Macrophages, but not neutrophils, are critical for proliferation of Burkholderia cenocepacia and ensuing host-damaging inflammation.

Bacteria of the Burkholderia cepacia complex (Bcc) can cause devastating pulmonary infections in cystic fibrosis (CF) patients, yet the precise mechanisms underlying inflammation, recurrent exacerbations and transition from chronic stages to acute infection and septicemia are not known. Bcc bacteria are generally believed to have a predominant extracellular biofilm life style in infected CF lungs, similar to Pseudomonas aeruginosa, but this has been challenged by clinical observations which show Bcc bacteria predominantly in macrophages. More recently, Bcc bacteria have emerged in nosocomial infections of patients hospitalized for reasons unrelated to CF. Research has abundantly shown that Bcc bacteria can survive and replicate in mammalian cells in vitro, yet the importance of an intracellular life style during infection in humans is unknown. Here we studied the contribution of innate immune cell types to fatal pro-inflammatory infection caused by B. cenocepacia using zebrafish larvae. In strong contrast to the usual protective role for macrophages against microbes, our results show that these phagocytes significantly worsen disease outcome. We provide new insight that macrophages are critical for multiplication of B. cenocepacia in the host and for development of a fatal, pro-inflammatory response that partially depends on Il1-signalling. In contrast, neutrophils did not significantly contribute to disease outcome. In subcutaneous infections that are dominated by neutrophil-driven phagocytosis, the absence of a functional NADPH oxidase complex resulted in a small but measurably higher increase in bacterial growth suggesting the oxidative burst helps limit bacterial multiplication; however, neutrophils were unable to clear the bacteria. We suggest that paradigm-changing approaches are needed for development of novel antimicrobials to efficiently disarm intracellular bacteria of this group of highly persistent, opportunistic pathogens.

Evolving serodiagnostics by rationally designed peptide arrays: the Burkholderia paradigm in Cystic Fibrosis.

Efficient diagnosis of emerging and novel bacterial infections is fundamental to guide decisions on therapeutic treatments. Here, we engineered a novel rational strategy to design peptide microarray platforms, which combines structural and genomic analyses to predict the binding interfaces between diverse protein antigens and antibodies against Burkholderia cepacia complex infections present in the sera of Cystic Fibrosis (CF) patients. The predicted binding interfaces on the antigens are synthesized in the form of isolated peptides and chemically optimized for controlled orientation on the surface. Our platform displays multiple Burkholderia-related epitopes and is shown to diagnose infected individuals even in presence of superinfections caused by other prevalent CF pathogens, with limited cost and time requirements. Moreover, our data point out that the specific patterns determined by combined probe responses might provide a characterization of Burkholderia infections even at the subtype level (genomovars). The method is general and immediately applicable to other bacteria.

Distinct colicin M-like bacteriocin-immunity pairs in Burkholderia.

The Escherichia coli bacteriocin colicin M (ColM) acts via degradation of the cell wall precursor lipid II in target cells. ColM producers avoid self-inhibition by a periplasmic immunity protein anchored in the inner membrane. In this study, we identified colM-like bacteriocin genes in genomes of several ß-proteobacterial strains belonging to the Burkholderia cepacia complex (Bcc) and the Burkholderia pseudomallei group. Two selected Burkholderia ambifaria proteins, designated burkhocins M1 and M2, were produced recombinantly and showed antagonistic activity against Bcc strains. In their considerably sequence-diverged catalytic domain, a conserved aspartate residue equally proved pivotal for cytotoxicity. Immunity to M-type burkhocins is conferred upon susceptible strains by heterologous expression of a cognate gene located either upstream or downstream of the toxin gene. These genes lack homology with currently known ColM immunity genes and encode inner membrane-associated proteins of two distinct types, differing in predicted transmembrane topology and moiety exposed to the periplasm. The addition of burkhocins to the bacteriocin complement of Burkholderia reveals a wider phylogenetic distribution of ColM-like bacteriotoxins, beyond the γ-proteobacterial genera Escherichia, Pectobacterium and Pseudomonas, and illuminates the diversified nature of immunity-providing proteins.

Residence in biofilms allows Burkholderia cepacia complex (Bcc) bacteria to evade the antimicrobial activities of neutrophil-like dHL60 cells.

