43 research outputs found
BarA-UvrY two-component system regulates virulence of uropathogenic E. coli CFT073.
No full textUropathogenic Escherichia coli (UPEC), a member of extraintestinal pathogenic E. coli, cause ∼80% of community-acquired urinary tract infections (UTI) in humans. UPEC initiates its colonization in epithelial cells lining the urinary tract with a complicated life cycle, replicating and persisting in intracellular and extracellular niches. Consequently, UPEC causes cystitis and more severe form of pyelonephritis. To further understand the virulence characteristics of UPEC, we investigated the roles of BarA-UvrY two-component system (TCS) in regulating UPEC virulence. Our results showed that mutation of BarA-UvrY TCS significantly decreased the virulence of UPEC CFT073, as assessed by mouse urinary tract infection, chicken embryo killing assay, and cytotoxicity assay on human kidney and uroepithelial cell lines. Furthermore, mutation of either barA or uvrY gene reduced the production of hemolysin, lipopolysaccharide (LPS), proinflammatory cytokines (TNF-α and IL-6) and chemokine (IL-8). The virulence phenotype was restored similar to that of wild-type by complementation of either barA or uvrY gene in trans. In addition, we discussed a possible link between the BarA-UvrY TCS and CsrA in positively and negatively controlling virulence in UPEC. Overall, this study provides the evidences for BarA-UvrY TCS regulates the virulence of UPEC CFT073 and may point to mechanisms by which virulence regulations are observed in different ways may control the long-term survival of UPEC in the urinary tract
CD23 MEDIATED IGE TRANSCYTOSIS IN AIRWAY INFLAMMATION
No full textCD23 (FceRII), a C-type lectin type II membrane glycoprotein, plays an important role in IgE homeostasis and development of allergic inflammation. I showed that CD23 was constitutively expressed in the established or primary human airway epithelial cells and its expression was significantly up-regulated by IL-4 stimulation. In a transcytosis assay, human IgE or IgE derived immune complex was transported and enhanced by IL-4 stimulation across a polarized Calu-3 monolayer. A CD23 specific antibody or soluble CD23 significantly reduced the transcytosis, suggesting a specific receptor-mediated transport by CD23. Transcytosis of both IgE and the immune complex was further verified in primary human airway epithelial cell monolayers. Furthermore, the transcytosed antigen-IgE complexes were competent in inducing degranulation of the cultured human mast cells. This study implies CD23-mediated IgE transcytosis in human airway epithelial cells may play a critical role in initiating and contributing to the perpetuation of airway allergic inflammation.
To verify the above results in a mouse model, CD23 expression was detected in epithelial cells lining mouse airway and enhanced by IL-4 exposure as well as in ovalbumin (OVA) sensitized mouse. I showed that CD23 transported IgE and OVA-IgE derived immune complex across airway epithelial cells in wild-type, but not CD23 knockout (KO), mice. The chimeric CD23KO mice repopulated with wild-type myeloid cells, sensitized and challenged with OVA showed significant reduction in siglec-F+ cells, eosinophils, macrophages and IL-4 in bronchoalveolar lavage fluid recovered 24 hours later compared to the wild-type mice.
Our finding of CD23-mediated IgE transport in airway epithelial cells suggest a possibility of CD23 transporting an IgE Fc-fused protein for immunotherapy. CTLA-4 (Cytotoxic T-Lymphocyte Antigen 4) which competitively binds CD80 and CD86 expressed on antigen presenting cells and inhibits CD28 mediated co-stimulation of T cell activation. A CTLA4-Fc (IgE) fusion protein produced in Chinese hamster ovary cells was intranasally administrated into mouse airway for assessing its specific transport by CD23. The effect of this fusion protein on the development of allergic inflammation is being fully investigated in wild-type, CD23-KO, and chimeric mouse model
Regulation of Virulence by BarA-UvrY Two-Component system and LuxS in Extraintestinal Pathogenic Escherichia coli.
No full textPathogenic E. coli cause intestinal or extraintestinal infections in many host species. E. coli strains enter the intestinal tract through food and colonize the intestinal epithelium to cause infections. In animals and humans, E. coli causes gastroenteritis, neonatal meningitis and urinary tract infections. In birds, E. coli causes a complex syndrome called avian colibacillosis.
