Characterization of an interplay between a Mycobacterium tuberculosis MazF homolog, Rv1495 and its sole DNA topoisomerase I

Abstract

The MazEF systems are thought to contribute to the capacity for long-term dormancy observed in the human pathogen, Mycobacterium tuberculosis. However, except for their functions as mRNA interferases, little is known regarding any additional cellular functions of these systems in the pathogen. In the present study, we observed a negative interplay between MazF protein Rv1495 and the sole M. tuberculosis DNA topoisomerase I (MtbTopA) with respect to protein functions. Through its C-terminal domain, MtbTopA physically interacted with and inhibited the mRNA cleavage activity of Rv1495. Rv1495, in turn, inhibited the DNA cleavage activity of MtbTopA as well as its function of relaxation of supercoiled DNA. An N-terminus fragment of Rv1495, designated Rv1495-N(29-56), lost mRNA cleavage activity, but retained a significant physical interaction and inhibitory effect on TopA proteins from both M. tuberculosis and M. smegmatis. This fragment, although less effective than the full-length protein, was able to inhibit mycobacterial growth when expressed through a recombinant plasmid in M. smegmatis. The Rv1495 physically interacted with the M. smegmatis TopA both in vitro and in vivo. Our findings imply that MazEF systems can affect bacterial survival by a novel mechanism that allows direct modulation of M. tuberculosis topoisomerase I.

Figure 1.

Physical interactions of Rv1495 with MtbTopA. (A) Bacterial two-hybrid assays (Stratagene) for the interaction of Rv1495 with MtbTopA, which were performed as described under ‘Materials and Methods’ section. Left panel, plate minus streptomycin (str) and 5 mM 3-amino-1,2,4-triazole (3-AT), Middle panel, plate plus str and 5 mM 3AT. Right panel, an outline of the plates. CK⁺, co-transformant containing pBT-LGF2 and pTRG-Gal11P as a positive control. CK⁻, co-transformant containing pBT and pTRG as a negative control. (B) Pull-down assays for examining the interaction between Rv1495 and MtbTopA. Equimolar amounts of 6× His-MtbTopA combined with GST-Rv1495 were used for pull-down assays as described in the ‘Materials and Methods’ section. GST was used as the negative control. One predicted size of his-tagged MtbTopA protein, pulled down by GST-tagged Rv1495 protein, was further examined by a western blotting assay.

Figure 2.

Effects of Rv1495 on topoisomerase activities of MtbTopA. Topoisomerase activity assays were performed as described under ‘Materials and Methods’ section. The plasmid pBluscript was used as DNA substrate in all reaction mixtures. The reaction was treated with 6% SDS and 4 mg/ml proteinase K analyzed on a 0.8% agarose gel and then stained with ethidium bromide. Lanes 1, no topoisomerase as negative control. L, linearized plasmid; R, relaxed plasmid; S, supercoiled plasmid. (A) Different amounts of topoisomerase I (0–0.15 μM) were added to 200 ng of supercoiled pBluscript DNA. (B) Effects of different amounts of Rv1495 (0–6 μM) on the topoisomerase I (0.125 μM) activities on the supercoiled pBluscript DNA. (C) Effects of different amounts of heated-Rv1495 on the topoisomerase I activities. (D) Effects of different amounts of GST on the topoisomerase I activities. (E) EMSA assays for the plasmid DNA-binding activities of RV1495, Lsr2, or Rv0054. Lsr2 is a positive control. Rv0054 (MtbSSB) is a negative control.

Figure 3.

Effects of Rv1495 on the single-stranded DNA cleavage activity of MtbTopA. 5′-end-labeled 32-mer oligonucleotide (5′-CAGTGAGCGAGCTTCCGCTTGACATCCC AATA-3′) (27) was used as a specific single-stranded DNA substrate. DNA-binding and cleavage assays of MtbTopA were performed as described under ‘Materials and Methods’ section. (A) DNA-binding activity assays for MtbTopA, Rv1495 and Rv0054 (a positive control). The protein concentrations were indicated on top of the panels. (B) ssDNA cleavage assays for MtbtopA (left panel) and the effects of Rv1495 (right panel). The 32-mer substrates and 19-mer cleavage products were indicated by arrows. (C) The effects of the denatured Rv1495 protein (left panel) or GST (right panel) on the cleavage activity of MtbTopA.

