Published ahead of print on January 31, 2003, doi:10.1164/rccm.200207-736BC
© 2003 American Thoracic Society
Role of the Quorum-sensing System in Experimental Pneumonia due to Pseudomonas aeruginosa in RatsINSERM EMI-U 9933, and Service d'Anatomie Pathologique, Hôpital Bichat, Paris, France; Département de Santé Publique, Hôpital Henri Mondor, Créteil; and Laboratoire d'Ingénierie des Systèmes Macromoléculaires, Centre National de la Recherche Scientifique, Marseille, France Correspondence and requests for reprints should be addressed to Dr. Philippe Lesprit, Service d'Immunologie Clinique, Hôpital Henri Mondor, 51 avenue du Maréchal de Lattre, 94010 Créteil, France. E-mail: phillipe.lesprit{at}hmn.ap-hop-paris.fr
The virulence of Pseudomonas aeruginosa is partly controlled by the las quorum-sensing system. A rat model of acute pneumonia was used to investigate the pathophysiological impact of this system by comparing the virulence of the wild-type virulent laboratory strain PAO1 with that of its lasR-deleted mutant PAOR. In comparison with PAO1, PAOR was avirulent after an instillation of 106 cfu (mortality rates, 72 versus 0%, respectively; p < 0.0001). A ten-fold higher inoculum slightly increased the mortality rate induced by PAOR (25%), which remained lower than that induced by PAO1 (75%, p = 0.0001). In addition, with both inocula lung and bronchoalveolar lavage bacterial counts were significantly lower in rats infected with PAOR than with PAO1 (p  0.01). Histopathological analysis showed that PAO1 induced a drastic vascular congestion and neutrophil infiltration of the lungs, whereas lung injury in rats infected with PAOR was mild with predominantly macrophage infiltration. This study adds evidence that the quorum-sensing system has an important role in the pathophysiology of P. aeruginosa pulmonary infection.
Key Words: cell-to-cell signaling • experimental pneumonia • Pseudomonas aeruginosa Pseudomonas aeruginosa is an opportunistic pathogen that is responsible for acute pneumonia in immunocompromised or mechanically ventilated patients and for chronic pulmonary infection in patients with cystic fibrosis. Many P. aeruginosa virulence factors have been shown to be controlled by a complex regulatory circuit involving cell-to-cell signaling (or quorum-sensing, QS) system that allow the bacteria to sense their own cell density and to communicate with each other (1). This results in a coordinated, cell density-dependent production of virulence factors. The main QS systems in gram-negative bacteria are composed of homoserine lactone (HSL)–based autoinducer molecules, which are synthesized by a LuxI-type autoinducer synthase, and a LuxR-type transcriptional activator of target genes (2). Two QS systems have been described in P. aeruginosa. The lasR/lasI system is activated by N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) and is required for optimal production of LasA and LasB elastases, alkaline protease, exotoxin A, and proteins of the secretory pathway (3). The second signaling system, referred to as rhl (for rhamnolipid production), is also necessary for optimal production of elastases, protease, pyocyanin, and hemolysin (4). Although both systems are highly specific for their respective autoinducers, there is a QS signaling hierarchy, because the las system activates the expression of rhlR/rhlI. In vitro studies have shown that production of elastase and other virulence factors are reduced in P. aeruginosa mutants defective in the las or the rhl systems (3, 5), but only a few studies have confirmed this role in vivo. The aim of this study was to determine the role of QS in the physiopathology of acute pulmonary infection by comparing the wild-type laboratory strain P. aeruginosa PAO1, which possesses two functional QS systems, and the lasR-deleted isogenic mutant PAOR. For this purpose, we used a rat model of acute pneumonia derived from Cash and coworkers (6). Strains were compared for their ability to induce death following bacterial challenge at two different inocula, and physiopathological changes were studied by bacterial and semiquantitative histological parameters.
Bacterial Strains PAO1 is an invasive virulent laboratory strain that possesses two functional QS systems (7). PAOR is a lasR-deleted isogenic mutant produced by gene replacement. Insertional inactivation of lasR, obtained by homologous recombination between a suicide vector and the PAO1 chromosome at the lasR locus, results in a nonfunctional las system that affects the production of elastase and alkaline protease (3, 8).
