Identification of Serine 119 as an Effective Inhibitor Binding Site of M. tuberculosis Ubiquitin-like Protein Ligase PafA Using Purified Proteins and M. smegmatis

Jiang, He‐wei; Czajkowsky, Daniel M.; Wang, Tao; Wang, Xude; Wang, Jia-Bin; Zhang, Hainan; Liu, Cheng-Xi; Wu, Fanlin; He, Xiang; Xu, Zhaowei; Chen, Hong; Guo, Shujuan; Li, Yang; Bi, Lijun; Deng, Jiao-Yu; Xie, Jin; Pei, Jianfeng; Zhang, Xian-En; Tao, Sheng-Ce

doi:10.1016/j.ebiom.2018.03.025

Cited by 14 publications

(10 citation statements)

References 56 publications

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“…Bacterial infection in macrophage was performed as described previously with some modification . Human THP-1 cell lines were cultured in RPMI 1640 medium (Hyclone) with 10% fetal bovine serum (Gibco) for cell accumulation.…”

Section: Methodsmentioning

confidence: 99%

“…Bacterial infection in macrophage was performed as described previously with some modification. 47 Human THP-1 cell lines were cultured in RPMI 1640 medium (Hyclone) with 10% fetal bovine serum (Gibco) for cell accumulation. For the induction of a macrophage-like state, cells were treated with 10 ng/mL polymethyl acrylate (PMA) for 12 h and then infected with M. smegmatis strains at a multiplicity of infection (MOI) of 10 for 2 h. Infected cells were washed three times with RPMI 1640 medium to remove free bacteria and then incubated in fresh complete RPMI 1640 medium with 15 μg/mL gentamicin.…”

Section: ■ Methodsmentioning

confidence: 99%

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RegX3 Controls Glyoxylate Shunt and Mycobacteria Survival by Directly Regulating the Transcription of Isocitrate Lyase Gene in Mycobacterium smegmatis

You

2021

ACS Infect. Dis.

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The glyoxylate shunt is a pathway associated with the assimilation of fatty acids and is implicated in the resistance of M. tuberculosis (Mtb). Isocitrate lyase (ICL), the first enzyme in the glyoxylate shunt, mediates Mtb infections and its survival in mice via fatty acids, metabolism, and physiological functions. Here, we found that in Mycobacterium smegmatis (M. smegmatis) the two-component system SenX3-RegX3 regulated the glyoxylate shunt in response to phosphate starvation by controlling the transcription of icl. In response to phosphate availability, the phosphate regulator RegX3 directly bound to the upstream regulatory region of icl and repressed its transcription. The inactivation of regX3 increased icl transcription and ICL activity, causing a growth defect in M. smegmatis with fatty acids as the sole source of carbon and energy. The growth defect was partly due to the toxicity of the excess glyoxylate produced by ICL. A decrease in glyoxylic acid levels, overexpression of regX3, or the chemical inhibition (IA or 3-NP) of ICL restored the growth of the Regx3-deficient M. smegmatis. Thus, we established a genetic network between the phosphate stress response and glyoxylate shunt based on the amount of intracellular ICL during mycobacterial survival on short-chain fatty acids, which contributed to its antimicrobial arsenal.

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Section: Methodsmentioning

confidence: 99%

Section: ■ Methodsmentioning

confidence: 99%

RegX3 Controls Glyoxylate Shunt and Mycobacteria Survival by Directly Regulating the Transcription of Isocitrate Lyase Gene in Mycobacterium smegmatis

You

2021

ACS Infect. Dis.

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“…Bacterial infection model of macrophages was mainly modified with reference to the previous report ( Jiang et al, 2018 ). Human THP-1 cell lines (Conservation Genetics, CAS Cell Bank) were grown in 1640 media (Hyclone, United States) containing 10% FBS (Gibco, United States), and adjusted to a final total of 2 × 10 6 cells.…”

Section: Methodsmentioning

confidence: 99%

RegX3-Mediated Regulation of Methylcitrate Cycle in Mycobacterium smegmatis

Pei

et al. 2021

Front. Microbiol.

