Papel das Yops secretadas por Yersinia sobre a resposta imune do hospedeiro

The genus Yersinia contains three species pathogenic to humans: Y. pestis, Y. enterocolitica e Y. pseudotuberculosis. The pathogenicity of Yersinia is linked to the presence of a 70-kb virulence plasmid (pYV) that is common to the three species and codifies a type III secretion system and a set of virulence proteins, including those known as Yersinia outer proteins (Yops), that are exported by this system when the bacteria encounter host cells. Two Yops translocators (YopB and YopD) are inserted into the host plasma membrane and transport six effectors (YopO, YopH, YopM, YopJ and YopT) across the membrane into the cytosol of the host cell. The Yops effectors interfere with multiple signaling pathways of the infected cell, affecting both the innate and adaptive immune responses. This article focuses on the role of Yops in the modulation of the host immune response.

Does a 10-valent pneumococcal-Haemophilus influenzae protein D conjugate vaccine prevent respiratory exacerbations in children with recurrent protracted bacterial bronchitis, chronic suppurative lung disease and bronchiectasis : protocol for a randomised controlled trial

Background Recurrent protracted bacterial bronchitis (PBB), chronic suppurative lung disease (CSLD) and bronchiectasis are characterised by a chronic wet cough and are important causes of childhood respiratory morbidity globally. Haemophilus influenzae and Streptococcus pneumoniae are the most commonly associated pathogens. As respiratory exacerbations impair quality of life and may be associated with disease progression, we will determine if the novel 10-valent pneumococcal-Haemophilus influenzae protein D conjugate vaccine (PHiD-CV) reduces exacerbations in these children. Methods A multi-centre, parallel group, double-blind, randomised controlled trial in tertiary paediatric centres from three Australian cities is planned. Two hundred six children aged 18 months to 14 years with recurrent PBB, CSLD or bronchiectasis will be randomised to receive either two doses of PHiD-CV or control meningococcal (ACYW(135)) conjugate vaccine 2 months apart and followed for 12 months after the second vaccine dose. Randomisation will be stratified by site, age (<6 years and >= 6 years) and aetiology (recurrent PBB or CSLD/bronchiectasis). Clinical histories, respiratory status (including spirometry in children aged >= 6 years), nasopharyngeal and saliva swabs, and serum will be collected at baseline and at 2, 3, 8 and 14 months post-enrolment. Local and systemic reactions will be recorded on daily diaries for 7 and 30 days, respectively, following each vaccine dose and serious adverse events monitored throughout the trial. Fortnightly, parental contact will help record respiratory exacerbations. The primary outcome is the incidence of respiratory exacerbations in the 12 months following the second vaccine dose. Secondary outcomes include: nasopharyngeal carriage of H. influenzae and S. pneumoniae vaccine and vaccine-related serotypes; systemic and mucosal immune responses to H. influenzae proteins and S. pneumoniae vaccine and vaccine-related serotypes; impact upon lung function in children aged >= 6 years; and vaccine safety. Discussion As H. influenzae is the most common bacterial pathogen associated with these chronic respiratory diseases in children, a novel pneumococcal conjugate vaccine that also impacts upon H. influenzae and helps prevent respiratory exacerbations would assist clinical management with potential short- and long-term health benefits. Our study will be the first to assess vaccine efficacy targeting H. influenzae in children with recurrent PBB, CSLD and bronchiectasis.

Increased surfactant protein D fails to improve bacterial clearance and inflammation in serpinB1-/- mice

