As a halophilic species, possesses the same TTSS gene organization as and [54, 55]. infections by or using immunoglobulins [13C24] and vaccines [25C27], from which several projects have progressed to human clinical trials [28C31]. We recently published an epidemiological study on serum titers against PcrV in human volunteers [32], and another showing how prophylactic administration of human serum-derived immunoglobulin with a high anti-PcrV titer significantly improves the survival rate, pulmonary edema, and inflammatory cytokine production of a pneumonia model [18]. The results of both studies imply that immunity against the V-antigen and its homologs might be necessary to prevent infections caused by pathogenic bacterial species employing the TTSS-virulence mechanism [18, 32]. V-antigen homologs have been recently reported in several Gram-negative bacteria, including spp., spp., and (hereafter referred to as porin F from the outer membrane (OprF) Five recombinant V-antigens and recombinant OprF were GSK2141795 (Uprosertib, GSK795) constructed. Details on the GSK2141795 (Uprosertib, GSK795) PCR primers and cloning sites are listed in Table 1. The coding regions of the V-antigens were amplified by polymerase chain reaction (PCR) with specific primers containing restriction enzyme sites for insertion into a protein expression vector. PCR-amplified genes were cloned into the pCR2.1 cloning vector and TOP10F cells via TOPO cloning (Thermo Fisher Scientific, Waltham, MA, USA). After digesting the purified plasmids containing each individual cloned gene with restriction enzymes, the inserted coding regions of each gene were transferred to the multiple cloning site of the expression vector pQE30 (Qiagen, Hilden, Germany) for expression of a hexahistidine-tagged protein in M15. The various endotoxin-free Gram-negative bacteria V-antigens were prepared as reported previously (Fig 1) [17, 20]. Open in a separate window Fig 1 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation of extracted recombinant hexahistidine-tagged V-antigen proteins.Recombinant PcrV from were separated by SDS-PAGE using a 10% Bis-Tris-gel. Table 1 Gene sources, primer sets for V-antigen and OprF gene cloning, and characteristics of the recombinant V-antigens and OprF used in this study. PA103pCD1 plasmidsubsp. subsp,?PAO1proteins, we performed an inhibition ELISA with a soluble fraction of M15 lysate, and no significant effect on titer measurement was observed. Except for human monoclonal anti-PcrV IgG mAb 6F5 as a standard to measure the anti-PcrV titer [32], there is no human anti-V-antigen IgG. Therefore, after optimization of the ELISA system for anti-PcrV titers using the mAb 6F5 standard [32], the OD measured under a consistent condition with the same secondary antibody was used to evaluate the titers. Inhibition ELISA Cross-reactivity was analyzed by an inhibition ELISA. Two human sera with relatively high anti-PcrV, LcrV, AcrV, VcrV, and LssV titers were diluted at Rabbit Polyclonal to GPR17 1:1000 and preincubated with either recombinant LssV or recombinant OprF (100 g/mL) overnight at 4C. The anti-V-antigen titers were measured in triplicate by an ELISA using recombinant V-antigen-coated plates. Immunizing mice with the V-antigens Certified pathogen-free, male ICR mice (4 weeks old) were purchased from Shimizu Laboratory Supplies, Co, Ltd (Kyoto, Japan). Mice were housed in cages with filter tops under pathogen-free conditions. The protocols for all animal experiments were approved GSK2141795 (Uprosertib, GSK795) GSK2141795 (Uprosertib, GSK795) by the Animal Research Committee of Kyoto Prefectural University of Medicine before undertaking the experiments (Authorization number: M29-592). Three mice per group were intradermally immunized with one of the five recombinant V-antigen proteins (10 g/dose) adjuvanted with complete Freunds adjuvant in the first injection, and four weeks later with incomplete Freunds adjuvant for GSK2141795 (Uprosertib, GSK795) the second injection. Eight weeks after the first injection, the immunized mice were euthanized with a large dose intraperitoneal injection of sodium pentobarbital, and peripheral blood samples were collected. Serum titers against the five V-antigens were individually measured by ELISAs, as described above. Phylogenetic and cluster analyses The five V-antigens were phylogenetically analyzed using ClustalW (Genome Net, https://www.genome.jp/tools-bin/clustalw) or RStudio (version 1.2, RStudio, Boston, MA, USA. https://www.rstudio.com) with R version 3.6.1 (The R Foundation, https://www.r-project.org). Unrooted trees were prepared using the neighbor-joining method, and rooted trees were prepared using the unweighted pair group method with arithmetic means applied to the ClustalW site and the standard R function the package ape.
