WO2014184805A2 - Biosensor for detection of mycothiol redox potential - Google Patents

Abstract

The disclosure provides a fusion construct that includes an engineered green fluorescent protein having a fluorescence spectrum that is sensitive to redox status and a mycothiol-dependent oxidoreductase linked to the engineered green fluorescent protein. The mycothiol-dependent oxidoreductase is a mycoredoxin identified from Mycobacterium tuberculosis strain H37Rv and the fusion construct can be used as a biosensor for detecting a change in a redox status of mycothiol in a cell.

Description

COMPLETE SPECIFICATION

Title of the Invention Biosensor for Detection of Mycothiol Redox Potential Cross Reference to a Related Application

This application takes priority from Indian Provisional Application having Application No. 1412/DEL/2013 filed on 13 May 2013. This application claims the benefit and priority of the above mentioned application, the contents of which are incorporated herein by reference.

Technical Field

The present disclosure relates generally to the field of biosensors proteins. More particularly, the disclosure relates to biosensor proteins capable of detecting a change in oxidation-reduction state of the mycothiol redox couple, present in various actinomycetes, particularly mycobacteria.

Background

It is estimated that nearly 2 billion people currently suffer from latent Mycobacterium tuberculosis {Mtb) infection and ~ 1.4 million people succumb to tuberculosis (TB) annually. The ability of Mtb to adapt and resist killing by immune system of the host facilitates its survival, replication, and persistence. Mtb continuously senses host environment to modulate its carbon metabolism, ATP pool, and redox balance.

Mtb faces a gradient of host-generated redox stresses such as reactive oxygen species (ROS), reactive nitrogen species (RNS), and hypoxia during infection. Additionally, bioreductive activation of anti-TB drugs such as isoniazid and PA-824 in Mtb also generate ROS and RNS. Research suggests that redox balance (redox homeostasis, signaling and dynamic reprogramming of intracellular redox metabolism) plays an important role in regulating intracellular survival and long term persistence of Mtb. Persistence of Mtb in host phagocytes depends on its ability to resist oxidant-mediated antibacterial responses. Mtb modulates multiple redox sensing pathways such as SigH/RshA, DosR S/T, and WhiB family for maintaining the redox homeostasis. Several cellular antioxidants such as mycothiol (MSH), alkyl hydroperoxide reductase, catalase, superoxide dismutase, thioredoxins, and ergothionine, assist Mtb in dissipating the redox stress.

In recent times, mycothiol redox couple (MSH/MSSM) has emerged as a key factor associated with redox homeostasis in Mtb. Mycothiol is a cysteine derivative produced only by actinomycetes. Among the actinomycetes, mycobacteria produce mycothiol in the greatest amounts (millimolar concentrations). Mycobacteria are the main group of actinomycetes that infect humans. Other genera of actinomycetes (Corynebacterium, Streptomyces, Nocardia and Rhodococcus) also produce mycothiol, but they are minor causes of animal/plant pathogenicity.

Thus, tracking the changes in the mycothiol redox potential (E_MSH) of Mtb during infection is central to understanding its mechanism of survival, replication, and persistence in the host. This will also aid in effective screening of anti-tuberculosis drugs and clinical diagnosis of mycobacterial infection. However, conventional approaches for mycothiol detection require cell disruption, precluding dynamic measurements and making the methodologies prone to oxidation artifacts. Alternative approaches, such as use of redox-sensitive dyes to detect ROS generation in cells, also suffer from non-specificity, irreversibility, and these dyes cannot deliver information regarding the redox potential of a specific redox couple.

A few non-invasive methods have been developed to measure the intracellular redox potential of glutathione (GSH) which is the major redox buffer in many organisms. For this, green fluorescent protein (GFP) indicators were genetically engineered to render them sensitive to redox changes in the glutathione redox couple (GSH/GSSG). The engineered green fluorescent protein (known as roGFP2) measures the intracellular redox potential of glutathione (EGSH) through interaction with endogenous glutaredoxins (Grxs; glutathione-dependent oxidoreductases) (as described in the articles titled 'Fluorescent protein-based redox probes' published in 'Antioxidants & Redox Signaling', Volume 13, Pages 621 -650 by Meyer and Dick in 2010 and 'Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the redox potential of the cellular glutathione redox buffer' published in 'Plant Journal', Volume 52, Pages 973-986 by Meyer et al. in 2007. These articles are incorporated herein as reference). roGFP2 had the disadvantage of slow response kinetics due to which absolute specificity towards GSH/GSSG redox couple could not be guaranteed. This disadvantage was circumvented by coupling of roGFP2 to human glutaredoxin 1 (Grxl). This coupled protein (Grxl -roGFP2) ensured complete specificity and rapid equilibration

described in the article titled 'Real-time imaging of the intracellular glutathione redox potential' published in 'Nature Methods', Volume 5, Pages 553-559 by Gutscher et. al. in 2008. This article is incorporated herein as reference). However, the specificity of roGFP2 and Grx l -roGFP2 based biosensors towards the glutathione redox couple render them ineffective in non-glutathione producers like gram positive bacteria (such as Bacillus species, Staphylococcus aureus and Deinoco.ccus radiodurans) and actinomycetes. Thus, roGFP2-based biosensors were unable to detect the mycothiol redox couple present in actinomycetes, particularly mycobacteria.

Therefore in light of the above discussion, there is a need for developing an improved non-invasive, sensitive, specific teclinology and corresponding biosensors to track changes in the mycothiol redox potential of actinomycetes, particularly mycobacteria (Mtb) that overcomes one or more problems associated with the prior art.

Accordingly, the present invention provides a novel method for mycothiol detection. The present invention addresses the problems of the prior art and provides a novel, non-invasive, sensitive and specific biosensor protein construct capable of detecting a change in oxidation- reduction state of the mycothiol redox couple present in actinomycetes, particularly mycobacteria.

Brief Summary

The present invention presents a novel biosensor protein construct capable of detecting a change in oxidation-reduction state of the mycothiol redox couple present in actinomycetes, particularly mycobacteria. In the present invention, the specificity and sensitivity of roGFP2 based biosensors has been completely altered by coupling oxidation and reduction of roGFP2 to the mycothiol redox couple via fusion of roGFP2 with a mycothiol-dependent oxidoreductase (mycoredoxin; Mrxl ) isolated from Mtb. Thus, the roGFP2 based sensors of the present invention have been endowed with a novel property of exclusively responding to perturbations in mycothiol redox potential (EMSH)- The fusion construct includes a first portion comprising an engineered green fluorescent protein having a fluorescence spectrum that is sensitive to redox status (roGFP2) and a second portion comprising a mycothiol-dependent oxidoreductase (Mrxl) linked to the engineered green fluorescent protein. Optionally, the first portion is linked to the second portion with a linker sequence to fuse the mycothiol-dependent oxidoreductase to the N- terminus of the engineered green fluorescent protein, wherein the linker sequence is a 30 amino acid linker comprising the sequence (Gly-Gly-Ser-Gly-Gly)₆. More specifically, the fusion construct may be a biosensor capable of detecting a change in a redox status of mycothiol in a gram-positive cell such as a member of taxa actinomycetes, selected from the group comprising Mycobacterium, Corynebacterium, Streptomyces, Nocardia and Rhodococcus. The fusion construct may be a biosensor cap_able_of _.identifying analytes selected from a group including drugs, antibodies, and chemical compounds that can trigger a change in a redox status of mycothiol in a cell. Further, the fusion construct may be a biosensor capable of detecting a cell comprising mycothiol redox couple in a sample. In further embodiments, the fusion construct may be a biosensor capable of detecting a level of drug resistance of a cell comprising mycothiol redox couple. It may be a biosensor capable of identifying a pathway that directly and/or indirectly communicates with mycothiol redox couple in a cell.

Also provided is a method of identifying an analyte that triggers a change in a redox status of mycothiol. In some embodiments of the disclosure, a method of detecting a cell having a mycothiol redox couple in a sample is provided.

Also provided is a kit for detecting a cell Having a mycothiol redox couple in a sample, wherein the kit includes a moiety capable of reacting with cells having mycothiol redox couple. The moiety has a fusion construct that includes an engineered green fluorescent protein having a fluorescence spectrum that is sensitive to redox status and a mycothiol-dependent oxidoreductase linked to the engineered green fluorescent protein. In other embodiments, the kit includes a processing system for lysing cells present in the sample and a fusion construct that includes an engineered green fluorescent protein having a fluorescence spectrum that is sensitive to redox status and a mycothiol-dependent oxidoreductase linked to the engineered green fluorescent protein.

Also provided is a method of detecting a level of drug resistance of a cell having mycothiol redox couple. Also provided is a method of identifying a pathway that directly and/or indirectly communicates with mycothiol redox couple in a cell. The present disclosure also presents a method of identifying a bacterial pathway that directly and/or indirectly communicates with mycothiol redox couple in a cell is provided. In yet other embodiments of the disclosure, a method of identifying a host pathway that directly and/or indirectly communicates with mycothiol redox couple in a cell is provided.

Brief Description of Drawings

The features of the present disclosure, which are believed to be novel, are set forth with particularity in the appended claims. The disclosure may best be understood by reference to the following description, taken in conjunction with the accompanying drawings. These drawings and the associated description are provided to illustrate some embodiments of the disclosure, and not to limit the scope of the disclosure.

FIG. 1 illustrates a molecular mechanism of MSSM sensing in Mrx l -roGFP2 expressing bacteria, in accordance with an embodiment of the disclosure;

FIG. 2 is a graph illustrating excitation spectra of purified Mrx l -roGFP2, in accordance with an embodiment of the disclosure;

FIG. 3 is a graph illustrating the sensitivity of Mrxl-roGFP2 towards small changes in OXDMSH, in accordance with an embodiment of the disclosure;

FIG. 4 is a graph illustrating specificity of Mrxl -roGFP2 towards MSSM and non- responsiveness towards non-specific oxidants, in accordance with an embodiment of the disclosure;

FIG. 5 is a graph illustrating the NADPH dependent reduction of MSSM by mycothiol reductase (Mtr), in accordance with an embodiment of the disclosure;

FIG. 6 is a graph illustrating MSH/Mtr ADPH electron transfer assay with oxidized Mrxl-roGFP2 as a substrate, in accordance with an embodiment of the disclosure;

FIG. 7 is a graph illustrating response of Mr l -roGFP2in cells of wild-type Msm, MsmAmshA, MsmAmshD and MsmAsigH on exposure to diamide and DTT, in accordance with an embodiment of the disclosure;

FIG. 8 is a graph depicting an in vitro redox calibration curve generated by treating MtbK l v the Mrxl-roGFP2 fusion construct with buffers of known redox potentials, in accordance with an embodiment of the disclosure; FIG. 9 is a graph illustrating response of uncoupled roGFP2, wild-type Mrx l -roGFP2 and variants Mr l(CGYA)-roGFP2 and Mrxl(AGYC)-roGFP2towards MSSM, in accordance with an embodiment of the disclosure;

FIG. 10 is a graph illustrating response of Msm expressing Mrxl -roGFP2 towards oxidation by varying concentrations (0.1 to l OmM) of H₂0₂, in accordance with an embodiment of the disclosure;

FIG. 1 1 is a graph illustrating oxidation of Mrxl -roGFP2 expressed in Msm upon stimulation with exogenous oxidants, in accordance with an embodiment of the disclosure;

FIG. 12 is a graph illustrating response of Msm cells expressing Mrx l -roGFP2 towards lower concentrations of H₂0₂, in accordance with an embodiment of the disclosure;

FIG. 13 is a graph illustrating response of Msm cells expressing Mrx l -roGFP2 upon treatment with dequalinium, cisplatin and 5-methoxyindole-2-carboxylic acid, in accordance with an embodiment of the disclosure;

FIG. 14 is a bar graph representing the intra-mycobacterial EMSHO? H37RV expressing Mrx l -roGFP2 inside na^'ive (resting) RAW 264.7 macrophages and IFN-y/LPS treated (activated) RAW 264.7 macrophages, in accordance with an embodiment of the disclosure;

FIG. 15 is a bar graph depicting influence of iNOS inhibition on intramycobacterial E_MSH in immune-activated RAW 264.7 macrophages, in accordance with an embodiment of the disclosure; FIG. 16are dot plots showing shift in population of Mf£H37Rv infected THP-1 cells towards oxidizing or reducing after treatment with CHP and DTT, in accordance with an embodiment of the disclosure;

FIG. 17 is bar graph representing the ratiometric sensor response of ¾H37Rv infected THP- 1 cells after treatment with CHP and DTT, in accordance with an embodiment of the disclosure;

FIG. 18 is a dot plot of THP- 1 cells infected with H37Rv expressing Mrx l -roGFP2 at 72h post-infection, in accordance with an embodiment of the disclosure; FIG. 19 is a bar graph representing percentages of basal, oxidized and reduced sub- populations of THP-1 cells infected with H37Rv expressing Mrxl -roGFP2 at 72h post-infection, in accordance with an embodiment of the disclosure;

FIG. 20 is a dot plot of H37Rv expressing Mrx 1 -roGFP2grown in 7H9 medium (control), in accordance with an embodiment of the disclosure;

FIG. 21 is a bar graph representing percentages of basal, oxidized and reduced sub- populations of H37Rv expressing Mrxl-roGFP2grown in 7H9 medium (control), in accordance with an embodiment of the disclosure;

FIG. 22 is a bar graph representing percentages of basal, oxidized and reduced sub- populations of t£H37Rvexpressing Mrxl -roGFP2inside THP-1 as analyzed by flow cytometry, in accordance with an embodiment of the disclosure;

FIG. 23 is a scatter plot depicting redox heterogeneity displayed by Mtb expressing Mrxl-roGFP2inside THP-1 at 24 h post-infection, wherein the scatter plot depicts quantification of microscopy data and each point on the plot represents a bacterium whilepercentage of bacilli in each sub-population is represented as a stacked bar graph, in accordance with an embodiment of the disclosure;

FIG. 24 are scatter plots depicting redox heterogeneity displayed by Mtb expressing Mrxl-roGFP2 inside (A) endosomes, (B) lysosomes, and (C) autophagosomes, wherein the scatter plot depicts quantification of microscopy data and each point on the plot represents a bacterium whilepercentage of.bacilli in each sub-population is represented as a stacked bar graph, in accordance with an embodiment of the disclosure;

FIG. 25 is a graph depicting induction of varying degrees of oxidative stress in

MtbH37Rv during infection in macrophages by various anti-TB drugs as analyzed by flow cytometry, in accordance with an embodiment of the disclosure;

FIG. 26 is a graph depicting survival percentage (on the basis of cfu counts) of r6H37Rv treated with INH, CFZ, RIF and ETH in the presence or absence of DTT (7H9 represents the untreated control group), in accordance with an embodiment of the disclosure.

