WO2020202188A1

WO2020202188A1 - In-vitro processes for screening candidate molecules

Info

Publication number: WO2020202188A1
Application number: PCT/IN2020/050290
Authority: WO
Inventors: Deepak Kumar Saini; Apoorva Bhatt; Chandrani THAKUR; Nagasuma Chandra; Vignesh Narayan HARIHARAN
Original assignee: Indian Institute Of Science
Priority date: 2019-03-29
Filing date: 2020-03-27
Publication date: 2020-10-08

Abstract

The present disclosure discloses in-vitro processes for screening candidate molecules capable of inhibiting growth of a bacterial pathogen. The present disclosure discloses strategies for screening candidate molecules having an ability to intervene the functional PdtaS-PdtaR signalling cascade, thereby inhibiting the growth of a bacterial pathogen. Further, the present disclosure discloses the use of PdtaS and c-di-GMP interaction in screening for candidate molecules capable of inhibiting growth of Mycobacterium species, along with the use of PdtaS and PdtaR interaction in screening for candidate molecules capable of inhibiting growth of Mycobacterium species, and the use of PdtaR and target RNA interaction in screening for candidate molecules capable of inhibiting growth of Mycobacterium species. The present disclosure also discloses recombinant strain of Mycobacterium species comprising a mutation in a gene encoding PdtaS protein or PdtaR protein.

Description

IN-VITRO PROCESSES FOR SCREENING CANDIDATE MOLECULES

FIELD OF INVENTION

[001] The present disclosure broadly relates to an in-vitro process for screening of candidate molecules for their ability to inhibit growth of a pathogenic bacteria. 5 Particularly, the present disclosure discloses an in-vitro process for screening of candidate molecules for their ability to inhibit growth of Mycobacterium species by targeting a specific Two-Component System (TCS).

BACKGROUND OF THE INVENTION

[002] As per a report from the World Health Organisation (WHO, 2016), number of deaths caused due to infectious diseases, such as tuberculosis and leprosy, is among the0 top 10 with respect to cause of deaths worldwide. As per the report, lower respiratory diseases due to infectious diseases caused around 3 million deaths worldwide in 2016. Owing to the high risk being faced by mankind, there is a constant pursuit for finding treatment methods to tackle such a huge loss of lives. Owing to the high occurrence of drug resistance in pathogens causing infectious diseases, the risk of losing a human life5 has increased manifolds.

[003] Presently, front line anti-tubercular therapy relies on drugs discovered almost fifty years ago, consisting of three to four drugs prescribed for a minimum of 6 months to a patient. The treatment for leprosy is also for a 6-12 months duration. The toxicity of these drugs and high patient non-compliance rates have resulted in the emergence0 of multi-, extensively-, and totally-drug resistant strains of M. tuberculosis and M. leprae. Given that these drugs target essential genes, resistance to them emerges during the course of natural selection, which may not be the case for genes that are not essential for cell viability but are important only under specific environmental conditions such as nutrient deprivation. New drugs against new drug targets, with minimal side effects, are the need of the hour to combat mycobacterial diseases.

[004] While elements of bacterial signalling such as two-component systems (TCS) have been proposed to be good anti-bacterial targets, success with using them have been limited due to lack of information about the cues they detect and the precise role they play in physiological adaptation of the bacterium against environmental cues. While many TCS have been shown to be critical for virulence and pathogenesis of various bacterial species, thereby serving as good growth and infection inhibition targets, for most, the lack of knowledge of the environmental cues they sense limits their utility as drug targets.

[005] TCS is an active field that has been investigated for its functional attributes to an organism. Therefore, there is a need to investigate further on the aspect of TCS and its potential as an anti-bacterial drug target. SUMMARY OF INVENTION

[006] In an aspect of the present disclosure, there is provided an in-vitro process for screening a candidate molecule capable of inhibiting growth of a bacterial pathogen, said process comprises: (a) obtaining PdtaS protein; (b) performing a phosphorylation assay by incubating PdtaS protein in the presence of a phosphor-donor molecule under in-vitro conditions, to obtain a product A, and analysing phosphorylation of the PdtaS protein of the product A; (c) obtaining a candidate molecule; (d) performing another phosphorylation assay by incubating PdtaS protein in the presence of a phosphor-donor molecule and the candidate molecule under in-vitro conditions, to obtain a product B, and analysing phosphorylation of the PdtaS protein of the product B; and (e) comparing the phosphorylation of the PdtaS protein of the product B to the phosphorylation of the PdtaS protein of the product A, wherein a decrease in the phosphorylation of the PdtaS protein of the product B as compared to the phosphorylation of the PdtaS protein of the product A indicates capability of the candidate molecule in inhibiting growth of a bacterial pathogen. [007] In a second aspect of the present disclosure, there is provided an in-vitro process for screening a candidate molecule capable of inhibiting growth of a bacterial pathogen, said process comprises: (a) obtaining PdtaS protein, and PdtaR protein; (b) performing phosphorylation assay by incubating PdtaS protein in the presence of ATP and PdtaR protein under in-vitro conditions, to obtain a product C, and analysing the interaction of PdtaS protein and PdtaR protein of the product C; (c) obtaining a candidate molecule; (d) performing phosphorylation assay by incubating PdtaS protein in the presence of a phosphor-donor molecule, PdtaR protein, and the candidate molecule under in-vitro conditions, to obtain a product D, and analysing interaction of PdtaS protein and PdtaR protein of the product D; and (e) comparing the interaction of PdtaS protein and PdtaR protein of the product D to the interaction of PdtaS protein and PdtaR protein of the product C, wherein a decrease in interaction of PdtaS protein and PdtaR protein of the roduct D as compared to the interaction of PdtaS and PdtaR protein of the incubated product C indicates capability of the candidate molecule in inhibiting growth of a bacterial pathogen.

[008] In a third aspect of the present disclosure, there is provided an in-vitro process for screening a candidate molecule capable of inhibiting growth of a bacterial pathogen, said process comprises: (a) obtaining PdtaS protein; (b) incubating PdtaS protein in the presence of c-di-GMP under in-vitro conditions, to obtain a product E, and analysing interaction between PdtaS protein and c-di-GMP in the product E; (c) obtaining a candidate molecule; (d) incubating PdtaS protein in the presence of c-di-GMP, and the candidate molecule under in-vitro conditions, to obtain a product F, and analysing interaction between PdtaS protein and c-di-GMP in the product F; and (e) comparing interaction between PdtaS protein and c-di-GMP in the product F with interaction between PdtaS protein and c-di-GMP in the product E for observing an increase or decrease in interaction of PdtaS protein and c-di-GMP in the product F as compared to the product E, wherein a decrease in the interaction between PdtaS protein and c-di- GMP in the product F as compared to the interaction between PdtaS protein and c-di- GMP in the product E indicates capability of the candidate molecule in inhibiting growth of a bacterial pathogen.

[009] In a fourth aspect of the present disclosure, there is provided an in-vitro process for screening a candidate molecule capable of inhibiting growth of a bacterial pathogen, said process comprises: (a) obtaining PdtaR protein; (b) obtaining a target RNA molecule, wherein the target RNA molecule is able to bind to PdtaR protein; (c) incubating PdtaR protein in the presence of the target RNA molecule under in-vitro conditions, to obtain a product G, and analysing interaction between PdtaR protein and the target RNA in the product G; (d) obtaining a candidate molecule; (e) incubating PdtaR protein in the presence of the target RNA molecule and the candidate molecule under in-vitro conditions, to obtain a product H, and analysing the interaction between PdtaR protein and the target RNA in the product H; and (f) comparing interaction between PdtaR protein and the target RNA in the product H with interaction between PdtaR protein and the target RNA in the product G for observing an increase or decrease in interaction of PdtaR protein and the target RNA in the product H as compared to the product G, wherein a decrease in interaction between PdtaR protein and the target RNA in the product H as compared to the interaction between PdtaR protein and the target RNA in the product G indicates capability of the candidate molecule in inhibiting growth of a bacterial pathogen.

[0010] In a fifth aspect of the present disclosure, there is provided an in-vitro process for screening a candidate molecule capable of inhibiting growth of a bacterial pathogen, said process comprises: (a) obtaining PdtaS protein, and PdtaR protein; (b) performing phosphorylation assay by incubating PdtaS protein in the presence of ATP and PdtaR protein under in-vitro conditions, to obtain a product I; (c) obtaining a target RNA molecule; (d) incubating the target RNA with the product I under in-vitro conditions, to obtain a product J, and analysing interaction between PdtaR protein and the target RNA in the product J; (e) obtaining a candidate molecule; (f) incubating the target RNA with the product I in presence of the candidate molecule under in-vitro conditions, to obtain a product K, and analysing interaction between PdtaR protein and the target RNA in the product K; and (g) comparing interaction between PdtaR protein and the target RNA in the product K with interaction between PdtaR protein and the target RNA in the product J for observing an increase or decrease in interaction of PdtaR protein and the target RNA in the product K as compared to the product J, wherein a decrease in interaction between PdtaR protein and the target RNA in the incubated product K as compared to the interaction between PdtaR protein and the target RNA in the product J indicates capability of the candidate molecule in inhibiting growth of a bacterial pathogen.

[0011] In a sixth aspect of the present disclosure, there is provided an in-vitro process for screening a candidate molecule capable of inhibiting growth of a bacterial pathogen, said process comprises: (a) obtaining PdtaS protein, and PdtaR protein; (b) incubating PdtaS protein in the presence of PdtaR protein under in-vitro conditions, to obtain a product L, and analysing interaction between PdtaS protein and PdtaR protein in the product L; and (c) obtaining a candidate molecule; (d) incubating PdtaS protein in the presence of PdtaR protein and the candidate molecule, to obtain a product M, and analysing interaction between PdtaS protein and PdtaR protein in the product M; and (e) comparing interaction between PdtaS protein and PdtaR protein in the product M to interaction between PdtaS protein and PdtaR protein in the product L for observing an increase or decrease in interaction of PdtaS protein and PdtaR protein in the product M as compared to the product L, wherein a decrease in interaction between PdtaS protein and PdtaR protein in the product M as compared to the interaction between PdtaS protein and PdtaR protein in the product L indicates capability of the candidate molecule in inhibiting growth of a bacterial pathogen.

[0012] In a seventh aspect of the present disclosure, there is provided a recombinant strain of Mycobacterium species comprising a mutation in a gene encoding PdtaS protein.

[0013] In an eighth aspect of the present disclosure, there is provided a recombinant strain of Mycobacterium species comprising a mutation in a gene encoding PdtaR protein. [0014] In a ninth aspect of the present disclosure, there is provided a use of PdtaS and c-di-GMP interaction in screening for candidate molecules capable of inhibiting growth of Mycobacterium species.

[0015] In a tenth aspect of the present disclosure, there is provided a use of PdtaS and PdtaR interaction in screening for candidate molecules capable of inhibiting growth of Mycobacterium species.

