RegX3 Activates whiB3 Under Acid Stress and Subverts Lysosomal Trafficking of Mycobacterium tuberculosis in a WhiB3-Dependent Manner

Abstract

Two-component systems (TCSs) are central to the ability of Mycobacterium tuberculosis to respond to stress. One such paired TCS is SenX3-RegX3, which responds to phosphate starvation. Here we show that RegX3 is required for M. tuberculosis to withstand low pH, one of the challenges encountered by the bacterium in the host environment, and that RegX3 activates the cytosolic redox sensor WhiB3 to launch an appropriate response to acid stress. We show that the whiB3 promoter of M. tuberculosis harbors a RegX3 binding motif. Electrophoretic mobility shift assays (EMSAs) show that phosphorylated RegX3 (RegX3-P) (but not its unphosphorylated counterpart) binds to this motif, whereas a DNA binding mutant, RegX3 (K204A) fails to do so. Mutation of the putative RegX3 binding motif on the whiB3 promoter, abrogates the binding of RegX3-P. The significance of this binding is established by demonstrating that the expression of whiB3 is significantly attenuated under phosphate starvation or under acid stress in the regX3-inactivated mutant, regX3. Green fluorescent protein (GFP)-based reporter assays further confirm the requirement of RegX3 for the activation of the whiB3 promoter. The compromised survival of regX3 under acid stress and its increased trafficking to the lysosomal compartment are reversed upon complementation with either regX3 or whiB3, suggesting that RegX3 exerts its effects in a WhiB3dependent manner. Finally, using an in vitro granuloma model, we show that granuloma formation is compromised in the absence of regX3, but restored upon complementation with either regX3 or whiB3. Our findings provide insight into an important role of RegX3 in the network that regulates the survival of M. tuberculosis under acid stress similar to that encountered in its intracellular niche. Our results argue strongly in favor of a role of the RegX3-WhiB3 axis in establishment of M. tuberculosis infection.

A systems approach to decipher a role of transcription factor RegX3 in the adaptation of Mycobacterium tuberculosis to hypoxic stress

“…RegX3 was phosphorylated using phospho-EnvZ as the phosphate donor as described by Mahatha et al . [13]. The reaction was carried out in kinase buffer with 2.5 µM phosphorylated EnvZ and His-RegX3.…”

“…The cells were lysed and DNA was sheared on a BIORUPTOR PLUS (Diagenode) with 25 cycles of 30 s/90 s on/off respectively, so as to generate fragment sizes of approximately 250 bp. Immunoprecipitation (IP) was carried out using RegX3 antibody (raised by Thermo Fisher Scientific) [13,22] and DNA was purified after decrosslinking with Proteinase K. The specificity of the RegX3 antibody was confirmed earlier by Western blotting using lysates from wild-type and ΔregX3 [22] qPCR validation of ChIP.…”

“…RegX3 belongs to the OmpR family of response regulators. SenX3-RegX3 regulates virulence [10][11][12], the ability of M. tuberculosis to survive acidic pH [13], the stringent response [14], and persister formation [15]. It is a redox sensing SK [16].…”

Two‐component sensor histidine kinases of Mycobacterium tuberculosis: Beacons for niche navigation

“…The sensor kinase autophosphorylates upon stimulus detection on a histidine (H) residue and relays this phosphoryl group (P) to an aspartate residue (D) of the receiver domain located on its cognate response regulator, subsequently acting as a transcriptional regulator to modulate the expression of genes required for successful adaptation. Twocomponent systems generally self-regulate in a positive manner, whereby the phosphorylated RR promotes the transcription of genes encoding the two-component system TA B L E 1 Mycobacterium tuberculosis TCS sensor kinase knowledge base; regulation, interaction, and inhibition (Singh & Kumar, 2015), phosphate starvation (Glover et al, 2007;Rifat et al, 2014;Tischler et al, 2013), pH (Mahatha et al, 2020) NCA RegX3 (Himpens et al, 2000) No (Agrawal et al, 2015) NCA NCA HK1/HK2 NCA NCA TcrA (Shrivastava et al, 2007) No (Agrawal et al, 2015) NCA NCA PhoR pH (Abramovitch et al, 2011;Baker et al, 2014;Feng et al, 2018), chloride (Tan et al, 2013), magnesium a (Walters et al, 2006) Yes (Xing et al, 2017) PhoP (Gupta et al, 2006) DosR, TcrX, TcrA (Agrawal et al, 2015) NCA Ethoxzolamide b (Johnson et al, 2015) NarS Nitrate/Nitrite (Malhotra et al, 2015) NCA NarL <...>…”

Assessment of Experimental Techniques That Facilitate Human Granuloma Formation in an In Vitro System: A Systematic Review

“…A majority of the studies were focused on mycobacteria antigens, including several mycobacterial strains such as M. tb H37Rv [ 5 , 22 , 27 , 28 , 38 , 39 , 43 , 44 , 45 , 47 , 49 , 50 , 55 , 66 , 71 , 72 , 73 , 74 , 75 , 76 ], M. tb Erdmann [ 63 , 65 , 77 , 78 ], and species such as M. leprae [ 12 , 79 ], M. abscessus [ 48 ] as well as BCG [ 24 , 29 , 30 , 56 , 57 , 62 , 69 , 80 ] and PPD [ 23 , 54 , 59 , 61 ]. Others used antigens of other infectious species such as Schistosoma [ 31 , 32 , 33 , 34 , 35 , 36 , 37 , 58 ] or Candida [ 2 , 26 , 42 , 81 ].…”