Figure 4.

Effects of MtbTopA on mRNA cleavage activities of Rv1495. (A) mRNA cleavage experiments were performed as described under ‘Materials and Methods’ section. A variant concentration of Rv1495 (0.5–2.0 μM) or MtbTopA (40–120 nM) proteins was incubated with 0.5 μM mRNA substrates in the reactions. The mRNA substrate, products and their sequences were indicated by arrows. Rv0054 was used a negative control. (B) Two mutant proteins of MtbTopA and their interactions with Rv1495. The names given to mutants (NTD and CTD) and their amino acid regions are indicated. Bacterial two-hybrid assays for all mutants were performed as described under ‘Materials and Methods’ section. + represents growth on the screening plate; − represents no growth on the screening plate. (C) Effects of N-terminal and C-terminal mutant proteins of MtbTopA on the mRNA cleavage activities of Rv1495. The protein concentrations were indicated on top of the panel.

Figure 5.

Interactions of Rv1495 with MsmTopA in vitro and in vivo. (A) Bacterial two-hybrid assays (Stratagene). Experiments were performed as described under ‘Materials and Methods’ section. Left panel, plate minus streptomycin (str) and 5 mM 3-amino-1,2,4-triazole (3-AT), Middle panel, plate plus str and 5 mM 3AT. Right panel, an outline of the plates. (B) Pull-down assays for examining the interaction between Rv1495 and MsmTopA. Equimolar amounts of 6× His-MsmTopA combined with GST-Rv1495 were used for pull-down assays as described in the ‘Materials and Methods’ section. GST was used as the negative control. One predicted size of his-tagged MsmTopA protein, pulled down by GST-tagged Rv1495 protein, was further examined by a western blotting assay. (C) Co-IP assays. Exponentially growing cells of the recombinant M. smegmatis containing Rv1495-expression plasmid were harvested, resuspended and lysed. Co-IPs were performed by incubating 10 μl of M. smegmatis cell extracts with 3 μl of MsmTopA antiserum for 3 h at 4°C. A 20-μl slurry of protein A Sepharose was added, and incubation was continued for another hour. Immune complexes were collected, and the beads were washed with buffer. Finally, the beads were resuspended in SDS–PAGE sample buffer. After boiling, the samples were analyzed by western blotting using anti-Rv1495 antibody.

Figure 6.

N-terminus of Rv1495 regulates the topoisomerase activities of MtbTopA. Topoisomerase activity and bacterial two-hybrid assays for all mutants were performed as described under ‘Materials and Methods’ section. + represents growth on the screening plate; −  represents no growth on the screening plate. (A) The N-terminal mutant fragments of Rv1495 containing 56 and 28 amino acid residues (left panel) and their interactions with MtbTopA assayed by the bacterial two-hybrid system (right panel). (B) Two synthesized short peptides and their amino acid sequences. (C) Effect of different amount of Rv1495-N28, Rv1495-N56, and Rv1495-N(29-56) (0–6.0 μM) on topoisomerase activity.

Figure 7.

Activities of Rv1494 and Rv1495-N(29-56) and their effects on the mycobacterial growth. (A) Effects of Rv1495 and Rv1495-N(29-56) on the topoisomerase activities of MsmTopA. The assays were performed as described under ‘Materials and Methods’ section. L, linearized plasmid; R, relaxed plasmid; S, supercoiled plasmid. The protein concentrations were indicated on top of the panels. (B) mRNA cleavage activity assays. Experiments were performed as described under ‘Materials and Methods’ section. A varying concentration of Rv1495 or Rv1495-N(29-56) proteins was incubated with 0.5 μM mRNA substrates in the reactions. The mRNA substrate and products were indicated by arrows. (C) Effects of the expressions of Rv1495 and Rv1495-N(29-56) on the mycobacterial growth. A TetR-controlled expression system was used to analyze the effects of Rv1495 genes on the growth of M. smegmatis mc² 155 as described in the ‘Materials and Methods’ section. The growth of these recombinant mycobacterial strains were examined in the presence (induction) or absence (no induction) of tetracycline (Tc). Aliquots were taken at the indicated times and the OD₆₀₀ was measured. Each analysis was performed in triplicate. The representative growth curves were plotted. The recombinant mycobacterial strains were indicated above the panels.