Combating Mycobacterium tuberculosis using electron microscopy

Mycobacterium tuberculosis is the deadliest bacterium in the history of mankind. Despite the development of antibiotics and vaccines, this aerosol transmitted human pathogen still causes 1.4 million deaths per year. Understanding the biology of these bacteria is even more relevant since the spread of multi and even total drug resistant strains. In the Van der Wel group, we are focusing on 3 aspects of the biology, mostly driven by high resolution analysis of these bacteria. By visualizing the bacteria in cells, but also in cultures we have a described a novel pathogenicity factor,  discovered the capsular layer is extremely labile and demonstrated a novel strategy to kill these bacteria.

Contact: N. van der Wel (n.n.vanderwel@amc.uva.nl)

Research overview

1. Subcellular trafficking of mycobacteria; a pathogenicity factor

Understanding the virulence mechanisms of Mycobacteria is essential for vaccine development. We have demonstrated that M. tuberculosis is able to translocate from the phagolysosomal compartment into the cytosol of host cells (van der Wel et al.,2007), which completely shifted the paradigm of the known virulence mechanisms of M. tuberculosis. The vaccine currently used to prevent M. tuberculosis infections is M. bovis BCG. This live vaccine was developed almost 1 century ago and has not been improved ever since. We collaborate with several groups worldwide (Amsterdam VU, Germany, France, Spain, Swiss, USA, South Africa) to improve this vaccine and understand the biology of tuberculosis infection (figure 1). The rationale behind the improving the vaccines lies in the analysis of the behavior of the bacteria in host cells. The subcellular localization within cultured cells is different for pathogenic bacteria such as M. tuberculosis compared to the nonpathogenic vaccines BCG (figure 2) and is subject of our ongoing research. We investigate different mutants for the subcellular trafficking in host cells and also the behavior of these bacteria in vivo using fluorescence and electron microscopy and Combined Light and Electron Microscopy (figure 3).

2. High resolution analysis of the lipid-rich capsular layer in Mycobacteria

The outermost layer of mycobacteria consists of a labile structure that contains several secreted products, which are involved in the immune responses of pathogenic bacteria (figure 4). In collaboration with Wilbert Bitters group (VUmc), we have investigated the capsular stability and demonstrated that the ESX5 secretion system is crucial. Now we are analyzing the regeneration of the capsular layer using cryo-EM and immunolabellings. The project serves both fundamental innovative research and has a clear application in the vaccine manufacturing as the capsular layer is the first encounter of the vaccine BCG with the host.

3. Condensation of DNA as a generic stress response in Mycobacterium tuberculosis

Multidrug-resistant tuberculosis is a serious threat to public health worldwide. Even in Europe, about 10% of the Mycobacterium tuberculosis cases are confronted with resistance. Recently we have discovered that M. tuberculosis condenses its DNA in response to antibiotic stress. Condensation is visualized using high resolution fluorescence and electron microscopy (figure 5). DNA-condensation of is an established mechanism to survive antibiotic stress, and in developing multidrug resistance. We have demonstrated that condensation appears rapidly after antibiotic stress and does not affect the viability of M. tuberculosis (figure 6). Interestingly patient derived, multidrug-resistant M. tuberculosis strains are more capable of condensing their DNA than drug-susceptible strains. These results imply that by condensing their DNA, mycobacteria can avoid the effects of antibiotics, which facilitates resistance development. Our study provides the first principal evidence that DNA-condensation is a reversible, generic stress response of viable M. tuberculosis, and we are currently screening for new therapeutic approaches to manipulate the DNA condensation and reduce survival specifically of latent and multidrug resistant TB.

4. Electron microscopy

At the Core facility Cellular Imaging, several different microscopy techniques are made available for the AMC-staff, and affiliated researchers (www.cellularimaging.nl). Since March 2014 Dr. Nicole van der Wel was appointed head Electron Microscopy. With a group of 3 research technicians (Daisy Picavet, Henk van der Veen,  and Anita Grootemaat) and 2 pathology technicians (Per Larsen and Jeannette Pankras) we assist researchers from the AMC and partners with performing Electron Microscopy. We give advice on the techniques needed, support performing and interpretation of the experiments.

