Observations on the mechanism of eggshell formation in the liver fluke, Fasciola hepatica

A mechanism for eggshell production in Schistosoma mansoni has been proposed (Wells & Cordingley, 1991), and suggests that the release of eggshell protein globules from the vitelline cells occurs under alkaline conditions within the ootype followed by their subsequent fusion to form the eggshell. Fusion and tanning of these components produces eggshell which autofluoresces. The present study was carried out to determine whether a similar process operates in Fasciola hepatica. A number of drug treatments were used to disrupt key steps in the maturation of vitelline cells. Treatment with the calcium ionophore lasalocid (1 x 10(-5) M) led to the premature release of eggshell globules from the vitelline cells but not their fusion. Incubation in monensin (1 x 10(-6) M), a sodium ionophore and ammonium chloride (NH4Cl) (5 x 10(-2) M), a weak base, resulted in the premature fusion of eggshell protein globules within the vitelline cells and premature tanning of the eggshell protein material. The copper-containing enzyme, phenol oxidase, is thought to be involved in the tanning process during the production of eggs. Diethyldithiocarbamate (DDC, 1 x 10(-3) M) is a phenol oxidase inhibitor and treatment with this compound, in combination treatments with monensin and NH4Cl, prevented fusion of the vitelline cell globules and tanning of the shell protein material. The results of the study suggest that the mechanism for eggshell formation in F. hepatica is similar to that proposed for S. mansoni and may be common to other trematodes as well.

The Respiratory Syncytial Virus G Protein Conserved Domain Induces a Persistent and Protective Antibody Response in Rodents

Respiratory syncytial virus (RSV) is an important cause of severe upper and lower respiratory disease in infants and in the elderly. There are 2 main RSV subtypes A and B. A recombinant vaccine was designed based on the central domain of the RSV-A attachment G protein which we had previously named G2Na (aa130–230). Here we evaluated immunogenicity, persistence of antibody (Ab) response and protective efficacy induced in rodents by: (i) G2Na fused to DT (Diphtheria toxin) fragments in cotton rats. DT fusion did not potentiate neutralizing Ab responses against RSV-A or cross-reactivity to RSV-B. (ii) G2Nb (aa130–230 of the RSV-B G protein) either fused to, or admixed with G2Na. G2Nb did not induce RSV-B-reactive Ab responses. (iii) G2Na at low doses. Two injections of 3 µg G2Na in Alum were sufficient to induce protective immune responses in mouse lungs, preventing RSV-A and greatly reducing RSV-B infections. In cotton rats, G2Na-induced RSV-reactive Ab and protective immunity against RSV-A challenge that persisted for at least 24 weeks. (iv) injecting RSV primed mice with a single dose of G2Na/Alum or G2Na/PLGA [poly(D,L-lactide-co-glycolide]. Despite the presence of pre-existing RSV-specific Abs, these formulations effectively boosted anti-RSV Ab titres and increased Ab titres persisted for at least 21 weeks. Affinity maturation of these Abs increased from day 28 to day 148. These data indicate that G2Na has potential as a component of an RSV vaccine formulation.

The Type VI secretion system of Burkholderia cenocepacia affects multiple Rho family GTPases disrupting the actin cytoskeleton and the assembly of NADPH oxidase complex in macrophages

Burkholderia cenocepacia is a Gram-negative opportunistic pathogen of patients with cystic fibrosis and chronic granulomatous disease. The bacterium survives intracellularly in macrophages within a membrane-bound vacuole (BcCV) that precludes the fusion with lysosomes. The underlying cellular mechanisms and bacterial molecules mediating these phenotypes are unknown. Here, we show that intracellular B. cenocepacia expressing a type VI secretion system (T6SS) affects the activation of the Rac1 and Cdc42 RhoGTPase by reducing the cellular pool of GTP-bound Rac1 and Cdc42. The T6SS also increases the cellular pool of GTP-bound RhoA and decreases cofilin activity. These effects lead to abnormal actin polymerization causing collapse of lamellipodia and failure to retract the uropod. The T6SS also prevents the recruitment of soluble subunits of the NADPH oxidase complex including Rac1 to the BcCV membrane, but is not involved in the BcCV maturation arrest. Therefore, T6SS-mediated deregulation of Rho family GTPases is a common mechanism linking disruption of the actin cytoskeleton and delayed NADPH oxidase activation in macrophages infected with B. cenocepacia.

