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.

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.

Plant nutraceuticals (Quercetrin and Afzelin) capped silver nanoparticles exert potent antibiofilm effect against food borne pathogen Salmonella enterica serovar Typhi and curtail planktonic growth in zebrafish infection model.

Purified plant nutraceuticals afzelin and quercetrin from an edible plant- Crotolaria tetragona was employed for the fabrication of silver nanoparticles (AgNPs) by a sunlight mediated process. From among a panel of strains tested, AgNPs displayed potent bacteriostatic and bactericidal effect against P. aeruginosa and S. Typhi. Time kill studies revealed green synthesized AgNPs displayed comparable bactericidal effect with chemically synthesized AgNPs against S. Typhi. Antibiofilm potential of AgNPs showed that they were highly effective at sub MIC concentrations in causing 50% biofilm inhibition against food borne pathogen S. Typhi implying that antibiofilm effect is independent of antibacterial effect, which was evidenced by fluorescent imaging and SEM imaging. Mechanistic studies revealed that reduced cell surface hydrophobicity, decreased surface adherence, loss of membrane potential contributed to antibiofilm potential of afzelin/quercetrin AgNPs. Green synthesized afzelin/quercetrin AgNPs were also relatively less toxic and more effective in curtailing bioburden of S. Typhi in infected zebrafish by > 3 log fold. Ability of sunlight reduced afzelin/quercetrin NPs to mitigate planktonic mode of growth in vitro and in vivo and curtail biofilm formation of S. Typhi in vitro demonstrates its potential to curtail food borne pathogen in planktonic and biofilm mode of growth.

Inhibitory Effect of the Ethanol Extract of a Rice Bran Mixture Comprising Angelica gigas, Cnidium officinale, Artemisia princeps, and Camellia sinensis on Brucella abortus Uptake by Professional and Nonprofessional Phagocytes.

In this study, we evaluated the inhibitory effect of a rice bran mixture extract (RBE) on Brucella abortus pathogenesis in professional (RAW 264.7) and nonprofessional (HeLa) phagocytes. We fermented the rice bran mixture and then extracted it with 50% ethanol followed by gas chromatography-mass spectrometry to identify the components in RBE. Our results clearly showed that RBE caused a significant reduction in the adherence of B. abortus in both cell lines. Furthermore, analysis of phagocytic signaling proteins by western blot assay revealed that RBE pretreatment resulted in inhibition of phosphorylation of JNK, ERK, and p38, leading to decline of internalization compared with the controls. Additionally, the intensity of F-actin observed by fluorescence microscopy and FACS was remarkably reduced in RBE-pretreated cells compared with control cells. However, the intracellular replication of B. abortus within phagocytes was not affected by RBE. Taken together, these findings suggest that the phagocytic receptor blocking and suppressive effects of RBE on the MAPK-linked phagocytic signaling pathway could negatively affect the invasion of B. abortus into phagocytes.

Antibiofilm activity of Vetiveria zizanioides root extract against methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a leading human pathogen responsible for causing chronic clinical manifestation worldwide. In addition to antibiotic resistance genes viz. mecA and vanA, biofilm formation plays a prominent role in the pathogenicity of S. aureus by enhancing its resistance to existing antibiotics. Considering the role of folk medicinal plants in the betterment of human health from the waves of multidrug resistant bacterial infections, the present study was intended to explore the effect of Vetiveria zizanioides root on the biofilm formation of MRSA and its clinical counterparts. V. zizanioides root extract (VREX) showed a concentration-dependent reduction in biofilm formation without hampering the cellular viability of the tested strains. Micrographs of scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) portrayed the devastating impact of VREX on biofilm formation. In addition to antibiofilm activity, VREX suppresses the production of biofilm related phenotypes such as exopolysaccharide, slime and α-hemolysin toxin. Furthermore, variation in FT-IR spectra evidenced the difference in cellular factors of untreated and VREX treated samples. Result of mature biofilm disruption assay and down regulation of genes like fnbA, fnbB, clfA suggested that VREX targets these adhesin genes responsible for initial adherence. GC-MS analysis revealed the presence of sesquiterpenes as a major constituent in VREX. Thus, the data of present study strengthen the ethnobotanical value of V. zizanioides and concludes that VREX contain bioactive molecules that have beneficial effect over the biofilm formation of MRSA and its clinical isolates.

