Quantitative proteomics (2-D DIGE) reveals molecular strategies employed by Burkholderia cenocepacia to adapt to the airways of cystic fibrosis patients under antimicrobial therapy

Chronic respiratory infections caused by Burkholderia cenocepacia in patients with cystic fibrosis (CF) are characterized by low responsiveness to antibiotic therapy and, in general, to a more rapid decline of lung function. To get clues into the molecular mechanisms underlying the adaptive strategies employed to deal with the stressing conditions of the CF lung including antibiotic therapy, quantitative proteomics (2-D DIGE) was used to compare the expression programs of two clonal isolates retrieved from a chronically infected CF patient. Isolate IST439 was the first bacterium recovered while the clonal variant IST4113 was obtained after 3 years of persistent infection and intravenous therapy with ceftazidime/gentamicin. This isolate exhibits higher resistance levels towards different classes of antimicrobials. Proteins of the functional categories Energy metabolism, Translation, Nucleotide synthesis, Protein folding and stabilization are more abundant in IST4113, compared with IST439, suggesting an increased protein synthesis, DNA repair and stress resistance in IST4113. The level of proteins involved in peptidoglycan, membrane lipids and lipopolysaccharide synthesis is also altered and proteins involved in iron binding and transport are more abundant in IST4113. The quantitative comparison of the two proteomes suggests a genetic adaptation leading to increased antimicrobial resistance and bacterial persistence in the CF airways.     

Structural Stability of Burkholderia cenocepacia Biofilms Is Reliant on eDNA Structure and Presence of a Bacterial Nucleic Acid Binding Protein

Cystic fibrosis (CF) is the most common lethal inherited genetic disorder affection Caucasians. Even with medical advances, CF is life-shortening with patients typically surviving only to age 38. Infection of the CF lung by Burkholderia cenocepacia presents exceptional challenges to medical management of these patients as clinically this microbe is resistant to virtually all antibiotics, is highly transmissible and infection of CF patients with this microbe renders them ineligible for lung transplant, often the last lifesaving option. Here we have targeted two abundant components of the B. cenocepacia biofilm for immune intervention: extracellular DNA and DNABII proteins, the latter of which are bacterial nucleic acid binding proteins. Treatment of B. cenocepacia biofilms with antiserum directed at one of these DNABII proteins (integration host factor or IHF) resulted in significant disruption of the biofilm. Moreover, when anti-IHF mediated destabilization of a B. cenocepacia biofilm was combined with exposure to traditional antibiotics, B. cenocepacia resident within the biofilm and thereby typically highly resistant to the action of antibiotics, were now rendered susceptible to killing. Pre-incubation of B. cenocepacia with anti-IHF serum prior to exposure to murine CF macrophages, which are normally unable to effectively degrade ingested B. cenocepacia, resulted in a statistically significant increase in killing of phagocytized B. cenocepacia. Collectively, these findings support further development of strategies that target DNABII proteins as a novel approach for treatment of CF patients, particularly those whose lungs are infected with B. cenocepacia.     

The genome of Burkholderia cenocepacia J2315, an epidemic pathogen of cystic fibrosis patients

Bacterial infections of the lungs of cystic fibrosis (CF) patients cause major complications in the treatment of this common genetic disease. Burkholderia cenocepacia infection is particularly problematic since this organism has high levels of antibiotic resistance, making it difficult to eradicate; the resulting chronic infections are associated with severe declines in lung function and increased mortality rates. B. cenocepacia strain J2315 was isolated from a CF patient and is a member of the epidemic ET12 lineage that originated in Canada or the United Kingdom and spread to Europe. The 8.06-Mb genome of this highly transmissible pathogen comprises three circular chromosomes and a plasmid and encodes a broad array of functions typical of this metabolically versatile genus, as well as numerous virulence and drug resistance functions. Although B. cenocepacia strains can be isolated from soil and can be pathogenic to both plants and man, J2315 is representative of a lineage of B. cenocepacia rarely isolated from the environment and which spreads between CF patients. Comparative analysis revealed that ca. 21% of the genome is unique in comparison to other strains of B. cenocepacia, highlighting the genomic plasticity of this species. Pseudogenes in virulence determinants suggest that the pathogenic response of J2315 may have been recently selected to promote persistence in the CF lung. The J2315 genome contains evidence that its unique and highly adapted genetic content has played a significant role in its success as an epidemic CF pathogen.     

