Targeting redox heterogeneity to counteract drug tolerance in replicating Mycobacterium tuberculosis

The capacity of Mycobacterium tuberculosis (Mtb) to tolerate multiple antibiotics represents a major problem in tuberculosis (TB) management. Heterogeneity in Mtb populations is one of the factors that drives antibiotic tolerance during infection. However, the mechanisms underpinning this variation in bacterial population remain poorly understood. Here, we show that phagosomal acidification alters the redox physiology of Mtb to generate a population of replicating bacteria that display drug tolerance during infection. RNA sequencing of this redox-altered population revealed the involvement of iron-sulfur (Fe-S) cluster biogenesis, hydrogen sulfide (H2S) gas, and drug efflux pumps in antibiotic tolerance. The fraction of the pH- and redox-dependent tolerant population increased when Mtb infected macrophages with actively replicating HIV-1, suggesting that redox heterogeneity could contribute to high rates of TB therapy failure during HIV-TB coinfection. Pharmacological inhibition of phagosomal acidification by the antimalarial drug chloroquine (CQ) eradicated drug-tolerant Mtb, ameliorated lung pathology, and reduced postchemotherapeutic relapse in in vivo models. The pharmacological profile of CQ (Cmax and AUClast) exhibited no major drug-drug interaction when coadministered with first line anti-TB drugs in mice. Our data establish a link between phagosomal pH, redox metabolism, and drug tolerance in replicating Mtb and suggest repositioning of CQ to shorten TB therapy and achieve a relapse-free cure.

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Biofilm formation in the lung contributes to virulence and drug tolerance of Mycobacterium tuberculosis

Nature Communications ◽

10.1038/s41467-021-21748-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Poushali Chakraborty ◽

Sapna Bajeli ◽

Deepak Kaushal ◽

Bishan Dass Radotra ◽

Ashwani Kumar

Keyword(s):

Mycobacterium Tuberculosis ◽

Immune System ◽

Biofilm Formation ◽

Antimycobacterial Activity ◽

Drug Tolerance ◽

Host Immune System ◽

Tissue Sections ◽

Slow Growing

AbstractTuberculosis is a chronic disease that displays several features commonly associated with biofilm-associated infections: immune system evasion, antibiotic treatment failures, and recurrence of infection. However, although Mycobacterium tuberculosis (Mtb) can form cellulose-containing biofilms in vitro, it remains unclear whether biofilms are formed during infection in vivo. Here, we demonstrate the formation of Mtb biofilms in animal models of infection and in patients, and that biofilm formation can contribute to drug tolerance. First, we show that cellulose is also a structural component of the extracellular matrix of in vitro biofilms of fast and slow-growing nontuberculous mycobacteria. Then, we use cellulose as a biomarker to detect Mtb biofilms in the lungs of experimentally infected mice and non-human primates, as well as in lung tissue sections obtained from patients with tuberculosis. Mtb strains defective in biofilm formation are attenuated for survival in mice, suggesting that biofilms protect bacilli from the host immune system. Furthermore, the administration of nebulized cellulase enhances the antimycobacterial activity of isoniazid and rifampicin in infected mice, supporting a role for biofilms in phenotypic drug tolerance. Our findings thus indicate that Mtb biofilms are relevant to human tuberculosis.

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Gran1: A Granulysin-Derived Peptide with Potent Activity against Intracellular Mycobacterium tuberculosis

International Journal of Molecular Sciences ◽

10.3390/ijms22168392 ◽

2021 ◽

Vol 22 (16) ◽

pp. 8392

Author(s):

Reiner Noschka ◽

Fanny Wondany ◽

Gönül Kizilsavas ◽

Tanja Weil ◽

Gilbert Weidinger ◽

...