Detailed Description The specification concludes with the claims defining the features of the disclosure that are regarded as novel, it is believed that the disclosure will be better understood from a consideration of the following description in conjunction with the drawings.

As required, detailed embodiments of the disclosure are disclosed herein; however, it is to be understood that the disclosed embodiments are merely exemplary of the disclosure, which can be embodied in various forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but merely as a basis for the claims and as a representative basis for teaching one skilled in the art to variously employ the disclosure as suitable. Further, the terms and phrases used herein are not intended to be limiting but rather to provide an understandable description of the disclosure.

The terms "a" or "an", as used herein, are defined as one or more than one. The term "another", as used herein, is defined as at least a second or more. The terms "including", "containing" and/or "having" as used herein, are defined as comprising (i.e. open transition).

In some embodiments of the disclosure, a fusion construct is provided. The fusion construct can include a first portion comprising an engineered green fluorescent protein having a fluorescence spectrum that is sensitive to redox status (roGFP2) and a second portion comprising a mycothiol-dependent oxidoreductase (Mrxl) linked to the engineered green fluorescent protein. In another embodiment, the first portion may be linked to the second portion with a linker sequence to fuse the mycothiol-dependent oxidoreductase to the N-terminus of the engineered green fluorescent protein, wherein the linker sequence is a 30 amino acid linker comprising the sequence (Gly-Gly-Ser-Gly-Gly)₆.

In further embodiments, the mycothiol-dependent oxidoreductase may be a mycoredoxin identified from Mycobacterium tuberculosis strain H37Rv.

In other embodiments, the fusion construct may be a biosensor capable of detecting a change in a redox status of mycothiol in a cell.

In further embodiments, the fusion construct may be a biosensor capable of detecting a change in a redox status of mycothiol in a gram-positive cell such as a member of taxa actinomycetes. In other embodiments, the member of the taxa actinomycetes is selected from the group comprising Mycobacterium, Corynebacterium, Streptomyces, Nocardia and Rhodococcus. In still other embodiments, the fusion construct may be a biosensor capable of detecting a change in a redox status of mycothiol in a strain of Mycobacterium tuberculosis.

In yet other embodiments, the fusion construct may be a biosensor capable of identifying analytes selected from a group including drugs, antibodies, and chemical compounds that can trigger a change in a redox status of mycothiol in a cell.

In further embodiments, the fusion construct may be a biosensor capable of detecting a cell comprising mycothiol redox couple in a sample.

In further embodiments, the fusion construct may be a biosensor capable of detecting a level of drug resistance of a cell comprising mycothiol redox couple.

In further embodiments, the fusion construct may be a biosensor capable of identifying a pathway that directly and/or indirectly communicates with mycothiol redox couple in a cell.

In some embodiments of the disclosure, a method of identifying an analyte that triggers a change in a redox status of mycothiol is provided. The method includes the step of inserting a fusion construct into a cell having a mycothiol redox couple, such that the fusion construct includes an engineered green fluorescent protein having a fluorescence spectrum that is sensitive to redox status and a mycothiol-dependent oxidoreductase linked to the engineered green fluorescent protein. Thereafter, the method includes the step of incubating the cell with a number of analytes for a time sufficient for the analytes to complete a reaction with at least one constituent of the cell. Thereafter, fluorescence responses corresponding to the analytes are detected, the fluorescence responses being indicated by the fusion construct. Further, the method is characterized such that the detection of a fluorescence response is indicative of a corresponding analyte being the analyte that triggers the change in the redox status of mycothiol.

In some embodiments of the disclosure, a method of detecting a cell having a mycothiol redox couple in a sample is provided.

In some embodiments, the method of detecting a cell in a sample includes the step of inserting a fusion construct into a moiety capable of reacting with cells having mycothiol redox couple, such that the fusion construct includes an engineered green fluorescent protein having a fluorescence spectrum that is sensitive to redox status and a mycothiol-dependent oxidoreductase linked to the engineered green fluorescent protein. Thereafter, the method includes the step of incubating the moiety with the sample for a time period sufficient for the moiety to complete a reaction with at least one constituent of the sample. The method also includes the step of detecting a fluorescence response indicated by the fusion construct, such that the detection of the fluorescence response is indicative of the presence of the cell comprising mycothiol redox couple.

In other embodiments, the method of detecting a cell in a sample includes the step of subjecting the sample to a processing system that causes lysis of cells present in the sample. Thereafter, the method includes the step of incubating the lysed sample with a fusion construct, such that the fusion construct includes an engineered green fluorescent protein having a fluorescence spectrum that is sensitive to redox status and a mycothiol-dependent oxidoreductase linked to the engineered green fluorescent protein. The lysed sample is incubated with the fusion construct for a time period sufficient for the fusion construct to complete a reaction with at least one constituent of the lysed sample. The method also includes the step of detecting a fluorescence response indicated by the fusion construct, such that the detection of the fluorescence response is indicative of the presence of the cell comprising mycothiol redox couple.

In some embodiments of the disclosure, a kit for detecting a cell having a mycothiol redox couple in a sample is provided. In some embodiments, the kit includes a moiety capable of reacting with cells having mycothiol redox couple. The moiety has a fusion construct that includes an engineered green fluorescent protein having a fluorescence spectrum that is sensitive to redox status and a mycothiol-dependent oxidoreductase linked to the engineered green fluorescent protein. In other embodiments, the kit includes a processing system for lysing cells present in the sample and a fusion construct that includes an engineered green fluorescent protein having a fluorescence spectrum that is sensitive to redox status and a mycothiol-dependent oxidoreductase linked to the engineered green fluorescent protein.

In some embodiments of the disclosure, a method of detecting a level of drug resistance of a cell having mycothiol redox couple is provided. The method includes the step of introducing into the cell a fusion construct that includes an engineered green fluorescent protein having a fluorescence spectrum that is sensitive to redox status and a mycothiol-dependent oxidoreductase linked to the engineered green fluorescent protein. Thereafter, the method includes the step of detecting a fluorescence response indicated by the fusion construct, such that a pre-defined intensity level of the fluorescence response of the fusion construct is indicative of a drug resistance level of the cell having mycothiol redox couple.

In some embodiments of the disclosure, a method of identifying a pathway that directly and/or indirectly communicates with mycothiol redox couple in a cell is provided.

In some embodiments of the disclosure, a method of identifying a bacterial pathway that directly and/or indirectly communicates with mycothiol redox couple in a cell is provided. The method includes the step of preparing an array of cells having a mycothiol redox couple. Thereafter, each cell of the array is modified by disrupting a unique pathway. Subsequently, a fusion construct that includes an engineered green fluorescent protein having a fluorescence spectrum that is sensitive to redox status and a mycothiol-dependent oxidoreductase linked to the engineered green fluorescent protein is introduced into each cell of the array. Once the fusion construct has been introduced into each cell of the array, a fluorescence response indicated by the fusion construct is detected, such that a pre-defined intensity level of the fluorescence response is indicative of the unique pathway being the pathway that directly and/or indirectly communicates with mycothiol redox couple.

In yet other embodiments of the disclosure, a method of identifying a host pathway that directly and/or indirectly communicates with mycothiol redox couple in a cell is provided. The method includes the step of preparing an array of host cells and modifying each host cell of the array by disrupting a unique pathway. Subsequently, each host cell of the array is infected with a mycothiol redox couple containing cell comprising a fusion construct that includes an engineered green fluorescent protein having a fluorescence spectrum that is sensitive to redox status and a mycothiol-dependent oxidoreductase linked to the engineered green fluorescent protein. In this way, the fusion construct and the mycothiol redox couple is incorporated into each host cell of the array. Subsequently, a fluorescence response indicated by the fusion construct is detected, such that a pre-defined intensity level of the fluorescence response is indicative of the unique pathway being the pathway that directly and/or indirectly communicates with mycothiol redox couple.

Methods

Measurement of ratiometric fluorescence response: Excitation scan (350 to 500nm) was performed at fixed emission (of for example .515nm) using a spectrofluorometer. The ratio of emitted light (at 515nm) after excitation at 390 and 490nm (hereinafter referred to as 390/490nm ratio or 390/490nm excitation ratio or ratiometric fluorescence response or fluorescence response) was then determined. In vitro measurements using various roGFP2 variants were performed on SpectraMax M3 microplate reader.

Cloning of fusion construct

Following PCR amplification, all the fusion constructs were sub cloned into the expression vector pET28b (Novagen), expressed in the E. coli strain BL21 DE3 (Stratagene), and fusion proteins were purified via hexahistidine affinity chromatography.

Effect of oxidants

To study the effect of oxidants, the fusion construct was first reduced using DTT. Unless otherwise specified, the fusion construct was reduced by incubation with l OmM DTT for30 minutes (min) on ice followed by desalting using with Zeba Desalt spin columns

(PierceBiotechnology). This was followed by incubation of the fusion construct with the oxidants for the required time periods.

Bacterial cells and culture conditions

The bacterial species and strains used in this study were Mycobacterium smegmatis

(Msm) mc2155, MsmAmshA, MsmAmshD, Msm SigH, M. bovis BCG Pasteur, M. tuberculosis H37Rv and the field isolates Jal 1934, Jal 2287, Jal 2261, BND 320 and MYC 43 1. Bacteria were grown in Middlebrook 7H9 broth (Difco) supplemented with 10% OADC (Becton Dickinson), 0.1% Glycerol and 0.1% Tween 80 until the mid-log phase (OD₆oo of 0.8). Bacilli were washed twice with 10% glycerol and resuspended in 1/10th the actual volume. The competent cells were electroporated using l-2μg of the plasmid in Bio Rad Gene Pulser with settings of 2.5kV voltage, 25μΡ capacitance and 1000Ω resistance. After overnight recovery in 7H9, selection was performed on 7H10 agar plates containing hygromycin (50μg/ml). After 21 days of selection, bacteria were grown in 7H9 broth till mid-log phase and used for further studies. Mammalian cells and infection

The human monocytic cell line THP-1 and mouse macrophage cell line RAW 264.7 were maintained in an atmospherecontaining 5% C0₂ at 37°C in the culture medium recommended by ATCC. THP-1 monocytes were differentiated into macrophages by a 24 hours (h) treatment with 20ng/ml phorbol 12-myristate 13-acetate (PMA). Cells were rested for 3 daysfoUowing chemical differentiation to ensure that they reverted to a restingphenotype before infection. RAW 264.7 macrophages were activated by treatment with lOOU/ml IFN-yD 12 h before infection and lOOng/ml LPS for 2h before infection. PMA-differentiated THP-1 cells or RAW 264.7 macrophages seeded at 2 χ 10⁵ cells per well in 24-well plates were infected with Mrxl -roGFP2 expressing mycobacteria at a multiplicities of infection (MOI) of 10 and incubated for 4h at 37°C in 5%C0₂. Extracellular bacteria were removed by washing twice with PBS.

Redox potential measurement

Mycobacterial strains grown till an OD₆oo of 0.6 to 0.8 were harvested, washed twice and resuspended in PBS. Fluorescence excitation scan (350-500nm) was performed at 515nm emission. For each experiment, the minimal and maximal fluorescence ratios were also determined, that correspond to 100% biosensor reduction and 100% biosensor oxidation, respectively. Diamide (400μΜ) was used as the oxidant and DTT (40mM) as the reductant. In case of Mtb, cumene hydroperoxide (CHP, ImM) was used instead of diamide. The observed fluorescence ratio was used to calculate the corresponding degree of biosensor oxidation using the equation given below.

