Type I IFNs facilitate innate immune control of the opportunistic bacteria Burkholderia cenocepacia in the macrophage cytosol.

The mammalian immune system is constantly challenged by signals from both pathogenic and non-pathogenic microbes. Many of these non-pathogenic microbes have pathogenic potential if the immune system is compromised. The importance of type I interferons (IFNs) in orchestrating innate immune responses to pathogenic microbes has become clear in recent years. However, the control of opportunistic pathogens-and especially intracellular bacteria-by type I IFNs remains less appreciated. In this study, we use the opportunistic, Gram-negative bacterial pathogen Burkholderia cenocepacia (Bc) to show that type I IFNs are capable of limiting bacterial replication in macrophages, preventing illness in immunocompetent mice. Sustained type I IFN signaling through cytosolic receptors allows for increased expression of autophagy and linear ubiquitination mediators, which slows bacterial replication. Transcriptomic analyses and in vivo studies also show that LPS stimulation does not replicate the conditions of intracellular Gram-negative bacterial infection as it pertains to type I IFN stimulation or signaling. This study highlights the importance of type I IFNs in protection against opportunistic pathogens through innate immunity, without the need for damaging inflammatory responses.

(R)-Roscovitine and CFTR modulators enhance killing of multi-drug resistant Burkholderia cenocepacia by cystic fibrosis macrophages.

Cystic fibrosis (CF) is characterized by chronic bacterial infections and heightened inflammation. Widespread ineffective antibiotic use has led to increased isolation of drug resistant bacterial strains from respiratory samples. (R)-roscovitine (Seliciclib) is a unique drug that has many benefits in CF studies. We sought to determine roscovitine's impact on macrophage function and killing of multi-drug resistant bacteria. Human blood monocytes were isolated from CF (F508del/F508del) and non-CF persons and derived into macrophages (MDMs). MDMs were infected with CF clinical isolates of B. cenocepacia and P. aeruginosa. MDMs were treated with (R)-roscovitine or its main hepatic metabolite (M3). Macrophage responses to infection and subsequent treatment were determined. (R)-roscovitine and M3 significantly increased killing of B. cenocepacia and P. aeruginosa in CF MDMs in a dose-dependent manner. (R)-roscovitine-mediated effects were partially dependent on CFTR and the TRPC6 channel. (R)-roscovitine-mediated killing of B. cenocepacia was enhanced by combination with the CFTR modulator tezacaftor/ivacaftor and/or the alternative CFTR modulator cysteamine. (R)-roscovitine also increased MDM CFTR function compared to tezacaftor/ivacaftor treatment alone. (R)-roscovitine increases CF macrophage-mediated killing of antibiotic-resistant bacteria. (R)-roscovitine also enhances other macrophage functions including CFTR-mediated ion efflux. Effects of (R)-roscovitine are greatest when combined with CFTR modulators or cysteamine, justifying further clinical testing of (R)-roscovitine or optimized derivatives.

Interferon-gamma-activated macrophages infected with Burkholderia cenocepacia process and present bacterial antigens to T-cells by class I and II major histocompatibility complex molecules.

Burkholderia cenocepacia is an emerging opportunistic pathogen for people with cystic fibrosis and chronic granulomatous disease. Intracellular survival in macrophages within a membrane-bound vacuole (BcCV) that delays acidification and maturation into lysosomes is a hallmark of B. cenocepacia infection. Intracellular B. cenocepacia induce an inflammatory response leading to macrophage cell death by pyroptosis through the secretion of a bacterial deamidase that results in the activation of the pyrin inflammasome. However, how or whether infected macrophages can process and present B. cenocepacia antigens to activate T-cells has not been explored. Engulfed bacterial protein antigens are cleaved into small peptides in the late endosomal major histocompatibility class II complex (MHC) compartment (MIIC). Here, we demonstrate that BcCVs and MIICs have overlapping features and that interferon-gamma-activated macrophages infected with B. cenocepacia can process bacterial antigens for presentation by class II MHC molecules to CD4+ T-cells and by class I MHC molecules to CD8+ T-cells. Infected macrophages also release processed bacterial peptides into the extracellular medium, stabilizing empty class I MHC molecules of bystander cells. Together, we conclude that BcCVs acquire MIIC characteristics, supporting the notion that macrophages infected with B. cenocepacia contribute to establishing an adaptive immune response against the pathogen.

