Blackwell Publishing LtdOxford, UKCMICellular Microbiology 1462-5814© 2006 The Authors; Journal compilation © 2006 Blackwell Publishing Ltd20068914561466Original ArticleIntracellular trafficking of B. cenocepaciaU. S. Sajjan, J. H. Yang, M. B. Hershenson and J. J. LiPuma Cellular Microbiology (2006) 8(9), 1456–1466 doi:10.1111/j.1462-5822.2006.00724.x First published online 13 April 2006 Intracellular trafficking and replication of Burkholderia cenocepacia in human cystic fibrosis airway epithelial cells Umadevi S. Sajjan, Jeffrey H. Yang, Although Pseudomonas aeruginosa is the most common Marc B. Hershenson and John J. LiPuma* opportunistic bacterial pathogen in CF, species of the Department of Pediatrics and Communicable Disease, Burkholderia cepacia complex (Bcc) also have emerged University of Michigan Medical School, Ann Arbor, as important respiratory pathogens in this population MI 48109, USA. (LiPuma, 1998). Patients infected with Bcc exhibit an unpredictable and variable clinical course ranging from asymptomatic carriage to fatal acute necrotizing pneumo- Summary nia and septicaemia (referred to as ‘cepacia syndrome’), We investigated the trafficking of Burkholderia ceno- which occurs in as many as 20% of infected patients (Isles cepacia, an opportunistic respiratory pathogen of et al., 1984; Liou et al., 2001). This type of fatal acute persons with cystic fibrosis (CF), in immortalized CF syndrome is rarely seen with other CF pathogens, includ- airway epithelial cells in vitro. Our results indicate ing P. aeruginosa (Govan and Deretic, 1996). The inherent that bacteria enter cells in a process involving actin resistance of Bcc strains to multiple antibiotics severely rearrangement. Whereas both live and heat-killed bac- limits treatment options and infection is typically impossi- teria reside transiently in early endosomes, only live ble to eradicate. bacteria escape from late endosomes to colocalize in The Bcc consists of at least nine genetically distinct vesicles positive for lysosomal membrane marker species (Coyene et al., 2001; Vandamme et al., 2003). LAMP1, endoplasmic reticulum (ER) membrane Although all nine species have been recovered from CF marker calnexin, and autophagosome marker mon- patients, B. cenocepacia and B. multivorans account for odansylcadavarine (MDC). Twenty-four hours after the great majority (∼85%) of infection in patients in North infection, microcolonies of live bacteria were America and Europe (Agodi et al., 2001; LiPuma et al., observed in the perinuclear area colocalizing with cal- 2001; Speert et al., 2002). Genotyping studies have also nexin. In contrast, after ingestion, dead bacteria colo- identified several so-called epidemic strains that are com- calized with late endosome marker Rab7, and mon to multiple CF patients. Most epidemic strains iden- lysosome markers LAMP1 and cathepsin D, but not tified to date, including strain ET12 (common among with calnexin or MDC. Six to eight hours after inges- infected patients in Canada and UK) (Johnson et al., tion of dead bacteria, degraded bacterial particles 1994; Pitt et al., 1996; Mahenthiralingam et al., 2002) and were observed in the cytoplasm and in vesicles pos- strain PHDC (widespread in the United States) (Chen itive for cathepsin D. These results indicate that live et al., 2001; Reik et al., 2005) are members of the B. cenocepacia gain entry into human CF airway cells B. cenocepacia species. by endocytosis, escape from late endosomes to enter Although the pathogenic mechanisms involved in autophagosomes that fail to fuse with mature lysos- human infection by B. cenocepacia are largely unknown, omes, and undergo replication in the ER. This survival several putative virulence factors have been described and replication strategy may contribute to the capac- (Mahenthiralingam et al., 2005). These include, cable pili ity of B. cenocepacia to persist in the lungs of and the associated adhesin (Sajjan et al., 2002; Urban infected CF patients. et al., 2005), flagella (Tomich et al., 2002; Urban et al., 2004), lipopolysaccharide and surface exopolysaccha- rides (Shaw et al., 1995; Chung et al., 2003), type III Introduction secretion system (Tomich et al., 2003), production of sid- Pulmonary infection-related inflammation is a major cause erophores (Visser et al., 2004), catalase and superoxide of morbidity and mortality in cystic fibrosis (CF) patients. dismutase (Lefebre and Valvano, 2001), proteases and lipases (McKevitt et al., 1989) and quorum sensing sys- tem (Sokol et al., 2003). To date the role of these factors Received 18 November, 2005; revised 15 February, 2006; accepted 5 March, 2006. *For correspondence. E-mail [email protected]; Tel. in contributing to lung disease has not been clearly (+1) 734 936 9767; Fax (+1) 734 764 4279. elucidated. © 2006 The Authors Journal compilation © 2006 Blackwell Publishing Ltd Intracellular trafficking of B. cenocepacia 1457 Growing evidence suggests that the capacity of B. cenocepacia to enter, survive and replicate intracellu- larly in human cells contributes significantly to