Keyword(s):

Antimicrobial Activity ◽

Mycobacterium Tuberculosis ◽

Developmental Toxicity ◽

Super Resolution ◽

In Vivo Models ◽

Large Size ◽

Acid Fragment ◽

Powerful Approach ◽

Target Effects

Granulysin is an antimicrobial peptide (AMP) expressed by human T-lymphocytes and natural killer cells. Despite a remarkably broad antimicrobial spectrum, its implementation into clinical practice has been hampered by its large size and off-target effects. To circumvent these limitations, we synthesized a 29 amino acid fragment within the putative cytolytic site of Granulysin (termed “Gran1”). We evaluated the antimicrobial activity of Gran1 against the major human pathogen Mycobacterium tuberculosis (Mtb) and a panel of clinically relevant non-tuberculous mycobacteria which are notoriously difficult to treat. Gran1 efficiently inhibited the mycobacterial proliferation in the low micro molar range. Super-resolution fluorescence microscopy and scanning electron microscopy indicated that Gran1 interacts with the surface of Mtb, causing lethal distortions of the cell wall. Importantly, Gran1 showed no off-target effects (cytokine release, chemotaxis, cell death) in primary human cells or zebrafish embryos (cytotoxicity, developmental toxicity, neurotoxicity, cardiotoxicity). Gran1 was selectively internalized by macrophages, the major host cell of Mtb, and restricted the proliferation of the pathogen. Our results demonstrate that the hypothesis-driven design of AMPs is a powerful approach for the identification of small bioactive compounds with specific antimicrobial activity. Gran1 is a promising component for the design of AMP-containing nanoparticles with selective activity and favorable pharmacokinetics to be pushed forward into experimental in vivo models of infectious diseases, most notably tuberculosis.

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Three-dimensional in situ morphometrics of Mycobacterium tuberculosis infection within lesions by optical mesoscopy and novel acid-fast staining

Scientific Reports ◽

10.1038/s41598-020-78640-4 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Robert J. Francis ◽

Gillian Robb ◽

Lee McCann ◽

Bhagwati Khatri ◽

James Keeble ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Three Dimensional ◽

Large Field ◽

Staining Procedure ◽

3D Analysis ◽

Mycobacterium Tuberculosis Infection ◽

In Vivo Models ◽

Novel Approach

AbstractTuberculosis (TB) preclinical testing relies on in vivo models including the mouse aerosol challenge model. The only method of determining colony morphometrics of TB infection in a tissue in situ is two-dimensional (2D) histopathology. 2D measurements consider heterogeneity within a single observable section but not above and below, which could contain critical information. Here we describe a novel approach, using optical clearing and a novel staining procedure with confocal microscopy and mesoscopy, for three-dimensional (3D) measurement of TB infection within lesions at sub-cellular resolution over a large field of view. We show TB morphometrics can be determined within lesion pathology, and differences in infection with different strains of Mycobacterium tuberculosis. Mesoscopy combined with the novel CUBIC Acid-Fast (CAF) staining procedure enables a quantitative approach to measure TB infection and allows 3D analysis of infection, providing a framework which could be used in the analysis of TB infection in situ.

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Preferential infection and depletion of Mycobacterium tuberculosis–specific CD4 T cells after HIV-1 infection

Journal of Experimental Medicine ◽

10.1084/jem.20100090 ◽

2010 ◽

Vol 207 (13) ◽

pp. 2869-2881 ◽

Cited By ~ 155

Author(s):

Christof Geldmacher ◽

Njabulo Ngwenyama ◽

Alexandra Schuetz ◽

Constantinos Petrovas ◽

Klaus Reither ◽

...

