Strain RB51 does not produce detectable levels of O-PS, and animals vaccinated with this strain do not develop anti-O-PS antibodies (Schurig et al., 1991). in the O-polysaccharide synthesis and smooth phenotype. Our results demonstrate that overexpression of or Furin gene in RB51 does not result in O-polysaccharide expression as shown by Western blotting with ME0328 specific antibodies. However, challenge in mice. RB51, glycosyltransferase, overexpression, exopolysaccharide, clumping Introduction Members of the genus are Gram-negative, facultative intracellular coccobacilli that can cause chronic infections in several mammals, including humans. Based on the structure of the lipopolysaccharide (LPS) molecule and the colony morphology, spp. can be separated into smooth and rough phenotypes. Smooth ME0328 colony morphology of strains is determined by the presence of LPS containing the O-polysaccharide (O-PS) in their cell wall structure. spp. with LPS that is devoid of the O-PS display rough colony morphology. Based on the reactive specificities of antibodies, the O-PS of smooth LPS is defined to contain A (for Abortus), M (for Melitensis), and C (for Common) epitopes (Diaz et al., 1968; Hurvell and Lindberg, 1973). O-PS is a linear homopolymer of 4,6-dideoxy-4-formamido–D-mannopyranosyl (perosamine) subunits connected in -1,2 linkage in A-dominant smooth strains, with every fifth residue connected in -1,3 linkage in M-dominant smooth strains (Caroff et ME0328 al., 1984; Bundle et al., 1987). The O-PS is an immunodominant antigen, and infected animals usually develop robust antibodies to this antigen. Detection of anti-O-PS antibodies in the body fluids is the basis for several brucellosis diagnostic assays. At least in some animal species, anti-O-PS antibodies play a role in conferring enhanced protection against infections by (Araya and Winter, 1990; Ficht et al., 2009; Vitry et al., 2014). The O-PS also acts as a virulence factor by protecting the bacteria against complement-mediated lysis and the intracellular bactericidal milieu of phagocytic cells (Allen et al., 1998). Consequently, the smooth strains are generally more virulent than their rough counterparts, which are typically attenuated (Allen et al., 1998; McQuiston et al., 1999). RB51, a laboratory derived stable rough attenuated strain, is used as a licensed live vaccine in the control of bovine brucellosis in the US and several other countries. Strain RB51 does not produce detectable levels of O-PS, and animals vaccinated with this strain do not develop anti-O-PS antibodies (Schurig et al., 1991). However, presence of low levels of M-like O-PS was detected in this strain (Cloeckaert et al., 2002). The complete biosynthetic pathway of smooth LPS is yet to be determined. However, several genes are known to be essential for the biosynthesis of the O-PS (Godfroid et al., 2000; Gonzales et al., 2008; Zygmunt et al., 2009). These genes are located in two loci, and chromosome (Zygmunt et al., 2009). Genes encoding four putative glycosyltransferases, RB51, the gene is disrupted by an ISelement (Vemulapalli et al., 1999). ME0328 Sequence analysis of the and loci of strain RB51 did not reveal any other gene-disrupting mutations (Adone et al., 2011). Complementation of strain RB51 with a functional gene (RB51WboA) did not restore smooth phenotype, but resulted in the production of low levels of O-PS which remained in the cytoplasm (Vemulapalli et al., 2000). In mouse models, strain RB51WboA vaccination induced low titers of anti-O-PS antibodies and conferred superior protection against virulent and challenge (Vemulapalli et al., 2000, 2004). The initial objective of this study was to determine if overexpression of or in strain RB51 would overcome the deficiency and lead to production of detectable levels of O-PS. We cloned each gene in a multi-copy plasmid under a strong synthetic promoter and used the resulting plasmids to transform strain RB51. Although we did not detect any O-PS production by the recombinant strains, unexpectedly, the overexpression of strains RB51 and 2308 were from our culture collection. were obtained from American Type Culture Collection. Generation of strain RB51WboA was described previously (Vemulapalli et al., 2000). strain DH5 (Invitrogen, Carlsbad, CA) was used for the preparation of the necessary plasmid constructs. All bacteria were grown in tryptic soy broth (TSB).
