Anti-pathogenic Potential of Coral Associated Bacteria Isolated from Gulf of Mannar Against Pseudomonas aeruginosa

Infections of Pseudomonas aeruginosa are of great concern because of its increasing resistance towards conventional antibiotics. Quorum sensing system of P. aeruginosa acts as a global regulator of almost all the virulence factors and majorly its biofilm formation. In the present study, quenching of QS system of P. aeruginosa has been explained with bioactives from bacteria associated with the coral Acropora digitifera. Isolated bioactives inhibited the expression of various virulence traits of P. aeruginosa like biofilm formation, and the production of extracellular enzymes like protease and elastase. This study also emphasises the potential of coral associated bacteria in producing bioactive agents with anti-pathogenic properties.     

Modelling antibiotic- and anti-quorum sensing treatment of a spatially-structured Pseudomonas aeruginosa population

The bacterial cell to cell signalling system known as quorum sensing (QS) is essential for the regulation of virulence in many pathogens and offers a specific biochemical target for novel antibacterial therapies. Expanding on earlier work, in which consideration was given to the primary QS system (lasR system) in a homogeneous population of the common human pathogen Pseudomonas aeruginosa, we build a simple spatial model of an early-stage P. aeruginosa biofilm subject to treatment with topically applied anti-QS drugs (of two specific kinds) and conventional antibiotics. In the case of a slowly growing biofilm we show that both kinds of anti-quorum sensing drug are effective in reducing the level of the relevant signal molecule (3-oxo-C12-homoserine lactone; henceforth AHL), in each case obtaining an explicit bound on the steady-state AHL profile in terms of a prescribed surface drug concentration. Using numerical methods, we are also able to reproduce the hysteretic phenomena exhibited by the homogeneous model, in particular showing that for each kind of anti-QS drug there is a parameter regime in which a catastrophic collapse occurs in the steady-state AHL concentration as the surface drug concentration passes some critical value; an alternative way of interpreting this result is to say that, for a prescribed surface drug concentration, there is a critical biofilm depth such that treatment is successful until this depth is reached, but fails thereafter. In the thick-biofilm limit we show that the critical concentration of each drug increases exponentially with the biofilm thickness (or, conversely, that the critical depth increases logarithmically with surface drug concentration); this is dramatically different to the behaviour observed in the corresponding homogeneous model, where the critical concentrations grow linearly with bacterial carrying capacity, and thus highlights the relative difficulty of treating a large, spatially-structured population with diffusing antibacterials.     

Pentachlorophenol degradation by Pseudomonas aeruginosa

Five Pseudomonas species were tested for ability to degrade pentachlorophenol (PCP). Pseudomonas aeruginosa completely degraded PCP up to 800 mg/l in 6 days with glucose as co-substrate. With 1000 mg PCP/l, 53% was degraded. NH₄ ⁺ salts were better at enhancing degradation than organic nitrogen sources and shake-cultures promoted PCP degradation compared with surface cultures. Degradation was maximal at pH 7.6 to 8.0 and at 30 to 37°C. Only PCP induced enzymes that degraded PCP and chloramphenicol inhibited this process. The PCP was degraded to CO₂, with release of Cl^-.The authors are with the Bacteriology Laboratory, Central Leather Research Institute, Madras-600 020, India.     

The Pseudomonas aeruginosa quorum-sensing molecule N-(3-oxododecanoyl)-L-homoserine lactone stimulates phagocytic activity in human macrophages through the p38 MAPK pathway

Quorum-sensing is an important mechanism for the regulation of bacteria-to-bacteria communication. Recent advances have demonstrated that the Pseudomonas aeruginosa signaling molecule N-(3-oxododecanoyl)-L-homoserine lactone (3O-C(12)-HSL) is also a potent modulator of eukaryotic cells and may thus play an important role in the host response during P. aeruginosa infections. Little is known, however, about specific effects of 3O-C(12)-HSL molecules on human macrophages. To address this issue, we investigated the influence of 3O-C(12)-HSL on the phagocytic activity, production of reactive oxygen species, and activation of p38 and p42/44 MAPK signaling pathways in human macrophages. We show an effect of 3O-C(12)-HSL on the phagocytic capacity in human macrophages, which depends on concentration and time of exposure. When cells were exposed to 100 microM 3O-C(12)-HSL for 30 min or 1 h, the phagocytic activity increased 1.8 and 1.6 times, respectively. The 3O-C(12)-HSL treatments had no significant effect on the level of reactive oxygen species production. Furthermore, the p38 MAPK, but not the p42/44 MAPK, signaling pathway was activated in response to 3O-C(12)-HSL. In addition, specific blocking of p38 MAPK activation with 10 microM SB 203580 prevented the 3O-C(12)-HSL-induced increase in the phagocytic activity. These findings demonstrate that the bacterial quorum-sensing can play a significant role also in regulation of macrophage activity during infections caused by P. aeruginosa.     

