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Survival strategies of Mycobacterium tuberculosis inside the human macrophage
Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences. (Maria Lerm)
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Mycobacterium tuberculosis (Mtb) is the bacterium responsible for tuberculosis (TB). For decades, it was believed that TB was a disease of the past, but the onset of the HIV epidemic resulting in a greatly increased number of TB cases, the emergence of antibiotic resistant Mtb strains, and the relative ineffectiveness of the BCG vaccine have put TB back on the agenda. With almost two million people being killed by TB each year, the World Health Organization has declared it a global emergency. TB is an especially big issue in low-income countries, where crowded living conditions accelerates spread of the disease, and where access to health care and medication is problematic. Mtb spreads by aerosol and infects its host through the airways. The bacterium is phagocytosed by resident macrophages in the lung, and when successful is able to replicate inside these cells, which are actually designed to kill invading microbes. Mtb is able to evade macrophage responses in part by inhibiting the fusion between the phagosome in which it resides and bactericidal lysosomes, as well as by dampening the acidification of the vacuole. The initial macrophage infection results in a pro-inflammatory response and the recruitment of other cells of the innate and adaptive immune systems, giving rise to the hallmark of Mtb infection – the granuloma. It is believed that in up to 50 % of exposed individuals, however, the infection is cleared without the involvement of the adaptive immune system, indicating that the innate immune system may be able to control or clear the infection if activated appropriately. This thesis focuses on the interaction between the host macrophage and Mtb, aiming to understand some of the mechanisms employed by the bacterium to evade macrophage responses to enable replication and spread to new host cells. Furthermore, mechanisms used by the macrophage to keep the infection under control were studied, and a method that could be used to measure the replication of the bacilli inside macrophages in vitro in an efficient way was developed. We found that a mycobacterial glycoprotein, mannose-capped lipoarabinomannan (ManLAM), which is shed from the bacilli during phagocytosis by macrophages, integrates into membrane raft domains of the host cell membrane via its GPI anchor. This integration leads to an inhibition of phagosomal maturation. Subsequently, we developed a luciferase-based method by which intracellular replication of Mtb as well as viability of the host macrophage could be measured in a rapid, inexpensive and quantitative way in a 96-well plate. This method could be used for drug screening as well as for studying the different host and bacterial factors that influence the growth of Mtb inside the host cell. Using this method, we discovered that infection of macrophages with Mtb at a low multiplicity of infection (MOI) led to effective control of bacterial growth by the cell, and that this was dependent on functional lysosomal proteases as well as phagosomal acidification. However, we found no correlation between controlled bacterial growth and the translocation of late endosomal membrane proteins to the phagosome, showing that these markers are poor indicators of phagosomal functionality. Furthermore, we discovered that infection of macrophages with Mtb at a higher MOI led to replication of the bacilli accompanied by host cell death within a few days. We characterized this cell death, and concluded that when replication of Mtb inside macrophages reaches a certain threshold and the bacteria secrete a protein termed ESAT-6, necrotic cell death of the host cell occurs. However, although the bacilli activated inflammasome complexes in the host cell and IL-1β was secreted during infection of macrophages, Mtb infection did not induce either of the recently characterized inflammasome-related cell death types pyroptosis or pyronecrosis. Thus, we have elucidated some of the strategies that Mtb uses to be able to survive and replicate inside the macrophage and spread to new cells, as well as studied the conditions under which the host cell is able to control infection. This knowledge could be used in the future for developing drugs that boost the innate immune system or targets bacterial virulence factors in the macrophage.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press , 2011. , 84 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1223
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-65452ISBN: 978-91-7393-251-6 (print)OAI: oai:DiVA.org:liu-65452DiVA: diva2:395814
Public defence
2011-03-04, Berzeliussalen, Hälsouniversitetet, Campus US, Linköpings universitet, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2011-02-22 Created: 2011-02-08 Last updated: 2011-02-22Bibliographically approved
List of papers
1. Incorporation of Mycobacterium tuberculosis lipoarabinomannan into macrophage membrane rafts is a prerequisite for the phagosomal maturation block.
Open this publication in new window or tab >>Incorporation of Mycobacterium tuberculosis lipoarabinomannan into macrophage membrane rafts is a prerequisite for the phagosomal maturation block.
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2008 (English)In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 76, no 7, 2882-2887 p.Article in journal (Refereed) Published
Abstract [en]

