Structure-Activity Relationship of Cinnamaldehyde Analogs as Inhibitors of AI-2 Based Quorum Sensing and Their Effect on Virulence of Vibrio spp

Background: Many bacteria, including Vibrio spp., regulate virulence gene expression in a cell-density dependent way through a communication process termed quorum sensing (QS). Hence, interfering with QS could be a valuable novel antipathogenic strategy. Cinnamaldehyde has previously been shown to inhibit QS-regulated virulence by decreasing the DNA-binding ability of the QS response regulator LuxR. However, little is known about the structure-activity relationship of cinnamaldehyde analogs. Methodology/Principal Findings: By evaluating the QS inhibitory activity of a series of cinnamaldehyde analogs, structural elements critical for autoinducer-2 QS inhibition were identified. These include an alpha, beta unsaturated acyl group capable of reacting as Michael acceptor connected to a hydrophobic moiety and a partially negative charge. The most active cinnamaldehyde analogs were found to affect the starvation response, biofilm formation, pigment production and protease production in Vibrio spp in vitro, while exhibiting low cytotoxicity. In addition, these compounds significantly increased the survival of the nematode Caenorhabditis elegans infected with Vibrio anguillarum, Vibrio harveyi and Vibrio vulnificus. Conclusions/Significance: Several new and more active cinnamaldehyde analogs were discovered and they were shown to affect Vibrio spp. virulence factor production in vitro and in vivo. Although ligands for LuxR have not been identified so far, the nature of different cinnamaldehyde analogs and their effect on the DNA binding ability of LuxR suggest that these compounds act as LuxR-ligands

Al-2 quorum-sensing inhibitors affect the starvation response and reduce virulence in several <i>Vibrio</i> species, most likely by interfering with LuxPQ

The increase of disease outbreaks caused by Vibrio species in aquatic organisms as well as in humans, together with the emergence of antibiotic resistance in Vibrio species, has led to a growing interest in alternative disease control measures. Quorum sensing (QS) is a mechanism for regulating microbial gene expression in a cell density-dependent way. While there is good evidence for the involvement of auto-inducer 2 (AI-2)-based interspecies QS in the control of virulence in multiple Vibrio species, only few inhibitors of this system are known. From the screening of a small panel of nucleoside analogues for their ability to disturb AI-2-based QS, an adenosine derivative with a p-methoxyphenylpropionamide moiety at C-3′ emerged as a promising hit. Its mechanism of inhibition was elucidated by measuring the effect on bioluminescence in a series of Vibrio harveyi AI-2 QS mutants. Our results indicate that this compound, as well as a truncated analogue lacking the adenine base, block AI-2-based QS without interfering with bacterial growth. The active compounds affected neither the bioluminescence system as such nor the production of AI-2, but most likely interfered with the signal transduction pathway at the level of LuxPQ in V. harveyi. The most active nucleoside analogue (designated LMC-21) was found to reduce the Vibrio species starvation response, to affect biofilm formation in Vibrio anguillarum, Vibrio vulnificus and Vibrio cholerae, to reduce pigment and protease production in V. anguillarum, and to protect gnotobiotic Artemia from V. harveyi-induced mortality

Copolymer-induced stabilizing effect of highly swollen hexagonal mesophases

We show quantitatively that tiny amounts of copolymer that decorate a oil/water interfaces can greatly enhance the stability of swollen surfactant hexagonal phases, comprising oil tubes regularly arranged in a water matrix. Such soft composite materials, whose both radius of the tubes and water channel between the tubes can be controlled independently over large ranges, offer a potential interest for the synthesis of mesoporous materials

Non-antibiotic quorum sensing inhibitors acting against N-acyl homoserine lactone synthase as druggable target

YesN-acylhomoserine lactone (AHL)-based quorum sensing (QS) is important for the regulation of proteobacterial virulence determinants. Thus, the inhibition of AHL synthases offers non-antibiotics-based therapeutic potentials against QS-mediated bacterial infections. In this work, functional AHL synthases of Pseudomonas aeruginosa LasI and RhlI were heterologously expressed in an AHL-negative Escherichia coli followed by assessments on their AHLs production using AHL biosensors and high resolution liquid chromatography–mass spectrometry (LCMS). These AHL-producing E. coli served as tools for screening AHL synthase inhibitors. Based on a campaign of screening synthetic molecules and natural products using our approach, three strongest inhibitors namely are salicylic acid, tannic acid and trans-cinnamaldehyde have been identified. LCMS analysis further confirmed tannic acid and trans-cinnemaldehyde efficiently inhibited AHL production by RhlI. We further demonstrated the application of trans-cinnemaldehyde inhibiting Rhl QS system regulated pyocyanin production in P. aeruginosa up to 42.06%. Molecular docking analysis suggested that trans-cinnemaldehyde binds to the LasI and EsaI with known structures mainly interacting with their substrate binding sites. Our data suggested a new class of QS-inhibiting agents from natural products targeting AHL synthase and provided a potential approach for facilitating the discovery of anti-QS signal synthesis as basis of novel anti-infective approach.University of Malaya High Impact Research (HIR) Grant (UM-MOHE HIR Grant UM.C/625/1/HIR/MOHE/CHAN/14/1, no. H-50001-A000027) given to K.G.C. and National Natural Science Foundation of China (no. 81260481) given to H.W

