In vivo-induced argininosuccinate lyase plays a role in the replication of Brucella abortus in RAW264.7 cells.

ABSTRACT

Brucellosis caused by Brucella species is a zoonotic disease with a serious impact on public health and the livestock industry. To better understand the pathogenesis of the disease, in vivo-induced antigen technology (IVIAT) was used to investigate the in vivo-induced antigens of Brucella abortus in this study. A genomic expression library of B. abortus was constructed and screened using pooled bovine B. abortus-positive sera by IVIAT. In total, 33 antigens were identified. Five antigens were further expressed and tested for their seroreactivity against 33 individual bovine B. abortus-positive sera by Western blot analysis. The results showed a highest positive rate of 32/33 for argininosuccinate lyase (ASL), indicating that ASL may be used as a candidate marker for serodiagnosis of brucellosis. Furthermore, an asl gene-deleted mutant strain S2308ΔASL was constructed, and the intracellular survival and replication of the mutant strain in RAW264.7 cells were investigated. Interestingly, the numbers of bacteria recovered from cells infected with mutant strain S2308ΔASL were similar at all time points observed from 0 h to 96 h post-infection, suggesting the asl gene plays an important role in the bacterial replication in RAW264.7 cells. Real-time quantitative PCR (qPCR) analysis showed that the mRNA levels in S2308ΔASL were decreased for BvrR, BvrS and virB5 when compared with those in S2308 (P<0.05). Our results not only expand the knowledge of Brucella intracellular replication but also expand the list of candidates for serodiagnostic markers of brucellosis.