The orthologs of BarA-UvrY two-component (TCS) system is known to regulate a number of phenotypic traits in gamma proteobacteria, although their role in Extraintestinal pathogenic Escherichia coli (ExPEC) virulence is yet to be determined. The barA gene is membrane bound sensor kinase protein and the uvrY gene encodes the cognate response regulator in E. coli. Work in this study has focused how the BarA-UvrY and LuxS system regulates in vivo virulence in uropathogenic E. coli (UPEC) and avian pathogenic E. coli (APEC) during infection. The main goal of this study is to look at how BarA-UvrY TCS and LuxS regulate virulence in APEC 7122 and UPEC CFT073. In this study, we studied the role of BarA-UvrY TCS system in regulation of virulence in the aforementioned ExPEC strains using animal model and tissue culture system and the role of LuxS in regulation of virulence determination in ExPEC. Our results indicate that BarA-UvrY regulates multiple virulence properties in APEC 7122 and UPEC CFT073 and that LuxS regulates partial virulence properties in APEC 7122 and UPEC CFT073
Reply to Lee and Howden
No full textGreen Open Access added to TU Delft Institutional Repository ‘You share, we take care!’ – Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public.Pattern Recognition and Bioinformatic
Orientation of <i>fim</i> switch in CFT073 (A) and MG1655 (B).
No full text<p>Direction of the <i>fim</i> switch was determined by using a PCR inversion assay. Densitometric analysis was performed to assess relative intensity of bands from agarose gels by using a software, ImageJ. The OFF or ON band intensity of the wild-type was set at 100%. Two independent replicates for each strain were used for this assay. This assay was repeated three times.</p
Expression of <i>fim</i> operon in K-12 strain MG1655.
No full text<p>β-galalactosidase assay was performed in relevant genetic background harboring a single copy plasmid with <i>fimA-lacZYA</i> transcriptional fusions either at invertible or at locked ON orientation. Bars represent means ± SEM of three experiments.</p
Biofilm formation on various abiotic surfaces in UPEC CFT073.
No full text<p>Crystal violet staining was performed to assess air-liquid biofilm formation on abiotic surfaces such as polystyrene (A) or PVC (B) at 48 hours, whereas crystal violet staining was performed to assess biofilm biomass on glass (C) at 6, 12, 24 and 48 hours. Strains were grown in LB broth with appropriate antibiotics without aeration at room temperature. Bars represent means of three experiments with three replicates per sample. The error bars represent standard errors of three replicates.</p
Effect of <i>csrA</i> on message stability of <i>fimA</i> (A) and <i>lrhA</i> (B) in K-12 strain MG1655.
No full text<p>Total RNA was harvested from late log growth phase. The mRNA stability of <i>fimA</i>, <i>lrhA</i> or <i>icd</i> (housekeeping control) was assessed for 10 mins after addition of rifampicin. The relative intensities of the wild-type and the mutant was compared to the intensity of <i>icd</i> mRNA. Normalized transcript levels at time zero prior to rifampicin treatment was set at 100%. This experiment was repeated two times.</p
Relative expression levels of <i>papA</i>, <i>hlyB</i> and <i>galU</i> in UPEC strain CFT073.
No full text<p>Bacterial strains were grown in LB broth for 48 hours at room temperature. Intensities of bands from agarose gels were subjected to image analysis post semi-quantitative RT-PCR from various strains. Transcript levels of <i>rrnA</i> were used as an internal control. Fold change was represented by the expression levels of genes in various strains relative to the wild-type, set at 100%. Bars represented means ± S.E.M. of three replicates.</p
Pleiotropic roles of uvrY on biofilm formation, motility and virulence in uropathogenic Escherichia coli CFT073.
No full textUrinary tract infections primarily caused by uropathogenic strains of Escherichia coli (E. coli) remain a significant public health problem in both developed and developing countries. An important virulence determinant in uropathogenesis is biofilm formation which requires expression of fimbriae, flagella, and other surface components such as lipopolysaccharides. In this study, we explored the regulation of uvrY and csrA genes in biofilm formation, motility and virulence determinants in uropathogenic E. coli. We found that mutation in uvrY suppressed biofilm formation on abiotic surfaces such as polyvinyl chloride, polystyrene and glass, and complementation of uvrY in the mutant restored the biofilm phenotype. We further evaluated the role of uvrY gene in expression of type 1 fimbriae, an important adhesin that facilitates adhesion to various abiotic surfaces. We found that phase variation of type 1 fimbriae between fimbriated and afimbriated mode was modulated by uvrY at its transcriptional level. Deletion mutant of uvrY lowered expression of fimbrial recombinase genes, such as fimB, fimE, and fimA, a gene encoding major fimbrial subunit. Furthermore, transcription of virulence specific genes such as papA, hlyB and galU was also reduced in the deletion mutant. Swarming motility and expression of flhD and flhC was also diminished in the mutant. Taken together, our findings unravel a possible mechanism in which uvrY facilitates biofilm formation, persistence and virulence of uropathogenic E. coli
