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WO2017068593A1 - Polypeptide pour la détection de mycobactéries pathogènes et utilisations de celui-ci - Google Patents

Polypeptide pour la détection de mycobactéries pathogènes et utilisations de celui-ci Download PDF

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Publication number
WO2017068593A1
WO2017068593A1 PCT/IN2015/050195 IN2015050195W WO2017068593A1 WO 2017068593 A1 WO2017068593 A1 WO 2017068593A1 IN 2015050195 W IN2015050195 W IN 2015050195W WO 2017068593 A1 WO2017068593 A1 WO 2017068593A1
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polypeptide
detection
post
rvl988
histone
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Sanjeev KHOSLA
Imtiyaz YASEEN
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Centre for DNA Fingerprinting and Diagnosis of Ministry of Science and Technology India
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Centre for DNA Fingerprinting and Diagnosis of Ministry of Science and Technology India
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/5695—Mycobacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/35—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycobacteriaceae (F)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
    • G01N2440/12—Post-translational modifications [PTMs] in chemical analysis of biological material alkylation, e.g. methylation, (iso-)prenylation, farnesylation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/26—Infectious diseases, e.g. generalised sepsis

Definitions

  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • a non-invasive method of determination of pathogenic mycobacterial infection in a subject 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.
  • 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.
  • 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.
  • Figure 1 depicts the identification of mycobacterial proteins that interact with Histone H3, in accordance with an embodiment of the present disclosure.
  • Figure 2A-B depicts the Rvl988 interaction with Histone H3, in an embodiment of the present disclosure.
  • FIG. 3A-E depicts the Histone methyltransferase (HMTase) activity of Rvl988, in accordance with an embodiment of the present disclosure.
  • 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.
  • Figure 5 depicts the morphology of macrophages infected with Rwl9SS::M.smegmatis , in accordance with an embodiment of the present disclosure.
  • 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.
  • 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.
  • Figure 8A-D depicts the Rvl988 mediated transcriptional repression, in accordance with an embodiment of the present disclosure.
  • Figure 9A-F depicts the effect of Rvl988: :M.smegmatis on repression of host genes, in accordance with an embodiment of the present disclosure.
  • 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.
  • a non-invasive method of determination of pathogenic mycobacterial infection in a subject 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.
  • 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.
  • 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.
  • ChIP Chromatin Immunoprecipitation
  • histone methylation-specific PCR histone methylation-specific PCR
  • pyrosequencing methylated DNA immunoprecipitation
  • Hpall tiny fragment Enrichment by Ligation-mediated PCR assay antibody recognition, and combinations thereof.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Histone methyltransferase assay in-vitro histone methyltransferase assay was performed by incubating 2 ⁇ g of recombinant Rvl988 protein with 1-2 ⁇ g of recombinant Histone H3 or its mutants and 0.2 mM S-adenosyl-methionine (cold SAM, NEB) or 2 ⁇ 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.
  • SAM S-adenosyl-methionine
  • H-AdoMet American Radiolabeled Chemicals
  • 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.
  • Enhancer Spray Perkin Elmer
  • 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).
  • 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).
  • the supernatant was passed through 0.45 ⁇ 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.
  • 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.
  • 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.
  • 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.
  • 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-immunoprecipitation-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.
  • the chromatin was eluted by adding 120 ⁇ 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.
  • 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).
  • 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).
  • MS/MS Tandem Mass Spectrometry
  • MS/MS Tandem Mass Spectrometry
  • 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.
  • 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.
  • M.smegmatis transformed with Rvl988 functions like pathogenic mycobacteria
  • 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.
  • Pathogenic mycobacteria have the ability to interfere with host cell production of reactive oxygen species (ROS), in contrast to non-pathogenic mycobacteria.
  • ROS reactive oxygen species
  • Table 1 depicts the genes and primers used for qRT-PCR while Table 2 depicts the genes and primers used for ChIP assay.
  • 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
  • NOX1, NOX4 NADPH Oxidases
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.

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Abstract

La présente invention concerne un polypeptide pour la détection de mycobactéries pathogènes dans un échantillon biologique. L'invention concerne également l'utilisation dudit polypeptide, et un procédé de détection de mycobactéries pathogènes dans un échantillon biologique.
PCT/IN2015/050195 2015-10-19 2015-12-09 Polypeptide pour la détection de mycobactéries pathogènes et utilisations de celui-ci Ceased WO2017068593A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004083448A2 (fr) * 2003-03-14 2004-09-30 The Board Of Trustees Of The Leland Stanford Junior University Differences moleculaires entre des especes du complexe m. tuberculosis
WO2014020343A1 (fr) * 2012-07-31 2014-02-06 Proteinlogic Limited Biomarqueurs pour diagnostiquer et/ou surveiller la tuberculose
CN104371013A (zh) * 2014-10-16 2015-02-25 东北农业大学 牛分枝杆菌cfp-10的迟发型变态反应抗原表位多肽及其应用
WO2015077541A2 (fr) * 2013-11-22 2015-05-28 Regents Of The University Of Minnesota Biomarqueurs de mycobacterium et procédés associés
CN103063837B (zh) * 2011-10-18 2015-06-17 复旦大学附属华山医院 一种检测分枝杆菌感染的试剂、方法和试剂盒

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004083448A2 (fr) * 2003-03-14 2004-09-30 The Board Of Trustees Of The Leland Stanford Junior University Differences moleculaires entre des especes du complexe m. tuberculosis
CN103063837B (zh) * 2011-10-18 2015-06-17 复旦大学附属华山医院 一种检测分枝杆菌感染的试剂、方法和试剂盒
WO2014020343A1 (fr) * 2012-07-31 2014-02-06 Proteinlogic Limited Biomarqueurs pour diagnostiquer et/ou surveiller la tuberculose
WO2015077541A2 (fr) * 2013-11-22 2015-05-28 Regents Of The University Of Minnesota Biomarqueurs de mycobacterium et procédés associés
CN104371013A (zh) * 2014-10-16 2015-02-25 东北农业大学 牛分枝杆菌cfp-10的迟发型变态反应抗原表位多肽及其应用

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
IMTIYAZ YASEEN ET AL: "Mycobacteria modulate host epigenetic machinery by Rv1988 methylation of a non-tail arginine of histone H3", NATURE COMMUNICATIONS, vol. 6, 16 November 2015 (2015-11-16), pages 8922, XP055286772, DOI: 10.1038/ncomms9922 *
JOCELYNE M LEW ET AL: "TubercuList 10 years after", TUBERCULOSIS, ELSEVIER, GB, vol. 91, no. 1, 30 September 2010 (2010-09-30), pages 1 - 7, XP028362825, ISSN: 1472-9792, [retrieved on 20101007], DOI: 10.1016/J.TUBE.2010.09.008 *
MAGEE ET AL., PLOS ONE, vol. 7, 2012, pages E32034
SORCI ET AL., PHILOS. TRANS. R. SOC. LOND. B. BIOL. SCI., vol. 364, 2009, pages 71 - 83
WYSOCKA J ET AL: "Histone arginine methylation and its dynamic regulation", FRONTIERS IN BIOSCIENCE, FRONTIERS IN BIOSCIENCE, ALBERTSON, NY, US, vol. 11, 1 January 2006 (2006-01-01), pages 344 - 355, XP002611758, ISSN: 1093-9946, DOI: 10.2741/1802 *

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