pathogen- esis. Indeed, B. cenocepacia has been detected inside airway and alveolar epithelial cells, and macrophages in the lungs of CF patients (Sajjan et al., 2001a). Intracellular replication also has been observed in the mouse model of infection (Chiu et al., 2001; Sajjan et al., 2001b), and in vitro studies have demonstrated that B. cenocepacia enter and replicate in both macrophages and epithelial cells (Burns et al., 1996; Saini et al., 1999; Martin and Mohr, 2000). Recently, we found that B. cenocepacia of the ET12 lineage preferentially enter CF airway epithelial cells Fig. 1. Uptake of B. cenocepacia by IB3 cells. IB3 cells were infected (as compared with normal airway epithelial cells) and with 5 × 105, 5 × 106 or 5 × 107 B. cenocepacia (moi of 1, 10 or 100 replicate efficiently (Sajjan et al., 2004). However, the respectively) and incubated for 2 h. Extracellular bacteria were killed, processes involved in this and the mechanism(s) by IB3 cells were lysed, and serial dilutions of lysates were plated to determine the quantity of intracellular bacteria. Each bar represents which B. cenocepacia escape host defences are not well mean (± SEM) of six to nine experiments. understood. In this study, we demonstrate that B. cenocepacia ited bacterial uptake by IB3 cells (30% inhibition with readily enters CF airway epithelial cells by endocytosis, − 10 µg ml 1 colchicine; data not shown). escapes from the classical endocytic pathway and repli- cates in the endoplasmic reticulum (ER). In contrast, heat- killed bacteria enter epithelial cells to a much lesser extent Burkholderia cenocepacia replicate intracellularly in CF compared with live bacteria, enter the classical endocytic epithelial cells pathway and are degraded in lysosomes. To determine whether internalized B. cenocepacia repli- cate in CF airway epithelial cells, IB3 cells were infected with a moi of 1 or 10 and incubated for 2 h. Extracellular Results bacteria were killed by the addition of ceftazidime and Uptake of B. cenocepacia by immortalized CF airway gentamicin. Cells were further incubated for 24 h, 48 h or epithelial cells 72 h, lysed and plated to enumerate intracellular bacte- ria. After 24 h of incubation <1% of the infecting inoculum IB3 cells were infected at a multiplicity of infection (moi) of viable bacteria was recovered from lysed cells of 1, 10 or 100. After 2 h cells were washed, extracellular (Table 2). The concentration of bacteria increased at 48 h bacteria were killed and intracellular bacteria were quan- and 72 h post infection indicating survival and replication tified by plating serial dilutions of cell lysates. The level of of ingested bacteria. Up to 48 h post infection no appar- B. cenocepacia uptake was inoculum dependent (Fig. 1). ent change in cell viability was observed by trypan blue To determine if actin and microtubule formation is uptake assay in cells infected at a moi of 1, whereas cells involved in bacterial entry, IB3 cells were treated with infected at a moi of 10 showed extensive damage by either cytochalasin D or colchicine for 1 h prior to infection 48 h. To determine whether CF cells are inherently defi- with bacteria at a moi of 50. Cytochalasin D, which inhibits cient in killing ingested live bacteria, thereby supporting actin rearrangement, inhibited bacterial uptake in a dose- survival and replication of intracellular bacteria, we exam- dependent manner; maximum inhibition was observed with a dose of 2 µg ml−1 of cytochalasin D (Table 1). Actin rearrangement with bacterial uptake was confirmed by Table 1. Effect of cytochalasin D on uptake of B. cenocepacia by IB3 cells. staining F-actin with phalloidin conjugated to Alexa Fluor- 488. Uninfected IB3 cells showed an even distribution of Concentration of cytochalasin D (µg ml−1) Intracellular bacteria (cfu well−1) filamentous actin throughout the cytoplasm (Fig. 2A), whereas cells infected with B. cenocepacia showed F- 0 5.02 × 104 ± 7.77 × 102 actin polymerization, resulting in the formation of actin 0 (vehicle control) 5.18 × 104 ± 1.26 × 103 0.5 1.88 × 104 ± 1.12 × 102 stress fibres (Fig. 2B). Actin rearrangement was also 1.0 6.08 × 103 ± 6.30 × 102a observed in cells incubated with heat-killed bacteria 2.0 1.09 × 103 ± 1.15 × 102a 3 2a (Fig. 2C), suggesting that CF cells take up viable and non- 5.0 1.895 × 10 ± 3.72 × 10 viable bacteria by a similar endocytic pathway. Colchicine, a. P < 0.05 compared with vehicle control. which inhibits microtubule rearrangement, partially inhib- Values represent mean ± SEM of triplicate experiments. © 2006 The Authors Journal compilation © 2006 Blackwell Publishing Ltd, Cellular Microbiology, 8, 1456–1466 1458 U. S. Sajjan, J. H. Yang, M. B. Hershenson and J. J. LiPuma Fig. 2. Actin rearrangement in IB3 cells infected with B. cenocepacia. IB3 cells were grown in collagen-coated multichambered slides, serum starved overnight, infected with live or heat-killed B. cenocepacia at a moi of 10, and incubated for 2 h. F-actin was detected by Alexa Fluor 488- labelled phalloidin (green) and bacteria were detected by using rabbit antibody to B.