scholarly journals Combined Bioinformatic and Rational Design Approach To Develop Antimicrobial Peptides against Mycobacterium tuberculosis

2016 ◽  
Vol 60 (5) ◽  
pp. 2757-2764 ◽  
Author(s):  
C. Seth Pearson ◽  
Zachary Kloos ◽  
Brian Murray ◽  
Ebot Tabe ◽  
Monica Gupta ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Antimicrobial Peptides ◽  
Rational Design ◽  
Pathogenic Bacteria ◽  
First Generation ◽  
De Novo ◽  
Specific Activity ◽  
Lung Epithelial Cells ◽  
Content Type ◽  
Low Cytotoxicity

ABSTRACTDrug-resistant pathogens are a growing problem, and novel strategies are needed to combat this threat. Among the most significant of these resistant pathogens isMycobacterium tuberculosis, which is an unusually difficult microbial target due to its complex membrane. Here, we design peptides for specific activity againstM. tuberculosisusing a combination of “database filtering” bioinformatics, protein engineering, andde novodesign. Several variants of these peptides are structurally characterized to validate the design process. The designed peptides exhibit potent activity (MIC values as low as 4 μM) againstM. tuberculosisand also exhibit broad activity against a host of other clinically relevant pathogenic bacteria such as Gram-positive bacteria (Streptococcus) and Gram-negative bacteria (Escherichia coli). They also display excellent selectivity, with low cytotoxicity against cultured macrophages and lung epithelial cells. These first-generation antimicrobial peptides serve as a platform for the design of antibiotics and for investigating structure-activity relationships in the context of theM. tuberculosismembrane. The antimicrobial peptide design strategy is expected to be generalizable for any pathogen for which an activity database can be created.

scholarly journals Targeting Mycobacterium tuberculosis and Other Microbial Pathogens Using Improved Synthetic Antibacterial Peptides

2013 ◽  
Vol 57 (5) ◽  
pp. 2295-2303 ◽  
Author(s):  
Santiago Ramón-García ◽  
Ralf Mikut ◽  
Carol Ng ◽  
Serge Ruden ◽  
Rudolf Volkmer ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mycobacterium Tuberculosis ◽  
Antimicrobial Peptides ◽  
Fungal Pathogens ◽  
Antimicrobial Activities ◽  
Microbial Pathogens ◽  
Cationic Antimicrobial Peptides ◽  
Content Type ◽  
Activity Spectrum ◽  
Low Cytotoxicity

ABSTRACTThe lack of effective therapies for treating tuberculosis (TB) is a global health problem. WhileMycobacterium tuberculosisis notoriously resistant to most available antibiotics, we identified synthetic short cationic antimicrobial peptides that were active at low micromolar concentrations (less than 10 μM). These small peptides (averaging 10 amino acids) had remarkably broad spectra of antimicrobial activities against both bacterial and fungal pathogens and an indication of low cytotoxicity. In addition, their antimicrobial activities displayed various degrees of species specificity that were not related to taxonomy. For example,Candida albicansandStaphylococcus aureuswere the best surrogates to predict peptide activity againstM. tuberculosis, whileMycobacterium smegmatiswas a poor surrogate. Principle component analysis of activity spectrum profiles identified unique features associated with activity againstM. tuberculosisthat reflect their distinctive amino acid composition; active peptides were more hydrophobic and cationic, reflecting increased tryptophan with compensating decreases in valine and other uncharged amino acids and increased lysine. These studies provide foundations for development of cationic antimicrobial peptides as potential new therapeutic agents for TB treatment.

scholarly journals De Novo Emergence of Genetically Resistant Mutants of Mycobacterium tuberculosis from the Persistence Phase Cells Formed against Antituberculosis Drugs In Vitro

2016 ◽  
Vol 61 (2) ◽  
Author(s):  
Jees Sebastian ◽  
Sharmada Swaminath ◽  
Rashmi Ravindran Nair ◽  
Kishor Jakkala ◽  
Atul Pradhan ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Hydroxyl Radical ◽  
Prolonged Exposure ◽  
De Novo ◽  
Random Mutagenesis ◽  
Content Type ◽  
Genome Wide ◽  
Resistant Mutants ◽  
Phase Population

