Characterization of G-Quadruplex Motifs in espB, espK, and cyp51 Genes of Mycobacterium tuberculosis as Potential Drug Targets

Mishra, Subodh Kumar; Shankar, Uma; Jain, Neha; Sikri, Kriti; Tyagi, Jaya Sivaswami; Sharma, Tarun Kumar; Mergny, Jean-Louis; Kumar, Amit

of macrophages and sequesters Beta-2-Microglobulin (b2M) protein, an essential component of MHC class I system. 21 This sequestration reduces MHC class I-mediated antigen presentation, an innate immune response of the host cell. 21 In addition to ESAT-6 and CFP-10, ESX-1 also secretes another ESX-substrate protein, EspB. EspB interacts with the C-terminal region of EspK protein and directly portrays an inhibitory role in the maturation of phagosome. 18, 20 Upon EspKassisted secretion of EspB outside the cytosol by ESX-1 secretion machinery ( Figure S1A ), EspB undergoes oligomerization, binds to phospholipids of host macrophages, and initiates a membranolytic or pore-forming phenomenon inside the phagosome that serves as a crucial initial step of immune evasion for bacteria. 22 Thus, secretion of EspB helps the bacteria in suppressing the innate immune response of the host cell and promotes its survival within the host 20 (Figure S1A ). Above insights into ESX-1 secretion machinery revealed the crucial role of EspB and EspK proteins in the pathogenesis of M. tuberculosis within the host and suggested that inhibition of espK and espB expression could lead to a synergistic reduction in M. tuberculosis virulence and pathogenesis, and thus represents a novel therapeutic approach. The cyp51 gene is another PGQ-possessing gene that has been previously reported to be involved in sterol biosynthesis pathway and membrane formation. [23] [24] [25] [26] Because our bioinformatics analysis revealed the PGQs in the espk, espB, and cyp51 genes, in the present work, we sought to investigate the regulatory role of these PGQs on the expression of PGQ-possessing genes. To accomplish this, we used various biophysical and biochemical techniques, and confirmed the formation of G-quadruplex structures by predicted PGQs. Isothermal titration calorimetry (ITC) and circular dichroism (CD) melting analysis allowed us to demonstrate the affinity of TMPyP4 to PGQs. Next, polymerase stop assay and qRT-PCR assay of RNA from a M. tuberculosis culture treated with TMPyP4 confirmed the downregulation of all PGQs possessing genes espK, espB, and cyp51. Therefore, this study demonstrates a novel approach for developing active therapeutics against M. tuberculosis infection by exploiting PGQ targets ( Figure S1B ).