Pseudomonas aeruginosa

Pseudomonas aeruginosa is a bacterium responsible for severe nosocomial infections, life-threatening infections in immunocompromised persons, and chronic infections in cystic fibrosis patients. The bacterium’s virulence depends on a large number of cell-associated and extracellular factors. Cell-to-cell signaling systems control the expression and allow a coordinated, cell-density–dependent production of many extracellular virulence factors. We discuss the possible role of cell-to-cell signaling in the pathogenesis of P. aeruginosa infections and present a rationale for targeting cell-to-cell signaling systems in the development of new therapeutic approaches.

Pseudomonas aeruginosa as a Human Pathogen

Pseudomonas aeruginosa, an increasingly prevalent opportunistic human pathogen, is the most common gram-negative bacterium found in nosocomial infections. P. aeruginosa is responsible for 16% of nosocomial pneumonia cases, 12% of hospital-acquired urinary tract infections, 8% of surgical wound infection, and 10% of bloodstream infections. Immunocompromised patients, such as neutropenic cancer and bone marrow transplant patients, are particularly susceptible to opportunistic infections. In this group of patients, P. aeruginosa is responsible for pneumonia and septicemia with attributable deaths reaching 30%. P. aeruginosa is also one of the most common and lethal pathogens responsible for ventilator-associated pneumonia in intubated patients, with directly attributable death rates reaching 38%. In burn patients, P. aeruginosa bacteremia has declined as a result of better wound treatment and dietary changes (removal of raw vegetables, which can be contaminated with P. aeruginosa, from the diet). However, P. aeruginosa outbreaks in burn units are still associated with high (60%) death rates. In the expanding AIDS population, P. aeruginosa bacteremia is associated with 50% of deaths. Cystic fibrosis (CF) patients are characteristically susceptible to chronic infection by P. aeruginosa, which is responsible for high rates of illness and death in this population. The capacity of P. aeruginosa to produce such diverse, often overwhelming infections is due to an arsenal of virulence factors. Many extracellular virulence factors secreted by P. aeruginosa have been shown to be controlled by a complex regulatory circuit involving cell-to-cell signaling systems that allow the bacteria to produce these factors in a coordinated, cell-density–dependent manner. In this article we describe major virulence factors of P. aeruginosa and the possible involvement of cell-to-cell signaling in the pathogenesis of acute P. aeruginosa infection. We also summarize data suggesting that these regulatory systems could be exploited for the design of therapeutic interventions.

Pathogenesis of P. aeruginosa Infections

P. aeruginosa (family Pseudomonadaceae), an aerobic, motile, gram-negative rod able to grow and survive in almost any environment, lives primarily in water, soil, and vegetation. However, despite abundant opportunities for spread, P. aeruginosa rarely causes community-acquired infections in immunocompetent patients. As a result, the pathogen is viewed as opportunistic. The different phases of P. aeruginosa infection are shown in Figure 2.

Colonization: The Predominant Role of Cell-Associated Virulence Factors

To initiate infection, P. aeruginosa usually requires a substantial break in first-line defenses. Such a break can result from breach or bypass of normal cutaneous or mucosal barriers (e.g., trauma, surgery, serious burns, or indwelling devices), disruption of the protective balance of normal mucosal flora by broad-spectrum antibiotics, or alteration of the immunologic defense mechanisms (e.g., in chemotherapy-induced neutropenia, mucosal clearance defects from cystic fibrosis, AIDS, and diabetes mellitus).

The first step in P. aeruginosa infections is colonization of altered epithelium. The pathogen colonizes the oropharynx of up to 6% and is recovered from the feces of 3% to 24% of healthy persons. In contrast, up to 50% of hospitalized patients are at high risk for P. aeruginosa colonization. Adherence of P. aeruginosa to epithelium is probably mediated by type 4 pili similar to those of Neisseria gonorrhoeae. Several other nonpilus adhesins responsible for the binding to mucin have been described, but their role in the infection process remains unclear. Flagella, which are primarily responsible for motility, may also act as adhesins to epithelial cells.

