A Nitrooxidoreductase Rv2466c-dependent Fluorescent Probe for

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A Nitrooxidoreductase Rv2466c-dependent Fluorescent Probe for Rapid Mycobacterium Tuberculosis Diagnosis and Drug Susceptibility Testing Ran Mu, Chengcheng Kong, Wenjun Yu, Hongyao Wang, Yao Ma, Xueyuan Li, Jie Wu, Selin Somersan-Karakaya, Haitao Li, Zhaogang Sun, and Gang Liu ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.9b00006 • Publication Date (Web): 27 Mar 2019 Downloaded from http://pubs.acs.org on March 28, 2019

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ACS Infectious Diseases

A Nitrooxidoreductase Rv2466c-dependent Fluorescent Probe for Rapid Mycobacterium Tuberculosis Diagnosis and Drug Susceptibility Testing Ran Mu1#, Chengcheng Kong2,3#, Wenjun Yu1#, Hongyao Wang1#, Yao Ma1, Xueyuan Li1, Jie Wu1, Selin Somersan-Karakaya4, Haitao Li5, Zhaogang Sun2,3*, Gang LIU1* 1. School of Pharmaceutical Sciences, Tsinghua University, Haidian Dist., Beijing 100084, P. R. China. 2. Beijing Key Laboratory in Drug Resistant Tuberculosis Research, Beijing Tuberculosis & Thoracic Tumor Research Institute, Beijing 101149, P. R. China. 3. Translational Medicine Center, Beijing Chest Hospital, Capital Medical University, Beijing 101149, P. R. China. 4. Department of Medicine, Division of Infectious Diseases, Weill Cornell Medicine, 1300 York Avenue, New York, NY, 10065, USA. 5. Department of Basic Medical Sciences, School of Medicine, Tsinghua University, Haidian Dist., Beijing 100084, P. R. China. Corresponding author addresses: [email protected] and [email protected] ABSTRACT: This study firstly demonstrated that natural product-inspired coumarin-based nitrofuranylcalanolides (NFCs) can form Rv2466c-mycothiol (MSH)-NFC (RvMN) ternary complex via NFC binding to W21, N51 and Y61 of Rv2466c and be specifically reduced by Rv2466c, which is accompanied with the generation of high level of fluorescence. Additionally, the results unveiled that the acetylated cysteine-glucosamine motif (AcCys-GlcN) of MSH is sufficient to interact with Rv2466c and adopt the active conformation that is essential for fully reducing NFCs. Further clinical translational investigation in this article indicated that the novel fluorescent NFC probe can serve as a much needed high-throughput and low-cost detection method for rapid detection of living Mtb and precise determination of MIC values of a full range of available drugs. This method can greatly facilitate the development of phenotypic drug-susceptibility testing (pDST) that will allow the point-of-care of treatment of TB within a week after diagnosis. KEYWORDS diagnosis; drug-susceptibility testing; Rv2466c; fluorescent probe

Tuberculosis (TB), resulting from Mycobacterium tuberculosis (Mtb), is the leading cause of death worldwide from a single infectious agent, ranking above HIV/AIDS.1, 2 According to estimates by the World Health Organization (WHO),1 there were about 10 million new cases of TB in 2017, and 1.57 million people died of TB. However, the actual number of new cases reported in 2017 was only 6.4 million, accounting for about 64% of the estimated cases. Additionally, the emergency of multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) is a serious and continuous threat to global health due to mismanagement of drug regimens, prevalence and complex nature of widely existed latent TB infection (LTBI) and the relapses of seemingly cured patients,3-6 alerting that there are large gaps between detection and proper treatment of TB patients, which directly contributes to high morbidity and substantial mortality. Therefore, a rapid, precise and reliable drug susceptibility test (DST) supplying reasonable regimens for drug treatment means a significance in TB chemotherapy. Several diagnostic and DST methods have been developed for Mtb, including phenotype- and genotype-based approaches.7-12 Although genotype-based methods such as Xpert MTB/RIF provide a simultaneous rapid Mtb-specific method with genotypic information on rifampicin resistance, 13 the prominent mechanisms of resistance are limited by the

knowledge on resistance conferring mutations and can result in false-resistance (mutations not conferring resistance) and falsesusceptibility (mutations different from the known mechanism). As a result, worldwide TB diagnostic rate and TB burden have not improved or eased, even with the expanding use of Xpert MTB/RIF.14, 15 Comparably, culture-based method, which directly detects living bacteria, is still the gold standard method. For instance, recent data showed that the accuracy of pDST, e.g., fast-culture Bactec MGIT 960 (MGIT 960) method, was above 90% in gaining a critical concentration (CC),16 whereas Xpert MTB/RIF was below 50%.17 Accumulated evidences recently indicate that tentative epidemiological cutoff values (ECOFFs) of many TB cases have not been established, which represent the drug’s highest concentration of the wild-type MIC value distribution based on pharmacokinetic, pharmacodynamic and clinical data defined by the European Committee on Antimicrobial Susceptibility Test (EUCAST),18 or similarity to consensus-based CCs. 19 Hence, obtaining these MIC value distributions of drugs is a task that remains to be solved, in addition to identifying all resistant mutations to profile drug resistance. Herein, we report a nitro-oxidoreductase Rv2466c-dependent fluorescent probe for TB diagnosis, rapid pDST and its clinical translation in a highthroughput manner with low cost.

