New chemotherapeutics active against multidrug-resistant Mycobacterium tuberculosis are urgently needed. We report on the identification of an adamantyl urea compound that shows potent bactericidal ...activity against M. tuberculosis and a unique mode of action, namely the abolition of the translocation of mycolic acids from the cytoplasm, where they are synthesized to the periplasmic side of the plasma membrane and are in turn transferred onto cell wall arabinogalactan or used in the formation of virulence-associated, outer membrane, trehalose-containing glycolipids. Whole-genome sequencing of spontaneous-resistant mutants of M. tuberculosis selected in vitro followed by genetic validation experiments revealed that our prototype inhibitor targets the inner membrane transporter MmpL3. Conditional gene expression of mmpL3 in mycobacteria and analysis of inhibitor-treated cells validate MmpL3 as essential for mycobacterial growth and support the involvement of this transporter in the translocation of trehalose monomycolate across the plasma membrane.
Bacteria use population heterogeneity, the presence of more than one phenotypic variant in a clonal population, to endure diverse environmental challenges - a 'bet-hedging' strategy. Phenotypic ...variants have been described in many bacteria, but the phenomenon is not well-understood in mycobacteria, including the environmental factors that influence heterogeneity. Here, we describe three reproducible morphological variants in
- smooth, rough, and an intermediate morphotype that predominated under typical laboratory conditions.
has two recognized morphotypes, smooth and rough. Interestingly,
exists in only a rough form. The shift from smooth to rough in both
and
was observed over time in extended static culture, however the frequency of the rough morphotype was high in pellicle preparations compared to planktonic culture, suggesting a role for an aggregated microenvironment in the shift to the rough form. Differences in growth rate, biofilm formation, cell wall composition, and drug tolerance were noted among
and
variants. Deletion of the global regulator
shifted the
intermediate morphotype to a smooth form but did not fully phenocopy the naturally generated smooth morphotype, indicating Lsr2 is likely downstream of the initiating regulatory cascade that controls these morphotypes. Rough forms typically correlate with higher invasiveness and worse outcomes during infection and our findings indicate the shift to this rough form is promoted by aggregation. Our findings suggest that mycobacterial population heterogeneity, reflected in colony morphotypes, is a reproducible, programmed phenomenon that plays a role in adaptation to unique environments and this heterogeneity may influence infection progression and response to treatment.
The sigmoid E
model was used to describe the rRNA synthesis ratio (RS ratio) response of Mycobacterium tuberculosis to antimicrobial concentration. RS-E
measures the maximal ability of a drug to ...inhibit the RS ratio and can be used to rank-order drugs based on their RS ratio effect. RS-EC
is the concentration needed to achieve 90% of the RS-E
, which may guide dose selection to achieve a maximal RS ratio effect
.
Isoxyl (ISO) and thiacetazone (TAC), two prodrugs once used in the clinical treatment of tuberculosis, have long been thought to abolish Mycobacterium tuberculosis (M. tuberculosis) growth through ...the inhibition of mycolic acid biosynthesis, but their respective targets in this pathway have remained elusive. Here we show that treating M. tuberculosis with ISO or TAC results in both cases in the accumulation of 3-hydroxy C18, C20, and C22 fatty acids, suggestive of an inhibition of the dehydratase step of the fatty-acid synthase type II elongation cycle. Consistently, overexpression of the essential hadABC genes encoding the (3R)-hydroxyacyl-acyl carrier protein dehydratases resulted in more than a 16- and 80-fold increase in the resistance of M. tuberculosis to ISO and TAC, respectively. A missense mutation in the hadA gene of spontaneous ISO- and TAC-resistant mutants was sufficient to confer upon M. tuberculosis high level resistance to both drugs. Other mutations found in hypersusceptible or resistant M. tuberculosis and Mycobacterium kansasii isolates mapped to hadC. Mutations affecting the non-essential mycolic acid methyltransferases MmaA4 and MmaA2 were also found in M. tuberculosis spontaneous ISO- and TAC-resistant mutants. That MmaA4, at least, participates in the activation of the two prodrugs as proposed earlier is not supported by our biochemical evidence. Instead and in light of the known interactions of both MmaA4 and MmaA2 with HadAB and HadBC, we propose that mutations affecting these enzymes may impact the binding of ISO and TAC to the dehydratases.
Background: The anti-TB prodrugs isoxyl (ISO) and thiacetazone (TAC) inhibit mycolic acid biosynthesis.
Results: We show that ISO and TAC both target the dehydration step of the FAS-II elongation system.
Conclusion: ISO and TAC share the same mode of action.
