Genetic analysis of the β-lactamases of Mycobacterium tuberculosis and Mycobacterium smegmatis and susceptibility to β-lactam antibiotics

Microbiology ◽  
2005 ◽  
Vol 151 (2) ◽  
pp. 521-532 ◽  
Author(s):  
Anthony R. Flores ◽  
Linda M. Parsons ◽  
Martin S. Pavelka,
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Inhibitory Concentration ◽  
Cell Envelope ◽  
Model Organism ◽  
Resistance Mechanisms ◽  
Disc Diffusion ◽  
Peptidoglycan Biosynthesis ◽  
Strain H37rv

Mycobacteria produce β-lactamases and are intrinsically resistant to β-lactam antibiotics. In addition to the β-lactamases, cell envelope permeability and variations in certain peptidoglycan biosynthetic enzymes are believed to contribute to β-lactam resistance in these organisms. To allow the study of these additional mechanisms, mutants of the major β-lactamases, BlaC and BlaS, were generated in the pathogenic Mycobacterium tuberculosis strain H37Rv and the model organism Mycobacterium smegmatis strain PM274. The mutants M. tuberculosis PM638 (ΔblaC1) and M. smegmatis PM759 (ΔblaS1) showed an increase in susceptibility to β-lactam antibiotics, as determined by disc diffusion and minimal inhibitory concentration (MIC) assays. The susceptibility of the mutants, as assayed by disc diffusion tests, to penicillin-type β-lactam antibiotics was affected most, compared to the cephalosporin-type β-lactam antibiotics. The M. tuberculosis mutant had no detectable β-lactamase activity, while the M. smegmatis mutant had a residual type 1 β-lactamase activity. We identified a gene, blaE, encoding a putative cephalosporinase in M. smegmatis. A double β-lactamase mutant of M. smegmatis, PM976 (ΔblaS1ΔblaE : : res), had no detectable β-lactamase activity, but its susceptibility to β-lactam antibiotics was not significantly different from that of the ΔblaS1 parental strain, PM759. The mutants generated in this study will help determine the contribution of other β-lactam resistance mechanisms in addition to serving as tools to study the biology of peptidoglycan biosynthesis in these organisms.

scholarly journals Characterization of Novel Mycobacterium tuberculosis and Mycobacterium smegmatis Mutants Hypersusceptible to β-Lactam Antibiotics

2005 ◽  
Vol 187 (6) ◽  
pp. 1892-1900 ◽  
Author(s):  
Anthony R. Flores ◽  
Linda M. Parsons ◽  
Martin S. Pavelka
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Cell Envelope ◽  
Novel Genes ◽  
Antibiotic Resistant ◽  
Peptidoglycan Biosynthesis ◽  
Isolation And Characterization ◽  
Lactam Antibiotic ◽  
Transposon Mutants

ABSTRACT Our laboratory previously constructed mutants of Mycobacterium tuberculosis and Mycobacterium smegmatis with deletions in the genes for their major β-lactamases, BlaC and BlaS, respectively, and showed that the mutants have increased susceptibilities to most β-lactam antibiotics, particularly the penicillins. However, there is still a basal level of resistance in the mutants to certain penicillins, and the susceptibilities of the mutants to some cephalosporin-based β-lactams are essentially the same as those of the wild types. We hypothesized that characterizing additional mutants (derived from β-lactamase deletion mutants) that are hypersusceptible to β-lactam antibiotics might reveal novel genes involved with other mechanisms of β-lactam resistance, peptidoglycan assembly, and cell envelope physiology. We report here the isolation and characterization of nine β-lactam antibiotic-hypersusceptible transposon mutants, two of which have insertions in genes known to be involved with peptidoglycan biosynthesis (ponA2 and dapB); the other seven mutants have insertions which affect novel genes. These genes can be classified into three groups: those involved with peptidoglycan biosynthesis, cell division, and other cell envelope processes. Two of the peptidoglycan-biosynthetic genes (ponA2 and pbpX) may encode β-lactam antibiotic-resistant enzymes proposed to be involved with the synthesis of the unusual diaminopimelyl linkages within the mycobacterial peptidoglycan.

scholarly journals Hypoxanthine-Guanine Phosphoribosyltransferase Is Dispensable for Mycobacterium smegmatis Viability

