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

Abstract

SUMMARY 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 E. 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.

Real-Time Bioluminescence Imaging of Mixed Mycobacterial Infections

“…Mycobacteria are primarily considered intracellular pathogens that can readily infect and replicate within macrophages [53] , [54] . Since metabolic changes could impact expression of luciferase by mycobacteria, we examined whether a similar correlation with bacterial numbers was observed when the bacteria are growing within macrophages to that observed in culture.…”

Genetic Strategies for Identifying New Drug Targets

“…There is a wealth of proof-ofconcept evidence in the literature showing that all of the above approaches could be implemented in mycobacteria (28,33,(79)(80)(81), but there has been no real effort to date to perform such screens in a systematic manner. One of the few attempts at generating a high-throughput system to use for gene overexpression is random inducible controlled expression (RICE), but it has not been applied to the identification of potential antibiotic targets (82).…”