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Importance of phagosomal functionality for growth restriction of Mycobacterium tuberculosis in primary human 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, 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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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. Vol. 3, no 5, 508-518 p.
National Category
Basic Medicine
Identifiers
URN: urn:nbn:se:liu:diva-65447DOI: 10.1159/000325297ISI: 000294572500008PubMedID: 21576918OAI: oai:DiVA.org:liu-65447DiVA: diva2:395797
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
In thesis
1. 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
2. Interplay of human macrophages and Mycobacterium tuberculosis phenotypes
Open this publication in new window or tab >>Interplay of human macrophages and Mycobacterium tuberculosis phenotypes
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Mycobacterium tuberculosis (Mtb) is the pathogen causing tuberculosis (TB), a disease most often affecting the lung. 1.5 million people die annually due to TB, mainly in low-income countries. Usually considered a disease of the poor, also developed nations recently put TB back on their agenda, fueled by the HIV epidemic and the global emergence of drug-resistant Mtb strains. HIV-coinfection is a predisposing factor for TB, and infection with multi-drug resistant and extremely drug resistant strains significantly impedes and lengthens antibiotic treatment, and increases fatality. Mtb is transmitted from a sick individual via coughing, and resident macrophages are the first cells to encounter the bacterium upon inhalation. These cells phagocytose intruders and subject them to a range of destructive mechanisms, aiming at killing pathogens and protecting the host. Mtb, however, has evolved to cope with host pressures, and has developed mechanisms to submerge macrophage defenses. Among these, inhibition of phagosomal maturation and adaptation to the intracellular environment are important features. Mtb profoundly alters its phenotype inside host cells, characterized by altered metabolism and slower growth. These adaptations contribute to the ability of Mtb to remain dormant inside a host during latent TB infection, a state that can last for decades. According to recent estimates, one third of the world’s population is latently infected with Mtb, which represents a huge reservoir for active TB disease. Mtb is also intrinsically tolerant to many antibiotics, and adaptation to host pressures enhances tolerance to first-line TB drugs. Therefore, TB antibiotic therapy takes 6 to 9 months, and current treatment regimens involve a combination of several antibiotics. Patient noncompliance due to therapeutic side effects as well as insufficient penetration of drugs into TB lesions are reasons for treatment failure and can lead to the rise of drug-resistant populations. In view of the global spread of drug-resistant strains, new antibiotics and treatment strategies are urgently needed.

In this thesis, we studied the interplay of the primary host cell of Mtb, human macrophages, and different Mtb phenotypes. A low-burden infection resulted in restriction of Mtb replication via phagolysosomal effectors and the maintenance of an inactive Mtb phenotype reminiscent of dormant bacteria. Macrophages remained viable for up to 14 days, and profiling of secreted cytokines mirrored a silent infection. On the contrary, higher bacterial numbers inside macrophages could not be controlled by phagolysosomal functions, and intracellular Mtb shifted their phenotype towards active replication. Although slowed mycobacterial replication is believed to render Mtb tolerant to antibiotics, we did not observe such an effect. Mtb-induced macrophage cell death is dependent on ESAT6, a small mycobacterial virulence factor involved in host cell necrosis and the spread of the pathogen. Although well-studied, the fate of ESAT6 inside infected macrophages has been enigmatic. Cultivation of Mtb is commonly carried out in broth containing detergent to avoid aggregation of bacilli due to their waxy cell wall. Altering cultivation conditions revealed the presence of a mycobacterial capsule, and ESAT6 situated on the mycobacterial surface. Infection of macrophages with this encapsulated Mtb phenotype resulted in rapid ESAT6-dependent host cell death, and ESAT6 staining was lost as bacilli were ingested by macrophages. These observations could reflect the earlier reported integration of ESAT6 into membranes followed by membrane rupture and host cell death.

In conclusion, the work presented in this thesis shows that the phenotype of Mtb has a significant impact on the struggle between the pathogen and human macrophages. Taking the bacterial phenotype into account can lead to the development of drugs active against altered bacterial populations that are not targeted by conventional antibiotics. Furthermore, deeper knowledge on Mtb virulence factors can inform the development of virulence blockers, a new class of antibiotics with great therapeutic potential.

Place, publisher, year, edition, pages
Linköping University Electronic Press, 2016. 87 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1537
Keyword
Mycobacterium tuberculosis, tuberculosis, macrophage, innate immunity, host-pathogen interaction, antibiotic tolerance, phagosomal maturation, bacterial phenotype, dormancy, persistence, virulence factor, ESAT-6, ESX-1
National Category
Infectious Medicine Microbiology in the medical area Immunology Microbiology Immunology in the medical area
Identifiers
urn:nbn:se:liu:diva-132321 (URN)10.3384/diss.diva-132321 (DOI)9789176856901 (ISBN)
Public defence
2016-11-30, Hasselquistsalen, Campus US, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2016-11-07 Created: 2016-10-31 Last updated: 2016-11-17Bibliographically approved

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