Preparation of the Inoculum
Infection of Animals
Survival
Bacterial Growth in Lung and Bronchoalveolar Lavage
Lung Histopathology
Statistical Analysis
Mortality Rates and Mean Time to Death The mortality rate of animals infected with PAO1 was 72% after the instillation of 106 cfu, whereas all animals infected with PAOR survived bacterial challenge (p < 0.0001) (Figure 1) . Instillation of 107 cfu slightly increased the mortality rate of animals infected with PAOR (25%), which remained lower than that of PAO1 (75%, p = 0.0001). In addition, the mean time to death after infection with PAO1 was significantly shorter than with PAOR (1.69 ± 0.73 versus 2.6 ± 1.2 days, p < 0.01). All control animals survived after injection of uninfected agar beads.
Bacterial Counts in Lungs and BAL Fluids Pseudomonas aeruginosa was isolated in pure culture from the lung or BAL samples. When rats were infected with an inoculum of 106 cfu, a 3 log-unit difference was observed between the lungs and BAL of PAO1- and PAOR-infected animals (Table 1) . For the inoculum of 107 cfu, a tenfold rise in bacterial counts was observed in rats infected with PAOR, but bacterial counts remained 100-fold smaller compared with samples isolated from rats infected with the same inoculum of PAO1. Bacterial counts in animals dying after the bacterial challenge were similar between those infected with PAO1 or PAOR (Table 2) . Persistent infection was demonstrated in lung samples and BAL fluids of animals killed 5 or 10 days after inoculation with 107 cfu of PAOR. Among animals killed 5 days after inoculation, BAL bacterial counts were significantly higher in the PAOR group (p = 0.0003 for comparison with the PAO1 group).
Lung Histopathological Analysis For both strains, the semiquantitative analysis showed that rats had significant lung injury after bacterial challenge, with numerous foci of necrosis and inflammatory infiltrates, as compared with controls (Figure 2) . Histology of the lungs infected with PAO1 demonstrated severe acute pneumonia with vascular congestion and dense consolidation consisting primarily of neutrophils (p = 0.03 for comparison of congestion with PAOR). In contrast, macrophage infiltration was a predominant finding in the lungs of rats infected with PAOR (p = 0.02 for comparison with PAO1). Inflammatory exudates surrounding beads containing bacteria were found in the adjacent bronchial wall and sometimes obliterated the distal airways. Increased numbers of alveolar macrophages infiltrating the alveolar septa were found in the surrounding parenchyma. Some of the macrophages had ingested bacteria. Macrophage infiltration was absent or moderate in the lungs of rats infected with PAO1 and in the lungs of controls.
Features of chronic pneumonia with predominance of macrophage infiltration were demonstrated in animals surviving inoculation with 107 cfu of PAOR (Figure 3) . This inflammatory response persisted throughout the 10-day period of observation.
Pseudomonas aeruginosa is associated with a broad spectrum of lung diseases, ranging from acute pneumonia in immunosuppressed or mechanically ventilated patients to chronic infection in patients with cystic fibrosis. Because the aim of our study was to evaluate the effects of QS expression on the pathophysiology of P. aeruginosa pneumonia, an animal model that closely reproduces the broad spectrum of pulmonary infection in humans was required. Because animal models of acute pneumonia that involve the administration of free P. aeruginosa to rats have shown that the animals either die or clear the inoculum within 48 hours, we used the agarose model described by Cash and coworkers, with several variations (6, 10). In this model, inoculated bacteria are enmeshed in agar beads, allowing the development of a typical bronchopneumonia without the use of any immunosuppressive agent or previous bronchopulmonary injury. Although the model was initially designed to induce a chronic respiratory infection with an inoculum of 104 cfu, dose–response studies performed in our laboratory with the strain PAO1 have demonstrated the influence of the bacterial inoculum on the outcome of lung infection. The mortality rate dramatically increased from 5 to 82% for inocula above a threshold of 106 cfu. Rats infected with inocula of 106 cfu or greater either died of acute pneumonia or survived with a low bacterial clearance within the lungs and pathological features of chronic pneumonia (11). We have previously shown that our experimental model of acute pneumonia is accurate to study the pathogenesis of P. aeruginosa pneumonia and the effect of therapies against this pathogen (10, 11). In this study, we used the same laboratory strain, PAO1, instead of a clinical isolate, because PAO1 is considered a reference laboratory strain and is characterized as having an invasive phenotype (12). Moreover, several well-defined mutants of PAO1 are available. Although genetic differences between clinical and laboratory strains may exist, studies have shown that clinical and environmental isolates are functionally equivalent in several traits relevant for their virulence properties (13). We first demonstrated that all animals survived after administration of 106 cfu of PAOR, a lasR-deleted isogenic mutant of PAO1. In in vitro studies this mutant demonstrated impaired production of several virulence factors depending on the las QS