New insights into the immunoproteome of B. cenocepacia J2315 using serum samples from cystic fibrosis patients.

Bacteria of the Burkholderia cepacia complex (Bcc) are ubiquitous multidrug resistant organisms and opportunistic pathogens capable of causing life threatening lung infections among cystic fibrosis (CF) patients. No effective therapies are currently available to eradicate Bcc bacteria from CF patients, as these organisms are inherently resistant to the majority of clinically available antimicrobials. An immunoproteomics approach was used to identify Bcc proteins that stimulate the humoral immune response of the CF host, using bacterial cells grown under conditions mimicking the CF lung environment and serum samples from CF patients with a clinical record of Bcc infection. 24 proteins of the Bcc strain B. cenocepacia J2315 were identified as immunoreactive, 19 here reported as immunogenic for the first time. Ten proteins were predicted as extracytoplasmic, 9 of them being conserved in Bcc genomes. The immunogenic Bcc extracytoplasmic proteins are potential targets for development of novel therapeutic strategies and diagnostic tools to protect patients against the onset of chronic Bcc lung infections.

Staphylococcus aureus products subvert the Burkholderia cenocepacia-induced inflammatory response in airway epithelial cells.

Introduction. Chronic pulmonary infection is associated with colonization with multiple micro-organisms but host-microbe and microbe-microbe interactions are poorly understood.Aim. This study aims to investigate the differences in host responses to mono- and co-infection with S. aureus and B. cenocepacia in human airway epithelial cells.Methodology. We assessed the effect of co-infection with B. cenocepacia and S. aureus on host signalling and inflammatory responses in the human airway epithelial cell line 16HBE, using ELISA and western blot analysis.Results. The results show that B. cenocepacia activates MAPK and NF-κB signalling pathways, subsequently eliciting robust interleukin (IL)-8 production. However, when airway epithelial cells were co-treated with live B. cenocepacia bacteria and S. aureus supernatants (conditioned medium), the pro-inflammatory response was attenuated. This anti-inflammatory effect was widely exhibited in the S. aureus isolates tested and was mediated via reduced MAPK and NF-κB signalling, but not via IL-1 receptor or tumour necrosis factor receptor modulation. The staphylococcal effectors were characterized as small, heat-stable, non-proteinaceous and not cell wall-related factors.Conclusion. This study demonstrates for the first time the host response in a S. aureus/B. cenocepacia co-infection model and provides insight into a staphylococcal immune evasion mechanism, as well as a therapeutic intervention for excessive inflammation.

CASP4/caspase-11 promotes autophagosome formation in response to bacterial infection.

CASP4/caspase-11-dependent inflammasome activation is important for the clearance of various Gram-negative bacteria entering the host cytosol. Additionally, CASP4 modulates the actin cytoskeleton to promote the maturation of phagosomes harboring intracellular pathogens such as Legionella pneumophila but not those enclosing nonpathogenic bacteria. Nevertheless, this non-inflammatory role of CASP4 regarding the trafficking of vacuolar bacteria remains poorly understood. Macroautophagy/autophagy, a catabolic process within eukaryotic cells, is also implicated in the elimination of intracellular pathogens such as Burkholderia cenocepacia. Here we show that CASP4-deficient macrophages exhibit a defect in autophagosome formation in response to B. cenocepacia infection. The absence of CASP4 causes an accumulation of the small GTPase RAB7, reduced colocalization of B. cenocepacia with LC3 and acidic compartments accompanied by increased bacterial replication in vitro and in vivo. Together, our data reveal a novel role of CASP4 in regulating autophagy in response to B. cenocepacia infection.

Alpha-kinase 1 is a cytosolic innate immune receptor for bacterial ADP-heptose.

