Infect Immun 68(1): 24-29

Invasion and Intracellular Survival of <em>Burkholderia cepacia</em>

Department of Microbiology and Immunology, East Carolina University School of Medicine, Greenville, North Carolina 27834,^{and Department of Microbiology, University of Minnesota, Minneapolis, Minnesota 55455}²

^{Corresponding author. Mailing address: Department of Microbiology, University of Minnesota, 1460 Mayo Memorial Bldg., Box 196, 420 Delaware St. SE, Minneapolis MN 55455-0312. Phone: (612) 625-7104. Fax: (612) 626-0623. E-mail:}ude.nmu.dem.itnel@rhom.

Received 1999 Jul 22; Revisions requested 1999 Sep 20; Accepted 1999 Oct 1.

Abstract

Burkholderia cepacia has emerged as an important pulmonary pathogen in immunocompromised patients and in patients with cystic fibrosis (CF). Little is known about the virulence factors and pathogenesis of B. cepacia, although the persistent and sometimes invasive infections caused by B. cepacia suggest that the organism possesses mechanisms for both cellular invasion and evasion of the host immune response. In this study, cultured human cells were used to analyze the invasion and intracellular survival of B. cepacia J2315, a highly transmissible clinical isolate responsible for morbidity and mortality in CF patients. Quantitative invasion and intracellular growth assays demonstrated that B. cepacia J2315 was able to enter, survive, and replicate intracellularly in U937-derived macrophages and A549 pulmonary epithelial cells. Transmission electron microscopy of infected macrophages confirmed the presence of intracellular B. cepacia and showed that intracellular bacteria were contained within membrane-bound vacuoles. An environmental isolate of B. cepacia, strain J2540, was also examined for its ability to invade and survive intracellularly in cultured human cells. J2540 entered cultured macrophages with an invasion frequency similar to that of the clinical strain, but it was less invasive than the clinical strain in epithelial cells. In marked contrast to the clinical strain, the environmental isolate was unable to survive or replicate intracellularly in either cultured macrophages or epithelial cells. Invasion and intracellular survival may play important roles in the ability of virulent strains of B. cepacia to evade the host immune response and cause persistent infections in CF patients.

Abstract

The gram-negative bacterium Burkholderia cepacia causes serious opportunistic infections in humans and has recently emerged as an important pulmonary pathogen in patients with cystic fibrosis (CF) (7, 8, 11, 24). In CF patients the clinical outcome of B. cepacia colonization can vary from maintenance of a normal respiratory function to a rapid and ultimately fatal clinical decline (11, 22). This latter condition, referred to as “B. cepacia syndrome,” occurs in approximately 25% of CF patients and is characterized by fever, acute necrotizing pneumonia and, in some cases, bacteremia (7). The specific mechanisms by which B. cepacia is able to subvert host defense mechanisms, invade deeper tissues of the lung, and ultimately become blood-borne are poorly understood. Compounding this lack of knowledge is the inherent resistance of B. cepacia to multiple antibiotics, which has made treatment of B. cepacia infections especially difficult (14, 21). Once a CF patient is colonized with B. cepacia, the organism is rarely eradicated.

There is growing evidence that the persistent infections caused by B. cepacia may be due, in part, to the ability of the organism to invade and survive intracellularly in human cells. Two of the main cell types encountered by B. cepacia infecting the CF lung are respiratory epithelial cells and pulmonary macrophages. B. cepacia organisms have been observed in tracheal epithelial cells harvested at the time of autopsy from a CF patient (J. L. Burns, D. K. Clark, and C. D. Wadsworth, Proc. 6th Annu. N. Am. Cystic Fibrosis Conf., abstr. 201, 1992). B. cepacia has also been shown to invade and survive in cultured respiratory epithelial cells (2). In contrast to epithelial cells, the interaction between B. cepacia and macrophages has received little attention (7). Since pulmonary macrophages represent a first line of defense within the CF lung, the ability of B. cepacia to enter and survive within macrophages could provide a mechanism for evasion of the host immune response and may help to explain the reported ability of B. cepacia to achieve prolonged pulmonary colonization despite a pronounced antibody response (17). Moreover, an intracellular niche may also explain the persistence of B. cepacia in the CF lung despite the use of antibiotics with demonstrated activity against the organism in vitro (5).

B. cepacia can be cultured from a range of natural environments, including soil, water, and plants (3). The pathogenic potential of environmental isolates and their genetic relationship to clinical strains responsible for severe and sometimes fatal pulmonary infections is an important, yet unresolved issue. One clinical strain in particular, J2315, has been responsible for epidemic outbreaks and increased mortality in CF patients (12, 20, 25). Strain J2315 expresses an unusual cable-like pilus that has been shown to play a role in adherence to CF mucin and airway respiratory epithelial cells (25). Other studies have demonstrated that J2315 exoproducts stimulate interleukin-8 (IL-8) release from cultured lung epithelial cells and peripheral blood monocytes (18). More recently, it has been shown that strain J2315 produces a hemolytic toxin that induces apoptosis (programmed cell death) in cultured macrophages (9). Taken together, these findings suggest that strain J2315 possesses mechanisms for both host cell invasion and evasion of the host immune response. A cell culture model for both invasion and intracellular survival would be a valuable tool to further define these processes and determine their role in the pathogenesis of B. cepacia.

In this study, we established a macrophage model of invasion and intracellular survival for B. cepacia. We examined the ability of B. cepacia strain J2315, as well as an environmental isolate of B. cepacia, to enter and survive intracellularly in cultured human macrophages, as well as in respiratory epithelial cells. Our findings suggest that invasion and intracellular survival may play important roles in the ability of virulent strains of B. cepacia to evade the host immune response and cause persistent and sometimes fatal infections in CF patients.

ACKNOWLEDGMENTS

We thank John Govan, Department of Medical Microbiology, University of Edinburgh, for kindly providing B. cepacia J2315 and J2540. We also thank Nafisa Ghori, Stanford University, for TEM preparation and sectioning. We are grateful to Stanley Falkow, Lucy Thompkins, and Lucy Shapiro, in whose laboratories the initial stages of this study were conducted.

C.D.M. was supported by University of Minnesota Grant-in-Aid grant 17929. D.W.M. was supported by a Fellowship Award from the Center for Indoor Air Research. Lucy Thompkins and Lucy Shapiro were supported by NIH grants AI30618 and GM32506/5120MZ, respectively.

ACKNOWLEDGMENTS

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