Maaike van Zon

M. tuberculosis and M. leprae Translocate from the Phagolysosome to the Cytosol in Myeloid Cells

M. tuberculosis and M. leprae are considered to be prototypical intracellular pathogens that have evolved strategies to enable growth in the intracellular phagosomes. In contrast, we show that lysosomes rapidly fuse with the virulent M. tuberculosis- and M. leprae-containing phagosomes of human monocyte-derived dendritic cells and macrophages. After 2 days, M. tuberculosis progressively translocates from phagolysosomes into the cytosol in nonapoptotic cells. Cytosolic entry is also observed for M. leprae but not for vaccine strains such as M. bovis BCG or in heat-killed mycobacteria and is dependent upon secretion of the mycobacterial gene products CFP-10 and ESAT-6. The cytosolic bacterial localization and replication are pathogenic features of virulent mycobacteria, causing significant cell death within a week. This may also reveal a mechanism for MHC-based antigen presentation that is lacking in current vaccine strains.

ESX-1-mediated translocation to the cytosol controls virulence of mycobacteria

Mycobacterium species, including Mycobacterium tuberculosis and Mycobacterium leprae, are among the most potent human bacterial pathogens. The discovery of cytosolic mycobacteria challenged the paradigm that these pathogens exclusively localize within the phagosome of host cells. As yet the biological relevance of mycobacterial translocation to the cytosol remained unclear. In this current study we used electron microscopy techniques to establish a clear link between translocation and mycobacterial virulence. Pathogenic, patient‐derived mycobacteria species were found to translocate to the cytosol, while non‐pathogenic species did not. We were further able to link cytosolic translocation with pathogenicity by introducing the ESX‐1 (type VII) secretion system into the non‐virulent, exclusively phagolysosomal Mycobacterium bovis BCG. Furthermore, we show that translocation is dependent on the C‐terminus of the early‐secreted antigen ESAT‐6. The C‐terminal truncation of ESAT‐6 was shown to result in attenuation in mice, again linking translocation to virulence. Together, these data demonstrate the molecular mechanism facilitating translocation of mycobacteria. The ability to translocate from the phagolysosome to the cytosol is with this study proven to be biologically significant as it determines mycobacterial virulence.

Direct Visualization by Cryo-EM of the Mycobacterial Capsular Layer: A Labile Structure Containing ESX-1-Secreted Proteins

The cell envelope of mycobacteria, a group of Gram positive bacteria, is composed of a plasma membrane and a Gram-negative-like outer membrane containing mycolic acids. In addition, the surface of the mycobacteria is coated with an ill-characterized layer of extractable, non-covalently linked glycans, lipids and proteins, collectively known as the capsule, whose occurrence is a matter of debate. By using plunge freezing cryo-electron microscopy technique, we were able to show that pathogenic mycobacteria produce a thick capsule, only present when the cells were grown under unperturbed conditions and easily removed by mild detergents. This detergent-labile capsule layer contains arabinomannan, α-glucan and oligomannosyl-capped glycolipids. Further immunogenic and proteomic analyses revealed that Mycobacterium marinum capsule contains high amounts of proteins that are secreted via the ESX-1 pathway. Finally, cell infection experiments demonstrated the importance of the capsule for binding to cells and dampening of pro-inflammatory cytokine response. Together, these results show a direct visualization of the mycobacterial capsular layer as a labile structure that contains ESX-1-secreted proteins.

The ESX-5 secretion system of Mycobacterium marinum modulates the macrophage response

The ESX-5 secretion system of pathogenic mycobacteria is responsible for the secretion of various PPE and PE-PGRS proteins. To better understand the role of ESX-5 effector proteins in virulence, we analyzed the interactions of Mycobacterium marinum ESX-5 mutant with human macrophages (Mφ). Both wild-type bacteria and the ESX-5 mutant were internalized and the ESX-5 mutation did not affect the escape of mycobacteria from phagolysosomes into the cytosol, as was shown by electron microscopy. However, the ESX-5 mutation strongly effected expression of surface Ags and cytokine secretion. Whereas wild-type M. marinum actively suppressed the induction of appreciable levels of IL-12p40, TNF-α, and IL-6, infection with the ESX-5 mutant resulted in strongly induced production of these proinflammatory cytokines. By contrast, infection with M. marinum wild-type strain resulted in a significant induction of IL-1β production as compared with the ESX-5 mutant. These results show that ESX-5 plays an essential role in the modulation of immune cytokine secretion by human Mφ. Subsequently, we show that an intact ESX-5 secretion system actively suppresses TLR signaling-dependent innate immune cytokine secretion. Together, our results show that ESX-5 substrates, directly or indirectly, strongly modulate the human Mφ response at various critical steps.