Keyword(s):

T Cells ◽

Mycobacterium Tuberculosis ◽

Hiv Infection ◽

Cd4 T Cells ◽

Staphylococcal Enterotoxin B ◽

Opportunistic Pathogens ◽

Rapid Loss ◽

Hiv 1

HIV-1 infection results in the progressive loss of CD4 T cells. In this study, we address how different pathogen-specific CD4 T cells are affected by HIV infection and the cellular parameters involved. We found striking differences in the depletion rates between CD4 T cells to two common opportunistic pathogens, cytomegalovirus (CMV) and Mycobacterium tuberculosis (MTB). CMV-specific CD4 T cells persisted after HIV infection, whereas MTB-specific CD4 T cells were depleted rapidly. CMV-specific CD4 T cells expressed a mature phenotype and produced very little IL-2, but large amounts of MIP-1β. In contrast, MTB-specific CD4 T cells were less mature, and most produced IL-2 but not MIP-1β. Staphylococcal enterotoxin B–stimulated IL-2–producing cells were more susceptible to HIV infection in vitro than MIP-1β–producing cells. Moreover, IL-2 production was associated with expression of CD25, and neutralization of IL-2 completely abrogated productive HIV infection in vitro. HIV DNA was found to be most abundant in IL-2–producing cells, and least abundant in MIP-1β–producing MTB-specific CD4 T cells from HIV-infected subjects with active tuberculosis. These data support the hypothesis that differences in function affect the susceptibility of pathogen-specific CD4 T cells to HIV infection and depletion in vivo, providing a potential mechanism to explain the rapid loss of MTB-specific CD4 T cells after HIV infection.

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IN VITRO MODELS OF MYCOBACTERIUM TUBERCULOSIS DORMANCY, AND IN VIVO MODELS OF LATENT TUBERCULOSIS INFECTION

Russian Clinical Laboratory Diagnostics ◽

10.18821/0869-2084-2019-64-5-299-307 ◽

2019 ◽

Vol 64 (5) ◽

pp. 299-307

Author(s):

M. V. Fursov ◽

I. A. Dyatlov ◽

V. D. Potapov

Keyword(s):

Mycobacterium Tuberculosis ◽

Latent Tuberculosis Infection ◽

Latent Tuberculosis ◽

In Vitro Models ◽

Tuberculosis Infection ◽

Published Data ◽

In Vivo Models ◽

Dormant State

Modeling of tuberculosis infection is carried out in order to clarify various aspects of the tuberculosis pathogenesis, as well as the testing of new anti-tuberculosis drugs. The characteristic of in vitro models (n = 16) for Mycobacterium tuberculosis dormant state and in vivo models (n = 14) for the latent tuberculosis infection involving several animal species published to date are presented in this review. A brief description of the models and the results obtained by the authors are presented. The analysis of the published data reflects the list of methodological procedures that allow researchers to study the mechanism of the transition of M. tuberculosis cells to a dormant state and reverse to metabolically active state, as well as the process of conversion of active tuberculosis infection to a latent tuberculosis and reactivation.

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Preclinical Testing of the Nitroimidazopyran PA-824 for Activity against Mycobacterium tuberculosis in a Series of In Vitro and In Vivo Models

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.6.2294-2301.2005 ◽

2005 ◽

Vol 49 (6) ◽

pp. 2294-2301 ◽

Cited By ~ 207

Author(s):

Anne J. Lenaerts ◽

Veronica Gruppo ◽

Karen S. Marietta ◽

Christine M. Johnson ◽

Diane K. Driscoll ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Bacterial Load ◽