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RESULTS AND DISCUSSION Natural product-inspired nitrofuranyl calanolides (NFCs) containing a coumarin core were recently presented to have high bactericidal activities against both replicating and non-replicating Mtb (R- and NR-Mtb), coupled with the absence of bactericidal activity against other tested gram-negative and grampositive bacteria.20 Recent studies reveal that the nitro group of NFCs can be specifically reduced to the corresponding amine group by a phylogenetically divergent Rv2466c as a novel mycothiol (MSH)-dependent nitro-oxidoreductase of Mtb.21, 22 With the knowledge that molecules bearing a heteroatom at the 7-position of coumarin structure are used as fluorophores,23 and its fluorescent ability can be quenched via attachment of electron-withdrawing group, e.g., nitro group, on the heteroatom at 7-position of coumarin structure,24 this property of coumarin derivatives prompted us to further explore the specific conversion of NFCs by Rv2466c and to expand the possibility of NFCs applications utilizing its reduced product as a reporter for monitoring the biological processes or interactions of interest in live Mtb, thereby facilitate TB diagnosis and pDST in a high throughput manner. Selection of fluorescently active NFCs via Rv2466c conversion. Thirty mycobactericidal compounds (Scheme S1) with bactericidal activity against R- and NR-Mtb and seven additional inactive compounds were selected to evaluate the change in relative fluorescent intensity before and after incubation with Rv2466c. The fluorescent fold change (FFC) of each compound was then calculated in the presence of Rv2466c and MSH (Figure S1). Nine compounds with highest fluorescencechange (Figure 1A) were identified for further evaluation of their fluorescent response to Mycobacterium bovis Bacillus Calmette–Guérin (BCG). Subsequently, compound 6 and 22 were found to show a high level of fluorescent activity, respectively (Figure 1B). Even though compound 22 has a better fluorescence turn-on ratio, a lower initial fluorescence intensity of compound 6 itself may benefit future clinical assay(Figure S2). Compound 6 was further incubated with the bacteria including wt Mtb, Rv2466c knock out strain Δrv2466c and the Rv2466c complemented Δrv2466c::rv2466c strain. As depicted in Figure 2A, Δrv2466c strain significantly reduced fluorescent ability of 6 in contrast to wt Mtb, however, the complemented Mtb Δrv2466c::rv2466c strain rescued the fluorescent response to NFCs as expected. This result confirmed that Rv2466c was specific to the fluorescent generation of NFCs. Mass spectrum study uncovered that 6 was efficiently converted into 6b after 3 hours incubation with recombinant protein Rv2466c (rRv2466c) in the presence of MSH, via an intermediate 6a (Figure 2B and Figure S3). Compound 6b was further hydrolyzed into 6d, possibly via 6c in medium of Mtb lysates for prolonged period, however, with a much less amount (< 1%, data not shown). The reduced products of compound 6 showed significantly fluorescent enhancements versus compound 6. In particular, as shown in Figure 2C, a 220-fold fluorescent intensity enhancement was seen with 6b excited at its maximum excitation wavelength of 390 nm (the excitation spectrum of 6b was shown in Figure S4), which was consistent with the results before and after incubation with rRv2466c (Figure 1B and Figure S1). Accordingly, the fluorescence quantum yield of compound 6b was 0.183 in methanol using quinine bisulphate as a reference while that of compound 6 was 0.00178. Therefore, the enhanced fluorescent effect of NFCs, whose self-fluorescence

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was quenched by the attached strong electron-withdrawing nitro group, encourages us to apply this fluorescent change as an indicator for measuring Rv2466c activity in viable bacilli.

Figure 1. NFCs can be reduced by Rv2466c and subsequently generated fluorescence. (A) The structures of 9 NFCs exhibited high fluorescent change in the presence of recombinant WT Rv2466c. (B) The fluorescent responses of 9 NFCs in BCG. Log phase bacteria were exposed to varied concentrations of compound 6. The reaction was monitored at 24 hours. Values are means ±SD of duplicate wells.

A good correlation was found between FFC, colony-forming unit (CFU) of BCG and tested concentrations of compound 6 as shown in Figure 3A. Accordingly, the favorable great relationship between FFC and CFU was curved for each clinical isolate including 2 drug-sensitive strains, 3 MDR-TB strains and 3 XDR-TB strains (Figure 3B and Figure S5) at 1.0 μg/mL of compound 6. Intriguingly, the detection sensitivity of Mtb clinical isolates was varied in a wide range (Figure 3B). In a previous study, 25 the rv2466c gene transcription under oxidative stress was associated with Mtb sigH that responded to stresses of heat shock and oxidation. Another study claimed that the oxygen depletion in non-replicating phase II of Mtb caused 4.97fold up-regulation of rv2466c in comparison to its replicating phase. 26 Therefore, it’s assumed that the expressions of


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Rv2466c were varied in individuals due to their different oxidative stress.

Figure 2. NFCs are fluorescent specifically reduced by Rv2466c. (A) Rv2466c in Mtb was specific for the generation of fluorescence of NFCs’ conversion at 0.5 and 1.0 μg/mL of compound 6. The fluorescent reaction was monitored by the FFC at excitation wavelength of 390 nm (Ex390) and emission wavelength of 470 nm (Em470). Data are means ±SD of triplicate wells. ***P < 0.001 (two-tailed Student’s t-test). (B) Overview of compound 6’s conversion. Compound 6 was first reduced by Rv2466c to 6b (major fluorescent compound) via 6a and finally hydrolyzed into 6c or 6d. (C) The emission spectrum of compound 6, and its synthetic reduced products 6a, 6b, 6c and 6d excited at 390 nm. Tested compounds were all at 1.0 mM. Mtb strains including two drug-sensitive strains (14897 and 14905), three MDR-TB strains (15027, 15038 and 15042), and three XDRTB strains (15272, 16630 and 17252). Values are means ± SD of duplicate wells.

