Brucella neotomae as a vector for developing brucellosis vaccines

Dina Abdel Wahid Moustafa, Purdue University

Abstract

Members of genus Brucella are the causative agent of brucellosis, one of the major public health concerns in several developing countries. The disease has been described as one of the world’s most widespread zoonotic infections. There are six well-recognized and four recently identified species in Brucella genus. B. melitensis , B. suis and B. abortus are known to be highly virulent for humans and are the most often identified causative agents of naturally acquired human brucellosis. Humans usually acquire the infection by consuming contaminated dairy or meat products or by coming in contact with the infected animal tissues and secretions. Ingestion, inhalation, and contamination of conjunctiva or broken skin by the infected animal products are the common modes by which humans are infected. Brucellosis is also considered an occupational disease for people who work in the livestock sector, abattoir workers, laboratory personnel, researchers and hunters. The zoonotic nature of the disease coupled with the ease of airborne transmission has increased the potential of Brucella being used as a bioterrorism weapon. B. abortus, B. melitensis and B. suis are classified to be ‘select agents’ by the United States departments of Agriculture and Human and Health Services. The first part of our study was undertaken to investigate the genetic basis for the disparity in Cu/Zn superoxide dismutase (SOD) expression in B. neotomae. SOD is a periplasmic enzyme known to be involved in protecting Brucella from oxidative bactericidal effects of host phagocytes. SOD is also a protective antigen of B. abortus. Previous research suggested that B. neotomae might not express detectable levels of SOD. Our nucleotide sequence analysis of upstream region to the sodC gene revealed a single-nucleotide insertion in the potential promoter region. A similar single-nucleotide insertion was detected in the sodC promoter of B. suis strain belonging to biovar 2 in which SOD expression was undetectable previously. Examination of the sodC promoter activities using translational fusion constructs with E. coli β-galactosidase demonstrated that the B. neotomae and B. suis biovar 2 promoters were very weak in driving gene expression. Site-directed mutation studies indicated that the single nucleotide insertion in the B. neotomae sodC promoter reduced the promoter activity. Increasing the level of SOD expression in B. neotomae through complementation with B. abortus sodC gene did not alter the bacterial survival in J774A.1 macrophage-like cells and in tissues of BALB/c and C57BL/6 mice. In addition, our results for the first time demonstrated the effect of single-nucleotide polymorphism on promoter function and gene expression in Brucella . The second part of this study was aimed at evaluating the immunogenic potential of B. neotomae as a vaccine vector for brucellosis. We generated two recombinant B. neotoame strains that overexpressed SOD and Bp26, a 26 kDa periplasmic protein that is previously shown to be immunogenic and a protective antigen of B. melitensis. To render the vaccine safer, we used gamma-radiation to disable the replication capacity of B. neotomae and its recombinant strains. Although unable to replicate, the irradiated bacteria remained metabolically active and persisted for 5 days in the spleens of immunized mice. For evaluation of the vaccine potential, female BALB/c mice were immunized by two intraperitoneal inoculations at 2-weeks interval with 108 CFU-equivalent of each of the irradiated bacteria. All three vaccines induced antigen-specific antibody and T cell responses. The vaccinated mice showed significant resistance against challenge with virulent B. abortus 2308, B. melitensis 16M, and B. suis 1330. Overexpression of SOD and the 26 kDa in B. neotomae did not enhance its vaccine efficacy against any one of the three virulent Brucella spp . Immunization with even one dose of the irradiated B. neotomae vaccine conferred significant protection against B. melitensis 16M challenge. (Abstract shortened by UMI.)

Degree

Ph.D.

Advisors

Vemulapalli, Purdue University.

Subject Area

Molecular biology

COinS