The phosphatidyl-myo-inositol dimannoside acyltransferase PatA is essential for Mycobacterium tuberculosis growth in vitro and in vivo

Mycobacterium tuberculosis comprises an unusual cell envelope dominated by unique lipids and glycans that provides a permeability barrier against hydrophilic drugs and is central for its survival and virulence. Phosphatidyl-myo-inositol mannosides (PIMs) are glycolipids considered not only key structural components of the cell envelope but also the precursors of lipomannan (LM) and lipoarabinomannan (LAM), important lipoglycans implicated in host-pathogen interactions. Here, we focus on PatA, a membrane associated acyltransferase that transfers a palmitoyl moiety from palmitoyl–CoA to the 6-position of the mannose ring linked to 2-position of inositol in PIM1/PIM2. We validate that the function of PatA is vital for M. tuberculosis in vitro and in vivo. We constructed a patA conditional mutant and showed that silencing patA is bactericidal in batch cultures. This phenotype was associated with significant reduced levels of Ac1PIM2, an important structural component of the mycobacterial inner membrane. The requirement of PatA for viability was also demonstrated during macrophage infection and in a mouse model of infection, where a dramatic decrease in viable counts was observed upon silencing of the patA gene. This is reminiscent of the behavior of PimA, the mannosyltransferase that initiates the PIM pathway, also found to be essential for M. tuberculosis growth in vitro and in vivo. Altogether, the experimental data highlights the significance of the early steps of PIM biosynthetic pathway for M. tuberculosis physiology and uncover that PatA is a novel target for drug discovery programs against this major human pathogen. IMPORTANCE TB is the leading cause of death from a single infectious agent. The emergence of drug resistance in strains of M. tuberculosis, the etiologic agent of TB, emphasizes the need of identifying new targets and antimicrobial agents. The mycobacterial cell envelope is a major factor in this intrinsic drug resistance. Here, we have focused on the biosynthesis of PIMs, key virulence factors and important components of the cell envelope. Specifically, we have determined PatA, the acyltransferase responsible for the first acylation step of the PIM synthesis pathway, is essential in M. tuberculosis. These results highlight the importance of early steps of PIM biosynthetic pathway for mycobacterial physiology and the suitability of PatA as a potential new drug target.

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The Effect of pH on Antibiotic Efficacy against Coxiella burnetii in Axenic Media

Scientific Reports ◽

10.1038/s41598-019-54556-6 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Cody B. Smith ◽

Charles Evavold ◽

Gilbert J. Kersh

Keyword(s):

Coxiella Burnetii ◽

Antimicrobial Agents ◽

Q Fever ◽

Antimicrobial Treatment ◽

Etiologic Agent ◽

Growth And Survival ◽

Intracellular Compartments ◽

The Impact

AbstractCoxiella burnetii, the etiologic agent of Q fever, replicates in an intracellular phagolysosome with pH between 4 and 5. The impact of this low pH environment on antimicrobial treatment is not well understood. An in vitro system for testing antibiotic susceptibility of C. burnetii in axenic media was set up to evaluate the impact of pH on C. burnetii growth and survival in the presence and absence of antimicrobial agents. The data show that C. burnetii does not grow in axenic media at pH 6.0 or higher, but the organisms remain viable. At pH of 4.75, 5.25, and 5.75 moxifloxacin, doxycycline, and rifampin are effective at preventing growth of C. burnetii in axenic media, with moxifloxacin and doxycycline being bacteriostatic and rifampin having bactericidal activity. The efficacy of doxycycline and moxifloxacin improved at higher pH, whereas rifampin activity was pH independent. Hydroxychloroquine is thought to inhibit growth of C. burnetii in vivo by raising the pH of typically acidic intracellular compartments. It had no direct bactericidal or bacteriostatic activity on C. burnetii in axenic media, suggesting that raising pH of acidic intracellular compartments is its primary mechanism of action in vivo. The data suggest that doxycycline and hydroxychloroquine are primarily independent bacteriostatic agents.

