FIELD OF INVENTION

[001] The present disclosure relates to the field of molecular biology and diagnostics. There is provided a polypeptide for differential detection of pathogenic mycobacteria in a biological sample, and methods of uses thereof.

BACKGROUND OF THE INVENTION

[002] Virulent species of mycobacteria are known to interact and modulate their host cell machinery at various subcellular levels and by different mechanisms. While there are several reports that have documented changes in the expression of host genes (Magee et al., PLoS One, 2012, 7, e32034), there is no description of any such pathogen specific protein, which directly influences host gene expression or possibility of such protein being used a potential biomarker for detection of presence of pathogenic mycobacteria.

[003] PCT/US2014/066782 describes a method for detecting infection of an animal by Mycobacterium bovis. The method generally includes obtaining a biological sample from a host animal at risk of being infected by Mycobacterium bovis and analysing the sample for the presence or absence of at least one M. bovis polypeptide.

[004] PCT/GB2012/050978 describes a method of determining the presence or absence in a sample of a biomarker, the method comprising: (a) linking an antigen to colloidal gold to provide a gold-antigen species; (b) contacting the gold-antigen species with the sample; (c) adding a diagnosis agent to the sample; and (d) observing the colour of the sample.

[005] PCT/GB2013/052055 relates to biomarkers for diagnosing and/or monitoring tuberculosis in both immunocompetent and immunocompromised individuals, monitoring the responses of individuals to anti-mycobacterial chemotherapy, monitoring the progression of latent tuberculosis to active tuberculosis, differentiating active tuberculosis from latent tuberculosis, and from other clinical conditions that mimic tuberculosis (TB).

SUMMARY OF THE INVENTION

[006] In an aspect of the present disclosure, there is provided a non-invasive method of determination of pathogenic mycobacterial infection in a subject, said method comprising: (a) obtaining a biological sample from said subject; and (b) detecting the presence of a post-translational modification in a polypeptide having amino acid sequence as set forth in SEQ ID NO: 1 in said biological sample, wherein detection of said post-translational modification in the polypeptide in said biological sample is indicative of pathogenic mycobacterial infection in said subject, and said post- translational modification in polypeptide is dimethylation of Arginine residue at position 42 in the N terminus direction.

[007] In an aspect of the present disclosure, there is provided a non-invasive method of detection of a post-translational modification in a polypeptide having amino acid sequence as set forth in SEQ ID NO: 1, said method comprising: (a) obtaining a biological sample comprising said polypeptide; and (b) detecting presence of said post- translational modification in said polypeptide, wherein said post-translational modification in said polypeptide is dimethylation of Arginine residue at position 42 in the N terminus direction.

[008] In an aspect of the present disclosure, there is provided a polypeptide for detection of pathogenic mycobacterial infection in a subject, said polypeptide being a polypeptide fragment having amino acid sequence as set forth in SEQ ID NO: 1, further post-transcriptionally modified at Arginine residue at position 42 in the N terminus direction, wherein said Arginine residue is dimethylated.

[009] In an aspect of the present disclosure, there is provided a polypeptide having amino acid sequence as set forth in SEQ ID NO: 1 for use in detection of pathogenic mycobacterial infection in a subject, wherein said polypeptide is post-transcriptionally modified at Arginine residue at position 42 in the N terminus direction, wherein said Arginine residue is dimethylated.

[0010] 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 THE ACCOMPANYING DRAWINGS

[0011] The following drawings form 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.

[0012] Figure 1 depicts the identification of mycobacterial proteins that interact with Histone H3, in accordance with an embodiment of the present disclosure.

[0013] Figure 2A-B depicts the Rvl988 interaction with Histone H3, in an embodiment of the present disclosure.

[0014] Figure 3A-E depicts the Histone methyltransferase (HMTase) activity of Rvl988, in accordance with an embodiment of the present disclosure.