Significance: ISO and TAC are the first antibiotics reported to target the FAS-II dehydratase(s) of Mycobacterium tuberculosis.
is a strict aerobe capable of prolonged survival in the absence of oxygen. We investigated the ability of anaerobic
to counter challenges to internal pH homeostasis in the absence of aerobic ...respiration, the primary mechanism of proton efflux for aerobic bacilli. Anaerobic
populations were markedly impaired for survival under a mildly acidic pH relative to standard culture conditions. An acidic environmental pH greatly increased the susceptibilities of anaerobic bacilli to the collapse of the proton motive force by protonophores, to antimicrobial compounds that target entry into the electron transport system, and to small organic acids with uncoupling activity. However, anaerobic bacilli exhibited high tolerance against these challenges at a near-neutral pH. At a slightly alkaline pH, which was near the optimum intracellular pH, the addition of protonophores even improved the long-term survival of bacilli. Although anaerobic
bacilli under acidic conditions maintained 40% lower ATP levels than those of bacilli under standard culture conditions, ATP loss alone could not explain the drop in viability. Protonophores decreased ATP levels by more than 90% regardless of the extracellular pH but were bactericidal only under acidic conditions, indicating that anaerobic bacilli could survive an extreme ATP loss provided that the external pH was within viable intracellular parameters. Acidic conditions drastically decreased the anaerobic survival of a DosR mutant, while an alkaline environment improved the survival of the DosR mutant. Together, these findings indicate that intracellular acidification is a primary challenge for the survival of anaerobic
and that the DosR regulon plays a critical role in sustaining internal pH homeostasis.
During infection,
bacilli are prevalent in environments largely devoid of oxygen, yet the factors that influence the survival of these severely growth-limited and metabolically limited bacilli remain poorly understood. We determined how anaerobic bacilli respond to fluctuations in environmental pH and observed that these bacilli were highly susceptible to stresses that promoted internal acidic stress, whereas conditions that promoted an alkaline internal pH promoted long-term survival even during severe ATP depletion. The DosR regulon, a major regulator of general hypoxic stress, played an important role in maintaining internal pH homeostasis under anaerobic conditions. Together, these findings indicate that in the absence of aerobic respiration, protection from internal acidification is crucial for long-term
survival.
To address the ongoing global tuberculosis crisis, there is a need for shorter, more effective treatments. A major reason why tuberculosis requires prolonged treatment is that, following a short ...initial phase of rapid killing, the residual
withstands drug killing. Because existing methods lack sensitivity to quantify low-abundance mycobacterial RNA in drug-treated animals, cellular adaptations of drug-exposed bacterial phenotypes
remain poorly understood. Here, we used a novel RNA-seq method called SEARCH-TB to elucidate the
transcriptome in mice treated for up to 28 days with standard doses of isoniazid, rifampin, pyrazinamide, and ethambutol. We compared murine results with
SEARCH-TB results during exposure to the same regimen. Treatment suppressed genes associated with growth, transcription, translation, synthesis of rRNA proteins, and immunogenic secretory peptides. Bacteria that survived prolonged treatment appeared to transition from ATP-maximizing respiration toward lower-efficiency pathways and showed modification and recycling of cell wall components, large-scale regulatory reprogramming, and reconfiguration of efflux pump expression. Although the pre-treatment
and
transcriptomes differed profoundly
genes differentially expressed following treatment
and
were similar, with differences likely attributable to immunity and drug pharmacokinetics in mice. These results reveal cellular adaptations of
that withstand prolonged drug exposure
demonstrating proof of concept that SEARCH-TB is a highly granular pharmacodynamic readout. The surprising finding that differential expression is concordant
and
suggests that insights from transcriptional analyses
may translate to the mouse. IMPORTANCE A major reason that curing tuberculosis requires prolonged treatment is that drug exposure changes bacterial phenotypes. The physiologic adaptations of
that survive drug exposure
have been obscure due to low sensitivity of existing methods in drug-treated animals. Using the novel SEARCH-TB RNA-seq platform, we elucidated
phenotypes in mice treated for with the global standard 4-drug regimen and compared them with the effect of the same regimen
. This first view of the transcriptome of the minority
population that withstands treatment
reveals adaptation of a broad range of cellular processes, including a shift in metabolism and cell wall modification. Surprisingly, the change in gene expression induced by treatment
and
was largely similar. This apparent "portability" from
to the mouse provides important new context for
transcriptional analyses that may support early preclinical drug evaluation.