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Glucan Is a Component of the Mycobacterium tuberculosis Surface That Is Expressed In Vitro and In Vivo

Infection and Immunity ◽

10.1128/iai.70.5.2566-2575.2002 ◽

2002 ◽

Vol 70 (5) ◽

pp. 2566-2575 ◽

Cited By ~ 41

Author(s):

J. Reid Schwebach ◽

Aharona Glatman-Freedman ◽

Leslie Gunther-Cummins ◽

Zhongdong Dai ◽

John B. Robbins ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Cell Envelope ◽

Tween 80 ◽

Enzyme Linked Immunosorbent Assay ◽

In Vitro Growth ◽

Comprehensive Picture ◽

The Media ◽

Surface Polysaccharide

ABSTRACT The outermost layer of Mycobacterium tuberculosis is composed primarily of two polysaccharides, glucan (GC) and arabinomannan. To analyze the surface polysaccharide composition of M. tuberculosis, we generated a monoclonal antibody (MAb) that binds M. tuberculosis GC and is known as MAb 24c5. Immunofluorescence and whole-mount immunoelectron microscopy indicated that GC is on the outermost portion of the bacteria. M. tuberculosis strains Erdman and CDC 1551 were analyzed for their ability to bind MAb 24c5 after in vitro growth in media with and without the detergent Tween 80. MAb 24c5 bound to Erdman and CDC 1551 at all culture times with only slightly greater apparent affinity after extended culture in the absence of Tween 80, indicating that a stable amount of GC polysaccharide antigen is associated with the cell surface of M. tuberculosis. An enzyme-linked immunosorbent assay indicated that GC is antigenically similar to glycogen, and the amount of GC antigen increased in the media of M. tuberculosis cultures grown either with or without the detergent Tween 80. Other nontuberculosis mycobacteria have antigenically similar GCs on their surfaces after in vitro growth. Inoculation of mice with live bacilli but not inoculation with dead bacilli elicited a strong antibody response to GC consistent with production of this antigen in vivo. Our results provide a more comprehensive picture of the M. tuberculosis cell envelope and the conditions that allow expression of M. tuberculosis GC.

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Fusidic Acid-Resistant Mutants of Salmonella enterica Serovar Typhimurium with Low Fitness In Vivo Are Defective in RpoS Induction

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.47.12.3743-3749.2003 ◽

2003 ◽

Vol 47 (12) ◽

pp. 3743-3749 ◽

Cited By ~ 33

Author(s):

Mirjana Macvanin ◽

Johanna Björkman ◽

Sofia Eriksson ◽

Mikael Rhen ◽

Dan I. Andersson ◽

...

Keyword(s):

Protein Synthesis ◽

Fusidic Acid ◽

Salmonella Enterica ◽

Antimicrobial Agents ◽

Relative Fitness ◽

Infection Model ◽

Macrophage Infection ◽

Serovar Typhimurium

ABSTRACT Mutants of Salmonella enterica serovar Typhimurium resistant to fusidic acid (Fusr) have mutations in fusA, the gene encoding translation elongation factor G (EF-G). Most Fusr mutants have reduced fitness in vitro and in vivo, in part explained by mutant EF-G slowing the rate of protein synthesis and growth. However, some Fusr mutants with normal rates of protein synthesis still suffer from reduced fitness in vivo. As shown here, Fusr mutants could be similarly ranked in their relative fitness in mouse infection models, in a macrophage infection model, in their relative hypersensitivity to hydrogen peroxide in vivo and in vitro, and in the amount of RpoS production induced upon entry into the stationary phase. We identify a reduced ability to induce production of RpoS (σs) as a defect associated with Fusr strains. Because RpoS is a regulator of the general stress response, and an important virulence factor in Salmonella, an inability to produce RpoS in appropriate amounts can explain the low fitness of Fusr strains in vivo. The unfit Fusr mutants also produce reduced levels of the regulatory molecule ppGpp in response to starvation. Because ppGpp is a positive regulator of RpoS production, we suggest that a possible cause of the reduced levels of RpoS is the reduction in ppGpp production associated with mutant EF-G. The low fitness of Fusr mutants in vivo suggests that drugs that can alter the levels of global regulators of gene expression deserve attention as potential antimicrobial agents.

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Vitamin C Potentiates the Killing of Mycobacterium tuberculosis by the First-Line Tuberculosis Drugs Isoniazid and Rifampin in Mice

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02165-17 ◽

2018 ◽

Vol 62 (3) ◽

Cited By ~ 9

Author(s):

Catherine Vilchèze ◽

John Kim ◽

William R. Jacobs

Keyword(s):

