Mycobacterium tuberculosis cords within lymphatic endothelial cells to evade host immunity
Thomas R. Lerner, Christophe J. Queval, Rachel P. Lai, Matthew R.G. Russell, Antony Fearns, Daniel J. Greenwood, Lucy Collinson, Robert J. Wilkinson, Maximiliano G. Gutierrez
Thomas R. Lerner, Christophe J. Queval, Rachel P. Lai, Matthew R.G. Russell, Antony Fearns, Daniel J. Greenwood, Lucy Collinson, Robert J. Wilkinson, Maximiliano G. Gutierrez
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Research Article Infectious disease Vascular biology

Mycobacterium tuberculosis cords within lymphatic endothelial cells to evade host immunity

Abstract

The ability of Mycobacterium tuberculosis to form serpentine cords is intrinsically related to its virulence, but specifically how M. tuberculosis cording contributes to pathogenesis remains obscure. Here, we show that several M. tuberculosis clinical isolates form intracellular cords in primary human lymphatic endothelial cells (hLECs) in vitro and in the lymph nodes of patients with tuberculosis. We identified via RNA-Seq a transcriptional program that activated, in infected-hLECs, cell survival and cytosolic surveillance of pathogens pathways. Consistent with this, cytosolic access was required for intracellular M. tuberculosis cording. Mycobacteria lacking ESX-1 type VII secretion system or phthiocerol dimycocerosates expression, which failed to access the cytosol, were indeed unable to form cords within hLECs. Finally, we show that M. tuberculosis cording is a size-dependent mechanism used by the pathogen to avoid its recognition by cytosolic sensors and evade either resting or IFN-γ–induced hLEC immunity. These results explain the long-standing association between M. tuberculosis cording and virulence and how virulent mycobacteria use intracellular cording as strategy to successfully adapt and persist in the lymphatic tracts.

Authors

Thomas R. Lerner, Christophe J. Queval, Rachel P. Lai, Matthew R.G. Russell, Antony Fearns, Daniel J. Greenwood, Lucy Collinson, Robert J. Wilkinson, Maximiliano G. Gutierrez

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Figure 6

Intracellular cords are not recognized by cytosolic immune sensors in both resting and IFN-γ–activated hLECs.

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Intracellular cords are not recognized by cytosolic immune sensors in bo...
(A–C) Live-cell imaging of hLECs expressing p62-RFP (red) infected with M. tuberculosis-GFP (green) for 115 hours. Imaging started 15 minutes after addition of the bacteria to the cells. Snapshots from the movies (Supplemental Videos 1–3) are shown, with the time point displayed above in hh:mm:ss format. Scale bars: 10 μm. (A) The pink arrow tracks an example of an intracellular cord forming from a single bacterium, which never interacts/associates with p62. (B) The blue arrow tracks an example of an individual M. tuberculosis bacterium becoming associated to p62 throughout which leads to restriction of growth. (C) The blue arrow tracks an example of M. tuberculosis associating and dissociating with p62 multiple times. Only after p62 association ceased completely, cord formation started. (C–E, right-hand side of panel) NIH ImageJ software quantification of the GFP intensity and the p62-RFP association of the arrowed bacteria over time. Letters a-f refer to the snapshots in A–C. (D) Representative image of hLECs infected with M. tuberculosis WT-EGFP (blue) for 72 hours and stained for the autophagy adaptor p62 (red) and the autophagy receptor ubiquitin (Ub) (green). Cell nuclei are stained with DAPI (blue). Scale bar: 10 μm. (E) Intracellular markers of autophagy pathogen sensing were assessed for their association to intracellular cords 72 hours after infection. Particles with a Feret diameter greater than 10 μM were considered cords, and a marker association score above 100 was considered positive. Points correspond to individual bacterial particles. (F and G) Representative image of hLECs infected with M. tuberculosis WT-EGFP (green) for 72 hours to let bacteria form intracellular cords before being treated or not with 200 ng/mL of human IFN-γ for an additional 24 hours. Cells were then fixed and stained for the autophagy adaptor p62 (F, left) or LC3 (G, left). Both p62 and LC3 markers are visualized in red; cell nuclei are stained with DAPI (blue). Scale bar: 10 μm. Intracellular markers p62 (F, right) and LC3 (G, right) were assessed for their association to intracellular bacteria in function of the size of the bacterial cluster. Particles with a Feret diameter greater than 10 μM were considered cords, and marker association scores above 15,000 for p62 and 10,000 for LC3 were considered positive. (D–G) Data were obtained from 3 independent experiments, each performed in duplicate. hLECs, images of human lymphatic endothelial cells.