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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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RT-PCR detection ofwbkAandwbkEmRNA in strains RB51, RB51WbkA, and RB51WbkE. Graph shows the mean values of fold change for wbkA and wbkE transcript levels in the recombinant strains RB51WbkA and RB51WbkE, respectively, relative to those of RB51, which were converted to 1. All the values are relative to those of internal control gene IF-1. Results are shown as mean ± standard deviation (n = 3).
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Figure 2: RT-PCR detection ofwbkAandwbkEmRNA in strains RB51, RB51WbkA, and RB51WbkE. Graph shows the mean values of fold change for wbkA and wbkE transcript levels in the recombinant strains RB51WbkA and RB51WbkE, respectively, relative to those of RB51, which were converted to 1. All the values are relative to those of internal control gene IF-1. Results are shown as mean ± standard deviation (n = 3).

Mentions: RT-PCR assay was performed to quantify the levels of wbkA and wbkE mRNA in different strains. An increase of ≈two-fold was observed in the expression level of wbkA in strain RB51WbkA relative to the RNA level observed in RB51 (Figure 2). Also, the expression level of wbkE was increased ≈1.5 fold in strain RB51WbkE when compared to the RNA expression level in strain RB51 (Figure 2). Due to the distinct mucoid phenotype of RB51WbkA, one colony was selected for further studies.


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)

RT-PCR detection ofwbkAandwbkEmRNA in strains RB51, RB51WbkA, and RB51WbkE. Graph shows the mean values of fold change for wbkA and wbkE transcript levels in the recombinant strains RB51WbkA and RB51WbkE, respectively, relative to those of RB51, which were converted to 1. All the values are relative to those of internal control gene IF-1. Results are shown as mean ± standard deviation (n = 3).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: RT-PCR detection ofwbkAandwbkEmRNA in strains RB51, RB51WbkA, and RB51WbkE. Graph shows the mean values of fold change for wbkA and wbkE transcript levels in the recombinant strains RB51WbkA and RB51WbkE, respectively, relative to those of RB51, which were converted to 1. All the values are relative to those of internal control gene IF-1. Results are shown as mean ± standard deviation (n = 3).
Mentions: RT-PCR assay was performed to quantify the levels of wbkA and wbkE mRNA in different strains. An increase of ≈two-fold was observed in the expression level of wbkA in strain RB51WbkA relative to the RNA level observed in RB51 (Figure 2). Also, the expression level of wbkE was increased ≈1.5 fold in strain RB51WbkE when compared to the RNA expression level in strain RB51 (Figure 2). Due to the distinct mucoid phenotype of RB51WbkA, one colony was selected for further studies.

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