Figure 3. The fluorescent response of NFCs to BCG and Mtb clinically isolates. (A) The FFC and CFU of BCG at varied concentrations of compound 6 (0.0625, 0.125, 0.25, 0.5, 1.0 and 2.0 μg/mL). (B) The FFC and CFU of Mtb H37Rv and 8 clinically isolated Mtb strains at 1.0 μg/mL of compound 6. The fluorescent reaction was monitored by the FFC at excitation wavelength of 390 nm (Ex 390) and emission wavelength of 470 nm (Em470). The clinically isolated

NFC compounds are fluorescence-specific to Rv2466c. Different types of nitro-containing substrates (Figure 4A) were evaluated accounting for the substrate specificity of Rv2466c (Figure 4B) and BCG (Figure 4C), meanwhile some nitro-free drugs were also used as reference to make sure that they would not affect the fluorescence of NFC compound in the presence of Rv2466c. These nitro group-containing compounds, representing by nitroimidazoles PA-824 that targets deazaflavin-dependent nitroreductase (Ddn),27 metronidazole targeting nucleic acid synthesis via disrupting DNA,28 benzothiazinone BTZ-04329 and IMMLG-694430 inhibiting decaprenylphosphoryl-β-D-ribose oxidase (DprE1), exhibited no difference in fluorescent changes before and after the treatment with either rRv2466c or BCG. Fluorescent enhancements were also absent from selected nitro group non-containing antiTB drugs including rifampicin (DNA-dependent RNA polymerase inhibitor), isoniazid (prodrug, enoyl-acyl carrier protein reductase InhA) and pyrazinamide (prodrug, intrabacterial acidification), as expected. It was worth noted that TP053, another substrate of Rv2466c,31, 32 exhibited much less fluorescent increase after the treatment with either rRv2466c or BCG compared to NFCs. Moreover, the fluorescence enhancements of compound 6 were recorded after incubation with the representative gram-positive bacteria and gram-negative bacteria (Figure 4D). As expected, compound 6 showed a weak or even no fluorescent enhancement with all tested gram-positive bacteria, i.e., Staphylococcus aureus (S. aureus), gram-negative bacteria, i.e., Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli) in contrast to BCG. Mycothiolation of Rv2466c resulted into an active conformation required for reducing NFCs. We previously modeled MSH in the active site of Rv2466c using the covalent docking protocol CovDock.22 In this article, a total of 28 mycobactericidal NFCs were further computationally docked



using the recombinant wild type Rv2466c (WT rRv2466c) crystal structure (PDB ID: 4NXI)32 and performed by a modified non-covalent docking method (Schrodinger, Inc.: Maestro v10.7, 2016). The results showed that Rv2466c may interact with NFCs via W21, N51, R54, Y61, H104 and T153 residues via non-covalent stabilizing interactions (Figure S6A). Accordingly, the docking result of compound 6 alone with Rv2466c also clearly showed the two hydrogen bonds interactions of N51 and Y61 residues with nitro group (Figure S6B). Based on the results, mutant proteins C19S, P20A, W21A, C22S, N51A, R54A, Y61A, H104A, T153A and Q205A were generated, and

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mutants R200A, D150A, and V151A were selected as controls. Subsequently, to study the specific interactions among NFCs, MSH or mycothione (oxidized MSH) (MSSM) and Rv2466c, differential scanning fluorimetry (DSF) assay of WT and mutant proteins at indicated conditions (Figure 5A-G, Figure S7) and functional fluorescent experiments (Figure 5H) were performed by means of a soluble NFC compound 11. The shifts of melting temperature (∆Tm) of the investigated proteins are summarized in Table S1 as an indicator of conformational change induced in the indicated conditions.

Figure 4. The fluorescent specificity of compound 6 to Rv2466c. (A) Representative anti-TB drug candidates and drugs used for the study of fluorescent specificity of Rv2466c. (B-C) Compound 6 showed prominent fluorescent response to rRv2466c and BCG. The reaction worked with 400 μg/mL rRv2466c in the presence of 1.0 mM MSH in the buffer of 50 mM Tris-Cl, 50 mM NaCl, pH 7.5 (B) or log phase bacteria diluted to the indicated OD580 value (OD580 = 0.1) in 7H9 broth with 10% OADC (C). Values are means ± SD of duplicate wells. (D) Compound 6 was incubated with log-phase bacteria for 24 hours, then the FFC values were measured and calculated. The fluorescent reaction was monitored at excitation wavelength of 390 nm (Ex390) and emission wavelength of 470 nm (Em470). Values are means ± SD of duplicate wells.

Insight of the ∆Tm in Table S1, mutants C19S (-90C), P20A (-50C), W21A (-140C), C22S (-80C) or R200A (-80C) showed significantly more negative ∆Tm compared to WT rRv2466c. This result revealed that the native conformation of WT Rv2466c was compromised by the replacement of these five amino acid residues by alanine (Figure 5B-D, Figure S7A， Figure S7H, Figure S7K-N and Figure S7V). It’s intriguing that neither compound 11 nor its corresponding nitro-reduced product 11b alone affected Tm of WT Rv2466c, pointing out that NFC or their corresponding reduced fluorescent compound does not directly affect the native conformation of Rv2466c. MSH or MSSM alone significantly changed ∆Tm value in comparison to WT Rv2466c (i.e., -13°C or -19°C, respectively), indicating that MSH and MSSM strongly bind to target enzyme and regulate the conformational change of Rv2466c to a specific state, due that the stability of most proteins decreases with

melting temperature. With the addition of compound 11, a further significantly negative shift of Tm was observed in the presence of MSH (-12°C, Figure 5A in green) or MSSM (-7°C, Figure S7J in green, Table S1). This result proved that MSH can bind to Rv2466c via the non-sulfhydryl group motif and subsequently occurred a mycothiolation via sulfhydryl group, thereby inducing an active conformation of Rv2466c to accommodate compound 11. With the further study, the result that MSH, but not MSSM, assisted Rv2466c to excite the fluorescence of compound 11 (Figure S7X) uncovered that the free sulfhydryl group of MSH is a key factor for mycothiolation of the canonical 19 CPWC22 motif of nitroreducase Rv2466c for enzymatic activity and that the 19CPWC22 motif should be a reduced form for subsequent mycothiolation, providing the 2H atoms necessary for fully reducing NFCs to promote the fluorescence of 11b. In addition, 1) compound 11, but not 11b, further negatively


