The Screening And Identification Of Novel Immunogenic Proteins And The Funcational Study Of Cytolethal Distending Toxin In The Haemophilus Parasuis

Haemophilus parasuis （HPS） is the causative agent of Glasser’s disease of pigs, a disease associated with fibrinous polyserositis, polyarthritis and meningitis. With the recent changes in production methods, diseases caused by H. parasuis have called people’s attention significantly world-wide. However there is no systematic proteomics study of Haemophilus parasuis. The obtained findings were as follows:1. A comprehensive proteome map of the Haemophilus parasuis serovar 5Systematic reference maps of outer membrane, intracellular and extracellular proteome fractions of the clinical isolate H. parasuis SH0165 were examined by 2-DE coupled with MALDI-TOF MS. A total of 539 proteins spots were successfully identified, corresponding to 317 different proteins which were classified into functional categories. The majority of these proteins were linked to housekeeping functions in amino acid transport and metabolism, secondary metabolites biosynthesis, transport and catabolism and posttranslational modification, protein turnover, chaperones. A significant number of outer membrane proteins were identified, such as Wza,Omp2,Omp5, D15 and PalA, which were supposed to play important roles in basic physiology of H. parasuis. Besides, several virulence-associated proteins involved in type I （TolC）, type III （DsbA, DsbC） and type V （Autotransporter adhesins） secretion systems, and solute-binding proteins participating in iron-uptake systems were also identified in the present study.2. Identification and characterization of novel immunogenic outer membrane proteins of Haemophilus parasuis serovar 5To develop more effective vaccines, an immunoproteome-based and reverse vaccinology approach was used to analyze the outer membrane proteins of H. parasuis serovar 5. A total of 15 proteins with high immunogenicity were identified by immunoproteomics method, and all were showed to be immunogens for the first time in H. parasuis. Further analyses of 8 selected proteins revealed that （1） significantly higher level of serum antibodies against 6 proteins was detected with convalescent sera and immunized sera; （2） antisera against 5 of the selected proteins could effectively inhibit H. parasuis growth in mouse blood; and （3） 4 proteins could induce protective response of the vaccinated mice against H. parasuis. The results suggest these 4 proteins （PalA, Omp2, D15 and HPS₀6257） have strong potential to be vaccine candidates. In addition, we also identified three immunogenic proteins （Hsp60, Hsp70 and HbpB） by reverse vaccinology approach. Vaccination with three immunogenic proteins resulted in a significant increase in mixed antibody response （IgGl and IgG2a）. Immunization of mice with GroEL and HbpB, respectively conferred 70% and 90% survival against lethal H. parasuis challenge. In addition, the hyperimmune sera against GroEL and HbpB could efficiently kill the bacteria by opsonized phagocytosis and significantly protect mice against H. parasuis infection in the experiment of passive immunization.3. The research on pathogenesis and immunity of cytolethal distending toxin in Haemophilus parasuisWe firstly report that the genome of H. parasuis contains two copies of cytolethal distending toxins （Cdts）, which these two Cdts shows the identifical function in vitro. Although CdtB is the only Cdt subunit that is capable of inducing cell cycle arrest by itself, CdtA and CdtC can significantly enhance the ability of CdtB to induce G2 arrest. Three Cdt peptides are able to interact with one another to form an active tripartite holotoxin that exhibits maximum cellular toxicity. Moreover, CdtA, CdtB and CdtC can bind to cell surface in PAM cell; while CdtA and CdtC, but not CdtB are able to bind to cell surface in Hep-2/Hela cells, and the association of Cdt toxicity with plasma membranes is dependent upon binding to cholesterol. Further analysis indicates that CdtC subunit contains an atypical cholesterol recognition/interaction amino acid consensus （CRAC） region. The mutation of CRAC site resulted in decreased cell toxicity. Western blot analysis show all the 15 H. parasuis reference strains and clinical isolates expressed CdtB subunit and demonstrated Cdt activity, indicating that Cdt is a conservative putative virulence factor for H. parasuis. Finally, Cdts toxoid could induce noticeable ELISA antibody and neutralizing antibodies in mice; Cdts toxoid also could induce the expression of IL-2, IL-4 and IFN-a. Cdts toxoid could significantly protect mice against H. parasuis infection, with the development of the potential active ingredients for HPS vaccine.