The Erwinia chrysanthemi Type III Secretion System Is Required for Multicellular Behavior

Enterobacterial animal pathogens exhibit aggregative multicellular behavior, which is manifested as pellicles on the culture surface and biofilms at the surface-liquid-air interface. Pellicle formation behavior requires production of extracellular polysaccharide, cellulose, and protein filaments, known as curli. Protein filaments analogous to curli are formed by many protein secretion systems, including the type III secretion system (TTSS). Here, we demonstrate that Erwinia chrysanthemi, which does not carry curli genes, requires the TTSS for pellicle formation. These data support a model where cellulose and generic protein filaments, which consist of either curli or TTSS-secreted proteins, are required for enterobacterial aggregative multicellular behavior. Using this assay, we found that hrpY, which encodes a two-component system response regulator homolog, is required for activity of hrpS, which encodes a σ(54)-dependent enhancer-binding protein homolog. In turn, hrpS is required for activity of the sigma factor homolog hrpL, which activates genes encoding TTSS structural and secreted proteins. Pellicle formation was temperature dependent and pellicles did not form at 36°C, even though TTSS genes were expressed at this temperature. We found that cellulose is a component of the E. chrysanthemi pellicle but that pellicle formation still occurs in a strain with an insertion in a cellulose synthase subunit homolog. Since the TTSS, but not the cellulose synthase subunit, is required for E. chrysanthemi pellicle formation, this inexpensive assay can be used as a high throughput screen for TTSS mutants or inhibitors

Jagn1 Is Induced in Response to ER Stress and Regulates Proinsulin Biosynthesis

<div><p>The Jagn1 protein was indentified in a SILAC proteomic screen of proteins that are increased in insulinoma cells expressing a folding-deficient proinsulin. Jagn1 mRNA was detected in primary rodent islets and in insulinoma cell lines and the levels were increased in response to ER stress. The function of Jagn1 was assessed in insulinoma cells by both knock-down and overexpression approaches. Knock-down of Jagn1 caused an increase in glucose-stimulated insulin secretion resulting from an increase in proinsulin biosynthesis. In contrast, overexpression of Jagn1 in insulinoma cells resulted in reduced cellular proinsulin and insulin levels. Our results identify a novel role for Jagn1 in regulating proinsulin biosynthesis in pancreatic β-cells. Under ER stress conditions Jagn1 is induced which might contribute to reducing proinsulin biosynthesis, in part by helping to relieve the protein folding load in the ER in an effort to restore ER homeostasis.</p></div

Jagn1 is expressed in insulinoma cells and islets and is induced by ER stress.

<p>(A) RT-PCR was performed to examine Jagn1 expression. Total RNA was isolated from control rat INS-1 832/13 insulinoma cells or cells treated with 2 μg/ml tunicamycin (Tm) for 16 h, 1 μM thapsigargin (Tg) for 6 h or from rat liver tissue. The (-) Control lane lacked one of the primers in the PCR reaction. (B) Total RNA was isolated from the indicated tissues and RT-PCR analysis was performed to detect Jagn1 expression. DC; dendritic cells. Lower panel shows rRNA in the total RNA detected by ethidium bromide (1 μg loaded for all samples except 100 ng islet RNA was loaded). (C-F) qPCR analysis for Jagn1 expression. (C) INS-1 insulin 2 (C96Y) cells treated or not with doxycycline (2 μg/ml) to induce mutant proinsulin expression for 24 h prior to total RNA isolation. (D) INS-1 832/13 cells treated with 2 μg/ml tunicamycin for 16 h. (E) Isolated mouse islets treated or not with tunicamycin as in D. (F) Islets isolated from mice fed a normal chow diet or a 45% high fat diet (HFD) for 12 weeks. In all cases results are from a minimum of N = 3 experiments. *p<0.05.</p

Overexpression of Jagn1 decreases steady-state insulin levels.

<p>(A) INS-1 832/13 cells were transfected with Myc-Jagn1 plasmid. After 24 h the cells were fixed and immunostained for insulin (green) and Myc-Jagn1 (red) and imaged by confocal microscopy. Scale bar: 50 μM. (B) Cellular insulin levels were quantified by measuring fluorescence intensity expressed as mean fluorescence in arbitrary units.</p

Jagn1 knock-down enhances proinsulin biosynthesis in INS-1 832/13 cells.