2019 ◽  
Vol 202 (5) ◽  
Author(s):  
Zdeněk Knejzlík ◽  
Klára Herkommerová ◽  
Dana Hocková ◽  
Iva Pichová
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Model Organism ◽  
Specific Inhibitor ◽  
Purine Salvage ◽  
Content Type ◽  
Acyclic Nucleoside Phosphonates ◽  
Hypoxanthine Guanine Phosphoribosyltransferase

ABSTRACT Purine metabolism plays a ubiquitous role in the physiology of Mycobacterium tuberculosis and other mycobacteria. The purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is essential for M. tuberculosis growth in vitro; however, its precise role in M. tuberculosis physiology is unclear. Membrane-permeable prodrugs of specifically designed HGPRT inhibitors arrest the growth of M. tuberculosis and represent potential new antituberculosis compounds. Here, we investigated the purine salvage pathway in the model organism Mycobacterium smegmatis. Using genomic deletion analysis, we confirmed that HGPRT is the only guanine and hypoxanthine salvage enzyme in M. smegmatis but is not required for in vitro growth of this mycobacterium or survival under long-term stationary-phase conditions. We also found that prodrugs of M. tuberculosis HGPRT inhibitors displayed an unexpected antimicrobial activity against M. smegmatis that is independent of HGPRT. Our data point to a different mode of mechanism of action for these inhibitors than was originally proposed. IMPORTANCE Purine bases, released by the hydrolytic and phosphorolytic degradation of nucleic acids and nucleotides, can be salvaged and recycled. The hypoxanthine-guanine phosphoribosyltransferase (HGPRT), which catalyzes the formation of guanosine-5′-monophosphate from guanine and inosine-5′-monophosphate from hypoxanthine, represents a potential target for specific inhibitor development. Deletion of the HGPRT gene (Δhgprt) in the model organism Mycobacterium smegmatis confirmed that this enzyme is not essential for M. smegmatis growth. Prodrugs of acyclic nucleoside phosphonates (ANPs), originally designed against HGPRT from Mycobacterium tuberculosis, displayed anti-M. smegmatis activities comparable to those obtained for M. tuberculosis but also inhibited the Δhgprt M. smegmatis strain. These results confirmed that ANPs act in M. smegmatis by a mechanism independent of HGPRT.

scholarly journals Brassicasterol with Dual Anti-Infective Properties against HSV-1 and Mycobacterium tuberculosis, and Cardiovascular Protective Effect: Nonclinical In Vitro and In Silico Assessments

Biomedicines ◽  
2020 ◽  
Vol 8 (5) ◽  
pp. 132
Author(s):  
Sherif T. S. Hassan
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Inhibitory Concentration ◽  
Molecular Targets ◽  
Biological Properties ◽  
Selectivity Index ◽  
Cell Wall Biosynthesis ◽  
Simplex Virus ◽  
Hsv 1

While few studies have revealed the biological properties of brassicasterol, a phytosterol, against some biological and molecular targets, it is believed that there are still many activities yet to be studied. In this work, brassicasterol exerts a therapeutic utility in an in vitro setting against herpes simplex virus type 1 (HSV-1) and Mycobacterium tuberculosis (Mtb) as well as a considerable inhibitory property against human angiotensin-converting enzyme (ACE) that plays a dynamic role in regulating blood pressure. The antireplicative effect of brassicasterol against HSV-1 is remarkably detected (50% inhibitory concentration (IC50): 1.2 µM; selectivity index (SI): 41.7), while the potency of its effect is ameliorated through the combination with standard acyclovir with proper SI (IC50: 0.7 µM; SI: 71.4). Moreover, the capacity of this compound to induce an adequate level of antituberculosis activity against all Mtb strains examined (minimum inhibitory concentration values ranging from 1.9 to 2.4 µM) is revealed. The anti-ACE effect (12.3 µg/mL; 91.2% inhibition) is also ascertained. Molecular docking analyses propose that the mechanisms by which brassicasterol induces anti-HSV-1 and anti-Mtb might be related to inhibiting vital enzymes involved in HSV-1 replication and Mtb cell wall biosynthesis. In summary, the obtained results suggest that brassicasterol might be promising for future anti-HSV-1, antituberculosis, and anti-ACE drug design.

