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Incorporation of Mycobacterium tuberculosis lipoarabinomannan into macrophage membrane rafts is a prerequisite for the phagosomal maturation block.
Linköping University, Department of Molecular and Clinical 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, Medical Microbiology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
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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.

Place, publisher, year, edition, pages
2008. Vol. 76, no 7, 2882-2887 p.
National Category
Microbiology in the medical area
Identifiers
URN: urn:nbn:se:liu:diva-20816DOI: 10.1128/IAI.01549-07PubMedID: 18426888OAI: oai:DiVA.org:liu-20816DiVA: diva2:236348
Available from: 2009-09-22 Created: 2009-09-22 Last updated: 2011-02-22Bibliographically approved
In thesis
1. Leukocyte responses to pathogens: integrins, membrane rafts and nitric oxide
Open this publication in new window or tab >>Leukocyte responses to pathogens: integrins, membrane rafts and nitric oxide
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

During microbial invasion, leukocytes of the innate immunity are rapidly recruited to the site of infection where they internalize (phagocytose), kill and digest the invaders. To aid this process, leukocytes express surface receptors such as Toll-like receptors, β2-integrins and Fc-receptors. The β2-integrins are also used for attachment to the extracellular matrix and are important for migration. When pro- vs. anti-inflammatory regulation of β2-integrins was investigated, it was found that chemotactic factors modulate neutrophil adhesion through altered affinity and/or avidity of β2-integrins. A bacteria-derived chemoattractant evoked a large increase in affinity as well as in mobility and clustering, while an early, host-derived chemotactic factor induced increased clustering and surface mobility, but only a slight increase in affinity. Anti-inflammatory lipoxin affected β2-integrin avidity, but not affinity.

The leukocyte membrane is composed of lipids and proteins, which are inhomogeneously distributed. Specific domains in the membrane, membrane rafts, are enriched in signaling proteins and receptors. It was found that lipophosphoglycan (LPG) a virulence factor and membrane component of the parasite Leishmania donovani, accumulated in macrophage rafts during infection, inhibited PKCα translocation to the membrane and halted phagosomal maturation. Membrane rafts were instrumental for LPG to exert its effect. We further showed that nitric oxide (NO) rescued phagosomal maturation halted by Leishmania donovani parasites, possibly through effects on actin dynamics. NO did not affect parasite virulence per se. Moreover, lipoarabinomannan (LAM), a virulence factor on Mycobacterium tuberculosis (Mtb) bacteria, also inserted itself into macrophage membrane rafts. LAM from a less virulent strain (PILAM) was less efficiently inserted. Insertion could to some extent be inhibited by phosphatidylinositol mannoside (PIM), another structural molecule from Mtb. LAM did not activate the p38 MAPK signaling pathway nor did LAM interfere with TLR 2 or 4 signaling. In neutrophil leukocytes we observed a simultaneous, calciumdependent up-regulation of membrane rafts and secretion of azurophilic granules at the site of phagocytosis. Rafts were also found in the phagosome membrane. Wild type Streptococcus pyogenes bacteria, which can survive phagocytosis, modulated raft delivery.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2008. 63 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1058
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:liu:diva-43383 (URN)73683 (Local ID)978-91-7393-921-8 (ISBN)73683 (Archive number)73683 (OAI)
Public defence
2008-05-28, Berzeliussalen, Hälsouniversitetet, Campus US, Linköpings universitet, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2015-11-19Bibliographically approved
2. Survival strategies of Mycobacterium tuberculosis inside the human macrophage
Open this publication in new window or tab >>Survival strategies of Mycobacterium tuberculosis inside the human macrophage
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:nbn:se:liu:diva-65452 (URN)978-91-7393-251-6 (ISBN)
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

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Welin, AmandaWinberg Tinnerfelt, MartinAbdalla, HanaSärndahl Lindblom, EvaRasmusson, BirgittaStendahl, OlleLerm, Maria

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