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Applied and Environmental Microbiology, August 2008, p. 4626-4635, Vol. 74, No. 15
0099-2240/08/$08.00+0     doi:10.1128/AEM.00104-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Engineering of a Human Vaginal Lactobacillus Strain for Surface Expression of Two-Domain CD4 Molecules{triangledown}

Xiaowen Liu,1,{dagger} Laurel A. Lagenaur,1,{dagger} Peter P. Lee,1,2 and Qiang Xu1*

Osel, Inc., 4008 Burton Drive, Santa Clara, California 95054,1 Department of Medicine, Stanford University, Stanford, California 943052

Received 13 January 2008/ Accepted 13 March 2008

Women are at significant risk of heterosexually transmitted human immunodeficiency virus (HIV) infection, with the mucosal epithelium of the cervix and vagina serving as a major portal of entry. The cervicovaginal mucosa naturally harbors dynamic microflora composed predominantly of lactobacilli, which may be genetically modified to serve as a more efficient protective barrier against the heterosexual transmission of HIV. We selected a vaginal strain of Lactobacillus, L. jensenii 1153, for genetic modification to display surface-anchored anti-HIV proteins. Genomic sequencing analyses revealed that L. jensenii 1153 encodes several unique high-molecular-weight cell wall-anchored proteins with a C-terminal cell wall sorting LPQTG motif. In this report, we employed these proteins to express a surface-anchored two-domain CD4 (2D CD4) molecule in L. jensenii 1153. Our studies indicated that the C-terminal cell wall sorting signal LPQTG motif alone is insufficient to drive the surface expression of heterologous proteins, and the display of surface-anchored 2D CD4 molecules required native sequences of a defined length upstream of the unique C-terminal LPQTG cell wall sorting signal and the positively charged C terminus in a Lactobacillus-based expression system. The modified L. jensenii strain displayed 2D CD4 molecules that were uniformly distributed on bacterial surfaces. The surface-anchored 2D CD4 molecule was recognized by a conformation-dependent anti-CD4 antibody, suggesting that the expressed proteins adopted a native conformation. The establishment of this Lactobacillus-based surface expression system, with potential broad applicability, represents a major step toward developing an inexpensive yet durable approach to topical microbicides for the mitigation of heterosexual transmission of HIV and other mucosally transmitted viral pathogens.

* Corresponding author. Mailing address: Osel, Inc., 4008 Burton Drive, Santa Clara, CA 95054. Phone: (408) 986-0012, ext. 205. Fax: (408) 986-0019. E-mail: qxu{at}oselinc.com

{triangledown} Published ahead of print on 21 April 2008.

{dagger} X.L. and L.A.L. contributed equally to this work.

Applied and Environmental Microbiology, August 2008, p. 4626-4635, Vol. 74, No. 15
0099-2240/08/$08.00+0     doi:10.1128/AEM.00104-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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