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

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 Ca²⁺ overload followed by necrosis of phosphatidylserine externalised neutrophils. This necrosis was dependent upon the Ca²⁺ activated protease calpain, as pharmacologic inhibition prevented this secondary necrosis. We also observed that the ESAT-6 induced increase in intracellular Ca²⁺, 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.Tuberculosis (TB) caused by Mycobacterium tuberculosis remains a leading source of mortality by infectious disease, with one-third of the world''s population infected, 8.6 million new cases of TB and 1.3 million deaths annually.¹ The fundamental feature of TB transmission is the generation of a pulmonary tubercle lesion that contains a cuff of immune cells surrounding a necrotic core laden with extracellular bacteria. This lesion may ‘cavitate'' into the airways of the lung releasing the bacteria to allow transmission via the respiratory route. The essential contribution of macrophage and neutrophil cell death to the generation of this pathology has been recognised in many studies; however, notably in a comprehensive study of TB lesion development, Medlar² observed that the polymorphonuclear cells were attracted to lesions following the death of mononuclear cells and that the bulk of necrotic tissue in human caseating tubercle lesions represented dead polymorphonuclear cells.We now know that the bacterium has evolved mechanisms to regulate the mode and timing of macrophage cell death.^{3, 4, 5, 6, 7} After initial infection into the lungs, it is supposed that the bacterium is phagocytosed by alveolar macrophages which migrate into the interstitium of the lung.⁸ The bacterium is able to replicate intracellularly in the macrophage, inhibiting apoptosis until at a certain bacterial load it induces necrosis of the macrophage.⁹ The ensuing inflammation attracts monocytes and neutrophils from post-capillary venules that engulf the released bacteria, and thus sequential rounds of replication and inflammation enable the generation of the tubercle lesion. However, although we are beginning to understand the mechanisms of macrophage cell death control,^{4, 5, 6} we know very little about how M. tuberculosis modulates neutrophil death.It is also clear that in some circumstances neutrophils have an antimycobacterial capacity,^10,11 which may be mediated by the direct generation of reactive oxygen species (ROS) or by apoptosis of the infected neutrophils and subsequent efferocytosis of the apoptotic body combined with ROS-dependent killing.^12,13 Additionally, neutrophil apoptosis has been linked to effective generation of adaptive immunity in M. tuberculosis infection.¹⁴ However, to counter this, M. tuberculosis has been recently shown to inhibit neutrophil apoptosis¹⁴ and furthermore, has been observed to induce necrosis.^11,12 Interestingly, neutrophil necrosis only occurs on exposure to virulent strains which express the region of difference 1 (RD1) which encodes a type VII secretion system (ESX) that secretes proteins including the abundant early secretory antigen-6 (ESAT-6).^{12,15, 16, 17} Thus the bacterial induction of pro-inflammatory neutrophil necrosis may have dual benefit to the pathogen by removing the antimicrobial threat of the neutrophil while simultaneously facilitating the generation of the necrotic cavitating lesions that drive TB transmission.The mechanism of necrosis in neutrophils can be varied and controlled. The most recent to be described is ‘NETosis'',^18,19 whereby death of the neutrophil results in formation of a structure made of DNA with a histone backbone which contains neutrophil elastase, myeloperoxidase (MPO) and metalloproteinases. These ‘traps'' are known to be produced in vivo and associate with bacteria in some infections.^{18,20, 21, 22} Importantly they have been shown to be produced by M. tuberculosis infected neutrophils in vitro²³ although with no bactericidal activity. As well as ‘NETosis'' there are also other described mechanisms of neutrophil death, one of which is ‘secondary necrosis''. In vitro aging, without any stimuli, results in the necrosis of neutrophils that have undergone apoptosis (termed secondary necrosis). Previous investigations have shown that neutrophils that have externalised phosphatidylserine, and are therefore ‘apoptotic'', can undergo secondary necrosis caused by a Ca²⁺ influx leading to the activation of a subtype of Ca²⁺ activated protease, calpain.²⁴ This we termed Ca²⁺ Induced Necrosis (CAIN). In the present study, we elucidate the molecular events that link the RD1 encoded ESX-1 type VII secretion system with neutrophil necrosis. We chose to investigate the ESAT-6 protein, which is secreted by ESX-1, because it interacts with lipid membranes and is thought to be pore-forming,^{25, 26, 27, 28} thus it has the potential to influence intracellular Ca²⁺. Furthermore, calpain has been shown previously to be active in M.bovis infections that were dependent on the RD1 locus.²⁹ ESAT-6 has also been shown to have cytotoxic effects to pneumocytes³⁰ and T lymphocytes.³¹ Therefore, this study aimed to determine if ESAT-6 had a leukocidin action, and if so, whether this was dependent on intracellular flux of Ca²⁺, activation of calpain, and further, if this resulted in the formation of neutrophil extracellular traps (NETs).