ABSTRACT Bacterial persisters are a subpopulation of cells that can tolerate lethal concentrations of antibiotics. However, the possibility of the emergence of genetically resistant mutants from antibiotic persister cell populations, upon continued exposure to lethal concentrations of antibiotics, remained unexplored. In the present study, we found that Mycobacterium tuberculosis cells exposed continuously to lethal concentrations of rifampin (RIF) or moxifloxacin (MXF) for prolonged durations showed killing, RIF/MXF persistence, and regrowth phases. RIF-resistant or MXF-resistant mutants carrying clinically relevant mutations in the rpoB or gyrA gene, respectively, were found to emerge at high frequency from the RIF persistence phase population. A Luria-Delbruck fluctuation experiment using RIF-exposed M. tuberculosis cells showed that the rpoB mutants were not preexistent in the population but were formed de novo from the RIF persistence phase population. The RIF persistence phase M. tuberculosis cells carried elevated levels of hydroxyl radical that inflicted extensive genome-wide mutations, generating RIF-resistant mutants. Consistent with the elevated levels of hydroxyl radical-mediated genome-wide random mutagenesis, MXF-resistant M. tuberculosis gyrA de novo mutants could be selected from the RIF persistence phase cells. Thus, unlike previous studies, which showed emergence of genetically resistant mutants upon exposure of bacteria for short durations to sublethal concentrations of antibiotics, our study demonstrates that continuous prolonged exposure of M. tuberculosis cells to lethal concentrations of an antibiotic generates antibiotic persistence phase cells that form a reservoir for the generation of genetically resistant mutants to the same antibiotic or another antibiotic. These findings may have clinical significance in the emergence of drug-resistant tubercle bacilli.

scholarly journals Whole-Genome Sequencing of Mycobacterium tuberculosis Provides Insight into the Evolution and Genetic Composition of Drug-Resistant Tuberculosis in Belarus

2016 ◽  
Vol 55 (2) ◽  
pp. 457-469 ◽  
Author(s):  
Kurt R. Wollenberg ◽  
Christopher A. Desjardins ◽  
Aksana Zalutskaya ◽  
Vervara Slodovnikova ◽  
Andrew J. Oler ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
De Novo ◽  
Acquired Resistance ◽  
Multiple Infections ◽  
Drug Resistant ◽  
Genetic Composition ◽  
Hiv Patients ◽  
Treatment Protocols ◽  
Content Type ◽  
Phenotypic Resistance

ABSTRACTThe emergence and spread of drug-resistantMycobacterium tuberculosis(DR-TB) are critical global health issues. Eastern Europe has some of the highest incidences of DR-TB, particularly multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB. To better understand the genetic composition and evolution of MDR- and XDR-TB in the region, we sequenced and analyzed the genomes of 138M. tuberculosisisolates from 97 patients sampled between 2010 and 2013 in Minsk, Belarus. MDR and XDR-TB isolates were significantly more likely to belong to the Beijing lineage than to the Euro-American lineage, and known resistance-conferring loci accounted for the majority of phenotypic resistance to first- and second-line drugs in MDR and XDR-TB. Using a phylogenomic approach, we estimated that the majority of MDR-TB was due to the recent transmission of already-resistantM. tuberculosisstrains rather than repeatedde novoevolution of resistance within patients, while XDR-TB was acquired through both routes. Longitudinal sampling ofM. tuberculosisfrom 34 patients with treatment failure showed that most strains persisted genetically unchanged during treatment or acquired resistance to fluoroquinolones. HIV+ patients were significantly more likely to have multiple infections over time than HIV− patients, highlighting a specific need for careful infection control in these patients. These data provide a better understanding of the genomic composition, transmission, and evolution of MDR- and XDR-TB in Belarus and will enable improved diagnostics, treatment protocols, and prognostic decision-making.

scholarly journals Rational Design of Engineered Cationic Antimicrobial Peptides Consisting Exclusively of Arginine and Tryptophan, and Their Activity against Multidrug-Resistant Pathogens

2013 ◽  
Vol 57 (6) ◽  
pp. 2511-2521 ◽  
Author(s):  
Berthony Deslouches ◽  
Jonathan D. Steckbeck ◽  
Jodi K. Craigo ◽  
Yohei Doi ◽  
Timothy A. Mietzner ◽  
...  
Keyword(s):  
Antimicrobial Peptides ◽  
Rational Design ◽  
Antimicrobial Agents ◽  
Divalent Cations ◽  
Multidrug Resistant ◽  
Resistance Mechanisms ◽  
Broth Culture ◽  
Cationic Antimicrobial Peptides ◽  
Content Type ◽  
Amphipathic Helices

