drug-resistant nosocomial infections. Sortase B (SrtB) is a crucial virulence factor in S. aureus and plays an important role during infection. In this study, we find that baicalin suppresses the activity of SrtB. Minimum inhibitory concentration and growth curve assays confirmed that baicalin has no anti-S. aureus properties. We performed live/dead, lactate dehydrogenase (LDH), adherence, and enzyme-linked immunosorbent assays to confirm that baicalin reduced human alveolar epithelial A549 cell injury caused by S. aureus, reduced the adherence of S. aureus to A549 cells, and significantly attenuated the inflammatory response of mouse macrophage J774 cells to S. aureus. Additionally, we were able to elucidate the binding mechanics and identify the interacting sites of baicalin and SrtB via a molecular dynamics simulation, site-directed mutagenesis, and fluorescence spectroscopy quenching. Finally, we confirmed that baicalin directly binds to the active center of SrtB, and the residues Asn92 and Tyr128 perform an important function in the interaction of SrtB and baicalin. Taken together, these data indicate that baicalin is a promising candidate to combat S. aureus infections.

Modulation of S. aureus and P. aeruginosa biofilm: an in vitro study with new coumarin derivatives.

Coumarin is an important heterocyclic molecular framework of bioactive molecules against broad spectrum pathological manifestations. In the present study 18 new coumarin derivatives (CDs) were synthesized and characterized for antibiofilm activity against two model bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa. It was observed that all the CDs executed significant effect in moderating activities against both planktonic and biofilm forms of these selected bacteria. Hence, to interpret the underlying probable reason of such antibiofilm effect, in-silico binding study of CDs with biofilm and motility associated proteins of these organisms were performed. All CDs have shown their propensity for occupying the native substrate binding pocket of each protein with moderate to strong binding affinities. One of the CDs such as CAMN1 showed highest binding affinity with these proteins. Interestingly, the findings of in-silico studies coincides the experimental results of antibiofilm and motility affect of CDs against both S. aureus and P. aeruginosa. Moreover, in-silico studies suggested that the antibiofilm activity of test CDs may be due to the interference of biofilm and motility associated proteins of the selected model organisms (PilT from P. aeruginosa and TarK, TarO from S. aureus). The detailed synthesis, characterization, methodology and results of biological screening along with computational studies have been reported. This study could be of greater interest in the context of the development of new anti-bacterial agent in the future.

Conformational analysis of Pneumococcal Surface Antigen A (PsaA) upon zinc binding by fluorescence spectroscopy.

PsaA (pneumococcal surface antigen A) is a S. pneumoniae virulence factor that belongs to the metal transport system. The Manganese PsaA binding has been associated with oxidative stress resistance becoming a pivotal element in the bacteria virulence. It has been shown that Zinc inhibits the Manganese acquisition and promotes bacteria toxicity. We have performed a PsaA conformational analysis both in the presence (Zn-rPsaA) and in the absence of Zinc (free-rPsaA). We performed experiments in the presence of different Zinc concentrations to determine the metal minimum concentration which induced a conformational change. The protein in free and Zn-binding condition was also studied in pH ranging 2.6-8.0 and in temperature ranging 25oC-85oC. pH experiments showed a decrease of fluorescence intensity only in acidic medium. Analysis of the heat-induced denaturation demonstrated that Zinc-binding promoted an increase in melting temperature from 55oC (free-rPsaA) to 78.8oC (Zn-rPsaA) according to fluorescence measurements. In addition, the rPsaA stabilization by Zinc was verified through analysis of urea and guanidine hydrochloride denaturation. Data showed that Zinc promoted an increase in the rPsaA stability and its removal by EDTA can lead to a PsaA intermediate conformation. These findings can be considered in the development of vaccines containing PsaA as antigen.