Genetic And Phenotypic Characterization Of Transposon Mutant Of Pseudomans Aeruginosa PAO Impaired – Essay Example
Genetic and Phenotypic Characterization of Transposon Mutanta of Pseudomonas aeruginosa PAO1 impaired in virulence factor production Introduction
The medical significance of Pseudomonas aeruginosa as an opportunistic pathogen is highlighted by its ability to cause a wide spectrum of infections (Brusset et al., 1998; Wareham and Curtis, 2007).In fact, P. aeruginosa infects more than 80% of patients with cystic fibrosis (CF) and this co-infection increases the morbidity and mortality of CF (Nemec et al., 2009). Goldberg and Pier (2000) suggested that the mutation in cystic fibrosis transmembrane conductance regulator increases the susceptibility of CF patients to P. aeruginosa infections.
In vivo and in vitro studies have shown that the mechanism by which P. aeruginosa establishes and sustains virulence is determined by the organism’s ability to interfere with the functions of host epithelial surfaces (Wareham and Curtis, 2007). This ability is a function of the various extracellular toxins such as, but not limited to LasA protease, hydrogen cyanide, rhamnolipids, and exotoxins produced by the organism (Kjelleberg et al., 2003; Bos-gelmez-Tınaz and Ulusoy, 2008; Tre-Hardy et al., 2008). Pier (2007) reported that P. aeruginosa lipipolysaccharide (LPS) serves as an endotoxin that kicks off an inflammatory response, contributing to high mortality in infected individuals. In addition, this pathogen holds an efficient secretion system that injects toxins directly into the host’s cytoplasm. (Corvecet al., 2008; Kerr and Snelling, 2009). Pseudomonas aeruginosa also exhibits a high propensity to develop and form biofilms, a functional community of cells, which, by virtue of its matrix and altered physiology, provides the organism protection against the penetration and detrimental effects of antibiotics and other biocides (Peyton 1996; Hoibyet al., 2001; Valdez et al., 2010). In general, microorganisms organized into biofilms are three orders of magnitude more resistant to conventional antibiotic treatment compared to its planktonic counterparts (Drenkard 2003; Tre-hardy et al.,2009) The expression of genes encoding for P. aeruginosa toxins, secretion systems, and biofilm formation are regulated and mediated in a population density-dependent way by a mechanism known as quorum sensing (Kjelleberg et al.,2003; Bos-gelmez-Tınaz and Ulusoy, 2008).
Pseudomonas aeruginosa has a dynamic genome and approximately 10% is regulated by a sophisticated multi-signal quorum sensing (QS) circuit. Quorum sensing is an intercellular communication strategy that coordinates microbial activities upon reaching a specific population density (Mathee et al., 2006; Meijler et al, 2009). This efficient cell-to-cell signaling is achieved through the production and subsequent detection of signaling molecules know as autoinducers (AIs) ((Mathee et al., 2006; Meijler et al, 2009).These autoinducers usually take the form of acylated homoserine lactones (Rumbaugh et al., 2000). When a threshold concentration of the autoinducer is achieved, usually during late log to early stationary phase, the microorganism detects the autoinducer through binding toreceptor or transcriptional activator. This binding initiates a signal transduction cascade that results in changes in the microorganism’s gene expression. In turn, the altered gene expression causes a change in the morphology and behaviorof the bacterial population. (Meijler et al., 2009)
Literature reviews and laboratory data indicated that the QS systems of P. aeruginosa are of two major types: las andrhl systems (Suga and Smith, 2003; Mathee.et al., 2006) Fig 1.According to Rumbaugh et al. (2000), the las system is a homologue of the Vibrio fisheri lux quorum sensing systemand specifically makes use of N-(3-oxododecanoyl)-Lhomoserine lactone, also called PAI-1 or OdDHL, as itsautoinducer (Pearson et al., 1994; Rambaugh et al., 2000). The lasI gene produces PAI-1 while lasR serves as the transcriptional activator. Coordination of these two components (lasI and lasR) effectively regulates the production of alkaline proteases, exotoxin A and elastase (Bos-gelmez-Tınaz and Ulusoy, 2008). Meanwhile, the rhl system, the other quorum sensing circuit of P. aeruginosa,relies on N-butanoyl-L-homoserine lactone (C4-HSL) to regulate production of rhamolipids, elastase, cyanide, and pyocianine (Pearson et al., 1995; Winson et al., 1995). Studies have provided strong evidence that the two QS systems acts in a hierarchical fashion: the las systems positively regulates the expression of rhl (Bos-gelmez-Tınaz and Ulusoy, 2008). Linked also to the two QS systems is another QS signal, 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS), which regulates iron (II) entrapment by P. aeruginosa (Diggle et al., 2007). PQS is also involved in the production of virulence determinants such as LecA, galactophilic lectin, elastase, rhamnolipids, and pyocyanin (Diggle et al., 2007). It was also observed that action of PQS is not cell-density dependent, which means to imply that a low population density culture will still produce virulence factors upon supplementation of PQS (Diggle et al., 2007).
In addition to controlling virulence factor and production of biofilm, the expression of genes for twitching motility, alginate, azurin, chitinase, lipase, catalase, rpoS, and superoxide dismutase are found to be governed by quorum sensing (Rumbaugh et al., 2000). Wu et al. (2004) successfully demonstrated that the antibody responses in the rat model of chronic P. aeruginosa infections are influenced by the ability of the bacteria to produce quorum sensing signal. In an attempt to determine the role of QS in proliferation and infection of P. aeruginosa in the lungs, Wu et al (2004) examined the serum anti-Pseudomonas aerugnoa IgG levels in rats infected with wild strain PAO1 and mutant strain PAO1, where lasI andrhlI are knocked-out. Results of the experiment revealed that the IgG levels of the mutant PAO1-infected rats are significantly lower compared to those infected with the wild strain. Even more interesting is that four out of 15 rats in the wild-type group still carried the infection on the 106th day, while the mutant PAO1 bacteria were no longer detected from the lungs of the mutant-infected group. Although the study did not involve the measurement of virulence factors such as ToxA, Wu and his colleagues provided a strong evidence that the absence of QS system in P. aeruginosa significantly lowers its ability to induce infection. Additionally, Wu and his team suggested that P. aeruginosa strains with anomalous quorum sensing circuits may lead to a significant decrease in its capability to colonize the lungs. The researchers argued that the low serum IgG detected in the mutant-infected group may be due to the significant decrease in the proteases expressed by the host, which downregulated the influence of QS on the host’s immune responses (Wu et al., 2004).
Goldberg and Pier. The role of the CFTR in susceptibility to Pseudomonas aeruginosa infections in cystic fibrosis. Trends in Microbiology. 2000;8:514-520
Nemec et al. High genotypic diversity of Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis in the Czech Republic. Research in Microbiology 2007; 158: 324-329