Bacteria of the Burkholderia cepacia complex (Bcc) persist in the airways of people with cystic fibrosis (CF) despite the continuous recruitment of neutrophils. Most members of Bcc are multidrug resistant and can form biofilms. As such, we sought to investigate whether biofilm formation plays a role in protecting Bcc bacteria from neutrophils. Using the neutrophil-like, differentiated cell line, dHL60, we have shown for the first time that Bcc biofilms are enhanced in the presence of these cells. Biofilm biomass was greater following culture in the presence of dHL60 cells than in their absence, likely the result of incorporating dHL60 cellular debris into the biofilm. Moreover, we have demonstrated that mature biofilms (cultured for up to 72 h) induced necrosis in the cells. Established biofilms also acted as a barrier to the migration of the cells and masked the bacteria from being recognized by the cells; dHL60 cells expressed less IL-8 mRNA and secreted significantly less IL-8 when cultured in the presence of biofilms, with respect to planktonic bacteria. Our findings provide evidence that biofilm formation can, at least partly, enable the persistence of Bcc bacteria in the CF airway and emphasize a requirement for anti-biofilm therapeutics.

Immunoproteomic analysis of proteins expressed by two related pathogens, Burkholderia multivorans and Burkholderia cenocepacia, during human infection.

Burkholderia cepacia complex (Bcc) is an opportunistic bacterial pathogen that causes chronic infections in people with cystic fibrosis (CF). It is a highly antibiotic resistant organism and Bcc infections are rarely cleared from patients, once they are colonized. The two most clinically relevant species within Bcc are Burkholderia cenocepacia and Burkholderia multivorans. The virulence of these pathogens has not been fully elucidated and the virulence proteins expressed during human infection have not been identified to date. Furthermore, given its antibiotic resistance, prevention of infection with a prophylactic vaccine may represent a better alternative than eradication of an existing infection. We have compared the immunoproteome of two strains each from these two species of Bcc, with the aim of identifying immunogenic proteins which are common to both species. Fourteen immunoreactive proteins were exclusive to both B. cenocepacia strains, while 15 were exclusive to B. multivorans. A total of 15 proteins were immunogenic across both species. DNA-directed RNA polymerase, GroEL, 38kDa porin and elongation factor-Tu were immunoreactive proteins expressed by all four strains examined. Many proteins which were immunoreactive in both species, warrant further investigations in order to aid in the elucidation of the mechanisms of pathogenesis of this difficult organism. In addition, identification of some of these could also allow the development of protective vaccines which may prevent colonisation.

Burkholderia multivorans survival and trafficking within macrophages.

Cystic fibrosis (CF) patients are at great risk of opportunistic lung infection, particularly by members of the Burkholderia cepacia complex (Bcc). This group of bacteria can cause damage to the lung tissue of infected patients and are difficult to eradicate due to their high levels of antibiotic resistance. Although the highly virulent Burkholderia cenocepacia has been the focus of virulence research for the past decade, Burkholderia multivorans is emerging as the most prevalent Bcc species infecting CF patients in North America. Despite several studies detailing the intramacrophage trafficking and survival of B. cenocepacia, no such data exist for B. multivorans. The results of this study demonstrated that the clinical CF isolates C5568 and C0514 and an environmental B. multivorans isolate, ATCC 17616, were able to replicate and survive within murine macrophages in a manner similar to that of B. cenocepacia strain K56-2. These strains were also able to survive but were unable to replicate within human THP-1 macrophages. Differences in macrophage uptake were observed among all three B. multivorans strains; these variances were attributed to major differences in O-antigen production. Unlike B. cenocepacia-containing vacuoles, which delay phagosomal maturation in murine macrophages by 6 h, all B. multivorans-containing vacuoles co-localized with lysosome-associated membrane protein-1, a late endosome/lysosomal marker, and the lysosomal marker dextran within 2 h of uptake. Together, these results indicated that, whilst both Bcc species were able to survive and replicate within macrophages, they utilized different intramacrophage survival strategies. To observe differences in virulence, the strains were compared using the Galleria mellonella (wax worm) model. When compared with the B. multivorans strains tested, B. cenocepacia K56-2 was highly virulent in this model and killed all worms within 24 h when injected at 10(7) c.f.u. B. multivorans clinical isolates C5568 and C0514 were significantly more virulent than the soil isolate ATCC 17616, which was avirulent even when worms were injected with 10(7) c.f.u. These results suggest strain differences in the virulence of B. multivorans isolates.