Phenolic plant extract enrichment of enzymatically mineralized hydrogels

Hydrogel mineralization with calcium phosphate (CaP) and antibacterial activity are desirable for applications in bone regeneration. Mineralization with CaP can be induced using the enzyme alkaline phosphatase (ALP), responsible for CaP formation in bone tissue. Incorporation of polyphenols, plant-derived bactericidal molecules, was hypothesized to provide antibacterial activity and enhance ALP-induced mineralization. Three phenolic rich plant extracts from: (i) green tea, rich in epigallocatechin gallate (EGCG) (herafter referred to as EGCG-rich extract); (ii) pine bark and (iii) rosemary were added to gellan gum (GG) hydrogels and subsequently mineralized using ALP. The phenolic composition of the three extracts used were analyzed by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MSn). EGCG-rich extract showed the highest content of phenolic compounds and promoted the highest CaP formation as corroborated by dry mass percentage meassurements and ICP-OES de-termination of mass of elemental Ca and P. All three extracts alone exhibited antibacterial activity in the following order EGCG-rich > PI > RO, respectively. However, extract-loaded and mineralized GG hydro-gels did not exhibit appreciable antibacterial activity by diffusion test. In conclusion, only the EGCG-rich extract promotes ALP-mediated mineralization

An in vitro collagen perfusion wound biofilm model; with applications for antimicrobial studies and microbial metabolomics

BackgroundThe majority of in vitro studies of medically relevant biofilms involve the development of biofilm on an inanimate solid surface. However, infection in vivo consists of biofilm growth on, or suspended within, the semi-solid matrix of the tissue, whereby current models do not effectively simulate the nature of the in vivo environment. This paper describes development of an in vitro method for culturing wound associated microorganisms in a system that combines a semi-solid collagen gel matrix with continuous flow of simulated wound fluid. This enables culture of wound associated reproducible steady state biofilms under conditions that more closely simulate the dynamic wound environment. To demonstrate the use of this model the antimicrobial kinetics of ceftazidime, against both mature and developing Pseudomonas aeruginosa biofilms, was assessed. In addition, we have shown the potential application of this model system for investigating microbial metabolomics by employing selected ion flow tube mass spectrometry (SIFT-MS) to monitor ammonia and hydrogen cyanide production by Pseudomonas aeruginosa biofilms in real-time. ResultsThe collagen wound biofilm model facilitates growth of steady-state reproducible Pseudomonas aeruginosa biofilms under wound like conditions. A maximum biofilm density of 1010 cfu slide-1 was achieved by 30 hours of continuous culture and maintained throughout the remainder of the experiment. Treatment with ceftazidime at a clinically relevant dose resulted in a 1.2 – 1.6 log reduction in biofilm density at 72 hours compared to untreated controls. Treatment resulted in loss of complex biofilm architecture and morphological changes to bacterial cells, visualised using confocal microscopy. When monitoring the biofilms using SIFT-MS, ammonia and hydrogen cyanide levels peaked at 12 hours at 2273 ppb (±826.4) and 138 ppb (±49.1) respectively and were detectable throughout experimentation. ConclusionsThe collagen wound biofilm model has been developed to facilitate growth of reproducible biofilms under wound-like conditions. We have successfully used this method to: (1) evaluate antimicrobial efficacy and kinetics, clearly demonstrating the development of antimicrobial tolerance in biofilm cultures; (2) characterise volatile metabolite production by P. aeruginosa biofilms, demonstrating the potential use of this method in metabolomics studies

Inhibition of Biofilm Formation, Quorum Sensing and Infection in Pseudomonas aeruginosa by Natural Products-Inspired Organosulfur Compounds

Using a microplate-based screening assay, the effects on Pseudomonas aeruginosa PAO1 biofilm formation of several S-substituted cysteine sulfoxides and their corresponding disulfide derivatives were evaluated. From our library of compounds, S-phenyl-L-cysteine sulfoxide and its breakdown product, diphenyl disulfide, significantly reduced the amount of biofilm formation by P. aeruginosa at levels equivalent to the active concentration of 4-nitropyridine-N-oxide (NPO) (1 mM). Unlike NPO, which is an established inhibitor of bacterial biofilms, our active compounds did not reduce planktonic cell growth and only affected biofilm formation. When used in a Drosophila-based infection model, both S-phenyl-L-cysteine sulfoxide and diphenyl disulfide significantly reduced the P. aeruginosa recovered 18 h post infection (relative to the control), and were non-lethal to the fly hosts. The possibility that the observed biofilm inhibitory effects were related to quorum sensing inhibition (QSI) was investigated using Escherichia coli-based reporters expressing P. aeruginosa lasR or rhIR response proteins, as well as an endogenous P. aeruginosa reporter from the lasI/lasR QS system. Inhibition of quorum sensing by S-phenyl-L-cysteine sulfoxide was observed in all of the reporter systems tested, whereas diphenyl disulfide did not exhibit QSI in either of the E. coli reporters, and showed very limited inhibition in the P. aeruginosa reporter. Since both compounds inhibit biofilm formation but do not show similar QSI activity, it is concluded that they may be functioning by different pathways. The hypothesis that biofilm inhibition by the two active compounds discovered in this work occurs through QSI is discussed