scholarly journals Mycobacterium tuberculosis Rv2700 Contributes to Cell Envelope Integrity and Virulence

2019 ◽  
Vol 201 (19) ◽  
Author(s):  
Edward R. Ballister ◽  
Marie I. Samanovic ◽  
K. Heran Darwin
Keyword(s):  
Growth Rate ◽  
Mycobacterium Tuberculosis ◽  
Cell Envelope ◽  
Bacterial Species ◽  
Transmembrane Helix ◽  
Antibiotic Sensitivity ◽  
Uncharacterized Protein ◽  
Content Type ◽  
Cell Envelopes ◽  
Envelope Functions

ABSTRACT The cell envelope of Mycobacterium tuberculosis is a key target for antibiotics, yet its assembly and maintenance remain incompletely understood. Here we report that Rv2700, a previously uncharacterized M. tuberculosis gene, contributes to envelope integrity. Specifically, an Rv2700 mutant strain had a decreased growth rate, increased sensitivity to antibiotics that target peptidoglycan crosslinking, and increased cell envelope permeability. We propose that Rv2700 be named a “cell envelope integrity” gene (cei). Importantly, a cei mutant had attenuated virulence in mice. Cei shares predicted structural homology with another M. tuberculosis protein, VirR (Rv0431), and we found that a virR mutant had growth rate, antibiotic sensitivity, and envelope permeability phenotypes similar to those of the cei mutant. Both Cei and VirR are predicted to consist of a transmembrane helix and an extracellular LytR_C domain. LytR_C domains have no known function, but they are also found in a family of proteins, the LytR-Cps2A-Psr (LCP) enzymes, that perform important cell envelope functions in a range of bacteria. In mycobacteria, LCP enzymes attach arabinogalactan to peptidoglycan, and mycobacterial LCP enzyme mutants have phenotypes similar to those of virR- and cei-deficient strains. Collectively, our results suggest that LytR_C domain proteins may contribute to the cell envelope functions performed by LCP proteins. This study provides a framework for further mechanistic investigations of LytR_C proteins and, more broadly, for advancing our understanding of the cell envelopes of mycobacteria and other medically and economically important genera. IMPORTANCE Mycobacterium tuberculosis causes about 1.5 million deaths per year. The unique composition of the Mycobacterium tuberculosis cell envelope is required for this bacterium to cause disease and is the target for several critical antibiotics. By better understanding the mechanisms by which mycobacteria assemble and maintain their cell envelope, we might uncover new therapeutic targets. In this work, we show that a previously uncharacterized protein, Rv2700, is important for cell envelope integrity in Mycobacterium tuberculosis and that loss of Rv2700 attenuates virulence in mice. This family of proteins is found in a broad group of bacterial species, so our work provides a first insight into their potential functions in many species important to the environment, industry, and human health.

scholarly journals Genetic Evidence for SecY Translocon-Mediated Import of Two Contact-Dependent Growth Inhibition (CDI) Toxins

mBio ◽  
2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Allison M. Jones ◽  
Petra Virtanen ◽  
Disa Hammarlöf ◽  
William J. Allen ◽  
Ian Collinson ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Target Cell ◽  
Bacterial Species ◽  
Genetic Evidence ◽  
Target Cells ◽  
Content Type ◽  
Sec Translocon ◽  
Dependent Growth ◽  
Cell Envelopes ◽  
Contact Dependent Growth Inhibition

