Mycobacterium tuberculosis secretory proteins CFP‐10, ESAT‐6 and the CFP10:ESAT6 complex inhibit lipopolysaccharide‐induced NF‐κB transactivation by downregulation of reactive oxidative species (ROS) production

Abstract

Mycobacterium tuberculosis (Mtb) causes death of 2-3 million people annually and is considered one of the most successful intracellular pathogens to persist inside the host macrophage. Recent studies have implicated the role of RD-1 region of Mtb genome in the mycobacterial pathogenesis. The role of RD-1-encoded secretory proteins of Mtb in modulation of macrophage function has not been investigated in detail. Here we show that RD-1 encoded two major secretory proteins, namely, culture filtrate protein-10 kDa (CFP-10) and early secreted antigenic target-6 kDa (ESAT-6), and their 1:1 CFP-10:ESAT6 complex inhibit production of reactive oxidative species (ROS) in RAW264.7 cells. These proteins also downregulated the bacterial lipopolysaccharide (LPS)-induced ROS production, which, in turn, downregulated LPS-induced nuclear factor-jB (NF-jB) p65 DNA-binding activity, as well as inhibited the NF-jB-dependent reporter gene (chloramphenicol acetyl transferase) expression in the treated macrophages. Moreover, addition of N-acetyl cysteine, which is a scavenger of ROS, also inhibited LPS-induced reporter gene expression by scavenging the ROS, thereby preventing NF-jB transactivation. These studies indicate that the secretory proteins CFP-10, ESAT-6 and the CFP10:ESAT6 complex of Mtb can inhibit LPS-induced NF-jB-dependent gene expression via downregulation of ROS production.

Safety of Recombinant Fusion Protein ESAT6-CFP10 as a Skin Test Reagent for Tuberculosis Diagnosis: an Open-Label, Randomized, Single-Center Phase I Clinical Trial

“…ESAT6-CFP10 manufacturing was overseen by regulators. The expression and purification of the recombinant protein were conducted by standard protocols (12,13). The purity of this recombinant fusion protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance liquid chromatography (HPLC) and was Ն95% purity in this study.…”

“…The study protocol was reviewed, approved, and registered at ClinicalTrials.gov under registration no. NCT01999231 (12).…”

Mycobacterium tuberculosis RD-1 Secreted Antigens as Protective and Risk Factors for Tuberculosis

“…Studies from our lab have shown that CFP-10 and ESAT-6 downregulates the production of reactive oxygen species (ROS) inside the macrophages; which in turn dampens the NF-ĸB transactivation property (Ganguly et al, 2008a, Ganguly et al, 2008b. The inhibition of ROS production was greater with the CFP10:ESAT6 complex compared to the individual proteins.…”

Adaptation of Mycobacterium smegmatis to nutrient depletion and its effect on esat-6 expression

“…Los bacilos tuberculosos responden a situaciones de estrés por agotamiento de nutrientes, pH ácido, especies reactivas de oxígeno y nitrógeno causado por su crecimiento en los macrófagos (7,8,14) . Para responder a estos estímulos, las micobacterias secretan enzimas como ESAT-6 y CFP-10 (2,15) , que en las células del hospedero, actúan en los procesos de necrosis, tales como la lisis del epitelio alveolar y membranas de los macrófagos, induce la fragmentación del ADN y la permeabilidad de la membrana de las mitocondrias (15) .…”

“…The bacilli respond to stress conditions due to nutrient depletion, acid pH, and reactive oxygen and nitrogen species, caused by their growth in macrophages (7,8,14) . In order to respond to these stimuli, mycobacteria secrete enzymes, such as ESAT-6 and CFP-10 (2,15) , which in the cells of the host, act in necrosis processes, such as lysis of alveolar epithelium and membranes of macrophages, induction of DNA fragmentation and permeability of mitochondrial membranes (15) .…”