A multi-phase mathematical model of quorum sensing in a maturing Pseudomonas aeruginosa biofilm

It is well known that sessile bacteria have a strong tendency to exist in a biofilm phenotype, whereby bacterial cells aggregate and produce a gel-like extracellular matrix, which, in an infection scenario, offers a significant barrier to attack by conventional antibiotics and the immune system. In this paper we develop a multi-phase model of a maturing Pseudomonas aeruginosa biofilm, allowing for the production and secretion of exopolysaccharide (EPS). The primary quorum-sensing system of P. aeruginosa (namely the lasR system) is believed to be required for full biofilm development, and we thus take the synthesis of EPS to be regulated by the cognate signal molecule, 3-oxo-C12-HSL. We also take EPS and signal production, along with bacterial growth, to be limited by oxygen availability, thus factoring in the nutrient poor conditions deep inside the biofilm. We use simulations to examine the role played by quorum sensing in the biofilm maturation process, and to investigate the effect of anti-quorum sensing and antibiotic treatments on EPS concentration, signal level, bacterial numbers and biofilm growth rate. In addition, we undertake analysis of the associated travelling-wave behaviour.     

Pseudomonas aeruginosa quorum sensing molecule N-(3 oxododecanoyl)-l-homoserine lactone disrupts epithelial barrier integrity of Caco-2 cells

Acyl-homoserine lactone (HSL) quorum sensing molecules play an important role in regulation of virulence gene expression in Pseudomonas aeruginosa. Here, we show that 3O-C(12)-HSL can disrupt barrier integrity in human epithelial Caco-2 cells as evidenced by decreased transepithelial electrical resistance (TER), increased paracellular flux, reduction in the expression and distribution of ZO-1 and occludin, and reorganization of F-actin. P. aeruginosa 3O-C(12)-HSL activate p38 and p42/44 kinases, and inhibition of these kinases partly prevented 3O-C(12)-HSL-induced changes in TER, paracellular flux and expression of occludin and ZO-1. These findings demonstrate that P. aeruginosa 3O-C(12)-HSL can modulate tight junction integrity of Caco-2 cells.     

Alginate acetylation influences initial surface colonization by mucoid Pseudomonas aeruginosa

Mucoid strains of Pseudomonas aeruginosa overproduce the exopolysaccharide alginate, which is substituted with O-acetyl groups. Under non-growing conditions in phosphate buffer, a mucoid clinical strain formed microcolonies on steel surfaces, while an acetylation-defective mutant was unable to form cell clusters. Enzymatic degradation of alginate by alginate lyase prevented microcolony formation of the mucoid parent strain. In a continuous-culture flow-cell system, using gluconate minimal medium, the mucoid strain with acetylated alginate formed microcolonies and grew into heterogenous biofilms, whereas the acetylation-defective mutant produced a thinner and more homogeneous biofilm. A lowered viscosity of extracellular material from the acetylation-defective mutant indicated a weakening of exopolymer interactions by loss of acetyl groups. These results suggest that acetyl substituents are necessary for the function of high-molecular-mass alginate to mediate cell aggregation into microcolonies in the early stages of biofilm development by mucoid P. aeruginosa, thereby determining the architecture of the mature biofilm.     

Alginate biosynthesis by Pseudomonas aeruginosa: effect of arsenite and other metabolic inhibitors

Biosynthesis of alginic acid in presence of metabolic inhibitors by resting cells of mucoid Pseudomonas aeruginosa was studied. Among the inhibitors tested, arsenite exhibited very interesting results, while the others showed no remarkable-effect. Firstly, arsenite stopped alginate production from all the substrates during initial hours of incubation; secondly, degradation of newly synthesized alginates to smaller molecular weight fragments took place if it was added after a few hours of incubation with the substrate; and thirdly, uncontrolled synthesis of alginate started after several hours of inhibition. Presence of arsenite was needed for the initial inhibitory phase of alginate synthesis; but once the cells were capable of synthesizing alginate after initial hours of inhibition, arsenite may be omitted from the medium.     