ABSTRACT The C-terminal (CT) toxin domains of contact-dependent growth inhibition (CDI) CdiA proteins target Gram-negative bacteria and must breach both the outer and inner membranes of target cells to exert growth inhibitory activity. Here, we examine two CdiA-CT toxins that exploit the bacterial general protein secretion machinery after delivery into the periplasm. A Ser281Phe amino acid substitution in transmembrane segment 7 of SecY, the universally conserved channel-forming subunit of the Sec translocon, decreases the cytotoxicity of the membrane depolarizing orphan10 toxin from enterohemorrhagic Escherichia coli EC869. Target cells expressing secYS281F and lacking either PpiD or YfgM, two SecY auxiliary factors, are fully protected from CDI-mediated inhibition either by CdiA-CTo10EC869 or by CdiA-CTGN05224, the latter being an EndoU RNase CdiA toxin from Klebsiella aerogenes GN05224 that has a related cytoplasm entry domain. RNase activity of CdiA-CTGN05224 was reduced in secYS281F target cells and absent in secYS281F ΔppiD or secYS281F ΔyfgM target cells during competition co-cultures. Importantly, an allele-specific mutation in secY (secYG313W) renders ΔppiD or ΔyfgM target cells specifically resistant to CdiA-CTGN05224 but not to CdiA-CTo10EC869, further suggesting a direct interaction between SecY and the CDI toxins. Our results provide genetic evidence of a unique confluence between the primary cellular export route for unfolded polypeptides and the import pathways of two CDI toxins. IMPORTANCE Many bacterial species interact via direct cell-to-cell contact using CDI systems, which provide a mechanism to inject toxins that inhibit bacterial growth into one another. Here, we find that two CDI toxins, one that depolarizes membranes and another that degrades RNA, exploit the universally conserved SecY translocon machinery used to export proteins for target cell entry. Mutations in genes coding for members of the Sec translocon render cells resistant to these CDI toxins by blocking their movement into and through target cell membranes. This work lays the foundation for understanding how CDI toxins interact with the protein export machinery and has direct relevance to development of new antibiotics that can penetrate bacterial cell envelopes.

scholarly journals RNA G-Quadruplex Structures Mediate Gene Regulation in Bacteria

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Xiaolong Shao ◽  
Weitong Zhang ◽  
Mubarak Ishaq Umar ◽  
Hei Yuen Wong ◽  
Zijing Seng ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Cell Envelope ◽  
Bacterial Species ◽  
Virulence Gene ◽  
Content Type ◽  
Cellular Processes ◽  
Wide Range ◽  
G Quadruplex ◽  
Eukaryotic Organisms

ABSTRACT Guanine (G)-rich sequences in RNA can fold into diverse RNA G-quadruplex (rG4) structures to mediate various biological functions and cellular processes in eukaryotic organisms. However, the presence, locations, and functions of rG4s in prokaryotes are still elusive. We used QUMA-1, an rG4-specific fluorescent probe, to detect rG4 structures in a wide range of bacterial species both in vitro and in live cells and found rG4 to be an abundant RNA secondary structure across those species. Subsequently, to identify bacterial rG4 sites in the transcriptome, the model Escherichia coli strain and a major human pathogen, Pseudomonas aeruginosa, were subjected to recently developed high-throughput rG4 structure sequencing (rG4-seq). In total, 168 and 161 in vitro rG4 sites were found in E. coli and P. aeruginosa, respectively. Genes carrying these rG4 sites were found to be involved in virulence, gene regulation, cell envelope synthesis, and metabolism. More importantly, biophysical assays revealed the formation of a group of rG4 sites in mRNAs (such as hemL and bswR), and they were functionally validated in cells by genetic (point mutation and lux reporter assays) and phenotypic experiments, providing substantial evidence for the formation and function of rG4s in bacteria. Overall, our study uncovers important regulatory functions of rG4s in bacterial pathogenicity and metabolic pathways and strongly suggests that rG4s exist and can be detected in a wide range of bacterial species. IMPORTANCE G-quadruplex in RNA (rG4) mediates various biological functions and cellular processes in eukaryotic organisms. However, the presence, locations, and functions of rG4 are still elusive in prokaryotes. Here, we found that rG4 is an abundant RNA secondary structure across a wide range of bacterial species. Subsequently, the transcriptome-wide rG4 structure sequencing (rG4-seq) revealed that the model E. coli strain and a major human pathogen, P. aeruginosa, have 168 and 161 in vitro rG4 sites, respectively, involved in virulence, gene regulation, cell envelope, and metabolism. We further verified the regulatory functions of two rG4 sites in bacteria (hemL and bswR). Overall, this finding strongly suggests that rG4s play key regulatory roles in a wide range of bacterial species.

scholarly journals Characterization of Guided Entry of Tail-Anchored Proteins 3 Homologues in Mycobacterium tuberculosis

2019 ◽  
Vol 201 (14) ◽  
Author(s):  
Kuan Hu ◽  
Ashley T. Jordan ◽  
Susan Zhang ◽  
Avantika Dhabaria ◽  
Amanda Kovach ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Structure Prediction ◽  
Bacterial Species ◽  
Normal Growth ◽  
Anion Transport ◽  
Content Type ◽  
Reading Frame ◽  
Anion Transporters ◽  
Anion Transporter

