Transcriptional Adaptation of Drug-tolerant Mycobacterium tuberculosis During Treatment of Human Tuberculosis

Abstract

Background: Treatment initiation rapidly kills most drug-susceptible Mycobacterium tuberculosis, but a bacterial subpopulation tolerates prolonged drug exposure. We evaluated drug-tolerant bacilli in human sputum by comparing messenger RNA (mRNA) expression of drug-tolerant bacilli that survive the early bactericidal phase with treatment-naive bacilli.

Methods: M. tuberculosis gene expression was quantified via reverse-transcription polymerase chain reaction in serial sputa from 17 Ugandans treated for drug-susceptible pulmonary tuberculosis.

Results: Within 4 days, bacterial mRNA abundance declined >98%, indicating rapid killing. Thereafter, the rate of decline slowed >94%, indicating drug tolerance. After 14 days, 16S ribosomal RNA transcripts/genome declined 96%, indicating slow growth. Drug-tolerant bacilli displayed marked downregulation of genes associated with growth, metabolism, and lipid synthesis and upregulation in stress responses and key regulatory categories-including stress-associated sigma factors, transcription factors, and toxin-antitoxin genes. Drug efflux pumps were upregulated. The isoniazid stress signature was induced by initial drug exposure, then disappeared after 4 days.

Conclusions: Transcriptional patterns suggest that drug-tolerant bacilli in sputum are in a slow-growing, metabolically and synthetically downregulated state. Absence of the isoniazid stress signature in drug-tolerant bacilli indicates that physiological state influences drug responsiveness in vivo. These results identify novel drug targets that should aid in development of novel shorter tuberculosis treatment regimens.

Keywords: Mycobacterium tuberculosis/genetics; Mycobacterium tuberculosis/physiology; gene expression profiling; pulmonary/epidemiology; sputum/microbiology; tuberculosis.

Figure 1.

Decline in Mycobacterium tuberculosis mRNA abundance among 17 patients during the first 56 days of tuberculosis treatment. The solid line represents the geometric mean percentage of mRNA abundance remaining at each day. The shaded area between dotted lines indicates 95% confidence intervals. Abbreviation: mRNA, messenger RNA.

Figure 2.

Change in expression of selected functional categories of genes after initiation of tuberculosis treatment. The percentage of genes in each category significantly up- (yellow) or down- (blue) regulated at each day relative to pretreatment expression (Day 0) is illustrated. Genes that did not display statistically significant change are not shown. Categories are ordered from most upregulated (top) to most downregulated (bottom). Category references are provided in the Supplementary Materials, Supplementary Table 5. Abbreviations: ATP, adenosine triphosphate; EHR, enduring hypoxic response; ESX, ESX/Type VII secretion system; IS & phages, insertion sequences and phage-related genes; TCA, tricarboxcylic acid cycle.

Figure 3.

Heat map of median expression fold-change of 82 genes differentially expressed (adjusted P value <.05) between the early bactericidal phase (Day 2) and sterilizing phase (Day 14). Yellow indicates increased expression relative to day 0; blue indicates decreased expression. Clustering by day (row) indicates similarity between expression patterns at different days. Clustering by gene (column) identifies 5 clusters of genes (a–e) with similar expression patterns over time.

Figure 4.

Comparison of enrichment of functional gene categories between drug-tolerant Mycobacterium tuberculosis in sputum and selected experimental models. A, Sputum after 14 days of drug treatment. B, Antibiotic selection model [11]. C, Hollow fiber artificial granuloma model [16]. D, Nutrient starvation model [13]. E, Oxygen deprivation nonreplicating persistence model [16], (F) Multiple-stress model [15]. Abbreviations: ATP, adenosine triphosphate; EHR, enduring hypoxic response; ESX, ESX/Type VII secretion system; IS & phages, insertion sequences and phage-related genes; TCA, tricarboxcylic acid cycle.