Alternate pathway to ascorbate induced inhibition of Mycobacterium tuberculosis

Accepted Manuscript Alternate pathway to ascorbate induced inhibition of Mycobacterium tuberculosis Harish Shukla, Shaheb Raj Khan, Rohit Shukla, Manju Yasoda Krishnan, Md. Sohail Akhtar, Timir Tripathi PII:

S1472-9792(17)30463-8

DOI:

10.1016/j.tube.2018.06.013

Reference:

YTUBE 1724

To appear in:

Tuberculosis

Received Date: 23 November 2017 Revised Date:

12 June 2018

Accepted Date: 16 June 2018

Please cite this article as: Shukla H, Khan SR, Shukla R, Krishnan MY, Akhtar MS, Tripathi T, Alternate pathway to ascorbate induced inhibition of Mycobacterium tuberculosis, Tuberculosis (2018), doi: 10.1016/j.tube.2018.06.013. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Alternate pathway to ascorbate induced inhibition of Mycobacterium tuberculosis Harish Shukla1, Shaheb Raj Khan2, Rohit Shukla1, Manju Yasoda Krishnan2, Md. Sohail

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Akhtar2*, Timir Tripathi1*

Molecular and Structural Biophysics Laboratory, Department of Biochemistry, North-Eastern

Hill University, Shillong 793022, INDIA

CSIR-Central Drug Research Institute, Sector 10, Jankipuram Extension, Sitapur Road,

Lucknow, Uttar Pradesh 226031, INDIA

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Running Title: Interaction of ascorbate with MtbICL

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To whom correspondence should be addressed: Dr. Timir Tripathi, Department of

Biochemistry,

North-Eastern

Hill

University,

Shillong-

793022,

India.

Email:

[email protected], [email protected]; Tel: +91-364-2722141; Fax: +91-364-2550108. Dr. Md. Sohail Akhtar, Molecular and Structural Biology Division, CSIR-Central Drug Research

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Institute, Jankipuram Extension, Lucknow 226031, India. Email: [email protected]; Tel.: +91

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522 2772450; Fax: +91 522 2772459.

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ABSTRACT Ascorbate has been demonstrated to interfere with the growth of Mycobacterium tuberculosis. It scavenges oxygen in the culture medium to induce dormancy of M. tuberculosis. It kills the mycobacteria by generating reactive oxygen intermediates via iron mediated Fenton reactions. In

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this study, we observed that ascorbate can inhibit M. tuberculosis isocitrate lyase (MtbICL) with an IC50 of 2.15 mΜ. MtbICL is an essential enzyme for the survival of M. tuberculosis under dormancy. We studied the effect of ascorbate on the growth of M. tuberculosis H37Rv metabolizing through citric acid cycle or glyoxylate cycle with glucose or acetate respectively as

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the sole carbon source. It was observed that 4 mM ascorbate inhibited ~89 % of the growth in glucose medium, which was confirmed to be mediated by Fenton reaction, as the inhibition was

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significantly lesser (61%) under low iron condition. On the other hand, in acetate medium, ~97% of the growth was inhibited and the inhibition was uninfluenced by the iron levels. 3nitropropionate, a known inhibitor of MtbICL, was seen to cause significantly higher inhibition in the acetate medium than in the glucose medium; however it was indifferent to iron levels in either medium. Molecular docking and dynamic simulation studies confirmed stable binding of ascorbate to MtbICL leading to its inhibition. These observations suggest an additional pathway

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for ascorbate induced inhibition of M. tuberculosis through inhibition of glyoxylate cycle. Since human immune cells can accumulate ascorbate in millimolar concentrations, the in vitro activity range (1-4 mM) of ascorbate against M. tuberculosis could be extrapolated in vivo. Our result

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supports the possible benefits of adding high vitamin C diet in TB-treated patients.

Key words: Ascorbate; fenton reaction; isocitrate lyase; mycobacteria; persistence; dormancy;

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molecular dynamic simulation.

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1. INTRODUCTION Tuberculosis (TB), caused by Mycobacterium tuberculosis, continues to be the leading infectious disease threat worldwide, killing more than 1.5 million people globally in 2013 1. In 2015, drug resistant TB was estimated to affect 480,000 people worldwide. Nearly 1.7 billion (24%) of the

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world population is latently infected and serves as the natural reservoir of this deadly pathogen 2. Eradication of TB is greatly hampered by the emergence of drug resistant strains and ability of the pathogen to persist as non-replicating dormant forms in the host. The non-replicating dormant population is phenotypically tolerant to anti-tubercular drugs interfering with bacterial

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growth and multiplication 3. Hence, the current anti-TB drug discovery programmes aim at discovering novel molecules that are effective against both active and dormant populations of M.

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tuberculosis. In addition to discovering new scaffolds, TB drug discovery programmes also invest in re-engineering or improving existing scaffolds. The various screening strategies mostly target whole bacteria and have been more successful in the past. Screening against selected pathways or processes or individual proteins are also pursued, especially after identifying the mechanism of drug action discovered through whole cell screening.