(R - R_red)

/ 490min

/ 490max

Where, R is the observed ratio, R_red and R_<,_x are the ratios of completely reduced and oxidized roGFP2, respectively. / 490min and / 490max are the fluorescence intensities measured with excitation at 490nm for fully oxidized and fully reduced roGFP2, respectively.

Next, the intracellular biosensor redox potential E_TOC «was calculated using the Nernst equation: PO' ^RT in ^{( 1'QxP} roGFP2 ^}

^EroGFP2 = roGFP2 ^ln ₀xD_roGFP2 roGFP2is known to have an average consensus midpoint redox potential of E°'_R0GFP2 ⁼ -280mV (as described in the article titled 'Investigating mitochondrial redox potential with redox-sensitive green fluorescent protein indicators' published in 'The Journal of biological chemistry', Volume 279, Pages 13044-13053 by Hanson et. al. in 2004. This article is incorporated herein as reference). Based on the equilibration between the biosensor and the mycothiol redox couple, the mycothiol redox potential is obtained {EMSH ⁼ E_R0GFP2)-

Generation of in vitro redox titration curve using Mrxl-roGFP2 protein

The Mrxl -roGFP2 protein was titrated in degassed PBS containing Ι μΜ protein and lOmM of total DTT (varying ratios of trans-4,5-dihydroxy-l ,2-dithiane (DTT_0Xd) and reduced DTP (DTTre_d)). The solutions were allowed to equilibrate for lh in an anaerobic glove box. Redox potential of the DTT standard solutions was calculated according to the Nernst equation:

E^' = E° _DTT - *I In ^[DTT-'

2F [DTT_0X<1]

where E° DTT ⁼ -330mV, R is the gas constant (8.313J/mol/K), T is temperature (K), n = 2 is the number of electrons exchanged, and F is Faraday's constant (96490 J/mol/volt). The Mrxl- roGFP2 ratios were normalized to the values measured using lOmM DTT_red as 0% oxidation and lOmM DTTox_d as 100% oxidation, and the normalized ratios were plotted against the calculated redox potentials of DTT standard solutions. Measurements were performed on spectraMaxM3 microplate reader (Molecular devices).

In vitro redox calibration curve using Mtb H37Rv

For estimating EMSH of Mtb during infection, a redox calibration curve^{^}was generated using Mtb cells grown in 7H9 medium in vitro. Because 7H9 grown Mtb does not show redox variations, exposure of cells to different ratios of oxidant and reductant would have uniform influence on the intracellular redox state of Mtb. In contrast, significant heterogeneity in Mtb redox state inside macrophages precludes generation of redox calibration curve in situ. Absolute redox potential values in cells expressing Mrx 1 -roGFP2 were obtained from in vitro calibration curve obtained by titrating cells with IX PBS containing different ratios of trans-4,5-dihydroxy- 1 ,2-dithiane (DTT_oxd) to reduced DTT (DTT_red) covering redox potential range of -330 to - 195mV. Excitation ratios (405/488nm) at 510nm emission were normalized to the range between 0% oxidation (lOmM reduced DTT) and 100% oxidation (ImM CHP) and the normalized ratios were plotted against the calculated redox potentials of the DTT standard solution which is calculated according to the Nernst equation:

E^< = E0_DTT . RI H≡^ )

2F [DTT_RED]

Where E° DTT = -330mV

Data were fit (Sigmaplot 10 statistical analysis software) by nonlinear regression to generate a calibration curve that was used to relate the Mrxl-roGFP2 ratios obtained from flow cytometry and/or confocal microscopy to midpoint potential in millivolts.

Mtr electron transfer assay

Mtb mtr ORF (Rv3198A) was cloned into pET28b (Novagen) and expressed in E. coli strain BL21 DE3. To perform Mtr electron transfer assay, a mixture of 2.5μΜ purified Mtr, 250μΜ MSSM and 500μΜ NADPH was prepared in 50mM HEPES pH 8.0 in a 96-well plate. In the control reaction, Mtr was absent. The mixture was incubated at 37°C and consumption of NADPH was monitored at 340nm for 60min. To check the specificity of Mrxl -roGFP2 fusion protein towards MSH, Mtr assay mixture was prepared and Mrxl -roGFP2 fusion protein (Ι Μ) wasadded after incubation at 37°C for 30min. Radiometric sensor response was monitored for 200min. A control reaction without MSSM was included.

Sensitivity of Mrxl-roGFP2 towards changes in OXDMSH

Pre-reduced uncoupled roGFP2 and Mrxl-roGFP2 (Ι μΜ) were incubated with mycothiol solutions (ImM total) containing increasing fractions of MSSM. The total concentration of MSH (MSH _totai) refers to MSH equivalents i.e. MSH ,₀₍₃ι= [MSH] + 2[MSSM]. OXDMSH is the fraction of MSH total that exists as [MSSM] and can be calculated using the following formula: Reduced from of mycothiol (MSH) was obtained by reducing MSSM with immobilized TCEP disulfide reducing gel (Thermo Scientific) under anaerobic conditions as per manufacturer's instructions.

Preparation οί Μώ cells for EMSH measurements by Flow Cytometry.

Downstream imaging analysis of the BSL3 class pathogens such as Mtb requires their chemical fixation by paraformaldehyde (PFA). However, PFA leads to oxidation of the biosensor. Therefore, the thiols of the Mrxl -roGFP2 fusion construct were alkylated using the cell permeable fast-acting thiol-modifier, N-ethyl maleimide (NEM) that prevents oxidation during PFA-fixation of Mtb cells To study EMSH of Mtb in vitro or ex vivo, Mtb grown in 7H9 or Mtb infected macrophages were washed with I xPBS and treated with l OmM NEM for 5min at room temperature followed by fixation with 4% PFA for 15min at room temperature. After washing thrice with PBS, bacilli or cells were scraped and analyzed using a BD FACS Verse Flow cytometer (BD Biosciences). Since the flow cytometric based measurements are dependent on fixed wavelength lasers, the Mrx l -roGFP2 biosensor was excited with the canonical 405 and 488nm laser wavelengths at a fixed emission wavelength of 510nm. The ratio of emission (510/10nm) after excitation at 405 and 488nm was calculated. Data was analyzed using the FACSuite software. Confocal microscopy

Infected THP- 1 cells were treated with l OmM NEM, fixed with 4% PFA and

permeabilized with 0.2% (w/v) Triton X- 100 in PBS for 20min. Cells were blocked with 3% (w/v) BSA and 0.5% Tween 20 in PBS for lh. For marking early endosomes, cells were stained with EEA 1 (Santa Cruz). For staining lysosomes, prior to fixing, the cells were pre-treated for l h with lysotracker (Invitrogen, Ι ΟΟηΜ). For autophagosomes, prior to fixing, cells were pre-treated with E64d and pepstatin A (Sigma, 10μg/ml each) followed by staining withprimary LC3 antibody (Cell Signaling Technology). For EEA1 and LC3 staining,cells were further stained with secondary (Alexafluor 568) antibody for lh. Ratiometric fluorescence response in Mtb co- localized within sub-vascular compartments was measured using confocal microscopy as follows. The coverslips were washed thoroughly with PBS and mounted onto glass slides with mounting media (Antifade reagent, Invitrogen). Seven fields were acquired randomly from each set with a Nikon Eclipse Ti-E laser-scanning confocal microscope equipped with a 60X/1.4 numerical aperture oil Plan-Apochromat differential interference contrast objective lens using the blue diode laser (excitation at 408nm and emission at 500/530nm), argon laser (excitation at 488nm and emission at 500/530nm) and helium neon laser (excitation at 543nm and emission at 567/642nm). Images were saved as 16-bit TIF files and analyzed by Image J software

(http://rsb.info.nih.gov/ij/). After subtracting the background, images were converted to 32-bit format. The intensities of the 488nm images were threshold and ratio images were created by dividing the 405nm image by the 488nm image pixel by pixel and displayed in false colors using the lookup table "Fire".

Propidium iodide (Pi) staining

For Pi staining, redox state of bacilli was first blocked by treatment with NEM followed by macrophage lysis by PBS containing 0.01 % SDS. Released bacilli were stained with Ι ΟμΜ propidium iodide (Pi) for 15min at room temperature in dark. After washing twice with l x PBS, bacilli were fixed in PFA, washed again with PBS and analyzed by flow cytometer. Mycothiol-specific fluorescent biosensor

This disclosure describes a new class of biosensor proteins (Mrx 1 -roGFP2) that allow non-invasive, real-time imaging of dynamic changes in the intra-cellular mycothiol redox potential (EMSH) with an unprecedented sensitivity and specificity. The biosensor includes a fusion construct having an engineered green fluorescent protein that has a fluorescence spectrum sensitive to redox status (roGFP2) coupled to a mycothiol-dependent oxidoreductase (m coredoxin; Mrx 1 ) isolated from Mtb. Thus, the invention has led to a complete alteration in the specificity and sensitivity of roGFP2 by coupling its oxidation and reduction to the mycothiol redox couple via fusion of roGFP2 with a mycothiol-dependent oxidoreductase. Also, the roGFP2 based sensors of the present invention have been endowed with a novel property of ^"* exclusively responding to perturbations in mycothiol redox potential (EMSH)- The engineered green fluorescent protein is a redox-sensitive green fluorescent protein (roGFP2) that ratiometrically changes fluorescence in response to defined redox conditions. The roGFP2 has two cysteines on either sides of a chromophore. The oxidation of the two cysteines generates a disulfide bond which leads to an increase in fluorescence intensity at ~400nm with a peak at ~490nm, while reduction of the two cysteines reverses the fluorescence spectrum. The ratio of fluorescence intensity at ~400nm and ~490nm (hereinafter referred to as the 400/490nm ratio or 400/490nm excitation ratio) indicates redox state of a cell/compartment in which the roGFP2 is expressed. A change in the 400/490 ratio displayed by the roGFP2 indicates a change in the redox state of the cell/compartment in which the roGFP2 is expressed (as described in the article titled 'Investigating mitochondrial redox potential with redox-sensitive green fluorescent protein indicators' published in 'The Journal of biological chemistry', Volume 279, Pages 13044- 13053 by Hanson et. al. in 2004. This article is incorporated herein as reference).

roGFP2 has been used in the past as a fluorescent partner of biosensors because it exhibits a large dynamic range (i.e. it exhibits maximum and minimum 400/490 excitation ratios at 100% and 0% oxidation, respectively), is brighter, pH insensitive, and is resistant to photoswitching. Further, since roGFP2 exhibits a ratiometric response, it eliminates errors due to variations in roGFP2 concentrations during different growth phases of an organism it is incorporated into. Moreover, the roGFP2 can measure the redox status of a cell/compartment in which it is expressed in real-time. Unlike conventional methods, there is no need to disrupt the cell/compartment and hence the measurement is non-invasive.

However, the specificity of roGFP2 based biosensors towards the glutathione redox couple render them ineffective in non-glutathione producers like gram positive bacteria (such as Bacillus species, Staphylococcus aureus and Deinococcus radiodurans) and actinomycetes. In other words, roGFP2-based biosensors were unable to detect the mycothiol redox couple. After much experimentation, the present group of inventors re-designed the roGFP2 to specifically measure mycothiol redox potential (EMS ) through interaction with a mycothiol-dependent oxidoreductase. An oxidoreductase enzyme, mycoredoxin- 1 (Mrx l ) identified from the genome of Mycobacterium tuberculosis can specifically interact with the mycothiol redox pair (MSH/MSSM). The Mrx l was coupled to the roGFP2 to generate an Mrx l -roGFP2 fusion construct, such that real-time equilibration between MSH/MSSM redox pair and roGFP2 could be facilitated by Mrx 1.

There is illustrated in Fig. 1 , a molecular mechanism of MSSM sensing in an Mrxl- roGFP2 expressing bacteria. When the bacteria, such as Mycobacterium tuberculosis, experiences oxidative stress, a nucleophilic cysteine of Mrx 1 specifically reacts with MSSM to generate a mixed Mrxl-MSSM intermediate. The Mrxl-MSSM interacts with one of the two proximal Cys thiols on roGFP2 and converts it into S-mycothionylatedroGFP2. In the final step, S-mycothionylatedroGFP2 re-arranges to form an intermolecular disulfide bond that results in an oxidative shift in £ s//leading to a change in the 400/490nm excitation ratio. Once oxidative stress depletes, EMSH normalizes predominantly via reduction of MSSM to MSH by action of MSH disulfide reductase (Mtr) enzyme. Thus, the Mrxl-roGFP2 fusion construct acts as a redox biosensor that allows dynamic real-time imaging of mycothiol redox potential {E_MSH) in diverse mycobacterial species and strains including drug-resistant clinical isolates under physiologically relevant conditions. In addition to mycobacteria, many other actinomycetes including, but not limited to, Corynebacterium, Streptomyces, Nocardia and Rhodococcus, also produce mycothiol and thus the Mrxl-roGFP2 fusion construct can be used in these bacteria to track their redox state.