Multidrug Resistant ◽

Infection Model ◽

Significant Activity ◽

Dependent Manner ◽

In Vivo Models ◽

Long Term Treatment

ABSTRACT This study extends earlier reports regarding the in vitro and in vivo efficacies of the nitroimidazopyran PA-824 against Mycobacterium tuberculosis. PA-824 was tested in vitro against a broad panel of multidrug-resistant clinical isolates and was found to be highly active against all isolates (MIC < 1 μg/ml). The activity of PA-824 against M. tuberculosis was also assessed grown under conditions of oxygen depletion. PA-824 showed significant activity at 2, 10, and 50 μg/ml, similar to that of metronidazole, in a dose-dependent manner. In a short-course mouse infection model, the efficacy of PA-824 at 50, 100, and 300 mg/kg of body weight formulated in methylcellulose or cyclodextrin/lecithin after nine oral treatments was compared with those of isoniazid, rifampin, and moxifloxacin. PA-824 at 100 mg/kg in cyclodextrin/lecithin was as active as moxifloxacin at 100 mg/kg and isoniazid at 25 mg/kg and was slightly more active than rifampin at 20 mg/kg. Long-term treatment with PA-824 at 100 mg/kg in cyclodextrin/lecithin reduced the bacterial load below 500 CFU in the lungs and spleen. No significant differences in activity between PA-824 and the other single drug treatments tested (isoniazid at 25 mg/kg, rifampin at 10 mg/kg, gatifloxacin at 100 mg/kg, and moxifloxacin at 100 mg/kg) could be observed. In summary, its good activity in in vivo models, as well as its activity against multidrug-resistant M. tuberculosis and against M. tuberculosis isolates in a potentially latent state, makes PA-824 an attractive drug candidate for the therapy of tuberculosis. These data indicate that there is significant potential for effective oral delivery of PA-824 for the treatment of tuberculosis.

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Attenuated replication and pathogenesis of SARS-CoV-2 B.1.1.529 Omicron

10.21203/rs.3.rs-1200124/v1 ◽

2021 ◽

Author(s):

Kwok-Yung Yuen ◽

Huiping Shuai ◽

Jasper Fuk-Woo Chan ◽

Bingjie Hu ◽

Yue Chai ◽

...

Keyword(s):

Immune Escape ◽

Vaccination Strategy ◽

Body Weight Loss ◽

Human Populations ◽

Lung Pathology ◽

Evolutionary Trajectory ◽

In Vivo Models ◽

Alpha Beta

Abstract SARS-CoV-2 Omicron emerged in November 2021 and is rapidly spreading among the human populations. The variant contains 34 changes in its spike protein including 15 substitutions at the receptor-binding domain (RBD). While recent reports reveal that the Omicron variant can robustly escape from vaccine and therapeutic neutralization antibodies, the pathogenicity of the virus remains unknown. Here, we investigate the virological features and pathogenesis of the Omicron variant using in vitro and in vivo models. Our results demonstrate that the replication of the Omicron variant is dramatically attenuated in Calu3 and Caco2 but not in VeroE6 cells. Further mechanistic investigations reveal that the Omicron variant is deficient in transmembrane serine protease 2 (TMPRSS2) usage in comparison to that of WT, Alpha, Beta, and Delta variant, which explained its inefficient replication in Calu3 and Caco2 cells. Importantly, the replication of the Omicron variant is markedly attenuated in both the upper and lower respiratory tract of infected K18-hACE2 mice in comparison to that of WT and Delta variant, which results in its dramatically ameliorated lung pathology. When compared with SARS-CoV-2 WT, Alpha, Beta, and Delta variant, infection by the Omicron variant causes the least body weight loss and mortality rate. Overall, our study demonstrates that the Omicron variant is significantly attenuated in virus replication and pathogenicity in comparison with WT and previous variants. Our data suggest the current global vaccination strategy has forced SARS-CoV-2 into a new evolutionary trajectory towards reduced replication fitness in exchange of better immune escape. These findings are critical for setting policy in the pandemic control and disease management of COVID-19.

