Pseudomonas aeruginosa Psl is a galactose- and mannose-rich exopolysaccharide

The Pseudomonas aeruginosa polysaccharide synthesis locus (psl) is predicted to encode an exopolysaccharide which is critical for biofilm formation. Here we used chemical composition analyses and mannose- or galactose-specific lectin staining, followed by confocal laser scanning microscopy and electron microscopy, to show that Psl is a galactose-rich and mannose-rich exopolysaccharide.     

PslD is a secreted protein required for biofilm formation by Pseudomonas aeruginosa

The function of pslD, which is part of the psl operon from Pseudomonas aeruginosa, was investigated in this study. The psl operon is involved in exopolysaccharide biosynthesis and biofilm formation. An isogenic marker-free pslD deletion mutant of P. aeruginosa PAO1 which was deficient in the formation of differentiated biofilms was generated. Expression of only the pslD gene coding region restored the wild-type phenotype. A C-terminal, hexahistidine tag fusion enabled the identification of PslD. LacZ and PhoA translational fusions with PslD indicated that PslD is a secreted protein required for biofilm formation, presumably via its role in exopolysaccharide export.     

Recent perspectives on the molecular basis of biofilm formation by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> and approaches for treatment and biofilm dispersal

Pseudomonas aeruginosa, a Gram-negative, rod-shaped bacterium causes widespread diseases in humans. This bacterium is frequently related to nosocomial infections such as pneumonia, urinary tract infections (UTIs) and bacteriaemia especially in immunocompromised patients. The current review focuses on the recent perspectives on biofilms formation by these bacteria. Biofilms are communities of microorganisms in which cells stick to each other and often adhere to a surface. These adherent cells are usually embedded within a self-produced matrix of extracellular polymeric substance (EPS). Pel, psl and alg operons present in P. aeruginosa are responsible for the biosynthesis of extracellular polysaccharide which plays an important role in cell surface interactions during biofilm formation. Recent studies suggested that cAMP signalling pathway, quorum-sensing pathway, Gac/Rsm pathway and c-di-GMP signalling pathway are the main mechanism that leads to the biofilm formation. Understanding the bacterial virulence depends on a number of cell-associated and extracellular factors and is very essential for the development of potential drug targets. Thus, the review focuses on the major genes involved in the biofilm formation, the state of art update on the biofilm treatment and the dispersal approaches such as targeting adhesion and maturation, targeting virulence factors and other strategies such as small molecule-based inhibitors, phytochemicals, bacteriophage therapy, photodynamic therapy, antimicrobial peptides and natural therapies and vaccines to curtail the biofilm formation by P. aeruginosa.     

SadC reciprocally influences biofilm formation and swarming motility via modulation of exopolysaccharide production and flagellar function

Pseudomonas aeruginosa has served as an important organism in the study of biofilm formation; however, we still lack an understanding of the mechanisms by which this microbe transitions to a surface lifestyle. A recent study of the early stages of biofilm formation implicated the control of flagellar reversals and production of an exopolysaccharide (EPS) as factors in the establishment of a stable association with the substratum and swarming motility. Here we present evidence that SadC (PA4332), an inner membrane-localized diguanylate cyclase, plays a role in controlling these cellular functions. Deletion of the sadC gene results in a strain that is defective in biofilm formation and a hyperswarmer, while multicopy expression of this gene promotes sessility. A ΔsadC mutant was additionally found to be deficient in EPS production and display altered reversal behavior while swimming in high-viscosity medium, two behaviors proposed to influence biofilm formation and swarming motility. Epistasis analysis suggests that the sadC gene is part of a genetic pathway that allows for the concomitant regulation of these aspects of P. aeruginosa surface behavior. We propose that SadC and the phosphodiesterase BifA (S. L. Kuchma et al., J. Bacteriol. 189:8165-8178, 2007), via modulating levels of the signaling molecule cyclic-di-GMP, coregulate swarming motility and biofilm formation as P. aeruginosa transitions from a planktonic to a surface-associated lifestyle.     

Influence of clove oil on certain quorum-sensing-regulated functions and biofilm of Pseudomonas aeruginosa and Aeromonas hydrophila

Quorum sensing (QS) plays an important role in virulence, biofilm formation and survival of many pathogenic bacteria including Pseudomonas aeruginosa. This signalling pathway is considered as novel and promising target for anti-infective agents. In the present investigation, effect of the Sub-MICs of clove oil on QS regulated virulence factors and biofilm formation was evaluated against P. aeruginosa PAO1 and Aeromonas hydrophila WAF-38 strain. Sub-inhibitory concentrations of the clove oil demonstrated statistically significant reduction of las- and rhl-regulated virulence factors such as LasB, total protease, chitinase and pyocyanin production, swimming motility and exopolysaccharide production. The biofilm forming capability of PAO1 and A. hydrophila WAF-38 was also reduced in a concentration-dependent manner at all tested sub-MIC values. Further, the PAO1-preinfected Caenorhabditis elegans displayed an enhanced survival when treated with 1.6% v/v of clove oil. The above findings highlight the promising anti-QS-dependent therapeutic function of clove oil against P. aeruginosa.     