ABSTRACT We characterized an operon in Mycobacterium tuberculosis, Rv3679-Rv3680, in which each open reading frame is annotated to encode “anion transporter ATPase” homologues. Using structure prediction modeling, we found that Rv3679 and Rv3680 more closely resemble the guided entry of tail-anchored proteins 3 (Get3) chaperone in eukaryotes. Get3 delivers proteins into the membranes of the endoplasmic reticulum and is essential for the normal growth and physiology of some eukaryotes. We sought to characterize the structures of Rv3679 and Rv3680 and test if they have a role in M. tuberculosis pathogenesis. We solved crystal structures of the nucleotide-bound Rv3679-Rv3680 complex at 2.5 to 3.2 Å and show that while it has some similarities to Get3 and ArsA, there are notable differences, including that these proteins are unlikely to be involved in anion transport. Deletion of both genes did not reveal any conspicuous growth defects in vitro or in mice. Collectively, we identified a new class of proteins in bacteria with similarity to Get3 complexes, the functions of which remain to be determined. IMPORTANCE Numerous bacterial species encode proteins predicted to have similarity with Get3- and ArsA-type anion transporters. Our studies provide evidence that these proteins, which we named BagA and BagB, are unlikely to be involved in anion transport. In addition, BagA and BagB are conserved in all mycobacterial species, including the causative agent of leprosy, which has a highly decayed genome. This conservation suggests that BagAB constitutes a part of the core mycobacterial genome and is needed for some yet-to-be-determined part of the life cycle of these organisms.

scholarly journals Regrowth of Mycobacterium tuberculosis Populations Exposed to Antibiotic Combinations Is Due to the Presence of Isoniazid and Not Bacterial Growth Rate

2019 ◽  
Vol 63 (12) ◽  
Author(s):  
Charlotte L. Hendon-Dunn ◽  
Henry Pertinez ◽  
Alice A. N. Marriott ◽  
Kim A. Hatch ◽  
Jon C. Allnutt ◽  
...  
Keyword(s):  
Growth Rate ◽  
Mycobacterium Tuberculosis ◽  
Time Course ◽  
Bacterial Population ◽  
Slow Growth ◽  
Bacterial Growth Rate ◽  
Content Type ◽  
Slow Growth Rate ◽  
Antibiotic Efficacy ◽  
Fast Growth Rate

ABSTRACT Modulation of the growth rate in Mycobacterium tuberculosis is key to its survival in the host, particularly with regard to its adaptation during chronic infection, when the growth rate is very slow. The resulting physiological changes influence the way in which this pathogen interacts with the host and responds to antibiotics. Therefore, it is important that we understand how the growth rate impacts antibiotic efficacy, particularly with respect to recovery/relapse. This is the first study that has asked how growth rates influence the mycobacterial responses to combinations of the frontline antimycobacterials, isoniazid (INH), rifampin (RIF), and pyrazinamide (PZA), using continuous cultures. The time course profiles of log-transformed total viable counts for cultures, controlled at either a fast growth rate (mean generation time [MGT], 23.1 h) or a slow growth rate (MGT, 69.3 h), were analyzed by the fitting of a mathematical model by nonlinear regression that accounted for the dilution rate in the chemostat and profiled the kill rates and recovery in culture. Using this approach, we show that populations growing more slowly were generally less susceptible to all treatments. We observed a faster kill rate associated with INH than with RIF or PZA and the appearance of regrowth. In line with this observation, regrowth was not observed with RIF exposure, which provided a slower bactericidal response. The sequential additions of RIF and PZA did not eliminate regrowth. We consider here that faster, early bactericidal activity is not what is required for the successful sterilization of M. tuberculosis, but instead, slower elimination of the bacilli followed by reduced recovery of the bacterial population is required.

scholarly journals Environment Shapes the Accessible Daptomycin Resistance Mechanisms in Enterococcus faecium

2019 ◽  
Vol 63 (10) ◽  
Author(s):  
Amy G. Prater ◽  
Heer H. Mehta ◽  
Abigael J. Kosgei ◽  
William R. Miller ◽  
Truc T. Tran ◽  
...  
Keyword(s):  
Enterococcus Faecium ◽  
Cell Envelope ◽  
Multidrug Resistant ◽  
Resistance Mechanisms ◽  
Clinical Strain ◽  
Content Type ◽  
Component Cell ◽  
Evolutionary Trajectories ◽  
High Level ◽  
Cell Envelopes

