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.

Fosfomycin and tobramycin in combination downregulate nitrate reductase genes narG and narH, resulting in increased activity against Pseudomonas aeruginosa under anaerobic conditions

The activity of aminoglycosides, used to treat Pseudomonas aeruginosa respiratory infection in cystic fibrosis (CF) patients, is reduced under the anaerobic conditions that reflect the CF lung in vivo. In contrast, a 4:1 (w/w) combination of fosfomycin and tobramycin (F:T), under investigation for use in the treatment of CF lung infection, has increased activity against P. aeruginosa under anaerobic conditions. The aim of this study was to elucidate the mechanisms underlying the increased activity of F:T under anaerobic conditions. Microarray analysis was used to identify the transcriptional basis of increased F:T activity under anaerobic conditions, and key findings were confirmed by microbiological tests including nitrate utilization assays, growth curves and susceptibility testing. Notably, growth in sub-inhibitory concentrations of F:T, but not tobramycin or fosfomycin alone, significantly downregulated (p <0.05) nitrate reductase genes narG and narH, essential for normal anaerobic growth of P. aeruginosa. Under anaerobic conditions, F:T significantly decreased (p

BIIL 284 reduces neutrophil numbers but increases P. aeruginosa bacteremia and inflammation in mouse lungs

BACKGROUND: A clinical study to investigate the leukotriene B(4) (LTB(4))-receptor antagonist BIIL 284 in cystic fibrosis (CF) patients was prematurely terminated due to a significantly increased risk of adverse pulmonary events. We aimed to establish the effect of BIIL284 in models of Pseudomonas aeruginosa lung infection, thereby contributing to a better understanding of what could have led to adverse pulmonary events in CF patients.

METHODS: P. aeruginosa DNA in the blood of CF patients during and after acute pulmonary exacerbations and in stable patients with non-CF bronchiectasis (NCFB) and healthy individuals was assessed by PCR. The effect of BIIL 284 treatment was tested in an agar bead murine model of P. aeruginosa lung infection. Bacterial count and inflammation were evaluated in lung and other organs.

RESULTS: Most CF patients (98%) and all patients with NCFB and healthy individuals had negative P. aeruginosa DNA in their blood. Similarly, the P. aeruginosa-infected mice showed bacterial counts in the lung but not in the blood or spleen. BIIL 284 treatment decreased pulmonary neutrophils and increased P. aeruginosa numbers in mouse lungs leading to significantly higher bacteremia rates and lung inflammation compared to placebo treated animals.

CONCLUSIONS: Decreased airway neutrophils induced lung proliferation and severe bacteremia in a murine model of P. aeruginosa lung infection. These data suggest that caution should be taken when administering anti-inflammatory compounds to patients with bacterial infections.

Relative Contribution of P5 and Hap Surface Proteins to Nontypable Haemophilus influenzae Interplay with the Host Upper and Lower Airways

Nontypable Haemophilus influenzae (NTHi) is a major cause of opportunistic respiratory tract disease, and initiates infection by colonizing the nasopharynx. Bacterial surface proteins play determining roles in the NTHi-airways interplay, but their specific and relative contribution to colonization and infection of the respiratory tract has not been addressed comprehensively. In this study, we focused on the ompP5 and hap genes, present in all H. influenzae genome sequenced isolates, and encoding the P5 and Hap surface proteins, respectively. We employed isogenic single and double mutants of the ompP5 and hap genes generated in the pathogenic strain NTHi375 to evaluate P5 and Hap contribution to biofilm growth under continuous flow, to NTHi adhesion, and invasion/phagocytosis on nasal, pharyngeal, bronchial, alveolar cultured epithelial cells and alveolar macrophages, and to NTHi murine pulmonary infection. We show that P5 is not required for bacterial biofilm growth, but it is involved in NTHi interplay with respiratory cells and in mouse lung infection. Mechanistically, P5NTHi375 is not a ligand for CEACAM1 or α5 integrin receptors. Hap involvement in NTHi375-host interaction was shown to be limited, despite promoting bacterial cell adhesion when expressed in H. influenzae RdKW20. We also show that Hap does not contribute to bacterial biofilm growth, and that its absence partially restores the deficiency in lung infection observed for the ΔompP5 mutant. Altogether, this work frames the relative importance of the P5 and Hap surface proteins in NTHi virulence.