Previously, we described the protective role of the neutrophil serine protease inhibitor serpinB1 in preventing early mortality of Pseudomonas aeruginosa lung infection by fostering bacterial clearance and limiting inflammatory cytokines and proteolytic damage. Surfactant protein D (SP-D), which maintains the antiinflammatory pulmonary environment and mediates bacterial removal, was degraded in infected serpinB1-deficient mice. Based on the hypothesis that increased SP-D would rescue or mitigate the pathological effects of serpinB1 deletion, we generated two serpinB1(-/-) lines overexpressing lung-specific rat SP-D and inoculated the mice with P. aeruginosa. Contrary to predictions, bacterial counts in the lungs of SP-D(low)serpinB1(-/-) and SP-D(high) serpinB1(-/-) mice were 4 logs higher than wild-type and not different from serpinB1(-/-) mice. SP-D overexpression also failed to mitigate inflammation (TNF-α), lung injury (free protein, albumin), or excess neutrophil death (free myeloperoxidase, elastase). These pathological markers were higher for infected SP-D(high)serpinB1(-/-) mice than for serpinB1(-/-) mice, although the differences were not significant after controlling for multiple comparisons. The failure of transgenic SP-D to rescue antibacterial defense of serpinB1-deficient mice occurred despite 5-fold or 20-fold increased expression levels, largely normal structure, and dose-dependent bacteria-aggregating activity. SP-D of infected wild-type mice was intact in 43-kD monomers by reducing SDS-PAGE. By contrast, proteolytic fragments of 35, 17, and 8 kD were found in infected SP-D(low)serpinB1(-/-), SP-D(high) serpinB1(-/-) mice, and serpinB1(-/-) mice. Thus, although therapies to increase lung concentration of SP-D may have beneficial applications, the findings suggest that therapy with SP-D may not be beneficial for lung inflammation or infection if the underlying clinical condition includes excess proteolysis.

Surfactant protein D modulates allergen particle uptake and inflammatory response in a human epithelial airway model

Background Allergen-containing subpollen particles (SPP) are released from whole plant pollen upon contact with water or even high humidity. Because of their size SPP can preferentially reach the lower airways where they come into contact with surfactant protein (SP)-D. The aim of the present study was to investigate the influence of SP-D in a complex three-dimensional human epithelial airway model, which simulates the most important barrier functions of the epithelial airway. The uptake of SPP as well as the secretion of pro-inflammatory cytokines was investigated. Methods SPP were isolated from timothy grass and subsequently fluorescently labeled. A human epithelial airway model was built by using human Type II-pneumocyte like cells (A549 cells), human monocyte derived macrophages as well as human monocyte derived dendritic cells. The epithelial cell model was incubated with SPP in the presence and absence of surfactant protein D. Particle uptake was evaluated by confocal microscopy and advanced computer-controlled analysis. Finally, human primary CD4+ T-Cells were added to the epithelial airway model and soluble mediators were measured by enzyme linked immunosorbent assay or bead array. Results SPP were taken up by epithelial cells, macrophages, and dendritic cells. This uptake coincided with secretion of pro-inflammatory cytokines and chemokines. SP-D modulated the uptake of SPP in a cell type specific way (e.g. increased number of macrophages and epithelial cells, which participated in allergen particle uptake) and led to a decreased secretion of pro-inflammatory cytokines. Conclusion These results display a possible mechanism of how SP-D can modulate the inflammatory response to inhaled allergen.

The role of inducible nitric oxide synthase for interstitial remodeling of alveolar septa in surfactant protein D-deficient mice

Surfactant protein D (SP-D) modulates the lung's immune system. Its absence leads to NOS2-independent alveolar lipoproteinosis and NOS2-dependent chronic inflammation, which is critical for early emphysematous remodeling. With aging, SP-D knockout mice develop an additional interstitial fibrotic component. We hypothesize that this age-related interstitial septal wall remodeling is mediated by NOS2. Using invasive pulmonary function testing such as the forced oscillation technique and quasistatic pressure-volume perturbation and design-based stereology, we compared 29-wk-old SP-D knockout (Sftpd(-/-)) mice, SP-D/NOS2 double-knockout (DiNOS) mice, and wild-type mice (WT). Structural changes, including alveolar epithelial surface area, distribution of septal wall thickness, and volumes of septal wall components (alveolar epithelium, interstitial tissue, and endothelium) were quantified. Twenty-nine-week-old Sftpd(-/-) mice had preserved lung mechanics at the organ level, whereas elastance was increased in DiNOS. Airspace enlargement and loss of surface area of alveolar epithelium coexist with increased septal wall thickness in Sftpd(-/-) mice. These changes were reduced in DiNOS, and compared with Sftpd(-/-) mice a decrease in volumes of interstitial tissue and alveolar epithelium was found. To understand the effects of lung pathology on measured lung mechanics, structural data were used to inform a computational model, simulating lung mechanics as a function of airspace derecruitment, septal wall destruction (loss of surface area), and septal wall thickening. In conclusion, NOS2 mediates remodeling of septal walls, resulting in deposition of interstitial tissue in Sftpd(-/-). Forward modeling linking structure and lung mechanics describes the complex mechanical properties by parenchymatous destruction (emphysema), interstitial remodeling (septal wall thickening), and altered recruitability of acinar airspaces.

