Characterization of Cell-to-Cell Signaling-Deficient Pseudomonas aeruginosa Strains Colonizing Intubated Patients

Abstract

Cell-to-cell signaling involving N-acyl-homoserine lactone compounds termed autoinducers (AIs) is instrumental to virulence factor production and biofilm development by Pseudomonas aeruginosa. In order to determine the importance of cell-to-cell signaling during the colonization of mechanically ventilated patients, we collected 442 P. aeruginosa pulmonary isolates from 13 patients. Phenotypic characterization showed that 81% of these isolates produced the AI-dependent virulence factors elastase, protease, and rhamnolipids. We identified nine genotypically distinct P. aeruginosa strains. Six of these strains produced AIs [N-butanoylhomoserine lactone or N-(3-oxo-dodecanoyl)-homoserine lactone] and extracellular virulence factors (elastase, total exoprotease, rhamnolipid, hydrogen cyanide, or pyocyanin) in vitro. Three of the nine strains were defective in the production of both AIs and extracellular virulence factors. Two of these strains had mutational defects in both the lasR and rhlR genes, which encode the N-acyl-homoserine lactone-dependent transcriptional regulators LasR and RhlR, respectively. The third of these AI-deficient strains was only mutated in the lasR gene. Our observations suggest that most, but not all, strains colonizing intubated patients are able to produce virulence factors and that mutations affecting the cell-to-cell signaling circuit are preferentially located in the transcriptional regulator genes.The gram-negative bacterium Pseudomonas aeruginosa is a major nosocomial pathogen responsible for severe pulmonary infections in mechanically ventilated patients, a complication associated with high mortality and excessive hospital costs (7,27). Among ventilator-associated pneumonia (VAP) caused by P. aeruginosa, 95% of cases are preceded by colonization of the respiratory tract, a well-recognized risk factor for pulmonary infection (20). The risk of colonization of intubated patients by P. aeruginosa is directly linked to the presence of the endotracheal intubation device and increases with the duration of intubation (29).P. aeruginosa regulates the production of several extracellular virulence factors by at least two cell-to-cell signaling systems, called lasRI and rhlRI (60). This circuit is organized in a hierarchical cascade in which the lasRI system is required for full expression of the rhlRI system (28). Accumulation of the two autoinducers (AIs) N-(3-oxododecanoyl)-homoserine lactone (3-oxo-C 12 -HSL) (39) and N-butanoyl-homoserine lactone (C 4 -HSL) (40), the products of the LasI and RhlI enzymes, respectively, allows the bacteria to sense their own cell density (17) in order to coordinate the production of virulence factors (9, 38, 60). As a result, the secretion of extracellular virulence factors such as elastase increases strongly once a threshold bacterial cell density has been reached (38). This

Social cheating in Pseudomonas aeruginosa quorum sensing

“…The result would be a heterogeneous community of QS proficient and QS deficient (cheater) variants. Intriguingly, in the only study to date that has examined the diversity of QS phenotypes of a large number of isolates from individual patients, such mixed populations have been found to exist (37). We suspect that cheating might also provide P. aeruginosa with a selective advantage in natural mixed-species environments in which other bacteria secrete extracellular degradative enzymes that generate the necessary nutrients for growth.…”

Exploiting Quorum Sensing To Confuse Bacterial Pathogens

“…For example, P. aeruginosa QS systems have been demonstrated to be important for pathogenesis in multiple animal infection models (325)(326)(327)(328), and AHLs and QS-regulated genes have been demonstrated to be expressed in vivo (329)(330)(331). However, analyses of clinical P. aeruginosa isolates have revealed that lasR, and less frequently rhlR, can be mutated during infection (332)(333)(334)(335)(336). In one study it was demonstrated that a single cystic fibrosis patient acquired at least four independent lasR mutants over an 8-year period (337), suggesting a strong selection pressure in vivo for abolishing LasR-mediated QS during chronic infections.…”

“…This growth advantage is at least in part due to increased expression of the regulator CbrB, which regulates various catabolic pathways, suggesting that aberrant expression of other regulators resulting from the loss of LasR function may provide a growth benefit during growth in vivo. Such a growth advantage could explain the emergence of lasR mutant populations during in vivo infection (332)(333)(334)(335)(336)(337).…”

“…Given that lasR mutations arise frequently during in vivo infection (332)(333)(334)(335)(336), it could also be reasoned that use of QSI compounds that target LasR may prevent the natural evolution of lasR mutant strains over time, given that the presence of the QSI compound would effectively make the bacteria lasR negative without genetic mutation. In agreement with this, azithromycin treatment of intubated patients colonized by P. aeruginosa, a treatment which inhibits both las and rhl QS systems, resulted in a decrease in the frequency of lasR mutants over time, whereas in the group treated with a placebo the frequency of lasR mutants increased.…”

Revisiting the quorum-sensing hierarchy in Pseudomonas aeruginosa: the transcriptional regulator RhlR regulates LasR-specific factors

“…Thus QS plays a predominant role in the regulation of virulence determinants in P. aeruginosa. Surprisingly, however, there are increasing reports that lasR mutants occur frequently in the natural environment (Cabrol et al, 2003), in airways from individuals with cystic fibrosis (D'Argenio et al, 2007;Smith et al, 2006), in intubated patients (Denervaud et al, 2004) and in individuals suffering from bacteraemia, pneumonia or wound infection (Hamood et al, 1996). This is intriguing, since the LasR regulator is widely considered essential for full P. aeruginosa virulence (Preston et al, 1997;Rumbaugh et al, 1999;Storey et al, 1998).…”