A Short Interfering Rna Molecular Beacon for the Attenuation of Mycobacterial Infection

Abstract

The ability of the pathogen Mycobacterium Tuberculosis (MTB) to invade and survive within macrophages of granulomas is attributed to the product of the Mammalian Cell Entry (MCE) operon whose gene, mce4A, encodes a cholesterol transporter that transports host lipids into the bacterium that allows the bacterium to survive during chronic infection. Here, we proposed and tested the hypothesis that a mce4A siRNA molecular beacon can be used to attenuate mycobacterial infection in macrophages. Mce4A gene was cloned and expressed in E. coli (E. coli-4A) and differentiated U937 cells were transduced with piLenti-siRNA-GFP phage expressing the mce4A siRNA for 24 h. This was followed by infection with either E. coli-4A or M. smegmatis for 3 h followed by incubation for 0, 3, 6, 24 and 48 h. The cells were lysed and the lysates were plated on LB agar plates containing ampicillin (100 µg mL −1 ) or on 7H11 media and incubated at 37°C overnight. Our results showed that the siRNA treatment attenuated E.coli-4A infection in macrophages at 3, 6, 24 and 48 h by 0, 77, 59.6 and 99.7%, respectively. Our results also showed that the siRNA treatment attenuated M. smegmatis infection in macrophages at 3, 6, 24 and 48 h. by 94.8, 70.3, 98.9 and 93.4%, respectively. In conclusion, a mce4A siRNA molecular beacon was successfully delivered and stably expressed in macrophages which attenuated E. coli expressing mce4A (E. coli-4A) and M. smegmatis infection in macrophages.

Use of siRNA molecular beacons to detect and attenuate mycobacterial infection in macrophages

“…The degree of attenuation of M. smegmatis infection was compared between 3, 6, 24, and 48 h against that at 0 h baseline and was found to be 94.8%, 70.3%, 98.9%, and 93.4%, respectively. Thus, our results showed that the mce4A siRNA was able to significantly attenuate both E. coli-4A and M. smegmatis infection in macrophages [159] . Separate set of experiments were conducted to further test the hypothesis that the mce4A siRNA molecular beacon can attenuate the mce4A mRNA levels in E. coli expressing mce4A gene within infected macrophages.…”

“…Antisense technology using synthetic siRNAs have been used as a powerful tool in knocking down genes in prokaryotes (and also in eukaryotes), including hepatitis G virus [151] , influenza virus [152] , picornavirus [153] , and Trypanosoma brucei [154] . When targeted to essential genes, siRNAs inhibit growth of E. coli [148,149,155,156] , S. enterica [157] , Staphylococcus aureus [158] , M. smegmatis [159] and MTB [61,160] . Antisense RNAs have been successfully used for the study of bacterial growth and metabolism since this approach allows conditional knock down of target genes [161165] .…”