In addition to the AMC work, we opened November 2015 the Electron Microscopy Centre Amsterdam (EMCA) (www.cellularimaging.nl/electron-microscopy/). The EMCA is a collaboration between several Amsterdam life science research institutes, including the Vumc, AMC, NKI, ACTA and NIN, and is housed at Core facility Cellular Imaging within the department of Medical Biology at the AMC. At the EMCA the operators and researchers from the different universities and institutes work on the various TEM and SEM microscopes and have shared work-discussions. The shared equipment is paid for by the involved partners as well as by various other academic and industrial users. As such we collaborate with most Amsterdam and various (inter)national research groups and have created an EM knowledge centre in Amsterdam. We have long standing expertise in transmission electron microscopy (TEM), scanning electron microscopy (SEM), but also apply new developments such as combined light electron microscopy (CLEM), tomography, immunoEM, and Cryo-EM

Figure 1. Schematic representation of the subcellular localization of pathogenic mycobacteria (in red) and the vaccine strain BCG and mutant BCG, designed to release its antigens into the cytosol.

Figure 2. Electron microscopy of 60 nm thin sections of  infected dendritic cells. Localization of pathogenic mycobacterium (A) M. tuberculosis (M.tub) differs from the nonpathogenic (B) M. bovis BCG (BCG) as pathogenic mycobacteria escape from the phagolysosomes (blue). Immunogold labelling for LAMP1 is present on lysosomes, and BCG containing phagolysosomes. Bottom images are a schematic cartoon of the lysosomes color coded in blue and the bacteria in red.

Figure 3. In vivo localization of pathogenic mycobacteria imaged with Transmission Electron Microscopy (TEM), Fluorescence Microscopy with nuclei in blue, vessel in red and mycobacteria in green.

Figure 4. Cryo-EM of intact mycobacteria cultured with (left panel) and without detergent (right panel). Bacteria clump without detergent but at high magnification, the capsular layer can be visualized.

Figure 5. Combined Light and Electron Microscopy of mycobacteria stained for lipids (red) and DNA (green). Mycobacteria can condense their DNA/nucleoid when viable.

Figure 6. Fluorescent microscopy of bacteria with (top) and without (bottom) antibiotics stained for DNA (blue) and lipids (red). After treatment with antibiotics, the bacteria appear to have a single clump of DNA while in the untreated a chain of smaller structure form the nucleoid.

People

Nicole van der Wel, M3-133, 64757, N.N.vanderwel@amc.nl
Head Electron Microscopy

Henk van Veen, M3-106-1, 64703, H.A.vanVeen@amc.nl
Manager Electron Microscopes, Epon, negative staining, SEM

Daisy Picavet, M3-131, 60080, D.I.Picavet@amc.nl
ImmunoEM, Epon, negative staining, SEM

Anita Grootemaat, M3-106-1, 64703, a.e.grootemaat@amc.uva.nl
ImmunoEM, Epon, negative staining

Per Larson, M3-131, 65634, p.w.larsen@amc.uva.nl
TEM diagnostics

Jeannette Pankras, M3-131, j.e.pankras@amc.uva.nl
TEM diagnostics

 

Students: from 2014
Larissa Verkroost
Arno Loef
Sadhana Khanal
Abel Overzier
Elif Karakoç
Zehui Zhang
Edwin Schol
Marcella de Boer
Enzo Scutigliani
Jessie Snoep

