Burkholderia cepacia (previously referred to as Pseudomonas cepacia) was recognized as an important pathogen in CGD in 1975 (Bottone et al., 1975). Bcc is now the leading cause of fatality from bacterial infections in CGD patients (Winkelstein et al., 2000). CGD is a genetic disorder caused by the inactivation of the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. Because of this defect, some cells (neutrophils, mononuclear cells, macrophages, and eosinophils) in CGD patients are unable to produce reactive oxygen species (ROS), which results in ineffective clearance of some pathogens. These patients present with recurrent bacterial and fungal infections, which is frequently accompanied by uncontrolled inflammation and granuloma formation. The rate of incidence in the United States is 1/200,000 live births, or at least 20 patients per year (Holland, 2010).

Chronic granulomatous disease can be caused by mutations in any of the four components of NADPH oxidase. Flavocytochrome b₅₅₈ is the catalytic center of the complex, and is a heterodimer composed of p22^phox and gp91^phox. It is contained on the membrane of specific granules and is incorporated into the phagosome membrane upon fusion (Segal, 2005). It provides a channel for electrons to be transferred through from NADPH in the cytosol to oxygen that is contained in the phagosome (Segal, 2005). The complex also contains two cytosolic components: p67^phox and p47^phox (Zarember and Malech, 2011). These cytoplasmic components are essential for stabilizing flavocytochrome b₅₅₈ and facilitating the transfer of electrons. A defect in any one of these four components leads to inactivation of the complex and an inability to produce ROS. X-linked recessive CGD is caused by defects in gp91^phox. Autosomal recessive CGD is caused by defects in p47^phox, p67^phox, or p22^phox. Approximately 65% of CGD cases are X-linked (defects in gp91^phox), 25% of cases are due to defects in p47^phox, and the rest are split evenly between defects in p67^phox and p22^phox (Holland, 2010). There are two other genetic disorders with similar presentations to CGD that are not caused by mutations in NADPH oxidase components. Inhibitory mutations in Rac or deficiencies of glucose-6-phosphate dehydrogenase can also result in an inability to produce ROS (Johnston, 2001).

Overall mortality for CGD patients is around 2–5% per year, but morbidity remains a major issue for these patients and their families, as most patients have at least one severe infection every 3–4years (Winkelstein et al., 2000; Marciano et al., 2004). In the initial cases of CGD diagnosed between 1957 and 1976, Staphylococcus was the most common infectious agent, followed by the common enteric pathogens Klebsiella species and Escherichia coli (Johnston, 2001). Now Aspergillus species are the most common causative infectious agents, followed by Bcc and Staphylococcus aureus (Winkelstein et al., 2000). The most common presentations of infection include pneumonia, infectious dermatitis, and recurrent/severe abscess formation beneath the skin and in organs (Winkelstein et al., 2000). Other symptoms have been described that are not caused directly by infection, including enteritis/colitis, discoid lupus, and chorioretinitis (Goldblatt et al., 1999; Segal et al., 2000; Winkelstein et al., 2000). The treatment regimen for these patients includes interferon-γ, antibacterial prophylaxis, and antifungal prophylaxis. Prophylactic treatment usually includes trimethoprim–sulfamethoxazole and itraconazole (Seger, 2008). Bone marrow transplantation can cure the disease if a histocompatible donor is available, which is not an option for most patients. As with other monogenetic diseases, gene therapy has the potential to be an effective curative treatment for CGD, however, to date, clinical trials have not been successful. The promise and problems associated with this approach have been recently reviewed (Grez et al., 2011).

Cystic fibrosis is caused by mutations in the CF transmembrane conductance regulator (CFTR), which functions as an ion channel. This defect results in the accumulation of mucus on epithelial surfaces, which contributes to ineffective mucociliary clearance in the lungs. CF is one of the most common inherited genetic diseases in the world with an annual frequency of approximately 1/3,000 live births (Hayes et al., 2011). In fact, one out of every 25 Caucasians is a heterozygous carrier of a mutated CFTR gene (Sheppard and Nicholson, 2002). The airway in CF patients is chronically colonized with potentially pathogenic bacteria, specifically Pseudomonas aeruginosa, S. aureus, and Haemophilus influenzae. Colonization and frequent infection of the CF airway leads to an excessive neutrophil (polymorphonuclear leukocyte, PMN) driven inflammatory response, which results in host tissue damage (McElvaney et al., 1992; Birrer et al., 1994). These patients experience recurrent respiratory infections and chronic lung inflammation that eventually culminates in respiratory failure (Dinwiddie, 2000). Pulmonary inflammation in CF is associated with high levels of inflammatory cytokines (IL-8, IL-6, TNF-alpha, and leukotriene B₄), reduced levels of anti-inflammatory cytokines and antiproteases, and a rapid and substantial influx of PMNs (Rowe et al., 2005).