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Bugs on Drugs: A Drosophila melanogaster Gut Model to Study In Vivo Antibiotic Tolerance of E. coli

Microorganisms ◽

10.3390/microorganisms10010119 ◽

2022 ◽

Vol 10 (1) ◽

pp. 119

Author(s):

Bram Van den Bergh

Keyword(s):

Drosophila Melanogaster ◽

In Vitro Assays ◽

Antibiotic Tolerance ◽

In Vivo Models ◽

Antibiotic Development ◽

E Coli ◽

Germ Free ◽

Flexible System

With an antibiotic crisis upon us, we need to boost antibiotic development and improve antibiotics’ efficacy. Crucial is knowing how to efficiently kill bacteria, especially in more complex in vivo conditions. Indeed, many bacteria harbor antibiotic-tolerant persisters, variants that survive exposure to our most potent antibiotics and catalyze resistance development. However, persistence is often only studied in vitro as we lack flexible in vivo models. Here, I explored the potential of using Drosophila melanogaster as a model for antimicrobial research, combining methods in Drosophila with microbiology techniques: assessing fly development and feeding, generating germ-free or bacteria-associated Drosophila and in situ microscopy. Adult flies tolerate antibiotics at high doses, although germ-free larvae show impaired development. Orally presented E. coli associates with Drosophila and mostly resides in the crop. E. coli shows an overall high antibiotic tolerance in vivo potentially resulting from heterogeneity in growth rates. The hipA7 high-persistence mutant displays an increased antibiotic survival while the expected low persistence of ΔrelAΔspoT and ΔrpoS mutants cannot be confirmed in vivo. In conclusion, a Drosophila model for in vivo antibiotic tolerance research shows high potential and offers a flexible system to test findings from in vitro assays in a broader, more complex condition.

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Stringent Response Factors PPX1 and PPK2 Play an Important Role in Mycobacterium tuberculosis Metabolism, Biofilm Formation, and Sensitivity to IsoniazidIn Vivo

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01139-16 ◽

2016 ◽

Vol 60 (11) ◽

pp. 6460-6470 ◽

Cited By ~ 19

Author(s):

Yu-Min Chuang ◽

Noton K. Dutta ◽

Chien-Fu Hung ◽

T.-C. Wu ◽

Harvey Rubin ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Biofilm Formation ◽

Tricarboxylic Acid Cycle ◽

Antibiotic Sensitivity ◽

Stringent Response ◽

Antibiotic Tolerance ◽

Inorganic Polyphosphate ◽

Response Factors ◽

Content Type

ABSTRACTMycobacterium tuberculosisremains a global health threat largely due to the lengthy duration of curative antibiotic treatment, contributing to medical nonadherence and the emergence of drug resistance. This prolonged therapy is likely due to the presence ofM. tuberculosispersisters, which exhibit antibiotic tolerance. Inorganic polyphosphate [poly(P)] is a key regulatory molecule in theM. tuberculosisstringent response mediating antibiotic tolerance. The polyphosphate kinase PPK1 is responsible for poly(P) synthesis inM. tuberculosis, while the exopolyphosphatases PPX1 and PPX2 and the GTP synthase PPK2 are responsible for poly(P) hydrolysis. In the present study, we show by liquid chromatography-tandem mass spectrometry that poly(P)-accumulatingM. tuberculosismutant strains deficient inppx1orppk2had significantly lower intracellular levels of glycerol-3-phosphate (G3P) and 1-deoxy-xylulose-5-phosphate. Real-time PCR revealed decreased expression of genes in the G3P synthesis pathway in each mutant. Theppx1-deficient mutant also showed a significant accumulation of metabolites in the tricarboxylic acid cycle, as well as altered arginine and NADH metabolism. Each poly(P)-accumulating strain showed defective biofilm formation, while deficiency ofppk2was associated with increased sensitivity to plumbagin and meropenem and deficiency ofppx1led to enhanced susceptibility to clofazimine. A DNA vaccine expressingppx1andppk2, together with two other members of theM. tuberculosisstringent response,M. tuberculosisrelandsigE, did not show protective activity against aerosol challenge withM. tuberculosis, but vaccine-induced immunity enhanced the killing activity of isoniazid in a murine model of chronic tuberculosis. In summary, poly(P)-regulating factors of theM. tuberculosisstringent response play an important role inM. tuberculosismetabolism, biofilm formation, and antibiotic sensitivityin vivo.

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