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shifted Tm of WT rRv2466c in the presence of MSH from -130C to -250C, which strongly implies that the nitro group of 11 participates directly in the interaction with active Rv2466c; 2) C19S mutant induced a negative Tm shift and also lost its ability to convert compound 11, which is consistent with absence of Tm change observed in all indicated conditions in the DSF assay and functional experiments (Figure 5H); 3) the side chain indole of W21 is thought to interact with the coumarin core structure of NFCs via a π-π bond interaction, as depicted in Figure S6. Accordingly, no extra negative Tm shifts were observed in mutant W21A in the presence of both MSH and compound 11 compared to MSH alone, which was consistent with the observation that mutant W21A lost ability to convert compound 11 in fluorescent functional experiments; 4) no significant Tm shifts were observed in mutants N51A (-3°C), Y61A (-4°C) and N51A/Y61A (-1°C) with both MSH and compound 11 compared to MSH alone, which was consistent with the docking results that the side chain carboxamide group of N51 and the phenolic hydroxyl group of Y61 maintained hydrogen-bond interactions with nitro group of compound 11 (Figure S6). Fluorescent functional experiments in Figure 5H further revealed

that mutants W21A, N51A, Y61A had a large impact on the reduced ability of Rv2466c to excite the fluorescence of 11. Therefore, the residues of W21A, N51A and Y61A play a pivotal role in both stabilization of the Rv2466c-MSH-NFC (RvMN) complex via interacting with NFC compound and converting NFC into the corresponding fluorescent product NFCNH2. Additional analyses using isothermal titration calorimetry (ITC) further support that the formation of RvMN complex (Figure 6). The pronounced but partial impact of R54A, H104A, R200A and Q205A mutations may reflect the solvent-exposed location of these polar residues, leading to a less stable RvMN complex. P20 is located in the conserved catalytic domain 19CPWC22. The substitution of this rigid amino acid with a more flexible one (alanine residue) could affect the conformation of the catalytic domain, which might subsequently lead to the disruption of formation of the RvMN complex. Nevertheless, the other mutations (D150A, V151A and T153A) had a less impact on both the formation of the RvMN complex and the conversion of NFCs.

Figure 5. Soluble NFC compound 11 binds Rv2466c through interactions with W21, N51 and Y61 residues. (A-G) DSF assays of WT and mutant proteins. WT or mutants of rRv2466c were incubated with MSH, compound 11 and compound 11b at indicated conditions. The impacts of these interactions were evaluated by shift in melting temperature. For a full range of DSF results, see Table S1 and Figure S7. (H) The effect of point mutations on Rv2466c’s ability to convert compound 11 to compound 11b in the presence of MSH. The reaction worked with 200 μg/mL rRv2466c in the presence of 0.5 mM MSH. The fluorescent reaction was monitored at excitation wavelength of 376 nm (Ex376) and emission wavelength of 454 nm (Em454). Values are means ±SD of duplicate wells.



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Figure 6. ITC titration study for compound 11 binding to Rv2466c. (A) ITC titration for compound 11 to rRv2466c with the absence of MSH, ligand solution: 1.5 mM 11 in 50 mM Tris-Cl, 50 mM NaCl buffer, protein solution: 0.09 mM 11 in 50 mM Tris-Cl, 50 mM NaCl buffer; (B) ITC titration and fitting curves for buffer to rRv2466c in the pres-ence of MSH, ligand solution: 50 mM Tris-Cl, 50 mM NaCl buffer containing 0.5 mM MSH, protein solution: 0.09 mM 11 in 50 mM Tris-Cl, 50 mM NaCl buffer containing 0.5 mM MSH; (C) ITC titra-tion and fitting curves for compound 11 in the presence of MSH, lig-and solution: 1.5 mM 11 in 50 mM Tris-Cl, 50 mM NaCl buffer con-taining 0.5 mM MSH, protein solution: 0.09 mM 11 in 50 mM Tris-Cl, 50 mM NaCl buffer containing 0.5 mM MSH.

The role of MSH’s motif. To further evaluate the role of MSH and its motifs, eight MSH analogs (shown in Figure 7A) were designed and synthesized. Inspection of MSH-1, in which D-inositol was replaced with the L-inositol, largely lost its ability to assist Rv2466c converting compound 11 into 11b (Figure 7B) while it caused a Tm shift (-19°C) as MSSM alone, and MSH-3 that lacked a 1D-myo-inositol motif still remained the same ∆Tm value as MSH (-12°C, Figure 7C) and caused a similar level of fluorescent response of 11 (Figure 7B) and all the other 8 active NFCs (Figure 7D) to that of MSH, demonstrating that D-configuration of inositol is useful for reduction of NFCs but not necessary. However,E MSH-2 (1D-myo-inositol replaced by cyclohexyl) and MSH-5 (1D-myo-inositol replaced by ethyoxyl group), retained the same Tm shift (-19°C) as MSSM and significantly decreased its ability assisting Rv2466c compared to MSH (Fig. 4B). This result indicated that a specific conformation of Rv2466c induced by MSH or MSH-3 (∆Tm = -12°C), but not by other MSH derivatives that resulted in a greater or less Tm shift, i.e., -19°C or 0°C, was necessary for NFC compound reduction. Moreover, MSH-4 and MSH-8, in which free sulfhydryl group was replaced with an isosteric hydroxyl group, showed no Tm shift (Figure 7C), and MSH-6 and MSH-7, in which the sulfhydryl group was blocked by an acetyl group or dimerization, showed the same Tm shift as MSSM (-19°C), all lost their ability to assist Rv2466c in converting NFC compound (Figure 7B). A competitive assay between MSH-3 and MSH-8 was carried out to further explore MSH binding to Rv2466c and mycothiolation (Figure 7E) accompanied with the observed no Tm shift for both MSH-4 and MSH-8 (Fig. 7C). The results showed that MSH-8 dose-dependently reduced the ability of MSH-3 to induce the fluorescence of 11 suggesting that the binding to Rv2466c does occur, but only thereafter mycothiolation can regulate Rv2466c into an active conformation for accommodating NFC. Because MSSM