<p>(A) INS-1 832/13 cells were transfected with Jagn1 siRNA (10 nM) or control siRNA (directed to firefly luciferase) for 72 h. The cells were then washed in Met and Cys-free media and incubated in media containing ³⁵[S]-Met/Cys for 20 min. The cells were then either lysed (time 0) or the cells were washed in PBS and incubated in regular media for 30 min (chase), prior to cell lysis. Cell lysates were immunoprecipitated with anti-insulin antibody, the immunoprecipitates were resolved by NuPage gels (Invitrogen) and newly synthesized proinsulin was detected by PhosphorImager analysis (result is representative of N = 3 experiments). (B) The proinsulin band was quantified by densitometry and expressed relative to levels in control siRNA treated cells at time 0. (C) Analysis of general protein translation after 20 min of ³⁵[S]-Met/Cys labelling in control and Jagn1 knock-down cells.</p

Knock-down of Jagn1 enhances insulin secretion and increases insulin content.

<p>(A) INS-1 832/13 cells were transfected with Jagn1 siRNA (10 nM) or control siRNA (directed to firefly luciferase) for 72 h. Total RNA was isolated and qPCR analysis was performed for Jagn1 expression (N = 3, *p<0.05). (B) INS-1 832/13 cells were transfected with Jagn1 siRNA or control siRNA as in (A). The cells were then treated with basal glucose (2.8 mM) or stimulatory glucose (16.7 mM) for 1 h. Insulin in the media was measured by RIA. Results are expressed as secreted insulin normalized to control siRNA transfected cells at basal glucose (N = 4, *p<0.05). (C) Cellular insulin content measured by RIA in the experiment in (B). (D) Cells were transfected with control or Jagn1 siRNA as in (A), cell lysates were prepared and western blot analysis was performed to detect proinsulin or tubulin (loading control protein). Representative of N = 2 experiments. (E) qPCR analysis of insulin2 gene expression in control or Jagn1 siRNA treated cells (10 nM for 72 h) (N = 3). (F) INS-1 832/13 cells were transfected with control siRNA or Jagn1 siRNA for 72 h. Total RNA was then isolated and levels of unspliced and spliced XBP1 mRNA were detected by RT-PCR. Cells treated with tunicamycin (2 μg/ml) (Tm) for 16 h was used as a positive control. Results from three independent experiments is shown (Expt.1–3).</p

Jagn1 is primarily an ER localized membrane protein.

<p>(A) N-terminally Myc-tagged Jagn1 was expressed in HeLa cells. Cells were fixed in paraformladehyde and permeabilized with either TX-100 or digitonin and immunostained for the Myc-tag. Representative confocal images. Scale bar: 10 μm. (B) N-terminal GFP-tagged Jagn1 expressed in INS1 832/13. Scale bar: 10 μm. (C) Myc-tagged Jagn1 was expressed in βTC3 cells, fixed, permeabilized with TX-100 and immunostained for the Myc-tag and protein disulfide isomerise (PDI). Scale bar: 10 μm. (D) Myc-tagged Jagn1 was expressed in INS-1 832/13 cells, fixed, permeabilized with digitonin and immunostained for the Myc-tag and COPI coatomer. Scale bar: 10 μm.</p

A serological survey of Dirofilaria immitis infection in pet dogs of Busan, Korea, and effects of chemoprophylaxis

The status of Dirofilaria immitis infection was assessed in pet dogs of Busan, Korea, and chemoprophylactic effects of microfilaricidal medication were evaluated. A total of 294 pet dogs older than 6 mo were examined, 217 of which had been maintained indoors, and 77 had been kept outdoors. The SnapR kit and direct microscopic examinations of the peripheral blood were used. The mean overall parasite positive rates were 10.2% and 6.5%, respectively. Outdoor dogs evidenced adult worm infection rate of 31.2% and microfilaria infection rate of 18.2%. The indoor dogs, however, evidenced adult worm infection rate of 2.8% and microfilaria infection rate of 2.3%. The prevalence in males was more than 2 times that of females. The changing pattern of infection rates by age evidenced a gradual increase, from 2- to 6-year-old dogs, after which, a decrease in infection rates was noted. With regard to chemoprophylaxis, the infection rates of complete and incomplete chemoprophylaxis groups were found to be 2-3 times lower than that of the non-chemoprophylaxis group. The results of the present study indicate that the risk of exposure to D. immitis in pet dogs is quite high, particularly in male outdoor dogs, and chemoprophylactic measures were quite effective