In an embodiment of the disclosure, the mycothiol-dependent oxidoreductase has an N- terminus Cysteine residue in its catalytic site. In another embodiment, the catalytic motif is one of a CGYC and a CGYA catalytic motif.

In an exemplary embodiment, coding sequence of the mycothiol-dependent oxidoreductase may be fused to N-terminus of the roGFP2 via a 30-amino acid linker, (GGSGG)₆ to obtain the Mrxl -roGFP2 fusion construct.

In another embodiment, the Mrx 1 may be identified from the genome of Mycobacterium tuberculosis strain H37Rv. In yet another embodiment, the Mrxl may be encoded by gene

Rv3198A of Mycobacterium tuberculosis strain H37Rv. In still another embodiment, the Mrxl may have the sequence depicted in Sequence ID No. 1.

In an embodiment of the disclosure, the nucleic acid sequence encoding the fusion construct Mrxl-roGFP2can be introduced in a suitable vector by methods known in the art. Examples of such a vector can include, but are not limited to, E.co//-mycobacteria shuttle vectors such as pMV762, pMV 761, pCV125, and pTetRO). The vector containing the nucleic acid sequence encoding the Mrxl-roGFP2can then be introduced into a host cell. Examples of such a host cell can include, but are not limited to, E.coli, Mycobacterium, Streptomyces, Corynebacterium, Nocardia, and Rhodococcus.

Provided below are details of the experiments conducted to design the mycothiol-specific fluorescent biosensor and establish its sensitivity and specificity.

Experiment 1 for designing of Mycothiol-specific fluorescent biosensor

Using in silico approaches, a mycothiol-dependent oxidoreductase with a CGYC catalytic motif (Rv3198A; mycoredoxin 1 [Mrxl ]) was identified in the genome of Mycobacterium tuberculosis {Mtb). Tor this, a homology based analysis was performed using the sequence of enzyme mycoredoxin-1 reported in a non-pathogenic saprophytic mycobacteria, Mycobacterium smegmatis {Msm). The mycoredoxin-1 from Msm exclusively interacts with the mycothiol redox couple (as described in the article titled 'Mycoredoxin- 1 is one of the missing links in the oxidative stress defense mechanism of Mycobacteria' published in 'Molecular Microbiology', Volume 86, Pages 787-804 by Van Laer et. al. in 2012. This article is incorporated herein as reference).The homology based analysis revealed three putative homologues of mycoredoxin-1 in Mtb namely, Rv3053c, Rv0508, and Rv3198A. Of the three, Rv3198A demonstrated the highest similarity with the mycoredoxin-1 from Msm (72% identity) as compared to Rv3053c and Rv0508. Therefore,Rv3198A open reading frame (ORF) was used as a putative

mycoredoxin-encoding gene and its product was named as Mrxl . Mrxl coding sequence was amplified from Mtb genome using a forward primer (5' ATGCCCATGGTGATCACCGCTGCG 3') and a reverse primer (5' ATGCACTAGTACCCGCGATCTTTAC 3')· The coding sequence of Mrxl has been depicted in Sequence ID No. 1. The Mrxl contained an active site (CGYC) similar to the mycoredoxin-1 from sT??.

Following this, coding sequence of the Mrxl was fused to N-terminus of the roGFP2 via a 30-amino acid linker, (GGSGG)₆ to obtain the Mrxl-roGFP2 fusion construct.

Experiment 2 for determining redox responsiveness of Mrxl-roGFP2 To determine the redox responsiveness of the Mrxl -roGFP2 fusion construct, the following experiments were carried out.

In a first sub-experiment, the Mrx 1 -roGFP2 fusion construct was purified using a Ni- NTA affinity column under aerobic conditions. Under the aerobic conditions, the Mr l-roGFP2 fusion construct was found to be in a fully oxidized state. Subsequently, 1 μΜ of the aerobically purified Mrxl-roGFP2 fusion construct was reduced using lOmM DTT. Thereafter, an excitation scan (390 to 490nm) was performed for oxidized and reduced Mrxl -roGFP2 fusion construct using a spectrophotometer (at 510nm) and the 390/490 excitation ratiowas plotted against time. As can be seen in Fig. 2, there was an increase in the relative fluorescence units (RFU)at ~ 390nm and a concomitant decrease at ~ 490nm in the oxidized Mrxl -roGFP2 fusion construct, whereas a reverse spectrum was obtained in the reduced Mrxl-roGFP2 fusion construct.

Findings from the above experiment thus show that the Mrx l-roGFP2 fusion construct displays ratiometric changes upon oxidation or reduction. Experiment 3 for determining specificity of Mrxl-roGFP2

To examine whether fusion of Mrxl with uncoupled roGFP2 provides the biosensor of the present disclosure with specificity to sense oxidized mycothiol (MSSM), the following experiment was carried out.

In a first sub-experiment, uncoupled roGFP2 and the Mrxl -roGFP2 fusion construct were fully reduced using l OmM DTT. In a first sub-experiment, the reduced uncoupled roGFP2 and the reduced Mrxl-roGFP2 fusion construct were incubated with mycothiol solutions (ImM total) containing increasing fractions of MSSM for 30 seconds (sec) and ratiometric sensor response (390/490 ratio) was measured. As can be seen in Fig. 3, the response of Mrxl-roGFP2 became exceedingly linear in the window between 10% to 90% oxidation, suggesting that the biosensor can effectively measure changes in EMSH within this range of probe oxidation. Further, a small increase in MSSM led to a significant increase in biosensor oxidation. For example, an increase in the amount of mycothiol oxidation (OXDMSH) from 0.00001 to 0.0001 (that corresponds to ~100nM increase in absolute MSSM) led to a larger increase in the biosensor oxidation (from -40% to 90%). These results confirmed that Mrxl -roGFP2 is capable of rapidly sensing nanomolar changes in MSSM against the backdrop of a highly reduced MSH pool (ImM). Further, it was seen that uncoupled roGFP2 remained completely non-responsive to changes in MSH/MSSM ratios.

In a second sub-experiment, different aliquots of the reduced Mrxl -roGFP2 fusion construct were each treated with ΙμΜ each of MSSM, GSSG, cystine (Cys₂) and 2-hydroxyethyl disulfide (HED) and ratiometric fluorescence response (390/490 ratio) was plotted against time. As can be seen in Fig. 4, response of the Mrxl-roGFP2 fusion construct was undetected towards non-specific oxidants such as cystine {Cysi), GSSG, and 2-hydroxyethyl disulfide (HED) but could be detected towards MSSM, thereby confirming specificity of the biosensor towards MSSM.

In a third sub-experiment, the response of Mrxl-roGFP2 towards reduced mycothiol

(MSH) was examined. To continuously maintain a reduced state of MSH, MSH disulfide reductase enzyme (Mtr) was used that is known to catalyze NADPH-dependent reduction of MSSM to MSH in mycobacterial cells. As a first step, the activity of Mtr was confirmed by monitoring NADPH oxidation in the presence of MSSM by tracking the rate of NADPH oxidation to NADP⁺ (at 340nm). As seen in Fig. 5, a time-dependent decrease in 340nm absorption due to NADPH consumption was observed which confirmed cycling of electrons from NADPH to MSSM by Mtr. Thereafter, oxidized Mrxl -roGFP2 was added as a substrate for MSH/Mtr NADPH electron transfer assay and ratiometric sensor response (390/490 ratio) was measured over time. As depicted in Fig. 6, a time-dependent decrease in 390/490 excitation ratio was observed which confirmed the reduction of Mrxl -roGFP2 by MSH. Further, no response was observed if MSH was omitted from the Mtr/NADPH/Mrxl -roGFP2 mixture.

In addition to MSH, mycobacteria are known to express several anti-oxidant systems that help in dissipating the redox stress. For example, mycobacteria express NADPH-dependent TRX system to efficiently counter oxidative stress (as described in the article titled 'The alternative sigma factor SigH regulates major components of oxidative and heat stress responses in

Mycobacterium tuberculosis' published in 'Journal of Bacteriology', Volume 183, Pages 61 19- 6125 by Raman et. al. in 2001. This article is incorporated herein as reference). Thus,

experiments were conducted to establish that the Mrx l-roGFP2 fusion construct was specific towards redox changes mediated by the MSH redox couple and not by other anti-oxidant systems present in mycobacteria. For this, Mrxl-roGFP2 fusion construct was expressed in wild-type Msm and three different mutant strains of Msm, namely MsmAmshA (MSH-negative strain lacking the mycothiol redox couple), MsmAmshD (MSH-depleted strain having low levels of mycothiol redox couple) and MsmAsigH (Trx-repressed strain having an intact mycothiol redox couple). Different samples of the cells were oxidized using diamide and reduced using DTT. Ratiometric fluorescence response was measured for untreated, oxidized and reduced cells.

Percent oxidation was calculated as the untreated 390/490 ratio relative to oxidized as 100% (diamide treated) and reduced (DTT treated) as 0% oxidized. P-values were also calculated by independently comparing MsmAmshA and MsmAmshD groups with the Msm group. As can be seen in Fig. 7, oxidation of the biosensor was nearly quantitative (~95%) in MsmAmshA as compared to -20% in both wild-type Msm and MsmAsigH, and -50% in MsmAmshD. These findings rule out the interaction of the Mrx 1 -roGFP2 fusion construct with the TRX system inside mycobacteria and establish that the biosensor specifically interacts with the mycothiol redox couple.

The findings from the above experiments clearly establish that the Mrxl-roGFP2 fusion construct is specific for the MSH redox couple and does not respond to other anti-oxidant systems prevalent in mycobacteria. Thus, the biosensor of the present disclosure can selectively track changes in the mycothiol redox couple.

Experiment 4 for establishing the use of the biosensorfor measurement of EMSH

The following experiments were performed to establish the applicability of the biosensor of the present disclosure for measurement of redox potential of various strains of mycobacteria.

In a first sub-experiment, the Mrxl -roGFP2 fusion construct was used for measurement of EMSH in various strains of Mycobacterium smegmatis(Msm), namely wild-type Msm,

MsmAmshA, MsmAmshD, and MsmAsigH, using the Nernst equation. More details on this step are provided in the methods section. EMSH in wildtype Msm, MsmAmshA, MsmAmshD, and MsmAsigH was found to be -300±2mV, -239±7mV, -275±7mV, and -300±3mV, respectively.

In a second sub-experiment, the basal redox potential differences between various slow growing strains of Mtb were determined using the Mrx-l-roGFP2 fusion construct. As a first step, the Mrxl-roGFP2 fusion construct was expressed in various slow growing lab-adapted and clinical mycobacterial strains, namely, a vaccine strain (M. bovis BCG), a virulent laboratory strain (Mtb H37Rv), and several Indian clinical isolates of Mtb including a single-drug resistant strain (BND 320), multi-drug resistant strains (Jal 2261 , Jal 1934 and Jal 2287), and an extensively-drug resistant strain (MYC 431) for EMSH measurements using Flow Cytometry. More details on this step are provided in the methods section. The resulting data (provided in table 1 below) indicated that there is relatively little variation in the redox state within and between drug-resistant clinical and drug-sensitive laboratory (Mtb H37Rv, M bovis BCG) strains, as exemplified by E^sw alues around -273mV to -280mV.

Table 1 .Drug-resistance patterns and E_MSH for H37Rv and different field isolates. In a third sub-experiment, the Mrxl-roGFP2 fusion construct was used to quantify redox changes that occur during physiological challenges in the natural context of infection such as intra-macrophage environment. For this, the Mrxl-roGFP2 fusion construct was expressed in Mtb H37Rv and the cells were used to infect THP-1 cells differentiated into macrophages by PMA treatment at multiplicity of infection (MOI) of 10. Thereafter, EMSH of the mycobacterial cells was monitored using NEM-PFA based fixation technique followed by ratiometric fluorescence analysis by flow cytometry. To measure changes in EMSH during infection, an in vitro redox calibration curve was generated by treating H37Rv with buffers of known redox potentials (Fig. 8). For this, Mtb H37Rv expressing the Mrxl-roGFP2 fusion construct was treated with l OmM DTT (for 100% Mrxl -roGFP2 reduction), ImM cumene hydroperoxide (CHP) (for 100 % Mrxl-roGP2 oxidation) and DTT_red:DTT_oxd solutions (final concentration of DTT_red+DTTox_d > lOmM in PBS) that had the redox potentials ranging from -330 to -195mV. The resulting change in the Mrxl-roGFP2 ratios were normalized to the ratio with l OmM DTT_red giving 0% oxidation and ratio with l mM CHP giving 100% oxidation. Apparent redox potential values were determined by plotting average Mrx 1 -roGFP2 ratios versus the equivalent redox potential values and fitting the data to a titration curve. Thus, EMSH oiMtb inside macrophages was precisely calculated. More details on various steps of this experiment are provided in the methods section.