Burkholderia cepacia complex infections in both the CGD and CF patient populations initially present as a respiratory tract infection. An interesting distinction between infections in these two patient populations is the clinical presentation of this infection. In CF, pulmonary infection with Bcc generally causes endobronchial disease, and diagnosis is made by identification of bacteria in the sputum. In CGD, Bcc lung infection results in a pneumonic process associated with nodular infiltrates and diagnosis requires isolation following lung biopsy. Antibiotic treatment is generally able to clear Bcc in CGD, although recurrent re-infections do occur. In CF, Bcc infections are usually seen in older patients that are already chronically colonized with other CF pathogens, notably P. aeruginosa (Sheppard and Nicholson, 2002). According to the 2009 Cystic Fibrosis Foundation Registry Report, about 2.7% of CF patients are infected with Bcc, and these infections are associated with earlier fatality than non-Bcc-infected patients. Potentially due to the chronic nature of Bcc infections in CF and the sustained presence of the microbial community in the form of biofilms in the CF lung, Bcc isolates associated with CF show a higher degree of antibiotic resistance than Bcc isolates from CGD infections (Greenberg et al., 2009). Bcc infections in CF patients can result in a severe, necrotizing pneumonia, referred to as “cepacia syndrome,” that is not observed in Bcc infections in CGD patients. However, Bcc infections in CGD patients can become invasive and result in septicemia.

Polymorphonuclear leukocytes are highly motile phagocytes that comprise the first line of defense of the innate immune system against bacteria and play a crucial role in host defense in the lung. Four percent of the adult body mass is devoted to the production of PMNs, and as a result, PMNs are the most common nucleated cell in blood. Although PMNs are terminally differentiated once they leave bone marrow, they are still able to act autonomously as wandering phagocytes in all tissues, requiring highly specific and specialized responses (Zarember and Malech, 2011). They kill bacteria by fusing granules containing antimicrobial compounds with phagocytic compartments containing bacteria. These granules can also fuse with the plasma membrane to kill extracellular bacteria.

There are three types of granules that PMNs synthesize, and these different granule types contain molecules with different antimicrobial activities that often work synergistically to kill microbes once fused with the phagosome. Granules contain components that kill bacteria through both oxidative and non-oxidative mechanisms. Azurophil or primary granules contain myeloperoxidase, three neutral proteinases (cathepsin G, elastase, and proteinase 3), bactericidal permeability-increasing protein (BPI), and defensins. Specific or secondary granules contain lactoferrin, transcobalamin II, lysozyme, gelatinase-associated lipocalin, and flavocytochrome b₅₅₈. Gelatinase or tertiary granules are classified by the presence of gelatinase but not lactoferrin and can contain any of the other components in specific granules. The matrix in granules contains negatively charged sulfated proteoglycans that sequester antimicrobial components until the granule is fused with the phagosome (Borregaard and Cowland, 1997; Segal, 2005).

Oxidative killing relies on the generation of ROS, primarily through NADPH oxidase. Electron transfer to oxygen through NADPH oxidase generates superoxide in the phagosome. Superoxide is rapidly degraded into hydrogen peroxide (H₂O₂) by superoxide dismutase. Hydrogen peroxide can readily cross bacterial membranes and cause intracellular damage. CGD PMNs are defective in their ability to assemble functional NADPH oxidase complexes and therefore are unable to utilize oxidative killing mechanisms.

In addition to oxidative killing mechanisms, normal PMNs can also use non-oxidative killing, which includes interactions between cationic regions of the granule proteins and the negatively charged bacterial membrane, as well as bacterial degradation by granule proteases. Examples of cationic peptides and proteins involved in non-oxidative killing of bacteria are BPI, LL-37, defensins, and azurocidin. These cationic granule peptides and proteins bind to and accumulate on the bacterial surface, leading to membrane permeabilization and osmotic lysis. These components are extremely important in non-oxidative killing of Gram-negative pathogens by PMNs (Hancock and Diamond, 2000). Granules also contain other components that possess different antimicrobial activities. Serine proteases degrade phagocytosed bacteria (Wiedow and Meyer-Hoffert, 2005). Lactoferrin sequesters iron away from pathogens, but also possesses direct antimicrobial activity (Ellison and Giehl, 1991). Many of the factors that function in non-oxidative killing strategies act synergistically or depend on each other for full activity after fusion or exocytosis. The lack of functional NADPH oxidase in CGD results in the reduced flux of potassium ions into the phagolysosome (Zarember and Malech, 2011). This influx plays a role in the release of cationic proteins from the matrix, and because of this CGD PMNs may also exhibit defects in non-oxidative killing. In CGD PMNs, the phagolysosome additionally does not increase pH and does not acidify as profoundly, which also has negative effects on the activity of antimicrobial peptides and enzymes (Dri et al., 2002).