Publications
  1. Bedussi B, van der Wel NN, de Vos J, van Veen H, Siebes M, VanBavel E, Bakker ENTP, Paravascular channels, cisterns, and the subarachnoid space in the rat brain: A single compartment with preferential pathways. J CEREBR BLOOD F MET 2017;37 (4):1374-1385 [PubMed]
  2. Beldman TJ, Senders ML, Alaarg A, Pérez-Medina C, Tang J, Zhao Y, Fay F, Deichmöller J, Born B, Desclos E, van der Wel NN, Hoebe RA, Kohen F, Kartvelishvily E, Neeman M, Reiner T, Calcagno C, Fayad ZA, de Winther MPJ, Lutgens E, Mulder WJM, Kluza E, Hyaluronan Nanoparticles Selectively Target Plaque-Associated Macrophages and Improve Plaque Stability in Atherosclerosis. ACS NANO 2017;11 (6):5785-5799 [PubMed]
  3. Gao Y, Vidal-Itriago A, Kalsbeek MJ, Layritz C, García-Cáceres C, Tom RZ, Eichmann TO, Vaz FM, Houtkooper RH, van der Wel N, Verhoeven AJ, Yan J, Kalsbeek A, Eckel RH, Hofmann SM, Yi CX, Lipoprotein Lipase Maintains Microglial Innate Immunity in Obesity. CELL REP 2017;20 (13):3034-3042 [PubMed]
  4. Maat DS, Biggs T, Evans C, van Bleijswijk JDL, van der Wel NN, Dutilh BE, Brussaard CPD, Characterization and Temperature Dependence of Arctic Micromonas polaris Viruses. VIRUSES-BASEL 2017;9 (6):134 [PubMed]
  5. van Zon M, de Punder K, van der Sar A, Brosch R, Pando RH, Grootemaat A , Tigchelaar-Gutter W, van der Wel N, Subcellular localization of M. tuberculosis in vivo and effect of the adaptive immunity. ULTRASTRUCT PATHOL 2017;41 (1):133 [PubMed]
  6. Abhyankar WR, Kamphorst K, Swarge BN, van Veen H, van der Wel NN, Brul S, de Koster CG, de Koning LJ, The Influence of Sporulation Conditions on the Spore Coat Protein Composition of Bacillus subtilis Spores. FRONT MICROBIOL 2016;7:1636 [PubMed]
  7. Ates LS, van der Woude AD, Bestebroer J, van Stempvoort G, Musters RJP, Garcia-Vallejo JJ, Picavet DI, Weerd Rvd, Maletta M, Kuijl CP, van der Wel NN, Bitter W, The ESX-5 System of Pathogenic Mycobacteria Is Involved In Capsule Integrity and Virulence through Its Substrate PPE10. PLOS PATHOG 2016;12 (6):e1005696 [PubMed]
  8. Potze L, Di Franco S, Grandela C, Pras-Raves ML, Picavet DI, van Veen HA, van Lenthe H, Mullauer FB, van der Wel NN, Luyf A, van Kampen AHC, Kemp S, Everts V, Kessler JH, Vaz FM, Medema JP, Betulinic acid induces a novel cell death pathway that depends on cardiolipin modification. ONCOGENE 2016;35 (4):427-437 [PubMed]
  9. Ribeiro CMS, Sarrami-Forooshani R, Setiawan LC, Zijlstra-Willems EM, van Hamme JL, Tigchelaar W, van der Wel NN, Kootstra NA, Gringhuis SI, Geijtenbeek TBH, Receptor usage dictates HIV-1 restriction by human TRIM5α in dendritic cell subsets. NATURE 2016;540 (7633):448-452 [PubMed]
  10. van der Spek AH, Bloise FF, Tigchelaar W, Dentice M, Salvatore D, van der Wel NN, Fliers E, Boelen A, The Thyroid Hormone Inactivating Enzyme Type 3 Deiodinase is Present in Bactericidal Granules and the Cytoplasm of Human Neutrophils. ENDOCRINOLOGY 2016;157 (8):3293-3305 [PubMed]
  11. Zheng L, Abhyankar W, Ouwerling N, Dekker HL, van Veen H, van der Wel NN, Roseboom W, de Koning LJ, Brul S, de Koster CG, Bacillus subtilis Spore Inner Membrane Proteome. J PROTEOME RES 2016;15 (2):585-594 [PubMed]
  12. Daleke-Schermerhorn MH, Felix T, Soprova Z, ten Hagen-Jongman CM, Vikström D, Majlessi L, Beskers J, Follmann F, de Punder K, van der Wel NN, Baumgarten T, Pham TV, Piersma SR, Jiménez CR, van Ulsen P, de Gier JW, Leclerc C, Jong WSP, Luirink J, Decoration of outer membrane vesicles with multiple antigens by using an autotransporter approach. APPL ENVIRON MICROB 2014;80 (18):5854-5865 [PubMed]
  13. Jong WSP, Daleke-Schermerhorn MH, Vikström D, ten Hagen-Jongman CM, de Punder K, van der Wel NN, van de Sandt CE, Rimmelzwaan GF, Follmann F, Agger EM, Andersen P, de Gier JW, Luirink J, An autotransporter display platform for the development of multivalent recombinant bacterial vector vaccines. MICROB CELL FACT 2014;13:162 [PubMed]