[0016] In an eleventh aspect of the present disclosure, there is provided a use of PdtaS and target RNA interaction in screening for candidate molecules capable of inhibiting growth of Mycobacterium species.

[0017] In a twelfth aspect of the present disclosure, there is provided a use of Two-

Component System (TCS), PdtaS-PdtaR, for in-vitro screening of a candidate molecule capable of inhibiting growth of a bacterial pathogen.

[0018] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS

[0019] The following drawings form a part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.

[0020] Figure 1 illustrates a schematic of PdtaS-PdtaR Two-Component System (TCS) and its function, in accordance with an implementation of the present disclosure.

[0021] Figure 2 illustrates the growth analysis of wildtype and mutant (ApdtaS) in nutrient-deprived conditions, in accordance with an implementation of the present disclosure. [0022] Figure 3 illustrates docking of c-di-GMP to the GAF domain of PdtaS, in accordance with an implementation of the present disclosure.

[0023] Figure 4 illustrates that growth of M. smegmatis requires functional c-di-GMP binding to PdtaS, in accordance with an implementation of the present disclosure.

[0024] Figure 5 illustrates binding affinity of c-di-GMP to PdtaS-EGFP using MST

(MicroScale thermophoresis), in accordance with an implementation of the present disclosure.

[0025] Figure 6A illustrates dose response time-course of c-di-GMP mediated phosphorylation (activation) of PdtaS, and Figure 6B illustrates gel and autoradiography of in-vitro phosphorylation of PdtaS, in accordance with an implementation of the present disclosure.

[0026] Figure 7 illustrates dilution spot assay for cells with active and kinase-deficient PdtaS, in accordance with an implementation of the present disclosure.

[0027] Figure 8 illustrates kinetics of PdtaS to PdtaR phosphor-transfer, in accordance with an implementation of the present disclosure.

[0028] Figure 9 illustrates FRET measurement of PdtaS -PdtaR interaction, in accordance with an implementation of the present disclosure.

[0029] Figure 10 illustrates interaction of PdtaS and PdtaR using Biolayer interferometry (BLI), in accordance with an implementation of the present disclosure.

[0030] Figure 11 illustrates dilution spot assay of wildtype, ApdtaS, A:mtPdtaR and

A:BM cultures, in accordance with an implementation of the present disclosure.

[0031] Figure 12 depicts in-vitro phosphorylation assays using mutated and unmutated PdtaS and PdtaR proteins, in accordance with an implementation of the present disclosure.

[0032] Figure 13 illustrates the effect of cyclic-di-GMP on the kinase activity of PdtaS sensor kinase measure through radioactive phosphoryl group incorporation where ATP is the donor molecule at pH 6.5 and pH 7.5, in accordance with an implementation of the present disclosure. [0033] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps, features, compositions, and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features.

SEQUENCES:

[0034] SEQ ID NO: 1 depicts amino acid sequence of PdtaS protein of Mycobacterium tuberculosis H37Rv.

[0035] MSTLGDLLAEHTVLPGS A VDHLHA VV GEW QLLADLSFAD YLMWVR

RDDG VL V C V AQCRPNTGPT V VHTD A V GT V V A ANSMPLV A ATFS GG VPGRE GAVGQQNSCQHDGHSVEVSPVRFGDQVVAVLTRHQPELAARRRSGHLETAY RLCATDLLRMLAEGTFPDAGDVAMSRSSPRAGDGFIRLDVDGVVSYASPNA LSAYHRMGLTTELEGVNLIDATRPLISDPFEAHEVDEHVQDLLAGDGKGMR MEVDAGGATVLLRTLPLVVAGRNVGAAILIRDVTEVKRRDRALISKDATIREI HHRVKNNLQTVAALLRLQARRTSNAEGREALIESVRRVSSIALVHDALSMSV DEQVNLDEVIDRILPIMNDVASVDRPIRINRVGDLGVLDSDRATALIMVITELV QNAIEHAFDPAAAEGSVTIRAERSARWLDVVVHDDGLGLPQGFSLEKSDSLG LQIVRTLVSAELDGSLGMRDARERGTDVVLRVPVGRRGRLML

[0036] SEQ ID NO: 2 depicts nucleic acid sequence of pdtaS gene of Mycobacterium tuberculosis H37Rv.

[0037] ATGTCCACACTCGGTGATCTGCTCGCCGAACACACGGTGCTGCCGG GCAGCGCGGTGGACCACCTGCATGCGGTGGTCGGGGAGTGGCAGCTCCTT GCCGACTTGTCGTTTGCCGATTACCTGATGTGGGTTCGCCGCGACGACGG TGTCCTGGTGTGCGTTGCGCAATGCCGGCCGAACACCGGGCCGACGGTGG TGCATACCGACGCGGTAGGCACCGTCGTCGCCGCCAATAGCATGCCGCTG GTCGCCGCGACCTTCTCCGGTGGTGTCCCGGGACGGGAAGGCGCTGTCGG CCAACAGAATTCATGTCAACACGACGGCCACAGTGTCGAAGTCTCCCCGG TGCGCTTTGGCGATCAGGTGGTGGCGGTGCTGACACGGCATCAACCCGAA CTGGCGGCGCGACGTAGATCCGGCCACCTGGAGACCGCCTATCGGTTGTG CGCCACAGATCTTCTCCGGATGCTGGCGGAGGGCACCTTTCCCGACGCAG GGGACGTGGCGATGTCGCGATCTAGCCCGCGCGCGGGTGACGGCTTCATC CGTCTCGATGTCGACGGTGTGGTCTCTTACGCCAGCCCCAATGCCCTATCG GCTTACCACCGAATGGGTTTGACCACCGAGTTGGAGGGCGTCAATCTCAT TGACGCGACGCGCCCGCTGATCTCGGACCCGTTCGAGGCGCACGAGGTAG ACGAGCATGTGCAGGACTTGCTGGCCGGGGATGGAAAGGGTATGCGGAT GGAGGTCGACGCCGGCGGCGCCACGGTGCTGCTGCGGACTCTGCCGCTGG TGGTAGCTGGTCGCAATGTCGGCGCCGCGATATTGATCCGCGACGTGACC GAGGTGAAGCGGCGCGACCGAGCCCTGATATCCAAGGACGCCACGATCC GGGAAATCCATCATCGGGTTAAGAACAACCTGCAGACGGTGGCCGCGCT GTTGCGGCTGCAGGCTCGCCGGACGTCCAACGCCGAGGGGCGGGAAGCG CTGATCGAGTCGGTGCGCCGAGTGTCGTCGATTGCCTTGGTCCACGATGC GTTGTCGATGTCGGTGGACGAGCAGGTGAACCTTGACGAGGTCATCGACC GGATTCTGCCGATCATGAACGATGTGGCATCGGTGGACAGGCCGATCCGG ATAAATCGGGTTGGCGACCTCGGTGTGCTCGACTCCGACCGCGCCACGGC GCTGATCATGGTGATCACCGAGCTGGTGCAGAACGCGATCGAGCATGCGT TCGACCCGGCGGCGGCGGAAGGGTCCGTGACGATTCGAGCGGAACGCTC TGCGCGTTGGCTCGATGTCGTCGTGCACGACGACGGGCTTGGTCTGCCGC AAGGTTTCAGCCTGGAGAAGTCGGACAGCCTGGGCCTGCAGATCGTGCGG ACCTTGGTCTCTGCGGAATTGGACGGCTCGTTAGGTATGCGGGACGCCCG CGAACGTGGCACCGATGTGGTGCTACGGGTACCGGTCGGACGCCGGGGA CGGTTGATGCTGTAG

[0038] SEQ ID NO: 3 depicts amino acid sequence of PdtaR protein of Mycobacterium tuberculosis H37Rv.

[0039] MTGPTTDADAAVPRRVLIAEDEALIRMDLAEMLREEGYEIVGEAGDG QEAVELAELHKPDLVIMDVKMPRRDGIDAASEIASKRIAPIVVLTAFSQRDLV ERARDAGAMAYLVKPFSISDLIPAIELAVSRFREITALEGEVATLSERLETRKL VERAKGLLQTKHGMTEPDAFKWIQRAAMDRRTTMKRVAEVVLETLGTPKD

T

[0040] SEQ ID NO: 4 depicts the nucleic acid sequence of pdtaR gene of Mycobacterium tuberculosis H37Rv.

[0041] ATGACCGGCCCCACCACCGACGCCGATGCCGCTGTCCCACGTCGG

GTCTTGATCGCGGAAGATGAAGCGCTCATCCGCATGGACCTGGCCGAGAT GTTGCGAGAGGAGGGATATGAAATTGTCGGCGAGGCCGGCGACGGCCAG GAAGCCGTCGAGCTGGCCGAGCTGCACAAGCCCGACCTGGTGATCATGG ACGTGAAGATGCCGCGCCGGGACGGGATCGACGCCGCATCCGAAATCGC CAGCAAACGTATTGCCCCGATCGTGGTGCTGACCGCGTTCAGCCAGCGTG ATCTGGTCGAACGTGCGCGTGATGCCGGGGCGATGGCATACCTGGTAAAG CCTTTCAGCATCAGCGACCTGATTCCAGCGATTGAATTGGCGGTCAGCCG GTTCAGGGAGATCACCGCGTTGGAAGGCGAGGTGGCGACGCTATCTGAA CGGTTGGAAACCCGCAAGCTGGTGGAACGAGCAAAAGGCCTGCTGCAGA CCAAACATGGGATGACCGAGCCGGACGCTTTCAAGTGGATTCAACGTGCC GCCATGGATCGGCGCACCACCATGAAGCGGGTGGCCGAAGTCGTGCTGG AAACCCTCGGAACACCCAAAGACACCTGA

[0042] SEQ ID NO: 5 depicts a primer sequence PdtaSH303Qf.

[0043] CCGGGAAATCCATCAGCGGGTTAAGAACAAC.

[0044] SEQ ID NO: 6 depicts a primer sequence PDtaSH303Qr.

[0045] GTTGTTCTTAACCCGCTGATGGATTTCCCGG

[0046] SEQ ID NO: 7 depicts a primer sequence PdtaRD65Vf.

[0047] GACCTGGTGATCATGGTCGTGAAGATGCCGC.

[0048] SEQ ID NO: 8 depicts a primer sequence PdtaRD65Vr.

[0049] GCGGCATCTTCACGACCATGATCACCAGGTC

Definitions

[0050] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are delineated here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.

[0051] The articles“a”,“an” and“the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[0052] The terms“comprise” and“comprising” are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as “consists of only”.

[0053] Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as“comprises” and“comprising”, will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps.

[0054] The term “including” is used to mean “including but not limited to”. “Including” and“including but not limited to” are used interchangeably.

[0055] The term“candidate molecule” refers to any molecule used in the screening process, in the process as disclosed in the present disclosure. It is construed to cover peptides, proteins, small molecules, nucleic acid molecules, and combinations thereof.

[0056] The term“target RNA” refers to ribonucleic acid molecules which are bound directly by PdtaR protein.

