Mycobacterium tuberculosis precursor rRNA as a measure of treatment-shortening activity of drugs and regimens

Abstract

There is urgent need for new drug regimens that more rapidly cure tuberculosis (TB). Existing TB drugs and regimens vary in treatment-shortening activity, but the molecular basis of these differences is unclear, and no existing assay directly quantifies the ability of a drug or regimen to shorten treatment. Here, we show that drugs historically classified as sterilizing and non-sterilizing have distinct impacts on a fundamental aspect of Mycobacterium tuberculosis physiology: ribosomal RNA (rRNA) synthesis. In culture, in mice, and in human studies, measurement of precursor rRNA reveals that sterilizing drugs and highly effective drug regimens profoundly suppress M. tuberculosis rRNA synthesis, whereas non-sterilizing drugs and weaker regimens do not. The rRNA synthesis ratio provides a readout of drug effect that is orthogonal to traditional measures of bacterial burden. We propose that this metric of drug activity may accelerate the development of shorter TB regimens.

Comparative Analysis of Pharmacodynamics in the C3HeB/FeJ Mouse Tuberculosis Model for DprE1 Inhibitors TBA-7371, PBTZ169, and OPC-167832

“…4D and E ). In summary, rRNA synthesis was lower in caseum, indicating slowed M. tuberculosis rRNA synthesis, which, in the absence of any drug treatment, is consistent with a significantly reduced bacterial replication rate ( 40 ) in the hypoxic caseum core relative to the better-aerated cellular-caseum interface.…”

“…Whereas mature rRNA is stable, spacer regions are rapidly degraded; therefore, the abundance of pre-rRNA relative to mature rRNA (also defined as rRNA synthesis ratio [RS ratio]) serves as a measure of ongoing rRNA synthesis. The RS ratio has been validated as a molecular biomarker that strongly correlates with growth rate of M. tuberculosis in vitro and in vivo in the absence of drug treatment ( 40 ).…”

“…To assess the replication status of M. tuberculosis in C3HeB/FeJ mouse lungs, we used quantitative multiplexed RNA- in situ hybridization (ISH) to measure pre-rRNA and 23S rRNA signals in bacteria in sections of caseous necrotic type I lesions of untreated animals ( 40 ). Results demonstrated that bacterial populations in the C3HeB/FeJ mouse model producing new rRNA, a biomarker associated with active bacterial replication ( 40 ), are primarily located in the foamy macrophage and neutrophil layers directly inside the collagen rim in necrotic lesions (i.e., cellular-caseum interface; Fig. 4D and E ).…”

“…Mouse lungs were fixed with formaldehyde, paraffin embedded, and stained by multiplexed-ISH using optimized protocols, as previously described ( 39 , 40 ). Briefly, mouse lungs were directly placed into 4% PFA prepared in 1× PBS upon excision, fixed for 48 h at 4°C, and paraffin embedded.…”

Lipid droplets and the transcriptome of Mycobacterium tuberculosis from direct sputa: a literature review

“…However, such techniques are inadequate for the detecting non-replicating drug-tolerant Mtb, which is important for predicting treatment duration, treatment outcome and drug resistance. Techniques measuring 16 s rRNA or pre-rRNA promise to add new depth to our understanding of the efficacy of drug combinations in patients [100,101]. Demirci and colleagues assessed the diagnostic accuracy of Mtb-mRNA-based RT-qPCR technique, with the BACTEC MGIT 960 method used as the gold standard.…”