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Tuberculosis (Edinb). 2011 Dec;91 Suppl 1:S66-8. doi: 10.1016/j.tube.2011.10.013. Epub 2011 Nov 13.

Random inducible controlled expression (RICE) for identification of mycobacterial virulence genes.

Author information

1
Department of Microbial and Molecular Pathogenesis, Texas A&M Health Science Center, Medical Research & Education Building, 8447 State Hwy 47, Bryan, TX 77807, USA.

Abstract

We have developed Random Inducible Controlled Expression (RICE), a high throughput genetic approach to identify regulated virulence pathways in pathogenic mycobacteria. RICE allows expression of bacterial genes under conditions where they are normally off, e.g. under laboratory growth conditions, via the use of an inducible or constitutive promoter as well as gene dosage effects due to the presence of the gene on a plasmid. Mycobacterial genomic DNA can be digested to yield random fragments for cloning into a suicide expression vector downstream of a mycobacterial promoter or with their own promoter on a replicating plasmid increasing expression by gene dosage effects. The plasmid DNA is normally amplified in Escherichia coli and delivered into mycobacteria to select for recombinants or plasmid transformants. The resulting library is then directly screened for enhanced host cell interactions in functional assays that evaluate the efficiency of adherence, entry and replication inside host cells. This approach has resulted in identification of several virulence factors from pathogenic mycobacteria. Our analysis of one such locus identified by RICE, the mycobacterial enhanced entry locus (mel2), found that the genes present facilitate bacterial persistence inside the host by protecting the pathogen against oxidative damage. Thus, we have developed a genetic strategy that offers several advantages: (i) it allows identification of bacterial genetic elements that have a direct role during host-pathogen interactions (ii) it can be used to identify virulence factors in a broad range of pathogens and (iii) it can reveal genes that are only induced at specific stages of infection.

PMID:
22079212
PMCID:
PMC3248965
DOI:
10.1016/j.tube.2011.10.013
[Indexed for MEDLINE]
Free PMC Article

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