[0057] The term“nutrient deprived” refers to any medium that is constituted by base medium and lacking one or more additives (primarily carbon or nitrogen source) used in the culture of mycobacteria such as glycerol, oleic acid, dextrose, albumin, L- arginine, casamino acids, and catalase.

[0058] The term“product” as mentioned in the present disclosure, for example, product A, product B, and so on, refers to a mixture of more than one molecules which either has a phosphorylation or a general interaction involved among the molecules.

[0059] The proteins referred to in the present disclosure“PdtaS” and“PdtaR” refer to the sensor kinase and response regulator of the Two-Component System (TCS), PdtaS- PdtaR. Although, the present disclosure is exemplified using the sequence of proteins from Mycobacterium, however, the present disclosure is intended to cover any other homologs of such proteins which might be present in any bacterial organism. A non limiting list of such genus includes Mycobacterium and Corynebacterium, and any other species harbouring homologs of PdtaS protein and PdtaR protein belonging to suborder Corynebacterineae.

[0060] The term“bacterial pathogen” as per the present disclosure refers to any disease-causing bacterial organism selected from a group consisting of Mycobacterium species, Corynebacterium species, any other species harbouring homologs of PdtaS protein and PdtaR protein belonging to suborder Corynebacterineae.

[0061] The term“phosphor-donor” refers to any molecule which is capable of donating a phosphate moiety to a sensor kinase during in-vitro phosphorylation assay. One of the well-established phosphor-donor molecule is ATP.

[0062] The term“inhibiting growth of a bacterial pathogen” relates to an action which is performed by the candidate molecule, which is screened by the method as disclosed in the present disclosure. The action of inhibiting growth can be in the form of either killing bacterial cells, or preventing the reactivation of the bacterial pathogen or inhibiting bacterial pathogen from entering into latency. The candidate molecule thus screened can have any one or a combination of the above-mentioned properties, which effectively would provide medical intervention for treating infections caused by the bacterial pathogen.

[0063] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference.

[0064] As discussed in the background section, to circumvent the problem arising due to the present practice of selecting a drug target, the present disclosure discloses a new and effective drug target i.e., the Two-Component System, PdtaS-PdtaR signalling cascade that can be used as a target to combat the bacterial pathogens. In a broad sense, targeting PdtaS-PdtaR signalling cascade would help in inhibiting any bacterial pathogen which has a functional PdtaS-PdtaR signalling cascade. The PdtaS-PdtaR signalling cascade can be targeted not only to combat the causative agent of tuberculosis, but also leprosy and other mycobacterial diseases. Due to the highly conserved and important nature of the PdtaSR signalling cascade, drugs against this pathway may also be effective against drug resistant strains of other pathogenic bacteria as well. Two-Component Signalling systems (TCSs) are one of the key pathways used by bacteria to sense and respond to changes in both internal and external environments. In the human pathogen M. tuberculosis, TCSs are involved in host cell invasion, intracellular survival, intracellular multiplication as well as dormancy during the infection process. Many of these TCSs are also conserved across multiple species of Mycobacteria and perform many similar functions in these species as well.

[0065] One such TCS, the PdtaS-PdtaR system is conserved across all species of Mycobacteria as well as soil-dwelling non-pathogenic species of Actinobacteria. The presence of this TCS across multiple species underscores the importance of this signalling system in the basic biology of these bacteria.

[0066] The signalling system disclosed as a target here is completely absent in all eukaryotic organisms and in any other bacteria apart from Mycobacteria, Corynebacterium, and any species belonging to suborder Corynebacterineae, thereby having the advantage of limited side effects during targeted therapy. Furthermore, as mentioned above, the major limitation in using TCS as targets is lack of knowledge about their ligands, which is addressed in the present disclosure. The process as disclosed in the present disclosure also imparts further specificity in a manner since the TCS disclosed as a target in the present disclosure is absent in other bacterial species apart from Mycobacterium and Corynebacterium species, and any species belonging to suborder Corynebacterineae. Therefore, the candidate molecule screened herein shall selectively target only Mycobacterium and Corynebacterium species and not any other bacterial species. TCS typically is made up of a sensor kinase (SK) and a response regulator (RR). The disclosure also reports multiple assays based on typical TCS which can be utilized for screening inhibitors, including inhibitors to the ligand-binding site of the SK, which has been disclosed in the present disclosure.

[0067] The present disclosure discloses different in-vitro processes targeting the functionality of the PdtaS-PdtaR signalling system. The present disclosure further establishes that interfering with the functionality of the TCS (PDtaS-PdtaR) interferes with the ability of the growth of the organism under nutrient-deprived conditions. The intervention is with respect to either autophosphorylation of PdtaS, or transphosphorylation of PdtaR, or interaction between PdtaS and PdtaR, or interaction between activated PdtaR and a target RNA, including any combination of these steps. In one aspect of the present disclosure, the comparison is made to analyse the level of phosphorylation of PdtaS, and/or PdtaR under respective conditions. The analysis is made by observing the band intensity obtained in the autoradiogram.

[0068] In an embodiment of the present disclosure, there is provided an in-vitro process for screening a candidate molecule capable of inhibiting growth of a bacterial pathogen, said process comprises: (a) obtaining PdtaS protein; (b) performing phosphorylation assay by incubating PdtaS protein in the presence of ATP under in-vitro conditions, to obtain a product A, and analysing phosphorylation of the PdtaS protein of the product A; (c) obtaining a candidate molecule; (d) performing phosphorylation assay by incubating PdtaS protein in the presence of ATP and the candidate molecule under in- vitro conditions, to obtain a product B, and analysing phosphorylation of the PdtaS protein of the product B; and (e) comparing the phosphorylation of the PdtaS protein of the product B to the phosphorylation of the PdtaS protein of the product A, wherein a decrease in the phosphorylation of the PdtaS protein of the product B as compared to the phosphorylation of the PdtaS protein of the product A indicates capability of the candidate molecule in inhibiting growth of a bacterial pathogen. In another embodiment, the decrease in the phosphorylation is at least 1.5 folds.

[0069] In an embodiment of the present disclosure, there is provided an in-vitro process for screening a candidate molecule capable of inhibiting growth of a bacterial pathogen, said process comprises: (a) obtaining PdtaS protein; (b) performing phosphorylation assay by incubating PdtaS protein in the presence of at least one phosphor-donor molecule under in-vitro conditions, to obtain a product A, and analysing phosphorylation of the PdtaS protein of the product A; (c) obtaining a candidate molecule; (d) performing phosphorylation assay by incubating PdtaS protein in the presence of at least one phosphor-donor molecule and the candidate molecule under in- vitro conditions, to obtain a product B, and analysing phosphorylation of the PdtaS protein of the product B; and (e) comparing the phosphorylation of the PdtaS protein of the product B to the phosphorylation of the PdtaS protein of the product A, wherein a decrease in the phosphorylation of the PdtaS protein of the product B as compared to the phosphorylation of the PdtaS protein of the product A indicates capability of the candidate molecule in inhibiting growth of a bacterial pathogen.

[0070] In an embodiment of the present disclosure, there is provided an in-vitro process for screening a candidate molecule capable of inhibiting reactivation of the bacterial pathogen, said process comprises: (a) obtaining PdtaS protein; (b) performing phosphorylation assay by incubating PdtaS protein in the presence of at least one phosphor-donor molecule under in-vitro conditions, to obtain a product A, and analysing phosphorylation of the PdtaS protein of the product A; (c) obtaining a candidate molecule; (d) performing phosphorylation assay by incubating PdtaS protein in the presence of at least one phosphor-donor molecule and the candidate molecule under in-vitro conditions, to obtain a product B, and analysing phosphorylation of the PdtaS protein of the product B; and (e) comparing the phosphorylation of the PdtaS protein of the product B to the phosphorylation of the PdtaS protein of the product A, wherein a decrease in the phosphorylation of the PdtaS protein of the product B as compared to the phosphorylation of the PdtaS protein of the product A indicates capability of the candidate molecule in inhibiting reactivation of the bacterial pathogen.

[0071] In an embodiment of the present disclosure, there is provided an in-vitro process for screening a candidate molecule capable of inhibiting a bacterial pathogen from entering into latency, said process comprises: (a) obtaining PdtaS protein; (b) performing phosphorylation assay by incubating PdtaS protein in the presence of at least one phosphor-donor molecule under in-vitro conditions, to obtain a product A, and analysing phosphorylation of the PdtaS protein of the product A; (c) obtaining a candidate molecule; (d) performing phosphorylation assay by incubating PdtaS protein in the presence of at least one phosphor-donor molecule and the candidate molecule under in-vitro conditions, to obtain a product B, and analysing phosphorylation of the PdtaS protein of the product B; and (e) comparing the phosphorylation of the PdtaS protein of the product B to the phosphorylation of the PdtaS protein of the product A, wherein a decrease in the phosphorylation of the PdtaS protein of the product B as compared to the phosphorylation of the PdtaS protein of the product A indicates capability of the candidate molecule in inhibiting the bacterial pathogen from entering into latency.

[0072] In an embodiment of the present disclosure, there is provided an in-vitro process for screening a candidate molecule capable of inhibiting growth of a bacterial pathogen, said process comprises: (a) obtaining PdtaS protein, and PdtaR protein; (b) performing phosphorylation assay by incubating PdtaS protein in the presence of ATP and PdtaR protein under in-vitro conditions, to obtain a product C, and analysing the interaction of PdtaS protein and PdtaR protein of the product C; (c) obtaining a candidate molecule; (d) performing phosphorylation assay by incubating PdtaS protein in the presence of ATP, PdtaR protein, and the candidate molecule under in-vitro conditions, to obtain a product D, and analysing interaction of PdtaS protein and PdtaR protein of the product D; and (e) comparing the interaction of PdtaS protein and PdtaR protein of the product D to the interaction of PdtaS protein and PdtaR protein of the product C, wherein a decrease in interaction of PdtaS protein and PdtaR protein of the roduct D as compared to the interaction of PdtaS and PdtaR protein of the incubated product C indicates capability of the candidate molecule in inhibiting growth of a bacterial pathogen.

[0073] In an embodiment of the present disclosure, there is provided an in-vitro process for screening a candidate molecule capable of inhibiting growth of a bacterial pathogen, said process comprises: (a) obtaining PdtaS protein, and PdtaR protein; (b) performing phosphorylation assay by incubating PdtaS protein in the presence of at least one phosphor-donor molecule and PdtaR protein under in-vitro conditions, to obtain a product C, and analysing interaction of PdtaS protein and PdtaR protein of the product C; (c) obtaining a candidate molecule; (d) performing phosphorylation assay by incubating PdtaS protein in the presence of at least one phosphor-donor molecule, PdtaR protein, and the candidate molecule under in-vitro conditions, to obtain a product D, and analysing interaction of PdtaS protein and PdtaR protein of the product D; and (e) comparing the interaction of PdtaS protein and PdtaR protein of the product D to the interaction of PdtaS protein and PdtaR protein of the product C, wherein a decrease in interaction of PdtaS protein and PdtaR protein of the product D as compared to the interaction of PdtaS and PdtaR protein of the product C indicates capability of the candidate molecule in inhibiting growth of a bacterial pathogen.