Vitamin C (L-ascorbic acid or ascorbate) has long been tested for efficacy in treating M.

tuberculosis

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tuberculosis infection, using guinea pig model 4 or antibacterial activity in in vitro cultures of M. In the recent times, two separate studies demonstrated different ways wherein

ascorbate can interfere with the growth and physiology of M. tuberculosis. In the first study, Taneja et al demonstrated that ascorbate induces the devR (dosR) dormancy regulon of M.

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tuberculosis, causing growth arrest and dormancy of M. tuberculosis axenic cultures 7. They further demonstrated that the induction of dormancy was due to the oxygen scavenging property

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of ascorbate. In the second study by Vilcheze et al, ascorbate was demonstrated to kill M. tuberculosis by acting as a pro-oxidant, generating reactive oxygen intermediates (ROI) via the Fenton reaction, which in turn cause lipid alterations, redox unbalance and DNA damage. They showed that M. tuberculosis was killed as rapidly with daily addition of 1 mM ascorbate as with one dose of 4 mM at day 0 8. Isocitrate lyase (ICL, EC 4.1.3.1) catalyses the conversion of isocitrate to succinate and glyoxylate. The ICL of M. tuberculosis (MtbICL) is a proposed drug target during bacterial survival on host fatty acids as a sole carbon source, a condition encountered by persistant bacteria 9. In M. tuberculosis there are two known isoforms of ICL: ICL1 and ICL2, which are 3

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encoded by the genes icl1 and icl2, respectively

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. Both ICL1 and ICL2 are jointly required

for growth, survival, and virulence of M. tuberculosis in macrophages and mice

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. Sequence

analyses indicate that, although ICL1 and ICL2 share 27% sequence identity, ICL1 is a prokaryotic-like ICL isoform whereas ICL2 is a eukaryotic-like ICL isoform

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. The most

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studied isoform of M. tuberculosis ICLs is ICL1. It has a stronger binding affinity to the substrate D-isocitrate than ICL2, and is more active than ICL2 in vitro

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. In vitro experiments

with purified ICL1 and ICL2 proteins showed that both enzymes could use isocitrate and methylisocitrate as substrates, although isocitrate is the preferred substrate over methylisocitrate

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for ICL1 and ICL2.

While testing certain natural products against purified MtbICL, we found ascorbate

effects in the biological system

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inhibited the activity of MtbICL. Ascorbate, being a vitamin and natural product, has pleiotropic . It is well known for its roles as anti-oxidant or pro-oxidant,

enzyme co-factor, and immune booster. Hence, it was worth investigating whether ascorbate could interfere with mycobacterial growth through inhibition of MtbICL, in addition to the already known mechanism. We studied both in vitro and in silico aspects of the ascorbateMtbICL interaction and tested ascorbate against M. tuberculosis growing in vitro on fatty acid

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substrate. Our observations suggest that ascorbate interacts favorably with MtbICL and inhibits its activity. In this study, we propose an alternate pathway for ascorbate induced killing of M. tuberculosis.

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2. RESULTS AND DISCUSSION

2.1 Ascorbate binds to and inhibits MtbICL activity

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We observed optimum binding, specificity, and inhibition of MtbICL by ascorbate. The fractional change in the fluorescence intensity of MtbICL vs ascorbate concentration showed a binding constant (Kd) of 2.651 mM (Fig. 1A). We also performed binding analysis using fluorescence anisotropy that showed almost similar Kd (3.069 mM) (Fig. 1B). The activity of recombinant MtbICL was inhibited by ascorbate with an IC50 value of 2.155 mΜ (Fig. 1C). The recombinant MtbICL binding and inhibition by ascorbate suggested that MtbICL is a potential target of ascorbate during persistence on fatty acids. Ascorbate was also found to bind to M. tuberculosis catalase-peroxidase (katG), a Reactive oxygen species (ROS) scavenger (Kd- 0.665 mM) (Fig. 2A) and completely inhibit the enzymatic activity at 1 mM (Fig. 2B). Our results is in 4

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coherence with earlier studies showing that ascorbate has inhibitory effect on catalase activity 1517

. Thus, ascorbate may act as a dual sword on M. tuberculosis, generating ROS and preventing

its neutralization through katG.

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2.2 Ascorbate inhibits both ICL-dependent and ICL-independent in vitro growth of M. tuberculosis

Two earlier studies have reported the mechanism of the action of ascorbate-based inhibition of M. tuberculosis. In the first one, ascorbate was demonstrated to induce dormancy phenotype on

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in vitro growing cultures of M. tuberculosis by scavenging dissolved oxygen in the medium 7. In the more recent study, ascorbate was found to act as a pro-oxidant, generating ROI through

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Fenton reaction, a reaction that is limited by dissolved iron concentration in the medium 8. Our observations suggest another potential mechanism by which ascorbate can influence the growth of mycobacteria, which is by the inhibition of MtbICL activity. Expression of MtbICL is dependent on the carbon source in the growth medium and was found to be minimal in the presence of glucose and succinate and high in the presence of a fatty acid substrate like acetate or palmitate 18. We tested ascorbate against bacilli metabolizing through the citric acid cycle or the

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glyoxylate shunt by growing on either glucose or acetate respectively as sole carbon source. 3nitropropionate (3-NP), a standard inhibitor of MtbICL, showed only 34% inhibition in glucose medium, whereas 97 % of the growth was inhibited in acetate medium. This confirmed the ICLdependent growth of bacilli in acetate medium. For ascorbate, the percentage growth inhibition

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was similar for both substrates (Fig. 3A). Ascorbate at 4 mM inhibited ~89 % and 94% of bacterial growth compared to untreated control culture in glucose and acetate medium

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respectively. Ascorbate showed bactericidal activity at higher concentrations in both media, causing nearly 2log10 reduction in Colony forming units (CFUs) at 64 mM (Fig. 3B).

