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Mycobacterium tuberculosis ESAT-6 is a leukocidin causing Ca2+ influx, necrosis and neutrophil extracellular trap formation.

Francis RJ, Butler RE, Stewart GR - Cell Death Dis (2014)

Bottom Line: This inflammatory pathology is essential for disease transmission and M. tuberculosis has evolved to stimulate inflammatory granuloma development while simultaneously avoiding destruction by the attracted phagocytes.This necrosis was dependent upon the Ca(2+) activated protease calpain, as pharmacologic inhibition prevented this secondary necrosis.Thus we conclude that ESAT-6 has a leukocidin function, which may facilitate bacterial avoidance of the antimicrobial action of the neutrophil while contributing to the maintenance of inflammation and necrotic pathology necessary for granuloma formation and TB transmission.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbial and Cellular Sciences and Bioimaging and Flow Cytometry Core Facility, Faculty of Health and Medical Sciences, University of Surrey, Guildford, GU2 7XH, UK.

ABSTRACT
Mycobacterium tuberculosis infection generates pulmonary granulomas that consist of a caseous, necrotic core surrounded by an ordered arrangement of macrophages, neutrophils and T cells. This inflammatory pathology is essential for disease transmission and M. tuberculosis has evolved to stimulate inflammatory granuloma development while simultaneously avoiding destruction by the attracted phagocytes. The most abundant phagocyte in active necrotic granulomas is the neutrophil. Here we show that the ESAT-6 protein secreted by the ESX-1 type VII secretion system causes necrosis of the neutrophils. ESAT-6 induced an intracellular Ca(2+) overload followed by necrosis of phosphatidylserine externalised neutrophils. This necrosis was dependent upon the Ca(2+) activated protease calpain, as pharmacologic inhibition prevented this secondary necrosis. We also observed that the ESAT-6 induced increase in intracellular Ca(2+), stimulated the production of neutrophil extracellular traps characterised by extruded DNA and myeloperoxidase. Thus we conclude that ESAT-6 has a leukocidin function, which may facilitate bacterial avoidance of the antimicrobial action of the neutrophil while contributing to the maintenance of inflammation and necrotic pathology necessary for granuloma formation and TB transmission.

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Intracellular Ca2+ increases in ESAT-6 treated neutrophils and precedes necrosis. (a) Sequential fluorescence micrographs showing accumulation of calcium (green, Fluo4) in Annexin V positive (purple)/PS externalised aged neutrophils after exposure to exogenous ESAT-6 (20 μg/ml). Uptake of propidium iodide (PI) (red) indicates necrosis of the cell. Arrow tracks an individual cell. White bar=10 μm. Panel (b) shows a representative quantitation of intracellular Ca2+ levels of a PS externalised (Annexin V+ve) cell exposed to ESAT-6 (16 responses in N=3 experiments). Panel (c) shows representative intracellular Ca2+ in a non-PS externalised neutrophil exposed to ESAT-6. (21 responses in N=4)
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fig1: Intracellular Ca2+ increases in ESAT-6 treated neutrophils and precedes necrosis. (a) Sequential fluorescence micrographs showing accumulation of calcium (green, Fluo4) in Annexin V positive (purple)/PS externalised aged neutrophils after exposure to exogenous ESAT-6 (20 μg/ml). Uptake of propidium iodide (PI) (red) indicates necrosis of the cell. Arrow tracks an individual cell. White bar=10 μm. Panel (b) shows a representative quantitation of intracellular Ca2+ levels of a PS externalised (Annexin V+ve) cell exposed to ESAT-6 (16 responses in N=3 experiments). Panel (c) shows representative intracellular Ca2+ in a non-PS externalised neutrophil exposed to ESAT-6. (21 responses in N=4)

Mentions: Previous work had identified increased intracellular Ca2+ as a mechanism of secondary necrosis in human neutrophils.24 To examine the effect of ESAT-6 on neutrophil Ca2+ we loaded 24- h aged cells with fluo4-AM, a fluorescent Ca2+ indicator that stays within intact cells following uptake and conversion from its AM ester to the hydrophilic fluo4 molecule.32 Using confocal microscopy we observed that the addition of ESAT-6 caused an increase in intracellular Ca2+ levels in a proportion of neutrophils within 20 min of exposure. The sequential micrographs in Figure 1a illustrate the Ca2+ influx as increasing Fluo4 fluorescence (green) in a phosphatidylserine externalised (Annexin V positive, purple) cell that ultimately undergoes necrosis (propidium iodide, red) at 60 min post-ESAT-6 addition. A movie of this response is available (Supplementary Figure 1). A representative quantitation of the Ca2+ increase followed by simultaneous Ca2+ release and uptake of propidium iodide at the point of cell lysis is shown in Figure 1b. Interestingly ESAT-6 did not induce significant calcium uptake in cells without externalised phosphatidylserine (annexin V negative) (Figure 1c).