Genetic Diversity and Multihost Pathogenicity of Clinical and Environmental Strains of Burkholderia cenocepacia

A collection of 54 clinical and agricultural isolates of Burkholderia cenocepacia was analyzed for genetic relatedness by using multilocus sequence typing (MLST), pathogenicity by using onion and nematode infection models, antifungal activity, and the distribution of three marker genes associated with virulence. The majority of clinical isolates were obtained from cystic fibrosis (CF) patients in Michigan, and the agricultural isolates were predominantly from Michigan onion fields. MLST analysis resolved 23 distinct sequence types (STs), 11 of which were novel. Twenty-six of 27 clinical isolates from Michigan were genotyped as ST-40, previously identified as the Midwest B. cenocepacia lineage. In contrast, the 12 agricultural isolates represented eight STs, including ST-122, that were identical to clinical isolates of the PHDC lineage. In general, pathogenicity to onions and the presence of the pehA endopolygalacturonase gene were detected only in one cluster of related strains consisting of agricultural isolates and the PHDC lineage. Surprisingly, these strains were highly pathogenic in the nematode Caenorhabditis elegans infection model, killing nematodes faster than the CF pathogen Pseudomonas aeruginosa PA14 on slow-kill medium. The other strains displayed a wide range of pathogenicity to C. elegans, notably the Midwest clonal lineage which displayed high, moderate, and low virulence. Most strains displayed moderate antifungal activity, although strains with high and low activities were also detected. We conclude that pathogenicity to multiple hosts may be a key factor contributing to the potential of B. cenocepacia to opportunistically infect humans both by increasing the prevalence of the organism in the environment, thereby increasing exposure to vulnerable hosts, and by the selection of virulence factors that function in multiple hosts.The betaproteobacterium Burkholderia cenocepacia, 1 of now 17 classified species belonging to the Burkholderia cepacia complex (BCC), is ubiquitous and extremely versatile in its metabolic capabilities and interactions with other organisms (38, 40, 57, 58). Strains of B. cenocepacia are pathogens of onion and banana plants, opportunistic pathogens of humans, symbionts of numerous plant rhizospheres, contaminants of pharmaceutical and industrial products, and inhabitants of soil and surface waters (14, 29, 33, 34, 37, 45). Originally described as a pathogen of onions (8), organisms of the BCC emerged in the past 3 decades as serious human pathogens, capable of causing devastating chronic lung infections in persons with cystic fibrosis (CF) or chronic granulomatous disease (21, 24, 28). Infections due to BCC are a serious concern to CF patients due to their inherent antibiotic resistance and high potential for patient-to-patient transmission (23). Although 16 of the BCC species have been recovered from respiratory secretions of CF patients in many countries (46, 58), B. cenocepacia has been the most common species isolated in North America, detected in 50% of 606, 83% of 447, and 45.6% of 1,218 patients in recent studies (35, 46, 52).The epidemiology of infectious disease caused by B. cenocepacia appears to involve patient-to-patient spread of genetically distinct lineages. B. cenocepacia lineages, such as ET12, Midwest, and PHDC, have been identified from large numbers of individuals in disease outbreaks in North America and Europe (11, 32, 54). A recently developed multilocus sequence typing (MLST) scheme has been shown to be a reliable epidemiologic tool for differentiating between the five subgroups (IIIA to IIIE) of B. cenocepacia, and strains representing three of these subgroups (IIIA, IIIB, and IIID) have been recovered from CF patients (2). Outside of the patient-to-patient transmission of clonal lineages, the mode of acquisition of strains causing sporadic cases of B. cenocepacia in CF patients remains unclear, although environmental sources are a logical reservoir for infection. Previously, an isolate of B. cenocepacia indistinguishable from the PHDC epidemic clonal lineage by using standard typing methods (e.g., repetitive-sequence-based PCR, randomly amplified polymorphic DNA, pulsed-field gel electrophoresis) was detected in an agricultural soil sample (34). Similarly, three distinct MLST sequence types containing both clinical and environmental (plant and soil) B. cenocepacia isolates were identified (1). These findings suggest that natural populations of B. cenocepacia in soil or associated with plants are a potential reservoir for the emergence of new human pathogenic lineages.Experimental models for the study of virulence potential and traits of B. cenocepacia include mouse and rat models with genetic defects allowing chronic lung infections to be established (e.g., see reference 48). Nematode (Caenorhabditis elegans), alfalfa (Medicago sativa), and onion (Allium cepa) models have also been routinely utilized for the identification of virulence factors (5, 29, 31). C. elegans has been extensively used to study the pathogenesis and virulence factors of a wide variety of bacterial and fungal pathogens (9, 15, 42, 51, 56). In several pathogens, including Pseudomonas (56) and Burkholderia (20), putative virulence factors important for the pathogenesis in mammalian systems (15, 51) have been identified using the C. elegans model. The C. elegans model might be limited in the detection of host-specific virulence factors; however, several attributes, such as small size and rapid development, make it an excellent whole animal model for pathogenesis research (16, 51).The evidence that individual strains of B. cenocepacia can be pathogenic to both plants and humans and are prevalent in various environmental niches has provoked particular interest in elucidating the clinical pathogenic potential of environmental isolates. The basis of this study was to examine whether genetically related B. cenocepacia strains exhibit shared characteristics that contribute to their pathogenicity in multiple hosts and to examine the potential for circulating environmental isolates to emerge as new clinical pathogens. Here, we tested the degree of virulence in animal (nematode) and plant (onion) infection models, the production of antifungal activity, and the genetic relatedness of clinical and environmental B. cenocepacia subgroup IIIB strains predominantly isolated from Michigan.     

