Global changes in gene expression by the opportunistic pathogen Burkholderia cenocepacia in response to internalization by murine macrophages

Burkholderia cenocepacia is an opportunistic pathogen causing life-threatening infections in patients with cystic fibrosis. The bacterium survives within macrophages by interfering with endocytic trafficking and delaying the maturation of the B. cenocepacia-containing phagosome. We hypothesize that B. cenocepacia undergoes changes in gene expression after internalization by macrophages, inducing genes involved in intracellular survival and host adaptation.

Burkholderia cenocepacia O antigen lipopolysaccharide prevents phagocytosis by macrophages and adhesion to epithelial cells

Chronic respiratory infections by the Burkholderia cepacia complex (Bcc) are of great concern to patients with cystic fibrosis. Bcc isolates may survive intracellularly within amoebae, respiratory epithelial cells and macrophages. The molecular mechanisms facilitating colonization and pathogenesis remain unclear. Given the importance of bacterial adhesion to host surfaces in microbial pathogenesis, we investigated the role of the O antigen LPS in the interaction of Burkholderia cenocepacia, a member of the Bcc, with macrophages and epithelial cells. Our results demonstrated that the O antigen modulates phagocytosis but does not affect intracellular survival of B. cenocepacia. Internalization of strains that lack O antigen was significantly increased compared to that of their isogenic smooth counterparts. However, no differences between rough and smooth strains were found in their ability to delay phagosomal maturation. We also found that the O antigen interfered with the ability of B. cenocepacia to adhere to bronchial epithelial cells, suggesting that this polysaccharide may mask one or more bacterial surface adhesins.

Burkholderia cenocepacia-induced delay of acidification and phagolysosomal fusion in cystic fibrosis transmembrane conductance regulator (CFTR)-defective macrophages

The Burkholderia cepacia complex (Bcc) is a group of opportunistic bacteria chronically infecting the airways of patients with cystic fibrosis (CF). Several laboratories have shown that Bcc members, in particular B. cenocepacia, survive within a membrane-bound vacuole inside phagocytic and epithelial cells. We have previously demonstrated that intracellular B. cenocepacia causes a delay in phagosomal maturation, as revealed by impaired acidification and slow accumulation of the late phagolysosomal marker LAMP-1. In this study, we demonstrate that uninfected cystic fibrosis transmembrane conductance regulator (CFTR)-defective macrophages or normal macrophages treated with a CFTR-specific drug inhibitor display normal acidification. However, after ingestion of B. cenocepacia, acidification and phagolysosomal fusion of the bacteria-containing vacuoles occur in a lower percentage of CFTR-negative macrophages than CFTR-positive cells, suggesting that loss of CFTR function contributes to enhance bacterial intracellular survival. The CFTR-associated phagosomal maturation defect was absent in macrophages exposed to heat-inactivated B. cenocepacia and macrophages infected with a non-CF pathogen such as Salmonella enterica, an intracellular pathogen that once internalized rapidly traffics to acidic compartments that acquire lysosomal markers. These results suggest that not only a defective CFTR but also viable B. cenocepacia are required for the altered trafficking phenotype. We conclude that CFTR may play a role in the mechanism of clearance of the intracellular infection, as we have shown before that B. cenocepacia cells localized to the lysosome lose cell envelope integrity. Therefore, the prolonged maturation arrest of the vacuoles containing B. cenocepacia within cftr(-/-) macrophages could be a contributing factor in the persistence of the bacteria within CF patients.