Mycobacterium tuberculosis ESAT-6 is a leukocidin causing Ca2+ influx, necrosis and neutrophil extracellular trap formation.

Francis RJ, Butler RE, Stewart GR - Cell Death Dis (2014)

Intracellular Ca2+ increases in ESAT-6 treated neutrophils and precedes necrosis. (a) Sequential fluorescence micrographs showing accumulation of calcium (green, Fluo4) in Annexin V positive (purple)/PS externalised aged neutrophils after exposure to exogenous ESAT-6 (20 μg/ml). Uptake of propidium iodide (PI) (red) indicates necrosis of the cell. Arrow tracks an individual cell. White bar=10 μm. Panel (b) shows a representative quantitation of intracellular Ca2+ levels of a PS externalised (Annexin V+ve) cell exposed to ESAT-6 (16 responses in N=3 experiments). Panel (c) shows representative intracellular Ca2+ in a non-PS externalised neutrophil exposed to ESAT-6. (21 responses in N=4)
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4237235&req=5

fig1: Intracellular Ca2+ increases in ESAT-6 treated neutrophils and precedes necrosis. (a) Sequential fluorescence micrographs showing accumulation of calcium (green, Fluo4) in Annexin V positive (purple)/PS externalised aged neutrophils after exposure to exogenous ESAT-6 (20 μg/ml). Uptake of propidium iodide (PI) (red) indicates necrosis of the cell. Arrow tracks an individual cell. White bar=10 μm. Panel (b) shows a representative quantitation of intracellular Ca2+ levels of a PS externalised (Annexin V+ve) cell exposed to ESAT-6 (16 responses in N=3 experiments). Panel (c) shows representative intracellular Ca2+ in a non-PS externalised neutrophil exposed to ESAT-6. (21 responses in N=4)
Mentions: Previous work had identified increased intracellular Ca2+ as a mechanism of secondary necrosis in human neutrophils.24 To examine the effect of ESAT-6 on neutrophil Ca2+ we loaded 24- h aged cells with fluo4-AM, a fluorescent Ca2+ indicator that stays within intact cells following uptake and conversion from its AM ester to the hydrophilic fluo4 molecule.32 Using confocal microscopy we observed that the addition of ESAT-6 caused an increase in intracellular Ca2+ levels in a proportion of neutrophils within 20 min of exposure. The sequential micrographs in Figure 1a illustrate the Ca2+ influx as increasing Fluo4 fluorescence (green) in a phosphatidylserine externalised (Annexin V positive, purple) cell that ultimately undergoes necrosis (propidium iodide, red) at 60 min post-ESAT-6 addition. A movie of this response is available (Supplementary Figure 1). A representative quantitation of the Ca2+ increase followed by simultaneous Ca2+ release and uptake of propidium iodide at the point of cell lysis is shown in Figure 1b. Interestingly ESAT-6 did not induce significant calcium uptake in cells without externalised phosphatidylserine (annexin V negative) (Figure 1c).

Bottom Line: This inflammatory pathology is essential for disease transmission and M. tuberculosis has evolved to stimulate inflammatory granuloma development while simultaneously avoiding destruction by the attracted phagocytes.This necrosis was dependent upon the Ca(2+) activated protease calpain, as pharmacologic inhibition prevented this secondary necrosis.Thus we conclude that ESAT-6 has a leukocidin function, which may facilitate bacterial avoidance of the antimicrobial action of the neutrophil while contributing to the maintenance of inflammation and necrotic pathology necessary for granuloma formation and TB transmission.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbial and Cellular Sciences and Bioimaging and Flow Cytometry Core Facility, Faculty of Health and Medical Sciences, University of Surrey, Guildford, GU2 7XH, UK.

ABSTRACT
Mycobacterium tuberculosis infection generates pulmonary granulomas that consist of a caseous, necrotic core surrounded by an ordered arrangement of macrophages, neutrophils and T cells. This inflammatory pathology is essential for disease transmission and M. tuberculosis has evolved to stimulate inflammatory granuloma development while simultaneously avoiding destruction by the attracted phagocytes. The most abundant phagocyte in active necrotic granulomas is the neutrophil. Here we show that the ESAT-6 protein secreted by the ESX-1 type VII secretion system causes necrosis of the neutrophils. ESAT-6 induced an intracellular Ca(2+) overload followed by necrosis of phosphatidylserine externalised neutrophils. This necrosis was dependent upon the Ca(2+) activated protease calpain, as pharmacologic inhibition prevented this secondary necrosis. We also observed that the ESAT-6 induced increase in intracellular Ca(2+), stimulated the production of neutrophil extracellular traps characterised by extruded DNA and myeloperoxidase. Thus we conclude that ESAT-6 has a leukocidin function, which may facilitate bacterial avoidance of the antimicrobial action of the neutrophil while contributing to the maintenance of inflammation and necrotic pathology necessary for granuloma formation and TB transmission.

Show MeSH
Related in: MedlinePlus