ABSTRACTThe emergence of multidrug-resistant (MDR) pathogens underscores the need for new antimicrobial agents to overcome the resistance mechanisms of these organisms. Cationic antimicrobial peptides (CAPs) provide a potential source of new antimicrobial therapeutics. We previously characterized a lytic base unit (LBU) series of engineered CAPs (eCAPs) of 12 to 48 residues demonstrating maximum antibacterial selectivity at 24 residues. Further, Trp substitution in LBU sequences increased activity against bothP. aeruginosaandS. aureusunder challenging conditions (e.g., saline, divalent cations, and serum). Based on these findings, we hypothesized that the optimal length and, therefore, the cost for maximum eCAP activity under physiologically relevant conditions could be significantly reduced using only Arg and Trp arranged to form idealized amphipathic helices. Hence, we developed a novel peptide series, composed only of Arg and Trp, in a sequence predicted and verified by circular dichroism to fold into optimized amphipathic helices. The most effective antimicrobial activity was achieved at 12 residues in length (WR12) against a panel of both Gram-negative and Gram-positive clinical isolates, including extensively drug-resistant strains, in saline and broth culture and at various pH values. The results demonstrate that the rational design of CAPs can lead to a significant reduction in the length and the number of amino acids used in peptide design to achieve optimal potency and selectivity against specific pathogens.

scholarly journals Rational Design of Biosafety Level 2-Approved, Multidrug-Resistant Strains ofMycobacterium tuberculosisthrough Nutrient Auxotrophy

mBio ◽  
2018 ◽  
Vol 9 (3) ◽  
Author(s):  
Catherine Vilchèze ◽  
Jacqueline Copeland ◽  
Tracy L. Keiser ◽  
Torin Weisbrod ◽  
Jacqueline Washington ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Rational Design ◽  
Multidrug Resistant ◽  
New Drugs ◽  
Drug Resistant ◽  
Content Type ◽  
Auxotrophic Mutants ◽  
Level 3 ◽  
Resistant Mutants ◽  
Biosafety Level

ABSTRACTMultidrug-resistant (MDR) tuberculosis, defined as tuberculosis resistant to the two first-line drugs isoniazid and rifampin, poses a serious problem for global tuberculosis control strategies. Lack of a safe and convenient model organism hampers progress in combating the spread of MDR strains ofMycobacterium tuberculosis. We reasoned that auxotrophic MDR mutants ofM. tuberculosiswould provide a safe means for studying MDRM. tuberculosiswithout the need for a biosafety level 3 (BSL3) laboratory. Two different sets of triple auxotrophic mutants ofM. tuberculosiswere generated, which were auxotrophic for the nutrients leucine, pantothenate, and arginine or for leucine, pantothenate, and methionine. These triple auxotrophic strains retained their acid-fastness, their ability to generate both a drug persistence phenotype and drug-resistant mutants, and their susceptibility to plaque-forming mycobacterial phages. MDR triple auxotrophic mutants were obtained in a two-step fashion, selecting first for solely isoniazid-resistant or rifampin-resistant mutants. Interestingly, selection for isoniazid-resistant mutants of the methionine auxotroph generated isolates with single point mutations inkatG, which encodes an isoniazid-activating enzyme, whereas similar selection using the arginine auxotroph yielded isoniazid-resistant mutants with large deletions in the chromosomal region containingkatG. TheseM. tuberculosisMDR strains were readily sterilized by second-line tuberculosis drugs and failed to kill immunocompromised mice. These strains provide attractive candidates forM. tuberculosisbiology studies and drug screening outside the BSL3 facility.IMPORTANCEElimination ofMycobacterium tuberculosis, the bacterium causing tuberculosis, requires enhanced understanding of its biology in order to identify new drugs against drug-susceptible and drug-resistantM. tuberculosisas well as uncovering novel pathways that lead toM. tuberculosisdeath. To circumvent the need for a biosafety level 3 (BSL3) laboratory when conducting research onM. tuberculosis, we have generated drug-susceptible and drug-resistant triple auxotrophic strains ofM. tuberculosissuitable for use in a BSL2 laboratory. These strains originate from a double auxotrophicM. tuberculosisstrain, H37Rv ΔpanCDΔleuCD, which was reclassified as a BSL2 strain based on its lack of lethality in immunocompromised and immunocompetent mice. A third auxotrophy (methionine or arginine) was introduced via deletion ofmetAorargB, respectively, sinceM. tuberculosisΔmetAandM. tuberculosisΔargBare unable to survive amino acid auxotrophy and infect their host. The resulting triple auxotrophicM. tuberculosisstrains retained characteristics ofM. tuberculosisrelevant for most types of investigations.