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Mycobacterium tuberculosis is a global human pathogen that infects macrophages and can establish a latent infection. Emerging evidence has established the nutrients metabolism as a key point to study the pathogenesis of M. tuberculosis and host immunity. It was reported that fatty acids and cholesterol are the major nutrient sources of M. tuberculosis in the period of infection. However, the mechanism by which M. tuberculosis utilizes lipids for maintaining life activities in nutrient-deficiency macrophages is poorly understood. Mycobacterium smegmatis is fast-growing and generally used to study its pathogenic counterpart, M. tuberculosis. In this work, we found that the phosphate sensing regulator RegX3 of M. smegmatis is required for its growing on propionate and surviving in macrophages. We further demonstrated that the expression of prpR and related genes (prpDBC) in methylcitrate cycle could be enhanced by RegX3 in response to the phosphate-starvation condition. The binding sites of the promoter region of prpR for RegX3 and PrpR were investigated. In addition, cell morphology assay showed that RegX3 is responsible for cell morphological elongation, thus promoting the proliferation and survival of M. smegmatis in macrophages. Taken together, our findings revealed a novel transcriptional regulation mechanism of RegX3 on propionate metabolism, and uncovered that the nutrients-sensing regulatory system puts bacteria at metabolic steady state by altering cell morphology. More importantly, since we observed that M. tuberculosis RegX3 also binds to the prpR operon in vitro, the RegX3-mediated regulation might be general in M. tuberculosis and other mycobacteria for nutrient sensing and environmental adaptation.

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“…A recent study showed that a well-characterized serine protease inhibitor, (4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), could inhibit the activities of PafA. 26 inhibition. Ultimately our goal will be to screen for inhibitors that will be able to better penetrate the Mtb cell envelope and have minimal to no toxicity in mammalian cells.…”

Section: Testing the Compounds On Nativementioning

confidence: 99%

Discovery and Optimization of Inhibitors for the Pup Proteasome System in Mycobacterium tuberculosis

Janssen

Zhang

Merkx

et al. 2019

Preprint

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Tuberculosis is a global health problem with the existence and spreading of multidrug resistant and extensive drug resistant strains. The development of new drugs for tuberculosis that inhibit different activities than the current drugs is thus urgent. The prokaryotic ubiquitin like protein proteasome system is an attractive target for the development of new drugs. Using a Pup-based fluorogenic substrate, we screened for inhibitors of Dop, a depupylase, and identified I-OMe-Tyrphostin AG538 (1) and Tyrphostin AG53 (2). The hits were validated and determined to be fast reversible non-ATP competitive inhibitors. The SAR was established by testing 27 synthesized analogs of 1 and 2. Several of the synthesized compounds also inhibited the depupylation of a native substrate, FabD∼Pup. Importantly, the pupylation and depupylation activities of PafA, the sole Pup ligase in M. tuberculosis, was also inhibited by some of these compounds. With the identification of the first described lead compounds for Dop and PafA inhibition, this study shows that high throughput screening can be a successful strategy for this purpose.

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Identification of Serine 119 as an Effective Inhibitor Binding Site of M. tuberculosis Ubiquitin-like Protein Ligase PafA Using Purified Proteins and M. smegmatis

Cited by 14 publications

References 56 publications

RegX3 Controls Glyoxylate Shunt and Mycobacteria Survival by Directly Regulating the Transcription of Isocitrate Lyase Gene in Mycobacterium smegmatis

RegX3 Controls Glyoxylate Shunt and Mycobacteria Survival by Directly Regulating the Transcription of Isocitrate Lyase Gene in Mycobacterium smegmatis

RegX3-Mediated Regulation of Methylcitrate Cycle in Mycobacterium smegmatis

Discovery and Optimization of Inhibitors for the Pup Proteasome System in Mycobacterium tuberculosis

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