2.3 Ascorbate-mediated growth inhibition requires iron in the growth medium for M. tuberculosis surviving on glucose but not on fatty acids It has been established that the effect of ascorbate on M. tuberculosis is mediated by Fenton reaction and hence dependent on the presence of iron in the medium 8. To test if ICL is also a target of ascorbate in mycobacteria, iron dependent activity of ascorbate must be masked. Hence, we performed the growth inhibition experiments at reduced iron levels reported to be sufficient 5

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for bacterial growth, but inadequate for ascorbate to drive Fenton reaction, as reported earlier8. This was achieved by adding deferoxamine at a concentration of 200 µg.mL-1 to the cultures containing glucose or acetate and different concentrations of ascorbate. In glucose medium, the percentage growth inhibition by 4 mM and 16 mM ascorbate

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showed significant difference between normal and reduced iron concentrations, that is 89% vs 61% and 99% vs75%, respectively (p<0.001) (Fig. 3C). The difference was absent at higher concentration (64 mM) of ascorbate. 3-NP- mediated inhibition showed no difference in iron levels, emphasizing the role of iron in ascorbate mediated inhibition. On the other hand, in

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acetate medium, growth inhibition by ascorbate at any given concentration was similar under normal and reduced iron concentrations (Fig. 3D). Additionally, 3-NP mediated inhibition in

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acetate medium also was independent of the iron concentration.

The results suggest that the Fenton reaction-mediated growth inhibition by ascorbate is more prominent under glucose metabolizing conditions. When the growth is fatty acid dependent or ICL dependent as in acetate medium, there seems additional mechanism(s) through which growth is inhibited by ascorbate. The increased growth inhibition with increasing concentrations of ascorbate despite the low but constant iron levels, in both glucose and acetate media, also

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suggests the same. Our observations reinstate the Fenton reaction-mediated ascorbate action on M. tuberculosis only when bacilli are metabolizing glucose. When the bacilli are dependent totally on fatty acids, acetate in this study, there seems at least one additional mechanism through which ascorbate inhibits the growth. Mechanisms of action of ascorbate other than through

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Fenton reaction become relevant for M. tuberculosis residing in a low-iron environment of the macrophage phagosome19. Since human immune cells can accumulate vitamin C in millimolar 20

, the in vitro activity range (1-4 mM) of ascorbate against M. tuberculosis as

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concentrations

reported by us and earlier studies could be extrapolated in vivo.

2.4 Molecular docking suggests binding of ascorbate to MtbICL The molecular docking was performed for prediction of binding interaction of ligands (3-NP and ascorbate) with MtbICL. The binding cavity of ligands was defined in the crystallized structure. Both ligands were docked in the defined binding site using Autodock, one of the automated docking tools. Based on the study observations, 3-NP showed -3.49 Kcal.mol-1 docking score. The MtbICL-3-NP complex was stabilized by seven H-bonds with Gly192, His193, Arg228, 6

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Asn313, Ser317, Thr347 and five hydrophobic interactions with Asp108, Lys189, Ser191, Asn313, Ser315 (Fig. 4A). The MtbICL-ascorbate complex showed -6.72 Kcal.mol-1 docking score. The MtbICL-ascorbate complex was stabilized by ten H-bonds with Trp93, Asp108, Asp153, Gly192, Arg228, Asn313, Thr347 and seven van der Waals interactions with Ser91,

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Gly92, His180, Lys189, Gly192, His193, Arg228 and Ser315 (Fig. 4B). All these residues were also reported in Protein Data Bank (PDB) structure for catalytic activity of the MtbICL 21. It was observed that ascorbate could also bind to the above-mentioned residues and inhibit the activity.

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2.5 Molecular dynamic simulations suggest highly stable nature of MtbICL-ascorbate complex

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Molecular dynamic simulations (MDS) were performed to predict the stability of protein-ligand complexes. MDS provides the atomic level interaction details of protein-ligand interaction. All the systems (apo-MtbICL, MtbICL-3-NP, MtbICL-ascorbate) were employed for 40 ns MDS study. The systems were well equilibrated after 30 ns and the last 10 ns trajectory were considered for further analysis.

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2.5.1 Root mean square deviation

The Root mean square deviation (RMSD) describes the difference between the initial backbone conformation of the protein and last backbone conformation. The stability of the protein relative to its conformation can be determined by the deviations produced during simulation. Smaller

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deviations are known to indicate more stable protein structure. The value of RMSD was calculated for predicting the structural stability of the apo-protein and ligand bound complexes.