ABSTRACT Daptomycin binds to bacterial cell membranes and disrupts essential cell envelope processes, leading to cell death. Bacteria respond to daptomycin by altering their cell envelopes to either decrease antibiotic binding to the membrane or by diverting binding away from septal targets. In Enterococcus faecalis, daptomycin resistance is typically coordinated by the three-component cell envelope stress response system, LiaFSR. Here, studying a clinical strain of multidrug-resistant Enterococcus faecium containing alleles associated with activation of the LiaFSR signaling pathway, we found that specific environments selected for different evolutionary trajectories, leading to high-level daptomycin resistance. Planktonic environments favored pathways that increased cell surface charge via yvcRS upregulation of dltABCD and mprF, causing a reduction in daptomycin binding. Alternatively, environments favoring complex structured communities, including biofilms, evolved both diversion and repulsion strategies via divIVA and oatA mutations, respectively. Both environments subsequently converged on cardiolipin synthase (cls) mutations, suggesting the importance of membrane modification across strategies. Our findings indicate that E. faecium can evolve diverse evolutionary trajectories to daptomycin resistance that are shaped by the environment to produce a combination of resistance strategies. The accessibility of multiple and different biochemical pathways simultaneously suggests that the outcome of daptomycin exposure results in a polymorphic population of resistant phenotypes, making E. faecium a recalcitrant nosocomial pathogen.

scholarly journals Prediction of Acquired Antimicrobial Resistance for Multiple Bacterial Species Using Neural Networks

mSystems ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
D. Aytan-Aktug ◽  
P. T. L. C. Clausen ◽  
V. Bortolaia ◽  
F. M. Aarestrup ◽  
O. Lund
Keyword(s):  
Machine Learning ◽  
Escherichia Coli ◽  
Mycobacterium Tuberculosis ◽  
Antimicrobial Resistance ◽  
Bacterial Species ◽  
Individual Species ◽  
Learning Models ◽  
Content Type ◽  
Multiple Species ◽  
Machine Learning Models

ABSTRACT Machine learning has proven to be a powerful method to predict antimicrobial resistance (AMR) without using prior knowledge for selected bacterial species-antimicrobial combinations. To date, only species-specific machine learning models have been developed, and to the best of our knowledge, the inclusion of information from multiple species has not been attempted. The aim of this study was to determine the feasibility of including information from multiple bacterial species to predict AMR for an individual species, since this may make it easier to train and update resistance predictions for multiple species and may lead to improved predictions. Whole-genome sequence data and susceptibility profiles from 3,528 Mycobacterium tuberculosis, 1,694 Escherichia coli, 658 Salmonella enterica, and 1,236 Staphylococcus aureus isolates were included. We developed machine learning models trained by the features of the PointFinder and ResFinder programs detected to predict binary (susceptible/resistant) AMR profiles. We tested four feature representation methods to determine the most efficient way for introducing features into the models. When training the model only on the Mycobacterium tuberculosis isolates, high prediction performances were obtained for the six AMR profiles included. By adding information on ciprofloxacin from the additional 3,588 isolates, there was no reduction in performance for the other antimicrobials but an increased performance for ciprofloxacin AMR profile prediction for Mycobacterium tuberculosis and Escherichia coli. In conclusion, the species-independent models can predict multi-AMR profiles for multiple species without losing any robustness. IMPORTANCE Machine learning is a proven method to predict AMR; however, the performance of any machine learning model depends on the quality of the input data. Therefore, we evaluated different methods of representing information about mutations as well as mobilizable genes, so that the information can serve as input for a robust model. We combined data from multiple bacterial species in order to develop species-independent machine learning models that can predict resistance profiles for multiple antimicrobials and species with high performance.

scholarly journals Evolution of Mycolic Acid Biosynthesis Genes and Their Regulation during Starvation in Mycobacterium tuberculosis

2015 ◽  
Vol 197 (24) ◽  
pp. 3797-3811 ◽  
Author(s):  
Stevie Jamet ◽  
Yves Quentin ◽  
Coralie Coudray ◽  
Pauline Texier ◽  
Françoise Laval ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Outer Membrane ◽  
Cell Envelope ◽  
Stringent Response ◽  
Mycolic Acid ◽  
Content Type ◽  
New Targets ◽  
Key Enzyme ◽  
Unique Cell ◽  
Translational Apparatus

