Burkholderia cenocepacia ET12 strain activates TNFR1 signalling in cystic fibrosis airway epithelial cells

Burkholderia cenocepacia is an important pulmonary pathogen in individuals with cystic fibrosis (CF). Infection is often associated with severe pulmonary inflammation, and some patients develop a fatal necrotizing pneumonia and sepsis ('cepacia syndrome'). The mechanisms by which this species causes severe pulmonary inflammation are poorly understood. Here, we demonstrate that B. cenocepacia BC7, a potentially virulent representative of the epidemic ET12 lineage, binds to tumour necrosis factor receptor 1 (TNFR1) and activates TNFR1-related signalling pathway similar to TNF-α, a natural ligand for TNFR1. This interaction participates in stimulating a robust IL-8 production from CF airway epithelial cells. In contrast, BC45, a less virulent ET12 representative, and ATCC 25416, an environmental B. cepacia strain, do not bind to TNFR1 and stimulate only minimal IL-8 production from CF cells. Further, TNFR1 expression is increased in CF airway epithelial cells compared with non-CF cells. We also show that B. cenocepacia ET12 strain colocaizes with TNFR1 in vitro and in the lungs of CF patients who died due to infection with B. cenocepacia, ET12 strain. Together, these results suggest that interaction of B. cenocepacia , ET12 strain with TNFR1 may contribute to robust inflammatory responses elicited by this organism.     

Adherence and autoaggregation phenotypes of a Burkholderia cenocepacia cable pilus mutant

Cable pili are unique peritrichous adherence organelles expressed by certain strains of the opportunistic human pathogen Burkholderia cenocepacia. Cable pili have been proposed to facilitate binding to human epithelial cells and mucin, and may play a role in the ability of B. cenocepacia to colonise the respiratory tract of compromised hosts. In this study, a genetic approach was undertaken to assess the role of cable pili in mediating adherence as well as bacterial cell-cell interactions. The cblA gene, encoding the major pilin subunit, was insertionally inactivated, and the resulting mutant was shown to be blocked in CblA expression and in cable pilus morphogenesis. Although non-piliated, the cblA mutant was not defective in adherence to either porcine mucin or to cultured A549 human respiratory epithelial cells. Microscopic and flow cytometric analyses of B. cenocepacia cultures revealed that cable pilus expression facilitated the formation of diffuse cell networks, whereas disruption of cable pilus biogenesis enhanced autoaggregation and the formation of compact cell aggregates. Autoaggregation was observed both in culture and during B. cenocepacia infection of A549 epithelial cell monolayers. These findings indicate that cable pilus expression plays an important role in mediating B. cenocepacia cell-cell interactions, and that both cable pilus-dependent and cable pilus-independent mechanisms may contribute to B. cenocepacia adherence to cellular and acellular surfaces.     

Activation of MMP-9 by human lung epithelial cells in response to the cystic fibrosis-associated pathogen Burkholderia cenocepacia reduced wound healing in vitro

Burkholderia cepacia complex is a group of bacterial pathogens that cause opportunistic infections in cystic fibrosis (CF). The most virulent of these is Burkholderia cenocepacia. Matrix metalloproteinases (MMPs) are upregulated in CF patients. The aim of this work was to examine the role of MMPs in the pathogenesis of B. cepacia complex, which has not been explored to date. Real-time PCR analysis showed that B. cenocepacia infection upregulated MMP-2 and MMP-9 genes in the CF lung cell line CFBE41o- within 1 h, whereas MMP-2, -7, and -9 genes were upregulated in the non-CF lung cell line 16HBE14o-. Conditioned media from both cell lines showed increased MMP-9 activation following B. cenocepacia infection. Conditioned media from B. cenocepacia-infected cells significantly reduced the rate of wound healing in confluent lung epithelia (P < 0.05), in contrast to conditioned media from Pseudomonas aeruginosa-infected cells, which showed predominant MMP-2 activation. Treatment of control conditioned media from both cell lines with the MMP activator 4-aminophenylmercuric acetate (APMA) also resulted in clear activation of MMP-9 and to a much lesser extent MMP-2. APMA treatment of control media also delayed the repair of wound healing in confluent epithelial cells. Furthermore, specific inhibition of MMP-9 in medium from cells exposed to B. cenocepacia completely reversed the delay in wound repair. These data suggest that MMP-9 plays a role in the reduced epithelial repair observed in response to B. cenocepacia infection and that its activation following B. cenocepacia infection contributes to the pathogenesis of this virulent pathogen.     