Thus, the biosensor of the present disclosure can efficiently measure EMSH of various species and strains of mycobacteria including slow-growing strains. Further, the biosensor can effectively measure redox potential under physiologically challenging conditions such as intra- macrophage environment.

Experiment 5 for determining importance of N-terminus Cysteine residue in the catalytic site of Mrxl

Two cysteines present in catalytic site of a wild-type Mrxl were independently replaced by alanines to generate two variants, Mrxl (CGYA) and Mrxl (AGYC). Each of the two variants wasfused to N-termini of roGFP2 proteins to obtain Mr l(CGYA)-roGFP2, and Mrxl (AGYC)- roGFP2 fusion constructs.

Uncoupled roGFP2 and the three fusions constructs (Mrxl -roGFP2, Mrxl (CGYA)- roGFP2, and Mrxl(AGYC)-roGFP2) were fully reduced using l OmM DTT. Following this, they were exposed to 50μΜ of MSSM for lOmin and ratiometric fluorescence response (390/490 ratio) was measured. As can be seen in Fig. 9, 390/490 ratios of the Mrx l-roGFP2 fusion construct and the Mrxl(CGYA)-roGFP2 fusion construct increased upon addition of MSSM whereas uncoupled roGFP2 and the Mrxl(AGYC)-roGFP2 fusion construct remained non- responsive.

The response of Mrxl(CGYA)-roGFP2, and Mrxl (AGYC)-roGFP2 towards reduced mycothiol (MSH) was also examined in an experiment similar to the third sub-experiment of Experiment 3 described above. The results indicated that no response was observed if Mrxl (AGYC)-roGFP2 was used as the substrate whereas a response was readily detected in the case of Mrxl (CGYA)-roGFP2.

Based on these findings, it can be concluded that N-terminus Cysteine residue in the catalytic site of Mrxl is essential for this function. Experiment 6 for determining sensitivity of Mrxl-roGFP2

To examine whether fusion of the Mrxl with roGFP2 provides the biosensor of the present disclosure with enhanced sensitivity towards transient changes in the mycothiol redox status the following experiments were carried out.

In a first sub-experiment, the Mrxl -roGFP2 fusion construct was reduced using DTT. Thereafter, Ι μ of the reduced Mrxl-roGFP2 fusion construct was exposed to different concentrations (0.5, 1 and 5mM) of H₂0₂m vitro. Ratiometric fluorescence response (390/490 ratio) was measured after 60sec. No significant change in the 390/490 ratio for the Mrxl-roGFP2 fusion construct exposed to H₂0₂ was observed. Thus, it can be concluded that H₂0₂ alone cannot oxidize the Mrxl-roGFP2 fusion construct/^ vitro.

In a second sub-experiment, the Mrx 1 -roGFP2 fusion construct was expressed in cells of Msm. The cells were then treated with varying concentrations (0.1 to lOmM) of H₂0₂for 2min and ratiometric fluorescence response (390/490 ratio) was measured. As seen in Fig. 10, application of H₂0₂ to cells of Msm expressing the Mrxl -roGFP2 fusion construct led to oxidation of the biosensor within Msm. Combined with results from the first sub-experiment, it can be concluded that that oxidation of MSH to MSSM is necessary for oxidation of the biosensor.

In a third sub-experiment, cells of Msm expressing the Mrx l -roGFP2 fusion construct were separately treated with 250μΜ of three different oxidants, namely diamide, aldrithiol, and menadione and ratiometric fluorescence response (390/490 ratio) was measured. As can be seen in Fig. 1 1 , the 390/490 ratio of the Mrx 1 -roGFP2 fusion construct increased upon exposureto the different oxidants.

In a fourth sub-experiment, Msm cells expressing the Mrx 1 -roGFP2 fusion construct were separately exposed to lower concentrations of H₂0₂ (such as Ι ΟΟμΜ, 500μΜ and ImM). The ratiometric fluorescence response (390/490 ratio) was measured and plotted against time. As can be seen in Fig. 12, exposure to lower concentrations of H₂0₂ resulted in a rapid, but shortlived (~5min) increase in the 390/490 excitation ratios. Thus, it can be concluded that Msm has an efficient mobilization of anti-oxidant response mechanisms. A similar experiment was performed using Msm cells expressing uncoupled roGFP2, the Mrx l (AGYC)-roGFP2 fusion construct and the Mr l(CGYA)-roGFP2 fusion construct. A rapid short-lived increase in the excitation ratio was detected in the Mrxl (CGYA)-roGFP2 fusion construct but not in uncoupled roGFP2 or the Mrxl(AGYC)-roGFP2 fusion construct. Thus, it can be concluded that Mrxl promotes a rapid and reversible equilibration of the biosensor of the present disclosure with intracellular MSH/MSSM redox buffer.

Results from the above experiments prove that the biosensor of the present disclosure is highly sensitive in detecting changes in the mycothiol redox potential. Combined with findings from the first sub-experiment of Experiment 3, it can be concluded that the biosensor is capable of rapidly sensing nanomolar changes in MSSM concentration.

Findings from the experiments 2-6 clearly demonstrate that the Mrxl-roGFP2 fusion construct can be used as a biosensor for detecting redox changes in mycothiol redox couple and sensing intracellular EMS W^' various mycobacterial species and strains. Further, the biosensor can measure EMSH under diverse physiological conditions. The coupling of the roGFP2 with the Mrx- 1 ensures that the roGFP2 functions as a substrate for the Mrx- 1 and dynamically oxidizes and reduces in response to EMSH- Further, the biosensor is specific towards MSH/MSSM redox couple and has enhanced sensitivity towards transient changes in mycothiol redox status It was also concluded that N-terminus Cysteine residue in the catalytic site of Mrxl is essential for this function. Detection of analytes using mycothiol-specific fluorescent biosensor

In experiments 2-6, it was seen that the biosensor of the present disclosure can detect redox changes in mycothiol redox couple triggered by various oxidizing and reducing

agents. Thus, it was proposed that the biosensor may be used for identification of analytes that trigger a change in the redox status of mycothiol in a cell. The analytes that trigger a change in the redox status of mycothiol in a cell can include, but are not limited to, drugs, antibodies, inhibitors and chemical compounds.

Provided below are details of the experiments conducted to establish the application of the biosensor for identification of relevant analytes. Experiment 7 for establishing the application of Mrxl-roGFP2 in detecting analytes that trigger a change in redox status of mycothiol

In a first sub-experiment, cells of Msm expressing the Mrxl-roGFP2 fusion construct were exposed to an oxidant (0.4mM of diamide) and a reductant (l OmM of DTT) for 5min and an excitation scan (390 to 490nm) was performed using a spectrophotometer (at 515nm). Results indicated that the Mr l -roGFP2 fusion construct responded ratiometrically upon exposure to diamide or DTT.

In a second sub-experiment, cells of Msm expressing theMrxl-roGFP2 fusion construct were exposed to three drug molecules, namely, dequalinium (a redox-drug), cisplatin (an inhibitor of Trx reductase) and 5-methoxyindole-2-carboxylic acid (MICA; an inhibitor of dihydrolipoamide dehydrogenase) at a concentration of

response (390/490 ratio) was measured after 2h and 24h post exposure. Of the three drug molecules, dequalinium is a well-established small-molecule inhibitor of MSH ligase while the remaining two drugs do not affect the cellular MSH pool. As expected, only dequalinium treatment led to a substantial time-dependent increase in the 390/490 ratio (Fig. 13). Fig. 13also depicts Pvalues calculated by comparing untreated group and dequalinium treated group.

Based on the findings from the above experiments, it can be concluded that the Mrxl - roGFP2 fusion constructemits a fluorescence response when exposed to molecules that perturb the redox status of mycothiol in a cell. Thus, biosensor of the present disclosure can be used in detecting analytes that trigger a change in the redox status of mycothiol in a cell. Further, the biosensor of the present disclosure can be used to differentiate analytes that trigger a change in the redox status of mycothiol in a cell from analytes that do not trigger such a change.

Experiment 8 for establishing the applicability of Mrxl-roGFP2 in detecting analytes that trigger a change in redox status of mycothiol under varied physiological conditions

Immunologically activated murine macrophages (RAW 264.7), known to control mycobacterial proliferation by producing ROS and RNS, were infected with Mtb H37Rv expressing the Mrxl-roGFP2 fusion construct to establish the applicability of the biosensor for detecting analytes under varied physiological conditions such as oxidant-mediated anti- mycobacterial stresses. RAW 264.7 macrophages were activated with IFN-γ and LPS prior to infection with Mtb H37Rv at MOI of 10. As evident from Fig. 14, a significant and sustained oxidative . shift in EMSH of Mtb H37Rv inside activated macrophages as compared to naive macrophageswas observed. Further, it is known that nitric oxide (NO), generated via inducible NO synthase (iNOS) pathway, is one of the major contributors of redox stress in Mtb inside immune-activated murine macrophages (as decribed in Chan et al. in 1992 in the article titled 'Killing of virulent Mycobacterium tuberculosis by reactive nitrogen intermediates produced by activated murine macrophages' published in 'Journal of Experimental Medicine', Volume 175, Pages 1 1 1 1 -1 122. This article is incorporated herein as reference). Therefore, the IFN-y/LPS activated RAW 264.7 macrophages were treated with a well established iNOS inhibitor N°- methyl-L-arginine (NMLA) and intramycobacterial EMSH was measured. Results showed that there was a substantial reduction in EA_/sy/following treatment with NMLA (Fig. 15). Thus, it was concluded that the Mrxl-roGFP2 fusion construct was very effective in dissecting redox signaling during infection. These results clearly demonstrate that the Mrxl -roGFP2 fusion construct can effectively detect analytes that trigger a change in the mycothiol redox status of a cell under varied physiological conditions.

Findings from the above experiments establish the applicability of the biosensor of the present disclosure in detecting and differentiating analytes that trigger a change in the redox status of mycothiol in a cell. In an embodiment, detection and/or identification of the analytes may be undertaken by inserting the biosensor into a cell having mycothiol redox couple, for example Mtb. Thereafter, an array of such cells may be prepared and each cell of the array may be separately incubated with an analyte from a plurality of analytes. The incubation may be done for a defined time period that is sufficient to allow the analyte of interest to cause the change in the redox status of the cell.

In an embodiment, the plurality of analytes may be those that need to be screened and differentiated based on their capability to trigger a change in the redox status of the cell. Based on the detection of a fluorescence response indicated by the biosensor incorporated in each cell of the array the relevant analyte can be identified. For example, in a scenario where the cell is incubated with such an analyte that can trigger a change in the redox status of the fusion . ! construct, fluorescence response will be detected, whereas when any other analyte is used, no fluorescence response will be detected.

Further, in an embodiment, a pre-defined intensity of the fluorescence response may be indicative of an effectiveness of the analyte on the cell comprising mycothiol redox couple.

In another embodiment, this method of detection of analytes using mycothiol-specific fluorescent biosensor may be a high throughput screening method.

Detection of cells using mycothiol-specific fluorescent biosensor

In the above experiments, it was seen that the biosensor of the present disclosure can be expressed in cells containing mycothiol redox couple. Further, the biosensor emits a response when there is a redox change in the mycothiol redox status of the cell. Further, results of Experiment 3 establish that the Mrxl-roGFP2 fusion construct can be used in detecting cells with varying levels of mycothiol redox couple. Cells having mycothiol redox couple can interact with the biosensor of the disclosure which may then emit a fluorescence response. Thus, the biosensor may enable differentiation between cells having mycothiol redox couple and those that do not have mycothiol redox couple.

In an embodiment, the cell having mycothiol redox couple may be a member of the taxa actinomycetes including, but not limited to, Mycobacterium, Corynebacterium, Streptomyces, Nocardia and Rhodococcus. In a preferred embodiment, the cell having mycothiol redox couple can be a strain of Mycobacterium tuberculosis.

In an embodiment, detection of cells having mycothiol redox couple in a sample to be to be tested for presence of the cells may be undertaken by inserting the biosensor into a moiety capable of reacting with the cells having mycothiol redox couple. Examples of such a moiety can include, but are not limited to, bacteriophages and mycobacteriophages. Thereafter, the moiety may be incubated with a sample which may or may not contain the cells having mycothiol redox couple. The incubation may be done for a defined time period that is sufficient, to allow the moiety to interact with the cells of interest. For example, in a scenario where the moiety is a bacteriophage or a mycobacteriophage, incubation may be done for a time period that is sufficient to allow the bacteriophage or the mycobacteriophage to infect the cells and transfer the biosensor to the infected cells. Based on the detection of a fluorescence response indicated by the biosensor incorporated in the moiety, it can be determined whether or not the sample contains the cells of interest. For example, in a scenario where the moiety is incubated with a sample containing the cells of interest, the mycothiol redox couple will interact with the biosensor and a fluorescence response will be detected, whereas when the cells of interest are not present, no fluorescence response will be detected.