Extracellular Glutathione Decreases the Ability of Burkholderia cenocepacia to Penetrate into Epithelial Cells and to Induce an Inflammatory Response

Background

The airway surface liquid (ASL) of Cystic Fibrosis (CF) patients contains a lower concentration of reduced glutathione (GSH) with respect to healthy people. It is not known whether this defect may favor lung colonization by opportunistic pathogens.

Principal Findings

We have analyzed the effects of extracellular GSH on the ability of Burkholderia cenocepacia to penetrate and multiply in epithelial respiratory cells. Extracellular GSH proved to be able to drastically reduce the pathogen ability to adhere and invade airway epithelial cells. This effect is correlated to a GSH-dependent increase in the number of free thiols on the surface of epithelial cells, suggestive of a change in the oxidoreductive status of membrane proteins involved in B. cenocepacia recognition. Moreover, treatments with GSH led to a consistent reduction of the expression of IL-8, TNF-α and IL-1β in response to B. cenocepacia infection.

Conclusions and Significance

Extracellular GSH modulates the interaction between B. cenocepacia and epithelial respiratory cells and inhibits the bacterial invasion into these cells. This suggests that therapies aimed at restoring normal levels of GSH in the ASL might be beneficial to control CF lung infections.     

Adaptation of Iron Homeostasis Pathways by a Pseudomonas aeruginosa Pyoverdine Mutant in the Cystic Fibrosis Lung

