Resuscitation of Dormant “Non-culturable” Mycobacterium tuberculosis Is Characterized by Immediate Transcriptional Burst

Abstract

Under unfavorable conditions such as host immune responses and environmental stresses, human pathogen Mycobacterium tuberculosis may acquire the dormancy phenotype characterized by “non-culturability” and a substantial decrease of metabolic activity and global transcription rates. Here, we found that the transition of M. tuberculosis from the dormant “non-culturable” (NC) cells to fully replicating population in vitro occurred not earlier than 7 days after the start of the resuscitation process, with predominant resuscitation over this time interval evidenced by shortening apparent generation time up to 2.8 h at the beginning of resuscitation. The early resuscitation phase was characterized by constant, albeit low, incorporation of radioactive uracil, indicating de novo transcription immediately after the removal of the stress factor, which resulted in significant changes of the M. tuberculosis transcriptional profile already after the first 24 h of resuscitation. This early response included transcriptional upregulation of genes encoding enzymes of fatty acid synthase system type I (FASI) and type II (FASII) responsible for fatty acid/mycolic acid biosynthesis, and regulatory genes, including whiB6 encoding a redox-sensing transcription factor. The second resuscitation phase took place 4 days after the resuscitation onset, i.e., still before the start of active cell division, and included activation of central metabolism genes encoding NADH dehydrogenases, ATP-synthases, and ribosomal proteins. Our results demonstrate, for the first time, that the resuscitation of dormant NC M. tuberculosis is characterized by immediate activation of de novo transcription followed by the upregulation of genes controlling key metabolic pathways and then, cell multiplication.

Applications of Transcriptomics and Proteomics for Understanding Dormancy and Resuscitation in Mycobacterium tuberculosis

“…Under these conditions, the bacterium underwent growth arrest and a decrease in respiration rate. Salina et al (2014Salina et al ( , 2019 have studied growth in potassium-deficient media which generates non-culturable (NC) bacilli, tolerant to cell wall targeting antimicrobials. Potassium supplementation enables resuscitation of growth.…”

“…TCS components mprAB, kdpD, prrA; whiB1, whiB6; kstR (involved in cholesterol degradation pathway); icl1, mutA (methylmalonyl pathway). bkdA, bkdB, bkdC, fadE2, fadE13, accD2 (implicated in the catabolism of branched-chain keto and amino acids); Proteases and peptidases: pepD, pepR, htrA and clpC2; arcA (arginine deiminase); gcvB (glycine dehydrogenase), which are involved in degradation of arginine and glycine, respectively; hsaG (involved in the degradation of aromatic compounds); nuoA-N; sigA, sigB, sigE, sigF, sigG, sigH, sigI, sigL, and sigM Salina et al, 2014Salina et al, , 2019 2019) and whiB6 was upregulated during potassium depletion (Salina et al, 2014(Salina et al, , 2019. Analysis of the transcriptional regulatory network that underlies the response to vitamin C, showed an early response (upto 1 h), an intermediate response (between 2 and 8 h) and a late response (24 h).…”

“…The genes pdhABC encoding subunits of the pyruvate dehydrogenase enzyme complex, the fumarate reductase complex (frdABCD),were upregulated under nutrient starvation. Salina et al (2014Salina et al ( , 2019 have analyzed transcriptional signatures generated for bacilli cultured in potassiumdepleted medium to show that genes linked to glycolysis and gluconeogenesis are repressed in the non-culturable bacilli, with pgi, fba, tpi, gap, pgk, pgmA, eno, pykA, aceE, and lpdC downregulated compared to bacteria grown in potassiumsufficient media (Salina et al, 2014(Salina et al, , 2019. Four genes implicated in the pentose phosphate shunt were also repressed (fgd1, zwf2, tkt, and tal).…”

MTS1338, A Small Mycobacterium tuberculosis RNA, Regulates Transcriptional Shifts Consistent With Bacterial Adaptation for Entering Into Dormancy and Survival Within Host Macrophages

“…Overexpression of MTS1338 dramatically changes the bacterial growth rate (Arnvig et al, 2011; Ignatov et al, 2015). Our experiments with M. tuberculosis dormancy and resuscitation in vitro demonstrated that MTS1338 participates in entering dormancy (Ignatov et al, 2015), but is not involved in the resuscitation process (Salina et al, 2019). In vivo , high levels of MTS1338 transcription were reported for M. tuberculosis residing in chronically infected mouse lungs (Arnvig et al, 2011; Ignatov et al, 2014).…”

Advancing Immunotherapeutic Vaccine Strategies Against Pulmonary Tuberculosis

“…In addition, targeting M.tb populations resuscitating from dormancy would also be critical in the development of therapeutic vaccines, as to eliminate/minimize reactivation. Mycobacterial resuscitation is a complex process and is regulated in a coordinated transcriptional burst preceding expression of metabolic and growth-related pathways ( 92 ). Resuscitation promoting factors (RPFs) represent a major class of antigens involved in this process and may represent promising vaccine antigen targets ( 92 – 94 ).…”