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The RMSD value for backbone for the 40 ns trajectory was calculated. The RMSD showed that after 30 ns all the systems attained the equilibration state. The RMSD for apo-MtbICL, MtbICL3-NP, and MtbICL-ascorbate was 0.46, 0.62, and 0.42 nm respectively (Fig. 5A). The MtbICL showed increasing value till 30 ns, following which it showed a stable pattern. On the other hand, the MtbICL-3-NP showed higher RMSD increment till 10 ns, but thereafter it showed constant RMSD peak. Finally, we observed that ascorbate showed lowest RMSD value and a stable pattern after 20 ns. Based on the RMSD result, we can say that all the complexes in this study produced stable trajectory for further analysis, and MtbICL-ascorbate complex is much more stable as compared to MtbICL-3-NP complex. 7

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2.5.2 Root mean square fluctuation The Root mean square fluctuation (RMSF) was calculated for prediction of flexible regions in proteins or how structural changes occur in protein upon ligand binding. The loop and coils

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showed more fluctuations because these are flexible in nature while helix and sheets showed fewer fluctuations due to their rigidity. The figure 5B showed that MtbICL-ascorbate complex showed less fluctuations as compared to control compound 3-NP during binding. The average RMSF value for apo-MtbICL, MtbICL-3-NP, and MtbICL-ascorbate were recorded as 0.15,

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0.14, and 0.11 nm, respectively. This RMSF indicated that binding of MtbICL-ascorbate induced fewer fluctuations in the protein, which implied that the MtbICL-ascorbate complex is much

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more stable as compared to MtbICL-3-NP complex. The flexibility of a protein or an individual residue is critical for performing its functions, we also identified the RMSF value for each residue after ligand binding and shown in Table 1.

The catalytically important residues in E. coli ICL are Lys193, Lys194, Gly196, and His197, and mutation in these conserved residues resulted in loss of enzyme activity

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Therefore, almost all the bacterial ICLs are thought to exhibit a similar behavioral pattern.

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Ligand binding in apo-MtbICL altered the RMSF, and this fluctuation suggests that the binding of ligand altered the conformation of these residues. Besides the conserved residues of active site, Ser319 also played an important role in catalytic activity

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MtbICL, we have mutated residues and predicted that His46, Phe345 and Leu418 play important

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role in maintaining the structure and catalytic activity of the enzyme 24-26.

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2.5.3 Radius of gyration

The Radius of gyration (Rg) of the apo-MtbICL, MtbICL-3-NP, and MtbICL-ascorbate complexes were determined to describe the compactness of the apo-protein and ligand bound complexes. The Rg is assigned as the mass weighted RMSD fit of a collection of atoms from their common center of mass. From the Figure 6A, it can be clearly seen that MtbICL-ascorbate formed a more compact complex as compared to apo-MtbICL and 3-NP bound complex. The average Rg value for apo-MtbICL, MtbICL-3-NP, and MtbICL-ascorbate were found to be 2.29, 2.27, and 2.23 nm, respectively. The result indicated that apo-MtbICL showed higher Rg value, implying that it is not well compact while our predicted compound MtbICL-ascorbate showed 8

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less Rg value as compared to control compound and showed that this complex is much compact than MtbICL-3-NP complex.

2.5.4 Hydrogen bonds

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Hydrogen bonds play a pivotal role in ligand and protein interactions. The ligands can easily detach from protein after its action due to its non-covalent binding. The number of hydrogen bonds was calculated for the last 10 ns time period trajectory for MtbICL-3-NP and MtbICLascorbate complexes. The number of hydrogen bonds was calculated in a specific time frame.

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Figure 6B showed that MtbICL-ascorbate complex showed higher number of hydrogen bonds as compared to MtbICL-3-NP complex. The average number of hydrogen bonds for MtbICL-3-NP

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and MtbICL-ascorbate was 0-1 and 0-2, respectively.

The key amino acids that play a very important role in ligand stabilization were also identified. The last 10 ns trajectory was considered for calculation of percent occupancy of hydrogen bonds here as well. Asp108 (3.99%) and Ser110 (0.20%) were the key residues taking part in hydrogen bond interaction in MtbICL-3-NP. Ser91 (0.20%), Trp93 (21.55%), Tyr89 (0.20%), Asp108 (0.20%), His180 (0.20%), Glu182 (0.20%) and Arg228 (0.40) were the key

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residues taking part in hydrogen bond interaction in MtbICL-ascorbate complex. Result reveals that both the inhibitors interact with key catalytic residues. MtbICL-ascorbate complex showed binding with more catalytic residues as compared to MtbICL-3-NP complex. The number of hydrogen bonds and percent occupancy of H-bonds also revealed that MtbICL-ascorbate

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complex is more stable as compared to MtbICL-3-NP complex.

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2.6 Principal component analysis supports the stable interaction of MtbICL-ascorbate complex

Principal component analysis (PCA) was carried out for the prediction of large concerted motions during ligand binding. Apo-MtbICL, MtbICL-3-NP, and MtbICL-ascorbate complex were employed for prediction of correlated motions. From the Figure 7A, it can been seen that eigenvalues obtained from the diagonalization of the covariance matrix of atomic fluctuations were plotted in decreasing order vs the corresponding eigenvector indices for apo-MtbICL, MtbICL-3-NP, and MtbICL-ascorbate. It is a well-known fact that the first few eigenvectors are very important for the prediction of concerted motions. The first 10 eigenvectors accounted for 9

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84.75%, 85.87%, and 76.31% of the motions observed for the last 10 ns trajectory for the apoMtbICL, MtbICL-3-NP, and MtbICL-ascorbate complexes, respectively. The percent of motions clearly indicated that the concerted motions decreased after ascorbate binding. Similar

stable as compared to MtbICL-3-NP complex.