Cystic fibrosis (CF) patients suffer from chronic bacterial lung infections, most notably by Pseudomonas aeruginosa, which persists for decades in the lungs and undergoes extensive evolution. P. aeruginosa requires iron for virulence and uses the fluorescent siderophore pyoverdine to scavenge and solubilize ferric iron during acute infections. Pyoverdine mutants accumulate in the lungs of some CF patients, however, suggesting that the heme and ferrous iron acquisition pathways of P. aeruginosa are more important in this environment. Here, we sought to determine how evolution of P. aeruginosa in the CF lung affects iron acquisition and regulatory pathways through the use of longitudinal CF isolates. These analyses demonstrated a significant reduction of siderophore production during the course of CF lung infection in nearly all strains tested. Mass spectrometry analysis of one of these strains showed that the later CF isolate has streamlined the metabolic flux of extracellular heme through the HemO heme oxygenase, resulting in more-efficient heme utilization. Moreover, gene expression analysis shows that iron regulation via the PrrF small RNAs (sRNAs) is enhanced in the later CF isolate. Finally, analysis of P. aeruginosa gene expression in the lungs of various CF patients demonstrates that both PrrF and HemO are consistently expressed in the CF lung environment. Combined, these results suggest that heme is a critical source of iron during prolonged infection of the CF lung and that changes in iron and heme regulatory pathways play a crucial role in adaptation of P. aeruginosa to this ever-changing host environment.     

Bacterial cis-2-unsaturated fatty acids found in the cystic fibrosis airway modulate virulence and persistence of Pseudomonas aeruginosa

There is an increasing appreciation of the polymicrobial nature of many bacterial infections such as those associated with cystic fibrosis (CF) and of the potentially important role for interspecies interactions in influencing both bacterial virulence and response to therapy. Patients with CF are often co-infected with Pseudomonas aeruginosa and other pathogens including Burkholderia cenocepacia and Stenotrophomonas maltophilia. These latter bacteria produce signal molecules of the diffusible signal factor (DSF) family, which are cis-2-unsaturated fatty acids. We have previously shown by in vitro studies that DSF from S. maltophilia leads to altered biofilm formation and increased resistance to antibiotics by P. aeruginosa; these responses of P. aeruginosa require the sensor kinase PA1396. Here we show that DSF signals are present in sputum taken from patients with CF. Presence of these DSF signals was correlated with patient colonization by S. maltophilia and/or B. cenocepacia. Analysis of 50 clinical isolates of P. aeruginosa showed that each responded to the presence of synthetic DSF by increased antibiotic resistance and these strains demonstrated little sequence variation in the PA1396 gene. In animal experiments using CF transmembrane conductance regulator knockout mice, the presence of DSF promoted P. aeruginosa persistence. Furthermore, antibiotic resistance of P. aeruginosa biofilms grown on human airway epithelial cells was enhanced in the presence of DSF. Taken together, these data provide substantial evidence that interspecies DSF-mediated bacterial interactions occur in the CF lung and may influence the efficacy of antibiotic treatment, particularly for chronic infections involving persistence of bacteria.     

Cystic fibrosis-niche adaptation of Pseudomonas aeruginosa reduces virulence in multiple infection hosts

The opportunistic pathogen Pseudomonas aeruginosa is able to thrive in diverse ecological niches and to cause serious human infection. P. aeruginosa environmental strains are producing various virulence factors that are required for establishing acute infections in several host organisms; however, the P. aeruginosa phenotypic variants favour long-term persistence in the cystic fibrosis (CF) airways. Whether P. aeruginosa strains, which have adapted to the CF-niche, have lost their competitive fitness in the other environment remains to be investigated. In this paper, three P. aeruginosa clonal lineages, including early strains isolated at the onset of infection, and late strains, isolated after several years of chronic lung infection from patients with CF, were analysed in multi-host model systems of acute infection. P. aeruginosa early isolates caused lethality in the three non-mammalian hosts, namely Caenorhabditis elegans, Galleria mellonella, and Drosophila melanogaster, while late adapted clonal isolates were attenuated in acute virulence. When two different mouse genetic background strains, namely C57Bl/6NCrl and Balb/cAnNCrl, were used as acute infection models, early P. aeruginosa CF isolates were lethal, while late isolates exhibited reduced or abolished acute virulence. Severe histopathological lesions, including high leukocytes recruitment and bacterial load, were detected in the lungs of mice infected with P. aeruginosa CF early isolates, while late isolates were progressively cleared. In addition, systemic bacterial spread and invasion of epithelial cells, which were detected for P. aeruginosa CF early strains, were not observed with late strains. Our findings indicate that niche-specific selection in P. aeruginosa reduced its ability to cause acute infections across a broad range of hosts while maintaining the capacity for chronic infection in the CF host.     