ABSTRACTMycobacterium tuberculosis, the etiological agent of tuberculosis, is a Gram-positive bacterium with a unique cell envelope composed of an essential outer membrane. Mycolic acids, which are very-long-chain (up to C100) fatty acids, are the major components of this mycomembrane. The enzymatic pathways involved in the biosynthesis and transport of mycolates are fairly well documented and are the targets of the major antituberculous drugs. In contrast, only fragmented information is available on the expression and regulation of the biosynthesis genes. In this study, we report that thehadA,hadB, andhadCgenes, which code for the mycolate biosynthesis dehydratase enzymes, are coexpressed with three genes that encode proteins of the translational apparatus. Consistent with the well-established control of the translation potential by nutrient availability, starvation leads to downregulation of thehadABCgenes along with most of the genes required for the synthesis, modification, and transport of mycolates. The downregulation of a subset of the biosynthesis genes is partially dependent on RelMtb, the key enzyme of the stringent response. We also report the phylogenetic evolution scenario that has shaped the current genetic organization, characterized by the coregulation of thehadABCoperon with genes of the translational apparatus and with genes required for the modification of the mycolates.IMPORTANCEMycobacterium tuberculosisinfects one-third of the human population worldwide, and despite the available therapeutic arsenal, it continues to kill millions of people each year. There is therefore an urgent need to identify new targets and develop a better understanding of how the bacterium is adapting itself to host defenses during infection. A prerequisite of this understanding is knowledge of how this adaptive skill has been implanted by evolution. Nutrient scarcity is an environmental condition the bacterium has to cope with during infection. In many bacteria, adaptation to starvation relies partly on the stringent response.M. tuberculosis's unique outer membrane layer, the mycomembrane, is crucial for its viability and virulence. Despite its being the target of the major antituberculosis drugs, only scattered information exists on how the genes required for biosynthesis of the mycomembrane are expressed and regulated during starvation. This work has addressed this issue as a step toward the identification of new targets in the fight againstM. tuberculosis.

scholarly journals Therv1184cLocus Encodes Chp2, an Acyltransferase in Mycobacterium tuberculosis Polyacyltrehalose Lipid Biosynthesis

2014 ◽  
Vol 197 (1) ◽  
pp. 201-210 ◽  
Author(s):  
Megan H. Touchette ◽  
Cynthia M. Holsclaw ◽  
Mary L. Previti ◽  
Viven C. Solomon ◽  
Julie A. Leary ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Cell Envelope ◽  
Selective Inhibition ◽  
Serine Hydrolase ◽  
Acyltransferase Activity ◽  
Content Type ◽  
Regulatory Interactions ◽  
Similar Phenotype ◽  
Lipid Transporter

Trehalose glycolipids are found in many bacteria in the suborderCorynebacterineae, but methyl-branched acyltrehaloses are exclusive to virulent species such as the human pathogenMycobacterium tuberculosis. InM. tuberculosis, the acyltransferase PapA3 catalyzes the formation of diacyltrehalose (DAT), but the enzymes responsible for downstream reactions leading to the final product, polyacyltrehalose (PAT), have not been identified. The PAT biosynthetic gene locus is similar to that of another trehalose glycolipid, sulfolipid 1. Recently, Chp1 was characterized as the terminal acyltransferase in sulfolipid 1 biosynthesis. Here we provide evidence that the homologue Chp2 (Rv1184c) is essential for the final steps of PAT biosynthesis. Disruption ofchp2led to the loss of PAT and a novel tetraacyltrehalose species, TetraAT, as well as the accumulation of DAT, implicating Chp2 as an acyltransferase downstream of PapA3. Disruption of the putative lipid transporter MmpL10 resulted in a similar phenotype. Chp2 activity thus appears to be regulated by MmpL10 in a relationship similar to that between Chp1 and MmpL8 in sulfolipid 1 biosynthesis. Chp2 is localized to the cell envelope fraction, consistent with its role in DAT modification and possible regulatory interactions with MmpL10. Labeling of purified Chp2 by an activity-based probe was dependent on the presence of the predicted catalytic residue Ser141 and was inhibited by the lipase inhibitor tetrahydrolipstatin (THL). THL treatment ofM. tuberculosisresulted in selective inhibition of Chp2 over PapA3, confirming Chp2 as a member of the serine hydrolase superfamily. Efforts to producein vitroreconstitution of acyltransferase activity using straight-chain analogues were unsuccessful, suggesting that Chp2 has specificity for native methyl-branched substrates.

scholarly journals Wild-Type and Non-Wild-Type Mycobacterium tuberculosis MIC Distributions for the Novel Fluoroquinolone Antofloxacin Compared with Those for Ofloxacin, Levofloxacin, and Moxifloxacin

