Effect of the luxS gene on biofilm formation and antibiotic resistance by Salmonella serovar Dublin.

Biofilms are communities of bacterial cells that serve to protect them from external adverse influences and enhance bacterial resistance to antibiotics and sanitizers. Here, we studied the regulatory effects of glucose and sodium chloride on biofilm formation in Salmonella serovar Dublin (S. Dublin). To analyze expression levels of the quorum sensing gene luxS, we created a luxS knockout mutant. Also, antimicrobial resistance, hydrophobicity and autoinducer-2 (AI-2) activity of both the wild-type (WT) and the mutant strain were investigated. Our results revealed that glucose was not essential for S. Dublin biofilm formation but had an inhibitory effect on biofilm formation when the concentration was over 0.1%. NaCl was found to be indispensable in forming biofilm, and it also exerted an inhibitory effect at high concentrations (>1.0%). Both the WT and the mutant strains displayed significant MIC growth after biofilm formation. An increase of up to 32,768 times in the resistance of S. Dublin in biofilm phonotype against antibiotic (ampicillin) compared to its planktonic phonotype was observed. However, S. Dublin luxS knockout mutant only showed slight differences compared to the WT strain in the antimicrobial tests although it displayed better biofilm-forming capacity than the WT strain. The mutant strain also exhibited higher hydrophobicity than the WT strain, which was a feature related to biofilm formation. The production of the quorum sensing autoinducer-2 (AI-2) was significantly lower in the mutant strain than in the WT strain since the LuxS enzyme, encoded by the luxS gene, plays an essential role in AI-2 synthesis. However, the limited biofilm-forming ability in the WT strain indicated AI-2 was not directly related to S. Dublin biofilm formation. Furthermore, gene expression analysis of the WT and mutant strains revealed upregulation of genes related to biofilm stress response and enhanced resistance in the luxS mutant strain, which may provide evidence for the regulatory role of the luxS gene in biofilm formation.

Inhibition of methionine gamma lyase deaminase and the growth of Porphyromonas gingivalis: A therapeutic target for halitosis/periodontitis.

BACKGROUND AND OBJECTIVES: Pathogenic infections caused by Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia can result in the production of volatile sulfur compounds (VSC's) and other toxic compounds from methionine catabolism that can lead to halitosis and periodontitis. Our aim is to block the activity of methionine gammalyase-deaminase (Mgld) of methionine catabolism to prevent halitosis/periodontitis. DESIGNS: Cloned, expressed, Mgld protein was tested for purity by SDS-PAGE and western blotting. Mgld activity was tested by UV-vis spectroscopy and DTNB assay. Effects of Mgld inhibitor propargylglycine (PGLY) was tested on P. gingivalis growth by turbidity measurements. The effects of PGLY on oral epithelial and periodontal ligament cells in culture at different concentrations and time were tested for cell viability by MTT and Live-Dead assays. Amino acid comparisons of Mgld from different oral pathogens were done using standard bioinformatics program. RESULTS: Propargylglycine (PGLY) inhibited purified Mgld activity completely. In vivo, PGLY is a potent inhibitor on the growth of the P. gingivalis over 24 h, grown at 25 °C and 37 °C. Correspondingly in vivo Mgld activity was also affected by PGLY. Amino acid comparisons of oral pathogens showed 100% identity on the key residues of Mgld catalysis. Mammalian oral cell lines with PGLY, showed no difference in cell death over untreated controls assessed by MTT and Live-Dead assays. CONCLUSIONS: PGLY arrest's VSC's production by P. gingivalis. Since initial Mgld activity is inhibited subsequent enzymatic and nonenzymatic products formed will be prevented. PGLY showed no toxicity towards cultured mammalian oral cells. Thus, PGLY can serve as a mouthwash ingredient to prevent halitosis/periodontitis.

Cinnamon Oil Inhibits Shiga Toxin Type 2 Phage Induction and Shiga Toxin Type 2 Production in Escherichia coli O157:H7.