N2-Succinylornithine in ornithine catabolism of Pseudomonas aeruginosa

Most Pseudomonas aeruginosa PAO mutants which were unable to utilize l-arginine as the sole carbon and nitrogen source (aru mutants) under aerobic conditions were also affected in l-ornithine utilization. These aru mutants were impaired in one or several enzymes involved in the conversion of N²-succinylornithine to glutamate and succinate, indicating that the latter steps of the arginine succinyltransferase pathway can be used for ornithine catabolism. Addition of aminooxyacetate, an inhibitor of the N²-succinylornithine 5-aminotransferase, to resting cells of P. aeruginosa in ornithine medium led to the accumulation of N²-succinylornithine. In crude extracts of P. aeruginosa an ornithine succinyltransferase (l-ornithine:succinyl-CoA N²-succinyltransferase) activity could be detected. An aru mutant having reduced arginine succinyltransferase activity also had correspondingly low levels of ornithine succinyltransferase. Thus, in P. aeruginosa, these two activities might be due to the same enzyme, which initiates aerobic arginine and ornithine catabolism.Abbreviations OAT ornithine 5-aminotransferase - SOAT N²-succinylornithine 5-aminotransferase - Oru ornithine utilization - Aru arginine utilization     

Biodegradation of 8-anilino-1-naphthalenesulfonic acid by Pseudomonas aeruginosa

Pseudomonas aeruginosa, isolated from soil near tannery effluent was able to degrade 8-anilino-1-naphthalenesulfonic acid (ANSA), a sulfonated aromatic amine. The organism degraded this amine up to a concentration of 1,200 mg l⁻¹ using glucose and ammonium nitrate as carbon and nitrogen sources respectively. The degradation started when the organism reached its late exponential growth phase. Salicylic acid and β-ketoadipic acid were identified as intermediate compounds using HPLC and GC–MS and provide evidence for ortho pathway reactions. Further proof for the pathway is obtained from the dioxygenase activity of the strain growing exponentially in medium with ANSA and glucose.     

Bioelectricity enhancement via overexpression of quorum sensing system in Pseudomonas aeruginosa-inoculated microbial fuel cells

Low electron transfer efficiency from bacteria to electrodes remains one of the major bottlenecks that limit industrial applications of microbial fuel cells (MFCs). Elucidating biological mechanism of the electron transfer processes is of great help in improving the efficiency of MFCs. Here, we reported that Pseudomonas aeruginosa could use different electron shuttles in a MFC under different quorum sensing (QS) expression patterns. An electron shuttle (rather than phenazines) with a high mid-point potential of 0.20 V (vs. Ag/AgCl–KCl saturated electrode) was found to be the dominating shuttle in a wild-type P. aeruginosa strain. Strikingly, upon genetic overexpression of rhl QS system in this wild-type strain, the electron shuttle was substituted by phenazines (pyocyanin and phenazine-1-carboxylate, with a low mid-point potential of −0.17 V and −0.28 V, respectively), which directly resulted in an increase of about 1.6 times of the maximum current of the rhl overexpressed strain over the wild-type strain. Our result implied that manipulating electron transfer pathways to improve MFCs’ efficiency could be achieved by rewiring gene regulatory circuits, thus synthetic biology strategies would be adopted.     

Isolation and characterization of mutant Pseudomonas aeruginosa strains unable to assimilate nitrate

Single-site mutants of Pseudomonas aeruginosa that lack the ability aerobically to assimilate nitrate and nitrite as sole sources of nitrogen have been isolated. Twentyone of these have been subdivided into four groups by transductional analysis. Mutants in only one group, designated nis, lost assimilatory nitrite reductase activity. Mutants in the other three transductional groups, designated ntmA, ntmB, ntmC, display a pleiotropic phenotype: utilization of a number of nitrogen-containing compounds including nitrite as sole nitrogen sources is impaired. Assimilatory nitrite reductase was shown to be the major route by which hydroxylamine is reduced in aerobically-grown cells.In memoriam of Professor R. Y. Stanier     

Structural basis of calcium and galactose recognition by the lectin PA-IL of Pseudomonas aeruginosa

The structure of the tetrameric Pseudomonas aeruginosa lectin I (PA-IL) in complex with galactose and calcium was determined at 1.6 A resolution, and the native protein was solved at 2.4 A resolution. Each monomer adopts a beta-sandwich fold with ligand binding site at the apex. All galactose hydroxyl groups, except O1, are involved in a hydrogen bond network with the protein and O3 and O4 also participate in the co-ordination of the calcium ion. The stereochemistry of calcium galactose binding is reminiscent of that observed in some animal C-type lectins. The structure of the complex provides a framework for future design of anti-bacterial compounds.     

