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Interplay of human macrophages and Mycobacterium tuberculosis phenotypes
Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences.ORCID iD: 0000-0001-8871-2560
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 [en]
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: urn:nbn:se:liu:diva-132321DOI: 10.3384/diss.diva-132321ISBN: 9789176856901 (Print)OAI: oai:DiVA.org:liu-132321DiVA: diva2:1043676
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
List of papers
1. 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: 2016-10-31Bibliographically approved
2. Replication Rates of Mycobacterium tuberculosis in Human Macrophages Do Not Correlate with Mycobacterial Antibiotic Susceptibility
Open this publication in new window or tab >>Replication Rates of Mycobacterium tuberculosis in Human Macrophages Do Not Correlate with Mycobacterial Antibiotic Susceptibility
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2014 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 9, no 11, e112426- p.Article in journal (Refereed) Published
Abstract [en]

The standard treatment of tuberculosis (TB) takes six to nine months to complete and this lengthy therapy contributes to the emergence of drug-resistant TB. TB is caused by Mycobacterium tuberculosis (Mtb) and the ability of this bacterium to switch to a dormant phenotype has been suggested to be responsible for the slow clearance during treatment. A recent study showed that the replication rate of a non-virulent mycobacterium, Mycobacterium smegmatis, did not correlate with antibiotic susceptibility. However, the question whether this observation also holds true for Mtb remains unanswered. Here, in order to mimic physiological conditions of TB infection, we established a protocol based on long-term infection of primary human macrophages, featuring Mtb replicating at different rates inside the cells. During conditions that restricted Mtb replication, the bacterial phenotype was associated with reduced acid-fastness. However, these phenotypically altered bacteria were as sensitive to isoniazid, pyrazinamide and ethambutol as intracellularly replicating Mtb. In support of the recent findings with M. smegmatis, we conclude that replication rates of Mtb do not correlate with antibiotic tolerance.

Place, publisher, year, edition, pages
Public Library of Science, 2014
National Category
Clinical Medicine Basic Medicine
Identifiers
urn:nbn:se:liu:diva-113014 (URN)10.1371/journal.pone.0112426 (DOI)000345250400061 ()25386849 (PubMedID)
Note

Funding Agencies|Bill and Melinda Gates Foundation; Swedish Research Council [2009-3821, 2012-3349]; Swedish International Development Cooperation Agency; Swedish Heart-Lung Foundation; King Oscar II Foundation; Carl Trygger Foundation; Clas Groschinsky Foundation

Available from: 2015-01-12 Created: 2015-01-08 Last updated: 2016-10-31

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