Investigating the Link Between Imipenem Resistance and Biofilm Formation by Pseudomonas aeruginosa

Pseudomonas aeruginosa, a ubiquitous environmental organism, is a difficult-to-treat opportunistic pathogen due to its broad-spectrum antibiotic resistance and its ability to form biofilms. In this study, we investigate the link between resistance to a clinically important antibiotic, imipenem, and biofilm formation. First, we observed that the laboratory strain P. aeruginosa PAO1 carrying a mutation in the oprD gene, which confers resistance to imipenem, showed a modest reduction in biofilm formation. We also observed an inverse relationship between imipenem resistance and biofilm formation for imipenem-resistant strains selected in vitro, as well as for clinical isolates. We identified two clinical isolates of P. aeruginosa from the sputum of cystic fibrosis patients that formed robust biofilms, but were sensitive to imipenem (MIC?≤?2 μg/ml). To test the hypothesis that there is a general link between imipenem resistance and biofilm formation, we performed transposon mutagenesis of these two clinical strains to identify mutants defective in biofilm formation, and then tested these mutants for imipenem resistance. Analysis of the transposon mutants revealed a role for previously described biofilm factors in these clinical isolates of P. aeruginosa, including mutations in the pilY1, pilX, pilW, algC, and pslI genes, but none of the biofilm-deficient mutants became imipenem resistant (MIC?≥?8 μg/ml), arguing against a general link between biofilm formation and resistance to imipenem. Thus, assessing biofilm formation capabilities of environmental isolates is unlikely to serve as a good predictor of imipenem resistance. We also discuss our findings in light of the limited literature addressing planktonic antibiotic resistance factors that impact biofilm formation.     

Characterization of Regulatory Pathways in Xylella fastidiosa: Genes and Phenotypes Controlled by algU

Many virulence genes in plant bacterial pathogens are coordinately regulated by “global” regulatory genes. Conducting DNA microarray analysis of bacterial mutants of such genes, compared with the wild type, can help to refine the list of genes that may contribute to virulence in bacterial pathogens. The regulatory gene algU, with roles in stress response and regulation of the biosynthesis of the exopolysaccharide alginate in Pseudomonas aeruginosa and many other bacteria, has been extensively studied. The role of algU in Xylella fastidiosa, the cause of Pierce's disease of grapevines, was analyzed by mutation and whole-genome microarray analysis to define its involvement in aggregation, biofilm formation, and virulence. In this study, an algU::nptII mutant had reduced cell-cell aggregation, attachment, and biofilm formation and lower virulence in grapevines. Microarray analysis showed that 42 genes had significantly lower expression in the algU::nptII mutant than in the wild type. Among these are several genes that could contribute to cell aggregation and biofilm formation, as well as other physiological processes such as virulence, competition, and survival.     

A Systems-Level Approach for Investigating Pseudomonas aeruginosa Biofilm Formation

Prevention of the initiation of biofilm formation is the most important step for combating biofilm-associated pathogens, as the ability of pathogens to resist antibiotics is enhanced 10 to 1000 times once biofilms are formed. Genes essential to bacterial growth in the planktonic state are potential targets to treat biofilm-associated pathogens. However, the biofilm formation capability of strains with mutations in these essential genes must be evaluated, since the pathogen might form a biofilm before it is eliminated. In order to address this issue, this work proposes a systems-level approach to quantifying the biofilm formation capability of mutants to determine target genes that are essential for bacterial metabolism in the planktonic state but do not induce biofilm formation in their mutants. The changes of fluxes through the reactions associated with the genes positively related to biofilm formation are used as soft sensors in the flux balance analysis to quantify the trend of biofilm formation upon the mutation of an essential gene. The essential genes whose mutants are predicted not to induce biofilm formation are regarded as gene targets. The proposed approach was applied to identify target genes to treat Pseudomonas aeruginosa infections. It is interesting to find that most essential gene mutants exhibit high potential to induce the biofilm formation while most non-essential gene mutants do not. Critically, we identified four essential genes, lysC, cysH, adk, and galU, that constitute gene targets to treat P. aeruginosa. They have been suggested by existing experimental data as potential drug targets for their crucial role in the survival or virulence of P. aeruginosa. It is also interesting to find that P. aeruginosa tends to survive the essential-gene mutation treatment by mainly enhancing fluxes through 8 metabolic reactions that regulate acetate metabolism, arginine metabolism, and glutamate metabolism.     