A novel, potent dual inhibitor of Arg-gingipains and Lys-gingipain as a promising agent for periodontal disease therapy.

The periodontal pathogen Porphyromonas gingivalis produces a unique class of cysteine proteinases termed gingipains that comprises Arg-gingipain (Rgp) and Lys-gingipain (Kgp). Growing evidence indicates that these 2 types of gingipains synergistically contribute to the entire virulence of the organism and increase the risk of periodontal disease (PD) by disrupting the host immune system and degrading the host tissue and plasma proteins. Therefore, a dual inhibitor of both gingipains would have attractive clinical potential for PD therapy. In this study, a novel, potent, dual inhibitor of Rgp and Kgp was developed through structure-based drug design, and its biological potency was evaluated in vitro and in vivo. This inhibitor had low nanomolar inhibitory potency (Ki=40 nM for Rgp, Ki=0.27 nM for Kgp) and good selectivity for host proteases and exhibited potent antibacterial activity against P. gingivalis by abrogating its manifold pathophysiological functions. The therapeutic potential of this inhibitor in vivo was also verified by suppressing the vascular permeability that was enhanced in guinea pigs by the organism and the gingival inflammation in beagle dog PD models. These findings suggest that a dual inhibitor of Rgp and Kgp would exhibit noteworthy anti-inflammatory activity in the treatment of PD.

Inhibitory effects of sword bean extract on alveolar bone resorption induced in rats by Porphyromonas gingivalis infection.

BACKGROUND: The domesticated legume, Canavalia gladiata (commonly called the sword bean), is known to contain canavanine. The fruit is used in Chinese and Japanese herbal medicine for treating the discharge of pus, but its pharmacological mechanisms are still unclear. OBJECTIVES: This study examined the effect of sword bean extract (SBE) on (i) oral bacteria and human oral epithelial cells in vitro, and (ii) the initiation and progression of experimental Porphyromonas gingivalis-induced alveolar bone resorption in rats. MATERIAL AND METHODS: A high-performance liquid chromatography/ultraviolet method was applied to quantitate canavanine in SBE. By assessing oral bacterial growth, we estimated the minimum inhibitory concentration and minimum bactericidal concentration of SBE, canavanine, chlorhexidine gluconate (CHX) solution. The cytotoxicity of SBE, canavanine, CHX, leupeptin and cystatin for KB cells was determined using a trypan blue assay. The effects of SBE, canavanine, leupeptin and cystatin on Arg-gingipain (Rgp) and Lys-gingipain (Kgp) were evaluated by colorimetric assay using synthetic substrates. To examine its effects on P. gingivalis-associated periodontal tissue breakdown, SBE was orally administered to P. gingivalis-infected rats. RESULT: Sword bean extract contained 6.4% canavanine. SBE and canavanine inhibited the growth of P. gingivalis and Fusobacterium nucleatum. The cytotoxicity of SBE, canavanine and cystatin on KB cells was significantly lower than that of CHX. Inhibition of Rgp with SBE was comparable to that with leupeptin, a known Rgp inhibitor, and inhibition of Kgp with SBE was significantly higher than that with leupeptin at 500 µg/mL ( p < 0.05). P. gingivalis-induced alveolar bone resorption was significantly suppressed by administration of SBE, with bone levels remaining comparable to non-infected animals ( p < 0.05). CONCLUSION: The present study suggests that SBE might be effective against P. gingivalis-associated alveolar bone resorption.

Combating Helicobacter pylori infections with mucoadhesive nanoparticles loaded with Garcinia mangostana extract.