system. In particular, the elastolytic activity of PAOR is about 5% of that of PAO1 (3). This result is in agreement with previous studies that used different animal models of pulmonary infection with P. aeruginosa QS mutants. In the neonatal mouse model of pneumonia, an inoculum of 5 x 106 cfu of the same PAOR strain resulted in no mortality. In contrast with our study, in which all animals infected with PAOR displayed significant bacterial counts in lungs 5 and 10 days after inoculation, only 48% of mice showed airway colonization 24 hours after infection, suggesting that PAOR was rapidly cleared from the airways in this model (14). In another study, Smith and coworkers compared the ability of lasI- and lasI/rhlI-deleted mutants to colonize the lungs of adult mice. For an inoculum of 106 cfu, bacterial counts were 4 and 3 log units lower in the lungs of mice infected with mutants than with PAO1. However, long-term survival and lung pathophysiological effects of QS-deleted mutants were not assessed in that study, because all animals were studied 24 hours after inoculation (15). Our results demonstrate that the lower mortality observed after PAOR administration cannot be explained by a rapid clearance of bacteria, and the model strongly suggests that the las system play a critical role in the establishment of pneumonia in adult rats. A tenfold higher inoculum of PAOR (107 cfu) slightly increased the ability of the mutant to induce a lethal pneumonia. The tenfold increase in bacterial counts and histopathological changes confirmed that, under these conditions, PAOR is able to induce significant lung damage. The modest increase in mortality rate (25%) suggests that the virulence of PAOR can be partially restored by increasing the bacterial inoculum despite an inactive las system. Whether the rhl system can operate in the absence of a functional las system is a critical point with regard to the development of antimicrobial agents that interfere with the QS system (16). Indeed, a previous study has shown that production of QS-controlled virulence factors by PAOR can be restored in vitro by starvation (17). When the strain was incubated under these specific culture conditions, upregulation of the rhl QS system was demonstrated. Of note, starvation could not restore the virulence of the lasI/rhlI double mutant. Because we did not incubate PAOR in a specific medium, it is unlikely that nutrient starvation played a role in the restoration of PAOR virulence factors. Increasing bacterial density with an inoculum of 107 cfu may allow the concentration of rhl-ASL molecules to reach a threshold level that is sufficient to activate independently the rhl system. Whether overexpression of the rhl system is sufficient to compensate for the absence of the las system at a high inoculum remains to be investigated in our model by measuring the production of virulence factors and HSL molecules in BAL fluids or lung tissues (18). Of note, when the relative virulence of lasI, rhlI, and lasI/rhlI mutants of PAO1 was compared in the neonatal mouse model of lung infection, the most significant reduction in virulence was seen with the double mutant (19). Our data confirm that therapeutic inhibition of both las and rhl systems will be required for optimal therapeutic efficacy of agents that interfere with the QS system. Another result of our study is the comparison of lung injury induced by an inoculum of 107 cfu of PAO1 and PAOR. Pulmonary lesions induced by PAO1 were typical of those observed in acute pulmonary infection in humans, with severe necrosis of bronchoalveolar structures, vascular congestion, and neutrophil infiltration. By contrast, two major differences were noted with PAOR. First, a lower bacterial clearance within the airways was noted 5 days after sacrifice of rats infected with PAOR, when BAL results were compared with those of rats infected with PAO1. Second, lung injury in rats surviving infection with PAOR was characterized by the absence of congestion and a predominance of macrophage infiltration, as demonstrated by hematoxylin–eosin staining. The ability of PAOR to colonize the lungs without inducing a significant inflammatory response can be explained by the lack of a functional las system, because it has been shown in vitro that the lasR autoinducer 3-oxo-C12-HSL has immunomodulatory activity (20). In particular, this molecule is a potent inducer of the neutrophil chemokine interleukin-8 and of the inflammatory mediator Cox-2 enzyme, which is involved in the pathophysiological processes of inflammation and edema (20, 21). In conclusion, our study provides evidence that the las QS system plays an important role in the pathophysiology of P. aeruginosa acute pulmonary infection, as demonstrated by the reduced ability of the mutant to induce mortality, a high lung bacterial load, and lung injury.
The authors are grateful to Christian Brun-Buisson, M.D., for reviewing the manuscript and providing comments.
Supported in part by grants from the Association Française de Lutte contre la Mucoviscidose, Paris, France. This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournal.org Received in original form July 23, 2002; accepted in final form January 24, 2003
This article has been cited by other articles:
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TOP
ABSTRACT
METHODS
factor RpoS. Mol Microbiol 1996;21:1137–1146.