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to our previous results, PCA analysis also suggested that MtbICL-ascorbate complex is more

The Figure 7A clearly shows that first few eigenvectors play a key role in overall concerted motions. For better representation of the result, we selected only first two eigenvectors and created a 2D principal components plot. It is clearly predicted from the Figure 7B that

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MtbICL-ascorbate complex forms a more stable cluster and occupies less phase space. Thus, it is clear from the 2D PCA result that MtbICL-ascorbate complex is more stable as compared to

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MtbICL-3-NP complex.

In order to study the effect of ligand binding on the motions described by PC1, the displacements of PC1 for the MtbICL, MtbICL-3-NP, and MtbICL-ascorbate were calculated (Figure 7C). This provided a clearer picture on the ligand binding induced correlated motions. Average fluctuation for MtbICL, MtbICL-3-NP, and MtbICL-ascorbate showed a value of 0.07, 0.07, and 0.05 nm, respectively. This indicated that MtbICL-ascorbate complex does not induce

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correlated motions after binding. It forms a stable complex as compared to MtbICL-3-NP. MtbICL-3-NP shows similar average motions as MtbICL, like high fluctuations in the Cterminal residues. Not only is this result is consistent with the RMSF analysis, it is in complete

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appropriation with our previously predicted RMSD, RMSF, and Rg results.

2.7 Binding energy analysis

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The binding free energy of both the complexes (MtbICL-3-NP and MtbICL-ascorbate) was estimated using the Molecular Mechanics Poisson−Boltzmann surface area (MM-PBSA) method. Last 10 ns equilibrated trajectory was considered for prediction of binding free energy. The binding free energy and its interrelated constituents achieved from the MM-PBSA estimation of the predicted hits are illustrated in Table 2. MtbICL-3-NP and MtbICL-ascorbate showed binding free energy of -35.70 and -27.98 kJ.mol-1, respectively. It was observed that electrostatic interactions, non-polar solvation energy, and Van der Waals negatively complimented the overall interaction energy, while polar solvation energy positively enriched the

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binding energy. The binding free energy of both the complexes revealed comparatively less binding affinity of ascorbate than MtbICL-3-NP complex.

2.8 Gibbs free energy analysis

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The Gibbs energy plot for PC1 and PC2 was also calculated and shown in Figure 8. The plot showed energy value ranging from 0 to 6.71, 0 to 6.94, and 0 to 5.82 kJ.mol−1 for MtbICL, MtbICL-3-NP, and MtbICL-ascorbate complexes, respectively. The MtbICL-ascorbate complex showed lowest energy compared to MtbICL and MtbICL-3-NP, which suggests that the complex

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followed energetically more favorable transition from one conformation to another. The energy minima region (blue region) is more in the MtbICL-ascorbate complex, suggesting that this

3. MATERIALS AND METHODS

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complex is thermodynamically more favorable.

The molecular biology kits and Ni-NTA agarose were purchased from Qiagen, CA, USA. The dNTPs and enzymes were purchased from New England Biolabs, MA, USA. All other reagents and chemicals were of the highest purity available and were purchased either from Sigma-

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Aldrich Chemical Company, St. Louis, MO, USA or Sisco Research Laboratories, Mumbai, India. Bacterial culture media was purchased from Himedia Laboratories, Mumbai, India.

3.1 Preparation of proteins and enzymatic assay

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The cloning of full-length MtbICL has been described previously and was a kind gift from Lt Dr. Vinod Bhakuni, CDRI. MtbICL was expressed and purified as earlier 27, 28. The full-length katG

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gene encoding for catalase–peroxidase was amplified using gene specific primers (forward-5′CAAGAATTCATGGTTCCCGAGCAACACCCACCCATTACA-3′

and

reverse-5′-

CCCAAGCTTGCGCACGTCAAATCTGTCGAGGTTCATCAC-3′). PCR was carried out in a total volume of 50 µL with Platinum Pfx DNA polymerase (Invitrogen). The PCR conditions used included 94 °C for 3 min followed by 30 cycles (94 °C for 30 s, 53 °C for 1 min, and 68 °C for 1 min), and a final elongation at 68 °C for 10 min. The amplified fragment was in pET23a(+) vector at EcoR1 and HindIII. The resultant construct was transformed into E. coli C41(DE3) cells for expression. Recombinant protein was overexpressed and purified as follows. A single colony from transformed plates was inoculated in 5 mL Luria Bertini (LB) broth containing 100 11

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μg/mL ampicillin. The cells were grown for 12 h at 30°C with continuous shaking at 160 rpm. Subsequently, two 500 mL LB broth tubes containing the 100 μg/mL ampicillin was inoculated with 1% (v/v) of grown culture and incubated at 30°C with shaking. Cultures were grown until

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the OD600 reached a value of 0.6; at this stage, the culture was induced with 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). The other un-induced culture was used as a control. After 12 h of induction at 20°C, both the cultures were pelleted by centrifugation at 8000 rpm for 10 min at 4°C. The pellet was then resuspended in lysis buffer that contained 20 mM Tris-Cl (pH 8.0) containing 300 mM NaCl and a cocktail of protease inhibitors. The dissolved cells were

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lysed by sonication, and the lysate was centrifuged at 13,000 rpm for 30 min at 4°C and the supernatant was collected. Ni-NTA affinity matrix was equilibrated with equilibration buffer.