Akt-Mediated Proinflammatory Response of Mononuclear Phagocytes Infected with Burkholderia cenocepacia Occurs by a Novel GSK3 beta-Dependent, I kappa B 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-kappa B 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 I kappa B kinase or NF-kappa B p65 phosphorylation, but rather inhibited GSK3 beta, a negative regulator of NF-kappa B transcriptional activity. This novel mechanism of modulation of NF-kappa B activity may provide a unique therapeutic target for controlling excessive inflammation upon B. cenocepacia infection. The Journal of Immunology, 2011, 187: 635-643.

Selective extracellular vesicle-mediated export of an overlapping set of microRNAs from multiple cell types

Background: MicroRNAs (miRNAs) are a class of small RNA molecules that regulate expression of specific mRNA targets. They can be released from cells, often encapsulated within extracellular vesicles (EVs), and therefore have the potential to mediate intercellular communication. It has been suggested that certain miRNAs may be selectively exported, although the mechanism has yet to be identified. Manipulation of the miRNA content of EVs will be important for future therapeutic applications. We therefore wished to assess which endogenous miRNAs are enriched in EVs and how effectively an overexpressed miRNA would be exported.

Results: Small RNA libraries from HEK293T cells and vesicles before or after transfection with a vector for miR-146a overexpression were analysed by deep sequencing. A subset of miRNAs was found to be enriched in EVs; pathway analysis of their predicted target genes suggests a potential role in regulation of endocytosis. RT-qPCR in additional cell types and analysis of publicly available data revealed that many of these miRNAs tend to be widely preferentially exported. Whilst overexpressed miR-146a was highly enriched both in transfected cells and their EVs, the cellular:EV ratios of endogenous miRNAs were not grossly altered. MiR-451 was consistently the most highly exported miRNA in many different cell types. Intriguingly, Argonaute2 (Ago2) is required for miR-451 maturation and knock out of Ago2 has been shown to decrease expression of other preferentially exported miRNAs (eg miR-150 and miR-142-3p).

Conclusion: The global expression data provided by deep sequencing confirms that specific miRNAs are enriched in EVs released by HEK293T cells. Observation of similar patterns in a range of cell types suggests that a common mechanism for selective miRNA export may exist.

Burkholderia cenocepacia Type VI Secretion System Mediates Escape of Type II Secreted Proteins into the Cytoplasm of Infected Macrophages

Burkholderia cenocepacia is an opportunistic pathogen that survives intracellularly in macrophages and causes serious respiratory infections in patients with cystic fibrosis. We have previously shown that bacterial survival occurs in bacteria-containing membrane vacuoles (BcCVs) resembling arrested autophagosomes. Intracellular bacteria stimulate IL-1ß secretion in a caspase-1-dependent manner and induce dramatic changes to the actin cytoskeleton and the assembly of the NADPH oxidase complex onto the BcCV membrane. A Type 6 secretion system (T6SS) is required for these phenotypes but surprisingly it is not required for the maturation arrest of the BcCV. Here, we show that macrophages infected with B. cenocepacia employ the NLRP3 inflammasome to induce IL-1ß secretion and pyroptosis. Moreover, IL-1ß secretion by B. cenocepacia-infected macrophages is suppressed in deletion mutants unable to produce functional Type VI, Type IV, and Type 2 secretion systems (SS). We provide evidence that the T6SS mediates the disruption of the BcCV membrane, which allows the escape of proteins secreted by the T2SS into the macrophage cytoplasm. This was demonstrated by the activity of fusion derivatives of the T2SS-secreted metalloproteases ZmpA and ZmpB with adenylcyclase. Supporting this notion, ZmpA and ZmpB are required for efficient IL-1ß secretion in a T6SS dependent manner. ZmpA and ZmpB are also required for the maturation arrest of the BcCVs and bacterial intra-macrophage survival in a T6SS-independent fashion. Our results uncover a novel mechanism for inflammasome activation that involves cooperation between two bacterial secretory pathways, and an unanticipated role for T2SS-secreted proteins in intracellular bacterial survival.