Immune recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors often activates proinflammatory NF-κB signalling1. Recent studies indicate that the bacterial metabolite D-glycero-ß-D-manno-heptose 1,7-bisphosphate (HBP) can activate NF-κB signalling in host cytosol2-4, but it is unclear whether HBP is a genuine PAMP and the cognate pattern recognition receptor has not been identified. Here we combined a transposon screen in Yersinia pseudotuberculosis with biochemical analyses and identified ADP-ß-D-manno-heptose (ADP-Hep), which mediates type III secretion system-dependent NF-κB activation and cytokine expression. ADP-Hep, but not other heptose metabolites, could enter host cytosol to activate NF-κB. A CRISPR-Cas9 screen showed that activation of NF-κB by ADP-Hep involves an ALPK1 (alpha-kinase 1)-TIFA (TRAF-interacting protein with forkhead-associated domain) axis. ADP-Hep directly binds the N-terminal domain of ALPK1, stimulating its kinase domain to phosphorylate and activate TIFA. The crystal structure of the N-terminal domain of ALPK1 and ADP-Hep in complex revealed the atomic mechanism of this ligand-receptor recognition process. HBP was transformed by host adenylyltransferases into ADP-heptose 7-P, which could activate ALPK1 to a lesser extent than ADP-Hep. ADP-Hep (but not HBP) alone or during bacterial infection induced Alpk1-dependent inflammation in mice. Our findings identify ALPK1 and ADP-Hep as a pattern recognition receptor and an effective immunomodulator, respectively.

Human Cystic Fibrosis Macrophages Have Defective Calcium-Dependent Protein Kinase C Activation of the NADPH Oxidase, an Effect Augmented by Burkholderia cenocepacia.

Macrophage intracellular pathogen killing is defective in cystic fibrosis (CF), despite abundant production of reactive oxygen species (ROS) in lung tissue. Burkholderia species can cause serious infection in CF and themselves affect key oxidase components in murine non-CF cells. However, it is unknown whether human CF macrophages have an independent defect in the oxidative burst and whether Burkholderia contributes to this defect in terms of assembly of the NADPH oxidase complex and subsequent ROS production. In this article, we analyze CF and non-CF human monocyte-derived macrophages (MDMs) for ROS production, NADPH assembly capacity, protein kinase C expression, and calcium release in response to PMA and CF pathogens. CF MDMs demonstrate a nearly 60% reduction in superoxide production after PMA stimulation compared with non-CF MDMs. Although CF MDMs generally have increased total NADPH component protein expression, they demonstrate decreased expression of the calcium-dependent protein kinase C conventional subclass α/ß leading to reduced phosphorylation of NADPH oxidase components p47 phox and p40 phox in comparison with non-CF MDMs. Ingestion of B. cenocepacia independently contributes to and worsens the overall oxidative burst deficits in CF MDMs compared with non-CF MDMs. Together, these results provide evidence for inherent deficits in the CF macrophage oxidative burst caused by decreased phosphorylation of NADPH oxidase cytosolic components that are augmented by Burkholderia These findings implicate a critical role for defective macrophage oxidative responses in persistent bacterial infections in CF and create new opportunities for boosting the macrophage immune response to limit infection.

Using dendritic cells to evaluate how Burkholderia cenocepacia clonal isolates from a chronically infected cystic fibrosis patient subvert immune functions.

Infection with Burkholderia cepacia complex (Bcc) bacteria is a threat to cystic fibrosis (CF) patients, commonly leading to a fatal pneumonia, the cepacia syndrome. It causes a massive production of pro-inflammatory cytokines and leucocyte recruitment to airway epithelium without resolving infection and contributing to tissue lesion. To dissect how Bcc bacteria subvert the immune response, we developed a co-culture model with human dendritic cells (DCs) and B. cenocepacia clonal variants isolated from a chronically infected CF patient, who died with cepacia syndrome. We demonstrated that the two late variants were sevenfold and 17-fold (respectively) more internalized by DCs than the variant that initiated infection. The late variants showed improved survival within DCs (60.29 and 52.82 CFU/DC) compared to the initial variant (0.38 CFU/DC). All clonal isolates induced high expression of inflammatory cytokines IL-8, IL-6, IL-1ß, IL-12, IL-23, TNF-α and IL-1ß. This pro-inflammatory trait was significantly more pronounced in DCs infected with the late variants than in DCs infected with the variant that initiated patient's infection. All infected DCs failed to upregulate maturation markers, HLA-DR, CD80, CD86 and CD83. Nevertheless, these infected DCs activated approximately twice more T cells than non-infected DCs. Similar T cell activation was observable with respective conditioned media, suggesting a non-antigen-specific activation. Our data indicate that during prolonged infection, B. cenocepacia acquires ability to survive intracellularly, inducing inflammation, while refraining DC's maturation and stimulating non-antigen-specific T cell responses. The co-culture model here developed may be broadly applied to study B. cenocepacia-induced immunomodulation.