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.     

Polyphasic characterisation of Burkholderia cepacia complex species isolated from children with cystic fibrosis

Cystic fibrosis (CF) patients with Burkholderia cepacia complex (Bcc) pulmonary infections have high morbidity and mortality. The aim of this study was to compare different methods for identification of Bcc species isolated from paediatric CF patients. Oropharyngeal swabs from children with CF were used to obtain isolates of Bcc samples to evaluate six different tests for strain identification. Conventional (CPT) and automatised (APT) phenotypic tests, polymerase chain reaction (PCR)-recA, restriction fragment length polymorphism-recA, recAsequencing, and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) were applied. Bacterial isolates were also tested for antimicrobial susceptibility. PCR-recA analysis showed that 36 out of the 54 isolates were Bcc. Kappa index data indicated almost perfect agreement between CPT and APT, CPT and PCR-recA, and APT and PCR-recA to identify Bcc, and MALDI-TOF and recAsequencing to identify Bcc species. The recAsequencing data and the MALDI-TOF data agreed in 97.2% of the isolates. Based on recA sequencing, the most common species identified were Burkholderia cenocepacia IIIA (33.4%),Burkholderia vietnamiensis (30.6%), B. cenocepaciaIIIB (27.8%), Burkholderia multivorans (5.5%), and B. cepacia (2.7%). MALDI-TOF proved to be a useful tool for identification of Bcc species obtained from CF patients, although it was not able to identify B. cenocepacia subtypes.     

Characterization of Burkholderia cepacia complex from cystic fibrosis patients in China and their chitosan susceptibility

A survey of Burkholderia cepacia complex (Bcc) species was conducted in sputum from cystic fibrosis (CF) patients in China. One hundred and four bacterial isolates were recovered on B. cepacia selective agar and 42 of them were assigned to Bcc by PCR assays. The species composition of the Bcc isolates from CF sputum was analyzed by a combination of recA-restriction fragment length polymorphism assays, species-specific PCR tests and recA gene sequencing. The results revealed that the 42 Bcc isolates belong to B. cepacia, B. cenocepacia and B. contaminans while predominant Bcc species was B. cenocepacia. This is the first report of B. contaminans from CF sputum in China. In addition, results from this study showed that chitosan solution at 10, 25, 50 and 100 μg/ml markedly inhibited the growth of the 16 representative isolates from the three different Bcc species, which indicated that chitosan was a potential bactericide against Bcc bacteria.     

IFN-γ Stimulates Autophagy-Mediated Clearance of Burkholderia cenocepacia in Human Cystic Fibrosis Macrophages

Burkholderia cenocepacia is a virulent pathogen that causes significant morbidity and mortality in patients with cystic fibrosis (CF), survives intracellularly in macrophages, and uniquely causes systemic infections in CF. Autophagy is a physiologic process that involves engulfing non-functional organelles and proteins and delivering them for lysosomal degradation, but also plays a role in eliminating intracellular pathogens, including B. cenocepacia. Autophagy is defective in CF but can be stimulated in murine CF models leading to increased clearance of B. cenocepacia, but little is known about autophagy stimulation in human CF macrophages. IFN-γ activates macrophages and increases antigen presentation while also inducing autophagy in macrophages. We therefore, hypothesized that treatment with IFN-γ would increase autophagy and macrophage activation in patients with CF. Peripheral blood monocyte derived macrophages (MDMs) were obtained from CF and non-CF donors and subsequently infected with B. cenocepacia. Basal serum levels of IFN-γ were similar between CF and non-CF patients, however after B. cenocepacia infection there is deficient IFN-γ production in CF MDMs. IFN-γ treated CF MDMs demonstrate increased co-localization with the autophagy molecule p62, increased autophagosome formation, and increased trafficking to lysosomes compared to untreated CF MDMs. Electron microscopy confirmed IFN-γ promotes double membrane vacuole formation around bacteria in CF MDMs, while only single membrane vacuoles form in untreated CF cells. Bacterial burden is significantly reduced in autophagy stimulated CF MDMs, comparable to non-CF levels. IL-1β production is decreased in CF MDMs after IFN-γ treatment. Together, these results demonstrate that IFN-γ promotes autophagy-mediated clearance of B. cenocepacia in human CF macrophages.     