scholarly journals Novel Antimicrobials from Uncultured Bacteria Acting against Mycobacterium tuberculosis

mBio ◽  
2020 ◽  
Vol 11 (4) ◽  
Author(s):  
Jeffrey Quigley ◽  
Aaron Peoples ◽  
Asel Sarybaeva ◽  
Dallas Hughes ◽  
Meghan Ghiglieri ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Natural Product ◽  
Protein Secretion ◽  
Specific Activity ◽  
Therapeutic Options ◽  
Content Type ◽  
Natural Product Discovery ◽  
Uncultured Bacteria ◽  
New Compounds ◽  
Drug Resistant Strains

ABSTRACT Mycobacterium tuberculosis, which causes tuberculosis (TB), is estimated to infect one-third of the world’s population. The overall burden and the emergence of drug-resistant strains of Mycobacterium tuberculosis underscore the need for new therapeutic options against this important human pathogen. Our recent work demonstrated the success of natural product discovery in identifying novel compounds with efficacy against Mycobacterium tuberculosis. Here, we improve on these methods by combining improved isolation and Mycobacterium tuberculosis selective screening to identify three new anti-TB compounds: streptomycobactin, kitamycobactin, and amycobactin. We were unable to obtain mutants resistant to streptomycobactin, and its target remains to be elucidated. We identify the target of kitamycobactin to be the mycobacterial ClpP1P2C1 protease and confirm that kitamycobactin is an analog of the previously identified compound lassomycin. Further, we identify the target of amycobactin to be the essential protein secretion pore SecY. We show further that amycobactin inhibits protein secretion via the SecY translocon. Importantly, this inhibition is bactericidal to nonreplicating Mycobacterium tuberculosis. This is the first compound, to our knowledge, that targets the Sec protein secretion machinery in Mycobacterium tuberculosis. This work underscores the ability of natural product discovery to deliver not only new compounds with activity against Mycobacterium tuberculosis but also compounds with novel targets. IMPORTANCE Decreasing discovery rates and increasing resistance have underscored the need for novel therapeutic options to treat Mycobacterium tuberculosis infection. Here, we screen extracts from previously uncultured soil microbes for specific activity against Mycobacterium tuberculosis, identifying three novel compounds. We further define the mechanism of action of one compound, amycobactin, and demonstrate that it inhibits protein secretion through the Sec translocation machinery.

scholarly journals Oxadiazoles Have Butyrate-Specific Conditional Activity against Mycobacterium tuberculosis

2016 ◽  
Vol 60 (6) ◽  
pp. 3608-3616 ◽  
Author(s):  
Julie V. Early ◽  
Allen Casey ◽  
Maria Angeles Martinez-Grau ◽  
Isabel C. Gonzalez Valcarcel ◽  
Michal Vieth ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Carbon Source ◽  
Mammalian Cells ◽  
Sole Carbon Source ◽  
Specific Activity ◽  
Antitubercular Activity ◽  
Content Type ◽  
Phenotypic Screen

Mycobacterium tuberculosisis a global pathogen of huge importance which can adapt to several host niche environments in which carbon source availability is likely to vary. We developed and ran a phenotypic screen using butyrate as the sole carbon source to be more reflective of the host lung environment. We screened a library of ∼87,000 small compounds and identified compounds which demonstrated good antitubercular activity againstM. tuberculosisgrown with butyrate but not with glucose as the carbon source. Among the hits, we identified an oxadiazole series (six compounds) which had specific activity againstM. tuberculosisbut which lacked cytotoxicity against mammalian cells.

scholarly journals A Diagnostic Algorithm To Investigate Pyrazinamide and Ethambutol Resistance in Rifampin-Resistant Mycobacterium tuberculosis Isolates in a Low-Incidence Setting

2018 ◽  
Vol 63 (2) ◽  
pp. e01798-18 ◽  
Author(s):  
Söenke Andres ◽  
Matthias I. Gröschel ◽  
Doris Hillemann ◽  
Matthias Merker ◽  
Stefan Niemann ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Sanger Sequencing ◽  
Rational Design ◽  
Clinical Decision Making ◽  
Susceptibility Testing ◽  
Diagnostic Algorithm ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Content Type ◽  
Phenotypic Resistance