Protein crosslinking studies suggest that Rhizobium meliloti C4-dicarboxylic acid transport protein D, a sigma 54-dependent transcriptional activator, interacts with sigma 54 and the beta subunit of RNA polymerase.

Rhizobium meliloti C4-dicarboxylic acid transport protein D (DCTD) activates transcription by a form of RNA polymerase holoenzyme that has sigma 54 as its sigma factor (referred to as E sigma 54). DCTD catalyzes the ATP-dependent isomerization of closed complexes between E sigma 54 and the dctA promoter to transcriptionally productive open complexes. Transcriptional activation probably involves specific protein-protein interactions between DCTD and E sigma 54. Interactions between sigma 54-dependent activators and E sigma 54 are transient, and there has been no report of a biochemical assay for contact between E sigma 54 and any activator to date. Heterobifunctional crosslinking reagents were used to examine protein-protein interactions between the various subunits of E sigma 54 and DCTD. DCTD was crosslinked to Salmonella typhimurium sigma 54 with the crosslinking reagents succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate and N-hydroxysulfosuccinimidyl-4-azidobenzoate. Cys-307 of sigma 54 was identified by site-directed mutagenesis as the residue that was crosslinked to DCTD. DCTD was also crosslinked to the beta subunit of Escherichia coli core RNA polymerase with succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, but not with N-hydroxysulfosuccinimidyl-4-azidobenzoate. These data suggest that interactions of DCTD with sigma 54 and the beta subunit may be important for transcriptional activation and offer evidence for interactions between a sigma 54-dependent activator and sigma 54, as well as the beta subunit of RNA polymerase.

Detection of surfactant protein A (SP-A) and surfactant protein D (SP-D) in equine synovial fluid with immunoblotting

Once considered unique to the lung, surfactant proteins have been clearly identified in the intestine and peritoneum and are suggested to exist in several other organs. In the lung, surfactant proteins assist in the formation of a monolayer of surface-active phospholipid at the liquid-air interface of the alveolar lining, reducing the surface tension at this surface. In contrast, surface-active phospholipid adsorbed to articular surfaces has been identified as the load-bearing boundary lubricant of the joint. This raises the question of whether surfactant proteins in synovial fluid (SF) are required for the formation of the adsorbed layer in normal joints. Proteins from small volumes of equine SF were resolved by 1- and 2-dimensional polyacrylamide gel electrophoresis and detected by Western blotting to investigate the presence of surfactant proteins. The study showed that surfactant proteins A and D (SP-A and SP-D) are present in the SF of normal horses. We suggest that, like surface-active phospholipid, SP-A and SP-D play a significant role in the functioning of joints. Next will be clarification of the roles of surfactant proteins as disease markers in a variety of joint diseases, such as degenerative joint disease and inflammatory problems.