or MSH-7, which is the corresponding dimer via formation a disulfide bond, are unable to functionally induce the fluorescence by converting NFCs (Figure 7B), we speculate that only MSH or MSH-3 with free sulfhydryl group, but not MSSM or MSH-7, can provide 2H atoms for the full reduction of nitro group of NFCs via mycothiolation of Rv2466c. MSSM or MSH-7 alone induced a significant ∆Tm (the former was -19°C and the later was -23°C), pointing out that a disulfide bond exchange occurred between these two molecules and Rv2466c, but such mycothiolation can only provide 1H atom that is insufficient to fully reduce NFC into highly fluorescent NFC-NH2. All above results indicated that sulfhydryl group of MSH plays a key role in reduction of NFCs while 1D-inositol doesn’t. Moreover, a specific conformation is required to reduce NFCs, which is regulated by mycothiolation of MSH. This understanding of the interactions of the RvMN complex offers possibilities to apply MSH-3 to discover inhibitors of actinomycetes including Mtb that specifically generates low-weight thiol molecule MSH. Taken together in Figure 7F, considering that MSH functions in widespread protein S-mycothiolation as an important thiol-protection mechanism, 33 we speculated that MSH binds to Rv2466c in the first step, and then the free sulfhydryl group of MSH forms a disulfide bond (mycothiolation) with C19 amino acid of reduced Rv2466c in the second step. Simultaneously, the functional conformation of Rv2466c is set in the second step, accommodating the NFC to form the RvMN ternary complex and subsequently reducing NFC to the corresponding fluorescent NFC-NH2, while mycothiolation provides 2H to achieve full NFC’s reduction. After NFC is reduced, the emerging amino group of NFC derivative lose its benefit from nitro group strongly bound to N51A and Y61A, and the fluorescent product dissociates from the RvMN ternary complex. Meanwhile, the C19-C22 disulfide bond is reformed.


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Figure 7. The role of MSH’s motif in Rv2466c converting NFC compound 11 to stimulate fluorescence. (A) The structure of 8 synthesized MSH analogs. (B) The impact of MSH and MSH analogs on the function of Rv2466c’s ability to convert compound 11 into compound 11b determined by the FFC of compound 11. The fluorescent reaction was monitored at excitation wavelength of 376 nm (Ex 376) and emission wavelength of 454 nm (Em454). Values are means ± SD of duplicate wells. (C) The change in melting temperature of WT rRv2466c in the presence of MSH or MSH analogs by DSF assays. (D) The evaluation of MSH-3’s impact on the function of Rv2466c’s ability to convert 9 high bactericidal NFCs. WT rRv2466c (200 μg/mL) was incubated with MSH-3 determined by 2.5 μg/mL NFCs, the maximum excitation and emission wavelengths of each NFC compound were shown in brackets. Values are means ±SD of duplicate wells. (E) The comparative evaluation of MSH-8 with MSH-3 on the function of Rv2466c’s ability to convert compound 6. The fluorescent reaction was monitored at excitation wavelength of 390 nm (Ex390) and emission wavelength of 470 nm (Em470).Values are means ± SD of duplicate wells. (F) The speculated MSH-dependent mechanism of Rv2466c in converting NFCs. MSH binds to Rv2466c in the first step, and then the free sulfhydryl group of MSH forms a disulfide bond (mycothiolation) with C19 residue of reduced Rv2466c in the second step. Simultaneously, the functional conformation of Rv2466c is set in the second step, accommodating the NFCs and subsequently reducing NFCs to the corresponding fluorescent NFC-NH2, while mycothiolation provides 2H to achieve full NFC’s reduction.



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Figure 8. Clinical translational investigations of NFC method. (A) Experimental design of diagnostic evaluation by NFC method. Outpatients’ sputum specimens were processed according to MGIT 960 method and cultured in MGIT 960 platform. During the whole process culture, the once diagnosed specimens by MGIT 960 were immediately determined by NFC method (n = 71, group A), an alternative group B (n = 102) or group C (n = 103) was performed when sputum specimens were cultured in the MGIT 960 for 10 days or 20 days. (B) NFC test results with group A, B and C. a Sensitivity is calculated from the percentage of NFC method-positive samples in the total MGIT 960-positive samples; b Specificity is calculated from the percentage of NFC method-negative samples in the total MGIT 960-negative samples. (C) The positive rate of NFC method and MGIT 960 at the same detection time (10 days/group B and 20 days/group C) compared with the whole process determination of MGIT 960 till the EPT (8 weeks). At indicated detection time, the specimens were both diagnosed by MGIT 960 and NFC method. c Positive rate is calculated from the percentage of NFC method-positive or MGIT 960-positive samples in the total test outpatients samples in the above indicated due date. (D) ROC curve analysis of NFC method in MGIT 960-positive specimens (n = 82) versus MGIT 960-negative specimens (n = 194), total n = 276. CI, confidence interval. (E) The coincidence (%) of NFC method versus MDSDT method and MABA method on DST results. CI, confidence interval. For fluorometric MICs in (E), inhibition rate was defined as (1-(test well RFU-M well RFU)/(mean RFU of B wells-M well RFU)), where M and B wells indicate additional control wells consisted of medium only (M) and bacteria only wells (B), the lowest drug concentration effecting an inhibition of ≥90% was considered the MIC. Data and error bars shown represent the means and standard deviations, respectively, of triplicate samples for all strains.