[0015] Figure 4 depicts the MS/MS spectra for Histone H3 after in-vitro methyltransferase assay with Rvl988, in accordance with an embodiment of the present disclosure.

[0016] Figure 5 depicts the morphology of macrophages infected with Rwl9SS::M.smegmatis , in accordance with an embodiment of the present disclosure.

[0017] Figure 6A-B depicts the LDH release or ROS generation by macrophages infected with Rwl9SS: :M.smegmatis , in accordance with an embodiment of the present disclosure. [0018] Figure 7A-B depicts the LDH release or ROS generation by PMA treated THP1 cells infected with Rwl9SS::M.smegmatis , in accordance with an embodiment of the present disclosure.

[0019] Figure 8A-D depicts the Rvl988 mediated transcriptional repression, in accordance with an embodiment of the present disclosure.

[0020] Figure 9A-F depicts the effect of Rvl988: :M.smegmatis on repression of host genes, in accordance with an embodiment of the present disclosure.

[0021] Figure 10 depicts the enrichment of H3 dimethyl arginine levels in THP1 cells infected with Rvl9SSS-G¥P::M.smegmatis or G¥P::M.smegmatis , in accordance with an embodiment of the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION

[0022] 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.

Definitions

[0023] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are collected 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.

[0024] 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. [0025] 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".

[0026] 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.

[0027] The term "including" is used to mean "including but not limited to". "Including" and "including but not limited to" are used interchangeably.

[0028] 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.

[0029] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally-equivalent products, compositions, and methods are clearly within the scope of the disclosure, as described herein.

[0030] In an embodiment of the present disclosure, there is provided a non-invasive method of determination of pathogenic mycobacterial infection in a subject, said method comprising: (a) obtaining a biological sample from said subject; and (b) detecting the presence of a post-translational modification in a polypeptide having amino acid sequence as set forth in SEQ ID NO: 1 in said biological sample, wherein detection of said post-translational modification in the polypeptide in said biological sample is indicative of pathogenic mycobacterial infection in said subject, and said post-translational modification in polypeptide is dimethylation of Arginine residue at position 42 in the N terminus direction. [0031] In an embodiment of the present disclosure, there is provided a non-invasive method of determination of pathogenic mycobacterial infection in a subject as described herein, wherein said polypeptide is encoded by a polynucleotide fragment having sequence as set forth in SEQ ID NO: 2.

[0032] In an embodiment of the present disclosure, there is provided a non-invasive method of determination of pathogenic mycobacterial infection in a subject as described herein, wherein said detection is carried out by a technique selected from the group consisting of mass spectrometry, Chromatin Immunoprecipitation (ChIP), histone methylation-specific PCR, pyrosequencing, methylated DNA immunoprecipitation, Hpall tiny fragment Enrichment by Ligation-mediated PCR assay, antibody recognition, and combinations thereof.

[0033] In an embodiment of the present disclosure, there is provided a non-invasive method of determination of pathogenic mycobacterial infection in a subject as described herein, wherein said detection technique is immunoprecipitation of histone H3 protein followed by detection of said post-translational modification by using antibody recognizing dimethylated Arginine.

[0034] In an embodiment of the present disclosure, there is provided a non-invasive method of determination of pathogenic mycobacterial infection in a subject as described herein, wherein said detection techniques is DNA enrichment followed by detection of post-translational modification of Histone H3 by using antibody recognizing dimethylated Arginine.

[0035] In an embodiment of the present disclosure, there is provided a non-invasive method of detection of a post-translational modification in a polypeptide having amino acid sequence as set forth in SEQ ID NO: 1, said method comprising: (a) obtaining a biological sample comprising said polypeptide; and (b) detecting presence of said post- translational modification in said polypeptide, wherein said post-translational modification in said polypeptide is dimethylation of Arginine residue at position 42 in the N terminus direction. [0036] In an embodiment of the present disclosure, there is provided a non-invasive method of detection of a post-translational modification in a polypeptide having amino acid sequence as set forth in SEQ ID NO: 1 as described herein, wherein said polypeptide is encoded by an oligonucleotide fragment having sequence as set forth in SEQ ID N: 2.

