CD23 MEDIATED IGE TRANSCYTOSIS IN AIRWAY INFLAMMATION

CD23 (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.

Pathogenic 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

Human cytomegalovirus evades antibody-mediated immunity through endoplasmic reticulum-associated degradation of the FcRn receptor

Partial funding for Open Access provided by the UMD Libraries' Open Access Publishing Fund.Human cytomegalovirus (HCMV) can persistently infect humans, but how HCMV avoids humoral immunity is not clear. The neonatal Fc receptor (FcRn) controls IgG transport from the mother to the fetus and prolongs IgG half-life. Here we show that US11 inhibits the assembly of FcRn with β2m and retains FcRn in the endoplasmic reticulum (ER), consequently blocking FcRn trafficking to the endosome. Furthermore, US11 recruits the ubiquitin enzymes Derlin-1, TMEM129 and UbE2J2 to engage FcRn, consequently initiating the dislocation of FcRn from the ER to the cytosol and facilitating its degradation. Importantly, US11 inhibits IgGFcRn binding, resulting in a reduction of IgG transcytosis across intestinal or placental epithelial cells and IgG degradation in endothelial cells. Hence, these results identify the mechanism by which HCMV infection exploits an ER-associated degradation pathway through US11 to disable FcRn functions. These results have implications for vaccine development and immune surveillance

BarA-UvrY Two-Component System Regulates Virulence of Uropathogenic E. coli CFT073

Uropathogenic 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

Pleiotropic roles of uvrY on biofilm formation, motility and virulence in uropathogenic Escherichia coli CFT073.

Urinary 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

Antibiotic Susceptibility Patterns and Virulence-Associated Factors of Vancomycin-Resistant Enterococcal Isolates from Tertiary Care Hospitals

This study explored the prevalence of multi-drug resistance and virulence factors of enterococcal isolates obtained from various clinical specimens (n = 1575) including urine, blood, pus, tissue, catheter, vaginal wash, semen, and endotracheal secretions. Out of 862 enterococcal isolates, 388 (45%), 246 (29%), 120 (14%), and 108 (13%) were identified as Enterococcus faecalis, Enterococcus faecium, Enterococcus durans, and Enterococcus hirae, respectively, using standard morphological and biochemical methods. The antibiotic resistance profile of all these enterococcal isolates was checked using the disc diffusion technique. High-level resistance was observed for benzylpenicillin (70%) and vancomycin (43%) among E. faecalis and E. faecium isolates, respectively. This study also revealed the prevalence of ‘multi-drug resistance (resistant to 3 antibiotic groups)’ among the vancomycin-resistant enterococcal strains, and this was about 11% (n = 91). The virulence determinants associated with vancomycin resistance (VR) were determined phenotypically and genotypically. About 70 and 39% of E. faecalis and E. faecium isolates showed to be positive for all four virulence factors (gelatinase, protease, hemolysin, and biofilm). Among the several virulence genes, gelE was the most common virulence gene with a prevalence rate of 76 and 69% among E. faecalis and E. faecium isolates, respectively. More than 50% of VRE isolates harbored other virulence genes, such esp, asa, ace, and cylA. Similarly, the majority of the VR enterococcal isolates (n = 88/91) harbored vanA gene and none of them harbored vanB gene. These results disclose the importance of VR E. faecalis and E. faecium and the associated virulence factors involved in the persistence of infections in clinical settings

Effect of <i>uvrY</i> and <i>csrA</i> on <i>fimB</i>, <i>fimE</i> and <i>fimA</i> transcription in UPEC strain CFT073.

<p><b>A</b>. Transcript levels of <i>fimB</i>, <i>fimE</i> and <i>fimA</i> were evaluated by quantitative RT-PCR. The <i>rrnA</i> was used as an internal control. <b>B</b>. Relative expression levels of various transcripts were calculated by normalization of the actual intensity of bands with <i>rrnA</i> control and expressed as fold change relative to the wild-type. The wild-type was set at one. Bars represent mean ± S.E.M of three replicates.</p

Expression of <i>fim</i> operon in K-12 strain MG1655.

<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

Effect of <i>csrA</i> on message stability of <i>fimA</i> (A) and <i>lrhA</i> (B) in K-12 strain MG1655.

<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

Orientation of <i>fim</i> switch in CFT073 (A) and MG1655 (B).

<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