scholarly journals In VitroCombination Studies of Benzothiazinone Lead Compound BTZ043 against Mycobacterium tuberculosis

2012 ◽  
Vol 56 (11) ◽  
pp. 5790-5793 ◽  
Author(s):  
Benoit Lechartier ◽  
Ruben C. Hartkoorn ◽  
Stewart T. Cole
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Inhibitory Concentration ◽  
Bactericidal Effect ◽  
Lead Compound ◽  
Content Type ◽  
Drug Candidates ◽  
New Class ◽  
Novel Targets ◽  
Essential Enzyme ◽  
Strain H37rv

ABSTRACTBenzothiazinones (BTZ) are a new class of drug candidates to combat tuberculosis that inhibit decaprenyl-phosphoribose epimerase (DprE1), an essential enzyme involved in arabinan biosynthesis. Using the checkerboard method and cell viability assays, we have studied the interaction profiles of BTZ043, the current lead compound, with several antituberculosis drugs or drug candidates againstMycobacterium tuberculosisstrain H37Rv, namely, rifampin, isoniazid, ethambutol, TMC207, PA-824, moxifloxacin, meropenem with or without clavulanate, and SQ-109. No antagonism was found between BTZ043 and the tested compounds, and most of the interactions were purely additive. Data from two different approaches clearly indicate that BTZ043 acts synergistically with TMC207, with a fractional inhibitory concentration index of 0.5. TMC207 at a quarter of the MIC (20 ng/ml) used in combination with BTZ043 (1/4 MIC, 0.375 ng/ml) had a stronger bactericidal effect onM. tuberculosisthan TMC207 alone at a concentration of 80 ng/ml. This synergy was not observed when the combination was tested on a BTZ-resistantM. tuberculosismutant, suggesting that DprE1 inhibition is the basis for the interaction. This finding excludes the possibility of synergy occurring through an off-target mechanism. We therefore hypothesize that sub-MICs of BTZ043 weaken the bacterial cell wall and allow improved penetration of TMC207 to its target. Synergy between two new antimycobacterial compounds, such as TMC207 and BTZ043, with novel targets, offers an attractive foundation for a new tuberculosis regimen.

Complexes of the uracil-DNA glycosylase inhibitor protein, Ugi, with Mycobacterium smegmatis and Mycobacterium tuberculosis uracil-DNA glycosylases

Microbiology ◽  
2003 ◽  
Vol 149 (7) ◽  
pp. 1647-1658 ◽  
Author(s):  
Narottam Acharya ◽  
Pradeep Kumar ◽  
Umesh Varshney
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Three Dimensional ◽  
Model Organism ◽  
Dna Glycosylase ◽  
Structural Elements ◽  
Dna Glycosylases ◽  
Hydrophobic Pocket ◽  
Uracil Dna Glycosylase ◽  
Simplex Virus

Uracil, a promutagenic base, appears in DNA either by deamination of cytosine or by incorporation of dUMP by DNA polymerases. This unconventional base in DNA is removed by uracil-DNA glycosylase (UDG). Interestingly, a bacteriophage-encoded short polypeptide, UDG inhibitor (Ugi), specifically inhibits UDGs by forming a tight complex. Three-dimensional structures of the complexes of Ugi with UDGs from Escherichia coli, human and herpes simplex virus have shown that two of the structural elements in Ugi, the hydrophobic pocket and the β1-edge, establish key interactions with UDGs. In this report the characterization of complexes of Ugi with UDGs from Mycobacterium tuberculosis, a pathogenic bacterium, and Mycobacterium smegmatis, a widely used model organism for the former, is described. Unlike the E. coli (Eco) UDG-Ugi complex, which is stable to treatment with 8 M urea, the mycobacterial UDG-Ugi complexes dissociate in 5–6 M urea. Furthermore, the Ugi from the complexes of mycobacterial UDGs can be exchanged by the DNA substrate. Interestingly, while EcoUDG sequestered Ugi into the EcoUDG-Ugi complex when incubated with mycobacterial UDG-Ugi complexes, even a large excess of mycobacterial UDGs failed to sequester Ugi from the EcoUDG-Ugi complex. However, the M. tuberculosis (Mtu) UDG-Ugi complex was seen when MtuUDG was incubated with M. smegmatis (Msm) UDG-Ugi or EcoUDG(L191G)-Ugi complexes. The reversible nature of the complexes of Ugi with mycobacterial UDGs (which naturally lack some of the structural elements important for interaction with the β1-edge of Ugi) and with mutants of EcoUDG (which are deficient in interaction with the hydrophobic pocket of Ugi) highlights the significance of both classes of interaction in formation of UDG-Ugi complexes. Furthermore, it is shown that even though mycobacterial UDG-Ugi complexes dissociate in 5–6 M urea, Ugi is still a potent inhibitor of UDG activity in mycobacteria.