Relative contributions of lipooligosaccharide inner and outer core modifications to nontypeable Haemophilus influenzae pathogenesis

Nontypeable Haemophilus influenzae (NTHi) is a frequent commensal of the human nasopharynx that causes opportunistic infection in immunocompromised individuals. Existing evidence associates lipooligosaccharide (LOS) with disease, but the specific and relative contributions of NTHi LOS modifications to virulence properties of the bacterium have not been comprehensively addressed. Using NTHi strain 375, an isolate for which the detailed LOS structure has been determined, we compared systematically a set of isogenic mutant strains expressing sequentially truncated LOS. The relative contributions of 2-keto-3-deoxyoctulosonic acid, the triheptose inner core, oligosaccharide extensions on heptoses I and III, phosphorylcholine, digalactose, and sialic acid to NTHi resistance to antimicrobial peptides (AMP), self-aggregation, biofilm formation, cultured human respiratory epithelial infection, and murine pulmonary infection were assessed. We show that opsX, lgtF, lpsA, lic1, and lic2A contribute to bacterial resistance to AMP; lic1 is related to NTHi self-aggregation; lgtF, lic1, and siaB are involved in biofilm growth; opsX and lgtF participate in epithelial infection; and opsX, lgtF, and lpsA contribute to lung infection. Depending on the phenotype, the involvement of these LOS modifications occurs at different extents, independently or having an additive effect in combination. We discuss the relative contribution of LOS epitopes to NTHi virulence and frame a range of pathogenic traits in the context of infection.

Activation of human TLR4/MD-2 by hypoacylated lipopolysaccharide from a clinical isolate of Burkholderia cenocepacia

Lung infection by Burkholderia species, in particular B. cenocepacia, accelerates tissue damage and increase post-lung transplant mortality in cystic fibrosis patients. Host- microbes interplay largely depends on interactions between pathogen specific molecules and innate immune receptors such as the Toll-like receptor 4 (TLR4), which recognizes the lipid A moiety of the bacterial lipopolysaccharide (LPS). The human TLR4/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. Further, 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 pro- inflammatory potential of B. cenocepacia penta-acylated lipid A. Molecular modeling, combined with mutagenesis of TLR4-MD2 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 pro- inflammatory responses associated to the severe pathogenicity of this opportunistic bacterium.

Production of extended-spectrum β-lactamases and the potential indirect pathogenic role of Prevotella isolates from the cystic fibrosis respiratory microbiota

Extended-spectrum β-lactamase (ESBL) production and the prevalence of the β-lactamase-encoding gene blaTEM were determined in Prevotella isolates (n=50) cultured from the respiratory tract of adults and young people with cystic fibrosis (CF). Time-kill studies were used to investigate the concept of passive antibiotic resistance and to ascertain whether a β-lactamase-positive Prevotella isolate can protect a recognised CF pathogen from the action of ceftazidime in vitro. The results indicated that approximately three-quarters (38/50; 76%) of Prevotella isolates produced ESBLs. Isolates positive for ESBL production had higher minimum inhibitory concentrations (MICs) of β-lactam antibiotics compared with isolates negative for production of ESBLs (P<0.001). The blaTEM gene was detected more frequently in CF Prevotella isolates from paediatric patients compared with isolates from adults (P=0.002), with sequence analysis demonstrating that 21/22 (95%) partial blaTEM genes detected were identical to blaTEM-116. Furthermore, a β-lactamase-positive Prevotella isolate protected Pseudomonas aeruginosa from the antimicrobial effects of ceftazidime (P=0.03). Prevotella isolated from the CF respiratory microbiota produce ESBLs and may influence the pathogenesis of chronic lung infection via indirect methods, including shielding recognised pathogens from the action of ceftazidime.

Eradication and phenotypic tolerance of Burkholderia cenocepacia biofilms exposed to atmospheric pressure non-thermal plasma.