  14. Stoop EJ, Mishra AK, Driessen NN, van Stempvoort G, Bouchier P, Verboom T, van Leeuwen LM, Sparrius M, Raadsen SA, van Zon M, van der Wel NN, Besra GS, Geurtsen J, Bitter W, Appelmelk BJ, van der Sar AM. Mannan core branching of lipo(arabino)mannan is required for mycobacterial virulence in the context of innate immunity. Cell Microbiol. 2013 Dec;15(12):2093-108. www.ncbi.nlm.nih.gov/pubmed/23902464
  15. van der Woude AD, Mahendran KR, Ummels R, Piersma SR, Pham TV, Jiménez CR, de Punder K, van der Wel NN, Winterhalter M, Luirink J, Bitter W, Houben EN. Differential detergent extraction of mycobacterium marinum cell envelope proteins identifies an extensively modified threonine-rich outer membrane protein with channel activity. J Bacteriol. 2013 May;195(9):2050-9. www.ncbi.nlm.nih.gov/pubmed/23457249
  16. Jong WS, Soprova Z, de Punder K, ten Hagen-Jongman CM, Wagner S, Wickström D, de Gier JW, Andersen P, van der Wel NN, Luirink J. A structurally informed autotransporter platform for efficient heterologous protein secretion and display. Microb Cell Fact. 2012 Jun 18;11:85. www.ncbi.nlm.nih.gov/pubmed/22709508
  17. Houben D, Demangel C, van Ingen J, Perez J, Baldeón L, Abdallah AM, Caleechurn L, Bottai D, van Zon M, de Punder K, van der Laan T, Kant A, Bossers-de Vries R, Willemsen P, Bitter W, van Soolingen D, Brosch R, van der Wel N, Peters PJ. ESX-1-mediated translocation to the cytosol controls virulence of mycobacteria. Cell Microbiol. 2012 Aug;14(8):1287-98. www.ncbi.nlm.nih.gov/pubmed/22524898
  18. Weerdenburg EM, Abdallah AM, Mitra S, de Punder K, van der Wel NN, Bird S, Appelmelk BJ, Bitter W, van der Sar AM. ESX-5-deficient Mycobacterium marinum is hypervirulent in adult zebrafish. Cell Microbiol. 2012 May;14(5):728-39. www.ncbi.nlm.nih.gov/pubmed/22256857
  19. Abdallah AM, Bestebroer J, Savage ND, de Punder K, van Zon M, Wilson L, Korbee CJ, van der Sar AM, Ottenhoff TH, van der Wel NN, Bitter W, Peters PJ. Mycobacterial secretion systems ESX-1 and ESX-5 play distinct roles in host cell death and inflammasome activation. J Immunol. 2011 Nov 1;187(9):4744-53. www.ncbi.nlm.nih.gov/pubmed/21957139 Free Article
  20. Daleke MH, Cascioferro A, de Punder K, Ummels R, Abdallah AM, van der Wel N, Peters PJ, Luirink J, Manganelli R, Bitter W. Conserved Pro-Glu (PE) and Pro-Pro-Glu (PPE) protein domains target LipY lipases of pathogenic mycobacteria to the cell surface via the ESX-5 pathway. J Biol Chem. 2011 May 27;286(21):19024-34. www.ncbi.nlm.nih.gov/pubmed/21471225
  21. Sani M, Houben EN, Geurtsen J, Pierson J, de Punder K, van Zon M, Wever B, Piersma SR, Jiménez CR, Daffé M, Appelmelk BJ, Bitter W, van der Wel N, Peters PJ. Direct visualization by cryo-EM of the mycobacterial capsular layer: a labile structure containing ESX-1-secreted proteins. PLoS Pathog. 2010 Mar 5;6. www.ncbi.nlm.nih.gov/pubmed/20221442
  22. Weerdenburg EM, Peters PJ, van der Wel NN. How do mycobacteria activate CD8+ T cells? Trends Microbiol. 2010 Jan;18(1):1-10. www.ncbi.nlm.nih.gov/pubmed/19962899
  23. Abdallah AM, Savage ND, van Zon M, Wilson L, Vandenbroucke-Grauls CM, van der Wel NN, Ottenhoff TH, Bitter W. The ESX-5 secretion system of Mycobacterium marinum modulates the macrophage response. J Immunol. 2008 Nov 15;181(10):7166-75. www.ncbi.nlm.nih.gov/pubmed/18981138
  24. Hava DL, van der Wel N, Cohen N, Dascher CC, Houben D, León L, Agarwal S, Sugita M, van Zon M, Kent SC, Shams H, Peters PJ, Brenner MB. Evasion of peptide, but not lipid antigen presentation, through pathogen-induced dendritic cell maturation. Proc Natl Acad Sci U S A. 2008 Aug 12;105(32):11281-6. www.ncbi.nlm.nih.gov/pubmed/18685099
  25. van der Wel N, Hava D, Houben D, Fluitsma D, van Zon M, Pierson J, Brenner M, Peters PJ. M. tuberculosis and M. leprae translocate from the phagolysosome to the cytosol in myeloid cells. Cell. 2007 Jun 29;129(7):1287-98. www.ncbi.nlm.nih.gov/pubmed/17604718