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The supernatant was poured on the affinity column and was allowed to bind slowly. The protein was eluted with a linear gradient of 20-400 mM imidazole buffer and the desired peaks were dialyzed against 50 mM phosphate buffer (pH 8.0) containing 100 mM NaCl. Protein concentration was determined by Bradford method using bovine serum albumin (BSA) as a standard. The purity and molecular weight determination of the recombinant proteins were carried out by SDS-PAGE as well as with size exclusion chromatography (SEC) on a

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SuperdexTM 200, 10/300GL column (manufacturer’s exclusion limit 600 kDa) on AKTA-FPLC (GE Healthcare Biosciences). The column was pre-equilibrated and run with 50 mM phosphate buffer (pH 8.0) containing 100 mM NaCl at 25 ºC with a flow rate of 0.5 mL min-1 and detection at 280 nm.

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The MtbICL activity was performed as reported earlier27, 29. Inhibition of MtbICL activity was done by using fixed concentration of DL-isocitrate with varying concentration of ascorbate.

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The data were presented as log plot, and linear regression than employed to calculate the IC50. Catalase activity was measured spectrophotometrically by the method described earlier

with slight modification 15. 0.036mM H2O2 in phosphate buffer (50 mM, pH 8.0) was taken and the catalase activity was measured by adding 0.5 µM M. tuberculosis katG. The activity was observed by decrease in absorbance at 240 nm which implies that rate of disappearance of H2O2. The inhibition of catalase activity was measured in the presence of 1 mM ascorbate.

3.2 Binding of ascobate to MtbICL 12

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The affinity of MtbICL for ascorbate was determined by fluorescence titration using a Perkin Elmer LS-50B spectrofluorimeter with a slit band-width of 8 nm for excitation and 6 nm for emission. MtbICL was titrated with increasing concentrations of ascorbate. Fluorescence quenching was measured by excitation at 280 nm and recording the emission maxima at 340 nm.

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The spectra were normalized for corresponding ascorbate concentrations.

Kd values were determined by plotting ΔF/Fmax where Fmax is the Trp fluorescence in the absence of ascorbate and ΔF is the difference between Fmax and emission maxima at each ascorbate concentration 30. Fluorescence anisotropy is the another method used to determine the

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non-linear regression in GraphPad Prism software.

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Kd by plotting anisotropy vs ascorbate concentration. Kd was calculated by curve-fitting using

3.3 Determination of bactericidal activity of ascorbate on glucose/fatty acid substrate Five mL of a mid exponential phase (~OD600= 0.6) culture of M. tuberculosis H37Rv was washed twice with Middlebrook 7H9 broth containing 0.085% NaCl and 0.05% Tween-80 for removal of carbon source. The pellet was resuspended in the same medium, declumped by bead beating and allowed to stand for 15-20 min. The supernatant was adjusted to a turbidity

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equivalent to McFarland Standard No. 1. The suspension was further diluted 100-fold with medium containing 0.5% albumin and carbon source (10 mM glucose or 3 mM acetate).

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dilution plan included adding 100 µL of ascorbate (final concentration 64, 16 or 4 mM) or 3-NP (final concentration 200 µM) or PBS to 900 µL of this culture. To test under iron depleted

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conditions, deferoxamine (DFO) was added to a final concentration of 200 µg mL-1. Triplicate tubes for each test/control samples were incubated at 37 °C for eight days. The bacilli were

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pelleted by centrifugation, washed twice with and re-suspended in PBS containing tween-80 (0.05%). Serial dilutions of were plated on Middlebrook 7H10+ 10% oleic acid-albumindextrose-catalase (OADC) agar plates for determining CFU counts.

3.4 Molecular docking

The protein-ligand docking was performed by Autodock. The X-ray structure of MtbICL is in co-crystallized form with its natural substrates, thus the same binding site was selected for docking. Autodock uses the semi-empirical free energy force field for the calculation of ligand binding conformations. 3-NP (reference compound) and ascorbate were prepared by using MGL 13

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tools. All the polar and non-polar hydrogens were added in the receptor and ligands. After that the 3D grid box for docking was set into X = 52°, Y = 56°, Z = 52° grid points, and the grid spacing was 0.375 Å. Other docking parameters were population size (150), maximum number of evaluations (2,500,0000), maximum number of generations (2,70,000), rate of gene mutation

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(0.02), and rate of crossover (0.8). The Lamarckian Genetic Algorithm was used for generation of binding poses. Here the docking parameters were increased for prediction of accurate binding poses of ascorbate. We generated 2000 binding conformations and the results were clustered on the basis of RMSD, of which 1805 binding poses showed conformation in the active site region.

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From this analysis, the best pose complex was selected for MD study.

