Altered expression of isoniazid-regulated genes in drug-treated dormant Mycobacterium tuberculosis (original) (raw)
Frontiers in Microbiology, 2018
The development of antibiotic tolerance is believed to be a major factor in the lengthy duration of current tuberculosis therapies. In the current study, we have modeled antibiotic tolerance in vitro by exposing Mycobacterium tuberculosis to two distinct stress conditions: progressive hypoxia and nutrient starvation [phosphate-buffered saline (PBS)]. We then studied the bacterial transcriptional response using RNA-seq and employed a bioinformatics approach to identify important transcriptional regulators, which was facilitated by a novel Regulon Enrichment Test (RET). A total of 17 transcription factor (TF) regulons were enriched in the hypoxia gene set and 16 regulons were enriched in the nutrient starvation, with 12 regulons enriched in both conditions. Using the same approach to analyze previously published gene expression datasets, we found that three M. tuberculosis regulons (Rv0023, SigH, and Crp) were commonly induced in both stress conditions and were also among the regulons enriched in our data. These regulators are worthy of further study to determine their potential role in the development and maintenance of antibiotic tolerance in M. tuberculosis following stress exposure.
Overview on mechanisms of isoniazid action and resistance in Mycobacterium tuberculosis
Infection, Genetics and Evolution, 2016
The tuberculosis (TB) is a disease caused by Mycobacterium tuberculosis (Mtb) and is considered a worldwide public health problem, classified as the leading cause of death by a single infectious agent. The conventional treatment is performed through the combination of drugs that exhibit precocious or sterilizing bactericidal activity. The commonly used drugs (knowing as first line drugs) are: izoniazide (INH), rifampicin (RIF), pyrazinamide (PZA) and ethambutol (ETH). However, the number of resistant TB cases (classified as RR-TB, MDR-TB and XDR-TB) to drugs has been growing in concern over the years. Of the total cases registered in 2016, about 600 thousand were resistant to one or more drugs used in the treatment. Resistance of Mtb strains to antituberculosis drugs is closely related to mutations in different bacillus genes. Multidrugresistant tuberculosis is a worldwide problem. A better understanding of the molecular mechanisms associated with the resistance to first-line drugs used in the TB treatment is of great importance for the development of new drugs, directly helping to reduce the number of resistant cases and bringing great benefits to public health. A review of the mechanisms of action of first-line drugs used in the treatment regimen to sensitive tuberculosis, as well as the main associated resistance mechanisms are.
Mycobacterium tuberculosis persistence mutants identified by screening in isoniazid-treated mice
Proceedings of the National Academy of Sciences, 2010
Tuberculosis (TB) is notoriously difficult to cure, requiring administration of multiple antibiotics for 6 mo or longer. Conventional anti-TB drugs inhibit biosynthetic processes involved in cell growth and division, such as DNA replication, RNA transcription, protein translation, and cell wall biogenesis. Although highly effective against bacteria cultured in vitro under optimal growth conditions, these antibiotics are less effective against bacteria grown in vivo in the tissues of a mammalian host. The factors that contribute to the antibiotic tolerance of bacteria grown in vivo are unknown, although altered metabolism and sluggish growth are hypothesized to play a role. To address this question, we identified mutations in Mycobacterium tuberculosis that impaired or enhanced persistence in mice treated with isoniazid (INH), a front-line anti-TB drug. Disruption of cydC , encoding a putative ATP-binding cassette transporter subunit, accelerated bacterial clearance in INH-treated mi...
Frontiers in Cellular and Infection Microbiology
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.
The Journal of infectious diseases, 2015
Treatment initiation rapidly kills most drug-susceptible Mycobacterium tuberculosis, but a bacterial sub-population tolerates prolonged drug exposure. We evaluated drug-tolerant bacilli in human sputum by comparing mRNA expression of drug-tolerant bacilli that survive the early bactericidal phase with treatment-naïve bacilli. M. tuberculosis gene expression was quantified via RT-PCR in serial sputa from 17 Ugandans treated for drug-susceptible pulmonary tuberculosis. Within four days, bacterial mRNA abundance declined >98%, indicating rapid killing. Thereafter, the rate of decline slowed >94%, indicating drug tolerance. After 14 days, 16S rRNA transcripts/genome declined 96%, indicating slow growth. Drug-tolerant bacilli displayed marked down-regulation of genes associated with growth, metabolism and lipid synthesis and up-regulation in stress responses and key regulatory categories - including stress-associated sigma factors, transcription factors, and toxin-antitoxin gene...