Recognition of mucin components by Pseudomonas aeruginosa

Pseudomonas aeruginosa remains one of the most important bacterial pathogens in lung diseases and especially in Cystic fibrosis. This unusual predilection is best explained by the existence of defects in host defense mechanisms as resulting from the genetic lesion and the presence of a specific colonization niche within the lungs. The niche has been identified as the mucus layer wherein mucin glycoproteins provide a substrate for binding and allows the persistence of this organism in this milieu by a number of possible mechanisms. While this organism is capable of binding to non CF mucins, it is perhaps a combination of factors e.g. increased binding and decreased mucociliary clearance that is responsible for this marked state of colonization in CF. The organism uses chiefly proteins of its flagellar apparatus to initiate this binding and recognizes a variety of oligosaccharides that have been identified in mucins. Among these are both, neutral oligosaccharides and several forms of acidic oligosaccharides derived from the Lewis antigens. There are more than likely a larger repertoire of receptors than those identified and certainly more adhesins present than those currently known. However, the information gathered to date provides an excellent example of the specificity of bacterial interactions with mucins that will certainly be expanded as we study more pulmonary pathogens.     

Heterologous expression of human paraoxonases in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> inhibits biofilm formation and decreases antibiotic resistance

Quorum sensing (QS) regulates virulence and biofilm formation in Pseudomonas aeruginosa and other medically relevant bacteria. Human paraoxonases (hPONs) are a family of closely related enzymes with multiple functions, including inactivation of the QS signal molecule in P. aeruginosa. However, there is no direct evidence to show the functions of hPONs on biofilm formation and antibiotic resistance in P. aeruginosa. In the present study, hPONs (hPON1, hPON2, and hPON3) genes were respectively cloned into the pMEKm12 shuttle vector and transformed into P. aeruginosa strain PAO1. Expression of the three recombinant proteins was confirmed by Western blotting, and growth of the recombinant strains was not affected by the hPONs gene expression. Biofilm formation and antibiotics resistance of the hPONs recombinant strains were analyzed. Our results showed that biofilm formation was significantly inhibited in all of the three hPONs recombinant strains. Interestingly, this inhibition can be reverted by addition of the corresponding hPONs polyclonal antibodies in the culture media, further indicating that the inhibition of biofilm formation was due to hPONs protein expression. In addition, we also demonstrated that hPONs expression decreased resistance of P. aeruginosa to gentamicin and ceftazidima, two antibiotics clinically used for the treatment of P. aeruginosa infection.     

The stereoisomers of pyochelin, a siderophore of Pseudomonas aeruginosa

The Pseudomonas aeruginosa siderophore pyochelin is obtained from the bacterial culture medium as a mixture of two epimers. Chromatically isolated pure stereoisomers equilibrate readily in most solvents. Experiments will be reported which allow to isolate one of the isomers in pure form and which shed some additional light on the epimerization reaction.     

Functional characterization of genes involved in alkane oxidation by Pseudomonas aeruginosa

Most clinical isolates identified as Pseudomonas aeruginosa grow on long-chain n-alkanes, while environmental P. aeruginosa isolates often grow on medium- as well as long-chain n-alkanes. Heterologous expression showed that the two alkane hydroxylase homologs of P. aeruginosa PAO1 (AlkB1 and AlkB2) oxidize C₁₂-C₁₆ n-alkanes, while two rubredoxin (RubA1 and RubA2) and a rubredoxin reductase (RubB) homologs can replace their P. putida GPo1 counterparts in n-octane oxidation. The two long-chain alkane hydroxylase genes are present in all environmental and clinical isolates of P. aeruginosa strains tested in this study. This revised version was published online in August 2006 with corrections to the Cover Date.