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Infection and Immunity, January 2009, p. 170-179, Vol. 77, No. 1
0019-9567/09/$08.00+0     doi:10.1128/IAI.00943-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Cytotoxic Necrotizing Factor Type 1-Neutralizing Monoclonal Antibody NG8 Recognizes Three Amino Acids in a C-Terminal Region of the Toxin and Reduces Toxin Binding to HEp-2 Cells {triangledown}

Kerian K. Grande,1 Karen C. Meysick,2 Susan B. Rasmussen,1 and Alison D. O'Brien1*

Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814,1 FDA/CBER, Bethesda, Maryland 208922

Received 29 July 2008/ Returned for modification 17 September 2008/ Accepted 22 October 2008

Cytotoxic necrotizing factor type 1 (CNF1) and CNF2 are toxins of pathogenic Escherichia coli that share 85% identity over 1,014 amino acids. Although both of these toxins modify GTPases, CNF1 is a more potent inducer of multinucleation in HEp-2 cells, binds more efficiently to HEp-2 cells, and, despite the conservation of amino acids (C866 and H881) required for enzymatic activity of the toxins, deamidates RhoA and Cdc42 better than CNF2. Here we exploited the differences between CNF1 and CNF2 to define the epitope on CNF1 to which the CNF1-specific neutralizing monoclonal antibody (MAb) (MAb NG8) binds and to determine the mechanism by which MAb NG8 neutralizes CNF1 activity on HEp-2 cells. For these purposes, we generated a panel of 21 site-directed mutants in which amino acids in CNF1 were exchanged for the amino acids in CNF2 between amino acids 546 and 869 and vice versa. This region of CNF1 not only is recognized by MAb NG8 but also is involved in binding of this toxin to HEp-2 cells. All the mutants retained the capacity to induce multinucleation of HEp-2 cells. However, the CNF1 double mutant with D591E and F593L mutations (CNF1D591E F593L) and the CNF1H661Q single mutant displayed drastically reduced reactivity with MAb NG8. A reverse chimeric triple mutant, CNF1E591D L593F Q661H, imparted MAb NG8 reactivity to CNF2. MAb NG8 neutralized CNF2E591D L593F Q661H activity in a dose-dependent manner and reduced the binding of this chimeric toxin to HEp-2 cells. Taken together, these results pinpoint three amino acids in CNF1 that are key amino acids for recognition by neutralizing MAb NG8 and further help define a region in CNF1 that is critical for full toxin binding to HEp-2 cells.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799. Phone: (301) 295-3400. Fax: (301) 295-3773. E-mail: aobrien{at}usuhs.mil

{triangledown} Published ahead of print on 27 October 2008.

Editor: V. J. DiRita

Infection and Immunity, January 2009, p. 170-179, Vol. 77, No. 1
0019-9567/09/$08.00+0     doi:10.1128/IAI.00943-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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