Burkholderia cenocepacia requires RpoE for growth under stress conditions and delay of phagolysosomal fusion in macrophages

Burkholderia cenocepacia is an opportunistic pathogen causing serious infections in patients with cystic fibrosis. The widespread distribution of this bacterium in the environment suggests that it must adapt to stress to be able to survive. We identified in B. cenocepacia K56-2 a gene predicted to encode RpoE, the extra-cytoplasmic stress response regulator. The rpoE gene is the first gene of a predicted operon encoding proteins homologous to RseA, RseB, MucD and a protein of unknown function. The genomic organization and the co-transcription of these genes were confirmed by PCR and RT-PCR. The mucD and rpoE genes were mutated, giving rise to B. cenocepacia RSF24 and RSF25, respectively. While mutant RSF24 did not demonstrate any growth defects under the conditions tested, RSF25 was compromised for growth under temperature (44 degrees C) and osmotic stress (426 mM NaCl). Expression of RpoE in trans could complement the osmotic growth defect but exacerbated temperature sensitivity in both RSF25 and wild-type K56-2. Inactivation of rpoE altered the bacterial cell surface, as indicated by increased binding of the fluorescent dye calcofluor white and by an altered outer-membrane protein profile. These cell surface changes were restored by complementation with a plasmid encoding rpoE. Macrophage infections in which bacterial colocalization with fluorescent dextran was examined demonstrated that the rpoE mutant could not delay the fusion of B. cenocepacia-containing vacuoles with lysosomes, in contrast to the parental strain K56-2. These data show that B. cenocepacia rpoE is required for bacterial growth under certain stress conditions and for the ability of intracellular bacteria to delay phagolysosomal fusion in macrophages.

Burkholderia cenocepacia requires the RpoN sigma factor for biofilm formation and intracellular trafficking within macrophages

Chronic respiratory infections by Burkholderia cenocepacia in cystic fibrosis patients are associated with increased morbidity and mortality, but virulence factors determining the persistence of the infection in the airways are not well characterized. Using a chronic pulmonary infection model, we previously identified an attenuated mutant with an insertion in a gene encoding an RpoN activator protein, suggesting that RpoN and/or components of the RpoN regulon play a role in B. cenocepacia virulence. In this study, we demonstrate that a functional rpoN gene is required for bacterial motility and biofilm formation in B. cenocepacia K56-2. Unlike other bacteria, RpoN does not control flagellar biosynthesis, as evidenced by the presence of flagella in the rpoN mutant. We also demonstrate that, in macrophages, the rpoN mutant is rapidly trafficked to lysosomes while intracellular wild-type B. cenocepacia localizes in bacterium-containing vacuoles that exhibit a pronounced delay in phagolysosomal fusion. Rapid trafficking to the lysosomes is also associated with the release of red fluorescent protein into the vacuolar lumen, indicating loss of bacterial cell envelope integrity. Although a role for RpoN in motility and biofilm formation has been previously established, this study is the first demonstration that the RpoN regulon in B. cenocepacia is involved in delaying phagolysosomal fusion, thereby prolonging bacterial intracellular survival within macrophages.

The mgtC gene of Burkholderia cenocepacia is required for growth under magnesium limitation conditions and intracellular survival in macrophages

Burkholderia cenocepacia, a bacterium commonly found in the environment, is an important opportunistic pathogen in patients with cystic fibrosis (CF). Very little is known about the mechanisms by which B. cenocepacia causes disease, but chronic infection of the airways in CF patients may be associated, at least in part, with the ability of this bacterium to survive within epithelial cells and macrophages. Survival in macrophages occurs in a membrane-bound compartment that is distinct from the lysosome, suggesting that B. cenocepacia prevents phagolysosomal fusion. In a previous study, we employed signature-tagged mutagenesis and an agar bead model of chronic pulmonary infection in rats to identify B. cenocepacia genes that are required for bacterial survival in vivo. One of the most significantly attenuated mutants had an insertion in the mgtC gene. Here, we show that mgtC is also needed for growth of B. cenocepacia in magnesium-depleted medium and for bacterial survival within murine macrophages. Using fluorescence microscopy, we demonstrated that B. cenocepacia mgtC mutants, unlike the parental isolate, colocalize with the fluorescent acidotropic probe LysoTracker Red. At 4 h postinfection, mgtC mutants expressing monomeric red fluorescent protein cannot retain this protein within the bacterial cytoplasm. Together, these results demonstrate that, unlike the parental strain, an mgtC mutant does not induce a delay in phagolysosomal fusion and the bacterium-containing vacuoles are rapidly targeted to the lysosome, where bacteria are destroyed.