[0037] In an embodiment of the present disclosure, there is provided a non-invasive method of detection of a post-translational modification in a polypeptide having amino acid sequence as set forth in SEQ ID NO: 1 as described herein, wherein said detection is carried out by a technique selected from the group consisting of mass spectrometry, methylation specific PCR, pyrosequencing, methylated DNA immunoprecipitation, Hpall tiny fragment Enrichment by Ligation-mediated PCR assay, antibody recognition, and combinations thereof.

[0038] In an embodiment of the present disclosure, there is provided a non-invasive method of detection of a post-translational modification in a polypeptide having amino acid sequence as set forth in SEQ ID NO: 1 as described herein, wherein said detection technique is immunoprecipitation of histone H3 protein followed by detection of said post-translational modification by using antibody recognizing dimethylated Arginine.

[0039] In an embodiment of the present disclosure, there is provided a non-invasive method of detection of a post-translational modification in a polypeptide having amino acid sequence as set forth in SEQ ID NO: 1 as described herein, wherein said detection technique is DNA enrichment followed by detection of post-translational modification of Histone H3 by using antibody recognizing dimethylated Arginine.

[0040] In an embodiment of the present disclosure, there is provided a polypeptide for detection of pathogenic mycobacterial infection in a subject, said polypeptide being a polypeptide fragment having amino acid sequence as set forth in SEQ ID NO: 1, further post-transcriptionally modified at Arginine residue at position 42 in the NH ₂ to COOH direction, wherein said Arginine residue is dimethylated.

[0041] In an embodiment of the present disclosure, there is provided a polypeptide for detection of pathogenic mycobacterial infection in a subject as described herein, wherein said polypeptide is encoded by a polynucleotide fragment having sequence as set forth in SEQ ID NO: 2.

[0042] In an embodiment of the present disclosure, there is provided a polypeptide having amino acid sequence as set forth in SEQ ID NO: 1 for use in detection of pathogenic mycobacterial infection in a subject, wherein said polypeptide is post- transcriptionally modified at Arginine residue at position 42 in the NH ₂ to COOH direction, wherein said Arginine residue is dimethylated.

[0043] In an embodiment of the present disclosure, there is provided a polypeptide having amino acid sequence as set forth in SEQ ID NO: 1 for use in detection of pathogenic mycobacterial infection in a subject as described herein, wherein said polypeptide is encoded by a polynucleotide fragment having sequence as set forth in SEQ ID NO: 2.

[0044] Although the subject matter has been described in considerable detail with reference to certain preferred embodiments thereof, other embodiments are possible.

EXAMPLES

[0045] 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.

Example 1

Materials and methods [0046] Affinity purification of H3 interacting proteins from the M. Bovis BCG lysate: To identify M. bovis BCG proteins that interact with Histone H3, E coli purified His- tagged Histone H3 was allowed to bind to pre-equilibrated Ni-NTA beads in buffer containing 50 mM Tris-HCl pH-8, 100 mM NaCl, 1% NP-40 and 10% glycerol, followed by incubation with pre-cleared M. bovis BCG lysate for 4 hrs. Ni-NTA beads alone were also incubated with M. Bovis BCG lysate as control. Beads were washed five times with the same buffer but containing 25 mM Imidazole. Bound proteins were resolved on a 12% SDS-PAGE. The prominent bands present only in H3 incubated fraction were cut and analysed by Mass Spectrometry.

