Identification of Rv3852 as an Agrimophol-Binding Protein in <i>Mycobacterium tuberculosis</i>

<div><p><i>Mycobacterial tuberculosis (Mtb)</i> is able to preserve its intrabacterial pH (pH_IB) near neutrality in the acidic phagosomes of immunologically activated macrophages and to cause lethal pathology in immunocompetent mice. In contrast, when its ability to maintain pH_IB homeostasis is genetically compromised, <i>Mtb</i> dies in acidic phagosomes and is attenuated in the mouse. Compounds that phenocopy the genetic disruption of <i>Mtb</i>’s pH_IB homeostasis could serve as starting points for drug development in their own right or through identification of their targets. A previously reported screen of a natural product library identified a phloroglucinol, agrimophol, that lowered <i>Mtb</i>’s pH_IB and killed <i>Mtb</i> at an acidic extrabacterial pH. Inability to identify agrimophol-resistant mutants of <i>Mtb</i> suggested that the compound may have more than one target. Given that polyphenolic compounds may undergo covalent reactions, we attempted an affinity-based method for target identification. The structure-activity relationship of synthetically tractable polyhydroxy diphenylmethane analogs with equivalent bioactivity informed the design of a bioactive agrimophol alkyne. After click-chemistry reaction with azido-biotin and capture on streptavidin, the biotinylated agrimophol analog pulled down the <i>Mtb</i> protein Rv3852, a predicted membrane protein that binds DNA <i>in vitro</i>. A ligand-protein interaction between agrimophol and recombinant Rv3852 was confirmed by isothermal calorimetry (ITC) and led to disruption of Rv3852’s DNA binding function. However, genetic deletion of rv3852 in <i>Mtb</i> did not phenocopy the effect of agrimophol on <i>Mtb</i>, perhaps because of redundancy of its function.</p></div

Overexpression and purification of Rv3852 in <i>E</i>. <i>coli</i>.

<p>Coomassie blue stained 12% SDS-PAGE. Lane 1: uninduced lysate; Lane 2: IPTG induced lysate; Lane 3: cytosolic fraction of IPTG induced lysate; Lane 4: membrane fraction of IPTG induced lysate (1% Triton-X100); Lane 5: purified Rv3852 (0.1% Triton-X100). Arrow indicates recombinant Rv3852.</p

Survival of WT and KO in Pcit-Tyl-4.5 at indicated time points.

<p>Means ± S. E. M. of triplicate samples represent three independent experiments. Some error bars are smaller than the symbols.</p

Specific binding between recombinant Rv3852 and agrimophol or a1 detected by ITC.

<p>Binding between recombinant Rv3852 with (A) DMSO, (B) a2, (C) agrimophol and (D) a1, respectively.</p

Construction of <i>rv3852</i> knockout <i>Mtb</i> and verification by Southern blot and PCR.

<p>(A) Upper panel displays the genetic organization of the <i>rv3852</i> region in <i>Mtb</i> (WT), Lower panel displays the same region with replacement of <i>rv3852</i> by hygromycin resistance gene in <i>rv3852</i> knockout <i>Mtb</i> (KO). Filled rectangle indicates the location of probe used in the Southern blot. Sites of digestion by BclI on genomic DNA from WT and KO as well as the sizes of the generated DNA fragment (a and b) are demonstrated under each panel. PCR products from genomic DNA from WT (1 and 2) and KO (3 and 4) are denoted. (B) Left, Southern blot of BclI digested genomic DNA from WT and KO. Calculated sizes of the fragments hybridizing with the probe were 3714 bp (WT) and 2916 bp (KO) as indicated in A. Right, PCR products from genomic DNA from WT and KO. The calculated sizes of the PCR products were 902 (Lane 1), 860 (Lane 2) for WT and 1322 (Lane 3), 1560 bp (Lane 4) for KO as indicated in A.</p

Specific binding between recombinant Rv3852 and a1b detected by fluorescently tagged streptavidin in Western blot.

<p>(A) Among DMSO, a1b, a2b, a1, a2 and biotin, only a1b demonstrated binding with recombinant Rv3852 as indicated in Western blot detecting by fluorescently tagged streptavidin. Rv2466c served as a control. (B) Equimolar recombinant Rv3852 (left) or Rv3852 and Rv2466c (right) were separated on 12% SDS-PAGE and stained by Coomassie Blue.</p

Identification of Mb3882.

<p>Detected sequences in MALDI-TOF MS are highlighted in bold. The 5 tetrapeptide repeats (4 PAKK and 1 KKAP) are italicized. Mb3882 was not identified in the DMSO treated group.</p><p>Identification of Mb3882.</p

EMSA.

<p>(A) Recombinant Rv3852 abrogated the mobility of proU2 on 1% agarose gel. (B) agrimophol or a1 interfered with recombinant Rv3852’s mobility shift of proU2 on 1% agarose gel in a concentration-dependent manner.</p

Activities of agrimophol and its analogs a1, a2, a1b and a2b on <i>BCG</i>.

<p>(A) pH_IB disruptive activity of agrimophol and its analogs a1 and a2 at 12.5 μM, a1b and a2b at 100 μM in Pcit-Tyl-4.5 at indicated time points. (B) CFU decreasing activity of agrimophol and its analogs a1 and a2 at 12.5 μM, a1b and a2b at 100 μM in Pcit-Tyl-4.5 at indicated time points. Means ± S. E. M. of triplicate samples represent three independent experiments. Some error bars are smaller than the symbols.</p

Synthesis of agrimophol analogs with alkyne group (a1 and a2) and corresponding biotinylated agrimophol analogs (a1b and a2b).

<p>Synthesis of agrimophol analogs with alkyne group (a1 and a2) and corresponding biotinylated agrimophol analogs (a1b and a2b).</p