Dehydroquinate Synthase Directly Binds to Streptomycin and Regulates Susceptibility of Mycobacterium bovis to Streptomycin in a Non-canonical Mode

Abstract

Antimicrobial resistance (AMR) represents one of the main challenges in Tuberculosis (TB) treatment. Investigating the genes involved in AMR and the underlying mechanisms holds promise for developing alternative treatment strategies. The results indicate that dehydroquinate synthase (DHQS) regulates the susceptibility of Mycobacterium bovis BCG to first-line anti-TB drug streptomycin. Perturbation of the expression of aroB encoding DHQS affects the susceptibility of M. bovis BCG to streptomycin. Purified DHQS impairs in vitro antibacterial activity of streptomycin, but did not hydrolyze or modify streptomycin. DHQS directly binds to streptomycin while retaining its own catalytic activity. Computationally modeled structure analysis of DHQS-streptomycin complex reveals that DHQS binds to streptomycin without disturbing native substrate binding. In addition, streptomycin treatment significantly induces the expression of DHQS, thus resulting in DHQS-mediated susceptibility. Our findings uncover the additional function of DHQS in AMR and provide an insight into a non-canonical resistance mechanism by which protein hijacks antibiotic to reduce the interaction between antibiotic and its target with normal protein function retained.

Keywords: DHQS; antimicrobial resistance; aroB; mycobacteria; streptomycin.

Figure 1

Dehydroquinate synthase (DHQS) expression level affects streptomycin susceptibility of Mycobacterium bovis BCG. (A) Overexpression of aroB reduced the streptomycin susceptibility of M. bovis BCG. The growth of aroB overexpressing strain and control strain (empty plasmid) on solid medium added with 0, 0.0625, and 0.125 μg/ml streptomycin was investigated. Freshly cultivated strains were grown on 7H9 medium until OD₆₀₀ reached 1.0. The serially diluted (10⁻⁰, 10⁻¹, 10⁻², 10⁻³, and 10⁻⁴) strains were spotted onto 7H10 plates containing OADC growth supplements and cultured for another 7 days at 37°C. (B) Growth of aroB overexpressing strain and control strain in liquid medium added with 0, 0.125 μg/ml streptomycin. Growth curves of control strain and aroB-overexpressing strain in the medium with 0.125 μg/ml streptomycin or without drug (No drug) during 10 days. Error bars represent the SD of three biological replicates. (C) Knock-down of aroB increased the streptomycin susceptibility of M. bovis BCG. Aliquots were sampled at the indicated times, and the CFU was measured. Error bars represent the SD of three biological replicates.

Figure 2

Dehydroquinate synthase impairs in vitro antibacterial activity of streptomycin. The 0.25 or 0.5 μg/ml streptomycin was incubated with 1.5 μM DHQS in the presence or absence of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) substrate for 30 min at 37°C. The reaction mixture was added to the 7H10 plate. Wild-type Mycobacterium smegmatis grown to the logarithmic phase was serially diluted (10⁻⁰, 10⁻¹, 10⁻², 10⁻³, and 10⁻⁴), and equal amount of culture was spotted on the solid plate and cultivated for 3 days.

Figure 3

Dehydroquinate synthase directly binds to streptomycin. (A) Binding analysis of streptomycin and DHQS by drug affinity response target stability (DARTS). About 40 μM DHQS was incubated with increasing concentrations of streptomycin (0.37, 0.75, and 1.5 mM) or 1.5 mM INH (negative control) for 30 min at 37°C, followed by digestion with 180 ng of pronase E for 30 min at 37°C. All the samples were subjected to a 12% w/v SDS-PAGE. The amount of protein degradation at each concentration was quantified with the non-digested lane as internal reference. Barplot shows the mean and SD of three biological replicates. p-values were calculated by GraphPad Prism 8 with an unpaired two-tailed Student’s t-test. Asterisk represents the significant difference between the two groups of data (^*p < 0.05, ^**p < 0.01). (B) Isothermal titration calorimetry (ITC) analysis of 50 μM DHQS binding with 1 mM streptomycin (left) or 1 mM isoniazid (right). Original titration data and integrated heat measurements are shown in the upper and lower panel, respectively. The solid line in the left lower panel represents the best fitting of a single-site binding model of DHQS and streptomycin, while right lower panel shows no interaction between DHQS and isoniazid.

Figure 4

Binding model of DHQS and streptomycin. (A) Streptomycin binds to the proposed site of DHQS. (B) Residues involved in the binding of streptomycin and DHQS. (C) ITC analysis of 1 mM streptomycin binding to 50 μM mutant proteins (S125A, Q161A, and S125AQ161A). ITC was carried out with 1 mM streptomycin and mutant protein concentration of 50 μM.

Figure 5

Streptomycin induces increased expression of aroB in Mycobacterium bovis BCG. (A) Expression of aroB at 2, 4, 6 h post-cultivation. The mid-log phase of M. bovis BCG (OD₆₀₀ = 1.0) was cultivated with 0.125 and 0.25 μg/ml streptomycin to induce the expression of aroB, and aroB expression in M. bovis BCG strain was detected by quantitative real-time PCR (qRT-PCR). (B) Expression of rpsL, gidB, eis, and ldtB genes at 6 h post-cultivation. The expression of streptomycin resistance-related genes rpsL and gidB, and unrelated genes eis and ldtB after streptomycin treatment were quantitatively detected. Relative expression levels of the genes were normalized with the sigA gene as internal reference. The data were analyzed using the 2^−ΔΔCt method. Barplot shows the mean and SD of three biological replicates.

Figure 6

A model in which DHQS mediates streptomycin susceptibility. Streptomycin inhibits bacteria by targeting 30S ribosome. The expression of DHQS protein was increased under streptomycin treatment. DHQS directly bound to streptomycin while retaining its own normal catalytic activity. As a result, DHQS “hijacks” streptomycin, thus reducing the binding of streptomycin to 30S ribosome target, eventually reducing the susceptibility of bacteria to streptomycin.