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 5

Access to the cytosol is required for M. tuberculosis intracellular cording.

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Access to the cytosol is required for M. tuberculosis intracellular cord...
(A) hLECs were infected for 72 hours with M. tuberculosis WT-RFP (red), M. tuberculosis-ΔPDIM-GFP (green), and M. tuberculosis-ΔRD1-E2-Crimson (green) either individually or as a coinfection WT-RFP/ΔPDIM-GFP or WT-RFP/ΔRD1-E2-Crimson. Cells were then fixed and stained for F-actin with AF633 or AF488-phalloidin (both visualized in white) and DAPI (blue). Scale bar: 10 μm. The images show that during single infection, M. tuberculosis WT exhibited intracellular cording, whereas M. tuberculosis ΔPDIM or ΔRD1 did not. However, in the coinfected sample, both M. tuberculosis ΔPDIM and ΔRD1 were able to form intracellular cords. (B) Feret diameter measurements from images in A were plotted. n represents the number of bacterial clusters analyzed. (C) Intracellular bacterial growth 72 hours after infection, expressed by the ratio bacterial area per cell 72 hours pi/5 hours pi. Values > 1 represent the bacterial growth. (A–C) Data ± SEM are representative of 3 independent experiments. One-way ANOVA with Tukey’s multiple comparisons tests: *P < 0.05; **P < 0.01; ***P < 0.001. (D and E) Coinfected hLEC samples were processed for CLEM to confirm at the ultrastructural level that M. tuberculosis ΔRD1-GFP cords were indeed present in the cytosol (E; magnifications of regions indicated in D, asterisks mark cytosolic bacteria). PDIM, phthiocerol dimycocerosate; hLECs, images of human lymphatic endothelial cells; pi, postinfection; CLEM, correlative light electron microscopy.