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Detection of clinical sputum specimens from outpatients by NFC method. The results described above revealed that the nitro group of NFCs was specifically reduced by a phylogenetically divergent Rv2466c assisted by MSH via RvMN ternary complex. This prompted us to further expand the possibility of NFCs applications utilizing its fully reduced product as a reporter for monitoring the biological processes or interactions of interest in live Mtb, thereby facilitates the diagnosis and pDST of TB. Several studies were then designed to develop a novel diagnostic method for pDST of Mtb by compound 6 as a fluorescent diagnosis probe and evaluate its value for application in clinical specimens. As shown in Figure 8A, a BD BACTEC™ MGIT™ 960 method (MGIT 960), a fully automated solution for mycobacterial liquid culture and susceptibility test method, was carried out for validation of the NFC method. Briefly, sputum specimens from total 276 outpatients, assigned into group A, B, and C, were processed according to MGIT 960, and cultivated in MGIT 960 platform. During the whole process culture, the once diagnosed specimens in group A (n = 71) by MGIT 960 were immediately determined by NFC method, and the group B (n = 102) or group C (n = 103) was performed in parallel by NFC method and MGIT 960. In group B, the sputum specimens were cultivated for 10 days, and alternatively, cultivated for 20 days in group C. To all evaluated specimens (n = 276), the diagnostic ability analysis (Figure 8B) indicated that the receiver operating characteristic (ROC) curves of NFC method showed a great classifier with an area under the curve (AUC) of 0.956 in comparison to MGIT 960 method, which exhibited a sensitivity of 87.8% and specificity of 99.0%, supporting its utility as a fluorescent detection method for TB. The cut-off value determined by Youden index was 1.559. The results shown in Figure 8C displayed that, 1) all of 25 MGIT 960-positive specimens in group A were immediately proved to be NFC method-positive, likewise, all of 46 MGIT 960-negative specimens on the 8-week ending point test (EPT) were NFC method-negative; 2) in group B, 20 specimens on 10-day due date in total 30 of MGIT 960positive specimens till 8-week EPT were shown to be NFC method-positive (sensitivity of 66.7% to MGIT 960), while 71 in total 72 of MGIT 960-negative specimens were NFC method-negative that disclosed a specificity of 98.6% to MGIT 960; 3) in group C, all of 27 MGIT 960-positive specimens till 8-week EPT were shown to be NFC method-positive (100% of sensitivity to MGIT 960), coupling with that 75 in total 76 of MGIT 960-negative specimens were NFC method-negative that revealed a specificity of 98.7% to MGIT 960. It was worth noticed that both 10 and 20-day groups have one MGIT 960 negative/ NFC positive case, which was speculated as indicated: 1) these two cases were possibly true positive but MGIT 960 failed to detect at 8-week EPT. Since the MGIT 960 was based on the fluorescence indicator that could be quenched by large amounts of dissolved oxygen, this failure to detect growth was possibly attributed to the granular growth pattern of Mtb, which produced less surface contact, making the consumption of oxygen below the detection limit; 2) the expressions of Rv2466c of these cases were high enough to be detected by NFC method even in low bacteria number below the detection limit of MGIT 960; 3) the detection positive threshold of NFC method was defined as 1.559 according to ROC analysis, whereas the false positive cases in 10 and 20-day groups were 1.585 and 1.573 respectively (not shown), which were close to the detection threshold and likely contributed to the real false positive cases.