[0047] Pull-down and Immunoprecipitation assays: Rvl988 having N-terminal SFB- tag was cloned into pCDNA3.1 and transfected into HEK293 (obtained from the Cell Culture Stock Centre at National Centre for Cell Science (NCCS), Pune) cells. 48 hrs after transfection, cells were lysed in a buffer containing 20 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 50 mM β-glycerophosphate, 10 mM NaF and protease inhibitors. Lysate was incubated with pre-equilibrated Streptavidin beads for 3 hrs, the beads were washed five times with the same buffer, loaded on a SDS-PAGE after resuspending them in 6X-SDS sample loading buffer and Western blotted. The blot was probed with H3 antibody (Abeam). For reverse IP, same procedure was followed except IP was done with H3 and IgG (Invitrogen) antibodies and probed with anti-FLAG (Sigma) antibody.

[0048] Histone methyltransferase assay: in-vitro histone methyltransferase assay was performed by incubating 2 μg of recombinant Rvl988 protein with 1-2 μg of recombinant Histone H3 or its mutants and 0.2 mM S-adenosyl-methionine (cold SAM, NEB) or 2 μθ ³ H-AdoMet (American Radiolabeled Chemicals) in buffer containing 50 mM Tris-HCl (pH 8.0), 10% glycerol, 5 mM MgC12, 20 mM KC1, and 1 mM PMSF at 30°C overnight. The reaction mixture was resolved on a SDS-PAGE, electroblotted on PVDF membrane (GE Healthcare) sprayed with Enhancer Spray (Perkin Elmer) and kept for exposure for 7 days at -80°C. For scintillation counts, reaction was stopped by adding 10% TCA and mixture was spotted on GF/C filters (Sigma) using vacuum manifold and washed thrice with 10%TCA and twice with absolute Ethanol. Filters were dried and put in vials containing scintillation fluid and counts were taken in a scintillation counter (Perkin Elmer).

[0049] Mass spectrometry: After the methyltransferase assay, samples were resolved on a SDS-PAGE, band were cut and send for MS/MS (Taplin Mass spectrometric Facility Harvard USA).

[0050] Preparation of Mycobacterium culture filtrate: The culture filtrate preparation for M. bovis BCG and M. smegmatis was done as described below. To obtain Rvl988 transformed M. smegmatis, electro competent M. smegmatis were transformed with ^g DNA construct of pVV16-Rvl988 as per standardized protocol. Colonies obtained after 3 days were inoculated in 5 ml Modified Sauton's media with Kanamycin and Hygromycin (Invitrogen). Secondary cultures were derived using 1% primary inoculum in Modified Sauton's media. Culture were harvested at 0.8 (OD ₆ oo) by centrifugation at 8000 rpm for 30 mins. The supernatant was passed through 0.45 μπι filters to remove cell contamination remains and concentrated 100X using 10 kDa Centricon (Millipore). Cell lysate was prepared by sonication. Glass beads were added to the lysate during the sonication for better yields. Rvl988 antibody was raised against the E. coli purified Rvl988 protein in mice. The specificity of the antibody was confirmed by Western Blotting.

[0051] Infection of THP1 cells and peritoneal macrophages: 0.3 X 10 ⁶ THP1 cells were seeded per chamber and treated for 12 hrs with 10 ng of PMA. The cells were collected from BALB/c mice as per approved IE AC guidelines (PCD/CDFD/15) and seeded in 12 well culture dishes or chamber slide at a density of 0.5 X 10 ⁶ and 0.3 X 10 ⁶ respectively. Infections were done as described above for THP1 cells. After 24 hrs of recovery in PMA free RPMI medium, infection was done with different strains of M. smegmatis or M. bovis BCG in antibiotic free media at an MOI of 30: 1 for 6 hrs. Cells were washed twice with PBS and cultured further in media containing antibiotics. Cells were fixed or assayed at different time points as indicated. Peritoneal exudates cells (PEC). [0052] Luciferase reporter gene assay: Rvl988 was cloned into pBind vector (Promega) containing Gal4 domain. pG5Luc vector was used as a reporter Luciferase construct that contains UAS sites (Gal4 binding) upstream of the Luciferase reporter gene.pEGFP-c3vector was used as a control for transfection efficiency.HEK293 cells were transfected using lipofectamine 2000 (Invitrogen), with either of the following three combinations 1) Rvl988pBind + pG5Luc + pEGFP-c3; 2) pBIND + pG5Luc + pEGFP-c3; 3) pG5Luc + pEGFP-c3. After 48hrs of transfection whole cell lysate was prepared and Luciferase activity was measured using Luciferase assay system as per the manufacturer's protocol (Promega). The same lysate was also probed with GFP antibody (Sigma) to monitor the transfection efficiency. Densitometry of the blot was done and values for GFP were used to normalize the respective luciferase values.