Lipoarabinomannan (LAM) is one of the key virulence factors for Mycobacterium tuberculosis, the etiological agent of tuberculosis. During uptake of mycobacteria, LAM interacts with the cell membrane of the host macrophage and can be detected throughout the cell upon infection. LAM can inhibit phagosomal maturation as well as induce a proinflammatory response in bystander cells. The aim of this study was to investigate how LAM exerts its action on human macrophages. We show that LAM is incorporated into membrane rafts of the macrophage cell membrane via its glycosylphosphatidylinositol anchor and that incorporation of mannose-capped LAM from M. tuberculosis results in reduced phagosomal maturation. This is dependent on successful insertion of the glycosylphosphatidylinositol anchor. LAM does not, however, induce the phagosomal maturation block through activation of p38 mitogen-activated protein kinase, contradicting some previous suggestions.

National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:liu:diva-20816 (URN)10.1128/IAI.01549-07 (DOI)18426888 (PubMedID)
Available from: 2009-09-22 Created: 2009-09-22 Last updated: 2011-02-22Bibliographically approved
2. Validation of a Medium-Throughput Method for Evaluation of Intracellular Growth of Mycobacterium tuberculosis
Open this publication in new window or tab >>Validation of a Medium-Throughput Method for Evaluation of Intracellular Growth of Mycobacterium tuberculosis
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2010 (English)In: Clinical and Vaccine Immunology, ISSN 1556-6811, E-ISSN 1556-679X, Vol. 17, no 4, 513-517 p.Article in journal (Refereed) Published
Abstract [en]

Intracellular pathogens such as Mycobacterium tuberculosis have adapted to a life inside host cells, in which they utilize host nutrients to replicate and spread. Ineffective methods for the evaluation of growth of intracellular pathogens in their true environment pose an obstacle for basic research and drug screening. Here we present the validation of a luminometry-based method for the analysis of intramacrophage growth of M. tuberculosis. The method, which is performed in a medium-throughput format, can easily be adapted for studies of other intracellular pathogens and cell types. The use of host cells in drug-screening assays dedicated to find antimicrobials effective against intracellular pathogens permits the discovery of not only novel antibiotics but also compounds with immunomodulatory and virulence-impairing activities, which may be future alternatives or complements to antibiotics.

Place, publisher, year, edition, pages
American Society for Microbiology, 2010
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-54868 (URN)10.1128/CVI.00446-09 (DOI)000276170900004 ()
Available from: 2010-04-16 Created: 2010-04-16 Last updated: 2017-10-31
3. Importance of phagosomal functionality for growth restriction of Mycobacterium tuberculosis in primary human macrophages
Open this publication in new window or tab >>Importance of phagosomal functionality for growth restriction of Mycobacterium tuberculosis in primary human macrophages
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2011 (English)In: Journal of Innate Immunity, ISSN 1662-811X, E-ISSN 1662-8128, Vol. 3, no 5, 508-518 p.Article in journal (Refereed) Published
Abstract [en]