Serum susceptibility in clinical isolates of Burkholderia cepacia complex bacteria: development of a growth-based assay for high throughput determination.

Burkholderia cepacia complex (BCC) bacteria can cause devastating chronic infections in people with cystic fibrosis. Of particular concern is "cepacia syndrome," a rapidly progressive and usually fatal decline in health, characterized by a necrotizing bacteremic pneumonia. An important component of defense against bloodstream infections is the bactericidal action of serum. Traditional methods to determine the capacity of bacterial isolates to resist the bactericidal effects of serum are relatively low-throughput viability assays. In this study, we developed a novel growth-based assay for serum susceptibility, which allows for high throughput analysis. We applied this assay to a range of clinical isolates of BCC as well as isolates comprising the BCC experimental strain panel. Our data demonstrate that isolates from all species of BCC examined can possess serum resistant or serum sensitive/intermediate phenotypes. Of particular clinical significance, we also found no direct link between the last saved pulmonary isolate from patients who subsequently developed "cepacia syndrome" and their capacity to resist the inhibitory effects of human serum, suggesting serum resistance cannot be used as a marker of an isolate's capacity to escape from the lung and cause bacteremia.

Targeting pan-resistant bacteria with antibodies to a broadly conserved surface polysaccharide expressed during infection.

BACKGROUND: New therapeutic targets for antibiotic-resistant bacterial pathogens are desperately needed. The bacterial surface polysaccharide poly-ß-(1-6)-N-acetyl-glucosamine (PNAG) mediates biofilm formation by some bacterial species, and antibodies to PNAG can confer protective immunity. By analyzing sequenced genomes, we found that potentially multidrug-resistant bacterial species such as Klebsiella pneumoniae, Enterobacter cloacae, Stenotrophomonas maltophilia, and the Burkholderia cepacia complex (BCC) may be able to produce PNAG. Among patients with cystic fibrosis patients, highly antibiotic-resistant bacteria in the BCC have emerged as problematic pathogens, providing an impetus to study the potential of PNAG to be targeted for immunotherapy against pan-resistant bacterial pathogens. METHODS: The presence of PNAG on BCC was assessed using a combination of bacterial genetics, microscopy, and immunochemical approaches. Antibodies to PNAG were tested using opsonophagocytic assays and for protective efficacy against lethal peritonitis in mice. RESULTS: PNAG is expressed in vitro and in vivo by the BCC, and cystic fibrosis patients infected by the BCC species B. dolosa mounted a PNAG-specific opsonophagocytic antibody response. Antisera to PNAG mediated opsonophagocytic killing of BCC and were protective against lethal BCC peritonitis even during coinfection with methicillin-resistant Staphylococcus aureus. CONCLUSIONS: Our findings raise potential new therapeutic options against PNAG-producing bacteria, including even pan-resistant pathogens.

IL-8 released from human lung epithelial cells induced by cystic fibrosis pathogens Burkholderia cepacia complex affects the growth and intracellular survival of bacteria.