[0053] Expression Analysis by Real Time PCR: THP 1 cells were infected with various strains of M. smegmatis and harvested 48 hrs post-infection followed by RNA isolation using TRI reagent (Sigma). 1 μg DNase treated RNA was used for cDNA preparation using Superscript III (Invitrogen). Expression analysis by Real Time RT-PCR using SYBER Green Master Mix (Thermo Scientific) in ABI Prism SDS 7500 system. C _t were normalized with C _t of GAPDH which was used as internal control. Each experiment was done in duplicates and repeated with three biological replicates. Primers used are as detailed in Table 1.

[0054] Chromatin Immunoprecipitation: For ChIP using various Histone H3 and histone modification antibodies we followed Abeam X-ChIP protocol (http://www.abcam.com/protocols/cross-linking-chromatin-immu noprecipitation-x- chip-protocol) with slight modifications. Briefly cultured cells were cross-linked with 0.75 % Formaldehyde for 10 minutes at room temperature followed by incubation with 125 mM of Glycine for 5 minutes. Cells were washed with PBS and sonicated in the ChIP lysis buffer containing 50 mM HEPES-KOH pH7.5, 140 mM NaCl, lmM EDTA pH8, 1% Triton X-1000, 1% Sodium Deoxycholate, 0.1% SDS and Protease Inhibitors using Bioruptor (Diagenode). 50 μg of protein supernatant was taken for each ChIP and diluted 10 times with RIPA buffer (50 mM Tris-HCl pH8, 150 mM NaCl, 2 mM EDTA pH8, 1% NP-40, 0.5% Sodium Deoxycholate, 0.1% SDS and Protease Inhibitors (added freshly each time)). 2 μg of the respective antibodies (5 μg for Dimethyl Arginine) were added to the respective samples and binding was done overnight. 20 μΐ of protein A/G (Santa Cruz) or protein-A magnetic beads (Diagenode) were added for 3 hrs. The beads were washed 3 times with wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA pH8, 150 mM NaCl, 20 mM Tris-HCl pH8)and once with final wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA pH8, 500 mM NaCl, 20 mM Tris-HCl pH 8). The chromatin was eluted by adding 120 μΐ of elution buffer (1% SDS and 100 mM NaHC0 ₃ ) and reverse cross linked by using Proteinase K at 65° C for 4 hrs. The DNA was extracted by Phenol: Chloroform and Ethanol precipitation (in presence of Glycogen) followed by Real Time qPCR using the primers as given in Table 2. Each experiment was done in duplicates and repeated with three biological replicates. Example 2

Identification of mycobacterial protein Rvl988 as interaction partner of host Histone H3