Intracellular survival of Burkholderia cenocepacia in macrophages is associated with a delay in the maturation of bacteria-containing vacuoles

Strains of the Burkholderia cepacia complex (Bcc) are opportunistic bacteria that can cause life-threatening infections in patients with cystic fibrosis and chronic granulomatous disease. Previous work has shown that Bcc isolates can persist in membrane-bound vacuoles within amoeba and macrophages without bacterial replication, but the detailed mechanism of bacterial persistence is unknown. In this study, we have investigated the survival of the Burkholderia cenocepacia strain J2315 within RAW264.7 murine macrophages. Strain J2315 is a prototypic isolate of the widespread and transmissible ET12 clone. Unlike heat-inactivated bacteria, which reach lysosomes shortly after internalization, vacuoles containing live B. cenocepacia J2315 accumulate the late endosome/lysosome marker LAMP-1 and start fusing with lysosomal compartments only after 6 h post internalization. Using fluorescent fluid-phase probes, we also demonstrated that B. cenocepacia-containing vacuoles continued to interact with newly formed endosomes, and maintained a luminal pH of 6.4 +/- 0.12. In contrast, vacuoles containing heat-inactivated bacteria had an average pH of 4.8 +/- 0.03 and rapidly merged with lysosomes. Additional experiments using concanamycin A, a specific inhibitor of the vacuolar H+-ATPase, revealed that vacuoles containing live bacteria did not exclude the H+-ATPase. This mode of bacterial survival did not require type III secretion, as no differences were found between wild type and a type III secretion mutant strain. Collectively, our results suggest that intracellular B. cenocepacia cause a delay in the maturation of the phagosome, which may contribute to facilitate bacterial escape from the microbicidal activities of the host cell.

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 very difficult to eradicate due to their high levels of antibiotic resistance. Though the highly virulent B. cenocepacia has been the focus of virulence research for the past decade, B. 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 exists for B. multivorans. Our results demonstrated that clinical CF isolates, C5568 and C0514, and an environmental B. multivorans isolate, ATCC17616, were able to replicate and survive within murine macrophages in a manner similar to B. cenocepacia 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 late endosome/lysosomal marker LAMP-1 and the lysosomal marker dextran within 2 h of uptake. Together, these results indicate that while both Bcc species are able to survive and replicate within macrophages, they utilize different intramacrophage survival strategies. To observe differences in virulence the strains were compared using the Galleria mellonella model. When compared to the B. multivorans strains tested, B. cenocepacia K56-2 is highly virulent in this model and killed all worms within 24 h when injected at 107 CFU. B. multivorans clinical isolates C5568 and C0514 were significantly more virulent than the soil isolate ATCC17616, which was avirulent, even when worms were injected with 107 CFU. These results suggest strain differences in the virulence of B. multivorans isolates.

Myeloid Angiogenic Cells Act as Alternative M2 Macrophages and Modulate Angiogenesis through Interleukin-8