Additionally, it was interesting that the diagnosis positive rate of NFC method was twice as that of MGIT 960 method on 10day due date, for instance, NFC method was 19.6% (20/102) and MGIT 960 method was 9.8% (10/102) in group B (Figure 8D). Evaluation of pDST by NFC method in a high throughput manner. Subsequently, the anti-TB drug screening potential of NFC method was assessed by collection of MIC values of 11 anti-TB first-line and second-line agents (Figure 8E) against 30 clinical isolated strains. A low-high CC drug resistant method (definition see Table S2) according to Clinical Microplate-based Drug Sensitive Detection Technology (MDSDT) was used to validate the results of NFC method. A microplate-based alamarBlue assay (MABA) was set up as a reference method using a chemical blue dye (resazurin) as an oxidized-reductive indicator for cell viability.34 All MICs of the 11 anti-TB agents were gained within 5 days (NFC method) and 8 days (MDSDT and MABA method), respectively. The distribution of MIC values of each drug was shown in Table S3 (3 first-line drugs) and Table S4 (8 second-line drugs). In terms of the MDSDT method, the report of results were only determined as “S” (sensitive) or “R” (resistant) on accounting of the setting CCs, therefore, MIC values determined by NFC method lower or higher than the CC resistant concentration of each drug were thought to be “S” or “R”. Accordingly, the consistency between NFC method and the MDSDT method were found for a series of drugs (Figure 8E), as indicated: rifampicin (RIF): 97%; isoniazid (INH): 100%; ethambutol (EMB): 77%; levofloxacin (LFX): 100%; capreomycin (CPM): 80%; moxifloxacin (MXF): 90%; amikacin (AMK): 93%; prothionamide (PTA): 90%; p-aminosalicylic acid (PAS): 97%; streptomycin (SM): 90%; kanamycin (KAN): 67%, respectively. Kappa test using to access the agreement between these two methods revealed a generally good consistency for any of the 30 Mtb clinical isolates. Moreover, Pearson and Spearman test were used to analyze the MIC differences (≤1 two-fold dilution) between NFC method and MABA method, and correlation coefficients were all close to 1.0 except for Pearson’s r of LFX that needs more clinical samples to be further determined, indicating a great correlation between these two methods. Additionally, a paired sample t-test confirmed again that no significantly statistical difference of each drug was observed between these two methods for any of the 30 Mtb clinical isolates. Therefore, NFC method offered a rapid, reliable and precise MIC value for each drug, which inspired us to apply this method directly in clinical sputum specimens after diagnosed rather than the clinical isolates. To determine the feasibility of NFC-based pDST method for full-range anti-TB drugs in clinical sputum specimens, a simulating retrospective study was carried out to evaluate the MICs of anti-TB drugs in combining the advantages of the typical pDST MGIT 960 method (on time diagnosis) and NFC method (rapid, high-throughput and low-cost). Briefly, in the first experiment, MGIT 960 vials were initially given inoculation of 9 clinically isolated drug-sensitive strains and MDR/XDR Mtb strains (3.0×103 cells/mL), respectively, followed by re-cultivated and re-diagnosed in MGIT 960 platform. Diagnosed positive TB specimens were immediately sonicated to disperse them to an OD580 value of 0.02, approximately equaling to 10 6 cells/mL of Mtb, then the MIC values were determined by NFC method within 5 days (4 days for Mtb growth plus additional 24-hour fluorescent accumulation) in a 96-well plate in which



the tested drugs had been previously added in two-fold serially dilutions higher and lower than the CC of each drug. The direct MICs determination of these isolated strains (no re-cultivation and re-diagnosis) were served as reference control that carried out by the same protocol. Figure 9A-C showed the curves of each first-line drug (RIF, INH and EMB) against these 9 re-cultivated and re-diagnosed clinical isolates, respectively. The detailed MIC values in Table S5 disclosed a fact that the MIC values of re-cultivated Mtb isolates against each tested drugs were completely reproduced to the referenced control that validated a direct pDST assay of clinical specimens. Finally, we performed the direct test of the drug screening potential of RIF, INH and EMB to clinical sputum specimens once diagnosed by MGIT 960 (n = 22 in each drug group) but not the clinical isolates. Results were illustrated in Figure 9D and Table S6. In RIF group, the MICs of 16 specimens in total 22 specimens were ranged from 0.125 µg/mL~2.0 µg/mL that

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were RIF-sensitive, while 6 RIF-resistant specimens in 22 patients were determined with MIC values ≥64 µg/mL, accounting for a resistant rate of 27.3% that approximately equaled to the average annual RIF-resistant rate (28.11%, n = 6724) during 2014-2016 in Beijing Chest Hospital (Figure 9D). For INH or EMB group, five drug resistant specimens in each 22 specimens group with the MICs ranging from 0.8 µg/mL ~>6.4 µg/mL (INH) or 10 µg/mL ~>320 µg/mL (EMB) were verified, indicating a resistant rate of 22.7% of both INH and EMB that was comparable to the average annual resistant rates (35.5% of INH and 16.8% of EMB, n = 6724) during 2014-2016 in Beijing Chest Hospital. Although more clinical tests are required to further determine the resistant rate by NFC method, recent data clearly prove that NFC-dependent method significantly shorten pDSTs determination time within 5 days after diagnosed in gaining precise MICs, which would tremendously decrease the cost in a high-throughput manner, regardless of the various drug resistance mechanisms, and possessed an ability to determine the MIC values of full range drugs within one week.

Figure 9. Retrospective study of Mtb clinically isolates by NFC method. (A-C) Drug inhibition curves of 9 Mtb clinical isolates towards RIF, INH and EMB determined by NFC method. MGIT 960 vials were initially given inoculation of clinically isolated Mtb strains (3.0×103 cells/mL) to simulate the clinical specimens, followed by re-cultivated and re-diagnosed in MGIT 960 platform and the re-detection date of each strain was as below in brackets: 14897 (9), 14899 (10), 14905 (7), 15027 (13), 15038 (9), 15042 (8), 15272 (8), 16630 (12) and 17252 (8). The clinical isolated strains 14897, 14899 and 14905 are drug-sensitive Mtb, 15027, 15038 and 15042 are MDR-Mtb, 15272, 16630 and 17252 are XDR-Mtb. Values are means ±SD of triplicate wells. (D) Resistant rate of NFC method (n = 22 in each drug group) and average resistant rate during 2014-2016 in Beijing Chest Hospital (n = 6724), the once diagnosed sputum specimens were evaluated by NFC method to determine the MIC values. a The resistant rate was defined as the ratio of drug resistant cases to the total culture-positive cases. b The DST assay of the whole positive cases in Beijing Chest Hospital were determined by proportion method and the resistant concentrations were defined as 40 μg/mL of RIF, 2.0 μg/mL of EMB and 0.2 μg/mL of INH, respectively, according to Laboratory Science Procedure of Diagnostic Bacteriology in Tuberculosis published by Chinese Antituberculosis Association. For fluorometric MICs in (A-D), inhibition rate was defined as (1-(test well RFU-M well RFU)/(mean RFU of B wells-M well RFU)), where M and B wells indicate additional control wells consisted of medium only (M) and bacteria only wells (B), the lowest drug concentration effecting an inhibition of ≥90% was considered the MIC. Data and error bars shown represent the means and standard deviations, respectively, of triplicate samples for all strains.