[0055] To identify mycobacterial proteins that could interact with Histone H3, recombinant His-tagged Histone H3 purified from E.coli was incubated with Mycobacterium bovis BCG lysate. The mycobacterial proteins interacting with Histone H3 were affinity purified using Ni-NTA beads and electrophoresed on an SDS-PAGE (Figure 1). Protein bands were cut out from the gel and identified by Mass Spectrometry. One of the mycobacterial proteins identified in this analysis was Rvl988. Listed as a probable methyltransferase in the Tuberculist database, Rvl988 is present in the pathogenic mycobacteria, M. tuberculosis and M. bovis but absent in the non-pathogenic M. smegmatis. To validate the interaction of Rvl988 with Histone H3, HEK293 cells were transfected with a pcDNA3.1 construct containing the SFB tagged Rvl988 gene. 48 hrs post transfection, Rvl988 and its interacting partners were affinity purified using Streptavidin beads from the protein lysate and analysed by Western blotting using Histone H3 antibody. Vector alone (pCDNA3.1) construct was used as a control. As can be seen from figure 2A, affinity purification of SFB-Rvl988 from the HEK293 protein lysate pulls down Histone H3, indicating that Rvl988 can indeed bind Histone H3. Western blot probed with FLAG antibody (FLAG peptide is part of the SFB tag) was used to control for the pull down efficiency (figure 2A, lower panel). Interaction of Rvl988 with Histone H3 was further confirmed by immunoprecipitation (IP) of H3 histone from the protein lysate of HEK293 cells transfected with SFB-Rvl988 followed by Western blotting and probing with FLAG antibody. As a control, IP was also done with IgG. FLAG tagged SFB-Rvl988 was co- immunoprecipitated with H3 but not with IgG (figure IB). IP efficiency was checked by probing the blots with antibody to Histone H3 (figure 2B, lower panel).

Example 3

Rvl988 as a functional Histone Methyltransferase

[0056] In-vitro methyltransferase assay was performed with recombinant MBP- Rvl988 using recombinant Histone H3 (purified from E.coli) as substrate and Tritiated SAM as methyl group donor and analyzed by autoradiography or Scintillation counting. Tritiated methyl groups were transferred to Histone H3 in presence of MBP- Rvl988 but not with MBP protein (Figure 3 A). Scintillation quantitation also indicated that statistically higher amount of tritiated methyl groups were transferred from tritiated SAM to Histone H3 in presence of MBP-Rvl988 than MBP indicating that Rvl988 can methylate Histone H3 (figure 3B). To identify the residues within Histone H3 that get methylated by Rvl988, Histone H3 incubated with Rvl988 was analyzed by Tandem Mass Spectrometry (MS/MS) that indicated Arginine at the 42 ^nd position of Histone H3 (H3R42) was methylated (figure 4). in vitro histone methyltransferase assay on recombinant Histone H3 using cold SAM and after Western blotting, probed with a Dimethyl Arginine antibody (Abeam ab413) revealed that dimethyl Arginine antibody detected Histone H3 that was incubated with MBP-Rvl988 but not with MBP (figure 3C). To find which of the Arginines was the target of Rvl988 methyltransferase activity, Arginines at H3R42 and H3R83 were changed to Alanine by site directed mutagenesis followed by in vitro Histone methyltransferase assay using tritiated SAM. The radiograph in figure 3D shows that Rvl988 was able to methylate the wild type H3 and H3R83A mutant but showed negligible methylation of the H3R42A mutant. The same result was obtained when in the in-vitro methyltransferase assay, cold SAM was used instead of tritiated SAM and probed with Dimethyl Arginine antibody (figure 3E). H3R42A showed negligible levels of Arginine methylation as compared to H3 and H3R83A suggesting that H3R42 was the primary target of Rv 1988.

Example 4

M.smegmatis transformed with Rvl988 functions like pathogenic mycobacteria

[0057] In survivability assay, it was observed that macrophages infected with Rvl988- GFP:: . smegmatis had a morphology resembling mature macrophages as compared to those infected with control GFP:: . smegmatis that resembled primary macrophages (figure 5). Pathogenic species of Mycobacterium are known to cause cytotoxicity by inducing necrosis of macrophages. Quantitation of lactose dehydrogenase (LDH) release from a cell is a well-established assay for necrosis. LDH release from Rvl9SS-G¥P::M. smegmatis was evaluated to see if it behaves like pathogenic mycobacteria and cause necrosis. As seen in Figure 6A, compared to control GFP:: . smegmatis , Rvl9SS-G¥P::M. smegmatis infected macrophages release significantly higher amount of LDH after 48 hours of infection.

