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Overexpression of Brucella putative glycosyltransferase WbkA in B. abortus RB51 leads to production of exopolysaccharide.

Dabral N, Jain-Gupta N, Seleem MN, Sriranganathan N, Vemulapalli R - Front Cell Infect Microbiol (2015)

Bottom Line: Our results demonstrate that overexpression of wbkA or wbkE gene in RB51 does not result in O-polysaccharide expression as shown by Western blotting with specific antibodies.However, wbkA, but not wbkE, overexpression leads to the development of a clumping phenotype and the production of exopolysaccharide(s) containing mannose, galactose, N-acetylglucosamine, and N-acetylgalactosamine.Moreover, we found that the clumping recombinant strain displays increased adhesion to polystyrene plates.

View Article: PubMed Central - PubMed

Affiliation: Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University West Lafayette, IN, USA.

ABSTRACT
Brucella spp. are Gram-negative, facultative intracellular bacteria that cause brucellosis in mammals. Brucella strains containing the O-polysaccharide in their cell wall structure exhibit a smooth phenotype whereas the strains devoid of the polysaccharide show rough phenotype. B. abortus strain RB51 is a stable rough attenuated mutant which is used as a licensed live vaccine for bovine brucellosis. Previous studies have shown that the wboA gene, which encodes a glycosyltransferase required for the synthesis of O-polysaccharide, is disrupted in B. abortus RB51 by an IS711 element. Although complementation of strain RB51 with a functional wboA gene results in O-polysaccharide synthesis in the cytoplasm, it does not result in smooth phenotype. The aim of this study was to determine if overexpression of Brucella WbkA or WbkE, two additional putative glycosyltransferases essential for O-polysaccharide synthesis, in strain RB51 would result in the O-polysaccharide synthesis and smooth phenotype. Our results demonstrate that overexpression of wbkA or wbkE gene in RB51 does not result in O-polysaccharide expression as shown by Western blotting with specific antibodies. However, wbkA, but not wbkE, overexpression leads to the development of a clumping phenotype and the production of exopolysaccharide(s) containing mannose, galactose, N-acetylglucosamine, and N-acetylgalactosamine. Moreover, we found that the clumping recombinant strain displays increased adhesion to polystyrene plates. The recombinant strain was similar to strain RB51 in its attenuation characteristic and in its ability to induce protective immunity against virulent B. abortus challenge in mice.

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Detection of the protective efficacy of RB51WbkA against challenge with the virulent strainB. abortus2308. Mice vaccinated with RB51, recombinant RB51WbkA and saline-inoculated mice were challenged by i.p. inoculation of 3 × 104 CFU-equivalent of virulent strain 2308. Two weeks post-challenge, the mice were euthanized and the Brucella CFUs in their spleens were determined. Results are shown as mean ± standard deviation (n = 5) of the log CFU of Brucella recovered from spleens. *Significantly different from the corresponding saline group (P < 0.01).
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Figure 12: Detection of the protective efficacy of RB51WbkA against challenge with the virulent strainB. abortus2308. Mice vaccinated with RB51, recombinant RB51WbkA and saline-inoculated mice were challenged by i.p. inoculation of 3 × 104 CFU-equivalent of virulent strain 2308. Two weeks post-challenge, the mice were euthanized and the Brucella CFUs in their spleens were determined. Results are shown as mean ± standard deviation (n = 5) of the log CFU of Brucella recovered from spleens. *Significantly different from the corresponding saline group (P < 0.01).

Mentions: Mice vaccinated with RB51 and RB51WbkA had significantly reduced number of virulent B. abortus 2308 in their spleens when compared with the saline-inoculated group of mice (Figure 12). However, there was no statistical difference in the splenic bacterial loads between the two vaccinated groups of mice.


Overexpression of Brucella putative glycosyltransferase WbkA in B. abortus RB51 leads to production of exopolysaccharide.

Dabral N, Jain-Gupta N, Seleem MN, Sriranganathan N, Vemulapalli R - Front Cell Infect Microbiol (2015)

Detection of the protective efficacy of RB51WbkA against challenge with the virulent strainB. abortus2308. Mice vaccinated with RB51, recombinant RB51WbkA and saline-inoculated mice were challenged by i.p. inoculation of 3 × 104 CFU-equivalent of virulent strain 2308. Two weeks post-challenge, the mice were euthanized and the Brucella CFUs in their spleens were determined. Results are shown as mean ± standard deviation (n = 5) of the log CFU of Brucella recovered from spleens. *Significantly different from the corresponding saline group (P < 0.01).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4478442&req=5

Figure 12: Detection of the protective efficacy of RB51WbkA against challenge with the virulent strainB. abortus2308. Mice vaccinated with RB51, recombinant RB51WbkA and saline-inoculated mice were challenged by i.p. inoculation of 3 × 104 CFU-equivalent of virulent strain 2308. Two weeks post-challenge, the mice were euthanized and the Brucella CFUs in their spleens were determined. Results are shown as mean ± standard deviation (n = 5) of the log CFU of Brucella recovered from spleens. *Significantly different from the corresponding saline group (P < 0.01).
Mentions: Mice vaccinated with RB51 and RB51WbkA had significantly reduced number of virulent B. abortus 2308 in their spleens when compared with the saline-inoculated group of mice (Figure 12). However, there was no statistical difference in the splenic bacterial loads between the two vaccinated groups of mice.

Bottom Line: Our results demonstrate that overexpression of wbkA or wbkE gene in RB51 does not result in O-polysaccharide expression as shown by Western blotting with specific antibodies.However, wbkA, but not wbkE, overexpression leads to the development of a clumping phenotype and the production of exopolysaccharide(s) containing mannose, galactose, N-acetylglucosamine, and N-acetylgalactosamine.Moreover, we found that the clumping recombinant strain displays increased adhesion to polystyrene plates.

View Article: PubMed Central - PubMed

Affiliation: Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University West Lafayette, IN, USA.

ABSTRACT
Brucella spp. are Gram-negative, facultative intracellular bacteria that cause brucellosis in mammals. Brucella strains containing the O-polysaccharide in their cell wall structure exhibit a smooth phenotype whereas the strains devoid of the polysaccharide show rough phenotype. B. abortus strain RB51 is a stable rough attenuated mutant which is used as a licensed live vaccine for bovine brucellosis. Previous studies have shown that the wboA gene, which encodes a glycosyltransferase required for the synthesis of O-polysaccharide, is disrupted in B. abortus RB51 by an IS711 element. Although complementation of strain RB51 with a functional wboA gene results in O-polysaccharide synthesis in the cytoplasm, it does not result in smooth phenotype. The aim of this study was to determine if overexpression of Brucella WbkA or WbkE, two additional putative glycosyltransferases essential for O-polysaccharide synthesis, in strain RB51 would result in the O-polysaccharide synthesis and smooth phenotype. Our results demonstrate that overexpression of wbkA or wbkE gene in RB51 does not result in O-polysaccharide expression as shown by Western blotting with specific antibodies. However, wbkA, but not wbkE, overexpression leads to the development of a clumping phenotype and the production of exopolysaccharide(s) containing mannose, galactose, N-acetylglucosamine, and N-acetylgalactosamine. Moreover, we found that the clumping recombinant strain displays increased adhesion to polystyrene plates. The recombinant strain was similar to strain RB51 in its attenuation characteristic and in its ability to induce protective immunity against virulent B. abortus challenge in mice.

Show MeSH
Related in: MedlinePlus