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Characterizing phenotypes of Mycobacterium tuberculosis and exploring anti-mycobacterial compounds through high content screening
Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences.
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Tuberculosis (TB), an airborne disease and one of the top 10 causes of death globally, is caused by Mycobacterium tuberculosis (Mtb). Current standard therapy for TB treatment includes multiple drugs for a period of at least 6 months. The long therapy duration is to sterilize a small sub-population of drug-tolerant bacteria, a characteristic related to biofilm formation, which otherwise responsible for disease relapse. On the other hand, because of such a long treatment period, patient adherence to therapy becomes difficult, which results in the emergence of multidrug-resistant (MDR) or, in worst cases, extensively drug-resistant (XDR)-TB. TB is primarily a disease of lungs and alveolar macrophages are one of the first host cell types to encounter Mtb following aerosol transmission. A well-established role of macrophages in immune defense is phagocytosis, but recent studies also demonstrated that upon interaction with large aggregates of microbes or cord-forming mycobacterial species, macrophages could produce extracellular traps known as macrophage extracellular traps (METs). METs have a DNA backbone with embeds histones and could trap a wide range of microorganisms, but may or may not be able to kill them. Natural products are always a promising starting point for drug discovery because of their wide range of activity. A large number of world’s population is still using extracts from different parts of plants as the primary source of medicines against diseases including TB. Today much effort is being invested by academia in screening campaigns that allows for fast discovery of new active compounds. Thanks to the use of automated technology such as automated microscopy or automated image analysis (known as high content screening, HCS) phenotypic drug discovery has become easier to perform. Therefore, the identification of highly effective compounds to combat infectious diseases like TB can be facilitated by the use of host-pathogen assays at the early stages of drug screening studies.

This thesis describes the characterization and antibiotic sensitivity of different phenotypes of Mtb namely planktonic, cord-forming and biofilm-producing phenotypes that arise due to different culture conditions. The culture of Mtb with a high percentage of a detergent (Tween-80) and standing condition promoted planktonic phenotype while a culture with a low amount of Tween-80 and more aeration due to shaking promoted cording and biofilm phenotypes. Primary human macrophages upon interaction with the shaken culture of wild-type Mtb died by releasing METs. Whereas, the shaken cultures of early secreted antigenic target-6 (ESAT-6), an important virulence factor of Mtb, deletion mutant strain could not induce MET formation showing that the cord formation is related to virulence. Moreover, the biofilm phenotype of Mtb is more tolerant to two first-line antibiotics isoniazid (INH) and rifampicin (RIF) as compared to cording and planktonic phenotypes which demand a search of more effective TB therapy. A screening campaign based on a whole-cell assay using different ethanolic crude extracts of many African plants lead to the discovery of a hit, i.e., a chloroform fraction of Khaya senegalensis bark, which showed non-significant inhibition of intracellular growth of a virulent strain of Mtb was selected for further purification and evaluation. Lastly, we have also developed and validated an HCS assay to explore new compounds against intracellular Mtb in human macrophages. INH and RIF, which were found most effective in our system were used in a combination as a positive control to calculate a Z’ factor value, which confirmed our assay to be suitable for HCS.

In conclusion, this thesis not only highlights the biology of TB infection, but also discusses the development of a pathophysiologically relevant assay that can be used in the identification of novel compound(s) that has either direct anti-mycobacterial activity (antibiotic), acts by stimulating the host cell immune mechanisms (immunomodulator) or acts by counteracting virulence factors (virulence blocker).  

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2018. , p. 76
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1657
National Category
Microbiology in the medical area
Identifiers
URN: urn:nbn:se:liu:diva-153735DOI: 10.3384/diss.diva-153735ISBN: 9789176851548 (print)OAI: oai:DiVA.org:liu-153735DiVA, id: diva2:1276000
Public defence
2019-02-08, Berzeliussalen, Campus US, Linköping, 09:00 (English)
Opponent
Supervisors
Funder
Ekhaga FoundationCarl Tryggers foundation Swedish Research CouncilSwedish Heart Lung Foundation
Note

Funding:

This work was funded by the Ekhaga Foundation, Carl Trygger Foundation, the SwedishResearch Council, the Swedish Heart-Lung Foundation and Olav Thon Foundation and in-kindsupport by the SciLifeLab platform Chemical Biology Consortium Sweden sponsored by theSwedish Research Council, the SciLifeLab and Karolinska Institute (www.cbcs.se).

