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Abstract Background Actinobacillus pleuropneumoniae (APP) is one of the most important swine pathogens worldwide. Identification and characterization of novel antigenic APP vaccine candidates are underway. In the present study, we use an immunoproteomic approach to identify APP protein antigens that may elicit an immune response in serotype 1 naturally infected swine and serotype 1 virulent strain S259-immunized rabbits. Results Proteins from total cell lysates of serotype 1 APP were separated by two-dimensional electrophoresis (2DE). Western blot analysis revealed 21 immunoreactive protein spots separated in the pH 4-7 range and 4 spots in the pH 7-11 range with the convalescent sera from swine; we found 5 immunoreactive protein spots that separated in the pH 4-7 range and 2 in the pH 7-11 range with hyperimmune sera from S259-immunized rabbits. The proteins included the known antigens ApxIIA, protective surface antigen D15, outer membrane proteins P5, subunit NqrA. The remaining antigens are being reported as immunoreactive proteins in APP for the first time, to our knowledge. Conclusions We identified a total of 42 immunoreactive proteins of the APP serotype 1 virulent strain S259 which represented 32 different proteins, including some novel immunoreactive factors which could be researched as vaccine candidates.

Abstract Background Extraintestinal pathogenic E. coli (ExPEC) represent a phylogenetically diverse group of bacteria which are implicated in a large range of infections in humans and animals. Although subgroups of different ExPEC pathotypes, including uropathogenic, newborn meningitis causing, and avian pathogenic E. coli (APEC) share a number of virulence features, there still might be factors specifically contributing to the pathogenesis of a certain subset of strains or a distinct pathotype. Thus, we made use of suppression subtractive hybridization and compared APEC strain IMT5155 (O2:K1:H5; sequence type complex 95) with human uropathogenic E. coli strain CFT073 (O6:K2:H5; sequence type complex 73) to identify factors which may complete the currently existing model of APEC pathogenicity and further elucidate the position of this avian pathoype within the whole ExPEC group. Results Twenty-eight different genomic loci were identified, which are present in IMT5155 but not in CFT073. One of these loci contained a gene encoding a putative autotransporter adhesin. The open reading frame of the gene spans a 3,498 bp region leading to a putative 124-kDa adhesive protein. A specific antibody was raised against this protein and expression of the adhesin was shown under laboratory conditions. Adherence and adherence inhibition assays demonstrated a role for the corresponding protein in adhesion to DF-1 chicken fibroblasts. Sequence analyses revealed that the flanking regions of the chromosomally located gene contained sequences of mobile genetic elements, indicating a probable spread among different strains by horizontal gene transfer. In accordance with this hypothesis, the adhesin was found to be present not only in different phylogenetic groups of extraintestinal pathogenic but also of commensal E. coli strains, yielding a significant association with strains of avian origin. Conclusions We identified a chromosomally located autotransporter gene in a highly virulent APEC strain which confers increased adherence of a non-fimbriated E. coli K-12 strain to a chicken fibroblast cell line. Even though flanked by mobile genetic elements and three different genetic regions upstream of the gene, most probably indicating horizontal gene transfer events, the adhesin gene was significantly linked with strains of avian origin. Due to the nucleotide sequence similarity of 98% to a recently published adhesin-related gene, located on plasmid pAPEC-O1-ColBM, the name aatA (APEC autotransporter adhesin A) was adopted from that study. Our data substantiate that AatA might not only be of relevance in APEC pathogenicity but also in facilitating their reservoir life style in the chicken intestine, which might pave the way for future intestinal preventive strategies.

