Generation and envelope protein analysis of internalization defective Brucella abortus mutants in professional phagocytes, RAW 264.7

Abstract

Brucella abortus is a facultative intracellular bacteria that replicates within a macrophage without producing any classical virulence factors. It can become internalized to cells by zipper-like and/or swimming internalization mechanisms. However, the bacterial proteins involved in internalization remain unclear. To define these bacterial proteins, random insertion mutants of B. abortus were generated by the Tn5 transposome complexes. In all, 132 mutants were screened, cellular internalization-defective mutants were selected, and these genomic and envelope proteomic features were identified. The transposon insertion sites were ccmC,ppk and BruAb2_0168 for the mutant C10, C29 and D7, respectively. Mutant C10 showed a deficiency in internalization without any changes in expression of the cell envelope proteins; however, mutant C29 showed a reduced expression of OMP25, and a mutant D7 also showed reduced expression of OMP25, OMP28 and Porin2b. These results suggest OMP25 is not an essential factor, but might be involved in host cellular internalization. We identified the ppk gene and BruAb2_0168 locus which are associated to expression of OMP25, OMP28 and Porin2b as well as pleiotropic effects of ccmC gene.

Expression, purification and immunochemical characterization of recombinant OMP28 protein of <i>Brucella</i> species

“…Furthermore, these results contradict previously published data, which described this antigen as of no diagnostic value (Xin et al, 2013). However, data from other studies (Cloeckaert et al, 2001;Liu et al, 2011;Cha et al, 2012) showed that the OMP28-based I-ELISA had high sensitivity and specificity in the diagnosis of brucellosis in bovine sera, conforming to our results. Fig.…”

“…have been the focus of vaccine development and the diagnosis of brucellosis (Cloeckaert et al, 2001;Gupta et al, 2010;Ko et al, 2012). OMP28 is considered as one of the outer membrane proteins of Brucella (Cha et al, 2012) and has been identified as an important diagnostic antigen in brucellosis (Seco-Mediavilla et al, 2003;Poester et al, 2010). OMP28 is highly conserved among B. abortus, B.…”

Early transcriptional responses of internalization defective Brucella abortus mutants in professional phagocytes, RAW 264.7

“…The internalization defective mutant C10, C29, D6 and D7 were derived from our previous study [8]. Brucellae were cultured in Brucella broth or agar (Difco, USA), and Kanamycin (30 μg/ml) was used when necessary.…”

“…RAW 264.7 cells were infected with each Brucella strain as described previously [8]. Briefly, RAW 264.7 cells were seeded (5 × 10 6 cells per flask) in T75 flasks one day before infection.…”

“…Previously, we generated B. abortus mutants with defective host cellular internalization by Tn5 transposome complexes. Their envelope (CE) proteins were analyzed regarding invasion of the macrophages that resulted in the ppk gene and BruAb2_0168 locus, which are associated with expression of the OMP25, OMP28 and Porin2b genes, as well as pleiotropic effects of the ccmC gene [8]. In the present study, we infected the professional phagocyte RAW 264.7 with the B. abortus mutants for four hours.…”