2016 ◽  
Vol 60 (9) ◽  
pp. 5232-5237 ◽  
Author(s):  
Xia Yu ◽  
Guirong Wang ◽  
Suting Chen ◽  
Guomei Wei ◽  
Yuanyuan Shang ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Critical Concentration ◽  
Bacterial Species ◽  
Drug Susceptibility Testing ◽  
World Health ◽  
Wild Type ◽  
Resistance Mutations ◽  
Content Type ◽  
Lowenstein Jensen

ABSTRACTAntofloxacin (AFX) is a novel fluoroquinolone that has been approved in China for the treatment of infections caused by a variety of bacterial species. We investigated whether it could be repurposed for the treatment of tuberculosis by studying itsin vitroactivity. We determined the wild-type and non-wild-type MIC ranges for AFX as well as ofloxacin (OFX), levofloxacin (LFX), and moxifloxacin (MFX), using the microplate alamarBlue assay, of 126 clinicalMycobacterium tuberculosisstrains from Beijing, China, of which 48 were OFX resistant on the basis of drug susceptibility testing on Löwenstein-Jensen medium. The MIC distributions were correlated with mutations in the quinolone resistance-determining regions ofgyrA(Rv0006) andgyrB(Rv0005). Pharmacokinetic/pharmacodynamic (PK/PD) data for AFX were retrieved from the literature. AFX showed lower MIC levels than OFX but higher MIC levels than LFX and MFX on the basis of the tentative epidemiological cutoff values (ECOFFs) determined in this study. All strains with non-wild-type MICs for AFX harbored known resistance mutations that also resulted in non-wild-type MICs for LFX and MFX. Moreover, our data suggested that the current critical concentration of OFX for Löwenstein-Jensen medium that was recently revised by the World Health Organization might be too high, resulting in the misclassification of phenotypically non-wild-type strains with known resistance mutations as wild type. On the basis of our exploratory PK/PD calculations, the current dose of AFX is unlikely to be optimal for the treatment of tuberculosis, but higher doses could be effective.

scholarly journals Usnic Acid Treatment Changes the Composition of Mycobacterium tuberculosis Cell Envelope and Alters Bacterial Redox Status

mSystems ◽  
2021 ◽  
Vol 6 (3) ◽  
Author(s):  
Elwira Sieniawska ◽  
Rafal Sawicki ◽  
Wieslaw Truszkiewicz ◽  
Andrey S. Marchev ◽  
Milen I. Georgiev
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
Acid Treatment ◽  
Cell Envelope ◽  
Redox Homeostasis ◽  
Usnic Acid ◽  
Lipid Synthesis ◽  
Redox Balance ◽  
Resistance Mechanisms ◽  
Content Type

ABSTRACT Mycobacterium tuberculosis developed efficient adaptation mechanisms in response to different environmental conditions. This resulted in the ability to survive in human macrophages and in resistance to numerous antibiotics. To get insight into bacterial responses to potent antimycobacterial natural compounds, we tested how usnic acid, a lichen-derived secondary metabolite, would influence mycobacteria at transcriptomic and metabolomic levels. The analysis of expression of sigma factors revealed a profound impact of usnic acid on one of the primary genetic regulatory systems of M. tuberculosis. Combined liquid chromatography-mass spectrometry and nuclear magnetic resonance analyses allowed us to observe the perturbations in metabolic pathways, as well as in lipid composition, which took place within 24 h of exposure. Early bacterial response was related to redox homeostasis, lipid synthesis, and nucleic acid repair. Usnic acid treatment provoked disturbances of redox state in mycobacterial cells and increased production of structural elements of the cell wall and cell membrane. In addition, to increase the number of molecules related to restoration of redox balance, the rearrangements of the cell envelope were the first defense mechanisms observed under usnic acid treatment. IMPORTANCE The evaluation of mechanisms of mycobacterial response to natural products has been barely studied. However, it might be helpful to reveal bacterial adaptation strategies, which are eventually crucial for the discovery of new drug targets and, hence, understanding the resistance mechanisms. This study showed that the first-line mycobacterial defense against usnic acid, a potent antimicrobial agent, is the remodeling of the cell envelope and restoring redox homeostasis. Transcriptomic data correlated with metabolomics analysis. The observed metabolic changes appeared similar to those exerted by antibiotics.

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