3.5 Molecular dynamics simulation

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GROMACS 4.6.5 31, 32 was used to perform MDS in an in house supercomputer as earlier 25, 33-36. Three systems were created and used for 40 ns MDS studies, one for predicting the stable structure of the apo-MtbICL and others for MtbICL-3-NP and MtbICL-ascorbate bound complexes. All the systems were solvated using simple point charge model in a cubic box. Ligand topology was generated by using ProDRG server

37

while protein topologies were

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generated by using GROMOS 9653a6 force field 38. 18 Na+ ions were added for neutralization of the systems. Steepest energy minimization was performed for all the systems to give the maximum force below 1000 kJ mol nm-1 for removing the steric clashes. Long range electrostatic interactions were calculated by Particle Mesh Ewald (PME) method

39

. For the

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computation of Lennard-Jones and Coulomb interactions, 1.0 nm radius cut-off was used. The LINCS algorithm was used to constrain the hydrogen bond lengths

40

. The time step was

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maintained at 2 fs for the MDS. For predicting the short-range non-bonded interactions, 10 Å cut-off distance was used. 1.6 Å Fourier grid spacing was used for the PME method for longrange electrostatics. All bonds were fixed by Shake algorithm 41. The systems were equilibrated after energy minimization. Then the position restraint simulation of 1 ns was carried out under NVT (the constant Number of particles, Volume and Temperature) and NPT (the constant Number of particles, Pressure and Temperature) conditions. Finally, all systems were submitted to 40 ns MDS. 2 fs interval was given for saving the coordinates. Then the RMSD, RMSF, Rg, hydrogen bonds and PCA were calculated by g_rms, g_rmsf, g_gyrate, g_hbond, g_cover and g_anaeig tools as described previously

25, 34, 36, 42

. The trajectories were analyzed by visual 14

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molecular dynamics 43 and Chimera 1.10.2 44. Origin 6.0 was used for generating and visualizing the plots.

3.6 Principal component analysis

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To investigate the conformational dynamics and collective motions upon ligand binding, PCA was performed. The PCA method was embedded in the GROMACS software package 45. After eliminating the rotational and translational movements, coordinates were superimposed onto a reference structure from which the positional covariance matrix of atomic coordinates and its

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eigenvectors were calculated. Then, diagonalization was performed on the calculated symmetric matrix by an orthogonal coordinate transformation matrix that generated the

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diagonal matrix of eigenvalues. In this diagonal matrix, columns were the eigenvectors corresponding to the direction of motion relative to the initial coordinates. Each eigenvector was associated with an eigenvalue that represented the total mean-square fluctuation of the system along the corresponding eigenvector. The g_anaeig and g_covar tools were used for PCA.

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3.7 Binding free energy calculation

The binding energy of protein-ligand complexes was calculated using the molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) method 46, 47. The free energy analysis is valuable in the advanced stage of drug discovery process. Free energy of solvation (polar + non-polar

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solvation energies) and molecular mechanics potential energy (electrostatic + Van der Waals interaction) were calculated by this tool. In this study, the last 10 ns of the MD trajectories were

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taken for the calculation of MM-PBSA.

4. CONCLUSION

ICL catalyses the conversion of isocitrate to succinate and glyoxylate, which is further oxidized to oxalate. Oxalate is also reported as the spontaneous degradation product of ascorbate in higher eukaryotes

48

. Since bacteria can also metabolize ascorbate

49

, there is a possibility of

spontaneous degradation of ascorbate to oxalate and feedback inhibition in addition to direct inhibition. A model of ascorbate mechanism of action on M. tuberculosis (Figure 9) is proposed based on the observations made during this study and in some previous studies 8. In iron-depleted 15

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microenvironments, ascorbate may act on the M. tuberculosis by generating ROS. It may even inhibit neutralization of ROS by the bacterial katG. On the other hand, under iron-depleted and fatty acid-rich microenvironments, ascorbate may inhibit the bacteria from metabolizing fatty

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acids through the inhibition of ICL.

Acknowledgments

This work was partly supported by CSIR network Project BSC0103 (UNDO). HS and SRK are

Conflict of interest

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The authors declare that there are no competing interests.

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grateful to CSIR, New Delhi while RS thank UGC, New Delhi for financial assistance.

Authors contributions

Conceived and designed the experiments: HS, RS, MYK, MSA, TT Performed the experiments: HS, SRK, RS

Analyzed the data: HS, RS, MYK, MSA, TT

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Contributed reagents/materials/analysis tools: HS, SRK, RS Wrote the paper: HS, RS, MYK, MSA, TT

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Table 1. RMSF value (nm) of catalytically important residues of apo-MtbICL, MtbICL-NP and MtbICL-ascorbic acid.

1

His46

0.0846

2

Lys189

0.1031

3

Lys190

0.1332

4

Ser191

0.1784

5

Gly192

0.2441

6

His193

0.2669

7

Ser315

0.1112

8

Leu418

0.2809

9

Glu423

0.4466

10

Glu424

MtbICL-NP 0.11 0.0916 0.0889 0.124

MtbICL-ascorbic acid

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apo-MtbICL

0.086

0.1067

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0.1227

0.12

0.1389

0.0863

0.142

0.0637

0.1151

0.3277

0.1398

0.2419

0.1554

0.2454

0.1472

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0.4568

0.1175

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Residues

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S. No.