Use of suppression-subtractive hybridization to identify genes in the Burkholderia cepacia complex that are unique to Burkholderia cenocepacia

We have previously shown differences in virulence between species of the Burkholderia cepacia complex using the alfalfa infection model and the rat agar bead chronic infection model. Burkholderia cenocepacia strains were more virulent in these two infection models than Burkholderia multivorans and Burkholderia stabilis strains. In order to identify genes that may account for the increased virulence of B. cenocepacia, suppression-subtractive hybridization was performed between B. cenocepacia K56-2 and B. multivorans C5393 and between B. cenocepacia K56-2 and B. stabilis LMG14294. Genes identified included DNA modification/phage-related/insertion sequences and genes involved in cell membrane/surface structures, resistance, transport, metabolism, regulation, secretion systems, as well as genes of unknown function. Several of these genes were present in the ET12 lineage of B. cenocepacia but not in other members of the B. cepacia complex. Virulence studies in a chronic lung infection model determined that the hypothetical YfjI protein, which is unique to the ET12 clone, contributes to lung pathology. Other genes specific to B. cenocepacia and/or the ET12 lineage were shown to play a role in biofilm formation and swarming or swimming motility.     

Neutrophils induce apoptosis of lung epithelial cells via release of soluble Fas ligand

Neutrophils release soluble Fas ligand (sFasL), which can induce apoptosis in certain Fas-bearing cell types (Liles WC, Kiener PA, Ledbetter JA, Aruffo A, and Klebanoff SJ. J Exp Med 184: 429-440, 1996). We hypothesized that neutrophils could induce alveolar epithelial apoptosis via release of sFasL. A549 pulmonary adenocarcinoma cells expressed surface Fas and underwent cell death (10 +/- 7% viability) and DNA fragmentation (354 +/- 98% of control cells) when incubated with agonistic CD95/Fas monoclonal antibody (P < 0.05). Coincubation with human neutrophils induced significant A549 cell death at 48 (51 +/- 9% viability; P < 0.05) and 72 h (25 +/- 10%; P < 0.05) and increased DNA fragmentation (178 +/- 42% of control cells; P < 0.05), with morphological characteristics of apoptosis. The addition of antioxidants did not inhibit apoptosis. sFasL concentrations were maximally increased in coculture medium at 24 h (4.9 +/- 0.7 ng/ml; P < 0.05). Neutrophil-induced A549 cell apoptosis was blocked by inhibitory anti-Fas (42 +/- 6% of control cells; P < 0.05) and anti-FasL monoclonal antibodies (29 +/- 3%; P < 0.05). Human neutrophils and Fas similarly affected murine primary alveolar epithelial cell bilayers, and caspase activation occurred in response to Fas exposure. We conclude that neutrophils undergoing spontaneous apoptosis induce A549 cell death and DNA fragmentation, independent of the oxidative burst, that is mediated by sFasL.     

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.     

Involvement of alpha5beta1-integrin and TNF-alpha in Staphylococcus aureus alpha-toxin-induced death of epithelial cells

Staphylococcus aureus causes suppurative infections which are often associated with tissue destruction and cell death. In the present study, we investigated the molecular and cellular basis of S. aureus-induced apoptosis and death in a human lung epithelial cell line (A549). We found that staphylococcal alpha-toxin is an important mediator of cytotoxicity in these epithelial cells. Specifically, we found that downregulating alpha-toxin production eliminated the cytotoxicity of S. aureus, whereas the addition of alpha-toxin to the cell culture medium significantly increased cell death in a dose-dependent manner. Importantly, we found that alpha-toxin-mediated cell death may partially function through alpha5beta1-integrin, because both the beta1-integrin antibody and the ligand fibronectin inhibited the cytotoxicity of alpha-toxin. Furthermore, we found that the overexpression of the inflammatory cytokine interferon (TNF)-alpha is associated with alpha-toxin-induced cell death, because both the TNF-alpha release inhibitor and antibody effectively inhibited the cytotoxicity of alpha-toxin. In contrast, the cytotoxicity of alpha-toxin was enhanced by the inhibition of the MAPK p38 and NF-kappaB pathways. Taken together, our results suggest that the activation of the MAPK p38 and NF-kappaB pathways are stress responses for survival, rather than direct contributes to alpha-toxin-induced cell death, and that the interaction of alpha-toxin with alpha5beta1-integrin and overproduction of TNF-alpha may contribute to destruction of epithelial cells during S. aureus infection.     