Endothelial progenitor cells (EPCs) promote angiogenesis, and clinical trials have shown such cell therapy to be feasible for treating ischemic disease. However, clinical outcomes have been contradictory owing to the diverse range of EPC types used. We recently characterized two EPC subtypes, and identified outgrowth endothelial cells as the only EPC type with true progenitor and endothelial characteristics. By contrast, myeloid angiogenic cells (MACs) were shown to be monocytic cells without endothelial characteristics despite being widely described as "EPCs." In the current study we demonstrated that although MACs do not become endothelial cells or directly incorporate into a microvascular network, they can significantly induce endothelial tube formation in vitro and vascular repair in vivo. MAC-derived interleukin-8 (IL-8) was identified as a key paracrine factor, and blockade of IL-8 but not vascular endothelial growth factor (VEGF) prevented MAC-induced angiogenesis. Extracellular IL-8 transactivates VEGFR2 and induces phosphorylation of extracellular signal-regulated kinases. Further transcriptomic and immunophenotypic analysis indicates that MACs represent alternative activated M2 macrophages. Our findings demonstrate an unequivocal role for MACs in angiogenesis, which is linked to paracrine release of cytokines such as IL-8. We also show, for the first time, the true identity of these cells as alternative M2 macrophages with proangiogenic, antiinflammatory and pro-tissue-repair properties.

A helminth cathelicidin-like protein suppresses antigen processing and presentation in macrophages via inhibition of lysosomal vATPase

We previously reported the identification of a novel family of immunomodulatory proteins, termed helminth defense molecules (HDMs), that are secreted by medically important trematode parasites. Since HDMs share biochemical, structural, and functional characteristics with mammalian cathelicidin-like host defense peptides (HDPs), we proposed that HDMs modulate the immune response via molecular mimicry of host molecules. In the present study, we report the mechanism by which HDMs influence the function of macrophages. We show that the HDM secreted by Fasciola hepatica (FhHDM-1) binds to macrophage plasma membrane lipid rafts via selective interaction with phospholipids and/or cholesterol before being internalized by endocytosis. Following internalization, FhHDM-1 is rapidly processed by lysosomal cathepsin L to release a short C-terminal peptide (containing a conserved amphipathic helix that is a key to HDM function), which then prevents the acidification of the endolysosomal compartments by inhibiting vacuolar ATPase activity. The resulting endolysosomal alkalization impedes macrophage antigen processing and prevents the transport of peptides to the cell surface in conjunction with MHC class II for presentation to CD4(+) T cells. Thus, we have elucidated a novel mechanism by which helminth pathogens alter innate immune cell function to assist their survival in the host.-Robinson, M. W., Alvarado, R., To, J., Hutchinson, A. T., Dowdell, S. N., Lund, M., Turnbull, L., Whitchurch, C. B., O'Brien, B. A., Dalton, J. P., Donnelly, S. A helminth cathelicidin-like protein suppresses antigen processing and presentation in macrophages via inhibition of lysosomal vATPase.

Docosahexaenoic acid induces an anti-inflammatory profile in lipopolysaccharide-stimulated human THP-1 macrophages more effectively than eicosapentaenoic acid

A number of studies have investigated the effects of fish oil on the production of pro-inflammatory cytokines using peripheral blood mononuclear cell models. The majority of these studies have employed heterogeneous blends of long-chain n-3 polyunsaturated fatty acids (PUFA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which preclude examination of the individual effects of LC n-3 PUFA. This study investigated the differential effects of pure EPA and DHA on cytokine expression and nuclear factor kappaB (NF-kappaB) activation in human THP-1 monocyte-derived macrophages. Pretreatment with 100 microM EPA and DHA significantly decreased lipopolysaccharide (LPS)-stimulated THP-1 macrophage tumor necrosis factor (TNF) alpha, interleukin (IL) 1beta and IL-6 production (P

Conjugated linoleic acid and atherosclerosis: no effect on molecular markers of cholesterol homeostasis in THP-1 macrophages

Macrophage cholesterol homeostasis is a key process involved in the initiation and progression of atherosclerosis. Peroxisome proliferator-activated receptors (PPARs) regulate the transcription of the genes involved in cholesterol homeostasis and thus represent an important therapeutic target in terms of reducing atherosclerosis. Conjugated linoleic acid (CLA) is a potent anti-atherogenic dietary fatty acid in animal models of atherosclerosis and is capable of activating PPARs in vitro and in vivo. Therefore, this study examined whether the anti-atherogenic effects of CLA in vivo could be ascribed to altered cholesterol homeostasis in macrophages and macrophage derived foam cells. Of several genes that regulate cholesterol homeostasis investigated, CLA had most effect on the class B scavenger receptor CD36. The cis-9,trans-11 CLA (c9,t11-CLA) and trans-10,cis-12 CLA (t10,c12-CLA) isomers augmented CD36 mRNA expression (P