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.

scholarly journals Inhibiting Mycobacterium tuberculosis CoaBC by targeting an allosteric site

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Vitor Mendes ◽  
Simon R. Green ◽  
Joanna C. Evans ◽  
Jeannine Hess ◽  
Michal Blaszczyk ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Drug Target ◽  
Biosynthetic Pathway ◽  
Model Organism ◽  
Allosteric Site ◽  
Cellular Processes ◽  
Coa Thioesters ◽  
Multiple Pathogens ◽  
Substantial Interest

AbstractCoenzyme A (CoA) is a fundamental co-factor for all life, involved in numerous metabolic pathways and cellular processes, and its biosynthetic pathway has raised substantial interest as a drug target against multiple pathogens including Mycobacterium tuberculosis. The biosynthesis of CoA is performed in five steps, with the second and third steps being catalysed in the vast majority of prokaryotes, including M. tuberculosis, by a single bifunctional protein, CoaBC. Depletion of CoaBC was found to be bactericidal in M. tuberculosis. Here we report the first structure of a full-length CoaBC, from the model organism Mycobacterium smegmatis, describe how it is organised as a dodecamer and regulated by CoA thioesters. A high-throughput biochemical screen focusing on CoaB identified two inhibitors with different chemical scaffolds. Hit expansion led to the discovery of potent and selective inhibitors of M. tuberculosis CoaB, which we show to bind to a cryptic allosteric site within CoaB.

scholarly journals ANTI-MYCOBACTERIUM TUBERCULOSIS STRAIN H37RV AND IRON CHELATION ACTIVITY OF SAPPAN WOOD EXTRACT (CAESALPINIA SAPPAN L.) IN VITRO

2018 ◽  
Vol 11 (6) ◽  
pp. 444
Author(s):  
Ratu Safitri ◽  
Ida Indrawati ◽  
Mas Rizky A A Syamsunarno ◽  
Mohammad Ghozali ◽  
Basri A Gani ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Inhibitory Concentration ◽  
Minimum Bactericidal Concentration ◽  
Iron Chelation ◽  
Iron Chelate ◽  
Caesalpinia Sappan ◽  
Lowenstein Jensen ◽  
Wood Extract ◽  
Strain H37rv

Objective: The objective of this study is to determine anti-Mycobacterium tuberculosis (MTB) strain H37Rv and iron chelation activities of sappan wood extract (SWE).Methods: The evaluation of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) by proportion methods. Whereas the iron chelate in Lowenstein–Jensen (LJ) medium as the indicator of Mycobacterium growth and SWE effect.Results: The SWE has bacteriocidal to MTB of 10−3 and 10−5 dilutions in of all concentrations (250, 500, 750, 1000, 2000, 4000, 8000, and 16000 part per millions [ppm]) also bacteriostatic in concentration 50 and 100 ppm.Conclusion: The SWE at 100 ppm could inhibit 87% of the MTB in 10−3 and 10−5 dilutions, respectively, also to reduce to growth the colony of MTB, and has chelating effects of iron expression of LJ medium and MTB.

scholarly journals Uji Aktivitas Ekstrak Daun Mengkudu (Morindra citrifolia Linn) dan Scopoletin secara In-Vitro terhadap Bakteri Tuberkulosis

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Estu Mahanani Dhilasari ◽  
Idha Kusumawati Kusumawati ◽  
Riesta Primaharinastiti Primaharinastiti
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Inhibitory Concentration ◽  
Ethanolic Extract ◽  
Morinda Citrifolia ◽  
Mycobacterium Tuberculosis H37rv ◽  
Ethanolic Extracts ◽  
Tuberculosis Activity ◽  
Strain H37rv ◽  
Mycobacterium Tuberculosis Strain