Mycobacterial Secretion Systems ESX-1 and ESX-5 Play Distinct Roles in Host Cell Death and Inflammasome Activation

During infection of humans and animals, pathogenic mycobacteria manipulate the host cell causing severe diseases such as tuberculosis and leprosy. To understand the basis of mycobacterial pathogenicity, it is crucial to identify the molecular virulence mechanisms. In this study, we address the contribution of ESX-1 and ESX-5—two homologous type VII secretion systems of mycobacteria that secrete distinct sets of immune modulators—during the macrophage infection cycle. Using wild-type, ESX-1– and ESX-5–deficient mycobacterial strains, we demonstrate that these secretion systems differentially affect subcellular localization and macrophage cell responses. We show that in contrast to ESX-1, the effector proteins secreted by ESX-5 are not required for the translocation of Mycobacterium tuberculosis or Mycobacterium marinum to the cytosol of host cells. However, the M. marinum ESX-5 mutant does not induce inflammasome activation and IL-1β activation. The ESX-5 system also induces a caspase-independent cell death after translocation has taken place. Importantly, by means of inhibitory agents and small interfering RNA experiments, we reveal that cathepsin B is involved in both the induction of cell death and inflammasome activation upon infection with wild-type mycobacteria. These results reveal distinct roles for two different type VII secretion systems during infection and shed light on how virulent mycobacteria manipulate the host cell in various ways to replicate and spread.

Evasion of peptide, but not lipid antigen presentation, through pathogen-induced dendritic cell maturation

Dendritic cells (DC) present lipid and peptide antigens to T cells on CD1 and MHC Class II (MHCII), respectively. The relative contribution of these systems during the initiation of adaptive immunity after microbial infection is not characterized. MHCII molecules normally acquire antigen and rapidly traffic from phagolysosomes to the plasma membrane as part of DC maturation, whereas CD1 molecules instead continually recycle between these sites before, during, and after DC maturation. We find that in Mycobacterium tuberculosis (Mtb)-infected DCs, CD1 presents antigens quickly. Surprisingly, rapid DC maturation results in early failure and delay in MHCII presentation. Whereas both CD1b and MHCII localize to bacterial phagosomes early after phagocytosis, MHCII traffics from the phagosome to the plasma membrane with a rapid kinetic that can precede antigen availability and loading. Thus, rather than facilitating antigen presentation, a lack of coordination in timing may allow organisms to use DC maturation as a mechanism of immune evasion. In contrast, CD1 antigen presentation occurs in the face of Mtb infection and rapid DC maturation because a pool of CD1 molecules remains available on the phagolysosome membrane that is able to acquire lipid antigens and deliver them to the plasma membrane.

Mannan core branching of lipo(arabino)mannan is required for mycobacterial virulence in the context of innate immunity

The causative agent of tuberculosis (TB), Mycobacterium tuberculosis, remains an important worldwide health threat. Although TB is one of the oldest infectious diseases of man, a detailed understanding of the mycobacterial mechanisms underlying pathogenesis remains elusive. Here, we studied the role of the α(1→2) mannosyltransferase MptC in mycobacterial virulence, using the Mycobacterium marinum zebrafish infection model. Like its M. tuberculosis orthologue, disruption of M. marinum mptC (mmar_3225) results in defective elongation of mannose caps of lipoarabinomannan (LAM) and absence of α(1→2)mannose branches on the lipomannan (LM) and LAM mannan core, as determined by biochemical analysis (NMR and GC‐MS) and immunoblotting. We found that the M. marinum mptC mutant is strongly attenuated in embryonic zebrafish, which rely solely on innate immunity, whereas minor virulence defects were observed in adult zebrafish. Strikingly, complementation with the Mycobacterium smegmatis mptC orthologue, which restored mannan core branching but not cap elongation, was sufficient to fully complement the virulence defect of the mptC mutant in embryos. Altogether our data demonstrate that not LAM capping, but mannan core branching of LM/LAM plays an important role in mycobacterial pathogenesis in the context of innate immunity.