Abstract Background Streptococcus suis serotype 2 (SS2) is a zoonotic agent that causes death and disease in both humans and swine. A better understanding of SS2-host molecular interactions is crucial for understanding SS2 pathogenesis and immunology. Conventional genetic and biochemical approaches used to study SS2 virulence factors are unable to take into account the complex and dynamic environmental stimuli associated with the infection process. In this study, in vivo-induced antigen technology (IVIAT), an immunoscreening technique, was used to identify the immunogenic bacterial proteins that are induced or upregulated in vivo during SS2 infection. Results Convalescent-phase sera from pigs infected with SS2 were pooled, adsorbed against in vitro antigens, and used to screen SS2 genomic expression libraries. Upon analysis of the identified proteins, we were able to assign a putative function to 40 of the 48 proteins. These included proteins implicated in cell envelope structure, regulation, molecule synthesis, substance and energy metabolism, transport, translation, and those with unknown functions. The in vivo-induced changes in the expression of 10 of these 40 genes were measured using real-time reverse transcription (RT)-PCR, revealing that the expression of 6 of the 10 genes was upregulated in the in vivo condition. The strain distribution of these 10 genes was analyzed by PCR, and they were found in the most virulent SS2 strains. In addition, protein sequence alignments of the newly identified proteins demonstrate that three are putative virulence-associated proteins. Conclusion Collectively, our results suggest that these in vivo-induced or upregulated genes may contribute to SS2 disease development. We hypothesize that the identification of factors specifically induced or upregulated during SS2 infection will aid in our understanding of SS2 pathogenesis and may contribute to the control SS2 outbreaks. In addition, the proteins identified using IVIAT may be useful potential vaccine candidates or virulence markers.

Abstract Background The M-like protein, also known as SzP, is expressed on the surface of Streptococcus equi subsp. zooepidemicus (S. zooepidemicus). Previous studies demonstrated that SzP is similar to M protein of group A Streptococcus in the structure and characteristics of antiphagocytosis. The M protein is an adhesin that can bind to the host cells, however it is not known whether the SzP of S. zooepidemicus also functions as an adhesin. We conducted an investigation to determine SzP as an adhesin, and one SzP epitope was identified to be responsible for mediating binding to HEp-2 cells. Methods The gene encoding SzP was expressed in E. coli, and the purified recombinant SzP (rSzP) was recognized by rabbit anti-S. zooepidemicus antibodies using immunoblot. Furthermore, the adherence of S. zooepidemicus to HEp-2 cells was inhibited by anti-rSzP antibodies in a dose-dependent manner. We employed a random 12-peptide phage display library for screening of immunodominant mimics of the SzP, which were recognized by an anti-SzP specific monoclonal antibody (mAb 2C8). Initial positive phage clones were identified by ELISA, followed by assays to determine the adherence-inhibiting ability of the peptide. Results Ten out of fourteen selected positive clones showed high reactivity that effectively inhibited the binding of mAb 2C8 to rSzP. The motif XSLSRX was highly conserved among six of the ten clones. Conclusion Collectively, our findings suggest that the motif XSLSRX may represent an immunodominant mimic epitope of the SzP of S. zooepidemicus strain ATCC 35246, and that the same epitope may be used to mediate SzP binding to HEp-2 cells.

Muramidase-released protein (MRP) of Streptococcus suis serotype 2 (SS2) is an important epidemic virulence marker with an unclear role in bacterial infection. To investigate the biologic functions of MRP, 3 mutants named Dmrp, Dmrp domain 1 (Δmrp-d1), and Δmrp domain 2 (Δmrp-d2) were constructed to assess the phenotypic changes between the parental strain and the mutant strains. The results indicated that MRP domain 1 (MRP-D1, the non-conserved region of MRP from a virulent strain, a.a. 242-596) played a critical role in adherence of SS2 to host cells, compared with MRP domain 1 (MRP-D1, the non-conserved region of MRP from a low virulent strain, a.a. 239-598) or MRP domain 2 (MRP-D2, the conserved region of MRP, a.a. 848-1222). We found that MRP-D1 but not MRP-D2, could bind specifically to fibronectin (FN), factor H (FH), fibrinogen (FG), and immunoglobulin G (IgG). Additionally, we confirmed that mrp-d1 mutation significantly inhibited bacteremia and brain invasion in a mouse infection model. The mrp-d1 mutation also attenuated the intracellular survival of SS2 in RAW246.7 macrophages, shortened the growth ability in pig blood and decreased the virulence of SS2 in BALB/c mice. Furthermore, antiserum against MRP-D1 was found to dramatically impede SS2 survival in pig blood. Finally, immunization with recombinant MRP-D1 efficiently enhanced murine viability after SS2 challenge, indicating its potential use in vaccination strategies. Collectively, these results indicated that MRP-D1 is involved in SS2 virulence and eloquently demonstrate the function of MRP in pathogenesis of infection. [ABSTRACT FROM AUTHOR]