Mycobacterium tuberculosis PE/PPE proteins enhance the production of reactive oxygen species and formation of neutrophil extracellular traps

Abstract

IntroductionNeutrophil granulocytes predominate in the lungs of patients infected with Mycobacterium tuberculosis (Mtb) in earlier stages of the disease. During infection, neutrophils release neutrophil extracellular traps (NETs), an antimicrobial mechanism by which a DNA-backbone spiked with antimicrobial components traps the mycobacteria. However, the specific mycobacterial factors driving NET formation remain unclear. Proteins from the proline-glutamic acid (PE)/proline-proline-glutamic acid (PPE) family are critical to Mtb pathophysiology and virulence.MethodsHere, we investigated NET induction by PE18, PPE26, and PE31 in primary human blood-derived neutrophils. Neutrophils were stimulated with the respective proteins for 3h, and NET formation was subsequently assessed using confocal fluorescence microscopy. Intracellular ROS levels and cell necrosis were estimated by flow cytometry. Additionally, the influence of phorbol-12-myristate-13-acetate (PMA), a known NADPH oxidase enhancer, on NET formation was examined. Neutrophil integrity following incubation with the PE/PPE proteins was evaluated using transmission electron microscopy.ResultsFor the first time, we report that stimulation of primary human blood-derived neutrophils with Mtb proteins PE18, PPE26, and PE31 resulted in the formation of NETs, which correlated with an increase in intracellular ROS levels. Notably, the presence of PMA further amplified this effect. Following incubation with the PE/PPE proteins, neutrophils were found to remain viable and structurally intact, as verified through transmission electron microscopy, indicating the occurrence of vital NET formation.DiscussionThese findings offer valuable insights that contribute to a better understanding of host-pathogen interactions during Mtb infection. Moreover, they underscore the significance of these particular Mtb antigens in triggering NET formation, representing a distinctive and previously unrecognized function of PE/PPE antigens.

Impact of bronchoalveolar lavage from influenza A virus diseased pigs on neutrophil functions and growth of co-infecting pathogenic bacteria

“…Bengoa et al. detected NET levels in human neutrophils at approximately 50% together with dead cells below 5% and confirmed vesicular NET formation with electron microscopy analysis ( 28 ). It needs to be considered that we have measured dead cells only in isolated neutrophils stimulated with BALF instead of in the in vivo -infected neutrophils, where we detected vesicular NET formation.…”

“…The method used was previously published by Bengoa et al. ( 28 ). Here, freshly isolated porcine neutrophils [2 × 10 5 dissolved in 75 µl of Roswell Park Memorial Institute (RPMI) 1640 medium] were seeded in a 96-well transparent, flat-bottom plate.…”

Immunogenicity of PE18, PE31, and PPE26 proteins from Mycobacterium tuberculosis in humans and mice

“…In order to investigate the immunogenicity and protective capacity of mycobacterial proteins we needed to generate quantities of pure protein. To do this, mycobacteria specific genes encoding proteins PE18, PE31 and PPE26 were expressed and purified as described before ( 28 ). Protein expression was induced with 1mM IPTG resulting in accumulation of insoluble proteins, and purity and precision of protein production confirmed by weight, with SDS-PAGE showing an apparent molecular weight of 9.8kDa for the PEs and 38kDa for PPE26.…”

“…The purification of the recombinant proteins was carried out as described before ( 28 ). Briefly, the plasmids containing genes of interest fused to 6xHis-tags were transformed and expressed in E. coli BL21(DE3) strain and the recombinant proteins were later purified by metal ion affinity chromatography.…”

“…In this study, we analysed the immunogenic potential of PE18 (Rv1788), PPE26 (Rv1789), and PE31 (Rv3477) proteins of Mtb in humans and in immunised mice. To do so, respective proteins were produced in recombinant form as described before ( 28 ), and tested for immunoreactivity with sera and PBMC from TB patients, IGRA + household contacts, and IGRA - BCG vaccinated healthy controls from the TB endemic country, Mozambique. We then tested their immunogenicity and protective potential in C57BL/6 mice after aerosol challenge with a low dose of Mycobacterium tuberculosis H37Rv.…”