[0074] In an embodiment of the present disclosure, there is provided an in-vitro process for screening a candidate molecule capable of inhibiting reactivation of a bacterial pathogen, said process comprises: (a) obtaining PdtaS protein, and PdtaR protein; (b) performing phosphorylation assay by incubating PdtaS protein in the presence of at least one phosphor-donor molecule and PdtaR protein under in-vitro conditions, to obtain a product C, and analysing interaction of PdtaS protein and PdtaR protein of the product C; (c) obtaining a candidate molecule; (d) performing phosphorylation assay by incubating PdtaS protein in the presence of at least one phosphor-donor molecule, PdtaR protein, and the candidate molecule under in-vitro conditions, to obtain a product D, and analysing interaction of PdtaS protein and PdtaR protein of the product D; and (e) comparing the interaction of PdtaS protein and PdtaR protein of the product D to the interaction of PdtaS protein and PdtaR protein of the product C, wherein a decrease in interaction of PdtaS protein and PdtaR protein of the product D as compared to the interaction of PdtaS and PdtaR protein of the product C indicates capability of the candidate molecule in inhibiting reactivation of a bacterial pathogen. [0075] In an embodiment of the present disclosure, there is provided an in-vitro process for screening a candidate molecule capable of inhibiting a bacterial pathogen from entering into latency, said process comprises: (a) obtaining PdtaS protein, and PdtaR protein; (b) performing phosphorylation assay by incubating PdtaS protein in the presence of at least one phosphor-donor molecule and PdtaR protein under in-vitro conditions, to obtain a product C, and analysing interaction of PdtaS protein and PdtaR protein of the product C; (c) obtaining a candidate molecule; (d) performing phosphorylation assay by incubating PdtaS protein in the presence of at least one phosphor-donor molecule, PdtaR protein, and the candidate molecule under in-vitro conditions, to obtain a product D, and analysing interaction of PdtaS protein and PdtaR protein of the product D; and (e) comparing the interaction of PdtaS protein and PdtaR protein of the product D to the interaction of PdtaS protein and PdtaR protein of the product C, wherein a decrease in interaction of PdtaS protein and PdtaR protein of the product D as compared to the interaction of PdtaS and PdtaR protein of the product C indicates capability of the candidate molecule in inhibiting the bacterial pathogen from entering into latency.

[0076] In an embodiment of the present disclosure, there is provided an in-vitro process for screening a candidate molecule capable of inhibiting growth of a bacterial pathogen, said process comprises: (a) obtaining PdtaS protein; (b) incubating PdtaS protein in the presence of c-di-GMP under in-vitro conditions, to obtain a product E, and analysing interaction between PdtaS protein and c-di-GMP in the product E; (c) obtaining a candidate molecule; (d) incubating PdtaS protein in the presence of c-di-GMP, and the candidate molecule under in-vitro conditions, to obtain a product F, and analysing interaction between PdtaS protein and c-di-GMP in the product F; and (e) comparing interaction between PdtaS protein and c-di-GMP in the product F with interaction between PdtaS protein and c-di-GMP in the product E for observing an increase or decrease in interaction of PdtaS protein and c-di-GMP in the product F as compared to the product E, wherein a decrease in the interaction between PdtaS protein and c-di- GMP in the product F as compared to the interaction between PdtaS protein and c-di- GMP in the product E indicates capability of the candidate molecule in inhibiting growth of a bacterial pathogen. In another embodiment, the interaction analysed between PdtaS protein and c-di-GMP is phosphorylation of PdtaS protein in the presence of c-di-GMP. In yet another embodiment, incubating under the in-vitro conditions refer to the incubation done to provide conducive environment for interaction of PdtaS protein and c-di-GMP.

[0077] In an embodiment of the present disclosure, there is provided an in-vitro process for screening a candidate molecule capable of inhibiting reactivation of a bacterial pathogen, said process comprises: (a) obtaining PdtaS protein; (b) incubating PdtaS protein in the presence of c-di-GMP under in-vitro conditions, to obtain a product E, and analysing interaction between PdtaS protein and c-di-GMP in the product E; (c) obtaining a candidate molecule; (d) incubating PdtaS protein in the presence of c-di- GMP, and the candidate molecule under in-vitro conditions, to obtain a product F, and analysing interaction between PdtaS protein and c-di-GMP in the product F; and (e) comparing interaction between PdtaS protein and c-di-GMP in the product F with interaction between PdtaS protein and c-di-GMP in the product E for observing an increase or decrease in interaction of PdtaS protein and c-di-GMP in the product F as compared to the product E, wherein a decrease in the interaction between PdtaS protein and c-di-GMP in the product F as compared to the interaction between PdtaS protein and c-di-GMP in the product E indicates capability of the candidate molecule in inhibiting reactivation of a bacterial pathogen.

[0078] In an embodiment of the present disclosure, there is provided an in-vitro process for screening a candidate molecule capable of inhibiting growth of a bacterial pathogen, said process comprises: (a) obtaining PdtaR protein; (b) obtaining a target RNA molecule, wherein the target RNA molecule is able to bind to PdtaR protein; (c) incubating PdtaR protein in the presence of the target RNA molecule under in-vitro conditions, to obtain a product G, and analysing interaction between PdtaR protein and the target RNA in the product G; (d) obtaining a candidate molecule; (e) incubating PdtaR protein in the presence of the target RNA molecule and the candidate molecule under in-vitro conditions, to obtain a product H, and analysing the interaction between PdtaR protein and the target RNA in the product H; and (f) comparing interaction between PdtaR protein and the target RNA in the product H with interaction between PdtaR protein and the target RNA in the product G for observing an increase or decrease in interaction of PdtaR protein and the target RNA in the product H as compared to the product G, wherein a decrease in interaction between PdtaR protein and the target RNA in the product H as compared to the interaction between PdtaR protein and the target RNA in the product G indicates capability of the candidate molecule in inhibiting growth of a bacterial pathogen. In another embodiment, the interaction between PdtaR protein and target RNA is in form of binding. In yet another embodiment, incubating under in-vitro conditions refer to the appropriate conditions conducive for PdtaR protein and target RNA to interact.

[0079] In an embodiment of the present disclosure, there is provided an in-vitro process for screening a candidate molecule capable of inhibiting growth of a bacterial pathogen, said process comprises: (a) obtaining PdtaS protein, and PdtaR protein; (b) performing phosphorylation assay by incubating PdtaS protein in the presence of ATP and PdtaR protein under in-vitro conditions, to obtain a product I; (c) obtaining a target RNA molecule; (d) incubating the target RNA with the product I under in-vitro conditions, to obtain a product J, and analysing interaction between PdtaR protein and the target RNA in the product J ; (e) obtaining a candidate molecule; (f) incubating the target RNA with the product I in presence of the candidate molecule under in-vitro conditions, to obtain a product K, and analysing interaction between PdtaR protein and the target RNA in the product K; and (g) comparing interaction between PdtaR protein and the target RNA in the product K with interaction between PdtaR protein and the target RNA in the product J for observing an increase or decrease in interaction of PdtaR protein and the target RNA in the product K as compared to the product J, wherein a decrease in interaction between PdtaR protein and the target RNA in the incubated product K as compared to the interaction between PdtaR protein and the target RNA in the product J indicates capability of the candidate molecule in inhibiting growth of a bacterial pathogen. In another embodiment, the interaction PdtaR protein and the target RNA is in form of binding. In yet another embodiment, incubating the target RNA with the product I under in-vitro conditions refer to the conditions conducive for target RNA to interact with the product I, and incubating the target RNA of step (c) with the product I of step (b) in the presence of the candidate molecule under appropriate conditions refer to the in-vitro conditions conducive for the target RNA to interact with the product I in presence of the candidate molecule.

[0080] In an embodiment of the present disclosure, there is provided an in-vitro process for screening a candidate molecule capable of inhibiting growth of a bacterial pathogen, said process comprises: (a) obtaining PdtaS protein, and PdtaR protein; (b) performing phosphorylation assay by incubating PdtaS protein in the presence of at least one phosphor-donor molecule and PdtaR protein under in-vitro conditions, to obtain a product I; (c) obtaining a target RNA molecule; (d) incubating the target RNA with the product I under in-vitro conditions, to obtain a product J, and analysing interaction between PdtaR protein and the target RNA in the incubated product J; (e) obtaining a candidate molecule; (f) incubating the target RNA with the product I in presence of the candidate molecule under in-vitro conditions, to obtain a product K, and analysing interaction between PdtaR protein and the target RNA in the product K; and (g) comparing interaction between PdtaR protein and the target RNA in the product K with interaction between PdtaR protein and the target RNA in the product J for observing an increase or decrease in interaction of PdtaR protein and the target RNA in the product K as compared to the product J, wherein a decrease in interaction between PdtaR protein and the target RNA in the incubated product K as compared to the interaction between PdtaR protein and the target RNA in the product J indicates capability of the candidate molecule in inhibiting growth of a bacterial pathogen.

[0081] In an embodiment of the present disclosure, there is provided an in-vitro process for screening a candidate molecule capable of inhibiting growth of a bacterial pathogen, said process comprises: (a) obtaining PdtaS protein, and PdtaR protein; (b) incubating PdtaS protein in the presence of PdtaR protein under in-vitro conditions, to obtain a product L, and analysing interaction between PdtaS protein and PdtaR protein in the product L; and (c) obtaining a candidate molecule; (d) incubating PdtaS protein in the presence of PdtaR protein and the candidate molecule, to obtain a product M, and analysing interaction between PdtaS protein and PdtaR protein in the product M; and (e) comparing interaction between PdtaS protein and PdtaR protein in the product M to interaction between PdtaS protein and PdtaR protein in the product L for observing an increase or decrease in interaction of PdtaS protein and PdtaR protein in the product M as compared to the product L, wherein a decrease in interaction between PdtaS protein and PdtaR protein in the product M as compared to the interaction between PdtaS protein and PdtaR protein in the product L indicates capability of the candidate molecule in inhibiting growth of a bacterial pathogen. In another embodiment, the interaction between PdtaS protein and PdtaR protein is in form of binding. In yet another embodiment, incubating PdtaS protein in the presence of PdtaR protein under in-vitro conditions refer to the conditions conducive for PdtaS protein to interact with PdtaR protein, and incubating PdtaS protein of step (a) in the presence of PdtaR protein of step (a) and the candidate molecule refer to the conditions conducive for PdtaS protein to interact with PdtaR protein in the presence of the candidate molecule.

[0082] In an embodiment of the present disclosure, there is provided an in-vitro process for screening a candidate molecule capable of inhibiting growth of a bacterial pathogen as described herein, wherein the bacterial pathogen is selected from a group consisting of Mycobacterium species, Corynebacterium species and any other genus containing homologs of PdtaS and PdtaR proteins.

