Discovery of the first macrolide antibiotic binding protein in ... antibiotics. One of these is

Discovery of the first macrolide antibiotic binding protein in ... antibiotics. One of these is
Discovery of the first macrolide antibiotic binding protein in ... antibiotics. One of these is
Discovery of the first macrolide antibiotic binding protein in ... antibiotics. One of these is
Discovery of the first macrolide antibiotic binding protein in ... antibiotics. One of these is
Discovery of the first macrolide antibiotic binding protein in ... antibiotics. One of these is
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  • LETTER

    Discovery of the first macrolide antibioticbinding protein in Mycobacteriumtuberculosis: a new antibiotic resistance drugtarget

    Dear Editor,

    The prevalence of multidrug-resistant Mycobacteriumtuberculosis (M. tuberculosis) is an increasing problemworldwide (Zumla et al., 2013; Dong et al., 2015). Accordingto a 2014 World Health Organization (WHO) report, 480,000individuals world-wide developed multidrug-resistant tuber-culosis (MDR-TB) and more than 100 countries have casesof extensively drug-resistant tuberculosis (XDR-TB). Com-pared with drug-susceptible TB, MDR-TB and XDR-TBrequire prolonged therapeutic treatment with a combinationof a number of second-line drugs (Chen et al., 2013). Forpatients where TB remains persistent despite prolongedtherapy with second-line TB drugs, the add-on agentsincluding bedaquiline and delamanid are recommended forsalvage therapy (Gnther, 2014; WHO, 2014; Shim and Jo,2013).

    The mechanisms of antibiotic resistance developed bybacteria are highly diverse (Alekshun and Levy, 2007),including protection of the antibiotic target by site-of-actionmutations or by chemical modification of the target/target site(e.g., methylation in the ribosomal 23S rRNA by ErmMTmethyl-transferase) (Buriankova et al., 2003). The directmodification or inactivation of antibiotics by specific enzymes(e.g., acetyltransferases, phosphotransferases) can beanother resistance mechanism. Alternatively, reduced per-meability or increased efflux (Black et al., 2014) of the drugscan also occur, thus preventing the drug from gaining accessto the target. M. tuberculosis has adopted several of thesestrategies to become resistant to the most widely usedantibiotics. One of these is the utilization of the ATP-bindingcassette (ABC) transporters, which are drug efflux pumpsthat lower the concentration of antibiotics within the bac-terium. Indeed, ABC transporters have been divided intothree classes based on phylogenetic analysis (Dassa andBouige, 2001): (i) the classical exporters (ii) importers whichare composed of hydrophobic transmembrane domains andhydrophilic nucleotide-binding domains, and (iii) the type-IIABC proteins that lack transmembrane domains (Nunez-

    Samudio and Chesneau, 2013), some of which mediateantibiotic resistance (Kerr et al., 2005; Sharkey et al., 2016).

    Bioinformatics analysis shows that M. tuberculosis pos-sesses as many as 87 ABC containing proteins. Amongstthese, Rv3197 is predicted to be a non-canonical ABC pro-tein with an N-terminal extension (1117), an ABC1 motif(118232) and an aminoglycoside phosphotransferase(APH) motif (256312) but lacking any transmembraneregions. The sequence characterization suggested Rv3197might be an antibiotic transporter. In our preliminary study,we evaluated the effect of Rv3197 in antibiotic susceptibilityby measuring the MICs of several antibiotics including iso-niazid, rifampicin, erythromycin, streptomycin, ampicillin,chloromycetin and tetracycline by overexpression of Rv3197in Mycobacterium smegmatis (M. smegmatis), which iscommonly used in lab as a model bacteria for M. tubercu-losis. Our results indicated that Rv3197 affected the sus-ceptibility of erythromycin (Table S1).

    Since a biosafety level 3 lab is needed to direct study ofM. tuberculosis and the amino acid sequence of Rv3197 inM. tuberculosis is equivalent to Mb3320 in M. bovis BCG, avaccine strain with low pathogenicity, we chose M. bovisBCG as a model for studying the function of Rv3197.Quantitative real-time PCR (qRT-PCR) analysis showed thatthe transcription level of rv3197 increases by 3.6 fold whenM. bovis BCG is treated with 6 mg/L erythromycin (Fig. 1A),showing that expression of rv3197 is erythromycin-inducible.To verify the effect of Rv3197 on erythromycin susceptibility,we first made an Mb3320-deletion mutant strain, denotedrv3197-BCG. This mutant strain grew at a similar rate to thewild-type strain in 7H9 medium (Fig. S1A), demonstratingthat, under the conditions where no antibiotic is added,Rv3197 has no effect on cell growth. However, upon expo-sure to 2 mg/L of erythromycin, cell growth was reduced inrv3197-BCG compared to wild-type BCG. This effect waspartially reversed in a complemented pMV361-rv3197/rv3197-BCG strain (Fig. 1B). In the presence of 3 mg/L oferythromycin, M. smegmatis, which contains the rv3197