The Burkholderia cenocepacia peptidoglycan-associated lipoprotein is involved in epithelial cell attachment and elicitation of inflammation.

The Burkholderia cepacia complex (Bcc) is a group of Gram-negative opportunistic pathogens causing infections in people with cystic fibrosis (CF). Bcc is highly antibiotic resistant, making conventional antibiotic treatment problematic. The identification of novel targets for anti-virulence therapies should improve therapeutic options for infected CF patients. We previously identified that the peptidoglycan-associated lipoprotein (Pal) was immunogenic in Bcc infected CF patients; however, its role in Bcc pathogenesis is unknown. The virulence of a pal deletion mutant (Δpal) in Galleria mellonella was 88-fold reduced (p < .001) compared to wild type. The lipopolysaccharide profiles of wild type and Δpal were identical, indicating no involvement of Pal in O-antigen transport. However, Δpal was more susceptible to polymyxin B. Structural elucidation by X-ray crystallography and calorimetry demonstrated that Pal binds peptidoglycan fragments. Δpal showed a 1.5-fold reduced stimulation of IL-8 in CF epithelial cells relative to wild type (p < .001), demonstrating that Pal is a significant driver of inflammation. The Δpal mutant had reduced binding to CFBE41o- cells, but adhesion of Pal-expressing recombinant E. coli to CFBE41o- cells was enhanced compared to wild-type E. coli (p < .0001), confirming that Pal plays a direct role in host cell attachment. Overall, Bcc Pal mediates host cell attachment and stimulation of cytokine secretion, contributing to Bcc pathogenesis.

A Burkholderia Type VI Effector Deamidates Rho GTPases to Activate the Pyrin Inflammasome and Trigger Inflammation.

Burkholderia cenocepacia is an opportunistic pathogen of the cystic fibrosis lung that elicits a strong inflammatory response. B. cenocepacia employs a type VI secretion system (T6SS) to survive in macrophages by disarming Rho-type GTPases, causing actin cytoskeletal defects. Here, we identified TecA, a non-VgrG T6SS effector responsible for actin disruption. TecA and other bacterial homologs bear a cysteine protease-like catalytic triad, which inactivates Rho GTPases by deamidating a conserved asparagine in the GTPase switch-I region. RhoA deamidation induces caspase-1 inflammasome activation, which is mediated by the familial Mediterranean fever disease protein Pyrin. In mouse infection, the deamidase activity of TecA is necessary and sufficient for B. cenocepacia-triggered lung inflammation and also protects mice from lethal B. cenocepacia infection. Therefore, Burkholderia TecA is a T6SS effector that modifies a eukaryotic target through an asparagine deamidase activity, which in turn elicits host cell death and inflammation through activation of the Pyrin inflammasome.

Structural Features Essential to the Antimicrobial Functions of Human SPLUNC1.

SPLUNC1 is an abundantly secreted innate immune protein in the mammalian respiratory tract that exerts bacteriostatic and antibiofilm effects, binds to lipopolysaccharide (LPS), and acts as a fluid-spreading surfactant. Here, we unravel the structural elements essential for the surfactant and antimicrobial functions of human SPLUNC1 (short palate lung nasal epithelial clone 1). A unique α-helix (α4) that extends from the body of SPLUNC1 is required for the bacteriostatic, surfactant, and LPS binding activities of this protein. Indeed, we find that mutation of just four leucine residues within this helical motif to alanine is sufficient to significantly inhibit the fluid spreading abilities of SPLUNC1, as well as its bacteriostatic actions against Gram-negative pathogens Burkholderia cenocepacia and Pseudomonas aeruginosa. Conformational flexibility in the body of SPLUNC1 is also involved in the bacteriostatic, surfactant, and LPS binding functions of the protein as revealed by disulfide mutants introduced into SPLUNC1. In addition, SPLUNC1 exerts antibiofilm effects against Gram-negative bacteria, although α4 is not involved in this activity. Interestingly, though, the introduction of surface electrostatic mutations away from α4 based on the unique dolphin SPLUNC1 sequence, and confirmed by crystal structure, is shown to impart antibiofilm activity against Staphylococcus aureus, the first SPLUNC1-dependent effect against a Gram-positive bacterium reported to date. Together, these data pinpoint SPLUNC1 structural motifs required for the antimicrobial and surfactant actions of this protective human protein.