Penyakit tuberkulosis disebabkan oleh bakteri Mycobacterium tuberculosis. Secara tradisional mengkudu digunakan untuk pengobatan tuberkulosis. Scopoletin merupakan komponen utama dalam mengkudu, oleh karena itu scopoletin sering dijadikan marker dalam studi farmakokinetik. Tujuan penelitian ini adalah untuk mengetahui aktivitas anti-Mycobacterium tuberculosis (H37RV) ekstrak daun mengkudu dan scopoletin melalui penentuan Konsentrasi Hambat Minimum (KHM). Uji aktivitas antibakteri dan penentuan KHM dari ekstrak etanol 50% daun mengkudu dilakukan dengan metode dilusi agar dengan konsentrasi 1,0×10-4 µg/ml – 5,1×10-11 µg/ml. Uji aktivitas menunjukan bahwa ekstrak etanol 50% daun mengkudu dapat menghambat pertumbuhan Mycobacterium tuberculosis dengan KHM 4,0×10-6 µg/ml. Sedangkan scopoletin dengan konsentrasi yang setara dengan kandungan pada ekstrak tidak menunjukan aktivitas anti-Mycobacterium tuberculosisnya.Kata kunci: Mengkudu, Scopoletin, tuberkulosisTuberculosis (TB) is a disease caused by a bacterium, Mycobacterium tuberculosis. Morinda citrifolia Linn has been found to kill Mycobacterium tuberculosis. Scopoletin is a major component in Morinda citrifolia Linn, therefore scopoletin often used as markers in studies of pharmacokinetic. This research purpose to determine anti-Mycobacterium tuberculosis (strain H37RV) activity based on the value of Minimum Inhibitory Concentration (MIC) used to ethanolic extracts from Morinda citrifolia Linn leaf and scopoletin. Experiment of anti-Mycobacterium tuberculosis activities tested by well dillution methods with a dose 1,0×10-4 µg/ml – 5,1×10-11 µg/ml . The results showed that ethanolic extract Morinda citrifolia Linn leaf  were found to be active to Mycobacterium tuberculosis activity with MIC 4×10-6 µg/ml while scopoletin at the same concentration with extract had no anti­- Mycobacterium tuberculosis activity.Keywords:  Noni, scopoletin, tuberculosis

scholarly journals Mycobacterium smegmatis whmD and its homologue Mycobacterium tuberculosis whiB2 are functionally equivalent

Microbiology ◽  
2006 ◽  
Vol 152 (9) ◽  
pp. 2735-2747 ◽  
Author(s):  
Tirumalai R. Raghunand ◽  
William R. Bishai
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Cell Division ◽  
Dna Sequences ◽  
Mycobacterium Smegmatis ◽  
Essential Gene ◽  
Cell Envelope ◽  
Protein Product ◽  
Polyclonal Antiserum ◽  
C Terminus ◽  
Gene Assay

Mycobacterium smegmatis whmD is is an essential gene involved in cell division. This paper shows that whmD and its homologue whiB2 in Mycobacterium tuberculosis are functionally equivalent. The genes are syntenous, and share significant homology in both their coding and non-coding DNA sequences. Transcription site mapping showed that the two genes possess near-identical promoter elements, and they displayed comparable promoter strengths in a reporter gene assay. The two proteins show near identity in their C-terminus, and polyclonal antiserum to WhmD specifically cross-reacts with a ∼15 kDa band in M. tuberculosis lysates. Following overexpression of sense and anti-sense constructs in their cognate mycobacterial hosts, whiB2 and whmD transformants displayed a small-colony phenotype, exhibited filamentation, and showed a reduction in viability. These observations reveal that the two proteins are functionally homologous and that their intracellular concentration is critical for septation in mycobacteria. Colonies of M. tuberculosis overexpressing whiB2 were spherical and glossy, suggesting a change in composition of the cell envelope. Filaments of the conditionally complemented M. smegmatis whmD mutant were non-acid-fast, also indicating changes in characteristics of surface lipids. M. smegmatis transformants carrying a whmD–gfp fusion showed a diffuse pattern of fluorescence, consistent with the putative role of WhmD as a regulator. These observations strongly suggest that M. tuberculosis whiB2 is an essential gene and its protein product in all likelihood regulates the expression of genes involved in the cell division cascade.

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