Drug Resistance ◽

Mycobacterium Tuberculosis ◽

Vitamin C ◽

Mouse Serum ◽

First Line ◽

Content Type ◽

Tb Treatment ◽

High Concentrations

ABSTRACT The treatment of drug-susceptible tuberculosis (TB) is long and cumbersome. Mismanagement of TB treatment can lead to the emergence of drug resistance in patients, so shortening the treatment duration could significantly improve TB chemotherapy and prevent the development of drug resistance. We previously discovered that high concentrations of vitamin C sterilize cultures of drug-susceptible and drug-resistant Mycobacterium tuberculosis . Here, we tested subinhibitory concentration of vitamin C in combination with TB drugs against M. tuberculosis in vitro and in a mouse model of M. tuberculosis infection. In vivo , we showed that the vitamin C level in mouse serum can be increased by intraperitoneal injection of vitamin C to reach vitamin C levels close to the concentrations required for activity in vitro . Although vitamin C had no activity by itself in M. tuberculosis -infected mice, the combination of vitamin C with the first-line TB drugs isoniazid and rifampin reduced the bacterial burden in the lungs of M. tuberculosis -infected mice faster than isoniazid and rifampin combined in two independent experiments. These experiments suggest that the addition of vitamin C to first-line TB drugs could shorten TB treatment. Vitamin C, an inexpensive and nontoxic compound, could easily be added to the TB pharmacopeia to substantially improve chemotherapy outcome, which would have a significant impact on the worldwide TB community.

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The Combination of Sulfamethoxazole, Trimethoprim, and Isoniazid or Rifampin Is Bactericidal and Prevents the Emergence of Drug Resistance in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00832-12 ◽

2012 ◽

Vol 56 (10) ◽

pp. 5142-5148 ◽

Cited By ~ 28

Author(s):

Catherine Vilchèze ◽

William R. Jacobs

Keyword(s):

Drug Resistance ◽

Mycobacterium Tuberculosis ◽

Multidrug Resistant ◽

New Drugs ◽

Drug Resistant ◽

Content Type ◽

The Past ◽

Tb Treatment

ABSTRACTThe challenges of developing new drugs to treat tuberculosis (TB) are indicated by the relatively small number of candidates entering clinical trials in the past decade. To overcome these issues, we reexamined two FDA-approved antibacterial drugs, sulfamethoxazole (SMX) and trimethoprim (TMP), for use in TB treatment. SMX and TMP inhibit folic acid biosynthesis and are used in combination to treat infections of the respiratory, urinary, and gastrointestinal tracts. The MICs of SMX and TMP, alone and in combination, were determined for drug-susceptible, multidrug-resistant (MDR), and extensively drug-resistantMycobacterium tuberculosisstrains. While TMP alone was not effective againstM. tuberculosis, the combination of TMP and SMX was bacteriostatic againstM. tuberculosis. Surprisingly, the combination of SMX and TMP was also active against a subset of MDRM. tuberculosisstrains. Treatment ofM. tuberculosiswith TMP-SMX and a first-line anti-TB drug, either isoniazid or rifampin, was bactericidal, demonstrating that the combination of TMP and SMX with isoniazid or rifampin was not antagonistic. Moreover, the addition of SMX-TMP in combination with either isoniazid or rifampin also prevented the emergence of drug resistancein vitro. In conclusion, this study further illustrates the opportunity to reevaluate the activity of TMP-SMXin vivoto prevent the emergence of drug-resistantM. tuberculosis.

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An essential mycolate remodeling program for mycobacterial adaptation in host cells

10.1101/354431 ◽

2018 ◽

Cited By ~ 1

Author(s):

Eliza J.R. Peterson ◽

Rebeca Bailo ◽

Alissa C. Rothchild ◽

Mario Arrieta-Ortiz ◽

Amardeep Kaur ◽

...

Keyword(s):

Cell Wall ◽

Mycobacterium Tuberculosis ◽

New Technology ◽

Mycolic Acid ◽

Host Cells ◽

Macrophage Infection ◽

Cell Wall Remodeling ◽

Mycobacterial Cell Wall

AbstractThe success of Mycobacterium tuberculosis (MTB) stems from its ability to remain hidden from the immune system within macrophages. Here, we report a new technology (Path-seq) to sequence miniscule amounts of MTB transcripts within up to million-fold excess host RNA. Using Path-seq we have discovered a novel transcriptional program for in vivo mycobacterial cell wall remodeling when the pathogen infects alveolar macrophages in mice. We have discovered that MadR transcriptionally modulates two mycolic acid desaturases desA1/A2 to initially promote cell wall remodeling upon in vitro macrophage infection and, subsequently, reduces mycolate biosynthesis upon entering dormancy. We demonstrate that disrupting MadR program is lethal to diverse mycobacteria making this evolutionarily conserved regulator a prime antitubercular target for both early and late stages of infection.One Sentence SummaryNovel technology (Path-seq) discovers cell wall remodeling program during Mycobacterium tuberculosis infection of macrophages