Subcellular localization of M. tuberculosis in vivo and effect of the adaptive immunity

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Abstract PL01-03: TIL therapy in the era of immune checkpoint blockade

Infusion of tumor-infiltrating T-lymphocytes was developed as treatment for metastatic melanoma by Dr. S. Rosenberg (Surgery Branch, NIH, Bethesda, MD, USA) in the late 909s of the past century. This therapy was based on the finding that melanomas oftentimes have infiltrates of CD8 T cells that show recognition of melanoma cells. Many tumor antigens have been characterized for which specific T cells can be found within the tumor or in peripheral blood. As tumors may use mechanisms to induce local T cell unresponsiveness, isolation of TILs and in vitro activation and expansion was shown to overcome this anergic state both in preclinical models and in clinical trials. In order for the TILs to survive, persist, and be cytolytic after infusion, culture time should be as short as possible (to obtain cells with a less terminally differentiated phenotype), patients should receive lymphodepleting treatment (chemotherapy +/- TBI) prior to infusion and interleukin-2 as a survival factor post infusion. Up to date several hundreds of metastatic melanoma patients have been treated with TIL therapy in centers within the US, Europe and Israel. Objective response rates vary between 38 and 72% depending on the phase I/II trial with 10-20% of patients acquiring a CR, which were most often very durable. Many of these oftentimes heavily pretreated patients have benefitted from TIL therapy. As melanomas are heavily mutated cancers, the idea that T cells may react to epitopes derived from mutated proteins could be investigated with new technologies developed at NKI. Using whole exome sequencing, the total mutational burden could be measured for each melanoma. Only those mutations present in expressed genes were used for epitope prediction for every MHC class I molecule of the patient using validated prediction algorithms. Filters were used to select those epitopes with high enough binding affinity for MHC that were subsequently synthesized for production of peptide-MHC multimers that could be used to screen for neo-antigen specific T cells among peripheral blood T- cells or TIL using a multiplex flow cytometric technology. Together with the NCI, using a somewhat different approach, we were able to show that in the vast majority of melanoma TIL, CD8 T cells specific for epitopes resulting from UV light induced DNA damage can be found, sometimes in high frequencies (up to 25-30%), strongly suggesting that neo-antigen-specific T cells play an important role in the success of TIL therapy and possibly other immunotherapies, including checkpoint inhibitors. So far TIL therapy has been mostly used in metastatic melanoma patients pretreated with BRAF inhibitors, anti-CTLA4, high-dose IL-2, and demonstrated activity irrespective of these treatments, although isolation of TIL appeared less successful in patients progressing upon BRAFi. Early results in patients pretreated with PD1 blocking antibodies are promising as well, although the objective response rate is still unknown. Next to metastatic melanoma, TIL can be grown from other cancers as well and promising results have been obtained in patients with cervical cancer and in a patient with cholangiocarcinoma. Trials are planned for patients with epithelial ovarian cancer as well. Citation Format: John B.A.G. Haanen, Joost Van den Berg, Maaike Van Zon, Noor Bakker, Marnix Geukes, Hans Van Thienen, Christian Blank, Pia Kvistborg, Ton Schumacher. TIL therapy in the era of immune checkpoint blockade. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr PL01-03.

Understanding the 3D architecture of organelle bound protein complexes using cryo-electron tomography of frozen hydrated sections and immunogold; our next great challenge

One of the focal points of our group is to reveal the macromolecular organisation of cells by means of cryo-electron tomography. This is the only method that can be used to obtain molecular resolution information of intact cells constituencies in a near-native situation. The tomogram contains a 3D map of the cellular proteome and we are just beginning to explore its potential after we worked for a few years on some technical hurdles of specimen preparation. The other central point of our group is to localize gene product in cellular structures by EM at the highest resolution with gold probes on cryosections. Our main focus is on understanding the molecular machinery and organization within the endomembrane system. We concentrate our work on the transport mechanism within the endocytic pathway and the link to pathogenesis. Cryo-electron tomography of unfixed frozen hydrated cryo-sections and cryo immunogold-EM of aldehyde fixed cells are our main techniques, which allow the subcellular detection and visualization of molecular machines at the highest resolution.