AIM: To combat the resistance of Helicobacter pylori to antibiotics through the use of Garcinia mangostana extract (GME) in the form that can be localized at stomach mucosa. MATERIALS & METHODS: GME and its major active component, α-mangostin, are encapsulated into the moderately acid stable mucoadhesive nanocarriers, and tested for anti-H. pylori and antiadhesion activities in vitro and their ability to eradicate H. pylori in infected mice. RESULTS: The two in vitro activities are observed and are enhanced when the materials are encapsulated into nanocarriers. Preliminary in vivo tests revealed the ability to combat H. pylori in mice following oral administration of the encapsulated GME, but not the unencapsulated GME. CONCLUSION: Nanoencapsulated GME is a potential anti-H. pylori agent.

Anti-adhesion methods as novel therapeutics for bacterial infections.

Anti-adhesion therapies for bacterial infections offer an alternative to antibiotics, with those therapies bacteria are not killed but are prevented from causing harm to a host by inhibiting adherence to host cells and tissues, a prerequisite for the majority of infectious diseases. The mechanisms of these potential therapeutic agents include inhibition of adhesins and their host receptors, vaccination with adhesins or analogs, use of probiotics and dietary supplements that interfere with receptor-adhesin interactions, subminimal inhibitory concentrations of antibiotics and manipulation of hydrophobic interactions. Once developed, these drugs will contribute to the arsenal for fighting infectious disease in the future, potentially subverting antibiotic resistance.

Antiadhesion as a functional concept for prevention of pathogens: N-Phenylpropenoyl-L-amino acid amides as inhibitors of the Helicobacter pylori BabA outer membrane protein.

SCOPE: Besides flavan-3-ols, a family of N-phenylpropenoyl-L-amino acids (NPAs) has been recently identified as polyphenol/amino acid conjugates in the seeds of Theobroma cacao as well as in a variety of herbal drugs. NPAs were shown to exhibit antiadhesive activities against Helicobacter pylori. METHODS AND RESULTS: For structure/activity relationship 24 homologous NPAs (2 mM) were investigated in a flow cytometric assay on potential antiadhesive effects against H. pylori adhesion to human gastric AGS cells. Dihydroxylation of the aromatic molecule part was shown to be necessary for activity; methoxylation decreases activity. High polarity of the amino acid is a prerequisite for activity. The model compound N-(E)-caffeoyl-L-glutamic acid 11 exerted a concentration-dependent inhibition of bacterial adhesion with saturation at 30% inhibition level. The antiadhesive effect was additionally confirmed by in situ adhesion assay on intact human gastric tissue. NPAs exhibited no cytotoxicity. Using immobilized ligands interaction 11 with bacterial adhesin BabA was demonstrated. RT-PCR indicated that the inhibition of BabA is not correlated with subsequent feed back regulations to express more adhesins or virulence factors (vacA, cagA, cagL, cagα, fucT, ureI, ureA, OMPs). The interaction of bacterial adhesins with the respective ligands does not automatically lead to a subsequent signal transduction towards induction of virulence processes. CONCLUSION: The nutritional use of NPA-containing food may justify a positive antiadhesive effect against the recurrence of H. pylori infections.

Polyphenols from Myrothamnus flabellifolia Welw. inhibit in vitro adhesion of Porphyromonas gingivalis and exert anti-inflammatory cytoprotective effects in KB cells.