This study evaluated the inhibitory effect of cinnamon oil against Escherichia coli O157:H7 Shiga toxin (Stx) production and further explored the underlying mechanisms. The MIC and minimum bactericidal concentration (MBC) of cinnamon oil against E. coli O157:H7 were 0.025% and 0.05% (vol/vol), respectively. Cinnamon oil significantly reduced Stx2 production and the stx2 mRNA expression that is associated with diminished Vero cell cytotoxicity. Consistently, induction of the Stx-converting phage where the stx2 gene is located, along with the total number of phages, decreased proportionally to cinnamon oil concentration. In line with decreased Stx2 phage induction, cinnamon oil at 0.75× and 1.0× MIC eliminated RecA, a key mediator of SOS response, polynucleotide phosphorylase (PNPase), and poly(A) polymerase (PAP I), which positively regulate Stx-converting phages, contributing to reduced Stx-converting phage induction and Stx production. Furthermore, cinnamon oil at 0.75× and 1.0× MIC strongly inhibited the qseBC and luxS expression associated with decreased AI-2 production, a universal quorum sensing signaling molecule. However, the expression of oxidative stress response genes oxyR, soxR, and rpoS was increased in response to cinnamon oil at 0.25× or 0.5× MIC, which may contribute to stunted bacterial growth and reduced Stx2 phage induction and Stx2 production due to the inhibitory effect of OxyR on prophage activation. Collectively, cinnamon oil inhibits Stx2 production and Stx2 phage induction in E. coli O157:H7 in multiple ways. IMPORTANCE: This study reports the inhibitory effect of cinnamon oil on Shiga toxin 2 phage induction and Shiga toxin 2 production. Subinhibitory concentrations (concentrations below the MIC) of cinnamon oil reduced Stx2 production, stx2 mRNA expression, and cytotoxicity on Vero cells. Subinhibitory concentrations of cinnamon oil also dramatically reduced both the Stx2 phage and total phage induction in E. coli O157:H7, which may be due to the suppression of RNA polyadenylation enzyme PNPase at 0.25× to 1.0× MIC and the downregulation of bacterial SOS response key regulator RecA and RNA polyadenylation enzyme PAP I at 0.75× or 1.0× MIC. Cinnamon oil at higher levels (0.75× and 1.0× MIC) eliminated quorum sensing and oxidative stress. Therefore, cinnamon oil has potential applications as a therapeutic to control E. coli O157:H7 infection through inhibition of bacterial growth and virulence factors.

Disruption of heterotypic community development by Porphyromonas gingivalis with small molecule inhibitors.

Porphyromonas gingivalis is one of the main etiological organisms in periodontal disease. On oral surfaces P. gingivalis is a component of multispecies biofilm communities and can modify the pathogenic potential of the community as a whole. Accumulation of P. gingivalis in communities is facilitated by interspecies binding and communication with the antecedent colonizer Streptococcus gordonii. In this study we screened a library of small molecules to identify structures that could serve as lead compounds for the development of inhibitors of P. gingivalis community development. Three small molecules were identified that effectively inhibited accumulation of P. gingivalis on a substratum of S. gordonii. The structures of the small molecules are derived from the marine alkaloids oroidin and bromoageliferin and contain a 2-aminoimidazole or 2-aminobenzimidazole moiety. The most active compounds reduced expression of mfa1 and fimA in P. gingivalis, genes encoding the minor and major fimbrial subunits, respectively. These fimbrial adhesins are necessary for P. gingivalis co-adhesion with S. gordonii. These results demonstrate the potential for a small molecular inhibitor-based approach to the prevention of diseases associated with P. gingivalis.

PLP-dependent enzymes as potential drug targets for protozoan diseases.

The chemical properties of the B(6) vitamers are uniquely suited for wide use as cofactors in essential reactions, such as decarboxylations and transaminations. This review addresses current efforts to explore vitamin B(6) dependent enzymatic reactions as drug targets. Several current targets are described that are found amongst these enzymes. The focus is set on diseases caused by protozoan parasites. Comparison across a range of these organisms allows insight into the distribution of potential targets, many of which may be of interest in the development of broad range anti-protozoan drugs. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology.