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 3

Intracellular cords are localized in the host cell cytosol and consist of chains of M. tuberculosis of a small size.

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Intracellular cords are localized in the host cell cytosol and consist o...
(A and B) CLEM images of hLECs infected with M. tuberculosis-GFP. Top left subpanel shows the light microscopy images, with the corresponding electron microscopy images in the top right subpanel. The larger subpanels show a composite of the fluorescence overlaid onto the electron microscopy. (A) M. tuberculosis intracellular cord, without any encapsulating host membrane, indicating that it is present in the cytosol. (B) M. tuberculosis encapsulated in a membranous compartment, as a control for confirming membrane preservation due to the sample preparation. Host cell membrane is highlighted in red. (C and D) To quantify the volume of M. tuberculosis, individual bacteria were manually segmented from slices of SBF SEM images and 3D reconstructions of selected bacteria were made (colored rods), using 3dmod. Representative reconstructions are shown, with corresponding fluorescence highlighted (matched manually with the corresponding SBF SEM slice in Z, and then aligned in xy with TurboReg in Fiji). Data set dimensions: (C) left panel, 8.7 × 8.7 × 50 nm pixels; right panel, 71.3 × 71.36 × 1 μm in xyz; (D) left panel, 6.3 × 6.3 × 50 nm pixels; right panel, 51.6 × 51.6 × 2.75 μm in xyz. (E) The volume of each bacterium reconstruction from 2 independent sample data sets was calculated in 3dmod, and a comparison between those in a membrane-bound compartment and those in an intracellular cord was made. The data ± SEM show that individual bacteria forming cords are significantly smaller. Student’s t test: **P < 0.01. CLEM, correlative light electron microscopy; hLECs, images of human lymphatic endothelial cells; SBF, serial block face; SEM, scanning electron microscopy.