ABSTRACT Phenotypic drug susceptibility testing (DST) for the two first-line tuberculosis drugs ethambutol and pyrazinamide is known to yield unreliable and inaccurate results. In this prospective study, we propose a diagnostic algorithm combining phenotypic DST with Sanger sequencing to inform clinical decision-making for drug-resistant Mycobacterium tuberculosis complex isolates. Sequencing results were validated using whole-genome sequencing (WGS) of the isolates. Resistance-conferring mutations obtained by pncA sequencing correlated well with phenotypic DST results for pyrazinamide. Phenotypic resistance to ethambutol was only partly explained by mutations in the embB 306 codon. Additional resistance-conferring mutations were found in the embB gene at codons 354, 406, and 497. In several isolates that tested ethambutol susceptibility by phenotypic DST, well-known resistance-conferring embB mutations were determined. Thus, targeted Sanger sequencing beyond the embB 306 codon or WGS together with phenotypic DST should be employed to ensure reliable ethambutol drug susceptibility testing, as a basis for the rational design of multidrug-resistant tuberculosis regimens with or without ethambutol.

scholarly journals Deciphering the Magainin Resistance Process of Escherichia coli Strains in Light of the Cytosolic Proteome

2012 ◽  
Vol 56 (4) ◽  
pp. 1714-1724 ◽  
Author(s):  
Simone Maria-Neto ◽  
Elizabete de Souza Cândido ◽  
Diana Ribas Rodrigues ◽  
Daniel Amaro de Sousa ◽  
Ezequiel Marcelino da Silva ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Peptides ◽  
Mechanism Of Action ◽  
De Novo ◽  
Metabolic Response ◽  
Content Type ◽  
Resistant Strains ◽  
Cell Commitment ◽  
Antibiotic Agents ◽  
Dimensional Electrophoresis

ABSTRACTAntimicrobial peptides (AMPs) are effective antibiotic agents commonly found in plants, animals, and microorganisms, and they have been suggested as the future of antimicrobial chemotherapies. It is vital to understand the molecular details that define the mechanism of action of resistance to AMPs for a rational planning of the next antibiotic generation and also to shed some light on the complex AMP mechanism of action. Here, the antibiotic resistance ofEscherichia coliATCC 8739 to magainin I was evaluated in the cytosolic subproteome. Magainin-resistant strains were selected after 10 subsequent spreads at subinhibitory concentrations of magainin I (37.5 mg · liter−1), and their cytosolic proteomes were further compared to those of magainin-susceptible strains through two-dimensional electrophoresis analysis. As a result, 41 differentially expressed proteins were detected byin silicoanalysis and further identified by tandem mass spectrometryde novosequencing. Functional categorization indicated an intense metabolic response mainly in energy and nitrogen uptake, stress response, amino acid conversion, and cell wall thickness. Indeed, data reported here show that resistance to cationic antimicrobial peptides possesses a greater molecular complexity than previously supposed, resulting in cell commitment to several metabolic pathways.

scholarly journals Mycobacterium tuberculosis Proteome Response to Antituberculosis Compounds Reveals Metabolic “Escape” Pathways That Prolong Bacterial Survival

2017 ◽  
Vol 61 (7) ◽  
Author(s):  
Lia Danelishvili ◽  
Natalia Shulzhenko ◽  
Jessica J. J. Chinison ◽  
Lmar Babrak ◽  
Jialu Hu ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
De Novo ◽  
Day Treatment ◽  
Bacterial Survival ◽  
Content Type ◽  
Treatment Groups ◽  
Metabolic Remodeling ◽  
Tuberculosis Therapy

ABSTRACTTuberculosis (TB) continues to be one of the most common bacterial infectious diseases and is the leading cause of death in many parts of the world. A major limitation of TB therapy is slow killing of the infecting organism, increasing the risk for the development of a tolerance phenotype and drug resistance. Studies indicate thatMycobacterium tuberculosistakes several days to be killed upon treatment with lethal concentrations of antibiotics bothin vitroandin vivo. To investigate how metabolic remodeling can enable transient bacterial survival during exposure to bactericidal concentrations of compounds,M. tuberculosisstrain H37Rv was exposed to twice the MIC of isoniazid, rifampin, moxifloxacin, mefloquine, or bedaquiline for 24 h, 48 h, 4 days, and 6 days, and the bacterial proteomic response was analyzed using quantitative shotgun mass spectrometry. Numerous sets ofde novobacterial proteins were identified over the 6-day treatment. Network analysis and comparisons between the drug treatment groups revealed several shared sets of predominant proteins and enzymes simultaneously belonging to a number of diverse pathways. Overexpression of some of these proteins in the nonpathogenicMycobacterium smegmatisextended bacterial survival upon exposure to bactericidal concentrations of antimicrobials, and inactivation of some proteins inM. tuberculosisprevented the pathogen from escaping the fast killingin vitroand in macrophages, as well. Our biology-driven approach identified promising bacterial metabolic pathways and enzymes that might be targeted by novel drugs to reduce the length of tuberculosis therapy.

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