Characterization and Protective Property of Brucella abortuscydCandlooPMutants

Abstract

bBrucella abortus readily multiplies in professional or nonprofessional phagocytes in vitro and is highly virulent in mice. Isogenic mutants of B. abortus biovar 1 strain IVKB9007 lacking the ATP/GDP-binding protein motif A (P-loop) (named looP; designated here the IVKB9007 looP::Tn5 mutant) and the ATP-binding/permease protein (cydC; designated here the IVKB9007 cydC::Tn5 mutant) were identified and characterized by transposon mutagenesis using the mini-Tn5Km2 transposon. Both mutants were found to be virtually incapable of intracellular replication in both murine macrophages (RAW264.7) and the HeLa cell line, and their virulence was significantly impaired in BALB/c mice. Respective complementation of the IVKB9007 looP::Tn5 and IVKB9007 cydC::Tn5 mutants restored their ability to survive in vitro and in vivo to a level comparable with that of the wild type. These findings indicate that the cydC and looP genes play important roles in the virulence of B. abortus. In addition, intraperitoneal immunization of mice with a dose of the live IVKB9007 looP::Tn5 and IVKB9007 cydC::Tn5 mutants provided a high degree of protection against challenge with pathogenic B. abortus strain 544. Both mutants should be evaluated further as a live attenuated vaccine against bovine brucellosis for their ability to stimulate a protective immune response. Brucella abortus, the causative agent of bovine brucellosis, causes abortion and reduced fertility in cattle and undulant fever in humans (1). Unlike other pathogenic bacteria, brucellae do not have classical virulence factors. A key aspect of the virulence of B. abortus is its ability to invade, survive, and proliferate within host phagocytic cells; it successfully bypasses the bactericidal activities of phagocytes and thereby establishes long-lasting chronic infections (2, 3). The B. abortus vaccine strains S19 and RB51 have been used worldwide for the prevention of brucellosis in cattle but have several drawbacks, including interference with diagnosis, residual virulence, and some pathogenicity for humans as well as cattle (4, 5). Therefore, understanding the virulence of B. abortus on a genetic level and the host response against B. abortus will provide important information for the development of a new vaccine for controlling brucellosis.Transposon mutagenesis is an extensively used approach for the identification of genes involved in the virulence of bacterial pathogens (6-8). To understand the sustained intracellular residence of brucellae in host cells, a transposon-based approach has been employed to identify genes in brucellae that are critically required for lipopolysaccharide biosynthesis, metabolic processes, stress responses, nutrient deprivation, and invasion of and survival in host cells. Mutations in these genes led to the attenuation of Brucella compared with the virulence of the parental strain; attenuation was recognized by the reduced intracellular survival and replication or rapid clearance of the mutants from both macrophages and mice (6-11). However, these atten...

Booster vaccination with safe, modified, live-attenuated mutants of Brucella abortus strain RB51 vaccine confers protective immunity against virulent strains of B. abortus and Brucella canis in BALB/c mice

“…Notably, RB51 mutants were more attenuated in these murine macrophages than the parental RB51. This observation was consistent with our previous finding that a transposon insertion in cydC of B. abortus crippled the ability of Brucella to survive inside macrophages by diminishing its resistance to host defence mechanisms (Truong et al, 2014). It is not surprising that a single deletion of cydD or deletion of both cydC and cydD in RB51 had the same attenuated phenotype in vitro as did the cydC mutant, because both genes encode an ATP-binding cassette (ABC)-type transporter that is required for the activity of cytochrome bd and c oxidases, similar to that seen in E. coli (Goldman et al, 1996;Poole et al, 1994).…”

“…In particular, cydC : : Tn5 and purD : : Tn5 mutants were found to be attenuated for virulence and survival in both RAW and HeLa cells and in BALB/c mice, and protected the mice against challenge with virulent B. abortus strain 544. These data indicate that cydC and purD can be used as the ideal candidate genes for the generation of new or improved vaccine strains (Truong et al, 2014(Truong et al, , 2015. In the present study, in an attempt to enhance the safety of RB51, cydC, cydD (ATPbinding protein) and purD were therefore selected for deletion to further attenuate strain RB51 vaccine.…”

“…Our previous studies using transposon mutagenesis have identified cydC and purD as being required for intramacrophage survival and virulence of Brucella in the purineor oxygen-limited environments inside hosts (Truong et al, 2014(Truong et al, , 2015. In particular, cydC : : Tn5 and purD : : Tn5 mutants were found to be attenuated for virulence and survival in both RAW and HeLa cells and in BALB/c mice, and protected the mice against challenge with virulent B. abortus strain 544.…”

“…The deletions of cydD (D5F/D6R), cydC (C5F/C6R) and purD (PD5F/ PD6R) in RB51 or RB51DcydC were then verified by PCR amplification and DNA sequencing analysis, and the resulting mutants referred to as RB51DcydD, RB51DcydC, RB51DpurD, RB51DcydCD-cydD and RB51DcydCDpurD. Genetic complementation strains corresponding to each single mutant were also constructed and characterization of these mutants was performed as described previously (Truong et al, 2014(Truong et al, , 2015.…”

“…Therefore, B. abortus mutants lacking the genes encoding cytochrome terminal oxidases or required for the biosynthesis of an essential nutrient are unlikely to survive in that harsh environment. Using transposon mutagenesis, previous work in our laboratory has identified the B. abortus genes cydC (encoding the ATP-binding/permease protein) and purD (encoding phosphoribosylamine-glycine ligase) as being required for intracellular survival and virulence in vitro and in vivo (Truong et al, 2014(Truong et al, , 2015. BALB/c mice immunized with the cydC : : Tn5 and purD : : Tn5 transposon mutants were protected against challenge with virulent B. abortus strain 544.…”