Pseudomonas aeruginosa utilizes the quorum sensing (QS) system to strategically coordinate virulence and biofilm formation. Targeting QS pathways may be a potential anti-infective approach to treat P. aeruginosa infections. In the present study, we define cephalosporins’ anti-QS activity using Chromobacterium violaceum CV026 for screening and QS-regulated mutants of P. aeruginosa for validation. We quantified the effects of three cephalosporins, cefepime, ceftazidime, and ceftriaxone, on (1) pyocyanin production using spectrophotometric assay, (2) bacterial motility using agar plate assay, and (3) biofilm formation using scanning electron microscopy. We also studied isogenic QS mutant strains of PAO1 (ΔlasR,ΔrhlR,ΔpqsA, and ΔpqsR) to compare and distinguish QS-mediated effects on the motility phenotypes and bacterial growth with and without sub-MIC concentrations of antibiotics. Results showed that cephalosporins have anti-QS activity and reduce bacterial motility, pyocyanin production, and biofilm formation for CV026 and PAO1. Also, sub-MICs of cefepime increased aminoglycosides’ antimicrobial activity against P. aeruginosa PAO1, suggesting the advantage of combined anti-QS and antibacterial treatment. To correlate experimentally observed anti-QS effects with the interactions between cephalosporins and QS receptors, we performed molecular docking with ligand binding sites of quorum sensing receptors using Autodock Vina. Molecular docking predicted cephalosporins’ binding affinities to the ligand-binding pocket of QS receptors (CviR, LasR, and PqsR). To validate our results using an infection model, we quantified the survival rate of Caenorhabditis elegans following P. aeruginosa PAO1 challenge at concentrations less than the minimum inhibitory concentration (MIC) of antibiotics. C. elegans infected with PAO1 without antibiotics showed 0% survivability after 72 h. In contrast, PAO1-infected C. elegans showed 65 ± 5%, 58 ± 4%, and 49 ± 8% survivability after treatment with cefepime, ceftazidime, and ceftriaxone, respectively. We determined the survival rates of C. elegans infected by QS mutant strains ΔlasR (32 ± 11%), ΔrhlR (27 ± 8%), ΔpqsA (27 ± 10%), and ΔpqsR (37 ± 6%), which suggest essential role of QS system in virulence. In summary, cephalosporins at sub-MIC concentrations show anti-QS activity and enhance the antibacterial efficacy of aminoglycosides, a different class of antibiotics. Thus, cephalosporins at sub-MIC concentrations in combination with other antibiotics are potential candidates for developing therapies to combat infections caused by P. aeruginosa.
Pseudomonas aeruginosa utilizes a chemical social networking system referred to 15 as quorum sensing (QS) to strategically co-ordinate the expression of virulence factors and biofilm 16 formation. Virulence attributes damage the host cells, impair the host immune system, and protect 17 bacterial cells from antibiotic attack. Thus, anti-QS agents may act as novel anti-infective 18 therapeutics to treat P. aeruginosa infections. The present study was performed to evaluate the 19 anti-QS, anti-biofilm, and anti-virulence activity of β-lactam antibiotics (carbapenems and 20 cephalosporins) against P. aeruginosa. The anti-QS activity was quantified using 21 Chromobacterium violaceum CV026 as a QS reporter strain. Our results showed that 22 cephalosporins including cefepime (CP), ceftazidime (CF), and ceftriaxone (CT) exhibited potent 23 anti-QS and anti-virulence activities against P. aeruginosa PAO1. These antibiotics significantly 24 impaired motility phenotypes, decreased pyocyanin production, and reduced the biofilm formation 25 by P. aeruginosa PAO1. In the present study, we studied isogenic QS mutants of PAO1: ∆LasR, 26 ∆RhlR, ∆PqsA, and ∆PqsR and found that the levels of virulence factors of antibiotic-treated 27 PAO1 were comparable to QS mutant strains. Molecular docking predicted high binding affinities 28 of cephalosporins for the ligand-binding pocket of QS receptors (CviR, LasR, and PqsR). In 29 addition, our results showed that the anti-microbial activity of aminoglycosides increased in the 30 presence of sub-inhibitory concentrations (sub-MICs) of CP against P. aeruginosa PAO1. Further, 31 utilizing Caenorhabditis elegans as an animal model for the in vivo anti-virulence effects of 32 antibiotics, cephalosporins showed a significant increase in C. elegans survival by suppressing 33 virulence factor production in P. aeruginosa. Thus, our results indicate that cephalosporins might 34 provide a viable anti-virulence therapy in the treatment of infections caused by multi-drug resistant 35 P. aeruginosa. 36 37 38 docking; Caenorhabditis elegans. 39 40 SHORT TITLE: Cephalosporins suppress virulence of Pseudomonas aeruginosa 41 42 43 44 45 46 47Pseudomonas aeruginosa causes nosocomial infections and multi-drug-resistant (MDR) P. 51 aeruginosa is increasingly problematic. Bacterial communication is critical for infection, 52 virulence, survival, antibiotic resistance, and biofilm formation in P. aeruginosa [1][2][3][4][5]. Cells 53 communicate by expressing specific chemical signal molecules that lead to coordinate their 54 population-wide behavior, a process known as quorum sensing (QS) [4]. QS involves the 55 expression of acyl-homoserine lactone (AHL) molecules and detecting these signal molecules 56 using specific protein receptors [6]. In the P. aeruginosa QS system, homo-serine lactone 57 autoinducer molecules (HSL) produced by LuxI-type enzymes bind to cognate transcriptional 58 regulator LuxR-type protein receptors enabling receptor dimerization and binding to DNA 59 promoter ...
With the ease of gene sequencing and the technology available to study and manipulate non-model organisms, the need to translate our understanding of model organisms to non-model organisms has become an urgent problem. For example, mining of large coral and their symbiont sequence data is a challenge, but also provides an opportunity for understanding functionality and evolution of these and other non-model organisms. Much more information than for any other eukaryotic species is available for humans, especially related to signal transduction and diseases. However, the coral cnidarian host and human have diverged over 700 million years ago and homologies between proteins are therefore often in the gray zone or undetectable with traditional BLAST searches. We introduce a two-stage approach to identifying putative coral homologues of human proteins. First, through remote homology detection using Hidden Markov Models, we identify candidate human homologues in the cnidarian genome. However, for many proteins, the human genome alone contains multiple family members with similar or even more divergence in sequence. In the second stage, therefore, we filter the remote homology results based on the functional and structural plausibility of each coral candidate, shortlisting the coral proteins likely to be true human homologues. We demonstrate our approach with a pipeline for mapping membrane receptors in humans to membrane receptors in corals, with specific focus on the stony coral, P. damicornis. More than 1000 human membrane receptors mapped to 335 coral receptors, including 151 G protein coupled receptors (GPCRs). To validate specific sub-families, we chose opsin proteins, representative GPCRs that confer light sensitivity, and Toll-like receptors, representative non-GPCRs, which function in the immune response, and their ability to communicate with microorganisms. Through detailed structure-function analysis of their ligand-binding pockets and downstream signaling cascades, we selected those candidate remote homologues likely to carry out related functions in the corals. This pipeline may prove generally useful for other non-model organisms, such as to support the growing field of synthetic biology.
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