AIM: Identification of anti-adhesive plant extracts against cell surface binding of Porphyromonas gingivalis and underlying mechanisms; investigation of potential cytoprotective effects of anti-adhesive extract on KB cells. MATERIALS AND METHODS: Polyphenol-enriched extract, fully characterized concerning flavan-3-ols and oligomeric proanthocyanidins, from Myrothamnus flabellifolia (MF), traditionally used for periodontitis, was tested for inhibition of P. gingivalis-mediated adhesion to KB cells by flow cytometry, for influence on gingipain activity (protease assay), haemagglutination and by microarray analysis for effects on bacterial transcriptome. The influence of MF on P. gingivalis-induced cytokine gene expression was monitored by RT-PCR and IL-6 titres by ELISA. RESULTS: MF (100 µg/ml) reduced P. gingivalis adhesion/invasion about 50% by interacting with bacterial OMPs. As shown by RT-PCR, fimbrillin and Arg-gingipain encoding genes were up-regulated by MF. On the protein level, inhibition (70%) of Arg-gingipain activity was observed, while the corresponding Lys-gingipain was hardly influenced. MF also inhibited haemagglutination. While exposure to P. gingivalis resulted in an increased expression of inflammation-related genes in KB cells, pre-treatment of KB cells with MF evoked cytoprotective effects concerning IL-1ß, IL-6, IL-8 and TNF-α gene expression as well as IL-6 release rates. Compounds from the plant extract belonging to the class of proanthocyanidins were shown to be responsible for the observed effects and were characterized for their respective structural features. CONCLUSIONS: While being cytoprotective, MF exerts anti-adhesive effects against P. gingivalis. Thus, MF may be useful for the prevention of P. gingivalis-associated periodontal diseases.

Development of biosensor-based assays to identify anti-infective oligosaccharides.

It is now well accepted that milk oligosaccharides can have a direct inhibitory effect on pathogenic microorganisms by interfering with their adhesion to human cells. Many free oligosaccharides from milk are considered to be soluble receptor analogs of epithelial cell surface carbohydrates and, thus, function as receptor decoys to which pathogens can bind instead of the host. In reality, there are few rapid methods to screen for such oligosaccharides, and much of the research in this area has centered on using human cell line models of infection that are time-consuming. Therefore, a quick and sensitive method is required for detecting the binding of microorganisms to milk oligosaccharides. Our study describes a number of biosensor-based methods to achieve these aims. Our approach involved the exposure of whole bacterial cells to the well-characterized human milk oligosaccharide, 2'-fucosyllactose, immobilized to a pretreated gold chip surface. The technique was validated by screening a range of pathogenic bacteria, including Campylobacter jejuni, to which 2'-fucosyllactose is known to bind. Where binding was detected, its specificity was confirmed by preincubation studies using unlabeled 2'-fucosyllactose. The techniques described represent a quick, cost-effective, and highly reproducible detection method for identifying anti-infective oligosaccharides.

Inhibition of Porphyromonas gingivalis proteinases (gingipains) by chlorhexidine: synergistic effect of Zn(II).

BACKGROUND/AIMS: Gingipains, proteolytic enzymes produced by the periodontal pathogen Porphyromonas gingivalis, are regarded as virulence factors in the pathogenesis of periodontitis. Inhibition of gingipain activity therefore may have therapeutic potential, and it has been suggested that chlorhexidine may inhibit the activities of these enzymes. The purposes of the present study were to examine systematically the inhibitory effects of chlorhexidine on three purified gingipains and to determine the effect of Zn(II) on chlorhexidine inhibition. METHODS: The activities of lys-gingipain (Kgp) and two forms of arg-gingipain (RgpB and HRgpA) were measured in the presence of varying concentrations of chlorhexidine and with chlorhexidine supplemented with Zn(II). Inhibition constants (K(i)'s) were determined for chlorhexidine alone and in the presence of Zn(II). Fractional inhibitory constant indices were calculated to assess the synergy of the chlorhexidine-Zn(II) inhibition. RESULTS: RgpB, HRgpA, and Kgp were all inhibited by chlorhexidine with K(i)'s in the micromolar range. For RgpB and HRgpA, the inhibitory effects of chlorhexidine were enhanced 3-30-fold by Zn(II). The chlorhexidine-Zn(II) interaction was synergistic for inhibition of HRgpA and RgpB. For Kgp, the effect of Zn(II) on chlorhexidine inhibition was antagonistic. CONCLUSIONS: Chlorhexidine is an effective inhibitor of gingipains, and the inhibition of R-gingipains is enhanced by Zn(II). A mixture of chlorhexidine and Zn(II) may be useful as an adjunct in the treatment of periodontitis and in the post-treatment maintenance of periodontitis patients.