Depletion of the ubiquitin-binding adaptor molecule SQSTM1/p62 from macrophages harboring cftr ΔF508 mutation improves the delivery of Burkholderia cenocepacia to the autophagic machinery

Cystic fibrosis is the most common inherited lethal disease in Caucasians. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), of which the cftr ?F508 mutation is the most common. ?F508 macrophages are intrinsically defective in autophagy because of the sequestration of essential autophagy molecules within unprocessed CFTR aggregates. Defective autophagy allows Burkholderia cenocepacia (B. cepacia) to survive and replicate in ?F508 macrophages. Infection by B. cepacia poses a great risk to cystic fibrosis patients because it causes accelerated lung inflammation and, in some cases, a lethal necrotizing pneumonia. Autophagy is a cell survival mechanism whereby an autophagosome engulfs non-functional organelles and delivers them to the lysosome for degradation. The ubiquitin binding adaptor protein SQSTM1/p62 is required for the delivery of several ubiquitinated cargos to the autophagosome. In WT macrophages, p62 depletion and overexpression lead to increased and decreased bacterial intracellular survival, respectively. In contrast, depletion of p62 in ?F508 macrophages results in decreased bacterial survival, whereas overexpression of p62 leads to increased B. cepacia intracellular growth. Interestingly, the depletion of p62 from ?F508 macrophages results in the release of the autophagy molecule beclin1 (BECN1) from the mutant CFTR aggregates and allows its redistribution and recruitment to the B. cepacia vacuole, mediating the acquisition of the autophagy marker LC3 and bacterial clearance via autophagy. These data demonstrate that p62 differentially dictates the fate of B. cepacia infection in WT and ?F508 macrophages.

Nontypeable Haemophilus influenzae clearance by alveolar macrophages is impaired by exposure to cigarette smoke

Nontypeable Haemophilus influenzae (NTHI) is an opportunistic gram-negative pathogen that causes respiratory infections and is associated with progression of respiratory diseases. Cigarette smoke is a main risk factor for development of respiratory infections and chronic respiratory diseases. Glucocorticoids, which are anti-inflammatory drugs, are still the most common therapy for these diseases. Alveolar macrophages are professional phagocytes that reside in the lung and are responsible for clearing infections by the action of their phagolysosomal machinery and promotion of local inflammation. In this study, we dissected the interaction between NTHI and alveolar macrophages and the effect of cigarette smoke on this interaction. We showed that alveolar macrophages clear NTHI infections by adhesion, phagocytosis, and phagolysosomal processing of the pathogen. Bacterial uptake requires host actin polymerization, the integrity of plasma membrane lipid rafts, and activation of the phosphatidylinositol 3-kinase (PI3K) signaling cascade. Parallel to bacterial clearance, macrophages secrete tumor necrosis factor alpha (TNF-alpha) upon NTHI infection. In contrast, exposure to cigarette smoke extract (CSE) impaired alveolar macrophage phagocytosis, although NTHI-induced TNF-alpha secretion was not abrogated. Mechanistically, our data showed that CSE reduced PI3K signaling activation triggered by NTHI. Treatment of CSE-exposed cells with the glucocorticoid dexamethasone reduced the amount of TNF-alpha secreted upon NTHI infection but did not compensate for CSE-dependent phagocytic impairment. The deleterious effect of cigarette smoke was observed in macrophage cell lines and in human alveolar macrophages obtained from smokers and from patients with chronic obstructive pulmonary disease.