Characterization of BCAM0224, a Multifunctional Trimeric Autotransporter from the Human Pathogen Burkholderia cenocepacia

Members of the trimeric autotransporter adhesin (TAA) family play a crucial role in adhesion of Gram-negative pathogens to host cells. Moreover, these proteins are multifunctional virulence factors involved in several other biological traits, including invasion into host cells and evasion of the host immune system. In cystic fibrosis epidemic Burkholderia cenocepacia strain J2315, we identified a unique TAA (BCAM0224)-encoding gene, previously described as being implicated in virulence. Here, we characterized this multifunctional protein, trying to establish its role in B. cenocepacia pathogenicity. We show that BCAM0224 occurs on the bacterial surface and adopts a trimeric conformation. Furthermore, we demonstrated that BCAM0224 is needed for earlier stages of biofilm formation and is required for swarming motility. In addition, BCAM0224 plays an important role in evasion of the human innate immune system, providing resistance against the bactericidal activity of serum via the complement classical pathway. Finally, BCAM0224 mediates bacterial adhesion to and invasion of cultured human bronchial epithelial cells. Together, these data reveal the high versatility of the BCAM0224 protein as a virulence factor in the pathogenic bacterium B. cenocepacia.     

Impairment in inflammasome signaling by the chronic Pseudomonas aeruginosa isolates from cystic fibrosis patients results in an increase in inflammatory response

Pseudomonas aeruginosa is a common respiratory pathogen in cystic fibrosis (CF) patients which undergoes adaptations during chronic infection towards reduced virulence, which can facilitate bacterial evasion of killing by host cells. However, inflammatory cytokines are often found to be elevated in CF patients, and it is unknown how chronic P. aeruginosa infection can be paradoxically associated with both diminished virulence in vitro and increased inflammation and disease progression. Thus, we investigated the relationship between the stimulation of inflammatory cell death pathways by CF P. aeruginosa respiratory isolates and the expression of key inflammatory cytokines. We show that early respiratory isolates of P. aeruginosa from CF patients potently induce inflammasome signaling, cell death, and expression of IL-1β by macrophages, yet little expression of other inflammatory cytokines (TNF, IL-6 and IL-8). In contrast, chronic P. aeruginosa isolates induce relatively poor macrophage inflammasome signaling, cell death, and IL-1β expression but paradoxically excessive production of TNF, IL-6 and IL-8 compared to early P. aeruginosa isolates. Using various mutants of P. aeruginosa, we show that the premature cell death of macrophages caused by virulent bacteria compromises their ability to express cytokines. Contrary to the belief that chronic P. aeruginosa isolates are less pathogenic, we reveal that infections with chronic P. aeruginosa isolates result in increased cytokine induction due to their failure to induce immune cell death, which results in a relatively intense inflammation compared with early isolates.Subject terms: Cell death, Immune cell death     

Potential mechanisms underlying the acute lung dysfunction and bacterial extrapulmonary dissemination during Burkholderia cenocepacia respiratory infection

Background

Burkholderia cenocepacia, an opportunistic pathogen that causes lung infections in cystic fibrosis (CF) patients, is associated with rapid and usually fatal lung deterioration due to necrotizing pneumonia and sepsis, a condition known as cepacia syndrome. The key bacterial determinants associated with this poor clinical outcome in CF patients are not clear. In this study, the cytotoxicity and procoagulant activity of B. cenocepacia from the ET-12 lineage, that has been linked to the cepacia syndrome, and four clinical isolates recovered from CF patients with mild clinical courses were analysed in both in vitro and in vivo assays.