From Colonization to Acute Infection: The Role of Extracellular Virulence Factors

P. aeruginosa produces several extracellular products that after colonization can cause extensive tissue damage, bloodstream invasion, and dissemination (Figure 1). In vivo studies have shown that mutants defective in the production of exotoxin A, exoenzyme S, elastase, or alkaline protease are essential for maximum virulence of P. aeruginosa; however, the relative contribution of a given factor may vary with the type of infection. Many of these factors are controlled by regulatory systems involving cell-to-cell signaling. We will summarize the known biologic effects of the most-studied extracellular virulence factors associated with acute P. aeruginosa infection.

Exotoxin A is produced by most P. aeruginosa strains that cause clinical infections. Like diphtheria toxin, P. aeruginosa exotoxin A catalyzes ADP-ribosylation and inactivation of elongation factor 2, leading to inhibition of protein biosynthesis and cell death. Exotoxin A is responsible for local tissue damage, bacterial invasion, and (possibly) immunosuppression (19). Purified exotoxin A is highly lethal for mice which supports its role as a major systemic virulence factor of P. aeruginosa.

Exoenzyme S is also an ADP-ribosyl transferase, but unlike exotoxin A, it preferentially ribosylates GTP-binding proteins such as Ras. This exoproduct is responsible for direct tissue destruction in lung infection and may be important for bacterial dissemination.

Two hemolysins, phospholipase C and rhamnolipid, produced by P. aeruginosa, may act synergistically to break down lipids and lecithin. Both may contribute to tissue invasion by their cytotoxic effects. Rhamnolipid, a rhamnose-containing glycolipid biosurfactant, has a detergentlike structure and is believed to solubilize the phospholipids of lung surfactant, making them more accessible to cleavage by phospholipase C. The resulting loss of lung surfactant may be responsible for the atelectasis associated with chronic and acute P. aeruginosa lung infection. Rhamnolipid also inhibits the mucociliary transport and ciliary function of human respiratory epithelium. However, the relative role of rhamnolipid in acute or chronic infection is not known.

Proteases are assumed to play a major role during acute P. aeruginosa infection. P. aeruginosa produces several proteases including LasB elastase, LasA elastase, and alkaline protease. The role of alkaline protease in tissue invasion and systemic infections is unclear; however, its role in corneal infections may be substantial. The ability of P. aeruginosa to destroy the protein elastin is a major virulence determinant during acute infection. Elastin is a major part of human lung tissue and is responsible for lung expansion and contraction. Moreover, elastin is an important component of blood vessels, which rely on it for their resilience. The concerted activity of two enzymes, LasB elastase and LasA elastase, is responsible for elastolytic activity. Elastolytic activity is believed to destroy elastin-containing human lung tissue and cause the pulmonary hemorrhages of invasive P. aeruginosa infections. LasB elastase is a zinc metalloprotease that acts on a number of proteins including elastin. LasB elastase is highly efficient, with a proteolytic activity approximately 10 times that of P. aeruginosa alkaline protease and an activity toward casein approximately four times that of trypsin. The LasA elastase is a serine protease that acts synergistically with LasB elastase to degrade elastin. LasA elastase nicks elastin, rendering it sensitive to degradation by other proteases such as LasB elastase, alkaline protease, and neutrophil elastase. Both LasB elastase and LasA elastase have been found in the sputum of CF patients during pulmonary exacerbation. However, the role of LasB elastase in tissue destruction during the chronic phase of CF is less clear. It has been postulated that during this phase, antibodies present in high titers neutralize LasB elastase, and elastin damaged by minute amounts of LasA is degraded mostly by neutrophil elastase. LasB elastase degrades not only elastin but also fibrin and collagen. It can inactivate substances such as human immunoglobulins G and A, airway lysozyme, complement components, and substances involved in protecting the respiratory tract against proteases such as a-1-proteinase inhibitor and bronchial mucus proteinase inhibitor. Therefore, LasB elastase not only destroys tissue components but also interferes with host defense mechanisms. Studies in animal models show that mutants defective in LasB elastase production are less virulent than their parent strains, which supports the role of LasB elastase as a virulence factor.