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Susceptibility of Clinical Strains of Mycobacterium tuberculosis to Reactive Nitrogen Species in Activated Macrophages
Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Background: Nitric oxide (NO) is produced in macrophages by the inducible NO synthase (iNOS) upon activation by pro-inflammatory cytokines. NO has been shown to be essential for the control of Mycobacterium tuberculosis infection in murine models whereas its importance in man is not as clear. There is a lack of studies regarding the susceptibility to reactive nitrogen species (RNS) in clinical strains of M. tuberculosis and the relation to first-line drug resistance, such as to isoniazid (INH). The aim of this study was to explore susceptibility to RNS and intracellular survival of clinical strains of M. tuberculosis, with or without INH resistance.

Method: Seven clinical strains of M. tuberculosis were transformed with the pSMT1-plasmid encoding Vibrio harveyi luciferase. Survival was analysed by luminometry following exposure to the NO donor DETA/NO or peroxynitrite (SIN-1). Intracellular killing was studied in murine macrophages (RAW 264.7) activated with interferon gamma (IFN-γ) and lipopolysaccharide (LPS).

Results: There was a significant effect on growth control of M. tuberculosis strains upon macrophage activation, which showed variability among clinical isolates. In the cell-free system, all strains showed a dose-dependent susceptibility to DETA/NO and SIN-1, and clinical strains were in general more resistant than H37Rv to DETA/NO. INH-resistant strains with an inhA mutation were significantly more tolerant to DETA/NO than inhA wild type.

Conclusion: Reactive nitrogen species inhibited growth of clinical M. tuberculosis isolates both in an intra- and extracellular model with significant difference between strains. Increased tolerance to NO was associated with isoniazid resistance mediated by inhA.

National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-77131OAI: oai:DiVA.org:liu-77131DiVA: diva2:525145
Available from: 2012-05-07 Created: 2012-05-07 Last updated: 2012-05-07Bibliographically approved
In thesis
1. The Role of Nitric Oxide in Host Defence Against Mycobacterium tuberculosis: Clinical and Experimental Studies
Open this publication in new window or tab >>The Role of Nitric Oxide in Host Defence Against Mycobacterium tuberculosis: Clinical and Experimental Studies
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), responsible for significant morbidity and mortality worldwide, especially in low-income countries. Considering aggravating factors, such as HIV co-infection and emerging drug resistance, new therapeutic interventions are urgently needed. Following exposure to M. tuberculosis, surprisingly few individuals will actually develop active disease, indicating effective defence mechanisms. One such candidate is nitric oxide (NO). The role of NO in human TB is not fully elucidated, but has been shown to have a vital role in controlling TB in animal models.

The general aim of this thesis was to investigate the role of NO in the immune defence against M. tuberculosis, by combining clinical and experimental studies. In pulmonary TB patients, we found low levels of NO in exhaled air, and low levels of NO metabolites in urine. HIV coinfection decreased levels of exhaled NO even further, reflecting a locally impaired NO production in the lung. Low levels of exhaled NO were associated with a decreased cure rate in HIV-positive TB patients. Household contacts to sputum smear positive TB patient presented the highest levels of both urinary NO metabolites and exhaled NO. Malnutrition, a common condition in TB, may lead to deficiencies of important nutrients such as the amino acid L-arginine, essential for NO production. We therefore assessed the effect of an argininerich food supplement (peanuts) in a clinical trial including pulmonary TB patients, and found that peanut supplementation increased cure rate in HIV-positive TB patients.

We also investigated NO susceptibility of clinical strains of M. tuberculosis, and its association to clinical outcome and antibiotic resistance. Patients infected with strains of M. tuberculosis with reduced susceptibility to NO in vitro, showed a tendency towards lower rate of weight gain during treatment. Moreover, there was a clear variability between strains in the susceptibility to NO, and in intracellular survival within NO-producing macrophages. A novel finding, that can be of importance in understanding drug resistance and for drug development, was that reduced susceptibility to NO was associated with resistance to firstline TB drugs, in particular isoniazid and mutations in inhA.

Taken together, the data presented here show that NO plays a vital role  in human immune defence against TB, and although larger multicentre studies are warranted, arginine-rich food supplementation can be recommended to malnourished HIV co-infected patients on TB treatment.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2012. 86 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1304
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-77145 (URN)978-91-7519-911-5 (ISBN)
Public defence
2012-06-07, Berzeliussalen, Hälsouniversitetet, Campus US, Linköpings universitet, Linköping, 09:00 (English)
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Available from: 2012-05-07 Created: 2012-05-07 Last updated: 2012-05-14Bibliographically approved

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Idh, JonnaLerm, MariaRaffetseder, JohannaEklund, DanielLarsson, MariePienaar, ElsjeForslund, TonySundqvist, TommyStendahl, Olle

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