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 1

M. tuberculosis forms extensive intracellular cords in hLECs and lymph node biopsies.

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M. tuberculosis forms extensive intracellular cords in hLECs and lymph ...
(A) Images of primary hLECs infected with GFP expressing M. tuberculosis for 2 to 72 hours. Over time, M. tuberculosis grows and forms large intracellular cords. Nuclei are stained with DAPI (blue) and F-actin is stained by rhodamine phalloidin (red). (B) 3D reconstruction of Z-stacks taken of an intracellular cord from (A). Various angles are shown to confirm that the cord is completely encapsulated within the host cell. (C) Measurement of the intracellular cords over time in hLECs (using the Feret diameter; see Supplemental Figure 1) showing that the cords elongated up to a maximum of 150 μm. The numbers of bacterial clusters analyzed were 418 (2 hours), 233 (24 hours), 814 (48 hours), and 618 (72 hours), obtained from 3 independent experiments. One-way ANOVA with Tukey’s multiple comparisons tests: ***P < 0.001. (D) Image of A549 cells infected with M. tuberculosis-EGFP for 72 hours showing an intracellular cord looping around the nucleus. Nuclei are stained with DAPI (blue) and F-actin is stained with rhodamine phalloidin (red). (E) Intracellular cord formation after 72 hours was also observed in hLECs infected with representative strains from 3 other M. tuberculosis lineages: N0072 (lineage 1), N0145 (lineage 2), and N0024 (lineage 3). Images displayed in D and E are representative of at least 3 independent experiments. (F) Tissue section of a granuloma present in a human lymph stained for AFB. Zoomed region shows association of M. tuberculosis cords with cells (black boxes). Representative histological sections from human patients after lymph node tissue resection surgery were stained for PDPN, M. tuberculosis, and nuclei (DAPI). Scale bar: 1 mm. White boxes delimit the zoomed regions displayed on the right-hand side. Arrows indicate the presence of M. tuberculosis cords within PDPN+ cells. Scale bar: 20 μm. hLECs, human lymphatic endothelial cells; AFB, acid fast bacilli; PDPN, podoplanin.