Subcutaneous and intranasal immunization with Stx2B-Tir-Stx1B-Zot reduces colonization and shedding of Escherichia coli O157:H7 in mice.

The type III secretion system of Escherichia coli O157:H7 is involved in colonization of mammalian hosts by the organism. The translocated intimin receptor (Tir) is inserted into the mammalian host cell plasma membrane in a hairpin loop topology with the central loop of the molecule exposed to the host cell surface and accessible for interaction with an LEE-encoded bacterial outer membrane adhesin called intimin. Shiga toxin type 1 and 2 produced by E. coli O157:H7 are responsible for hemolytic uremic syndrome and able to promote intestinal colonization. Zonula occludens toxin (Zot) is a single polypeptide chain encoded by the filamentous bacteriophage CTXφ of Vibrio cholerae. Zot binds a receptor on intestinal epithelial cells and increases mucosal permeability by affecting the structure of epithelial tight junctions. Because of these properties, Zot is a promising tool for mucosal drug and antigen (Ag) delivery. In the current study, we constructed a novel fusion protein carrying both of the immunogenic B subunits derived from the two toxins, Tir and Zot, designated Stx2B-Tir-Stx1B-Zot, expressed in the E. coli BL21 and harvested the purified protein by a simple GST·Bind Resin chromatography method. We used a streptomycin-treated mouse model to evaluate the efficacy of subcutaneous vs. intranasal administration of the vaccine. Following immunization, mice were infected with E. coli O157:H7 and feces were monitored for shedding. Immune responses against Stx2B-Tir-Stx1B-Zot, Stx2B-Tir-Stx1B and control agent (GST/PBS) were also monitored. Subcutaneous immunization of mice with Stx2B-Tir-Stx1B-Zot induced significant Stx2B-Tir-Stx1B-Zot-specific serum IgG antibodies but did not significantly induce any antigen-specific IgA in feces, whereas intranasal immunization elicited significant Stx2B-Tir-Stx1B-Zot-specific serum IgG antibodies with some animals developing antigen-specific IgA in feces. Mice that were immunized intranasally with Stx2B-Tir-Stx1B-Zot showed dramatically decreased E. coli O157:H7 shedding compared to those of Stx2B-Tir-Stx1B and control agent following experimental infection. Mice immunized subcutaneously with Stx2B-Tir-Stx1B-Zot or Stx2B-Tir-Stx1B both showed reduced shedding in feces, moreover, Stx2B-Tir-Stx1B-Zot did better. These results demonstrate the perspective for the use of Stx2B-Tir-Stx1B-Zot to prevent colonization and shedding of E. coli O157:H7.

Identification and characterization of inosine 5-monophosphate dehydrogenase in Streptococcus suis type 2.

Streptococcus suis type 2 is a swine pathogen responsible for diverse diseases. Although many virulent factors have been identified and studied, relatively little is known about the pathogenic mechanisms of type 2. The aim of the study was to identify and understand the characterization of Inosine 5-monophosphate dehydrogenase (IMPDH). A 957-bp gene, impdh, was identified in the virulent S. suis serotype 2 (SS2), and analysis of the predicted IMPDH sequence revealed IMP dehydrogenase/GMP reductase domain. The gene encoding for the IMPDH of S. suis was cloned and sequenced. The DNA sequence contained an open reading frame encoding for a 318 amino acid polypeptide exhibiting 23% sequence identity with the IMPDH from Streptococcus pyogenes (YP281355) and Streptococcus pneumoniae (ZP00404150). Using the pET(32) expression plasmid, the impdh gene was inducibly overexpressed in Escherichia coli to produce IMPDH with a hexahistidyl N-terminus to permit its purification. The (His)6 IMPDH protein was found to possess functional IMPDH enzymatic activity after the purification. The impdh-knockout SS2 mutant ( Delta IMPDH) constructed in this study was slower in growth and one pH unit higher than SS2-H after 6 h of culturing, and found to be attenuated in mouse models of infection for 2.5 times and not be capable of causing death in porcine models of infection in contrast with the parent SS2-H.

[Effects of trabeculectomy combined with amniotic membrane and extracellular matrix of conjunctiva on filtering bleb in rabbits eyes].

OBJECTIVE: To investigate the effect of trabeculectomy combined with amniotic membrane and extracellular matrix (ECM) of conjunctiva on filtering bleb in rabbits. METHODS: Glaucoma was induced by injecting alpha-chymotrypsin into the posterior chamber of rabbit eyes. 27 rabbits were randomly divided into the following three groups: group (1) trabeculectomy alone as control; group (2) trabeculectomy combined with amniotic membrane transplantation; group (3) trabeculectomy combined with extracellular matrix (ECM) transplantation. IOPs were measured by a Schiötz tonometer and filtering bleb observed under a microscopy and electric microscopy at day 1, 7, 14, 21, 28, 35, 42, 56 after surgery. RESULTS: Compared with pre-operation, the IOP in the control group (group 1) was reduced from (34.59 +/- 4.44) mm Hg (1 mm Hg = 0.133 kPa) to (11.31 +/- 2.76) mm Hg, (19.20 +/- 5.27) mm Hg, (21.17 +/- 4.36) mm Hg, (22.22 +/- 1.39) mm Hg, (23.90 +/- 1.97) mm Hg, (23.67 +/- 1.73) mm Hg, respectively, after surgery. In the group 2, the IOP was decreased from (34.38 +/- 4.21) mm Hg to (10.48 +/- 2.45) mm Hg, (12.80 +/- 3.41) mm Hg, (13.50 +/- 2.25) mm Hg, (16.17 +/- 1.73) mm Hg, (17.22 +/- 1.32) mm Hg, (16.71 +/- 1.52) mm Hg, respectively. In the group 3, the IOP was decreased from (34.66 +/- 4.49) mm Hg to (10.94 +/- 2.75) mm Hg, (11.29 +/- 2.40) mm Hg, (13.93 +/- 3.55) mm Hg, (15.63 +/- 3.54) mm Hg, (15.70 +/- 2.44) mm Hg, (15.12 +/- 3.65) mm Hg, respectively, at day 1, 7, 14, 28, 42, 56, post-operatively. IOPs in the group 2 and 3 were significantly (P < 0.01) lower than that in the control group (group 1) at post-operative day 7, 14, 28, 42, 56. However, there was no IOP difference between the group 2 and 3 post-operatively at any time. Morphologically, the filtering blebs were flatded at day 28 in the group 1, while were normal in the group 2 and 3 at day 56. Less scar formation of filtering blebs was observed in group 2 and 3 compared with group 1. CONCLUSIONS: Trabeculectomy combined with the application of amniotic membrane or conjunctiva ECM can improve filtering bleb function and reduce scar formation that lead to a better IOP control after surgery.