Dual mechanism for Mycobacterium tuberculosis cytotoxicity on lung epithelial cells

Mycobacterium tuberculosis strains CDC1551 and Erdman were used to assess cytotoxicity in infected A549 human alveolar epithelial cell monolayers. Strain CDC1551 was found to induce qualitatively greater disruption of A549 monolayers than was strain Erdman, although total intracellular and cell-associated bacterial growth rates over the course of the infections were not significantly different. Cell-free culture supernatants from human monocytic cells infected with either of the 2 M. tuberculosis strains produced a cytotoxic effect on A549 cells, correlating with the amount of tumor necrosis factor alpha (TNF-α) released by the infected monocytes. The addition of TNF-α-neutralizing antibodies to the supernatants from infected monocyte cultures did prevent the induction of a cytotoxic effect on A549 cells overlaid with this mixture but did not prevent the death of epithelial cells when added prior to infection with M. tuberculosis bacilli. Thus, these data agree with previous observations that lung epithelial cells infected with M. tuberculosis bacilli are rapidly killed in vitro. In addition, the data indicate that some of the observed epithelial cell killing may be collateral damage; the result of TNF-α released from M. tuberculosis-infected monocytes.     

The antibacterial properties of docosahexaenoic omega-3 fatty acid against the cystic fibrosis multiresistant pathogen Burkholderia cenocepacia

Burkholderia cepacia complex (Bcc) bacteria are opportunistic pathogens that cause multiresistant pulmonary infections in patients with cystic fibrosis (CF). In this study, we evaluated the in vitro antimicrobial efficacy of eight unsaturated fatty acids against Burkholderia cenocepacia K56-2, a CF epidemic strain. Docosahexaenoic acid (DHA) was the most active compound. Its action can be either bacteriostatic or bactericidal, depending upon the concentration used. The effect of DHA was also evaluated on two others B.?cenocepacia clinical isolates and compared with one representative member of all the 17 Bcc species. To test whether DHA could have a therapeutic potential, we assessed its efficacy using a Galleria mellonella caterpillar model of B.?cenocepacia infection. We observed that the treatment of infected larvae with a single dose of DHA (50 mM) caused an increase in the survival rate as well as a reduced bacterial load. Moreover, DHA administration markedly increases the expression profile of the gene encoding the antimicrobial peptide gallerimycin. Our results demonstrate that DHA has in vitro and in vivo antibacterial activity against Bcc microorganisms. These findings provide evidence that DHA may be a useful nutraceutical for the treatment of CF patients with lung infections caused by antibiotic multiresistant Bcc microorganisms.     

Airborne fine particulate matter induces multiple cell death pathways in human lung epithelial cells

Our group was the first one reporting that autophagy could be triggered by airborne fine particulate matter (PM) with a mean diameter of less than 2.5 μm (PM_2.5) in human lung epithelial A549 cells, which could potentially lead to cell death. In the present study, we further explored the potential interactions between autophagy and apoptosis because it was well documented that PM_2.5 could induce apoptosis in A549 cells. Much to our surprise, we found that PM_2.5-exposure caused oxidative stress, resulting in activation of multiple cell death pathways in A549 cells, that is, the tumor necrosis factor-alpha (TNF-α)-induced pathway as evidenced by TNF-α secretion and activation of caspase-8 and -3, the intrinsic apoptosis pathway as evidenced by increased expression of pro-apoptotic protein Bax, decreased expression of anti-apoptotic protein Bcl-2, disruption of mitochondrial membrane potential, and activation of caspase-9 and -3, and autophagy as evidenced by an increased number of double-membrane vesicles, accompanied by increases of conversion and punctuation of microtubule-associated proteins light chain 3 (LC3) and expression of Beclin 1. It appears that reactive oxygen species (ROS) function as signaling molecules for all the three pathways because pretreatment with N-acetylcysteine, a scavenger of ROS, almost completely abolished TNF-α secretion and significantly reduced the number of apoptotic and autophagic cells. In another aspect, inhibiting autophagy with 3-methyladenine, a specific autophagy inhibitor, enhanced PM_2.5-induced apoptosis and cytotoxicity. Intriguingly, neutralization of TNF-α with an anti-TNF-α special antibody not only abolished activation of caspase-8, but also drastically reduced LC3-II conversion. Thus, the present study has provided novel insights into the mechanism of cytotoxicity and even pathogenesis of diseases associated with PM_2.5 exposure.     

Sequence divergence in type III secretion gene clusters of the Burkholderia cepacia complex

The Burkholderia cepacia complex (BCC) comprises a group of bacteria associated with opportunistic infections, especially in cystic fibrosis patients. B. cenocepacia J2315, of the transmissible ET12 lineage, contains a type III secretion (TTS) gene cluster implicated in pathogenicity. PCR and hybridisation assays indicate that the TTS gene cluster is present in all members of the BCC except B. cepacia (formerly genomovar I). The TTS gene clusters of B. cenocepacia J2315 and B. multivorans are similar in organisation but have variable levels of gene identity. Nucleotide sequence data obtained for the equivalent region of the B. cepacia genome indicate the absence of TTS structural genes due to a rearrangement likely to involve more than one step.     