Chronic lung infection with bacteria from the Burkholderia cepacia complex (BCC), and in particular B. cenocepacia, is associated with significant morbidity and mortality in patients with cystic fibrosis (CF). B. cenocepacia can spread from person to person and exhibits intrinsic broad-spectrum antibiotic resistance. Recently, atmospheric pressure non-thermal plasmas (APNTPs) have gained increasing attention as a novel approach to the prevention and treatment of a variety of hospital-acquired infections. In this study, we evaluated an in-house-designed kHz-driven plasma source for the treatment of biofilms of a number of clinical CF B. cenocepacia isolates. The results demonstrated that APNTP is an effective and efficient tool for the eradication of B. cenocepacia biofilms but that efficacy is highly variable across different isolates. Determination of phenotypic differences between isolates in an attempt to understand variability in plasma tolerance revealed that isolates which are highly tolerant to APNTP typically produce biofilms of greater biomass than their more sensitive counterparts. This indicates a potential role for biofilm matrix components in biofilm tolerance to APNTP exposure. Furthermore, significant isolate-dependent differences in catalase activity in planktonic bacteria positively correlated with phenotypic resistance to APNTP by isolates grown in biofilms.

Autophagy stimulation by rapamycin suppresses lung inflammation and infection by Burkholderia cenocepacia in a model of cystic fibrosis

Cystic fibrosis (CF) is the most common inherited lethal disease in Caucasians which results in multiorgan dysfunction. However, 85% of the deaths are due to pulmonary infections. Infection by Burkholderia cenocepacia (B. cepacia) is a particularly lethal threat to CF patients because it causes severe and persistent lung inflammation and is resistant to nearly all available antibiotics. In CFTR Delta F508 (Delta F508) mouse macrophages, B. cepacia persists in vacuoles that do not fuse with the lysosomes and mediates increased production of IL-1 beta. It is believed that intracellular bacterial survival contributes to the persistence of the bacterium. Here we show for the first time that in wild-type but not in Delta F508 macrophages, many B. cepacia reside in autophagosomes that fuse with lysosomes at later stages of infection. Accordingly, association and intracellular survival of B. cepacia are higher in CFTR-Delta F508 macrophages than in WT macrophages. An autophagosome is a compartment that engulfs nonfunctional organelles and parts of the cytoplasm then delivers them to the lysosome for degradation to produce nutrients during periods of starvation or stress. Furthermore, we show that B. cepacia downregulates autophagy genes in WT and Delta F508 macrophages. However, autophagy dysfunction is more pronounced in Delta F508 macrophages since they already have compromised autophagy activity. We demonstrate that the autophagy-stimulating agent, rapamycin markedly decreases B. cepacia infection in vitro by enhancing the clearance of B. cepacia via induced autophagy. In vivo, rapamycin decreases bacterial burden in the lungs of CF mice and drastically reduces signs of lung inflammation. Together, our studies reveal that if efficiently activated, autophagy can control B. cepacia infection and ameliorate the associated inflammation. Therefore, autophagy is a novel target for new drug development for CF patients to control B. cepacia infection and accompanying inflammation.

Type-I interferons induce lung protease responses following respiratory syncytial virus infection via RIG-I-like receptors

The role of proteases in viral infection of the lung is poorly understood. Thus, we examined matrix metalloproteinases (MMPs) and cathepsin proteases in respiratory syncytial virus (RSV)-infected mouse lungs. RSV-induced gene expression for MMPs -2, -3, -7, -8, -9, -10, -12, -13, -14, -16, -17, -19, -20, -25, -27, and -28 and cathepsins B, C, E, G, H, K, L1, S, W, and Z in the airways of Friend leukemia virus B sensitive strain mice. Increased proteases were present in the bronchoalveolar lavage fluid (BALF) and lung tissue during infection. Mitochondrial antiviral-signaling protein (MAVS) and TIR-domain-containing adapter-inducing interferon-β-deficient mice were exposed to RSV. Mavs-deficient mice had significantly lower expression of airway MMP-2, -3, -7, -8, -9, -10, -12, -13, and -28 and cathepsins C, G, K, S, W, and Z. In lung epithelial cells, retinoic acid-inducible gene-1 (RIG-I) was identified as the major RIG-I-like receptor required for RSV-induced protease expression via MAVS. Overexpression of RIG-I or treatment with interferon-β in these cells induced MMP and cathepsin gene and protein expression. The significance of RIG-1 protease induction was demonstrated by the fact that inhibiting proteases with batimastat, E64 or ribavirin prevented airway hyperresponsiveness and enhanced viral clearance in RSV-infected mice.