Assay Development for Image-Based Quantification of Intracellular Bacterial Replication and Analysis of the Innate Immune Response to Infection.

Severe bacterial infection can lead to inflammation, host tissue damage, and ultimately disseminated septic shock. The mammalian innate immune system responds to microbial infection through the detection of invariant pathogen-associated molecular patterns (PAMPs) by a range of pattern recognition receptors (PRRs) expressed by the host cell. A successful immune response involves tightly coordinated signaling from these receptors, leading to a robust transcriptional response producing cytokines and antimicrobial effectors. While the PRR-expressing phagocytes of the host innate immune system function to contain and degrade internalized bacteria through pathways such as selective autophagy, pathogenic bacteria may subvert this process to replicate in the host cell. We describe the development of imaging assays to investigate these host-pathogen interactions through gene perturbation screens, which could lead to the identification of novel effectors of the host response to bacterial infection. We identify markers of coordinated initial signaling in macrophages challenged with ligands to PRRs of the toll-like receptor (TLR) family and compare this response to that induced by intact bacteria of the Burkholderia cenocepacia complex (Bcc), an opportunistic pathogen that causes life-threatening infections in patients with cystic fibrosis and chronic granulomatous disease. Bcc has been shown to escape the endocytic pathway, activate selective autophagy, and replicate within human macrophages. We demonstrate robust image-based quantification of multiple stages of Bcc infection of macrophages: ubiquitin tagging of cytosolic bacteria, recruitment of selective autophagy effector proteins, and intracellular bacterial replication, and we show perturbation of bacterial replication using drug treatment or siRNA-based gene knockdown. The described panel of imaging assays can be extended to other bacterial infections and pathogenic ligand combinations where high-content siRNA screening could provide significant new insight into regulation of the innate immune response to infection.

Activation of Human Toll-like Receptor 4 (TLR4)·Myeloid Differentiation Factor 2 (MD-2) by Hypoacylated Lipopolysaccharide from a Clinical Isolate of Burkholderia cenocepacia.

Lung infection by Burkholderia species, in particular Burkholderia cenocepacia, accelerates tissue damage and increases post-lung transplant mortality in cystic fibrosis patients. Host-microbe interplay largely depends on interactions between pathogen-specific molecules and innate immune receptors such as Toll-like receptor 4 (TLR4), which recognizes the lipid A moiety of the bacterial lipopolysaccharide (LPS). The human TLR4·myeloid differentiation factor 2 (MD-2) LPS receptor complex is strongly activated by hexa-acylated lipid A and poorly activated by underacylated lipid A. Here, we report that B. cenocepacia LPS strongly activates human TLR4·MD-2 despite its lipid A having only five acyl chains. Furthermore, we show that aminoarabinose residues in lipid A contribute to TLR4-lipid A interactions, and experiments in a mouse model of LPS-induced endotoxic shock confirmed the proinflammatory potential of B. cenocepacia penta-acylated lipid A. Molecular modeling combined with mutagenesis of TLR4-MD-2 interactive surfaces suggests that longer acyl chains and the aminoarabinose residues in the B. cenocepacia lipid A allow exposure of the fifth acyl chain on the surface of MD-2 enabling interactions with TLR4 and its dimerization. Our results provide a molecular model for activation of the human TLR4·MD-2 complex by penta-acylated lipid A explaining the ability of hypoacylated B. cenocepacia LPS to promote proinflammatory responses associated with the severe pathogenicity of this opportunistic bacterium.

Burkholderia cenocepacia Lipopolysaccharide Modification and Flagellin Glycosylation Affect Virulence but Not Innate Immune Recognition in Plants.