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.

Inactivation of macrophage Rab7 by Burkholderia cenocepacia

Strains of the Burkholderia cepacia complex can survive within macrophages by arresting the maturation of phagocytic vacuoles. The bacteria preclude fusion of the phagosome with lysosomes by a process that is poorly understood. Using murine macrophages, we investigated the stage at which maturation is arrested and analyzed the underlying mechanism. Vacuoles containing B. cenocepacia strain J2315, an isolate of the transmissible ET12 clone, recruited Rab5 and synthesized phosphatidylinositol-3-phosphate, indicating progression to the early phagosomal stage. Despite the fact that the B. cenocepacia-containing vacuoles rarely fused with lysosomes, they could nevertheless acquire the late phagosomal markers CD63 and Rab7. Fluorescence recovery after photobleaching and use of a probe that detects Rab7-guanosine triphosphate indicated that activation of Rab7 was impaired by B. cenocepacia, accounting at least in part for the inability of the vacuole to merge with lysosomes. The Rab7 defect was not due to excessive cholesterol accumulation and was confined to the infected vacuoles. Jointly, these experiments indicate that B. cenocepacia express virulence factors capable of interfering with Rab7 function and thereby with membrane traffic.

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.

eNOS overexpression exacerbates vascular closure in the obliterative phase of OIR and increases angiogenic drive in the subsequent proliferative stage

Purpose: In ischemic retinopathies, the misdirection of reparative angiogenesis away from the hypoxic retina leads to pathologic neovascularization. Thus, therapeutic strategies that reverse this trend would be extremely beneficial. Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) is an important mediator of vascular endothelial growth factor (VEGF) function facilitating vascular growth and maturation. However, in addition to NO, eNOS can also produce superoxide (O), exacerbating pathology. Here, our aim was to investigate the effect of eNOS overexpression on vascular closure and subsequent recovery of the ischemic retina.

Methods: Mice overexpressing eNOS-GFP were subjected to oxygen-induced retinopathy (OIR) and changes in retinal vascularization quantified. Background angiogenic drive was assessed during vascular development and in aortic rings. NOS activity was measured by Griess assay or conversion of radiolabeled arginine to citrulline, nitrotyrosine (NT), and superoxide by immunolabeling and dihydroethidium fluorescence and VEGF by ELISA.

Results: In response to hyperoxia, enhanced eNOS expression led to increased NOS-derived superoxide and dysfunctional NO production, NT accumulation, and exacerbated vessel closure associated with tetrahydrobiopterin (BH) insufficiency. Despite worse vaso-obliteration, eNOS overexpression resulted in elevated hypoxia-induced angiogenic drive, independent of VEGF production. This correlated with increased vascular branching similar to that observed in isolated aortas and during development. Enhanced recovery was also associated with neovascular tuft formation, which showed defective NO production and increased eNOS-derived superoxide and NT levels.

Conclusions: In hyperoxia, reduced BH bioavailability causes overexpressed eNOS to become dysfunctional, exacerbating vaso-obliteration. In the proliferative phase, however, eNOS has important prorepair functions enhancing angiogenic growth potential and recovery in ischemia. © 2012 The Association for Research in Vision and Ophthalmology, Inc.

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.