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 4

M. tuberculosis lacking RD1 locus or PDIMs expression fail to cord within hLECs.

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M. tuberculosis lacking RD1 locus or PDIMs expression fail to cord with...
(A) hLECs were infected with RFP-expressing M. tuberculosis WT, GFP-expressing M. tuberculosis ΔPDIM, or E2-Crimson–expressing ΔRD1 for 72 hours at a MOI of 10, fixed and stained for F-actin with AF633 or AF488-phalloidin. Either deleting PDIM or the RD1 locus abolished cord formation. WT bacteria (red), ΔPDIM and ΔRD1-bacteria (green), F-actin (white), and nuclei (blue). Scale bar: 50 μm. (B) Feret diameter measurements from 3 independent experiments were plotted. For each condition tested, the number of bacterial clusters analyzed was between 600 and 1200. (C) hLECs were infected for 72 hours with RFP-expressing M. tuberculosis WT at a MOI of 10 or with E2-Crimson–expressing M. tuberculosis ΔRD1 at a MOI of 10, 20, or 40. WT bacteria (red), ΔRD1-bacteria (green), F-actin (white), and nuclei (blue). Scale bar: 50 μm. (D) Feret diameter measurements from images in C from 2 independent experiments were plotted. The numbers of bacterial clusters analyzed were 3,960 for WT and 6,470, 9,472, and 11,759 for ΔRD1 at MOI of 10, 20 and 40, respectively. (E) Quantification of the bacterial load per cell, expressed in bacterial area (μm2) per cell, following the uptake (5 hours after infection) and 72 hours after infection. (F) Intracellular bacterial growth after 72 hours infection, expressed by the ratio bacterial area per cell 72 hours pi/5 hours pi. Values > 1 represent the bacterial growth. (E and F) Data ± SEM are representative of 2 independent experiments performed in duplicate. (B–E) One-way ANOVA with Tukey’s multiple comparisons tests against WT, **P < 0.01; ***P < 0.001; ns, not significant. PDIM, phthiocerol dimycocerosate; hLECs, images of human lymphatic endothelial cells; pi, postinfection.