  26. Touret N, Paroutis P, Terebiznik M, Harrison RE, Trombetta S, Pypaert M, Chow A, Jiang A, Shaw J, Yip C, Moore HP, van der Wel N, Houben D, Peters PJ, de Chastellier C, Mellman I, Grinstein S. Quantitative and dynamic assessment of the contribution of the ER to phagosome formation. Cell. 2005 Oct 7;123(1):157-70. www.ncbi.nlm.nih.gov/pubmed/16213220
  27. Boes M, van der Wel N, Peperzak V, Kim YM, Peters PJ, Ploegh H. In vivo control of endosomal architecture by class II-associated invariant chain and cathepsin S. Eur J Immunol. 2005 Sep;35(9):2552-62. www.ncbi.nlm.nih.gov/pubmed/16094690
  28. van der Wel NN, Fluitsma DM, Dascher CC, Brenner MB, Peters PJ. Subcellular localization of mycobacteria in tissues and detection of lipid antigens in organelles using cryo-techniques for light and electron microscopy. Curr Opin Microbiol. 2005 Jun;8(3):323-30. Review. www.ncbi.nlm.nih.gov/pubmed/15939357
  29. Angénieux C, Fraisier V, Maître B, Racine V, van der Wel N, Fricker D, Proamer F, Sachse M, Cazenave JP, Peters P, Goud B, Hanau D, Sibarita JB, Salamero J, de la Salle H. The cellular pathway of CD1e in immature and maturing dendritic cells. Traffic. 2005 Apr;6(4):286-302. www.ncbi.nlm.nih.gov/pubmed/15752135
  30. Baas AF, Kuipers J, van der Wel NN, Batlle E, Koerten HK, Peters PJ, Clevers HC. Complete polarization of single intestinal epithelial cells upon activation of LKB1 by STRAD Cell. 2004 Feb 6;116(3):457-66. www.ncbi.nlm.nih.gov/pubmed/15016379
  31. Griparic L, van der Wel NN, Orozco IJ, Peters PJ, van der Bliek AM. Loss of the intermembrane space protein Mgm1/OPA1 induces swelling and localized constrictions along the lengths of mitochondria. J Biol Chem. 2004 Apr 30;279(18):18792-8. www.ncbi.nlm.nih.gov/pubmed/14970223
  32. Cernadas M, Sugita M, van der Wel N, Cao X, Gumperz JE, Maltsev S, Besra GS, Behar SM, Peters PJ, Brenner MB. Lysosomal localization of murine CD1d mediated by AP-3 is necessary for NK T cell development. J Immunol. 2003 Oct 15;171(8):4149-55. www.ncbi.nlm.nih.gov/pubmed/14530337
  33. van der Wel NN, Sugita M, Fluitsma DM, Cao X, Schreibelt G, Brenner MB, Peters PJ. CD1 and major histocompatibility complex II molecules follow a different course during dendritic cell maturation. Mol Biol Cell. 2003 Aug;14(8):3378-88. www.ncbi.nlm.nih.gov/pubmed/12925770
  34. Puertollano R, van der Wel NN, Greene LE, Eisenberg E, Peters PJ, Bonifacino JS. Morphology and dynamics of clathrin/GGA1-coated carriers budding from the trans-Golgi network. Mol Biol Cell. 2003 Apr;14(4):1545-57. www.ncbi.nlm.nih.gov/pubmed/12686608
  35. Bertens P, Heijne W, van der Wel N, Wellink J, van Kammen A. Studies on the C-terminus of the Cowpea mosaic virus movement protein. Arch Virol. 2003 Feb;148(2):265-79. www.ncbi.nlm.nih.gov/pubmed/12556992
  36. Cao X, Sugita M, Van Der Wel N, Lai J, Rogers RA, Peters PJ, Brenner MB. CD1 molecules efficiently present antigen in immature dendritic cells and traffic independently of MHC class II during dendritic cell maturation. J Immunol. 2002 Nov 1;169(9):4770-7.www.ncbi.nlm.nih.gov/pubmed/12391186
  37. Sugita M, van Der Wel N, Rogers RA, Peters PJ, Brenner MB. CD1c molecules broadly survey the endocytic system. Proc Natl Acad Sci U S A. 2000 Jul 18;97(15):8445-50. www.ncbi.nlm.nih.gov/pubmed/10890914
  38. van der Wel NN, Goldbach RW, van Lent JW. The movement protein and coat protein of alfalfa mosaic virus accumulate in structurally modified plasmodesmata. Virology. 1998 May 10;244(2):322-9. www.ncbi.nlm.nih.gov/pubmed/9601503
  39. Kasteel DT, van der Wel NN, Jansen KA, Goldbach RW, van Lent JW. Tubule-forming capacity of the movement proteins of alfalfa mosaic virus and brome mosaic virus. J Gen Virol. 1997 Aug;78 ( Pt 8):2089-93. www.ncbi.nlm.nih.gov/pubmed/9267012