Characterization of Anti-ECA Antibodies in Rabbit Antiserum Against Rough Yersinia enterocolitica O:3*

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K. Rabsztyn^1,2, K. Kasperkiewicz¹, K. A. Duda³, C-M. Li², M. Łukasik¹, J. Radziejewska-Lebrecht¹, and M. Skurnik^2,4**

¹Department of Microbiology, University of Silesia, Jagiellońska 28, PL-40-032 Katowice, Poland; fax: +48-32-200-9361; E-mail: kamila.rabsztyn@us.edu.pl

²Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, PO Box 21, Haartmaninkatu 3, FIN-00014 Helsinki, Finland; fax: +358-9-191-26382; E-mail: mikael.skurnik@helsinki.fi

³Research Center Borstel, Leibniz Center for Medicine and Biosciences, Division of Structural Biochemistry, Parkallee 4a/c, D-23845 Borstel, Germany; fax: +4537-188-745; E-mail: kduda@fz-borstel.de

⁴Helsinki University Central Hospital Laboratory Diagnostics, PO Box 21, Haartmaninkatu 3, FIN-00014 Helsinki, Finland

^* This paper is based on a presentation made at the 4th Baltic Conference on Microbial Carbohydrates, Hyytiala Forestry Field Station, Finland, September 19-22, 2010.

^** To whom correspondence should be addressed.

Received February 14, 2011; Revision received February 25, 2011

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Enterobacterial common antigen (ECA) is a characteristic surface component in bacteria belonging to the Enterobacteriaceae family. It is generally integrated in the outer membrane via a linkage to phosphatidylglycerol (ECA_PG) and at the same time in some special cases via a linkage to lipopolysaccharide (ECA_LPS); the latter form is immunogenic. Yersinia enterocolitica O:3 expresses both ECA_PG and ECA_LPS. To study whether ECA-immunogenicity of Y. enterocolitica O:3 is temperature-regulated, rabbits were immunized with ECA-expressing Y. enterocolitica O:3 bacteria grown at 22 and 37ºC. To induce minimal amount of anti-LPS antibodies, immunization was performed with YeO3-c-trs8-R, an LPS mutant missing both O-polysaccharide and the outer core hexasaccharide. However, abundant antibodies specific for LPS core were still present in the obtained antisera such that the reactivity of ECA-specific antibodies could not be detected. To obtain “monovalent” anti-ECA antisera, the sera were absorbed with ECA-negative bacteria. Absorption with live bacteria removed efficiently the anti-LPS antibodies, whereas this was not the case with boiled bacteria. Western blotting revealed that the specificity of the monovalent anti-ECA antiserum was different from that of a monoclonal anti-ECA antibody (mAb 898) as it did not react with ECA_PG, and this suggested that in Y. enterocolitica O:3 ECA_LPS only one or two ECA repeat unit(s) is/are linked to LPS. Both ECA_PG and ECA_LPS expression were found to be regulated by temperature and repressed at 37ºC.

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KEY WORDS: Yersinia enterocolitica O:3, enterobacterial common antigen, lipopolysaccharide, monovalent antiserum

DOI: 10.1134/S0006297911070145

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