Differential interaction of bacterial species from the Burkholderia cepacia complex with human airway epithelial cells

To increase knowledge of the pathogenic potential of the Burkholderia cepacia complex (BCC), we investigated the effects of reference strains of the nine BCC species on human bronchial epithelial cells in vitro. B. multivorans exhibited the highest rates of adherence to and internalization by host cells. Two out of three clinical isolates recovered from cystic fibrosis patients confirmed the B. multivorans high adhesiveness. All four B. multivorans isolates exhibited an aggregated pattern of adherence but any of them expressed cable pili. When bacteria were centrifuged onto cell cultures to circumvent their poor adhesiveness, B. pyrrocinia exhibited the highest internalization rate, followed by B. multivorans. The percentages of apoptotic cells in cultures infected with B. cepacia, B. multivorans, B. cenocepacia (subgroups IIIA and IIIB), B. stabilis and B. vietnamiensis were significantly higher than in control non-infected cultures. All nine BCC species triggered a similar release of the inflammatory cytokine IL-8, that was not reduced by cell treatment with cytochalasin D. Hence, our data demonstrate, for the first time, that all BCC species exhibit a similar ability to induce the expression of host immune mediators whereas they differ on their ability to adhere to, invade and kill airway epithelial cells.     

Culture filtrates of Aspergillus fumigatus induce different modes of cell death in human cancer cell lines

Aspergillus fumigatus culture filtrate (CF) has a potent cytotoxic effect on three human cancer cell lines (DLKP, A549 and HEp-2) and initiates cell death by apoptosis but the execution of the apoptotic process is incomplete. DLKP cells treated with A. fumigatus CF demonstrate features associated with apoptosis but cytoplasmic and nuclear fragmentation were not observed and cells ultimately underwent necrosis. The apoptotic process commenced in A549 and HEp-2 cells upon exposure to CF, cell shrinkage was observed but membrane blebbing and apoptotic body formation were not detected and detached cells died by necrosis. In contrast, extensive nuclear fragmentation and apoptotic body formation were evident in DLKP and A549 cells treated with anti-neoplastic agents. This work indicates that A. fumigatus CF is cytotoxic to cancer cells and can initiate apoptosis but that the complete apoptotic pathway is not followed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Survivin and p53 modulate quercetin-induced cell growth inhibition and apoptosis in human lung carcinoma cells

Quercetin, a ubiquitous bioactive plant flavonoid, has been shown to inhibit the proliferation of cancer cells. However, the regulation of survivin and p53 on the quercetin-induced cell growth inhibition and apoptosis in cancer cells remains unclear. In this study, we investigated the roles of survivin and p53 in the quercetin-treated human lung carcinoma cells. Quercetin (20-80 mum for 24 h) induced the cytotoxicity and apoptosis in both A549 and H1299 lung carcinoma cells in a concentration-dependent manner. Additionally, quercetin inhibited the cell growth, increased the fractions of G(2)/M phase, and raised the levels of cyclin B1 and phospho-cdc2 (threonine 161) proteins. Moreover, quercetin induced abnormal chromosome segregation in H1299 cells. The survivin proteins were highly expressed in mitotic phase and were located on the midbody of cytokinesis; however, the survivin proteins were increased and concentrated on the nuclei following quercetin treatment in the lung carcinoma cells. Transfection of a survivin antisense oligodeoxynucleotide enhanced the quercetin-induced cell growth inhibition and cytotoxicity. Subsequently, quercetin increased the levels of total p53 (DO-1), phospho-p53 (serine 15), and p21 proteins, which were translocated to the nuclei in A549 cells. Treatment with a specific p53 inhibitor, pifithrin-alpha, or transfection of a p53 antisense oligodeoxynucleotide enhanced the cytotoxicity of the quercetin-treated cells. Furthermore, transfection of a small interfering RNA of p21 enhanced the quercetin-induced cell death in A549 cells. Together, our results suggest that survivin can reduce the cell growth inhibition and apoptosis, and p53 elevates the p21 level, which may attenuate the cell death in the quercetin-treated human lung carcinoma cells.     