Burkholderia cepacia complex (Bcc) is a group of Gram-negative pulmonary pathogens associated with life-threatening infections in patients with cystic fibrosis (CF). The airway epithelium plays a crucial role in the initiation and modulation of inflammatory responses to these pathogens. Interleukin (IL)-8 released from epithelial cells is a potent chemoattractant for neutrophils. The aims of this study were to compare the IL-8 response to Bcc infection in different epithelial cell types and to investigate the impact of IL-8 on Bcc growth and intracellular survival. To compare epithelial cell IL-8 responses, 4 human epithelial cell lines were used in the study; A549 cells, an alveolar epithelial cell line, Calu-3 cells, a sub-bronchial epithelial cell line, 16HBE14o- cells, and CFBE41o- cells, which are CFTR-positive and CFTR-negative bronchial epithelial cell lines, respectively. Two B. multivorans and 2 B. cenocepacia strains all induced a significant IL-8 response by 12 h and further increased in all cell lines at 24 h. Furthermore, the levels of IL-8 from Calu-3 and A549 cells were approximately 3 times that of 16HBE14o- or CFBE41o- cells. In 2 of the cell lines examined (16HBE14o- and CFBE41o-), B. cenocepacia LMG 16656 (J2315), an epidemic strain, induced greater levels of IL-8 (P<0.01) compared to other Bcc strains tested. The CFTR-positive and -negative cell lines secreted similar levels of IL-8 indicating a CFTR-independent induction of IL-8. However, the CFTR-negative cells did secrete constitutive levels of IL-8 greater than that of CFTR-positive cells. An investigation of the effect of IL-8 on Bcc extracellular and intracellular growth found that at low concentrations (<10 ng/ml) of recombinant human (rh) IL-8, the growth of B. cenocepacia LMG 16656 and B. multivorans LMG 13010 was enhanced, whereas at higher concentrations (10 ng/ml), growth of both strains was significantly reduced. Growth of both non-CF Bcc strains remained unchanged in the presence of rhIL-8. In contrast to extracellular growth, higher concentrations (10ng/ml) of rhIL-8 enhance the intracellular growth and survival of both LMG 16656 and LMG 13010 in 16HBE14o- and CFBE41o- cell lines. Although LMG 13010 uptake by epithelial cells was higher than LMG 16656 (P<0.01), the intracellular growth of LMG 16656 is greater than LMG 13010 (P<0.05). These studies demonstrated that the type of epithelial cells encountered by Bcc strains determines the extent of the IL-8 responses triggered and that this cytokine in addition to its well-established proinflammatory properties can enhance both the extracellular and intracellular growth of Bcc strains.

Macrophage responses to CF pathogens: JNK MAP kinase signaling by Burkholderia cepacia complex lipopolysaccharide.

Chronic bacterial colonization of the airways with opportunistic pathogens is the primary cause of morbidity and mortality in cystic fibrosis (CF) patients. Burkholderia cepacia complex (Bcc) organisms pose a particular challenge in CF lung disease, due in part to their ability to trigger a fulminant pneumonia. This study compares the U937 macrophage response to two Bcc species, B. cenocepacia and Burkholderia multivorans, against Pseudomonas aeruginosa and Staphylococcus aureus. The two Bcc strains demonstrated higher levels of U937 macrophage internalization compared with both P. aeruginosa and S. aureus. Both the Bcc strains also stimulated significantly greater levels of tumor necrosis factor-α and interleukin-1ß from macrophages when compared with P. aeruginosa. Further examination of the macrophage response to B. multivorans demonstrated that the lipopolysaccharide component of these bacteria was a potent inducer of proinflammatory cytokines and was shown to signal predominantly through the c-Jun N-terminal kinase mitogen-activated protein kinase pathway. These studies further characterize the host response to Bcc and in particular B. multivorans, now the predominant Bcc species in many CF populations.

Induction of immune response to the 17 kDa OMPA Burkholderia cenocepacia polypeptide and protection against pulmonary infection in mice after nasal vaccination with an OMP nanoemulsion-based vaccine.

Burkholderia cepacia complex (Bcc) are opportunistic bacteria associated with life-threatening illness in persons with cystic fibrosis. Once Bcc colonization is established, these antimicrobial-resistant and biofilm-forming bacteria are difficult to eradicate and are associated with increased rates of morbidity and mortality. At present, no vaccines are available to prevent the Bcc infection. There is currently a paucity of published information regarding the development of vaccines designed to prevent Burkholderia colonization. This work expands on the recent studies published by Bertot et al. [Infect Immun 75(6):2740-2752, 2007], where successful protective immune responses were generated in mice using a B. multivorans OMP-based vaccine. Here, we evaluate an experimental mucosal vaccine against Bcc using a novel mucosal adjuvant (nanoemulsion) and a novel B. cenocepacia-based OMP antigen. The OMP antigen derived from B. cenocepacia was mixed with either nanoemulsion or with PBS and delivered intranasally to CD-1 mice. Serum analysis showed robust IgG and mucosal secretory IgA immune responses in vaccinated versus control mice. The antibodies had cross-neutralizing activity against both B. cenocepacia and B. multivorans species. We found that immunized mice were protected against pulmonary colonization with B. cenocepacia. We have also identified that a 17 kDa OmpA-like protein highly conserved between Burkholderia and Ralstonia species as a new immunodominant epitope in mucosal immunization.