[0083] In an embodiment of the present disclosure, there is provided an in-vitro process for screening a candidate molecule capable of inhibiting growth of a bacterial pathogen as described herein, wherein the bacterial pathogen is selected from a group consisting of Mycobacterium species, Corynebacterium species and any other genus containing homologs of PdtaS and PdtaR proteins, and wherein the Mycobacterium species is selected from a group consisting of any Mycobacterium species including but not limited to ones selected from a group consisting of Mycobacterium tuberculosis, Mycobacterium smegmatis, Mycobacterium leprae, and Mycobacterium bovis.

[0084] In an embodiment of the present disclosure, there is provided an in-vitro process for screening a candidate molecule capable of inhibiting growth of a bacterial pathogen as described herein, wherein the bacterial pathogen is selected from a group consisting of Mycobacterium species, Corynebacterium species and any other genus containing homologs of PdtaS and PdtaR proteins, and wherein the Mycobacterium species is selected from a group consisting of any Mycobacterium species including but not limited to ones selected from a group consisting of Mycobacterium tuberculosis, Mycobacterium smegmatis, Mycobacterium leprae, and Mycobacterium bovis, and wherein the Mycobacterium species is Mycobacterium tuberculosis.

[0085] In an embodiment of the present disclosure, there is provided an in-vitro process for screening a candidate molecule capable of inhibiting growth of a bacterial pathogen as described herein, wherein the candidate molecule is capable of inhibiting reactivation of the bacterial pathogen.

[0086] embodiment of the present disclosure, there is provided an in-vitro process for screening a candidate molecule capable of inhibiting growth of a bacterial pathogen as described herein, wherein the candidate molecule is capable of inhibiting the bacterial pathogen from entering latency.

[0087] In an embodiment of the present disclosure, there is provided an in-vitro process for screening a candidate molecule capable of inhibiting growth of a bacterial pathogen as described herein, wherein the in-vitro conditions for performing in-vitro phosphorylation is well-known in the art.

[0088] In an embodiment of the present disclosure, there is provided an in-vitro process for screening a candidate molecule capable of inhibiting growth of a bacterial pathogen as described herein, wherein the in-vitro conditions is in presence of kinase buffer (50mM tris-Cl pH 8, 50mM KC1, lOmM MgC12) containing l pCi of [g32R] ATP for 60min or for time points specified at 30°C. [0089] In an embodiment of the present disclosure, there is provided an in-vitro process for screening a candidate molecule capable of inhibiting growth of a bacterial pathogen as described herein, wherein the comparing the phosphorylation of the PdtaS protein of the product B to the phosphorylation of the PdtaS protein of the product A is done by methods well-known in the art.

[0090] In an embodiment of the present disclosure, there is provided an in-vitro process for screening a candidate molecule capable of inhibiting growth of a bacterial pathogen as described herein, wherein comparing the interaction of PdtaS protein and PdtaR protein of the product D to the interaction of PdtaS protein and PdtaR protein of the product C is done by methods well-known in the art.

[0091] In an embodiment of the present disclosure, there is provided an in-vitro process for screening a candidate molecule capable of inhibiting growth of a bacterial pathogen as described herein, wherein comparing interaction between PdtaS protein and c-di- GMP in the product F with interaction between PdtaS protein and c-di-GMP in the product E for observing an increase or decrease is done by methods well-known in the art.

[0092] In an embodiment of the present disclosure, there is provided an in-vitro process for screening a candidate molecule capable of inhibiting growth of a bacterial pathogen as described herein, wherein comparing interaction between PdtaR protein and the target RNA in the product H with interaction between PdtaR protein and the target RNA in the product G for observing an increase or decrease is done by methods well- known in the art.

[0093] In an embodiment of the present disclosure, there is provided an in-vitro process for screening a candidate molecule capable of inhibiting growth of a bacterial pathogen as described herein, wherein comparing interaction between PdtaR protein and the target RNA in the product K with interaction between PdtaR protein and the target RNA in the product J for observing an increase or decrease is done by methods well-known in the art. [0094] In an embodiment of the present disclosure, there is provided an in-vitro process for screening a candidate molecule capable of inhibiting growth of a bacterial pathogen as described herein, wherein comparing interaction between PdtaS protein and PdtaR protein in the product M to interaction between PdtaS protein and PdtaR protein in the product L for observing an increase or decrease is done by methods well- known in the art.

[0095] In an embodiment of the present disclosure, there is provided an in-vitro process for screening a candidate molecule capable of inhibiting growth of a bacterial pathogen as described herein, wherein PdtaS protein has an amino acid sequence as set forth in SEQ ID NO: 1.

[0096] In an embodiment of the present disclosure, there is provided an in-vitro process for screening a candidate molecule capable of inhibiting growth of a bacterial pathogen as described herein, wherein PdtaR protein has an amino acid sequence as set forth in SEQ ID NO: 3.

[0097] In an embodiment of the present disclosure, there is provided a recombinant strain of Mycobacterium species comprising a mutation in a gene encoding PdtaS protein.

[0098] In an embodiment of the present disclosure, there is provided a use of PdtaS and PdtaR interaction in screening for a candidate molecule capable of inhibiting growth of Mycobacterium species.

[0099] In an embodiment of the present disclosure, there is provided a use of PdtaR and target RNA interaction in screening for a candidate molecule capable of inhibiting growth of Mycobacterium species.

[00100] In an embodiment of the present disclosure, there is provided a use of PdtaS and ci-di-GMP interaction in screening for a candidate molecule capable of inhibiting growth of Mycobacterium species.

[00101] In an embodiment of the present disclosure, there is provided a use of PdtaS and PdtaR interaction in screening for a candidate molecule capable of inhibiting reactivation of Mycobacterium species. [00102] In an embodiment of the present disclosure, there is provided a use of PdtaR and target RNA interaction in screening for a candidate molecule capable of inhibiting reactivation of Mycobacterium species.

[00103] In an embodiment of the present disclosure, there is provided a use of PdtaS and ci-di-GMP interaction in screening for a candidate molecule capable of inhibiting reactivation of Mycobacterium species.

[00104] In an embodiment of the present disclosure, there is provided a use of PdtaS and PdtaR interaction in screening for a candidate molecule capable of inhibiting Mycobacterium species from entering latency.

[00105] In an embodiment of the present disclosure, there is provided a use of PdtaR and target RNA interaction in screening for a candidate molecule capable of inhibiting Mycobacterium species from entering latency.

[00106] In an embodiment of the present disclosure, there is provided a use of PdtaS and ci-di-GMP interaction in screening for a candidate molecule capable of inhibiting Mycobacterium species from entering latency.

[00107] In an embodiment of the present disclosure, there is provided a use of PdtaS and PdtaR interaction in screening for a candidate molecule capable of inhibiting growth of a bacterial pathogen.

[00108] In an embodiment of the present disclosure, there is provided a use of PdtaR and target RNA interaction in screening for a candidate molecule capable of inhibiting growth of a bacterial pathogen.

[00109] In an embodiment of the present disclosure, there is provided a use of PdtaS and ci-di-GMP interaction in screening for a candidate molecule capable of inhibiting growth of a bacterial pathogen.

[00110] In an embodiment of the present disclosure, there is provided a use of PdtaS and PdtaR interaction in screening for candidate molecule capable of inhibiting growth of a bacterial pathogen, wherein the bacterial pathogen is selected from a group consisting of Mycobacterium species, Corynebacterium species, and any other bacterial organism belonging to suborder Corynebacterineae harbouring homologs of PdtaS and PdtaR proteins.

[00111] In an embodiment of the present disclosure, there is provided a use of PdtaR and target RNA interaction in screening for candidate molecule capable of inhibiting growth of a bacterial pathogen, wherein the bacterial pathogen is selected from a group consisting of Mycobacterium species, Corynebacterium species, and any other bacterial organism belonging to suborder Corynebacterineae harbouring homologs of PdtaS proteins.

[00112] In an embodiment of the present disclosure, there is provided a use of PdtaS and ci-di-GMP interaction in screening for candidate molecule capable of inhibiting growth of a bacterial pathogen, the bacterial pathogen is selected from a group consisting of Mycobacterium species, Corynebacterium species, and any other bacterial organism belonging to suborder Corynebacterineae harbouring homologs of PdtaR proteins.

[00113] Although the subject matter has been described with reference to specific embodiments, this description is not meant to be construed in a limiting sense. Various modifications of the disclosed embodiments, as well as alternate embodiments of the subject matter, will become apparent to persons skilled in the art upon reference to the description of the subject matter. It is therefore contemplated that such modifications can be made without departing from the spirit or scope of the present subject matter as defined.

EXAMPLES

[00114] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices, and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.

[00115] The present section details various in-vitro screening methods that can be used for screening various candidate molecules. PdtaS-PdtaR system is largely uncharacterized from a signalling perspective. It is not known what the sensor kinase PdtaS senses, how PdtaS interacts with PdtaR or what adaptive changes are orchestrated by PdtaR upon activation through PdtaS? In essence, the function or importance of this TCS in the physiology of Mycobacteria and/or Corynebacterium species are yet to be completely delineated. Figure 1 shows a schematic representation of generalized TCS activation which is applicable to PdtaS (SK) and PdtaR (RR) as well. In a typical scheme, the sensor kinase (SK) senses a signal (Step I), undergoes autophosphorylation (Step II), and then transfers the phosphate to the response regulator (RR) protein (Step III) through a process of direct physical interaction. The

RR upon phosphorylation binds to DNA or RNA to change the gene expression or protein levels and thus bring about adaptive changes (Step IV). The cascade can be targeted at the following steps:

Step I: Binding of the ligand to the sensor kinase (akin to GPCR binding to ligand) Step II: Autophosphorylation of SK

Step III: Phosphor-transfer from SK to RR which necessitates interaction between them Step IV: Binding of RR to the target DNA/ RNA

[00116] The forthcoming Examples will relate to in-vitro processes which can be used to screen for candidate molecules capable of targeting the PdtaS-PdtaR signalling cascade by exploiting the above-mentioned steps.

Example 1

PdtaS is crucial for growth of M. smegmatis in nutrient-deprived conditions [00117] All cloning steps were carried out in Escherichia coli DH 10b (Thermo Fisher Scientific). For protein purification, the expression strain, Arctic Express (DE3) (Agilent Technologies) was used. All E. coli were grown in Luria-Bertani (LB) medium with appropriate antibiotics at 37°C with shaking at 150rpm. Antibiotics were used at the following concentrations - ampicillin lOOmg/1, kanamycin 25mg/l, anhydrotetracycline 100pg/l, and hygromycin lOOmg/1.

[00118] Mycobacterium smegmatis me²155 was grown in Middlebrook 7H9 Broth Base (HiMedia) or Tryptone Soya Broth (HiMedia) at 37°C with shaking at 150rpm. 7H9 was supplemented with 0.025% tyloxapol and 0.2% glycerol unless otherwise specified.