Available from: 2019-01-07 Created: 2019-01-07 Last updated: 2019-01-07Bibliographically approved
List of papers
1. The Cording Phenotype of Mycobacterium tuberculosis Induces the Formation of Extracellular Traps in Human Macrophages
Open this publication in new window or tab >>The Cording Phenotype of Mycobacterium tuberculosis Induces the Formation of Extracellular Traps in Human Macrophages
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2017 (English)In: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 7, article id 278Article in journal (Refereed) Published
Abstract [en]

The causative agent of tuberculosis, Mycobacterium tuberculosis, shares several characteristics with organisms that produce biofilms during infections. One of these is the ability to form tight bundles also known as cords. However, little is known of the physiological relevance of the cording phenotype. In this study, we investigated whether cord-forming M. tuberculosis induce the formation of macrophage extracellular traps (METs) in human monocyte-derived macrophages. Macrophages have previously been shown to produce extracellular traps in response to various stimuli. We optimized bacterial culturing conditions that favored the formation of the cord-forming phenotype as verified by scanning electron microscopy. Microscopy analysis of METs formation during experimental infection of macrophages with M. tuberculosis revealed that cord-forming M. tuberculosis induced significantly more METs compared to the non-cording phenotype. Deletion of early secreted antigenic target-6 which is an important virulence factor of M. tuberculosis, abrogated the ability of the bacteria to induce METs. The release of extracellular DNA from host cells during infection may represent a defense mechanism against pathogens that are difficult to internalize, including cord-forming M. tuberculosis.

Place, publisher, year, edition, pages
FRONTIERS MEDIA SA, 2017
Keywords
Mycobacterium tuberculosis; macrophage extracellular traps (METs); cording; Tween-80; virulence; early secreted antigenic target-6 (ESAT-6)
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:liu:diva-139276 (URN)10.3389/fcimb.2017.00278 (DOI)000404073500001 ()
Note

Funding Agencies|Swedish Research Council [2012-3349, 2015-02593]; Swedish Heart Lung Foundation [20130685, 20150709]

Available from: 2017-07-07 Created: 2017-07-07 Last updated: 2019-01-07
2. Antimycobacterial activity of selected medicinal plants traditionally used in Sudan to treat infectious diseases
Open this publication in new window or tab >>Antimycobacterial activity of selected medicinal plants traditionally used in Sudan to treat infectious diseases
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2014 (English)In: Journal of Ethnopharmacology, ISSN 0378-8741, E-ISSN 1872-7573, Vol. 157, p. 134-139Article in journal (Refereed) Published
Abstract [en]

Ethnopharmacological relevance: The emergence of multidrug-resistant strains of Mycobacterium tuberculosis underscores the need for continuous development of new and efficient methods to determine the susceptibility of isolates of Mycobacterium tuberculosis in the search for novel antimycobacterial agents. Natural products constitute an important source of new drugs, and design and implementation of antimycobacterial susceptibility testing methods are necessary to evaluate the different extracts and compounds. In this study we have explored the antimycobacterial properties of 50 ethanolic extracts from different parts of 46 selected medicinal plants traditionally used in Sudan to treat infectious diseases. Materials and methods: Plants were harvested and ethanolic extracts were prepared. For selected extracts, fractionation with hydrophilic and hydrophobic solvents was undertaken. A luminometry-based assay was used for determination of mycobacterial growth in broth cultures and inside primary human macrophages in the presence or absence of plant extracts and fractions of extracts. Cytotoxicity was also assessed for active fractions of plant extracts. Results: Of the tested extracts, three exhibited a significant inhibitory effect on an avirulent strain of Mycobacterium tubercluosis (H37Ra) at the initial screening doses (125 and 6.25 mu g/ml). These were bark and leaf extracts of Khaya senegalensis and the leaf extract of Rosmarinus officinalis L. Further fractions of these plant extracts were prepared with n-hexane, chloroform, ethyl acetate, n-butanol, ethanol and water, and the activity of these extracts was retained in hydrophobic fractions. Cytotoxicity assays revealed that the chloroform fraction of Khaya senegalensis bark was non-toxic to human monocyte-derived macrophages and other cell types at the concentrations used and hence, further analysis, including assessment of IC50 and intracellular activity was done with this fraction. Conclusion: These results encourage further investigations to identify the active compound(s) within the chloroform fraction of Khaya senegalensis bark. (C) 2014 Elsevier Ireland Ltd. All rights reserved.

Place, publisher, year, edition, pages
Elsevier, 2014
Keywords
Mycobacterium tuberculosis; Sudanese medicinal plants; Primary human macrophages; Luminescence reporter assay; Cytotoxicity assay
National Category
Clinical Medicine
Identifiers
urn:nbn:se:liu:diva-113785 (URN)10.1016/j.jep.2014.09.020 (DOI)000347022700016 ()25261689 (PubMedID)
Note

Funding Agencies|Ekhaga Foundation [2011-33]; Swedish Insitute

Available from: 2015-02-02 Created: 2015-01-30 Last updated: 2019-01-07

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7891011121312 of 13
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