Differential modulation of innate immune cell functions by the Burkholderia cepacia complex: Burkholderia cenocepacia but not Burkholderia multivorans disrupts maturation and induces necrosis in human dendritic cells.

Burkholderia cepacia complex (BCC) bacteria cause pulmonary infections that can evolve into fatal overwhelming septicemia in chronic granulomatous disease or cystic fibrosis patients. Burkholderia cenocepacia and Burkholderia multivorans are responsible for the majority of BCC infections in cystic fibrosis patients, but B. cenocepacia is generally associated with a poorer prognosis than B. multivorans. The present study investigated whether these pathogens could modulate the normal functions of primary human monocyte-derived dendritic cells (DCs), important phagocytic cells that act as critical orchestrators of the immune response. Effects of the bacteria on maturation of DCs were determined using flow cytometry. DCs co-incubated for 24 h with B. cenocepacia, but not B. multivorans, had reduced expression of costimulatory molecules when compared with standard BCC lipopolysaccharide-matured DCs. B. cenocepacia, but not B. multivorans, also induced necrosis in DCs after 24 h, as determined by annexin V and propidium iodide staining. DC necrosis only occurred after phagocytosis of live B. cenocepacia; DCs exposed to heat-killed bacteria, bacterial supernatant or those pre-treated with cytochalasin D then exposed to live bacteria remained viable. The ability of B. cenocepacia to interfere with normal DC maturation and induce necrosis may contribute to its pathogenicity in susceptible hosts.

Chemical and biological features of Burkholderia cepacia complex lipopolysaccharides.

The Burkholderia cepacia complex comprises 10 closely related Gram-negative organisms all of which appear capable of causing disease in humans. These organisms appear of particular relevance to patients with cystic fibrosis. Lipopolysaccharide (LPS) is an important virulence determinant in Gram-negative pathogens. In this review, we highlight important data within the field commenting on LPS/lipid A structure-to-function relationships and cytokine induction capacity of Burkholderia strains studied so far.

Burkholderia cenocepacia induces neutrophil necrosis in chronic granulomatous disease.

Burkholderia cepacia complex is a life-threatening group of pathogens for patients with chronic granulomatous disease (CGD), whose phagocytes are unable to produce reactive oxygen species (ROS). Unlike other CGD pathogens, B. cepacia complex is particularly virulent, characteristically causing septicemia, and is the bacterial species responsible for most fatalities in these patients. We found that a nonmucoid Burkholderia cenocepacia (a predominant species in the B. cepacia complex) isolate was readily ingested by normal human neutrophils under nonopsonic conditions and promoted apoptosis in these cells. The proapoptotic effect was not due to secreted bacterial products, but was dependent on bacterial viability. Phagocytosis was associated with a robust production of ROS, and the apoptotic neutrophils could be effectively cleared by monocyte-derived macrophages. The proapoptotic effect of B. cenocepacia was independent of ROS production because neutrophils from CGD patients were rendered apoptotic to a similar degree as control cells after challenge. More importantly, neutrophils from CGD patients, but not from normal individuals, were rendered necrotic after phagocytosis of B. cenocepacia. The extreme virulence of B. cepacia complex bacteria in CGD, but not in immunocompetent hosts, could be due to its necrotic potential in the absence of ROS.

Lipopolysaccharide of Burkholderia cepacia complex.

Burkholderia cepacia complex (Bcc) is a group of phenotypically similar, genetically distinct bacteria that are beneficial to the environment but can also cause severe human infections. Bcc are being exploited for use as bioremediation agents and as a way to combat agricultural plant diseases. However, Bcc can cause lung infections in patients with chronic granulomatous disease or cystic fibrosis often resulting in mortality of these patients. Since it is unclear what bacterial components are necessary for causing human infections, studies of Bcc have focused on identifying putative virulence factors. As in other Gram-negative bacteria, the lipopolysaccharide (LPS) of Bcc induces a strong immune response that can contribute to host cell damage. The unusual structure of Bcc LPS lowers the anionic charge of the Bcc cell surface, which inhibits the binding and subsequent effects of cationic antibiotics. These distinguishing features include the substitution of a Ko for a Kdo residue in the inner core oligosaccharide and Ara4N residues bound to phosphates of the lipid A backbone. The structures of O antigen subunits and the consequent serotypes will also be discussed, with particular reference to the O antigen biosynthetic loci of two Bcc strains.