    The Author(s) 2018. This article is an open access publication

    Protein Cell 2018, 9(11):971975https://doi.org/10.1007/s13238-017-0502-7 Protein&Cell

    Protein

    &Cell

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  • gene (pMV261-rv3197-mc2155) has a growth advantageover M. smegmatis that has an empty pMV261 vector(pMV261-mc2155) (Fig. 1C). Likewise, the M. bovis BCG

    pMV261-rv3197 construct also showed a growth advantageover wild type in the presence of 4 mg/L of erythromycin(Fig. 1D). In contrast, no growth difference was detected

    A

    E F

    B DCBCGrv3197-BCGpMV361-rv3197/rv3197-BCG

    pMV261-mc2155pMV261-rv3197-mc2155

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    R200

    R200D170

    D170

    Q58

    Q58 E65

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    Q191

    Q191

    D189D189

    K67

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    1

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    6

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    **

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    pMV261-BCGpMV261-rv3197-BCG

    Figure 1. The functional and structural studies of Rv3197 (MABP-1). MABP-1 confers inducible resistance to erythromycin in

    mycobacteria (AD). (A) mRNA levels of rv3197 in BCG determined by qRT-PCR after treatment with 6 mg/L erythromycin. (B) Thegrowth of wild type BCG, rv3197-BCG and pMV361-rv3197/rv3197-BCG in 7H9 medium in the presence of 2 mg/L erythromycin.

    (C) Growth curves of pMV261-mc2155 and pMV261-rv3197-mc2155 in 7H9 medium in the presence of 3 mg/L erythromycin.

    (D) Growth curves of pMV261-BCG and pMV261-rv3197-BCG in 7H9 medium in the presence of 4 mg/L erythromycin. All results are

    shown as the mean standard deviation of three individual experiments. *P < 0.05, **P < 0.01, ***P < 0.001. The crystal structure of

    MABP-1 (EF). (E) The domain arrangement of a MABP-1 monomer. The accessory domain is encircled by the dashed outline.(F) Left, surface representation of the MABP-1 dimer. Right, the dimer subunit interface. Each subunit is coloured differently. The 1

    and 2 helices from each subunit insert into each others accessory domain. The black oval represents a two-fold axis perpendicular

    to the given view. Hydrogen bonds are shown as thick dashed lines.

    LETTER Qingqing Zhang et al.

    972 The Author(s) 2018. This article is an open access publication

    Protein

    &Cell

  • between pMV261-rv3197-mc2155 and pMV261-mc2155 andbetween pMV261-rv3197-BCG and pMV261-BCG in theabsence of erythromycin (Fig. S1B and S1C). These datademonstrate that Rv3197 is a factor responsible for ery-thromycin resistance in both M. bovis and M. smegmatis.Based on sequence similarity, this protein is also expected toperform a similar role in other Mycobacteria (e.g., My-cobacterium tuberculosis, Mycobacterium kansasii, andMycobacterium avium). In consideration of its antibioticresistance phenotype, we henceforth refer to this protein asmacrolide antibiotic binding protein-1 (MABP-1), signifying itas the first characterization of a macrolide antibiotic bindingprotein in Mycobacterium.

    The crystal structure of apo-MABP-1 was determined at2.2 by single wavelength anomalous dispersion (SAD)(Table S2). MABP-1 is a homodimer with each subunitcontaining a bilobal kinase domain (Fig. S2) and an acces-sory domain (Figs. 1E and S2). According to a DALI searchthe kinase domain strongly resembles that of an ancientUbIB protein kinase (ADCK3) (Table S3). The accessorydomain consists of six -helices: 14 helices (residues 1120) and 67 helices (residues 161206). These regionsare insertions that are not found in the canonical kinasestructures. Crucially, the 1 and 2 helices in each subunitinsert into the accessory domain of its adjoining subunit,resulting in an N-terminal domain-swapped dimer (Fig. 1F).The accessible surface area that is buried upon dimerizationis extensive, at 2021 2 per subunit.

    Structural analysis confirmed that MABP-1 has an ATPbinding loop (P-loop) in its kinase domain suggesting ATP asa substrate. Indeed, a spectrophotometric assay shows thatit can hydrolyse ATP (kcat = 0.0137 s

    1, Km = 0.285 mmol/L)(Fig. S3A), values that are comparable to that for other ABCtransporters (Lin et al., 2015). Moreover, our data showedthat the presence of erythromycin does not affect theATPase activity of MABP-1 (Fig. S3B). To gain furtherinsights into how MABP-1 recognizes ATP, the crystalstructures of MABP-1 in complex with AMPPNP and withADP, were determined. Since AMPPNP and ADP bind toMABP-1 in a similar fashion, only the binding mode ofAMPPNP (Figs. 2A and S4) is discussed below. In thiscomplex (Figs. 2B and S5A), AMPPNP is stabilized byextensive hydrogen bonds and hydrophob