Apple- and hop-polyphenols protect periodontal ligament cells stimulated with enamel matrix derivative from Porphyromonas gingivalis.

BACKGROUND: Enamel matrix derivative (EMD) is a tissue regenerative agent used clinically as an adjunct to periodontal surgery. It was previously demonstrated that Porphyromonas gingivalis, a periodontal pathogen, significantly diminished the efficacy of EMD with periodontal ligament (PDL) cells through the proteolytic actions of Arg- and Lys-gingipains (Rgp and Kgp). Thus, antiproteolytic supplements are considered clinically desirable for effective periodontal regenerative therapies. In the present study, we examined apple- (AP) and hop-polyphenols to determine their ability to protect EMD-stimulated PDL cells from P. gingivalis. METHODS: AP, apple condensed tannin (ACT), hop bract polyphenol (HBP), high and low molecular weight fractions of HBP (HMW-HBP and LMW-HBP), and epigallocatechin gallate (EGCg) were used. PDL cells were grown on EMD-coated dishes and infected with P. gingivalis, and cellular migration and proliferation were evaluated with an in vitro assay of wound healing assay in the presence or absence of the polyphenols. RESULTS: Each polyphenol significantly enhanced the viability of PDL cells infected with P. gingivalis, whereas only EGCg demonstrated cytotoxicity. Further, all polyphenols significantly inhibited Rgp activity, with AP, ACT, and HBP more effective toward Kgp. P. gingivalis markedly diminished the migration and proliferation of EMD-stimulated PDL cells, whereas the addition of AP, ACT, HBP, and HMW-HBP significantly protected the cells from bacterial cytotoxicity. In contrast, EGCg and LMW-HBP did not show protective effects. CONCLUSION: These results suggest that AP, ACT, AP, HBP, and HMW-HBP protect EMD-stimulated PDL cells from P. gingivalis and may be therapeutically useful supplements for EMD therapy.

Inhibitory effect of green tea catechins on cysteine proteinases in Porphyromonas gingivalis.

The purpose of this study was to examine the effects of catechins and their derivatives on the activities of Arg-gingipain (Rgp) and Lys-gingipain (Kgp) in Porphyromonas gingivalis. Catechin derivatives, which included (-)-epigallocatechin gallate, (-)-epicatechin gallate, (-)-gallocatechin gallate, and (-)-catechin gallate, significantly inhibited the Rgp activity. The 50% inhibitory concentrations (IC50s) of these catechin derivatives for Rgp ranged from 3 to 5 microm. While (-)-epigallocatechin and (-)-gallocatechin moderately inhibited Rgp activity (IC50s, 20 microm), (-) -epicatechin, (+)-catechin, and gallic acid were not effective, with IC50s greater than 300 microm. Further, some of the catechin derivatives tested also inhibited the Kgp activity, though to a lesser extent than inhibition of the Rgp activity. These findings suggest that green tea catechins may have the potential to reduce periodontal breakdown resulting from the potent proteinase activity of P. gingivalis.

Efficacy of pertussis vaccines consisted of antigens detoxified with tea-leaf catechins.

The ability of tea-leaf catechins to detoxify agents was examined. Filamentous hemagglutinin (FHA) and pertussis toxin (PT) were detoxified by the catechins at an extraordinarily lower concentration compared with that of formalin. The sera from the mice immunized by the catechin-treated antigens recognized, not only catechin-treated, but also untreated antigens. Furthermore, catechin-treated PT induced the antibody to neutralize PT activity in the sera of the immunized mice. Pertussis vaccines were prepared including antigens detoxified by the treatment of catechins and intraperitoneally injected into mice. Protection against Bordetella pertussis infection was shown in mice immunized with the vaccines prepared by treatment with catechins. These data suggest that catechins are effective toxoiding agents for preparing a pertussis vaccine.