Epidemiology and comparative analysis of Yersinia in Ireland

Yersiniosis is an acute or chronic enteric zoonosis caused by enteropathogenic Yersinia species. Although yersiniosis is predominantly associated with gastroenteric forms of infection, extraintestinal forms are often reported from the elderly or patients with predisposing factors. Yersiniosis is often reported in countries with cold and mild climates (Northern and Central Europe, New Zealand and North of Russian Federation). The Irish Health Protection Surveillance Centre (HPSC) currently records only 3-7 notified cases of yersiniosis per year. At the same time pathogenic Yersinia enterocolitica is recovered from pigs (main source of pathogenic Y. enterocolitica) at the levels similar to that observed in Yersinia endemic countries. Introduction of Yersinia selective culture procedures may increase Yersinia isolation rates. To establish whether the small number of notifications of human disease was an underestimate due to lack of specific selective culture for Yersinia we carried out a prospective culture study of faecal samples from outpatients with diarrhoea, with additional culture of appendix and throat swabs. Higher levels of anti-Yersinia seroprevalence than yersiniosis notification rates in endemic countries suggests that most yersiniosis cases are unrecognised by culture. Subsequently, in addition to a prospective culture study of clinical specimens, we carried out serological screening of Irish blood donors and environmental screening of human sewage. Pathogenic Yersinia strains were not isolated from 1,189 faeces samples, nor from 297 throat swabs, or 23 appendix swabs. This suggested that current low notification rates in Ireland are not due to the lack of specific Yersinia culture procedures. Molecular screening detected a wider variety of Y. enterocolitica-specific targets in pig slurry than in human sewage. A serological survey for antibodies against Yersinia YOP (Yersinia Outer Proteins) proteins in Irish blood donors found antibodies in 25%, with an age-related trend to increased seropositivity, compatible with the hypothesis that yersiniosis may have been more prevalent in Ireland in the recent past. Y. enterocolitica is a heterogeneous group of microorganisms that comprises strains with different degree of pathogenicity. Although non-pathogenic Y. enterocolitica lack conventional virulence factors, these strains can be isolated from patients with diarrhoea. Insecticidal Toxin Complex (ITC) and Cytolethal Distending Toxins can potentially contribute to the virulence of non-pathogenic Y. enterocolitica in the absence of other virulence factors. We compared distribution of ITC and CDT loci among pathogenic and non-pathogenic Y. enterocolitica. Additionally, to demonstrate potential pathogenicity of non-pathogenic Y. enterocolitica we compared their virulence towards Galleria mellonella larvae (a non-mammalian model of human bacterial infections) with the virulence of highly and mildly pathogenic Y. enterocolitica strains. Surprisingly, virulence of pathogenic and non-pathogenic Y. enterocolitica in Galleria mellonella larvae observed at 37°C did not correlate with their pathogenic potential towards humans. Comparative phylogenomic analysis detects predicted coding sequences (CDSs) that define host-pathogen interactions and hence providing insights into molecular evolution of bacterial virulence. Comparative phylogenomic analysis of microarray data generated in Y. enterocolitica strains isolated in the Great Britain from humans with diarrhoea and domestic animals revealed high genetic heterogeneity of these species. Because of the extensive human, animal and food exchanges between the UK and Ireland the objective of this study was to gain further insight into genetic heterogeneity and relationships among clinical and non-clinical Y. enterocolitica strains of various pathogenic potential isolated in Ireland and Great Britain. No evidence of direct transfer of strains between the two countries was found.

TNF-alpha, H2O2 and NO response of peritoneal macrophages to Yersinia enterocolitica O : 3 derivatives

In this study, the effect of Yersinia derivatives on nitric oxide (NO), hydrogen peroxide (H2O2) and tumor necrosis factor-alpha (TNF-alpha) production by murine peritoneal macrophages was investigated. Addition of lipopolysaccharide (LPS) to the macrophage culture resulted in NO production that was dose dependent. on the other hand, bacterial cellular extract (CE) and Yersinia outer proteins (Yops) had no effect on NO production. The possible inhibitory effect of Yops on macrophage cultures stimulated with LPS was investigated. Yops partially inhibited NO production (67.4%) when compared with aminoguanidine. The effects of Yersinia derivatives on H2O2 production by macrophages were similar to those on NO production. LPS was the only derivative that stimulated H2O2 release in a dose-dependent manner. All Yersinia derivatives provoked the production of TNF-alpha, but LPS had the strongest effect, as observed for NO production. CE and Yops stimulated TNF-alpha production to a lesser extent than LPS. The results indicate the possibility that in vivo Yops may aid the evasion of the bacteria from the host defense mechanism by impairing the secretion of NO by macrophages. (C) 2003 Elsevier SAS. All rights reserved.

Influence of Yersinia pseudotuberculosis outer proteins (Yops) on interleukin-12, tumor necrosis factor alpha and nitric oxide production by peritoneal macrophages

An essential key to pathogenicity in Yersinia is the presence of a 70 kb plasmid (pYV) which encodes a type-III secretion system and several virulence outer proteins whose main function is to enable the bacteria to survive in the host. Thus, a specific immune response is needed in which cytokines are engaged. The aim of this study was to assess the influence of Yersinia outer proteins (Yops) released by Yersinia pseudotuberculosis on the production of the proinflammatory cytokines, interleukin-12 (IL-12), and tumor necrosis factor alpha (TNF-alpha), and nitric oxide (NO) by murine peritoneal macrophages. To this end, female Swiss mice were infected intravenously with wild-type Y pseudotuberculosis or with mutant strains unable to secrete specific Yops (YopE, YopH, YopJ, YopM, and YpkA). on the 7th, 14th, 21st, and 28th days after infection, the animals were sacrificed and the cytokines and NO were assayed in the peritoneal macrophages culture supernatants. A fall in NO production was observed during the course of infection with all the strains tested, though during the infection with the strains that did not secrete YopE and YopH, the suppression occurred later. There was, in general, an unchanged or sometimes increased production of TNF-alpha between the 7th and the 21st day after infection, compared to the control group, followed by an abrupt decrease on the last day of infection. The IL-12 production was also suppressed during the infection, with most of the strains tested, except with those that did not secrete YopJ and YopE. The results suggest that Yops may suppress IL-12, TNF-alpha, and NO production and that the most important proteins involved in this suppression are YopE and YopH. (C) 2004 Elsevier B.V. All rights reserved.