Factors and Global Gene Regulation in Mycobacterium tuberculosis
Journal of Bacteriology, 2004
Tuberculosis remains a worldwide threat despite the availability of the BCG vaccine and antibiotic treatment. It is estimated that its etiologic agent, Mycobacterium tuberculosis, infects almost a third of the human population and kills two million people every year (27). The recent human immunodeficiency virus pandemic, the selection of multidrug-resistant strains of M. tuberculosis, and the increased immigration from countries with a high tuberculosis incidence, coupled with increasing poverty and homelessness in these countries, have awakened the developed nations from the widespread apathy toward tuberculosis (36). Indeed, recent years have seen great progress in the molecular characterization of this efficient human pathogen (26, 61). However, much work is still needed to understand how M. tuberculosis copes with the numerous environments it encounters in the course of a successful infection. Adaptation to such conditions must require a complex regulation of gene expression. The main stresses faced during infection can be summarized as follows. The first stress is exposure to oxidizing agents, principally represented by the reactive oxygen intermediates and reactive nitrogen intermediates, produced by activated macrophages. The second is exposure to low pH. Even if M. tuberculosis is able to block phagosome acidification, this block is not complete as the mycobacterial phagosome undergoes a slight decrease in pH (21). The third is damage of surface structures. Alveolar surfactant is a mild detergent with antibacterial activity and could damage the structure of its fatty acid-rich cell envelope. In addition, toxic peptides and proteins like granulysin, thought to act at the level of the bacterial surface, are released by activated macrophages and NK cells. Specifically, granulysin has been recently shown to be essential for M. tuberculosis killing after apoptosis of infected macrophages induced by NK cells (22). Finally, toxic free fatty acids, secreted from macrophages both inside the mycobacterial phagosome and in the external environment, exhibit their toxicity when interacting with the mycobacterial surface (2). The fourth is hypoxia, especially inside granulomas but also inside the phagosome. This environmental condition is actually the best candidate for the induction of persistence (also called dormancy or latency), a phenomenon of great importance in
Virulence
The ability of Mycobacterium tuberculosis (M. tb) to survive and persist in the host for decades in an asymptomatic state is an important aspect of tuberculosis pathogenesis. Although adaptation to hypoxia is thought to play a prominent role underlying M. tb persistence, how the bacteria achieve this goal is largely unknown. Rv0081, a member of the DosR regulon, is induced at the early stage of hypoxia while Rv3334 is one of the enduring hypoxic response genes. In this study, we uncovered genetic interactions between these two transcription factors. RNA-seq analysis of ΔRv0081 and ΔRv3334 revealed that the gene expression profiles of these two mutants were highly similar. We also found that under hypoxia, Rv0081 positively regulated the expression of Rv3334 while Rv3334 repressed transcription of Rv0081. In addition, we demonstrated that Rv0081 formed dimer and bound to the promoter region of Rv3334. Taken together, these data suggest that Rv0081 and Rv3334 work in the same regulatory pathway and that Rv3334 functions immediately downstream of Rv0081. We also found that Rv3334 is a bona fide regulator of the enduring hypoxic response genes. Our study has uncovered a regulatory pathway that connects the early and the enduring hypoxic response, revealing a transcriptional cascade that coordinates the temporal response of M. tb to hypoxia.
Modeling metabolic adjustment in Mycobacterium tuberculosis upon treatment with isoniazid
Systems and Synthetic Biology, 2010
Complex biological systems exhibit a property of robustness at all levels of organization. Through different mechanisms, the system tries to sustain stress such as due to starvation or drug exposure. To explore whether reconfiguration of the metabolic networks is used as a means to achieve robustness, we have studied possible metabolic adjustments in Mtb upon exposure to isoniazid (INH), a
PLOS One, 2010
Background: Tubercle bacilli are thought to persist in a dormant state during latent tuberculosis (TB) infection. Although little is known about the host factors that induce and maintain Mycobacterium tuberculosis (M. tb) within latent lesions, O 2 depletion, nutrient limitation and acidification are some of the stresses implicated in bacterial dormancy development/ growth arrest. Adaptation to hypoxia and exposure to NO/CO is implemented through the DevRS/DosT two-component system which induces the dormancy regulon.
PLoS Pathogens, 2012
Intracellular pathogens such as Mycobacterium tuberculosis have evolved strategies for coping with the pressures encountered inside host cells. The ability to coordinate global gene expression in response to environmental and internal cues is one key to their success. Prolonged survival and replication within macrophages, a key virulence trait of M. tuberculosis, requires dynamic adaptation to diverse and changing conditions within its phagosomal niche. However, the physiological adaptations during the different phases of this infection process remain poorly understood. To address this knowledge gap, we have developed a multi-tiered approach to define the temporal patterns of gene expression in M. tuberculosis in a macrophage infection model that extends from infection, through intracellular adaptation, to the establishment of a productive infection. Using a clock plasmid to measure intracellular replication and death rates over a 14day infection and electron microscopy to define bacterial integrity, we observed an initial period of rapid replication coupled with a high death rate. This was followed by period of slowed growth and enhanced intracellular survival, leading finally to an extended period of net growth. The transcriptional profiles of M. tuberculosis reflect these physiological transitions as the bacterium adapts to conditions within its host cell. Finally, analysis with a Transcriptional Regulatory Network model revealed linked genetic networks whereby M. tuberculosis coordinates global gene expression during intracellular survival. The integration of molecular and cellular biology together with transcriptional profiling and systems analysis offers unique insights into the host-driven responses of intracellular pathogens such as M. tuberculosis. Citation: Rohde KH, Veiga DFT, Caldwell S, Balázsi G, Russell DG (2012) Linking the Transcriptional Profiles and the Physiological States of Mycobacterium tuberculosis during an Extended Intracellular Infection. PLoS Pathog 8(6): e1002769.