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Table 2. Table showing the Van der Waals, electrostatic, polar salvation, SASA and binding energy in kJ mol-1 for each complex.

Compound

Van der Waals

No.

Electrostatic

energy energy

Polar salvation

SASA

energy

energy

Binding energy

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S.

3-nitropropionate

−67.98 ± 8.78

−12.51 ± 6.89

52.46 ± 11.18

−7.67 ± .67

−35.70 ± 7.48

2.

Ascorbic acid

−91.79 ± 8.52

−29.86 ± 7.49

103.45 ± 13.83

−9.78 ± .56

−27.98 ± 11.98

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1.

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FIGURE LEGENDS

Figure 1. Binding of ascorbate to MtbICL. (A) Plot of ΔF/Fmax vs [ascorbate] to determine

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the binding affinity (Kd) between MtbICL and ascorbate.ΔF is the difference of maximal and minimal fluorescence at given concentrations and Fmax is the maximum fluorescence before quenching. (B) Plot of Fluorescence anisotropy vs [ascorbate] to determine the binding affinity (Kd) between MtbICL and ascorbate. (C) Plot of percent MtbICL activity vs log of [ascorbate] to

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determine the IC50.

Figure 2. Binding of ascorbate to M. tuberculosis catalase-peroxidase (katG). (A) Plot of

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Fluorescence anisotropy vs [ascorbate] to determine the binding affinity (Kd) between M. tuberculosis katG and ascorbate. (B) Bar graph showing the inhibition of katG by ascorbate. The enzyme activity values are mean of values from three independent experiments.

Figure 3. Influence of carbon source and soluble iron on mycobacterial growth inhibitory property of ascorbate. M. tuberculosis H37Rv was allowed to grow for eight days in Middle

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brook 7H9 medium containing glucose or acetate as sole source of carbon and in presence or absence of deferoxamine, a biological iron chelating agent. The cultures were left untreated or treated with ascorbate at different concentrations. The known ICL-inhibitor 3-nitropropionate was used to indicate the ICL-dependence of culture. (A) Percent growth inhibition in eight days

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compared to untreated cultures (0% inhibition). (B) Log10 CFU/mL at the end of eight days. The dotted line across the graph indicates the CFUs at the start of experiment. (C) and (D) Percent

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growth inhibition of glucose utilizing and acetate utilizing bacilli respectively in presence of optimal and suboptimal levels of iron in eight days compared to untreated cultures (0% inhibition). The data shown are the mean ± SD of three independent experiments. Statistical significance (p<0.001, denoted by ***) was determined by 2-way ANOVA with Bonferroni post tests.

Figure 4. The potential binding poses for the 3-nitropropionate and ascorbate. (A) 3nitropropionate, and, (B) Ascorbate, binding modes in the active site of MtbICL .

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Figure 5. Molecular dynamic simulations. (A) RMSD of the Cα backbone of apo-MtbICL, MtbICL-3-NP and MtbICL-ascorbate over 40 ns MDS at 300 K. (B) RMSF of residues during MDS between apo-MtbICL, MtbICL-3-NP and MtbICL-ascorbate. In both the panel black, red and green colour represents apo-MtbICL, MtbICL-3-NP and MtbICL-ascorbate complex

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respectively.

Figure 6. Structural stability parameters. (A) Number of intramolecular hydrogen bonds of apo-MtbICL, MtbICL-3-NP and MtbICL-ascorbate during the last 10 ns simulation time. (B)

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Plot of Radius of gyration vs time for apo-MtbICL, MtbICL-3-NP and MtbICL-ascorbate. In both the panel black , red and green colour represents apo-MtbICL, MtbICL-3-NP and MtbICL-

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ascorbate complex respectively.

Figure 7. Principal component analysis. (A) Plot of eigenvalues was plotted vs eigenvector index. First 50 eigenvectors were considered and shown for apo-MtbICL, MtbICL-3-NP and MtbICL-ascorbate. (B) Projection of the motion of the protein in phase space along the PC1 and PC2 for apo-MtbICL, MtbICL-3-NP and MtbICL-ascorbate. In all panel panel black, red and

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green colour represents apo-MtbICL, MtbICL-3-NP and MtbICL-ascorbate complex respectively. (C) Projection of the motion of the protein in phase space along the PC1 and PC2 for apo-MtbICL, MtbICL-3-NP and MtbICL-ascorbate. The black, red and green colour

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represents apo-MtbICL, MtbICL-3-NP and MtbICL- ascorbate complex respectively.

Figure 8. Gibbs free energy landscape. PC1 and PC2 free energy landscape for (A) apo-

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MtbICL, (B) MtbICL-3-NP, and (C) MtbICL-ascorbate.

Figure 9. Model of the M. tuberculosis inhibition by L-ascorbate. Ascorbate in presence of ferric ions generates reactive oxygen intermediates (ROI) via Fenton reaction. Ascorbate may moderately inhibit M. tuberculosis katG and thereby lead to ROI build-up. The ROI are detrimental to the growth and viability of M. tuberculosis. In a low iron environment, Fenton reaction-mediated ascorbate action does not work. When the bacteria are growing on fatty acids alone they become dependent on ICL, which is a potential target of ascorbate. Inhibition of ICL by ascorbate bypasses the need for sufficient iron in the environment. 25

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