Normal PMNs also use neutrophil extracellular traps (NETs) as a mechanism to kill extracellular microbes. These NETs are composed of PMN chromatin coated in granule components that have been expelled from the cell. The chromatin binds extracellular bacteria and fungi, trapping them in close contact with high concentrations of antimicrobial granule contents. NET formation is dependent on the generation of ROS by NADPH oxidase, and therefore CGD PMNs are also deficient in their ability to produce NETs (Fuchs et al., 2007). It has been shown that this defect also contributes to the inability of CGD PMNs to kill pathogens commonly associated with infections in CGD, specifically S. aureus and Aspergillus species (Brinkmann et al., 2004; Bianchi et al., 2009).

Most prior research has focused on the lung epithelial cell defects in CF (Conese and Assael, 2001; Machen, 2006). However more recently, there has been an emphasis in the field on defective immune cell function in the CF lung environment, particularly on potential defects in PMN function. These studies may assist in bridging the gap in understanding the specific susceptibilities of these two patient populations to Bcc infections. Defects in CF PMN function could contribute to ineffective clearance of lung pathogens in the presence of zealous PMN recruitment and inflammation (Hayes et al., 2011). It is already well established that PMN necrosis and release of proteolytic enzymes contribute significantly to the tissue damage in the CF lung. It has been shown that PMNs can become trapped in bacterial biofilms in the CF lung and undergo necrosis; after necrosis, their biomass, NETs, and intracellular leakage can contribute biofilm formation and lung injury (Jesaitis et al., 2003; Brinkmann et al., 2004; Walker et al., 2005). CF PMNs display excessive and prolonged superoxide production when stimulated, are constitutively primed (likely via the presence of IL-8, TNF-alpha, and P. aeruginosa alginate in the CF airway), and are unresponsive to anti-inflammatory signals through IL-10 (Pedersen et al., 1990; Koller et al., 1995; Taggart et al., 2000). Another observed potential defect is an alteration in degranulation; greater levels of primary granule components are secreted extracellularly by CF PMNs than by normal PMNs, possibly due to effects of intracellular pH on signaling (Koller et al., 1995; Coakley et al., 2000, 2002; Taggart et al., 2000). This release of toxic granule contents contributes to the extensive lung damage characteristic of CF. The absence of CFTR has also been shown to have direct effects on PMN function. The addition of chloride ion to hydrogen peroxide to produce hypochlorous acid (HOCl) through myeloperoxidase is another ROS-dependent killing mechanism, and CF PMNs display a decreased ability to chlorinate bacteria and a decreased ability to kill P. aeruginosa that is exacerbated by a chloride-deficient microenvironment (Painter et al., 2006, 2008).

There are also indications that the environment in the CF lung may significantly impair PMN function. High concentrations of neutrophil elastase present in the CF lung cleave CXCR1, Fcγ receptors, and iC3b, as well as lead to the loss of CD16 and CD14 expression (Tosi et al., 1990; Birrer et al., 1994; Alexis et al., 2006; Hartl et al., 2007; Tirouvanziam et al., 2008). The loss of these receptors leads to deceased complement-dependent phagocytosis. Hypersalinity of the airway surface fluid has been shown to inhibit the killing actions of epithelial cells in the CF lung through inactivation of β-defensin-1 and potentially other antimicrobials, although these findings are considered somewhat controversial in the CF field (Smith et al., 1996; Goldman et al., 1997; Krouse, 2001). Peripheral PMNs isolated from CF patients show comparable NET formation to normal PMNs, but there is also evidence that PMNs in the CF lung environment display increased NET formation that contributes to the pulmonary obstruction typical in CF (Marcos et al., 2010; Young et al., 2011). Further investigation into defective phagocyte function in CF could provide insight into links between the susceptibilities of both these patient populations to Bcc infections.

The autosomal recessive mouse model of CGD lacks the mouse equivalent of p47^phox, which is the complex component most commonly associated with human cases of autosomal recessive CGD. Characterization of this model showed a phenotype similar to human CGD; these mice spontaneously developed severe infections, usually by the same or related pathogens that are associated with infections in human CGD hosts (Jackson et al., 1995). Phagocytes from these animals display an inability to produce an oxidative burst and are unable to kill S. aureus and other pathogens characteristic of CGD (Jackson et al., 1995). This model has also been used to study the virulence of Bcc in CGD. B. multivorans clinical isolates from both CGD and CF patients were shown to establish lethal infections when injected intraperitoneally, while an environmental isolate was avirulent (Zelazny et al., 2009).