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Host- and age-dependent transcriptional changes in Mycobacterium tuberculosis cell envelope biosynthesis genes after exposure to human alveolar lining fluid

10.1101/2021.09.08.459334 ◽

2021 ◽

Author(s):

Anna Allué-Guardia ◽

Andreu Garcia-Vilanova ◽

Angélica M. Olmo-Fontánez ◽

Jay Peters ◽

Diego J. Maselli ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Close Contact ◽

Cell Envelope ◽

Redox Homeostasis ◽

Transcriptional Response ◽

Before And After ◽

Transcriptional Changes ◽

Host Cell Interactions

AbstractTuberculosis (TB) infection, caused by the airborne pathogen Mycobacterium tuberculosis (M.tb), resulted in almost 1.4 million deaths in 2019 and the number of deaths is predicted to increase by 20% over the next 5 years due to the COVID-19 pandemic. Upon reaching the alveolar space, M.tb comes in close contact with the lung mucosa before and after its encounter with host alveolar compartment cells. Our previous studies show that homeostatic innate soluble components of the alveolar lining fluid (ALF) can quickly alter the cell envelope surface of M.tb upon contact, defining subsequent M.tb-host cell interactions and infection outcomes in vitro and in vivo. We also demonstrated that ALF from 60+ year old elders (E-ALF) vs. healthy 18- to 45-year-old adults (A-ALF) is dysfunctional with loss of homeostatic capacity and impaired innate soluble responses linked to high local oxidative stress. In this study, a targeted transcriptional assay demonstrates that M.tb exposure to human ALF alters the expression of its cell envelope genes. Specifically, our results indicate that A-ALF-exposed M.tb upregulates cell envelope genes associated with lipid, carbohydrate, and amino acid metabolism, as well as genes associated with redox homeostasis and transcriptional regulators. Conversely, M.tb exposure to E-ALF shows lesser transcriptional response, with most of the M.tb genes unchanged or downregulated. Overall, this study indicates that M.tb responds and adapts to the lung alveolar environment upon contact, and that the host ALF status determined by factors such as age might play an important role in determining infection outcome.

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The Mycobacterium tuberculosis Sigma Factor σB Is Required for Full Response to Cell Envelope Stress and Hypoxia In Vitro, but It Is Dispensable for In Vivo Growth

Journal of Bacteriology ◽

10.1128/jb.00510-09 ◽

2009 ◽

Vol 191 (18) ◽

pp. 5628-5633 ◽

Cited By ~ 48

Author(s):

P. A. Fontán ◽

M. I. Voskuil ◽

M. Gomez ◽

D. Tan ◽

M. Pardini ◽

...

Keyword(s):

Oxidative Stress ◽

Mycobacterium Tuberculosis ◽

Sigma Factor ◽

Cell Envelope ◽

Hypoxic Conditions ◽

Envelope Stress ◽

Stress Oxidative ◽

In Vivo Growth

ABSTRACT The numerous sigma (σ) factors present in Mycobacterium tuberculosis are indicative of the adaptability of this pathogen to different environmental conditions. In this report, we describe the M. tuberculosis σB regulon and the phenotypes of an M. tuberculosis sigB mutant strain exposed to cell envelope stress, oxidative stress, and hypoxia. The sigB mutant was especially defective in survival under hypoxic conditions in vitro, but it was not attenuated for growth in THP-1 cells or during mouse and guinea pig infection.

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Tuberkulose - Screening

Therapeutische Umschau ◽

10.1024/0040-5930/a000181 ◽

2011 ◽

Vol 68 (7) ◽

pp. 381-387

Author(s):

Otto Schoch

Keyword(s):

