Latent tuberculosis infection (LTBI) represents a major challenge to curing TB disease. Current guidelines for LTBI management include only three older drugs and their combinations-isoniazid and ...rifamycins (rifampicin and rifapentine). These available control strategies have little impact on latent TB elimination, and new specific therapeutics are urgently needed. In the present mini-review, we highlight some of the alternatives that may potentially be included in LTBI treatment recommendations and a list of early-stage prospective small molecules that act on drug targets specific for
latency.
Both latent and active TB infections are caused by a heterogeneous population of mycobacteria, which includes actively replicating and dormant bacilli in different proportions. Dormancy substantially ...affects
drug tolerance and TB clinical management due to a significant decrease in the metabolic activity of bacilli, which leads to the complexity of both the diagnosis and the eradication of bacilli. Most diagnostic approaches to latent infection deal with a subpopulation of active
, underestimating the contribution of dormant bacilli and leading to limited success in the fight against latent TB. Moreover, active TB appears not only as a primary form of infection but can also develop from latent TB, when resuscitation from dormancy is followed by bacterial multiplication, leading to disease progression. To win against latent infection, the identification of the Achilles' heel of dormant
is urgently needed. Regulatory mechanisms and metabolic adaptation to growth arrest should be studied using
and
models that adequately imitate latent TB infection in macroorganisms. Understanding the mechanisms underlying
dormancy and resuscitation may provide clues to help control latent infection, reduce disease severity in patients, and prevent pathogen transmission in the population.
Dormant Mycobacterium tuberculosis bacilli are believed to play an important role in latent tuberculosis infection. Previously, we have demonstrated that cultivation of M. tuberculosis in ...K(+)-deficient medium resulted in generation of dormant cells. These bacilli were non-culturable on solid media (a key feature of dormant M. tuberculosis in vivo) and characterized by low metabolism and tolerance to anti-tuberculosis drugs. The dormant bacteria demonstrated a high potential to reactivation after K(+) reintroduction even after prolonged persistence under rifampicin. In this work, we studied the transcriptome and stability of transcripts in persisting dormant bacilli under arrest of mRNA de novo synthesis.
RNA-seq-based analysis of the dormant non-culturable population obtained under rifampicin exposure revealed a 30-50-fold decrease of the total mRNA level, indicating global transcriptional repression. However, the analysis of persisting transcripts displayed a cohort of mRNA molecules coding for biosynthetic enzymes, proteins involved in adaptation and repair processes, detoxification, and control of transcription initiation. This 'dormant transcriptome' demonstrated considerable stability during M. tuberculosis persistence and mRNA de novo synthesis arrest. On the contrary, several small non-coding RNAs showed increased abundance on dormancy. Interestingly, M. tuberculosis entry into dormancy was accompanied by the cleavage of 23S ribosomal RNA at a specific point located outside the ribosome catalytic center.
Dormant non-culturable M. tuberculosis bacilli are characterized by a global transcriptional repression. At the same time, the dormant bacilli retain low-abundant mRNAs, which are considerably stable during in vitro persistence, reflecting their readiness for translation upon early resuscitation steps. Increased abundance of non-coding RNAs on dormancy may indicate their role in the entry into and maintenance of M. tuberculosis dormant non-culturable state.
Regulatory small non-coding RNAs play a significant role in bacterial adaptation to changing environmental conditions. Various stresses such as hypoxia and nutrient starvation cause a reduction in ...the metabolic activity of
, leading to entry into dormancy. We investigated the functional role of F6, a small RNA of
, and constructed an F6 deletion strain of
. Using the RNA-seq approach, we demonstrated that gene expression changes that accompany F6 deletion contributed to bacterial resistance against oxidative stress. We also found that F6 directly interacted with 5'-UTR of
mRNA encoding RpfE2, a resuscitation-promoting factor, which led to the downregulation of RpfE2 expression. The F6 deletion strain was characterized by the reduced ability to enter into dormancy (non-culturability) in the potassium deficiency model compared to the wild-type strain, indicating that F6 significantly contributes to bacterial adaptation to non-optimal growth conditions.