[00119] The gene coding for PdtaS from M. tuberculosis H37Rv was PCR amplified using isolated genomic DNA as a template, Pfu polymerase and gene-specific primers containing appropriate restriction sites at their ends. PCR amplified genes were cloned into appropriate vectors for E. coli and mycobacterial expression. All constructs were verified by sequencing after site-directed mutagenesis for the generation of point mutations as well as domain deletions, which were performed using Quick Change Site-Directed Mutagenesis protocol using Phusion polymerase (New England BioLabs, USA). Mutations/deletions introduced were verified by DNA sequencing.

[00120] Figure 2 depicts the growth analysis of wildtype and ApdtaS M. smegmatis (mutant strain) in a nutrient-deprived medium (consisting of 7H9 base medium supplemented with 0.2% glycerol and 0.025% tyloxapol). It was observed that the mutant strain was not able to grow well in the nutrient-deprived medium as compared to the wildtype strain. Therefore, it suggests that a functional PdtaS protein is required for the growth of the strain under nutrient-deprived conditions. The present Example forms the basis of the intervention of PdtaS protein, wherein the intervention is done at the step of autophosphorylation of PdtaS protein. Hence, any candidate molecule capable of reducing the phosphorylation of PdtaS protein in Mycobacterium species will result in reducing the growth of the pathogen. Example 2

Growth of M. smegmatis requires functional c-di-GMP binding PdtaS

[00121] The docking of c-di-GMP to the GAF ligand-binding pocket (Figure 3) provided details of stabilizing interactions between the GAF pocket residues and c-di- GMP. In order to see if c-di-GMP binding was essential for PdtaS -mediated adaptation to nutrient deprivation, the Trp43 residue was mutated to Ala in the complementation plasmid and electroporated into AmspdtaS to create A:W43A strain. The strain A:mtpdtaS refers to a complement strain having unmutated pdaS.

[00122] Figure 4 depicts growth of various strains under different dilution factors in nutrient-deprived medium. It can be observed that the mutated strain was not able to grow beyond a dilution of -4, whereas the wildtype was able to grow much further beyond that. It can also be observed that the complement strain having mutated pdtaS was not able to grow beyond a dilution factor of -4, whereas the strain having unmutated pdtaS gene showed a growth pattern similar to that of the wildtype. This experiment proves that PdtaS having a functional c-di-GMP binding site is required for growth under nutrient-deprived conditions. The present Example forms the basis of intervention of growth in the step of interaction of c-di-GMP and PdtaS. A candidate molecule capable of reducing or inhibiting the interaction of c-di-GMP and PdtaS will inhibit the growth of the pathogen.

Example 3

Analysis of binding affinity of c-di-GMP to PdtaS-EGFP using MicroScale Thermophoresis (MST)

Methodology

[00123] MicroScale Thermophoresis (MST) experiments were performed using the

Monolith NT.115 system (NanoTemper Technologies). The laser power was set at 20% and the LED at 100%. Equilibrated mixtures containing 5mM of PdtaS-EGFP and varying concentrations of c-di-GMP were loading onto hydrophobic capillaries (NanoTemper Technologies). The laser on and off times were set at 30s and 5s, respectively. Normalized fluorescence change was plotted against the concentration of c-di-GMP to get the binding curve. Curve fitting using NT analysis software was used to find KD values (htips://doi .org/1 (i.1016/j.molsttuc.2014.03.009).

[00124] Figure 5 depicts the binding of c-di-GMP to PdtaS-EGFP using MST. MicroScale thermophoresis of PdtaS-EGFP in the presence of increasing concentrations of cyclic-di-GMP. KD = 193 +/- 40 nM (n = 3). Identification of small molecule, cyclic di-guanosine monophosphate (c-di-GMP) as a ligand for PdtaS, which binds with very high affinity, 200 nM (Figure 5). This step can be used as a screen for inhibitors that compete with c-di-GMP binding, which have been shown to be important for PdtaS-mediated growth during nutrient deprivation. A decrease in interaction of c-di-GMP with PdtaS in presence of any candidate molecule reflects the ability of the candidate molecule in interfering with the signalling cascade, thereby inhibiting the growth of the organism. Example 4

Analysis of c-di-GMP activation of PdtaS

Methodology

[00125] Autophosphorylation and phosphor-transfer assays: Purified SKs (PdtaR) (150pmol unless stated otherwise) were incubated in kinase buffer (50mM tris-Cl pH 8, 50mM KC1, lOmM MgCb) containing l pCi of [g³²R] ATP for 60m in or for time points specified at 30°C. Reactions were terminated by the addition of IX SDS-PAGE loading buffer (2% SDS, 50mM Tris-Cl pH 8, 0.02% bromophenol blue, 1% b- mercaptoethanol, 10% glycerol) and loaded onto the wells of an SDS-PAGE gel. Samples were resolved on a 15% SDS-PAGE gel, washed with distilled water and exposed to a phosphor screen (Fujifilm Bas cassette) overnight followed by imaging with a Typhoon 9210 phosphor-imager (GE Healthcare).

[00126] For the effect of c-di-GMP on autophosphorylation, 150pmol of PdtaS was mixed with various concentrations of c-di-GMP and incubated in kinase buffer containing [g32R] ATP. In order to study the phosphor-transfer from SK to RR, SK~P was prepared as described above, following which 150pmol of purified RR diluted in IX kinase buffer was added to the reaction. The reaction was incubated at 30°C for 15min unless otherwise specified followed by reaction termination by the addition of IX SDS-PAGE loading buffer. The proteins were resolved on 15% SDS-PAGE gels followed by autoradiography.

[00127] Figure 6A and 6B depicts the autophosphorylation of PdtaS in the presence of c-di-GMP. Figure 6A is the graphical representation of band intensity, and Figure 6B shows the autoradiograph and the respective CBB stained polyacrylamide gel.

[00128] The binding of c-di-GMP to PdtaS is activating in nature, and results in an increase in the autophosphorylation of PdtaS in a dose-dependent manner (Figure 6). Therefore, it can be appreciated that c-di-GMP is an activator of PdtaS phosphorylation.

[00129] Figure 13 depicts the effect of c-di-GMP on the kinase activity of PdtaS sensor kinase measure through radioactive phosphoryl group incorporation, where ATP is the donor molecule. Figure 13 depicts the reaction at pH 7.5 and pH 6.5. It can be observed that the in-vitro phosphorylation assay at pH 7.5 shows an increase in phosphorylation in the presence of cyclic-di-GMP as compared to the reaction at pH 6.5.

[00130] The step of binding of c-di-GMP to PdtaS can be a potential step for the screening of candidate molecules capable of interfering at this step. Therefore, a decrease in binding between c-di-GMP and PdtaS in the presence of a candidate molecule as compared to the binding between c-di-GMP and PdtaS in absence of any candidate molecule will expose the ability of the candidate molecule in interfering in the signalling cascade of PdtaS-PdtaR, therefore, using it as a valuable screening method. Further, it was investigated whether a functional PdtaS capable of autophosphorylation is required for the growth of M. smegmatis under nutrient- deprived conditions.

Example 5

Requirement of a functional PdtaS for growth under nutrient-deprived condition Methodology

[00131] Primary cultures of M. smegmatis strains were grown in TSB for 24h or until the O.D. reached 0.8- 1.0, after which they were sub-cultured at a starting ODeoo of 0.005 in the medium in which the experiment was carried out. For starvation experiments, primary cultures were washed thrice with 7H9 alone (no carbon source, no supplements) followed by resuspension in 7H9 alone for 8h, following which they were sub-cultured at a starting O.D. of 0.005 in the medium used for the growth curve. A single time point assay for the growth of M. smegmatis was standardized using spot dilutions on tryptone soy agar (TSA) (HiMedia) plates. Briefly, primary cultures of M. smegmatis strains were inoculated in tubes containing the appropriate medium at an O.D. of 0.005. Cultures were allowed to grow for 30h (mid-log phase for wildtype cells) following which serial ten-fold dilutions of cultures were made in 7H9 base medium. 20m1 of each dilution was spotted carefully on the surface of TSA plates containing appropriate antibiotics. Spots were allowed to dry (no visible moisture on the agar surface) following which they were incubated at 37°C for 2-3 days to allow for the growth of single colonies.

[00132] A mutant strain obtained by mutating histidine residue responsible for autophosphorylation was checked for its ability to grow under nutrient-deprived condition. Figure 7 depicts the growth ability of AmspdtaS (mutant strain), A:mtpdtaS (complement strain with functional pdtaS), A:H303Q (complement strain with mutated pdtaS) as compared to the wildtype strain. As was observed from Example 4, c-di- GMP activates PdtaS, the present Example shows that the activation is necessary for PdtaS mediated adaptation to nutrient deprivation. This is demonstrated by mutating the histidine residue of PdtaS that is involved in phosphorylation (strain A:H303Q ), which leads to impaired growth in nutrient-deprived conditions. The autophosphorylation reaction is also a potential screen as a His mutant of PdtaS, (defective phosphorylation protein), which is unable to rescue the KO phenotype, thereby confirming the essentiality of phosphorylation reaction and a viable step for drug-screening. [00133] The step of autophosphorylation can be used as a potential screening step. A decrease in autophosphorylation of PdtaS in the presence of a candidate molecule reveals the ability of the candidate molecule in interfering with the PdtaS -PdtaR signalling cascade, thereby, forming a potential screening strategy.

[00134] PdtaS mutants (H303Q) were generated using the following primers:

[00135] SEQ ID NO: 5 depicts a primer sequence PdtaSH303Qf.

CCGGGAAATCCATCAGCGGGTTAAGAACAAC.

[00136] SEQ ID NO: 6 depicts a primer sequence PDtaSH303Qr.

GTTGTTCTTAACCCGCTGATGGATTTCCCGG

Example 6

Transphosphorylation of PdtaR in the presence of PdtaS

[00137] The transphosphorylation reaction was performed in presence of PdtaS, to check whether PdtaS, after getting phosphorylated was able to transfer phosphate to PdtaR which is a cognate response regulator of the TCS (PdtaS-PdtaR).

[00138] Figure 8 depicts the autoradiograph and the corresponding CBB stained polyacrylamide gel. It was observed that there was a transphosphorylation reaction that took place from PdtaS to PdtaR and also, the band intensity of PdtaR increased with time going from 2.5 minutes to 20 minutes. This data supports the functionality of the TCS (PdtaS-PdtaR), therefore, the step being a potential screening step to screen for candidate molecules that affect the signaling cascade by intervening at PdtaS transphosphorylation to PdtaR.

[00139] Figure 9 depicts the FRET measurement of PdtaS-PdtaR interaction. It was observed that, in the presence of the response regulator - PdtaR, a significant interaction was observed between the two partners, PdtaS and PdtaR.