[0058] Pathogenic mycobacteria have the ability to interfere with host cell production of reactive oxygen species (ROS), in contrast to non-pathogenic mycobacteria. Peritoneal macrophages infected with Rvl9SS-G¥P::M. smegmatis produce significantly less ROS compared to control macrophages 24 hours post infection (Figure 6B). For both LDH and ROS assays, similar results were obtained when PMA treated THP1 cells are used as host (Figure 7). Example 5

Rvl988 mediates transcriptional repression through Arginine dimethylation

[0059] In order to ascertain whether Rvl988 is indeed associated with pathogenic mycobacterial infection, the effect of dimethylation of Histone H3 Arginine at position 42 (R42) on host gene expression was evaluated. Modification of Histones especially Histone H3 have been very well correlated with regulation of gene expression. To examine the role of Rvl988 mediated H3R42me ₂ in gene regulation, Luciferase reporter gene assay was performed in HEK293 cells. Luciferase reporter gene construct (in pG51uc), containing Gal4 binding sites within its promoter was co- transfected with a Gal4-Rvl988 construct (in pBIND vector) (Figure 8A). Gal4 would allow tethering of Rv 1988 to the Gal4 binding site in the luciferase promoter, allowing dimethylation of H3R42 in the promoter associated histones.

[0060] Upon transfection of pG51uc with Rvl988-pBIND, significant decrease of Luciferase activity (approximately 78%) was observed as compared to Luciferase activity when only pG51uc alone or pG51uc+pBIND vectors were transfected (figure 8B). Furthermore, to test whether Gal4 assisted tethering of Rvl988 to the Gal4 binding sites in the Luciferase promoter leads to H3R42 dimethylation, ChIP analysis using Dimethyl Arginine Antibody for the Luciferase promoter was performed. As a control, the profile of known repressive Histone H3 modifications including H3K9me ₃ and H3K27me ₃ was also performed. While no enrichment or depletion of H3K9me ₃ and H3K27me ₃ for the Luciferase promoter was observed, significant enrichment was observed for Dimethyl Arginine modification in cells co-transfected with Rvl988- pBIND + pG51uc as compared to Control pG51uc + pBIND co-transfection (figure 8C). The observed enrichment was indeed for Arginine dimethylation in Histone H3 as substantial increase in the level of Histone H3 dimethylated at Arginine was observed in HEK cells transfected with Rvl988 as compared to vector alone (figure 8D).

Example 6 Infection with Ryl988-M.smegmatis leads to Histone H3 Arginine Dimethylation associated repression of specific host genes

[0061] In order to ascertain the effect of Rvl988 in H3R42me ₂ mediated gene regulation during infection of macrophages by mycobacteria, transcription level of various host genes was evaluated by mRNA extraction and isolation followed by qRT- PCR or by ChIP with antibodies to dimethyl arginine.

[0062] Table 1 depicts the genes and primers used for qRT-PCR while Table 2 depicts the genes and primers used for ChIP assay.

[0063] Table 1

CDCBPB SEQ ID NO: 31 SEQ ID NO: 32

NOX4 SEQ ID NO: 33 SEQ ID N: 34

LUC SEQ ID NO: 15 SEQ ID NO: 36

[0065] TRAF3 and TNFAIP2 are two immunologically important genes. CDCBPB and ENSG00000250584/lincRNA are genes present in genomic loci to which Rvl988 binds. NOS2 (Nitric Oxide Synthase) is involved in formation of free radicals. NOX1, NOX4 (NADPH Oxidases) are involved in production of oxygen radicals, while NOXA1 is a NOX activating protein. Genes involved in ROSS activity were chosen as it was previously observed that there is a decrease in ROS activity of GFP-Rvl988:: . smegmatis infected THP1 cells.