Abstract
We report on phenomena observed in planar integrated networks obtained connecting superconducting island by Josephson tunnel junctions. These networks, identifiable as tree-like graphs, have ...branches consisting of series arrays of Josephson junctions which can be individually current biased and characterized. Both Josephson supercurrents and gap parameters of the arrays embedded in the graph structures display properties significantly different from those of “reference” arrays fabricated on the same chips and having identical geometrical shape. The temperature and magnetic field dependencies of the Josephson current of the embedded arrays both show a singular behavior when a critical value is reached by the Josephson characteristic energy. The gap parameter of the junctions generating the embedded arrays is higher than that of the junctions forming the reference geometrical arrays.
In the course of evolution,
(Mtb), the etiological agent of tuberculosis, has developed sophisticated strategies to evade host immune response, including the synthesis of small non-coding RNAs ...(sRNAs), which regulate post-transcriptional pathways involved in the stress adaptation of mycobacteria. sRNA MTS1338 is upregulated in Mtb during its infection of cultured macrophages and in the model of chronic tuberculosis, suggesting involvement in host-pathogen interactions. Here, we analyzed the role of MTS1338 in the Mtb response to macrophage-like stresses in vitro. The Mtb strain overexpressing MTS1338 demonstrated enhanced survival ability under low pH, nitrosative, and oxidative stress conditions simulating the antimicrobial environment inside macrophages. Transcriptomic analysis revealed that in MTS1338-overexpressing Mtb, the stress factors led to the activation of a number of transcriptional regulators, toxin-antitoxin modules, and stress chaperones, about half of which coincided with the genes induced in Mtb phagocytosed by macrophages. We determined the MTS1338 "core regulon", consisting of 11 genes that were activated in all conditions under MTS1338 overexpression. Our findings indicate that MTS1338 is a stress-induced sRNA that promotes Mtb survival in macrophages by triggering adaptive transcriptional mechanisms in response to host antimicrobial defense reactions.
Summary
The culturability of several actinobacteria is controlled by resuscitation‐promoting factors (Rpfs). These are proteins containing a c. 70‐residue domain that adopts a lysozyme‐like fold. The ...invariant catalytic glutamate residue found in lysozyme and various bacterial lytic transglycosylases is also conserved in the Rpf proteins. Rpf from Micrococcus luteus, the founder member of this protein family, is indeed a muralytic enzyme, as revealed by its activity in zymograms containing M. luteus cell walls and its ability to (i) cause lysis of Escherichia coli when expressed and secreted into the periplasm; (ii) release fluorescent material from fluorescamine‐labelled cell walls of M. luteus; and (iii) hydrolyse the artificial lysozyme substrate, 4‐methylumbelliferyl‐β‐d‐N,N′,N′′‐triacetylchitotrioside. Rpf activity was reduced but not completely abolished when the invariant glutamate residue was altered. Moreover, none of the other acidic residues in the Rpf domain was absolutely required for muralytic activity. Replacement of one or both of the cysteine residues that probably form a disulphide bridge within Rpf impaired but did not completely abolish muralytic activity. The muralytic activities of the Rpf mutants were correlated with their abilities to stimulate bacterial culturability and resuscitation, consistent with the view that the biological activity of Rpf results directly or indirectly from its ability to cleave bonds in bacterial peptidoglycan.
Under unfavorable conditions such as host immune responses and environmental stresses, human pathogen
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
from the dormant "non-culturable" (NC) cells to fully replicating population
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
transcription immediately after the removal of the stress factor, which resulted in significant changes of the
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
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
is characterized by immediate activation of
transcription followed by the upregulation of genes controlling key metabolic pathways and then, cell multiplication.
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