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Infection and Immunity, June 2009, p. 2294-2303, Vol. 77, No. 6
0019-9567/09/$08.00+0     doi:10.1128/IAI.01577-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Antibody-Enhanced, Fc Gamma Receptor-Mediated Endocytosis of Clostridium difficile Toxin A{triangledown}

Xiangyun He,1,{dagger} Xingmin Sun,1,{dagger} Jufang Wang,2 Xiaoning Wang,2 Quanshun Zhang,1 Saul Tzipori,1 and Hanping Feng1*

Division of Infectious Diseases, Department of Biomedical Sciences, Tufts University Cummings School of Veterinary Medicine, North Grafton, Massachusetts 01536,1 School of Bioscience and Bioengineering, South China University of Technology (SCUT), Guangzhou, China 5100062

Received 29 December 2008/ Returned for modification 5 February 2009/ Accepted 10 March 2009

Toxin A (TcdA) and toxin B (TcdB) are major virulence factors of Clostridium difficile. These two toxins intoxicate cultured cells by similar mechanisms, and TcdB generally is more potent than TcdA in cultured cells. The exact reason for this difference is unclear. Here, we report that the cellular effects of TcdA can be substantially enhanced via an opsonizing antibody through Fc gamma receptor I (Fc{gamma}RI)-mediated endocytosis. A TcdA-specific monoclonal antibody, A1H3, was found to significantly enhance the cytotoxicity of TcdA to macrophages and monocytes. The A1H3-dependent enhancement of glucosyltransferase activity, cytoskeleton disruption, and tumor necrosis factor alpha production induced by TcdA was further demonstrated using RAW 264.7 cells. Subsequent experiments indicated that the interaction of Fc{gamma}RI with A1H3 underlays the antibody-dependent enhancement of the cellular effects of TcdA. While blocking Fc{gamma}RII and Fc{gamma}RIII with anti-CD16/32 antibodies did not affect the TcdA-mediated glucosylation of Rac1 in RAW 264.7 cells, presaturation of Fc{gamma}RI with anti-CD64 antibodies in THP1 cells significantly reduced this activity. Incubation of a TcdA-A1H3 immune complex with recombinant mouse CD64 completely abrogated the A1H3-mediated enhancement of the glucosyltransferase activity of TcdA in RAW 264.7 cells. Moreover, expression of Fc{gamma}RI in CHO cells strikingly enhanced the sensitivity of these cells to TcdA complexed with A1H3. We showed that the presence of A1H3 facilitated cell surface recruitment of TcdA, contributing to the antibody-dependent, Fc{gamma}RI-mediated enhancement of TcdA activity. Finally, studies using chlorpromazine and endosomal acidification inhibitors revealed an important role of the endocytic pathway in the A1H3-dependent enhancement of TcdA activity.

* Corresponding author. Mailing address: Division of Infectious Diseases, Tufts University Cummings School of Veterinary Medicine, 200 Westboro Road, North Grafton, MA 01536. Phone: (508) 839-4252. Fax: (508) 839-7977. E-mail: hanping.feng{at}tufts.edu

{triangledown} Published ahead of print on 23 March 2009.

Editor: R. P. Morrison

{dagger} X.E. and X.S. contributed equally to this work.

Infection and Immunity, June 2009, p. 2294-2303, Vol. 77, No. 6
0019-9567/09/$08.00+0     doi:10.1128/IAI.01577-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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