[0066] Gene expression of the above mentioned genes was examined for THP1 cells infected with GFP:: . smegmatis or Rvl988-GFP:: . smegmatis by qRT-PCR. As shown in figure 9A, except for CDCBPB, all other genes including ENSG00000250584/lincRNA, were repressed in Rvl988-GFP: :M. smegmatis infected THP1 cells, confirming the repressive influence of Rvl988 on gene expression as earlier observed in the in vitro experiments (figure 8B).

[0067] To deduce whether the repression observed for these genes was due to H3 Arginine dimethyl ation, THP1 cells either infected with GFP:: . smegmatis or Rwl9SS-G¥P::M. smegmatis were subjected to ChIP with antibodies to Dimethyl Arginine, H3K4me ₃ (activating chromatin mark), H3K9me ₃ , and H3K27me ₃ (repressive chromatin marks) with the promoters of the above mentioned genes were analyzed. Except for NOXA1, promoters of all other genes that showed decrease in their expression in RV1988-GFP:: . smegmatis infected THP1 cells showed significantly more association with Dimethylated Arginine (figure 9B). Neither was expression of CDC42BPB affected upon RV1988-GFP:: . smegmatis infection nor did we observe change its promoter association with Dimethyl Arginine (figure 9B). On the other hand, no change was observed for the association of H3K9me ₃ and H3K27me ₃ , the known repressive chromatin marks, with the promoters of these gene upon infection with RV1988-GFP:: . smegmatis (figure 9D,E). In fact, NOS2 showed decrease in association with the repressive H3K27me ₃ mark even though its expression level had decreased (figure 9E). Only a few of the gene promoters (NOXA1, NOX4 and the ENSG00000250584 HncRNA) showed decreased association with H3K4me ₃ , the active chromatin associated histone mark, in RV1988-GFP:: . smegmatis infected THP1 cells (figure 9F). Both the Rvl988 bound regions B and C present within the introns of CDC42BPB and ENSG00000250584 lincRNA also showed significantly higher association with Dimethyl Arginine in Rvl988-GFP:: . smegmatis infected THP1 cells (figure 9B) but not with the other histone modifications tested (figure 9D,E,F). Immunoprecipitation of H3 followed by Western with Dimethyl Arginine antibody confirmed that the infection with Rvl988-GFP:: . smegmatis was affecting the levels of Histone H3 Dimethyl Arginine (figure 10).

[0068] Discussion: Histone H3 Arginine dimethylation by Rvl988 during mycobacterial infection of macrophages leads to repression of genes involved in ROS induced autophagy, considered to a the first line of defense against infectious agents (Sorci et al., Philos. Trans. R. Soc. Lond. B. Biol. Sci., 2009, 364, 71-83). While pathogenic M. tuberculosis and M. bovis can suppress this host response, M. smegmatis fails to suppress this first line of host defense response. However, the present disclosure provides a mycobacterial protein (pathogenic strain specific), Rvl988 that directly binds to, and dimethylates Arginine 42 residue in histone H3, thus repressing host immune mechanisms. This post-translational modification of host protein is specific for pathogenic mycobacterial infection, and can be used as a polypeptide for pathogenic mycobacterial infection.

[0069] Overall, the present disclosure provides identification, characterization, and utility of a polypeptide (host Histone H3 with dimethylated Arginine at amino acid position 42 in the N terminus direction) that can be reliably used to ascertain the presence of mycobacterial infection in a biological sample obtained from a subject suspected of having mycobacterial infection. The polypeptide, and methods of the instant disclosure are particularly useful as the polypeptide, and modification thereof is present only in instances of pathogenic mycobacterial infection, and not in presence of non-pathogenic strains of mycobacteria. Further, presence of the polypeptide of the instant disclosure can be ascertained in a subject in a non-invasive manner from a sample comprising genetic material.