[00140] Figure 10 further substantiates the interaction between the two partners by showing the interaction by Biolayer interferometry (BFI). This data further substantiates the functionality of the TCS, therefore, making the step of PdtaS and PdtaR interaction a screening strategy for a potential molecule that can be exploited for its ability to intervene the functional TCS. The present Example forms the basis of intervening the interaction of PdtaS with PdtaR for inhibiting the growth of the pathogen. A candidate molecule capable of inhibiting or reducing the transphosphorylation between PdtaS and PdtaR will inhibit the growth of the pathogen.

Example 7

RNA binding ability of PdtaR is essential for growing under nutrient-deprived conditions

[00141] Figure 11 establishes that the RNA binding ability of PdtaR is essential for the growth of M. smegmatis, where unmutated PdtaR (strain A:mtpdtaR) is required for the growth, whereas binding defective PdtaR (strain A:BM) is not able to grow (Figure 11). This confirms the role for the RR protein in the relay which can also be targeted by inhibitor screens.

[00142] Therefore, the step of RNA binding of PdtaR can also be exploited as a potential screening strategy for screening candidate molecules. The inhibition of PdtaR and RNA interaction in presence of any candidate molecule establishes the ability of candidate molecule to interfere in the PdtaS -PdtaR signalling system.

[00143] PdtaR mutants (mtpdtaR) were generated using the following primers:

[00144] SEQ ID NO: 7 depicts a primer sequence PdtaRD65Vf.

GACCTGGTGATCATGGTCGTGAAGATGCCGC.

[00145] SEQ ID NO: 8 depicts a primer sequence PdtaRD65Vr.

GCGGCATCTTCACGACCATGATCACCAGGTC

Example 8

Analysis of phosphorylation by performing studies with mutant PdtaS and mutant PdtaR proteins

[00146] The mutant proteins - PdtaS and PdtaR were used for auto- and trans phosphorylation studies by a method as described previously. Figure 12 A-C depicts the autoradiogram along with the stained Coomassie blue gel under various conditions. Lanes 1-6 depict the following conditions as per Table 1.

Table 1

1 2 3 4 5 6

[00147] Referring to Table 1 and Figure 12, it can be appreciated that in presence of a mutated PdtaS (mutated at the phosphorylation site - His³⁰³), there is no autophosphorylation of PdtaS and neither a transphosphorylation to PdtaR, whereas in the presence of unmutated PdtaS and PdtaR, both auto- and trans- phosphorylation were observed. The protein PdtaR was mutated at Asp⁶⁵ (phosphate acceptor site).

[00148] The present Example, therefore, proves the requirement of unmutated PdtaS and PdtaR for the functioning of the TCS, thereby, adding more evidence as to the feasibility of using this TCS for screening candidate molecules.

[00149] The present disclosure thus establishes that c-di-GMP binding to PdtaS and PdtaS phosphorylation is important for the growth of Mycobacteria during conditions of nutrient limitation. The RNA binding ability of PdtaR is also essential for growth during conditions of nutrient limitation. The present disclosure also identified the exact binding site of c-di-GMP on PdtaS. All the steps reported in the present disclosure, thereby establishes a detailed analysis of why PdtaSR system is a good target for targeting Mycobacterial species. [00150] The present disclosure discloses the following target, which can be used to intervene the growth of Mycobacterial species.

1. c-di-GMP binding and activation of PdtaS as a target

2. Signal transduction through PdtaS-PdtaR interaction as a target

3. Adaptation to nutrient deprivation through PdtaR-RNA interactions as a drug target.

[00151] In line with the above-mentioned targets and observation in relation with the functioning of the TCS, the following in-vitro processes can be followed for screening of candidate molecules.

1. Screening of inhibitors against c-di-GMP binding to PdtaS

2. Screening of inhibitors against PdtaS activation and autophosphorylation

3. Screening of inhibitors against PdtaS-PdtaR interaction

4. Screening of inhibitors against PdtaR-RNA interactions

[00152] The observations of the present disclosure can be used to develop a vaccine strain that is compromised in virulence and growth during infection but can still provide protection against all Mycobacterial infections through activation of the host immune system.

[00153] Screening of inhibitors against c-di-GMP binding to PdtaS: (a) obtaining PdtaS protein; (b) incubating PdtaS protein in the presence of c-di-GMP under in-vitro conditions, to obtain a product E, and analysing interaction between PdtaS protein and c-di-GMP in the product E; (c) obtaining a candidate molecule; (d) incubating PdtaS protein in the presence of c-di-GMP, and the candidate molecule under in-vitro conditions, to obtain a product F, and analysing interaction between PdtaS protein and c-di-GMP in the product F; and (e) comparing interaction between PdtaS protein and c-di-GMP in the product F with interaction between PdtaS protein and c-di-GMP in the product E for observing an increase or decrease in interaction of PdtaS protein and c- di-GMP in the product F as compared with the product E, wherein a decrease in interaction between PdtaS protein and c-di-GMP in the product F as compared to the interaction between PdtaS protein and c-di-GMP in the product E indicates capability of the candidate molecule in inhibiting growth of a bacterial pathogen. The in-vitro conditions used for incubating is well-known in the art. Also, methods for analysing the interaction of PdtaS protein and c-di-GMP and comparing their interaction in products E and F can be done my any methods known in the art.

[00154] Screening of inhibitors against PdtaS activation and autophosphorylation: (a) obtaining PdtaS protein; (b) performing phosphorylation assay by incubating PdtaS protein in the presence of ATP under in-vitro conditions, to obtain a product A, and analysing phosphorylation of the PdtaS protein of the product A; (c) obtaining a candidate molecule; (d) performing phosphorylation assay by incubating PdtaS protein in the presence of ATP and the candidate molecule under in-vitro conditions, to obtain a product B, and analysing phosphorylation of the PdtaS protein of the product B; and (e) comparing the phosphorylation of the PdtaS protein of the product B to the phosphorylation of the PdtaS protein of the product A, wherein a decrease in the phosphorylation of the PdtaS protein of the product B as compared to the phosphorylation of the PdtaS protein of the product A indicates capability of the candidate molecule in inhibiting growth of a bacterial pathogen. The in-vitro conditions for phosphorylation has been described in the Example 4.

[00155] Screening of inhibitors against PdtaS-PdtaR interaction: (a) obtaining PdtaS protein, and PdtaR protein; (b) performing phosphorylation assay by incubating PdtaS protein in the presence of ATP and PdtaR protein under in-vitro conditions, to obtain a product C, and analysing interaction of PdtaS protein and PdtaR protein of the product C; (c) obtaining a candidate molecule; (d) performing phosphorylation assay by incubating PdtaS protein in the presence of ATP, PdtaR protein, and the candidate molecule under in-vitro conditions, to obtain a product D, and analysing interaction of PdtaS protein and PdtaR protein of the product D; and (e) comparing the interaction of PdtaS protein and PdtaR protein of the product D to the interaction of PdtaS protein and PdtaR protein of the product C, wherein a decrease in interaction of PdtaS protein and PdtaR protein of the incubated product D as compared to the interaction of PdtaS and PdtaR protein of the incubated product C indicates capability of the candidate molecule in inhibiting growth of a bacterial pathogen. The in-vitro conditions for auto phosphorylation and trans-phosphorylation have been described in the Example 4.

[00156] Alternative strategy: (a) obtaining PdtaS protein, and PdtaR protein; (b) incubating PdtaS protein in the presence of PdtaR protein under in-vitro conditions, to obtain a product L, and analysing interaction between PdtaS protein and PdtaR protein in the product L; and (c) obtaining a candidate molecule; (d) incubating PdtaS protein in the presence of PdtaR protein and the candidate molecule, to obtain a product M, and analysing interaction between PdtaS protein and PdtaR protein in the product M; and (e) comparing interaction between PdtaS protein and PdtaR protein in the product M to interaction between PdtaS protein and PdtaR protein in the product L for observing an increase or decrease in interaction of PdtaS protein and PdtaR protein in the product M as compared to the product L, wherein a decrease in interaction between PdtaS protein and PdtaR protein in the product M as compared to the interaction between PdtaS protein and PdtaR protein in the product L indicates capability of the candidate molecule in inhibiting growth of a bacterial pathogen. The conditions for incubation are well known in the art for facilitating such reactions and also the methods for comparing such interactions are well known.

[00157] Screening of inhibitors against PdtaR-RNA interactions: (a) obtaining PdtaR protein; (b) obtaining a target RNA molecule, wherein the target RNA molecule is able to bind to PdtaR protein; (c) incubating PdtaR protein in the presence of the target RNA molecule under in-vitro conditions, to obtain a product G, and analysing interaction between PdtaR protein and the target RNA in the product G; (d) obtaining a candidate molecule; (e) incubating PdtaR protein in the presence of the target RNA molecule and the candidate molecule under in-vitro conditions, to obtain a product H, and analysing interaction between PdtaR protein and the target RNA in the product H; and (f) comparing interaction between PdtaR protein and the target RNA in the product H with interaction between PdtaR protein and the target RNA in the product G for observing an increase or decrease in interaction of PdtaR protein and the target RNA in the product H as compared to the product G, wherein a decrease in interaction between PdtaR protein and the target RNA in the product H as compared to the interaction between PdtaR protein and the target RNA in the product G indicates capability of the candidate molecule in inhibiting growth of a bacterial pathogen. In the present methodology, the interaction between PdtaR protein and target RNA is targeted, and a candidate molecule capable of intervening this interaction would inhibit the growth of the pathogen.

[00158] Alternate Strategy: (a) obtaining PdtaS protein, and PdtaR protein; (b) performing phosphorylation assay by incubating PdtaS protein in the presence of ATP and PdtaR protein under in-vitro conditions, to obtain a product I; (c) obtaining a target RNA molecule; (d) incubating the target RNA with the product I under in-vitro conditions, to obtain a product J, and analysing interaction between PdtaR protein and the target RNA in the incubated product J; (e) obtaining a candidate molecule; (f) incubating the target RNA with the product I in the presence of the candidate molecule under appropriate conditions, to obtain a product K, and analysing interaction between PdtaR protein and the target RNA in the product K; and (g) comparing interaction between PdtaR protein and the target RNA in the product K with interaction between PdtaR protein and the target RNA in the product J for observing an increase or decrease in interaction of PdtaR protein and the target RNA in the product K as compared to the product J, wherein a decrease in interaction between PdtaR protein and the target RNA in the incubated product K as compared to the interaction between PdtaR protein and the target RNA in the product J indicates capability of the candidate molecule in inhibiting growth of a bacterial pathogen. In the present methodology, the PdtaR protein receives phosphate from PdtaS (transphosphorylation) to get activated PdtaR. The activated PdtaR will in turn interact with the target RNA to effect downstream regulators. A candidate molecule capable of intervening this transmission would inhibit the growth of the pathogen.