In an embodiment, the sample may be a biological sample. Examples of which may include, but are not limited to, a blood sample, a serum sample, a urine sample, a fecal sample, a tissue biopsy, a cerebrospinal fluid sample, an ascites sample, a pleural fluid sample, respiratory secretions, a saliva sample, and a sputum sample.

In another embodiment of the disclosure, the moiety containing the biosensor may be incorporated into a kit for detecting cells having mycothiol redox couple in a sample.

In yet another embodiment, detection of cells having mycothiol redox couple may be undertaken by subjecting the sample to a processing system that causes lysis of cells present in the sample. Examples of such a processing system, can include, but are not limited to, an enzymatic treatment, a chemical treatment such as detergent treatment, and a physical treatment such as sonication. Thereafter, the lysed sample may be incubated with the biosensor for a defined time period that is sufficient to allow the biosensor to interact with the lysed cells and their constituents. For example, in a scenario where the sample contains cells having mycothiol redox couple, cells lysis may cause release of cellular constituents such as mycothiol redox couple. The lysed sample may then be incubated with the biosensor for a time period that is sufficient to allow the biosensor to react with the released mycothiol redox couple and emit a fluorescence response. Based on the detection of a fluorescence response indicated by the biosensor, it can be determined whether or not the sample contains the cells of interest. For example, in a scenario where the biosensor is incubated with a sample containing the cells of interest, the mycothiol redox couple will interact with the biosensor and a fluorescence response will be detected, whereas when the cells of interest are not present, no fluorescence response will be detected. In yet another embodiment of the disclosure, the processing system for lysing cells present in the sample and the biosensor may be incorporated into a kit for detecting cells having mycothiol redox couple in a sample. Detection of drug resistance in cells using mycothiol-specific fluorescent biosensor

It is known from recent studies that intracellular Mtb exists in different vacuolar compartments, which may contribute to significant heterogeneity in intrabacterial gene expression, metabolic state and survival (as described - in the article titled 'Linking the transcriptional profiles and the physiological states of Mycobacterium tuberculosis during an extended intracellular infection' published in 'PLoS pathogens', Volume 8, Page el 002769 by Rohde et. al. in 20,12. This article is incorporated herein as reference).

It has also been established that susceptibility or resistance to antibiotic-induced cell death is dependent on the oxido-reductive state of a cell (as described by Dwyer et. al. in 2009 in the article titled 'Role of reactive oxygen species in antibiotic action and resistance' published in 'Current opinion in microbiology', Volume 12, Pages 482-489. This article is incorporated herein as reference). Further, it is known that the intramacrophage environment creates variability in mycobacterial cells to generate drug tolerant sub-populations (as described by Adams et. al in 201 1 in the article titled 'Drug tolerance in replicating mycobacteria mediated by a macrophage-induced efflux mechanism' published in 'Cell', Volume 145, Pages 39-53. This article is incorporated herein as reference). Based on the above, it was proposedthat macrophages and other vacuolar compartments promote heterogeneity in the redox status of Mtb cells. Further, it was proposed that the redox heterogeneity creates phenotypic variants of Mtb cells that have varying degrees of resistance to anti-TB drugs and heterogeneity in intrabacterial EMS may be one of the factors that underlies emergence of Mtb populations with differential antibiotic susceptibility.

Provided below are details of the experiments conducted to support the above.

Experiment 9 for showing that macrophages induce redox heterogeneity within Mtb populations In Experiment 4, the Mrxl -roGFP2 fusion construct was used to quantify redox changes that occur during physiological challenges, such as intra-macrophage environment using the flow cytometry method. PMA-differentiated THP-1 cells were infected with Mtb H37Rv expressing the Mrxl-roGFP2 fusion construct at MOI of 10 and ~ 30,000 cells were analyzed by flow cytometry by exciting at 405 and 488nm lasers at a constant emission (5 lOnm). Flow cytometric analyses showed the presence of cells with a gradient of intramycobacterial EMSH ranging from a highly oxidized (>-240mV) to a highly reduced (<-320mV) EMSH- The program BD FACS suite software was used to analyze the population distribution of Mtb, and each population was represented by a unique color. The gradient in redox heterogeneity of Mtb cells inside macrophages was classified into three sub-populations with different EMSH'- an Ewwbasal population with an intermediate EMSH o -275±5mV, and two deflected populations. Deflected cells with a mean EM_SH of -240±3mV represented an E^s^-oxidized sub-population, based on the observation that CHP treatment of infected macrophages resulted in a significant fraction of these gated cells (~98%) while the population with an average EMSH of -300±6mV represented an iiAiOT-reduced sub-population, as treatment of infected macrophages with the DTT resulted in ~96% of the cells gating into this sub-population (Fig. 16-19). Mtb cells present in media alone and analyzed in parallel did not show redox heterogeneity (Fig. 20and 21), suggesting that the intramacrophage environment perturbs redox homeostasis to induce redox variability in Mtb.

Experiment 10 for showing oscillatory changes in redox heterogeneity during infection Having revealed the existence of redox heterogeneity during intramacrophage growth of

Mtb, time-resolved changes in redox heterogeneity among a large number of Mtb cells during infection was examined by flow cytometry.

PMA-differentiated THP-1 cells were infected with Mtb H37Rv expressing the Mrxl- roGFP2 fusion construct at MOI of 10. At pre-defined time intervals (0, 24, 48 and 72h post- infection [p.i.]), the cells were treated with NEM-PFA and -30,000 infected macrophages were analyzed by flow cytometry and intramycobacterial EMSH was measured. The "0"h time point refers to time immediately after initial infection with /6H37Rv for 4h. In a subsequent step, the bacterial sub-populations were gated into sub-populations with basal, pro-oxidizing and pro- reducing EMSH- The percentage of bacilli in each sub-population was calculated and plotted as a bar graph. As can be seen in Fig 22, the initial period (0-24h p.i.) of infection was associated with a gradual increase in cells with reduced EMSH (60±7%) followed by an oxidative shift (25±5%) at 48h p.i. and then a significant recovery from oxidative stress, as revealed by a decrease in the population with oxidized EMSH (7±3%) at 72h p.i. The figure also shows p-values calculated by comparing EMSY/-oxidized populations of resting and activated macrophages (* p<0.01).To further validate this, PMA-differentiated THP-1 cells were infected with Mtb H37Rv expressing the Mrxl -roGFP2 fusion construct at MOI of 1. Similar patterns of time-dependent heterogeneity and oscillations in intramycobacterial EMSH were observed.

A similar experiment was performed for various slow growing mycobacteria including BCG and Indian clinical isolates such as BND 320, Jal 2287, Jal 1934, Jal 2261 and MYC 431 to establish that macrophage environment induces strain-specific variations in redox

heterogeneity. Results showed that heterogeneity in E SH for BND 320 largely followed the reductive-oxidative-reductive oscillatory pattern of H37Rv while distinct redox deviations were displayed by the other strains. For example, Jal 2287, Jal 1934 and Jal 2261 displayed

overrepresentation of the pro-oxidized sub-population at 48h p.i. followed by a poor recovery at 48-72h p.i., as compared to H37Rv. Further, intra-macrophage growth of MYC 431 displayed a loss of redox oscillatory pattern and showed a steady increase in pro-oxidizing sub-population at 48h p.i. Furthermore, infection with BCG showed a continuous decrease in .E^Stf-reduced sub- population with a concomitant increase in E^^-oxidized sub-population over time. Together, these findings suggested that macrophage environment triggers heterogeneity in E_MSH of Mtb. Further, clinical field isolates of Mtb show clear redox variance.

Experiment 11 for showing that distinct sub-vacuolar environments play a role in generating Mtb sub-populations with a gradient of redox potentials

To establish whether trafficking into distinct vacuolar compartments could promote redox heterogeneity in Mtb population, the following experiment was conducted. In a first step, PMA differentiated THP-1 cells were infected with Mrxl -roGFP2 expressing Mtb H37Rv cells and EMSH was monitored using NEM-based fixation technique followed by ratiometric fluorescence analysis using confocal microscopy. Similar to results obtained with flow cytometry, confocal analyses also revealed a gradient in redox heterogeneity which could be classified into EMSH- basal (-277±5mV, 26%), E OT-oxidized (-242±6mV, 23%), and E_W5//-reduced (-304±10mV, 51%)) sub-populations (Fig. 23). Further, Mtb grown in media indicated an over-representation of the cells with uniform EMSH- More details on the use of confocal microscopy can be seen in the methods section. In the next step, intrabacterial EMS_H within early endosomes, lysosomes, and autophagosomes was measured by visualizing co-localization of Mtb H37Rv expressing Mrxl - roGFP2 with compartment specific fluorescent markers at 24h p.i. as detailed in the methods section. It was seen that the majority of Mtb cells within early endosomes were highly reduced (54%) as compared to oxidized (22%) or basal (24%) EMSH- Further, in lysosomes, deflected sub- populations EA 5_/ .oxidizedwere clearly higher in proportion (58%), whereas Ews//-reduced (12%) and Z s_/z-basal (30%) sub-populations were under-represented. Furthermore, 100% of the Mtb population inside autophagosomes displayed maximal oxidative shift in

24). In Fig. 23 and Fig. 24, EMSH of co-localized bacilli ( 50) was calculated and distribution shown in scatter plot that depicts quantification of microscopy data. Each point on the plot represents a bacterium. Bar represents mean values, p-values were calculated by one way ANOVA followed by Tukey's HSD statistical test (* pO.01). Percentage of bacilli in each sub-population is represented as a stacked bar graph.

A similar experiment was conducted for known drug-resistant strains Jal 2287 and MYC 431. It was seen that Jal 2287 displayed redox deviations similar to Mtb H37Rvwhile MYC 431 showed over-representation of sub-populations with pro-oxidizing EMSH within the macrophage and sub- vacuolar compartments at 24h p.i.

Thus, the above results suggested that distinct sub-vacuolar environments play an important role in generating Mtb sub-populations with a gradient of redox potentials.

Experiment 12 for showing that Redox heterogeneity regulates antibiotic tolerance in Mtb Having shown the induction of redox heterogeneity in Mtb population during intra- macrophage residence, the functional consequences of redox heterogeneity with respect to antibiotic tolerance was examined.

In a first sub-experiment, the effect of anti-TB drugs on redox heterogeneity in Mtb cells during intra-rnacrophage residence was characterized. For this, infected macrophages were exposed to four anti-TB drugs with different modes of action, namely, isoniazid [INH; mycolic acid inhibition], ethambutol [EMB; arabinogalactan inhibition], rifampicin [RIF; inhibition of transcription], and clofazimine [CFZ; redox cycling and ROS production] at 5-fold the in vitro MIC(i.e. INH O^g/ml, CFZ 0.5^g/ml, RIF O^g/ml and ETF^g/ml). The redox response was measured by FC at defined time points. Results showed that treatment with all the four antibiotics induced variable levels of oxidative shift in Mtb sub-populations at 12, 24 and 48h p.i. Further, the skew towards oxidizing EMSH was activated at an early time point (12h p.i.) and increased significantly at 48h p.i. (Fig. 25). In a parallel experiment, infected macrophages were lysed and released bacilli were counted by plating cfu to examine if enhanced oxidizing EMS_/ycoirela ed _wj_m the killing potential of anti-TB drugs during infection. It was seen that the Mtb survival rate was comparable to the untreated control at 12h post-antibiotic treatment. However, a modest (~1.5-fold) to a significant reduction (~5-fold) in intramacrophage bacillary load as compared to untreated control was detected at 24 and 48h p.i., respectively. These findings showed that antibiotics with different mechanisms of action induce oxidative changes in intramycobacterial EMSH during infection. To further validate these results, a second sub-experiment was conducted wherein macrophages were infected with two Mtb strains namely BND 320 and Jal 2287 which are known to be INH resistant and CFZ sensitive. Subsequently, the redox heterogeneity in response to INH and CFZ at 48h p.i. was measured using flow cytometry. Results showed that the intrabacterial EMSH of strains genetically resistant to INH remained uninfluenced in response to INH. However, CFZ exposure generated considerable oxidative stress in these strains. These findings show that bactericidal antibiotics with different mechanisms of action generate intrabacterial oxidative stress in Mtb during infection.

