Disruption of the mycobacterial cell entry gene of Mycobacterium bovis BCG results in a mutant that exhibits a reduced invasiveness for epithelial cells.

Mycobacteria belonging to the Mycobacterium tuberculosis complex have the ability to invade and replicate in non-phagocytic cells, an event that requires the presence of bacterial surface components capable of triggering a cell response and the subsequent internalization of the microorganism. In this study, we report the sequencing of the mycobacterial cell entry gene (mce) of Mycobacterium bovis bacillus Calmette-Guérin (BCG) and the generation and characterization of a mutant BCG strain with an inactivated mce gene, by homologous recombination with double cross-over. This mutant strain does not express the mycobacterial cell entry protein (Mce) and exhibits a reduced ability to invade the non-phagocytic epithelial cell line HeLa as compared to wild-type BCG.

Synergistic induction of promoters containing metal- and glucocorticoid-responsive elements.

We have shown that it is possible to synergistically activate gene transcription when several glucocorticoid responsive elements (GREs) and metal responsive elements (MREs) that coexist within the same promoter are induced simultaneously. To demonstrate this, additional GREs were introduced into a human metallothionein IIA (hMT-IIA) promoter in which some constitutive elements had been deleted. The transcriptional strength and inducibility of the modified hMT-IIA promoters were studied in transient expression experiments using the CAT gene as a reporter. As a result of synergistic activation of transcription by CdCl2 and dexamethasone, the induced expression levels of the modified promoters were significantly higher than those obtained with wild-type hMT-IIA. Since the increase in inducible expression was not accompanied by a concominant increase in basal levels, the inducibility of the modified MT promoters was up to 6-fold higher. The degree of transcriptional synergism was shown to depend on the position and the number of GREs introduced. Thus, the engineering of synthetic promoters that include both GREs and MREs should offer the opportunity to develop a new series of improved inducible mammalian expression vectors.