Methods

B. cenocepacia-infected BEAS-2B epithelial respiratory cells were used to investigate the bacterial cytotoxicity assessed by the flow cytometric detection of cell staining with propidium iodide. Bacteria-induced procoagulant activity in cell cultures was assessed by a colorimetric assay and by the flow cytometric detection of tissue factor (TF)-bearing microparticles in cell culture supernatants. Bronchoalveolar lavage fluids (BALF) from intratracheally infected mice were assessed for bacterial proinflammatory and procoagulant activities as well as for bacterial cytotoxicity, by the detection of released lactate dehydrogenase.

Results

ET-12 was significantly more cytotoxic to cell cultures but clinical isolates Cl-2, Cl-3 and Cl-4 exhibited also a cytotoxic profile. ET-12 and CI-2 were similarly able to generate a TF-dependent procoagulant environment in cell culture supernatant and to enhance the release of TF-bearing microparticles from infected cells. In the in vivo assay, all bacterial isolates disseminated from the mice lungs, but Cl-2 and Cl-4 exhibited the highest rates of recovery from mice livers. Interestingly, Cl-2 and Cl-4, together with ET-12, exhibited the highest cytotoxicity. All bacteria were similarly capable of generating a procoagulant and inflammatory environment in animal lungs.

Conclusion

B. cenocepacia were shown to exhibit cytotoxic and procoagulant activities potentially implicated in bacterial dissemination into the circulation and acute pulmonary decline detected in susceptible CF patients. Improved understanding of the mechanisms accounting for B. cenocepacia-induced clinical decline has the potential to indicate novel therapeutic strategies to be included in the care B. cenocepacia-infected patients.     

Identification of Sodium Chloride-Regulated Genes in <Emphasis Type="Italic">Burkholderia cenocepacia</Emphasis>

Previous studies have suggested that the airways of cystic fibrosis (CF) patients have elevated sodium chloride (NaCl) levels due to the malfunctioning of the CF transmembrane conductance regulator protein. For bacteria to survive in this high-salt environment, they must adjust by altering the regulation of gene expression. Among the different bacteria inhabiting the airways of CF patients is the opportunistic pathogen Burkholderia cenocepacia. Previous studies have indicated that B. cenocepacia produces a toxin and cable pili under high osmolar conditions. We used transposon mutagenesis to identify NaCl-regulated genes in the clinical strain B. cenocepacia K56-2. Six transconjugants were induced with increasing NaCl concentration. The DNA flanking the transposon was sequenced and five distinct open reading frames were identified encoding the following putative proteins: an integrase, an NAD-dependent deacetylase, TolB, an oxidoreductase, and a novel hypothetical protein. The collective results of this study provide important information about the physiology of B. cenocepacia when faced with osmotic stress and suggest the identity of significant virulence mechanisms in this opportunistic pathogen.     

Phenotypic characterization of trimeric autotransporter adhesin-defective bcaC mutant of Burkholderia cenocepacia: cross-talk towards the histidine kinase BCAM0218

Burkholderia cenocepacia is a virulent species belonging to the Burkholderia cepacia complex (Bcc) and one of the most problematic agents of chronic lung infection in cystic fibrosis patients. B. cenocepacia possesses a large panel of virulence traits that include trimeric autotransporter adhesins (TAAs). Such proteins are obligate homotrimeric anchored in the outer membrane. They are players in the adhesion events that occur between bacteria and biotic/abiotic surfaces. In this study, we constructed two insertional-mutants for TAA bcaC and Histidine kinase (HK) BCAM0218 genes, which are clustered together within the B. cenocepacia K56-2 TAA cluster. The bcaC-mutant affects B. cenocepacia adhesion to extracellular matrix proteins and red blood cells hemagglutination. BcaC contributes to enhancing B. cenocepacia K56-2 adhesion to bronchial epithelial cells. The expression of bcaC seems to affect biofilm formation negatively. Due to a BCAM0218 disruption, the bcaC expression increases significantly, indicating that they are functionally linked. The overexpression of bcaC in the BCAM0218-mutant background rescues at least part of the BcaC functions. Altogether, these findings reveal the multifunctionality of BcaC as a novel B. cenocepacia K56-2 virulence factor and postulate the involvement of a sensor HK (BCAM0218) in the control of this TAA gene.