Functional analysis of the Mycobacterium tuberculosis MprAB two-component signal transduction system

Abstract

The mechanisms utilized by Mycobacterium tuberculosis to establish, maintain, or reactivate from latent infection in the host are largely unknown but likely include genes that mediate adaptation to conditions encountered during persistence. Previously, a two-component signal transduction system, mprAB, was found to be required in M. tuberculosis for establishment and maintenance of persistent infection in a tissue- and stage-specific fashion. To begin to characterize the role of this system in M. tuberculosis physiology and virulence, a functional analysis of the mprA and mprB gene products was initiated. Here, evidence is presented demonstrating that sensor kinase MprB and response regulator MprA function as an intact signal-transducing pair in vitro and in vivo. Sensor kinase MprB can be autophosphorylated, can donate phosphate to MprA, and can act as a phospho-MprA phosphatase in vitro. Correspondingly, response regulator MprA can accept phosphate from MprB or from small phosphodonors including acetyl phosphate. Mutagenesis of residues His249 in MprB and Asp48 in MprA abolished the ability of these proteins to be phosphorylated in vitro. Introduction of these alleles into Mycobacterium bovis BCG attenuated virulence in macrophages in vivo. Together, these results support a role for the mprAB two-component system in M. tuberculosis physiology and pathogenesis. Characterization of two-component signal transduction systems will enhance our understanding of processes regulated by M. tuberculosis during acute and/or persistent infection in the host.

FIG. 1.

In vitro autophosphorylation of GST-cMprB derivatives. (A) Purified GST-cMprB was incubated in the presence of [γ-³²P]ATP (lanes 1, 3, 5, and 7) or [α-³²P]ATP (lanes 2, 4, 6, and 8) and divalent cations including Mg²⁺ (lanes 1 and 2), Mn²⁺ (lanes 3 and 4), and Ca²⁺ (lanes 5 and 6) or in the absence of metal (lanes 7 and 8). (B) Wild-type GST-cMprB (lanes 1 to 4) or the GST-cMprB (His249-Gln) mutant (lanes 5 to 8) was incubated in the presence of [γ-³²P]ATP and Mg²⁺ (lanes 1 and 5), Mn²⁺ (lanes 2 and 6), or Ca²⁺ (lanes 3 and 7) or in the absence of divalent cations (lanes 4 and 8). Phosphorylation of wild-type or mutant GST-cMprB was detected by autoradiography, and polyclonal antibody directed against cMprB was used in Western blotting to confirm similar loading amounts between reactions.

FIG. 2.

Transphosphorylation between GST-cMprB and M. tuberculosis response regulators. Wild-type GST-cMprB was autophosporylated with [γ-³²P]ATP and then incubated in the absence of other proteins (lanes 1 and 2) or in the presence of wild-type His-MprA (lanes 3 to 7), the His-MprA (Asp48-Ala) mutant (lanes 8 to 12), or wild-type His-MtrA (lanes 13 to 17). Transphosphorylation reactions were allowed to proceed for 0 min (lanes 1, 3, 8, and 13), 5 min (lanes 4, 9, and 14), 10 min (lanes 5, 10, and 15), 20 min (lanes 6, 11, and 16), or 30 min (lanes 2, 7, 12, and 17). Closed arrows indicate the locations of full-length GST-cMprB and response regulator proteins. Open arrows indicate the locations of truncated (trunc.) forms of GST-cMprB. The asterisk indicates the position of phosphorylated His-MprA species. Transfer of radiolabel from GST-cMprB to response regulator proteins was detected by autoradiography. WT, wild type.

FIG. 3.

Phosphorylation of His-MprA with acetyl phosphate and resolution by 2D gel electrophoresis. Wild-type His-MprA was incubated in the absence (A) or presence (B) of acetyl phosphate. His-MprA protein was separated according to charge (pI) and molecular mass by 2D gel electrophoresis. Unphosphorylated (closed arrowheads) and phosphorylated (open arrowhead) forms of His-MprA were quantitated by densitometry, and the amount of product formed was determined (C). The mean and standard error for each category were determined from results of three independent reactions, each run on individual 15% polyacrylamide gels. Significance refers to the comparison between the amount of His-MprA in the shifted form (pI 5.8) following incubation with acetyl phosphate and the amount of His-MprA in the shifted form (pI 5.8) following incubation in the absence of acetyl phosphate. WT, wild type.

FIG. 4.

Phospho-MprA dephosphorylation by GST-cMprB. Wild-type His-MprA was incubated in the presence of acetyl phosphate (AP) for 30 min to induce phosphorylation. Reaction mixtures containing His-phospho-MprA were then incubated alone (A), in the presence of wild-type GST-cMprB (B), or in the presence of the GST-cMprB (His249-Gln) mutant (C) for an additional 30 min. Unphosphorylated (closed arrowheads) and phosphorylated (open arrowheads) forms of His-MprA were quantitated by densitometry, and the amount of phosphorylated product remaining was determined (D). The mean and standard error for each category were determined from results of three independent reactions, each run on individual 15% polyacrylamide gels. Significance refers to the comparison between the amount of His-MprA remaining in the shifted form (pI 5.8) following incubation with wild-type GST-cMprB and the amount of His-MprA in the shifted form (pI 5.8) following incubation with the GST-cMprB (His 249-Gln) mutant. WT, wild type.

FIG. 5.

Survival of M. bovis BCG derivatives in macrophages. J774 macrophages were infected with various BCG derivatives for 2 h or 1, 3, or 5 days. Numbers of CFU were determined from infected macrophages at each time point and were normalized to the 2-h time point numbers that were set as 100%. Strains examined included wild-type BCG Pasteur (closed squares), the isogenic mprA::Km^r mutant (diamonds), the mprA::Km^r mutant complemented with wild-type mprA and wild-type mprB (pTZ215) in trans (circles), the mprA::Km^r mutant complemented with wild-type mprA and mprB (His249-Gln) (pTZ339) in trans (triangles), and the mprA::Km^r mutant complemented with mprA (Asp48-Ala) and wild-type mprB (pTZ336) in trans (open squares). The means and standard errors of results from three independent infections are shown.