Characterization of nontypable Haemophilus influenzae isolates recovered from adult patients with underlying chronic lung disease reveals genotypic and phenotypic traits associated with persistent infection

Nontypable Haemophilus influenzae (NTHi) has emerged as an important opportunistic pathogen causing infection in adults suffering obstructive lung diseases. Existing evidence associates chronic infection by NTHi to the progression of the chronic respiratory disease, but specific features of NTHi associated with persistence have not been comprehensively addressed. To provide clues about adaptive strategies adopted by NTHi during persistent infection, we compared sequential persistent isolates with newly acquired isolates in sputa from six patients with chronic obstructive lung disease. Pulse field gel electrophoresis (PFGE) identified three patients with consecutive persistent strains and three with new strains. Phenotypic characterisation included infection of respiratory epithelial cells, bacterial self-aggregation, biofilm formation and resistance to antimicrobial peptides (AMP). Persistent isolates differed from new strains in showing low epithelial adhesion and inability to form biofilms when grown under continuous-flow culture conditions in microfermenters. Self-aggregation clustered the strains by patient, not by persistence. Increasing resistance to AMPs was observed for each series of persistent isolates; this was not associated with lipooligosaccharide decoration with phosphorylcholine or with lipid A acylation. Variation was further analyzed for the series of three persistent isolates recovered from patient 1. These isolates displayed comparable growth rate, natural transformation frequency and murine pulmonary infection. Genome sequencing of these three isolates revealed sequential acquisition of single-nucleotide variants in the AMP permease sapC, the heme acquisition systems hgpB, hgpC, hup and hxuC, the 3-deoxy-D-manno-octulosonic acid kinase kdkA, the long-chain fatty acid transporter ompP1, and the phosphoribosylamine glycine ligase purD. Collectively, we frame a range of pathogenic traits and a repertoire of genetic variants in the context of persistent infection by NTHi.

Monocyte-dependent oncostatin M and TNFa synergize to stimulate unopposed matrix metalloproteinase-1/3 secretion from human lung fibroblasts in tuberculosis

Leukocyte-derived matrix metalloproteinases (MMP) are implicated in the tissue destruction characteristic of tuberculosis (TB). The contribution of lung stromal cells to MMP activity in TB is unknown. Oncostatin M (OSM) is an important stimulus to extrapulmonary stromal MMP induction, but its role in regulation of pulmonary MMP secretion or pathophysiology of TB is unknown. We investigated OSM secretion from Mycobacterium tuberculosis (Mtb)-infected human monocytes/macrophages and the networking effects of such OSM on lung fibroblast MMP secretion. Mtb increased monocyte OSM secretion dose dependently in vitro. In vivo tuberculous granulomas immunostained positively for OSM. Further, conditioned media from Mtb-infected monocytes (CoMTb) induced monocyte OSM secretion (670 ± 55 versus 166 ± 14 pg/mL in controls), implicating an autocrine loop. Mtb-induced OSM secretion was prostaglandin (PG) sensitive, and required activation of surface G-protein coupled receptors. OSM induction was ERK MAP kinase dependent, p38-requiring but JNK-independent. OSM synergized with TNF-, a key cytokine in TB granuloma formation, to stimulate pulmonary fibroblast MMP-1/-3 secretion, while suppressing secretion of tissue inhibitors of metalloproteinases-1/-2. In summary, Mtb infection of monocytes results in PG-dependent OSM secretion, which synergizes with TNF- to drive functionally unopposed fibroblast MMP-1/-3 secretion, demonstrating a previously unrecognized role for OSM in TB.