The best characterized survival mechanism of Mycobacterium tuberculosis inside the macrophage is the inhibition of phagosomal maturation. Phagosomal maturation involves several steps including fusion with lysosomes and acidification. However, it has not been elucidated which components of phagosomal maturation correlate with growth restriction of virulent mycobacteria in human macrophages, and we aimed to study this. We infected human monocyte-derived macrophages with M. tuberculosis and assessed bacterial replication, translocation of CD63 to the phagosome, and phagosomal acidification. We found that unstimulated macrophages were able to control infection with M. tuberculosis upon inoculation at a low, but not high, multiplicity of infection (MOI). H37Rv and H37Ra infection, at both high and low MOI, led to equally ineffective translocation of CD63 to the phagosome. This was true despite the impaired growth ability of H37Rv at the low MOI and of H37Ra even at the high MOI, indicating that inhibition of CD63 translocation was not sufficient for growth to occur. On the other hand, acidification of mycobacterial phagosomes was more efficient at a low MOI with both mycobacterial strains, consistent with a role for phagosomal acidification in restricting M. tuberculosis growth. Inhibition of the vacuolar H+-ATPase as well as of cathepsin D led to enhanced mycobacterial replication inside the macrophage. We conclude that acidification and related functional aspects of the mature phagosome are important factors for restriction of M. tuberculosis replication in human macrophages.

Place, publisher, year, edition, pages
S. Karger, 2011
National Category
Basic Medicine
Identifiers
urn:nbn:se:liu:diva-65447 (URN)10.1159/000325297 (DOI)000294572500008 ()21576918 (PubMedID)
Note

Funding Agencies|Swedish Research Council|529-2003-5994,2005-7046,2006-5968,2007-2673,2009-3821|Bill and Melinda Gates Foundation||SIDA/SAREC||Ekhaga Foundation||Carl Trygger Foundation||King Gustaf V 80-Year Memorial Foundation||County Council of Ostergotland||Swedish Heart Lung Foundation||Oskar II Jubilee Foundation||Clas Groschinsky Foundation||Soderbergs Foundation||Colorado State University, Fort Collins (NIH, NIAID)|HHSN26620040 0091C|

Available from: 2011-02-08 Created: 2011-02-08 Last updated: 2017-12-11Bibliographically approved
4. Human macrophages infected with virulent Mycobacterium tuberculosis undergo ESAT-6-dependent necrosis, but not pyroptosis or pyronecrosis
Open this publication in new window or tab >>Human macrophages infected with virulent Mycobacterium tuberculosis undergo ESAT-6-dependent necrosis, but not pyroptosis or pyronecrosis
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Mycobacterium tuberculosis (Mtb) infects lung macrophages, which instead of killing the pathogen can be manipulated by the bacilli, creating an environment suitable for intracellular replication and spread to adjacent cells. The role of host cell death during Mtb infection is debated because the bacilli have been shown to be both anti-apoptotic, keeping the host cell alive to avoid the antimicrobial effects of apoptosis, and pro-necrotic, killing the host macrophage to allow infection of neighboring cells. Since mycobacteria are able to activate the NLRP3 inflammasome, we investigated whether Mtb could induce one of the recently described inflammasome-linked cell death modes pyroptosis and pyronecrosis, in human monocyte-derived macrophages. Cells were infected with virulent (H37Rv) Mtb at a multiplicity of infection (MOI) of 1 or 10. The higher MOI resulted in strongly enhanced release of IL-1β, while a low MOI gave no IL-1β response. The infected macrophages were collected and cell viability in terms of the integrity of DNA, mitochondria and the plasma membrane was determined. We found that infection with H37Rv at MOI 10, but not MOI 1, over two days led to extensive DNA fragmentation, loss of mitochondrial membrane potential and loss of plasma membrane integrity. Although we observed plasma membrane permeabilization and IL-1 β release from infected cells, the cell death induced by Mtb was not pyroptosis or pyronecrosis, as it was independent of caspase-1 and cathepsin B. Instead, we conclude that as virulent Mtb reaches a threshold number of bacilli inside the macrophage, ESAT-6-dependent necrosis occurs, activating caspase-1 in the process.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-65451 (URN)
Note
Submitted manuscriptAvailable from: 2011-02-08 Created: 2011-02-08 Last updated: 2011-02-22Bibliographically approved

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