Formoxanthone C,isolated from Cratoxylum formosum ssp. pruniflorum, reverses anticancer drug resistance by inducing both apoptosis and autophagy in human A549 lung cancer cells

Multidrug resistance (MDR) cancer toward cancer chemotherapy is one of the obstacles in cancer therapy. Therefore, it is of interested to use formoxanthone C (1,3,5,6-tetraoxygenated xanthone; XanX), a natural compound, which showed cytotoxicity against MDR human A549 lung cancer (A549RT-eto). The treatment with XanX induced not only apoptosis- in A549RT-eto cells, but also autophagy-cell death. Inhibition of apoptosis did not block XanX-induced autophagy in A549RT-eto cells. Furthermore, suppression of autophagy by beclin-1 small interfering RNAs (siRNAs) did not interrupt XanX-induced apoptosis, indicating that XanX can separately induce apoptosis and autophagy. Of interest, XanX treatment reduced levels of histone deacetylase 4 (HDAC4) protein overexpressed in A549RT-etocells. The co-treatment with XanX and HDAC4 siRNA accelerated both autophagy and apoptosis more than that by XanX treatment alone, suggesting survival of HDAC4 in A549RT-eto cells. XanX reverses etoposide resistance in A549RT-eto cells by induction of both autophagy and apoptosis, and confers cytotoxicity through down-regulation of HDAC4.     

Differentiation-induced uroplakin III expression promotes urothelial cell death in response to uropathogenic E. coli

Uropathogenic E. coli (UPEC) expressing type 1 pili underlie most urinary tract infections (UTIs). UPEC adherence to the bladder urothelium induces a rapid apoptosis and exfoliation of terminally differentiated urothelial cells, a critical event in pathogenesis. Of the four major uroplakin proteins that are densely expressed on superficial urothelial cells, UPIa serves as the receptor for type 1-piliated UPEC, but the contributions of uroplakins to cell death are not known. We examined the role of differentiation and uroplakin expression on UPEC-induced cell death. Utilizing in vitro models of urothelial differentiation, we demonstrated induction of tissue-specific differentiation markers including uroplakins. UPEC-induced urothelial cell death was shown to increase with enhanced differentiation but required expression of uroplakin III: infection with an adenovirus encoding uroplakin III significantly increased cell death, while siRNA directed against uroplakin III abolished UPEC-induced cell death. In a murine model of UTI where superficial urothelial cells were selectively eroded to expose less differentiated cells, urothelial apoptosis was reduced, indicating a requirement for differentiation in UPEC-induced apoptosis in vivo. These data suggest that induction of uroplakin III during urothelial differentiation sensitizes cells to UPEC-induced death. Thus, uroplakin III plays a pivotal role in UTI pathogenesis.     

Carrageenan inhibits granzyme A-induced detachment of and interleukin-8 release from alveolar epithelial A549 cells

Granzyme A (GrA) is a lymphocyte serine protease that is believed to enter virus-infected cells and growing tumors and induce apoptosis. We found recently that recombinant rat GrA (rGrA) promotes detachment of and interleukin (IL)-8 release from alveolar epithelial A549 cells and suggested that this protease is involved in the pathogenesis of certain inflammatory lung diseases. In the present study, we found that λ-carrageenan (a sulfated oligosaccharide constituting the cell walls of seaweeds) potently inhibits rGrA-induced detachment and IL-8 release of A549 cells. This sulfated oligosaccharide might be useful for suppressing the development of inflammatory lung diseases in which GrA is thought to be involved.     

Dihydroartemisinin Sensitizes Human Lung Adenocarcinoma A549 Cells to Arsenic Trioxide via Apoptosis

Recent studies have shown that arsenic trioxide (ATO) is an effective anti-cancer drug for treatment of acute promyelocytic leukemia and other types of human cancer. However, we have found that lung cancer cells constantly develop a high level of resistance to ATO. In this study, we have explored a possibility of combination of dihydroartemisinin (DHA) and ATO treatments to reduce ATO resistance of lung cancer cells. We determined the combinatory effects of DHA and ATO on cytotoxicity of human lung adenocarcinoma (A549) cells. We showed that co-exposure to DHA and ATO of A549 cells synergistically increased the cytotoxicity and apoptotic cell death in the cells. We found that the synergistic effect of DHA and ATO in promoting apoptosis mainly resulted from increased cellular level of reactive oxygen species (ROS) and DNA damage. ATO alone only exerted moderate growth inhibitory effects on A549 cells. The results indicate that DHA can significantly sensitize ATO-induced cytotoxicity of A549 lung cancer cells through apoptosis mediated by ROS-induced DNA damage. Interestingly, we found that the combinatory treatment of DHA and ATO did not result in significant adverse effects in normal human bronchial epithelial (HBE) cells. Our results further provide evidence for the potential application of combinatory effects of DHA and ATO as a safe therapy for human lung cancer.