Papel das proteínas Yops de Yersinia pseudotuberculosis na ativação dos linfócitos B

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Papel das Yops de yersinia pseudotuberculosis na modulação da resposta imune celular durante infecção experimental

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Truncated recombinant human SP-D attenuates emphysema and type II cell changes in SP-D deficient mice

BACKGROUND: Surfactant protein D (SP-D) deficient mice develop emphysema-like pathology associated with focal accumulations of foamy alveolar macrophages, an excess of surfactant phospholipids in the alveolar space and both hypertrophy and hyperplasia of alveolar type II cells. These findings are associated with a chronic inflammatory state. Treatment of SP-D deficient mice with a truncated recombinant fragment of human SP-D (rfhSP-D) has been shown to decrease the lipidosis and alveolar macrophage accumulation as well as production of proinflammatory chemokines. The aim of this study was to investigate if rfhSP-D treatment reduces the structural abnormalities in parenchymal architecture and type II cells characteristic of SP-D deficiency. METHODS: SP-D knock-out mice, aged 3 weeks, 6 weeks and 9 weeks were treated with rfhSP-D for 9, 6 and 3 weeks, respectively. All mice were sacrificed at age 12 weeks and compared to both PBS treated SP-D deficient and wild-type groups. Lung structure was quantified by design-based stereology at the light and electron microscopic level. Emphasis was put on quantification of emphysema, type II cell changes and intracellular surfactant. Data were analysed with two sided non-parametric Mann-Whitney U-test. MAIN RESULTS: After 3 weeks of treatment, alveolar number was higher and mean alveolar size was smaller compared to saline-treated SP-D knock-out controls. There was no significant difference concerning these indices of pulmonary emphysema within rfhSP-D treated groups. Type II cell number and size were smaller as a consequence of treatment. The total volume of lamellar bodies per type II cell and per lung was smaller after 6 weeks of treatment. CONCLUSION: Treatment of SP-D deficient mice with rfhSP-D leads to a reduction in the degree of emphysema and a correction of type II cell hyperplasia and hypertrophy. This supports the concept that rfhSP-D might become a therapeutic option in diseases that are characterized by decreased SP-D levels in the lung.

Pseudomonas syringae Hrp type III secretion system and effector proteins

Pseudomonas syringae is a member of an important group of Gram-negative bacterial pathogens of plants and animals that depend on a type III secretion system to inject virulence effector proteins into host cells. In P. syringae, hrp/hrc genes encode the Hrp (type III secretion) system, and avirulence (avr) and Hrp-dependent outer protein (hop) genes encode effector proteins. The hrp/hrc genes of P. syringae pv syringae 61, P. syringae pv syringae B728a, and P. syringae pv tomato DC3000 are flanked by an exchangeable effector locus and a conserved effector locus in a tripartite mosaic Hrp pathogenicity island (Pai) that is linked to a tRNALeu gene found also in Pseudomonas aeruginosa but without linkage to Hrp system genes. Cosmid pHIR11 carries a portion of the strain 61 Hrp pathogenicity island that is sufficient to direct Escherichia coli and Pseudomonas fluorescens to inject HopPsyA into tobacco cells, thereby eliciting a hypersensitive response normally triggered only by plant pathogens. Large deletions in strain DC3000 revealed that the conserved effector locus is essential for pathogenicity but the exchangeable effector locus has only a minor role in growth in tomato. P. syringae secretes HopPsyA and AvrPto in culture in a Hrp-dependent manner at pH and temperature conditions associated with pathogenesis. AvrPto is also secreted by Yersinia enterocolitica. The secretion of AvrPto depends on the first 15 codons, which are also sufficient to direct the secretion of an Npt reporter from Y. enterocolitica, indicating that a universal targeting signal is recognized by the type III secretion systems of both plant and animal pathogens.