Effect of long-term starvation in salty microcosm on biofilm formation and motility in Pseudomonas aeruginosa

The development of antibiotic resistance in the opportunistic pathogen Pseudomonas aeruginosa is a major cause of the pathogen’s morbidity and is strongly correlated with the biofilm formation. Motility and adherence capacity in long-term stressed cells have not been extensively analyzed even though P. aeruginosa considered a model organism for the study of biofilm formation. In this investigation, P. aeruginosa ATCC 27853 strain has been stored for 12 months in LB broth with 0.5 M NaCl. Several experiments demonstrated that the strain recovery from the salty microcosm had the ability to increase the biofilm formation and to reduce motility comparing with that of the original strain. To identify genes involved in the regulation of biofilm and/or in stress response by the recovered P. aeruginosa, differential display “DDRT-PCR” technique was used. The genes speD and ccoN2, coding, respectively, for an S-adenosylmethionine decarboxylase and Cbb3-type cytochrome oxidase, were identified in recovered strain of P. aeruginosa ATCC 27853 as two differentially expressed gene fragments. A comparison of the biofilm produced by the wild-type strain PA14 and the transposon insertion mutant for speD gene suggested that spermidine has a potential role in the adaptive response in P. aeruginosa incubated in long-term stress conditions.     

Influence of O Polysaccharides on Biofilm Development and Outer Membrane Vesicle Biogenesis in Pseudomonas aeruginosa PAO1

Pseudomonas aeruginosa is a common opportunistic human pathogen known for its ability to adapt to changes in its environment during the course of infection. These adaptations include changes in the expression of cell surface lipopolysaccharide (LPS), biofilm development, and the production of a protective extracellular exopolysaccharide matrix. Outer membrane vesicles (OMVs) have been identified as an important component of the extracellular matrix of P. aeruginosa biofilms and are thought to contribute to the development and fitness of these bacterial communities. The goal of this study was to examine the relationships between changes in the cell surface expression of LPS O polysaccharides, biofilm development, and OMV biogenesis in P. aeruginosa. We compared wild-type P. aeruginosa PAO1 with three chromosomal knockouts. These knockouts have deletions in the rmd, wbpM, and wbpL genes that produce changes in the expression of common polysaccharide antigen (CPA), O-specific antigen (OSA), or both. Our results demonstrate that changes in O polysaccharide expression do not significantly influence OMV production but do affect the size and protein content of OMVs derived from both CPA⁻ and OSA⁻ cells; these mutant cells also exhibited different physical properties from wild-type cells. We further examined biofilm growth of the mutants and determined that CPA⁻ cells could not develop into robust biofilms and exhibit changes in cell morphology and biofilm matrix production. Together these results demonstrate the importance of O polysaccharide expression on P. aeruginosa OMV composition and highlight the significance of CPA expression in biofilm development.     

Role of Exopolysaccharide in Aggregatibacter actinomycetemcomitans–Induced Bone Resorption in a Rat Model for Periodontal Disease

Aggregatibacter actinomycetemcomitans a causative agent of periodontal disease in humans, forms biofilm on biotic and abiotic surfaces. A. actinomycetemcomitans biofilm is heterogeneous in nature and is composed of proteins, extracellular DNA and exopolysaccharide. To explore the role played by the exopolysaccharide in the colonization and disease progression, we employed genetic reduction approach using our rat model of A. actinomycetemcomitans-induced periodontitis. To this end, a genetically modified strain of A. actinomycetemcomitans lacking the pga operon was compared with the wild-type strain in the rat infection model. The parent and mutant strains were primarily evaluated for bone resorption and disease. Our study showed that colonization, bone resorption/disease and antibody response were all elevated in the wild-type fed rats. The bone resorption/disease caused by the pga mutant strain, lacking the exopolysaccharide, was significantly less (P < 0.05) than the bone resorption/disease caused by the wild-type strain. Further analysis of the expression levels of selected virulence genes through RT-PCR showed that the decrease in colonization, bone resorption and antibody titer in the absence of the exopolysaccharide might be due to attenuated levels of colonization genes, flp-1, apiA and aae in the mutant strain. This study demonstrates that the effect exerted by the exopolysaccharide in A. actinomycetemcomitans-induced bone resorption has hitherto not been recognized and underscores the role played by the exopolysaccharide in A. actinomycetemcomitans-induced disease.