Mycobacterium tuberculosis and M. smegmatis form drug-tolerant biofilms through dedicated genetic programs. In support of a stepwise process regulating biofilm production in mycobacteria, it was ...shown elsewhere that lsr2 participates in intercellular aggregation, while groEL1 was required for biofilm maturation in M. smegmatis. Here, by means of RNA-Seq, we monitored the early steps of biofilm production in M. bovis BCG, to distinguish intercellular aggregation from attachment to a surface. Genes encoding for the transcriptional regulators dosR and BCG0114 (Rv0081) were significantly regulated and responded differently to intercellular aggregation and surface attachment. Moreover, a M. tuberculosis H37Rv deletion mutant in the Rv3134c-dosS-dosR regulon, formed less biofilm than wild type M. tuberculosis, a phenotype reverted upon reintroduction of this operon into the mutant. Combining RT-qPCR with microbiological assays (colony and surface pellicle morphologies, biofilm quantification, Ziehl-Neelsen staining, growth curve and replication of planktonic cells), we found that BCG0642c affected biofilm production and replication of planktonic BCG, whereas ethR affected only phenotypes linked to planktonic cells despite its downregulation at the intercellular aggregation step. Our results provide evidence for a stage-dependent expression of genes that contribute to biofilm production in slow-growing mycobacteria.
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.
Im Rahmen der Debatten über die Tragfähigkeit des Said'schen Orientalismus- Konzepts wurde Ostmitteleuropa bislang bestenfalls als Objekt hegemonialer Diskurse in den Blick genommen. Im Kontrast dazu ...wird in diesem Band nach ostmitteleuropäischen Orientalismen von 1800 bis in die Zwischenkriegszeit gefragt.Die interdisziplinären Beiträge thematisieren die Wahrnehmungen und Deutungen des Orients in jener europäischen Großregion - vom Baltikum bis zur Krim, von Polen bis nach Bosnien -, die über Jahrhunderte durch die Nachbarschaft zum Osmanischen Reich geprägt wurde. Im Fokus stehen dabei sowohl die Akteure, Institutionen und Diskurse als auch die unterschiedlichen Orient-Topoi sowie deren Darstellung in der Literatur und in den bildenden Künsten.
Purpose: Perinatal exposure, either in utero or more often during delivery, is an important mode of hepatitis B virus (HBV) transmission that results in chronic disease in approximately 90% of ...infected infants. Immunoprophylaxis (consisting of hepatitis B vaccine and hepatitis B immune globulin HBIG) is recommended for infants born to hepatitis B-infected women within 12 hours of birth and is 95% effective in preventing perinatal HBV transmission. For women with high viral loads (>200,000 IU/mL), antiviral therapy during pregnancy has been recently recommended to further reduce perinatal transmission risk. We sought to characterize antiviral therapy use in hepatitis B-infected pregnant women. Methods: The Centers for Disease Control and Prevention (CDC) funds 64 state and local jurisdictions to collect information on pregnant women infected with hepatitis B and their infants as part of its Perinatal Hepatitis B Prevention Program. CDC provided auxiliary funding for five jurisdictions to collect additional information on maternal and infant demographic and clinical characteristics. We analyzed data collected retrospectively from hepatitis B-infected pregnant women in Georgia, Michigan, New York City, Philadelphia, and Wisconsin identified as having delivered live births during April 2016-December 2017. We assessed maternal antiviral therapy use during pregnancy; HBV DNA levels included in our analysis were from the last result available prior to delivery for each woman. Results: We identified 3,971 women with hepatitis B infection during pregnancy; of these, 803 (20.2%) had information regarding prescription of antiviral therapy during pregnancy. HBV DNA levels were known for 1907 women, of whom 9.1% (n=173) had HBV DNA >200,000 IU/mL nearest delivery. Antiviral therapy was prescribed for 26.5% (n=213) of women with information. Antiviral therapy was more commonly prescribed for women aged <30 years vs. ≥30 years (32.0% vs. 23.1%, p=0.0069), Asian/Pacific Island race vs. white or black (42.7% vs. 2.8% and 6.2%, respectively, p<0.0001), and those whose HBV was monitored by a gastroenterologist/hepatologist vs. maternal fetal medicine or infectious disease specialist (55.1% vs. 10.3% and 36.4%, respectively, p<0.0001). Tenofovir was prescribed for 92.9% of women prescribed antiviral therapy; lamivudine was prescribed for 3.8%. Mean birth weight was 3196.7 grams and 3224.7 grams for infants born to mothers prescribed and not prescribed antiviral therapy, respectively (p=0.4713). Conclusion: Antiviral therapy was prescribed for approximately one-fourth of hepatitis B-infected pregnant women with information. Antiviral therapy was prescribed more commonly for women who were younger, Asian/Pacific Island race, and who received hepatitis B care from a gastroenterologist/hepatologist. Although these are preliminary findings and data collection is ongoing, opportunities may exist to improve guideline-concordant antiviral therapy use among pregnant women.
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