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CONCLUSION In summary, poor compliance and programmatic failure of TB therapy had been thought to underpin the generation of drug resistance of Mtb. However, in addition to the site mutant, recent studies have demonstrated that pharmacokinetic variability, cellular efflux pumps and poor drug penetration into tuberculosis lesions are also crucial for the pathogenesis of drug-resistant tuberculosis, whereas the empirical regimens based on CC are still clinically used.35, 36 Whole genome sequencing (WGS) of Mtb has been recently proposed to provide resistance profiles for all drugs within a single analysis, while several key factors need to be further addressed for achieving the rapid drug susceptibility profiling of Mtb from clinical specimens directly. These include selective extraction of more Mtb DNA from scarce resource of human complex sputum, effective and affordable manufacturing of more DNA isolation device, the cost of throughput sequencing, allowing directly test of sputum samples and available a library of resistance mutations to full-range drug resistances, identification new markers of resistance, and good databases involving clinical regimen, phenotypic and sequence data for TB research and health care. This article for the first time revealed the direct interactions of RvMN ternary complex and offered a reliable fluorescent probe in TB detection and DST. The results suggest that C19 mycothiolation of reduced form of Rv2466c by MSH induces

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Detailed experimental procedures, additional figures, tables, and copies of NMR-spectra (PDF).

AUTHOR INFORMATION Corresponding Author For G. L. E-mail: [email protected] For Z.-G. S. E-mail: [email protected] #R. M., C.-C. K., W.-J. Y. and H.-Y. W. contributed equally to the work. Author Contributions The authors contributed as follows: conceptualization (G. L.); investigation (R. M., C.-C. K., W.-J. Y., H.-Y. W., Y. M., X.Y. L., J. W., S.-K. S., H.-T. L., Z.-G. S., and G. L.); resources (G.L., Z.-G. S., B. J., H.-T. L.); writing (G. L. and R. M.). Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS We thank Professor Babak Javid for his reading and as-sistance in student training on BCG assay. This article was supported by National Natural Science Foundation of China (No. 81773575, 81573289, 81161120402), funds from Beijing Advanced Innovation Center for Structural Biology, Tsinghua University and Beijing Municipal Science & Technology Commission No. Z181100001718181 and Beijing Municipal Science & Technology Commission No. Z181100001718181.

ABBREVIATIONS

an active catalytic conformation that allows the NFCs to access the binding sites and form an RvMN ternary complex via NFC binding to W21, N51 and Y61 of Rv2466c. N51 and Y61 interact with nitro group of NFCs via hydrogen bonds and W21 interacts with coumarin core via a π-π bond. The reduced fluorescent NFC-NH2 losing the interaction with Rv2466c, which is absent of the nitro group, dissociates from RvMN complex. The mycothiolation of MSH provides 2H atoms for the full reduction of NFCs and thus Rv2466c is believed to be a novel MSHdependent nitro-oxidoreductase. The NFCs, but not other antiTB drugs containing nitro groups, are specifically reduced by Rv2466c. It is found that the AcCys-GlcN motif of MSH is sufficient to interact with Rv2466c and generate the active conformation to reduce NFCs. Translational investigation indicates that NFC method, which can detect the bacteria from 1.5 × 104 cells/mL (17252, a XDR-TB strain) to 1.2 × 106 cells/mL (15042, a MDR-TB strain) when the cut-off value was determined as 1.559 (red dash dot lines in Figure 3B), is a potential novel, specific, high-throughput and low-cost fluorescent method in rapid detection of live Mtb from clinical sputum specimens and precise determination of pDST that is urgently needed for the MIC values determination of full range of available drugs after diagnosed, thereby obtaining breakpoint or a point-of-care of treatment of continuously increasing MDR/XDR-TB varied in multi-resistance mechanism. AcCys-GlcN, acetylated cysteine-glucosamine; AMK, amikacin; BCG, Mycobacterium bovis Bacillus Calmette–Guérin; CC, critical concentration; CFU, colony-forming unit; CPM, capreomycin; Ddn, deazaflavin-dependent nitroreductase; DprE1, decaprenylphosphoryl-β-D-ribose oxidase; DSF, differential scanning fluorimetry; DST, drug susceptibility test; ECOFFs, epidemiological cutoff values; E. coli, Escherichia coli; EMB, ethambutol; EPT, ending point test; EUCAST, European Committee on Antimicrobial Susceptibility Test; FFC, fluorescent fold change; INH, isoniazid; ITC, isothermal titration calorimetry; LFX, levofloxacin; LTBI, latent TB infection; MABA, microplate-based alamarBlue assay; MDR-TB, multidrug-resistant TB; MDSDT, clinical Microplate-based Drug Sensitive Detection Technology; MGIT 960, Bactec MGIT 960; MSH, mycothiol; MSSM, mycothione; Mtb, Mycobacterium tuberculosis; MXF, moxifloxacin; NFCs, nitrofuranyl calanolides; NR-Mtb, non-replicating Mtb; P. aeruginosa, Pseudomonas aeruginosa; PAS, p-aminosalicylic acid; pDST, phenotypic drug-susceptibility testing; PTA, prothionamide; RIF, rifampicin; R-Mtb, replicating Mtb; rRv2466c, recombinant protein Rv2466c; RvMN, Rv2466c-mycothiol-NFC; S. aureus, Staphylococcus aureus; SM, streptomycin; TB, Tuberculosis; WHO, World Health Organization; WGS, Whole genome sequencing; WT rRv2466c, recombinant wild type Rv2466c; XDR-TB, extensively drug-resistant TB; ∆Tm, melting temperature

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A Nitrooxidoreductase Rv2466c-dependent Fluorescent Probe for

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