UNLABELLED: Burkholderia cenocepacia causes opportunistic infections in plants, insects, animals, and humans, suggesting that "virulence" depends on the host and its innate susceptibility to infection. We hypothesized that modifications in key bacterial molecules recognized by the innate immune system modulate host responses to B. cenocepacia. Indeed, modification of lipopolysaccharide (LPS) with 4-amino-4-deoxy-L-arabinose and flagellin glycosylation attenuates B. cenocepacia infection in Arabidopsis thaliana and Galleria mellonella insect larvae. However, B. cenocepacia LPS and flagellin triggered rapid bursts of nitric oxide and reactive oxygen species in A. thaliana leading to activation of the PR-1 defense gene. These responses were drastically reduced in plants with fls2 (flagellin FLS2 host receptor kinase), Atnoa1 (nitric oxide-associated protein 1), and dnd1-1 (reduced production of nitric oxide) null mutations. Together, our results indicate that LPS modification and flagellin glycosylation do not affect recognition by plant receptors but are required for bacteria to establish overt infection. IMPORTANCE: Virulence and pathogenicity are properties ascribed to microbes, which actually require careful consideration of the host. Using the term "pathogen" to define a microbe without considering its host has recently been debated, since the microbe's capacity to establish a niche in a given host is a critical feature associated with infection. Opportunistic bacteria are a perfect example of microbes whose ability to cause disease is intimately related to the host's ability to recognize and respond to the infection. Here, we use the opportunistic bacterium Burkholderia cenocepacia and the host plant Arabidopsis thaliana to investigate the role of bacterial surface molecules, namely, lipopolysaccharide and flagellin, in contributing to infection and also in eliciting a host response. We reveal that both molecules can be modified by glycosylation, and although the modifications are critical for the bacteria to establish an infection, they do not impact the host's ability to recognize the pathogen.

Identification of the flagellin glycosylation system in Burkholderia cenocepacia and the contribution of glycosylated flagellin to evasion of human innate immune responses.

Burkholderia cenocepacia is an opportunistic pathogen threatening patients with cystic fibrosis. Flagella are required for biofilm formation, as well as adhesion to and invasion of epithelial cells. Recognition of flagellin via the Toll-like receptor 5 (TLR5) contributes to exacerbate B. cenocepacia-induced lung epithelial inflammatory responses. In this study, we report that B. cenocepacia flagellin is glycosylated on at least 10 different sites with a single sugar, 4,6-dideoxy-4-(3-hydroxybutanoylamino)-D-glucose. We have identified key genes that are required for flagellin glycosylation, including a predicted glycosyltransferase gene that is linked to the flagellin biosynthesis cluster and a putative acetyltransferase gene located within the O-antigen lipopolysaccharide cluster. Another O-antigen cluster gene, rmlB, which is required for flagellin glycan and O-antigen biosynthesis, was essential for bacterial viability, uncovering a novel target against Burkholderia infections. Using glycosylated and nonglycosylated purified flagellin and a cell reporter system to assess TLR5-mediated responses, we also show that the presence of glycan in flagellin significantly impairs the inflammatory response of epithelial cells. We therefore suggest that flagellin glycosylation reduces recognition of flagellin by host TLR5, providing an evasive strategy to infecting bacteria.

Burkholderia cenocepacia†J2315 escapes to the cytosol and actively subverts autophagy in human macrophages.

Selective autophagy functions to specifically degrade cellular cargo tagged by ubiquitination, including bacteria. Strains of the Burkholderia cepacia complex (Bcc) are opportunistic pathogens that cause life-threatening infections in patients with cystic fibrosis (CF) and chronic granulomatous disease (CGD). While there is evidence that defective macrophage autophagy in a mouse model of CF can influence B. cenocepacia susceptibility, there have been no comprehensive studies on how this bacterium is sensed and targeted by the host autophagy response in human macrophages. Here, we describe the intracellular life cycle of B. cenocepacia J2315 and its interaction with the autophagy pathway in human cells. Electron and confocal microscopy analyses demonstrate that the invading bacteria interact transiently with the endocytic pathway before escaping to the cytosol. This escape triggers theselective autophagy pathway, and the recruitment of ubiquitin, the ubiquitin-binding adaptors p62 and NDP52 and the autophagosome membrane-associated protein LC3B, to the bacterial vicinity. However, despite recruitment of these key autophagy pathway effectors, B. cenocepacia blocks autophagosome completion and replicates in the host cytosol. We find that a pre-infection increase in cellular autophagy flux can significantly inhibit B. cenocepacia replication and that lower autophagy flux in macrophages from immunocompromised CGD patients could contribute to increased B. cenocepacia susceptibility, identifying autophagy manipulation as a potential therapeutic approach to reduce bacterial burden in B. cenocepacia infections.