Experiment 13 for showing that redox heterogeneity induces differential susceptibility to anti-TB drugs Experiments were performed to determine the susceptibility of Mtb cells with basal, oxidized, and reduced EMSH to antibiotics during infection of THP-1 cells. For this, the membrane integrity of Mtb cells was analyzed by assessing their capacity to exclude fluorescent nucleic-acid binding dye, propidium iodide (Pi), upon treatment with antibiotics. In a first step, THP-1 cells were infected with Mrxl-roGFP2expressing Mtb H37Rv and the cells were treated with NEM and PFA 24h p.i. followed by flow cytometry. In a parallel experiment, infected macrophages were lysed and redox heterogeneity within released Mtb cells was analyzed by flow cytometry. It was seen that NEM treatment of infected macrophages fixed the redox state of intracellular Mtb such that bacterial cells released from macrophages retained redox variations comparable to bacteria within macrophages. Thus, bacterial viability could be quantified by Pi staining of Mtb cells released from infected macrophages at various time points post antibiotic exposure. In a second step, infected THP-1 cells were exposed to anti-TB drugs (at 5-fold the in vitro MIC) and intracellular bacteria were fixed with NEM at 12, 24, and 48h p.i. Infected macrophages were lysed and the released bacteria were stained with Pi. ~ 30,000 bacilli were analyzed by multi-parameter flow cytometry to simultaneously profile EMSH and viability status. Results indicated that bacteria with oxidized EMSH were more sensitive to killing as evident by a time-dependent increase in Pi staining across all antibiotic treatments within this sub-population as compared to other two sub-populations and 35-40% of EA_/s _/-oxidized bacilli were Pi+ at 48h p.i. In contrast, the EA/s//-basal sub-population demonstrated a modest increase in Pi+ staining (-5-10%) at 24 and 48h p.i. while the E^st_f-reduced sub-population remained completely unaffected by antibiotics as shown by the absence of Pi+ cells within this group. This clearly showed that bacteria with lower EMSH are capable of excluding Pi and therefore maintaining membrane integrity post-antibiotic treatment.

Experiment 14 for showing that Mtb with reduced EM may represent a drug tolerant sub- population

Findings from the above experiments suggested that Mtb with enhanced reductive capabilities (i.e., reduced EMSH) may represent a drug tolerant sub-population during infection. Accordingly, the following experiment was conducted to confirm this.

The sensitivity of Mtb towards anti-TB drugs in the presence of reducing agent DTT in vitro was measured. In a first step, it was confirmed that exogenous addition of 5mM DTT maintained a reductive EMSH (-320±2.9mV) equivalent to the EMSH of reducing sub-population and is non-deleterious for £H37Rv. Subsequently, 0H37Rv was treated with INH (0^g/ml), CFZ (0^g/ml), RIF (O^g/ml) and ETH ^g/ml) in the presence or absence of 5mM DTT (added at 0 and 48 h post-antibiotic treatment). Number of bacilli was counted by plating cfu. P-values were also calculated by comparing untreated (7H9 control) and DTT treated groups. As evident from Fig. 26, in the absence of DTT treatment, all anti-TB drugs led to a significant reduction (6-12-fold) in the survival of Mtb. By contrast, -80% of Mtb survived exposure to INH or ETH and -50% in the case of CFZ in the presence of DTT. The results indicated that cells with reduced EMSH can be phenotypically tolerant to anti-

TB drugs, whereas pro-oxidized EMSH potentiates antibiotic action. The findings also suggested that host-induced cell-to-cell variation in EMSH may be a novel mechanism by which Mtb resists antimicrobial treatment during infection.

Thus, the biosensor of the present disclosure may be used for detecting tolerance or drug resistance of a cell having mycothiol redox couple by measuring the intracellular EMSH- For example, if a cell population displays reduced EMSH, i can be concluded that the cell population will be tolerant to anti-TB drugs. In contrast, if a cell population displays oxidized EMSH, it can be concluded that the cell population will be susceptible to anti-TB drugs. Further, it can be concluded that an estimated value of the intracellular EMSH may be indicative of the level of drug tolerance or drug resistance of a cell population. For example, a cell population with a highly reduced E^^may display a higher level of tolerance to anti-TB drugs as compared to a cell population with a slightly reduced EMSH- Cells expressing the biosensor of the present disclosure will emit variable fluorescence responses indicative of their value of intracellular EMSH_. Thus, it can be concluded that an intensity level of the fluorescence response may be indicative of the level of drug resistance of the cells.

In an embodiment, detection of drug resistance of cells having mycothiol redox couple may be undertaken by inserting the biosensor into each cell of a plurality of cells having mycothiol redox couple. The mycothiol redox couple in the each cell of the plurality of cells may interact with the fusion construct of the biosensor incorporated into the each cell and emit a fluorescence response. Further, ah intensity level of the fluorescence response may be indicative of the level of drug resistance of the each cell.

In an embodiment of the invention, the drug resistance may be phenotypic and/or genotypic drug resistance. The cell having mycothiol redox couple may be a member of the taxa actinomycetes including, but not limited to, Mycobacterium, Corynebacterium, Streptomyces, Nocardia and Rhodococcus. In an embodiment, the cell having mycothiol redox couple may be a strain of Mycobacterium tuberculosis.

Detection of host and bacterial pathways using mycothiol-specific fluorescent biosensor

The above experiments showed that the biosensor of the present disclosure is specific towards changes in the redox status of the mycothiol redox couple. Further, when expressed in a plurality of cells, the biosensor can emit a response in only those cells that undergo a change in the mycothiol redox status. Thus, it can be concluded that the biosensor can differentiate between cells that have a perturbed mycothiol redox status and cells that do not undergo any changes in the mycothiol redox status. For example, the biosensor can detect cells wherein a gene or a pathway that affects the mycothiol redox status has been inhibited or deleted. This was also seen in Experiment 8 wherein inhibition of the iNOS pathway could be detected by the biosensor.

In view of the above, it may be appreciated by those skilled in the art that the biosensor may be used for identifying a gene and/ora pathway that affects the redox status of a cell. The gene and/or the pathway may directly and/or indirectly interact or communicate with mycothiol redox couple in a cell thereby triggering a change in the redox status. The cell may be a member of the taxa actinomycetes including, but not limited to, Mycobacterium, Corynebacterium, Streptomyces, Nocardia and Rhodococcus. In an embodiment, the cell may be a strain of Mycobacterium tuberculosis.

In an embodiment, the pathway can include one or more genes that directly and/or indirectly communicate with mycothiol redox couple. In another embodiment, the pathway may be one of a bacterial pathway and a host pathway.

In an embodiment, the pathway may be a bacterial virulence pathway, example of which may include, but are not limited to, mycothiol biosynthesis pathway, cysteine biosynthesis pathway, sulfur assimilation pathway and sulfur transport pathway. In yet another embodiment, the pathway may be a host pathway, example of which may include, but are not limited to, iNOS pathway and NADPH oxidase (NOX) pathway.

In an embodiment, identification of a bacterial pathway that directly and/or indirectly communicates with mycothiol redox couple in a cell may be undertaken by preparing an array of cells having mycothiol redox couple. For example, an array of cells of Mycobacterium

tuberculosis may be prepared. This may be followed by disruption of a unique pathway in each cell of the array of cells. For example, a unique gene may be disrupted in the each cell of the array of cells. Thereafter, the biosensor of the disclosure may be introduced into the each cell. Based on the detection of a fluorescence response indicated by the biosensor incorporated into the each cell, it can be determined whether or not pathway disrupted in the each cell is the pathway that directly and/or indirectly communicates with mycothiol redox couple. For example, in a cell where the disrupted gene is a part of a pathway that directly and/or indirectly

communicates with mycothiol redox couple, a fluorescence response will not be detected, whereas in a cell where the disrupted gene is not a part of a pathway that directly and/or indirectly communicates with mycothiol redox couple, a fluorescence response will be detected.

In an embodiment, identification of a host pathway that directly and/or indirectly communicates with mycothiol redox couple in a cell may be undertaken by preparing an array of host cells. This may be followed by disruption of a unique pathway in each host cell of the array of host cells. For example, a unique gene may be disrupted in the each host cell of the array of host cells. Thereafter, the each host cell of the array of host cells is infected with a mycothiol redox couple containing cell comprising the biosensor of the disclosure. For example, the each host cell of the array of host cells is infected with Mycobacterium tuberculosis comprising the biosensor of the disclosure. In this way, the biosensor and the Mycothiol redox couple is incorporated into the each host cell of the array of host cells. Based on the detection of a fluorescence response indicated by the biosensor incorporated into the each host cell, it can be determined whether or not pathway disrupted in the each host cell is the pathway that directly and/or indirectly communicates with mycothiol redox couple. For example, in a host cell where the disrupted gene is a part of a pathway that directly and/or indirectly communicates with mycothiol redox couple, a fluorescence response will not be detected, whereas in a host cell where the disrupted gene is not a part of a pathway that directly and/or indirectly communicates with mycothiol redox couple, a fluorescence response will be detected.

It should be appreciated by people skilled in the art, that the applications of the biosensor provided are exemplary embodiments of the disclosure and should not be construed as limiting. As will be obvious to a person skilled in the art, the biosensor may find applications in other similar areas. SEQUENCE ID No. 1 :

MITAALTIYTTSWCGYCLRLKTALTANRIAYDEVDIEHNRAAAEFVGSVNGGNRTVPTV KFADGSTLTNPSADEVKAKLVKIAG

Claims

We claim:-

1. A fusion construct comprising:

,a. a first portion comprising an engineered green fluorescent protein having a

fluorescence spectrum that is sensitive to redox status;

b. a second portion comprising a mycothiol-dependent oxidoreductase linked to the engineered green fluorescent protein, optionally with a linker sequence between the first portion and the second portion to fuse the mycothiol-dependent oxidoreductase to the N-terminus of the engineered green fluorescent protein, wherein the linker sequence is a 30 amino acid linker comprising the sequence (Gly-Gly-Ser-Gly-Gly)₆.

2. The fusion construct according to claim 1 , wherein the mycothiol-dependent

oxidoreductase is a mycothiol-dependent oxidoreductase with an N-terminus Cysteine residue in its catalytic site, siich that the catalytic motif is one of a CGYC and a CGYA catalytic motif.

3. The fusion construct according to claim 1 , wherein the mycothiol-dependent

oxidoreductase is a mycoredoxin.

4. The fusion construct according to claim 1 , wherein the mycothiol-dependent

oxidoreductase is a mycoredoxin Mrxl :Rv3198 A.

5. The fusion construct according to claim 1 , wherein the mycothiol-dependent

oxidoreductase is isolated from Mycobacterium tuberculosis strain H37Rv.

6. The fusion construct according to claim 1 , wherein the mycothiol-dependent

oxidoreductase has the sequence of Sequence ID No. 1.

7. The fusion construct according to claim 1 is a biosensor capable of detecting a change in a redox status of mycothiol in a cell wherein a change in the fluorescence pattern of the biosensor indicates the change in the redox status.

8. The fusion construct according to claim 1 is a biosensor, wherein the biosensor is capable of identifying analytes selected from the group comprising drugs, antibodies, inhibitors and chemical compounds that can trigger a change in a redox status of mycothiol in a cell.

9. The fusion construct according to claim 1 is a biosensor, wherein the biosensor is capable of detecting a cell comprising mycothiol redox couple in a sample.

10. The fusion construct according to claim 1 is a biosensor, wherein the biosensor is capable of detecting a level of drug resistance of a cell comprising mycothiol redox couple.

1 1. The fusion construct according to claim 1 is a biosensor, wherein the biosensor is capable of identifying a pathway that directly and/or indirectly communicates with mycothiol redox couple in a cell.

12. A vector comprising a nucleic acid sequence coding for the fusion construct of Claim 1.

13. A host cell comprising the vector of claim 12.

14. A method of identifying an analyte that triggers a change in a redox status of mycothiol, the method comprising:

a. inserting a fusion construct into a cell comprising mycothiol redox couple, the fusion construct comprising:

i. an engineered green fluorescent protein having a fluorescence spectrum that is sensitive to redox status; and

ii. a mycothiol-dependent oxidoreductase linked to the engineered green fluorescent protein;

b. preparing an array of the cells;

c. incubating each cell of the array of the cells with a separate analyte from a

plurality of analytes for a time sufficient for the separate analyte to complete a reaction with at least one constituent of the each cell; and

d. detecting a plurality of fluorescence signals indicated by the fusion construct, each fluorescence signal of the plurality of fluorescence signals corresponding to each of the plurality of analytes, wherein detection of a fluorescence signal is indicative of the corresponding analyte being the analyte that triggers the change in the redox status of mycothiol.

15. The method according to claim 14, wherein the mycothiol-dependent oxidoreductase is a mycothiol-dependent oxidoreductase with an N-terminus Cysteine residue in its catalytic site, such that the catalytic motif is one of a CGYC and a CGYA catalytic motif.

16. The method according to claim 14, wherein the mycothiol-dependent oxidoreductase is a mycoredoxin Mrxl :Rv3198A.

17. The method according to claim 14, wherein the mycothiol-dependent oxidoreductase is isolated from Mycobacterium tuberculosis strain H37Rv.

18. The method according to claim 14, wherein a pre-defined intensity level of the each fluorescence signal of the plurality of fluorescence signals is indicative of an

effectiveness of the analyte on the cell comprising mycothiol redox couple.

19. The method according to claim 14, wherein the analyte is selected from the group

comprising drugs, antibodies, inhibitors and chemical compounds.