Mouse models allow analysis of Bcc virulence in a CGD host, but additional approaches are necessary to directly investigate Bcc interactions with host phagocytes. Both cell lines and primary cells have been used in assessment of bacterial virulence in interactions with CGD phagocytes. PMNs and macrophages can be derived from CGD patients, when available, or from CGD animals (Morgenstern et al., 1997; Kaneda et al., 1999; Zelazny et al., 2009). Because these cells are isolated directly from patients or animals with CGD, these cells most closely replicate the CGD defect, but unfortunately are unable to be genetically modified or adequately controlled experimentally. Also, the use of primary cells adds an additional level of variation to experiments due to donor-specific genetic and environmental factors. Cell lines that are genetically modified to lack functional NADPH oxidase proteins have also been constructed. PBL-985, a human myeloid leukemia cell line, is deficient in gp91^phox and can be differentiated into monocytes or granulocytes (Zhen et al., 1993). These cells can be further genetically modified and lack the donor variability of primary cells. Unfortunately, PMNs are terminally differentiated cells, and precursor immortal cell lines must be differentiated in vitro into a PMN-like state. In a related promyelocytic cell line, HL-60, it has been demonstrated that in vitro differentiation from the promyelocytic stage results in an inability to synthesize secondary and tertiary granules (Le Cabec et al., 1997). Therefore, these precursor cell lines lack the full array of antimicrobial activities associated with mature, terminal PMNs. There are also variants of the J774.16 murine macrophage cell line that are deficient in gp91^phox, and do not need to be differentiated (Goldberg et al., 1990). These immortal macrophage lines can be used to broadly study phagocyte interactions but are unable to utilize the full repertoire of killing mechanisms employed by PMNs.

B. cenocepacia has been shown to be readily ingested by normal PMNs and phagocytosis is associated with a robust oxidative burst. In PMN bactericidal assays, Bcc is killed by normal PMNs but is able to persist when incubated with CGD PMNs (Zelazny et al., 2009; Greenberg et al., 2010). Similarly B. cenocepacia has been found to induce PMN apoptosis, an effect dependent on viable bacteria. CGD PMNs were also equally apoptotic in comparison to normal PMNs after phagocytosis of B. cenocepacia, but B. cenocepacia also induced PMN necrosis in a subset of CGD PMNs, which could contribute to increased virulence in this population. This was true in both primary CGD PMNs and PMNs treated with DPI (Bylund et al., 2005). Another study found that a CGD clinical isolate of B. multivorans showed a higher association with CGD PMNs than with PMNs from normal, healthy donors, which suggests an additional distinction between the interactions of Bcc with normal and CGD PMNs (Zelazny et al., 2009).

A majority of the work investigating Bcc–host phagocyte interactions up to now has focused on macrophages, mainly due to the availability of immortal cell lines. Due to the large influx of PMNs to the site of infection in both CF and CGD, PMNs are playing an extremely important role in pathogenesis. The bacterial killing mechanisms used by PMNs are more numerous and varied than that of macrophages, and the process of phagosomal maturation also varies greatly between PMNs and macrophages. It is thought that Bcc exists in an intracellular Bcc-containing vacuole (BcCV) in macrophages, but it is unknown whether Bcc is able to persist in a similar compartment in PMNs (Lamothe et al., 2007). It has been shown that both the type IV and type VI secretion systems play a role in interactions with macrophages and may be important for intracellular survival, but it is unknown what their role is in interactions with PMNs (Aubert et al., 2008; Sajjan et al., 2008). It has been demonstrated that Bcc is ineffectively killed and cleared by CGD PMNs (Zelazny et al., 2009; Greenberg et al., 2010), but it is not clear whether the surviving Bcc are intracellular or associated extracellularly with PMNs. This distinction is very important for understanding the mechanism in which Bcc is able to persist in this patient population.

Another avenue for future research is to identify mechanisms of effective killing and clearance of Bcc in normal individuals. Normal PMNs are able to effectively kill Bcc while CGD PMNs are unable to do so. Because of the defect in oxidative killing in CGD, this was suggestive of oxidative killing being important for clearance by normal PMNs. However, in vitro studies have demonstrated high resistance of Bcc to both oxidative and non-oxidative killing mechanisms. There are also other defects in non-oxidative killing associated with CGD PMNs, specifically in granule component activation and NET formation. It could also be the synergistic effects of all of these defects together that allow for persistence in this patient population as well as in CF patients. These mechanistic studies are difficult in a terminally differentiated cell that is not amenable to genetic manipulation but could be feasible in vitro using purified components or in cell-based assays using combinations of inhibitors.

A more broad implication of understanding Bcc pathogenesis in CGD is to search for commonalities in virulence strategies employed by all CGD pathogens. Since the spectrum of potential pathogens causing infections in this population is very narrow and most infectious agents are opportunistic, there may be very specific factors or strategies utilized by this group of microbes to cause infections in CGD. Understanding these overarching themes of resistance to killing in the CGD host could lead to the development of both preventative care and treatment options.