Mycobacterium Tuberculosis ◽

Interferon Gamma ◽

Wird Eine ◽

Interferon Gamma Release Assays

Das primäre Ziel der Aktivitäten zur bevölkerungsbezogenen Tuberkulosekontrolle ist die Identifizierung von Patienten mit sputummikroskopisch positiver Lungentuberkulose. Wenn diese Patienten umgehend therapiert werden, haben sie nicht nur eine optimale Heilungschance, sondern übertragen auch den Krankheitserreger nicht weiter auf andere Personen. Das Screening, die systematische Suche nach Tuberkulose, erfolgt in der Regel radiologisch bei der Suche nach Erkrankten, während immunologische Teste bei der Suche nach einer Infektion mit Mycobacterium tuberculosis zur Anwendung kommen. Diese Infektion, die ein erhöhtes Risiko für die Entwicklung einer Tuberkulose-Erkrankung mit sich bringt, wird im Rahmen der Umgebungsuntersuchungen oder bei Hochrisikogruppen gesucht. Neben dem traditionellen in vivo Mantoux Hauttest stehen heute die neueren in vitro Blutteste, die sogenannten Interferon Gamma Release Assays (IGRA) zur Verfügung, die unter anderem den Vorteil einer höheren Spezifität mit sich bringen, weil die verwendeten Antigene der Mykobakterien-Wand beim Impfstamm Bacille Calmitte Guerin (BCG) und bei den meisten atypischen Mykobakterien nicht vorhanden sind. Zudem kann bei Immunsupprimierten dank einer mitgeführten Positivkontrolle eine Aussage über die Wahrscheinlichkeit eines falsch negativen Testresultates gemacht werden. Bei neu diagnostizierter Infektion mit Mycobacterium tuberculosis wird eine präventive Chemotherapie mit Isoniazid während 9 Monaten durchgeführt.

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Synthesis of Novel 3(N,N-dialkylamino)alkyl/phenyl Substituted Thieno [2,3-d]pyrimidinones as H1-Anti-Histaminic and Antimicrobial Agents

Current Bioactive Compounds ◽

10.2174/1573407214666180226130957 ◽

2019 ◽

Vol 15 (1) ◽

pp. 63-70

Author(s):

Shiv Dev Singh ◽

Arvind Kumar ◽

Firoz Babar ◽

Neetu Sachan ◽

Arun Kumar Sharma

Keyword(s):

Antimicrobial Activity ◽

Potassium Hydroxide ◽

Antimicrobial Agents ◽

Pyrimidine Ring ◽

Antimicrobial Activities ◽

Screening Methods ◽

Chlorpheniramine Maleate ◽

In Vivo Screening

Background: Thienopyrimidines are the bioisoster of quinazoline and unlike quinazoline exist in three isomeric forms corresponding to the three possible types annulation of thiophene to the pyrimidine ring viz thieno[2,3-d] pyrimidine, thieno[3,2-d] pyrimidine and thieno[3,4-d]pyrimidine. Heterocyclic containing the thienopyrimidinone moiety exhibits various pronounced activities such as anti-hypertensive, analgesic and anti-inflammatory, antiviral, platelet aggregation inhibitory, antiprotozoal bronchodilatory, phosphodiesterase inhibitory, antihistaminic, antipsychotic and antimicrobial activity. Objective: Synthesis of novel 3(N,N-dialkylamino)alkyl/phenyl substituted thieno[2,3-d]pyrimidinones as H1-anti-histaminic and antimicrobial agents. Methods: A series of 3-[(N,N-dialkylamino)alkyl/phenyl]-2-(1H)thioxo-5,6,7,8-tetrahydrobenzo(b) thieno(2,3-d)pyrimidine-4(3H)-ones[4a-d], their oxo analogous [5a-d] and 3-[(N,N-dialkylamino)alkyl]- 2-chlorophenyl-5,6,7,8-tetrahydrobenzo(b)thieno(2,3-d)pyrimidine- 4 (3H)-ones[6a-d]derivative were synthesized from 2-amino-4,5,6,7-tetrahydrobenzo(b)thiophene-3-carboxylic acid by nucleophilic substitution of different N,N-dialkyl alkylene/phenylene diamines on activated 3-acylchloride moiety followed by cyclocondensation with carbon disulfide and ethanolic potassium hydroxide to get [4a-d] and in second reaction by condensation with 4-chlorobenzoyl chloride to get [6a-d] by single pot novel innovative route. The oxo analogous [5a-d] were prepared by treating derivatives [4a-d] with potassium permagnate in ethanolic KOH. The synthesized compound were evaluated for H1-antihistaminic and antimicrobial activities. Results: All synthesized compounds exhibited significant H1-antihistaminic activity by in vitro and in vivo screening methods and data were verified analytically and statistically. The compound 4a, 4b, 5a and 5b showed significant H1-antihistaminiic activity than the reference standard chlorpheniramine maleate. The compound 6d, 6c, 5c and 4c exhibited significant antimicrobial activity.

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