20. The method according to claim 14 is a high throughput screening method.

21. A method of detecting a cell comprising mycothiol redox couple in a sample, the method comprising:

a. inserting a fusion construct into a moiety capable of reacting with cells

comprising mycothiol redox couple, the fusion construct comprising: i. an engineered green fluorescent protein having a fluorescence spectrum that is sensitive to redox status; and

ii. a mycothiol-dependent oxidoreductase linked to the engineered green fluorescent protein;

b. incubating the moiety with the sample to be tested for presence of the cell

comprising mycothiol redox couple, incubation being done for a time period sufficient for the moiety to complete a reaction with at least one constituent of the sample; and

c. detecting a fluorescence signal indicated by the fusion construct, wherein

detection of the fluorescence signal is indicative of the presence of the cell comprising mycothiol redox couple in the sample.

22. The method according to claim 21, wherein the mycothiol-dependent oxidoreductase is a mycothiol-dependent oxidoreductase with an N-terminus Cysteine residue in its catalytic site, such that the catalytic motif is one of a CGYC and a CGYA catalytic motif.

23. The method according to claim 21 , wherein the mycothiol-dependent oxidoreductase is a mycoredoxin Mrx 1 : Rv3198 A . ¹

24. The method according to claim 21 , wherein the mycothiol-dependent oxidoreductase is isolated from Mycobacterium tuberculosis strain H37Rv.

25. The method according to claim 21 , wherein the cell comprising mycothiol redox couple is a member of the taxa actinomycetes, further wherein the member of the taxa actinomycetes is selected from the group comprising Mycobacterium, Corynebacterium, Streptomyces, Nocardia and Rhodococcus.

26. The method according to claim 21, wherein the cell comprising mycothiol redox couple is a strain of Mycobacterium tuberculosis.

27. The method according to claim 21 , wherein the moiety is selected from the group

comprising a bacteriophage and a mycobacteriophage.

28. The method according to claim 21 , wherein the sample is a biological sample selected from the group, comprising a blood sample, a serum sample, a urine sample, a fecal sample, a tissue biopsy, a cerebrospinal fluid sample, an ascites sample, a pleural fluid sample, respiratory secretions, a saliva sample, and a sputum sample.

29. A kit for detecting a cell comprising mycothiol redox couple in a sample, the kit

comprising a moiety capable of reacting with cells comprising mycothiol redox couple, the moiety comprising a fusion construct comprising:

a. an engineered green fluorescent protein having a fluorescence spectrum that is sensitive to redox status; and

b. a mycothiol-dependent oxidoreductase linked to the engineered green fluorescent protein.

30. The kit according to claim 29, wherein the mycothiol-dependent oxidoreductase is a mycothiol-dependent oxidoreductase with an N-terminus Cysteine residue in its catalytic site, such that the catalytic motif is one of a CGYC and a CGYA catalytic motif.

31. The kit according to claim 29, wherein the mycothiol-dependent oxidoreductase is a mycoredoxin Mrxl :Rv3198 A.

32. The kit according to claim 29, wherein the mycothiol-dependent oxidoreductase is

isolated from Mycobacterium tuberculosis strain H37Rv.

33. The kit according to claim 29, wherein the cell comprising mycothiol redox couple is a member of the taxa actinomycetes, further wherein the member of the taxa actinomycetes is selected from the group comprising Mycobacterium, Corynebacterium, Streptomyces, Nocardia and Rhodococcus.

34. The kit according to claim 29, wherein the cell comprising mycothiol redox couple is a strain of Mycobacterium tuberculosis.

35. The kit according to claim 29, wherein the moiety is selected from the group comprising a bacteriophage and a mycobacteriophage.

36. The kit according to claim 29, wherein the sample is a biological sample selected from the group comprising a blood sample, a serum sample, a urine sample, a fecal sample, a tissue biopsy, a cerebrospinal fluid sample, an ascites sample, a pleural fluid sample, respiratory secretions, a saliva sample, and a sputum sample.

37. A method of detecting a cell comprising mycothiol redox couple in a sample, the method comprising:

a. subjecting the sample to be tested for presence of the cell comprising mycothiol redox couple to a processing system that causes lysis of cells present in the sample;

b. incubating the lysed sample with a fusion construct for a time period sufficient for the fusion construct to complete a reaction with at least one constituent of the lysed sample, the fusion construct comprising:

i. an engineered green fluorescent protein having a fluorescence spectrum that is sensitive to redox status; and

ii. a mycothiol-dependent oxidoreductase linked to the engineered green fluorescent protein; and

c. detecting a fluorescence signal indicated by the fusion construct, wherein

detection of the fluorescence signal is indicative of the presence of the cell comprising mycothiol redox couple in the sample.

38. The method according to claim 37, wherein the mycothiol-dependent oxidoreductase is a mycothiol-dependent oxidoreductase with an N-terminus Cysteine residue in its catalytic site, such that the catalytic motif is one of a CGYC and a CGYA catalytic motif.

39. The method according to claim 37, wherein the mycothiol-dependent oxidoreductase is a mycoredoxin Mrx 1 :Rv3198 A.

40. The method according to claim 37, wherein the mycothiol-dependent oxidoreductase is isolated from Mycobacterium tuberculosis strain H37Rv.

41. The method according to claim 37, wherein the cell comprising mycothiol redox couple is a member of the taxa actinomycetes, further wherein the member of the taxa actinomycetes is selected from the group comprising Mycobacterium, Corynebacterium, Streptomyces, Nocardia and Rhodococcus.

42. The method according to claim 37, wherein the cell comprising mycothiol redox couple is a strain of Mycobacterium tuberculosis.

43. The method according to claim 37, wherein the sample is a biological sample selected from the group comprising a blood sample, a serum sample, a urine sample, a fecal ^■ sample, a tissue biopsy, a cerebrospinal fluid sample, an ascites sample, a pleural fluid sample, respiratory secretions, a saliva sample, and a sputum sample.

44. A kit for detecting a cell comprising mycothiol redox couple in a sample, the kit

comprising:

a. a processing system for lysing cells present in the sample;

b. a fusion construct comprising: _,

i. an engineered green fluorescent protein having a fluorescence spectrum that is sensitive to redox status; and

ii. a mycothiol-dependent oxidoreductase linked to the engineered green fluorescent protein.

45. The kit according to claim 44, wherein the mycothiol-dependent oxidoreductase is a mycothiol-dependent oxidoreductase with an N-terminus Cysteine residue in its catalytic site, such that the catalytic motif is one of a CGYC and a CGYA catalytic motif.

46. The kit according to claim 44, wherein the mycothiol-dependent¹ oxidoreductase is a mycoredoxin Mrx 1 :Rv3198 A.

47. The kit according to claim 44, wherein the mycothiol-dependent oxidoreductase is

isolated from Mycobacterium tuberculosis strain H37Rv.

48. The kit according to claim 44, wherein the cell comprising mycothiol redox couple is a member of the taxa actinomycetes, further wherein the member of the taxa actinomycetes is selected from the group comprising Mycobacterium, Corynebacterium, Streptomyces, Nocardia and Rhodococcus.

49. The kit according to claim 44, wherein the cell comprising mycothiol redox couple is a strain of Mycobacterium tuberculosis.

50. The kit according to claim 44, wherein the sample is a biological sample selected from the group comprising a blood sample, a serum sample, a urine sample, a fecal sample, a tissue biopsy, a cerebrospinal fluid sample, an ascites sample, a pleural fluid sample, respiratory secretions, a saliva sample, and a sputum sample.

51. A method of detecting a level of drug resistance of each cell of a plurality of cells

comprising mycothiol redox couple, the method comprising:

a. introducing into each cell of the plurality of cells a fusion construct comprising: i. an engineered green fluorescent protein having a fluorescence spectrum that is sensitive to redox status; and

ii. a mycothiol-dependent oxidoreductase linked to the engineered green fluorescent protein; and

b. detecting a fluorescence signal indicated by the fusion construct in eachcell of the plurality of cells, wherein a pre-defined intensity level of the fluorescence signal of the fusion construct is indicative of the level of drug resistance of each cell of the plurality of cells comprising mycothiol redox couple.

52. The method according to claim 51 , wherein the mycothiol-dependent oxidoreductase is a mycothiol-dependent oxidoreductase with an N-terminus Cysteine residue in its catalytic site, such that the catalytic motif is one of a CGYC and a CGYA catalytic motif.

53. The method according to claim 51 , wherein the mycothiol-dependent oxidoreductase is a mycoredoxin Mrxl :Rv3198A.

54. The method according to claim 51 , wherein the mycothiol-dependent oxidoreductase is isolated from Mycobacterium tuberculosis strain H37Rv.

55. The method according to claim 51 , wherein the cell comprising mycothiol redox couple is a member of the taxa actinomycetes, further wherein the member of the taxa actinomycetes is selected from the group comprising Mycobacterium, Corynebacterium, Streptomyces, Nocardia and Rhodococcus.

56. The method according to claim 51 , wherein the cell comprising mycothiol redox couple is a strain of Mycobacterium tuberculosis.

57. A method of identifying a bacterial pathway that directly and/or indirectly communicates with mycothiol redox couple in a cell, the method comprising:

a. preparing an array of cells, wherein each cell of the array of cells comprises a mycothiol redox couple;

b. modifying each cell of the array of cells by disrupting a unique pathway to be determined as a pathway that directly and/or indirectly communicates with mycothiol redox couple;

c. introducing into each cell of the array of cells a fusion construct comprising: i. an engineered green fluorescent protein having a fluorescence spectrum that is sensitive to redox status; and

ii. a mycothiol-dependent oxidoreductase linked to the engineered green fluorescent protein; and

d. detecting a fluorescence signal indicated by the fusion construct in the each cell of the array of cells, wherein a pre-defined intensity level of the fluorescence signal of the fusion construct is indicative of the unique pathway being the pathway that directly and/or indirectly communicates with mycothiol redox couple.

58. The method according to claim 57, wherein the mycothiol-dependent oxidoreductase is a mycothiol-dependent oxidoreductase with an N-terminus Cysteine residue in its catalytic site, such that the catalytic motif is one of a CGYC and a CGYA catalytic motif.

59. The method according to claim 57, wherein the mycothiol-dependent oxidoreductase is a mycoredoxin Mrxl :Rv3198A.

60. The method according to claim 57, wherein the mycothiol-dependent oxidoreductase is isolated from Mycobacterium tuberculosis strain H37Rv.

61. The method according to claim 57, wherein the cell comprising mycothiol redox couple is a member of the taxa actinomycetes, further wherein the member of the taxa actinomycetes is selected from the group comprising Mycobacterium, Corynebacterium, Streptomyces, Nocardia and Rhodococcus.

62. The method according to claim 57, wherein the cell comprising mycothiol redox couple is a strain of Mycobacterium tuberculosis.

63. The method according to claim 57, wherein the bacterial pathway comprises one or more genes that directly and/or indirectly communicate with mycothiol redox couple.

64. The method according to claim 57, wherein the bacterial pathway is a bacterial virulence pathway, the bacterial virulence pathway being selected from the group comprising mycothiol biosynthesis pathway, cysteine biosynthesis pathway, sulfur assimilation pathway and sulfur transport pathway.

65. A method of identifying a host pathway that directly and/or indirectly communicates with mycothiol redox couple in a cell, the method comprising:

a. preparing an array of host cells,

b. modifying each host cell of the array of host cells by disrupting a unique pathway to be determined as a pathway that directly and/or indirectly communicates with mycothiol redox couple;

c. infecting each host cell of the array of host cells with the mycothiol redox couple containing cell that comprises a fusion construct comprising:

i. an engineered green fluorescent protein having a fluorescence spectrum that is sensitive to redox status; and

ii. a mycothiol-dependent oxidoreductase linked to the engineered green fluorescent protein;

wherein, the fusion construct and the mycothiol redox couple is incorporated into each host cell of the array of host cells; and

d. detecting a fluorescence signal indicated by the fusion construct in the each host cell of the array of host cells, wherein a pre-defined intensity level of the fluorescence signal of the fusion construct is indicative of the unique pathway being the pathway that directly and/or indirectly communicates with mycothiol redox couple.

66. The method according to claim 65, wherein the mycothiol-dependent oxidoreductase is a mycothiol-dependent oxidoreductase with an N-terminus Cysteine residue in its catalytic site, such that the catalytic motif is one of a CGYC and a CGYA catalytic motif.

67. The method according to claim 65, wherein the mycothiol-dependent oxidoreductase is a mycoredoxin Mrx 1 :Rv3198 A.

68. The method according to claim 65, wherein the mycothiol-dependent oxidoreductase is isolated from Mycobacterium tuberculosis strain H37Rv.

69. The method according to claim 65, wherein the cell comprising mycothiol redox couple is a member of the taxa actinomycetes, further wherein the member of the taxa actinomycetes is selected from the group comprising Mycobacterium, Corynebacterium, Streptomyces, Nocardia and Rhodococcus.

70. The method according to claim 65, wherein the cell comprising mycothiol redox couple is a strain of Mycobacterium tuberculosis.

71. The method according to claim 65, wherein the host pathway comprises one or more genes that directly and/or indirectly communicate with mycothiol redox couple.

72. The method according to claim 65, wherein the. host pathway is selected from the group comprising inducible nitric oxide synthase pathway and NADPH oxidase pathway.