[00159] The present disclosure establishes that interfering with the functionality of PDtaS-PdtaR interferes with the ability of the growth of the organism under nutrient- deprived conditions. The intervention is with respect to either autophosphorylation of PdtaS, or transphosphorylation of PdtaR, or interaction between PdtaS and PdtaR, or interaction between activated PdtaR and a target RNA, including any combination of these steps. The present disclosure discloses that intervening with the functioning of PdtaS/PdtaR system leads to the following: (a) inhibition of growth of bacterial pathogen; (b) inhibiting reactivation of the bacterial pathogen; and (c) inhibiting the bacterial pathogen from entering into latency. The process as disclosed in the present disclosure leads to identification of candidate molecules which leads to inhibition of growth of a bacterial pathogen, and/or inhibition of reactivation of the bacterial pathogen, and /or inhibiting the bacterial pathogen from entering into latency. A candidate molecule which inhibits reactivation of the bacterial pathogen would ensure that the pathogen, such as M. tuberculosis remains into latency, which in turn would not show any infection. A candidate molecule that inhibits the bacterial pathogen such as M. tuberculosis from entering into latency will, in turn, would not allow the pathogen to hide from the drug or the immune system, thus increasing the chances of its elimination.

Advantages of the present disclosure:

[00160] The present disclosure discloses strategies for screening candidate molecules having an ability to intervene the functional PdtaS-PdtaR signalling cascade. As depicted in the Examples, in-vitro screening strategies could help in identifying a potential candidate molecule. Since, the PdtaS-PdtaR TCS is non-essential for Mycobacteria, therefore, chances of the organism gaining resistance to the candidate molecule obtained by the in-vitro process as disclosed herein is very minimal. Therefore, the present disclosure provides a significant advantage in terms of providing a solution to the problem of antimicrobial resistance, which is quite prevalent.

[00161] The present disclosure targets a two-component signalling system involved in adaptation to nutrient deprivation in Mycobacteria. Also, it discloses the functions of the system at the level of RNA binding and transcription anti -termination in Mycobacteria. The present disclosure also discloses c-di-GMP binding site of a sensor histidine kinase in Mycobacteria as a target for possible intervention of the TCS. [00162] The present disclosure further discloses the interaction interface between the sensor histidine kinase PdtaS and the response regulator PdtaR in Mycobacteria as a possible target. The present disclosure targets a single two-component system for infections caused by all members of the genus Mycobacterium, including but not limited to tuberculosis, leprosy, pulmonary, and soft tissue infections caused by Mycobacteria.

[00163] The present disclosure also discloses the use of a sensor histidine kinase (PdtaS) mutant of Mycobacteria as a live attenuated vaccine strain. The present disclosure also discloses the use of a sensor response regulator (PdtaR) mutant of Mycobacteria as a live attenuated vaccine strain.

Claims

I/We Claim:

1. An in-vitro process for screening a candidate molecule capable of inhibiting growth of a bacterial pathogen, said process comprises:

a) obtaining PdtaS protein;

b) performing phosphorylation assay by incubating PdtaS protein in the presence of a phosphor-donor molecule under in-vitro conditions, to obtain a product A, and analysing phosphorylation of the PdtaS protein of the product A;

c) obtaining a candidate molecule;

d) performing phosphorylation assay by incubating PdtaS protein of step

(a) in the presence of a phosphor-donor molecule and the candidate molecule under in-vitro conditions, to obtain a product B, and analysing phosphorylation of PdtaS protein of the product B; and e) comparing the phosphorylation of the PdtaS protein of the product B to the phosphorylation of PdtaS protein of the product A, wherein a decrease in the phosphorylation of PdtaS protein of the product B as compared to the phosphorylation of PdtaS protein of the product A indicates capability of the candidate molecule in inhibiting growth of a bacterial pathogen.

2. An in-vitro process for screening a candidate molecule capable of inhibiting growth of a bacterial pathogen, said process comprises:

a) obtaining PdtaS protein, and PdtaR protein;

b) performing phosphorylation assay by incubating PdtaS protein in the presence of a phosphor-donor molecule and PdtaR protein under in- vitro conditions, to obtain a product C, and analysing interaction of

PdtaS protein and PdtaR protein of the product C;

c) obtaining a candidate molecule; d) performing phosphorylation assay by incubating PdtaS protein of step (a) in the presence of a phosphor-donor molecule, PdtaR protein of step (a), and the candidate molecule under in-vitro conditions, to obtain a product D, and analysing interaction of PdtaS protein and PdtaR protein of the product D; and

e) comparing the interaction of PdtaS protein and PdtaR protein of the product D to the interaction of PdtaS protein and PdtaR protein of the product C,

wherein a decrease in interaction of PdtaS protein and PdtaR protein of the product D as compared to the interaction of PdtaS and PdtaR protein of the product C indicates capability of the candidate molecule in inhibiting growth of a bacterial pathogen.

3. An in-vitro process for screening a candidate molecule capable of inhibiting growth of a bacterial pathogen, said process comprises:

a) obtaining PdtaS protein;

b) incubating PdtaS protein in the presence of c-di-GMP under in-vitro conditions, to obtain a product E, and analysing interaction between PdtaS protein and c-di-GMP in the product E;

c) obtaining a candidate molecule;

d) incubating PdtaS protein of step (a) in the presence of c-di-GMP, and the candidate molecule under in-vitro conditions, to obtain a product F, and analysing interaction between PdtaS protein and c-di-GMP in the product F; and

e) comparing interaction between PdtaS protein and c-di-GMP in the product F with interaction between PdtaS protein and c-di-GMP in the product E for observing an increase or decrease in interaction of PdtaS protein and c-di-GMP in the product F as compared with the product E, wherein a decrease in interaction between PdtaS protein and c-di-GMP in the product F as compared to the interaction between PdtaS protein and c-di-GMP in the product E indicates capability of the candidate molecule in inhibiting growth of a bacterial pathogen.

4. An in-vitro process for screening a candidate molecule capable of inhibiting growth of a bacterial pathogen, said process comprises:

a) obtaining PdtaR protein;

b) obtaining a target RNA molecule, wherein the target RNA molecule is able to bind to PdtaR protein

c) incubating PdtaR protein in the presence of the target RNA molecule under in-vitro conditions, to obtain a product G, and analysing interaction between PdtaR protein and the target RNA in the product G; d) obtaining a candidate molecule;

e) incubating PdtaR protein of step (a) in the presence of the target RNA molecule of step (b) and the candidate molecule under in-vitro conditions, to obtain a product H, and analysing interaction between PdtaR protein and the target RNA in the product H; and

f) comparing interaction between PdtaR protein and the target RNA in the product H with interaction between PdtaR protein and the target RNA in the product G for observing an increase or decrease in interaction of PdtaR protein and the target RNA in the product H as compared to the product G,

wherein a decrease in interaction between PdtaR protein and the target RNA in the product H as compared to the interaction between PdtaR protein and the target RNA in the product G indicates capability of the candidate molecule in inhibiting growth of a bacterial pathogen.

5. An in- vitro process for screening of a candidate molecule capable of inhibiting growth of a bacterial pathogen, said process comprises:

a) obtaining PdtaS protein, and PdtaR protein; b) performing phosphorylation assay by incubating PdtaS protein in the presence of a phosphor-donor molecule and PdtaR protein under in- vitro conditions, to obtain a product I;

c) obtaining a target RNA molecule;

d) incubating the target RNA with the product I under in-vitro conditions, to obtain a product J, and analysing interaction between PdtaR protein and the target RNA in the product J ;

e) obtaining a candidate molecule;

f) incubating the target RNA of step (c) with the product I of step (b) in the presence of the candidate molecule under appropriate conditions, to obtain a product K, and analysing interaction between PdtaR protein and the target RNA in the product K; and

g) comparing interaction between PdtaR protein and the target RNA in the product K with interaction between PdtaR protein and the target RNA in the product J for observing an increase or decrease in interaction of PdtaR protein and the target RNA in the product K as compared to the product J,

wherein a decrease in interaction between PdtaR protein and the target RNA in the product K as compared to the interaction between PdtaR protein and the target RNA in the product J indicates capability of the candidate molecule in inhibiting growth of a bacterial pathogen.

6. An in-vitro process for screening a candidate molecule capable of inhibiting growth of a bacterial pathogen, said process comprises:

a) obtaining PdtaS protein, and PdtaR protein;

b) incubating PdtaS protein in the presence of PdtaR protein under in-vitro conditions, to obtain a product L, and analysing interaction between PdtaS protein and PdtaR protein in the product L; and

c) obtaining a candidate molecule; d) incubating PdtaS protein of step (a) in the presence of PdtaR protein of step (a) and the candidate molecule, to obtain a product M, and analysing interaction between PdtaS protein and PdtaR protein in the product M; and

e) comparing interaction between PdtaS protein and PdtaR protein in the product M with interaction between PdtaS protein and PdtaR protein in the product L for observing an increase or decrease in interaction of PdtaS protein and PdtaR protein in the product M as compared to the product L,

wherein a decrease in interaction between PdtaS protein and PdtaR protein in the product M as compared to the interaction between PdtaS protein and PdtaR protein in the product L indicates capability of the candidate molecule in inhibiting growth of a bacterial pathogen.

7. The process as claimed in any one of the claims 1-6, wherein the bacterial pathogen is selected from a group consisting of Mycobacterium species, and

Corynebacterium species, and any species belonging to suborder Corynebacterineae.

8. The process as claimed in claim 7, wherein the Mycobacterium species is selected from a group consisting Mycobacterium tuberculosis, Mycobacterium smegmatis, Mycobacterium leprae, and Mycobacterium bovis.

9. The process as claimed in claim 8, wherein the Mycobacterium species is Mycobacterium tuberculosis.

10. The process as claimed in any one of the claims 1-6, wherein the candidate molecule is capable of inhibiting reactivation of the bacterial pathogen.

11. The process as claimed in any one of the claims 1-6, wherein the candidate molecule is capable of inhibiting the bacterial pathogen from entering into latency.

12. The process as claimed in any one of the claims 1-3, 5 or 6, wherein PdtaS protein has an amino acid sequence as set forth in SEQ ID NO: 1.

13. The process as claimed in any one of the claims 2, 4, 5, or 6, wherein PdtaR protein has an amino acid sequence as set forth in SEQ ID NO: 3.

14. A recombinant strain of Mycobacterium species comprising a mutation in a gene encoding PdtaS protein.

15. A recombinant strain of Mycobacterium species comprising a mutation in a gene encoding PdtaR protein.

16. Use of PdtaS and c-di-GMP interaction in screening for candidate molecules capable of: (a) inhibiting growth of Mycobacterium species; and/or (b) inhibiting reactivation of Mycobacterium species; and/or (c) inhibiting Mycobacterium species from entering latency.

17. Use of PdtaS and PdtaR interaction in screening for candidate molecules capable of: (a) inhibiting growth of Mycobacterium species; and/or (b) inhibiting reactivation of Mycobacterium species; and/or (c) inhibiting Mycobacterium species from entering latency.

18. Use of PdtaR and target RNA interaction in screening for candidate molecules capable of: (a) inhibiting growth of Mycobacterium species; and/or (b) inhibiting reactivation of Mycobacterium species; and/or (c) inhibiting Mycobacterium species from entering latency.