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Infect. Immun. doi:10.1128/IAI.01877-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Genetic Alteration of Mycobacterium smegmatis to Improve Mycobacterial Bactofection and DNA Immunization

Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Observatory, 7925, Cape Town, South Africa, Division of Infectious Disease, Wadsworth Center, New York State Department of Health, Department of Biomedical Sciences, University at Albany, Albany, NY 12201, Division of Viral Pathogenesis, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115, Howard Hughes Medical Institute, Albert Einstein College of Medicine, Bronx, NY 10461, USA, Department of Pediatrics, Albert Einstein College of Medicine, Bronx, NY 10461, USA, The Lewis M. Fraad Department of Pediatrics, Jacobi Medical Center, Bronx, NY 10461, USA

^* To whom correspondence should be addressed. Email: fennelly{at}aecom.yu.edu.

	Abstract

Mycobacteria target and persist within phagocytic monocytes and are strong adjuvants, making them attractive candidate vectors for DNA vaccines. We characterized the ability of mycobacteria to deliver transgenes to mammalian cells and the effects of various bacterial chromosomal mutations on the efficiency of transfer in vivo and in vitro. First, we observed green fluorescent protein expression via microscopy and FACS analysis after infection of phagocytic and non-phagocytic cell lines by M. smegmatis or M. bovis BCG harboring a plasmid encoding the fluorescent gene under the control of a eukaryotic promoter. Next, we compared the efficiency of gene transfer using M. smegmatis or BCG that contain chromosomal insertions or deletions that cause early lysis, hyperconjugation, or an increased plasmid copy number. We observed a significant—albeit only 1.7-fold—increase in the level of plasmid transfer to eukaryotic cells infected with M. smegmatis hyperconjugation mutants. M. smegmatis strains that overexpressed replication proteins (Rep) of pAL5000, a plasmid whose replicon is incorporated in many mycobacterial constructs, generated a 10-fold increase in plasmid copy number and a 3.5-fold and 3-fold increase in gene transfer efficiency to HeLa cells and J774 cells, respectively. Although BCG strains overexpressing Rep could not be recovered, BCG harboring a plasmid with a copy number-up mutation in the oriM resulted in a 3-fold increase in gene transfer to J774 cells. Moreover, M. smegmatis strains over-expressing replication proteins enhanced gene transfer in vivo compared with a wild-type control. Immunization of mice with mycobacteria harboring a plasmid (pgp120_h^E) encoding HIV gp120 elicited gp120-specific CD8 T- cell responses among splenocytes and PBMCs that were up to 2-fold (P < 0.05) and 3-fold (P <0.001) higher, respectively, in strains supporting higher copy numbers. The magnitude of these responses was approximately half of that observed after intramuscular immunization with pgp120_h^E. M. smegmatis and other nonpathogenic mycobacteria are promising candidate vectors for DNA vaccine delivery.