Activation of the pyrin inflammasome by intracellular Burkholderia cenocepacia.

Burkholderia cenocepacia is an opportunistic pathogen that causes chronic infection and induces progressive respiratory inflammation in cystic fibrosis patients. Recognition of bacteria by mononuclear cells generally results in the activation of caspase-1 and processing of IL-1ß, a major proinflammatory cytokine. In this study, we report that human pyrin is required to detect intracellular B. cenocepacia leading to IL-1ß processing and release. This inflammatory response involves the host adapter molecule ASC and the bacterial type VI secretion system (T6SS). Human monocytes and THP-1 cells stably expressing either small interfering RNA against pyrin or YFP-pyrin and ASC (YFP-ASC) were infected with B. cenocepacia and analyzed for inflammasome activation. B. cenocepacia efficiently activates the inflammasome and IL-1ß release in monocytes and THP-1. Suppression of pyrin levels in monocytes and THP-1 cells reduced caspase-1 activation and IL-1ß release in response to B. cenocepacia challenge. In contrast, overexpression of pyrin or ASC induced a robust IL-1ß response to B. cenocepacia, which correlated with enhanced host cell death. Inflammasome activation was significantly reduced in cells infected with T6SS-defective mutants of B. cenocepacia, suggesting that the inflammatory reaction is likely induced by an as yet uncharacterized effector(s) of the T6SS. Together, we show for the first time, to our knowledge, that in human mononuclear cells infected with B. cenocepacia, pyrin associates with caspase-1 and ASC forming an inflammasome that upregulates mononuclear cell IL-1ß processing and release.

Use of 3-hydroxy fatty acid concentrations in a murine air pouch infection model as a surrogate marker for LPS activity: a feasibility study using environmental Burkholderia cenocepacia isolates.

Using a murine hypodermic air pouch infection model designed to mimic the release of bacterial products at physiological levels, 3-hydroxy fatty acid (3-OH FA) and endotoxin unit levels from Burkholderia cenocepacia isolates were assessed. The B. cenocepacia environmental isolates (n=35) survived in the hypodermic air pouch but did not invade across the peritoneal epithelial layer during a 72-h infection. For all 35 strains, when the molar ratio of C(14:0) 3-OH FA to C(16:0) 3-OH FA in the air pouch fluid wash samples was between 1.4 and 2.5, the concentrations of C(14:0) 3-OH FA were correlated with the endotoxin unit levels. However, both surrogate markers exhibited different correlations to the inflammatory response. The linear regression coefficient was 0.4234 for C(14:0) 3-OH FA concentrations vs. NO productions, 0.223 for endotoxin unit levels vs. NO productions, 0.5008 for C(14:0) 3-OH FA concentrations vs. TNF-alpha productions and 0.2869 for endotoxin unit levels vs. TNF-alpha productions. Therefore, C(14:0) 3-OH FA concentrations, rather than endotoxin unit levels, acted as an immunostimulatory indicator for LPS in the B. cenocepacia isolates.

Akt-mediated proinflammatory response of mononuclear phagocytes infected with Burkholderia cenocepacia occurs by a novel GSK3ß-dependent, IκB kinase-independent mechanism.

The environmental bacterium Burkholderia cenocepacia causes opportunistic lung infections in immunocompromised individuals, particularly in patients with cystic fibrosis. Infections in these patients are associated with exacerbated inflammation leading to rapid decay of lung function, and in some cases resulting in cepacia syndrome, which is characterized by a fatal acute necrotizing pneumonia and sepsis. B. cenocepacia can survive intracellularly in macrophages by altering the maturation of the phagosome, but very little is known on macrophage responses to the intracellular infection. In this study, we have examined the role of the PI3K/Akt signaling pathway in B. cenocepacia-infected monocytes and macrophages. We show that PI3K/Akt activity was required for NF-κB activity and the secretion of proinflammatory cytokines during infection with B. cenocepacia. In contrast to previous observations in epithelial cells infected with other Gram-negative bacteria, Akt did not enhance IκB kinase or NF-κB p65 phosphorylation, but rather inhibited GSK3ß, a negative regulator of NF-κB transcriptional activity. This novel mechanism of modulation of NF-κB activity may provide a unique therapeutic target for controlling excessive inflammation upon B. cenocepacia infection.