Role of PE proteins of Mycobacterium tuberculosis
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L/O/G/O
Sameer TiwariDivision of Microbiology
Studies on the implications of PE class proteins of Mycobacterium tuberculosis as determinants of pathogenicity and Immuno-prophylaxis
Studies on the implications of PE class proteins of Mycobacterium tuberculosis as determinants of pathogenicity and Immuno-prophylaxis
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M. tuberculosisM. tuberculosis
Robert Koch1882
~9 million new cases~2 million deaths worldwide2011
2011 ~2 million new cases in India
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M. tuberculosis genomeM. tuberculosis genome
Cole et al. (1998) Nature 393: 537-544
4,000 genes 40% orphans
100 highly homologous
PE/PPE genes
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General Classification of M. tuberculosis genesGeneral Classification of M. tuberculosis genes
CLINICAL MICROBIOLOGY REVIEWS, July 2003, p. 463–496
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PE/PPE proteinsPE/PPE proteins
100 highly homologous genes with signature sequence of Pro-Glu (PE) amino acid near amino terminus.
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PE-PGRS (polymorphic GC rich repetitive sequences)PE-PGRS (polymorphic GC rich repetitive sequences)
Larger proteins domain extremely rich in Gly (~ 40%) & Ala (~ 25%)
residues PGRS domain occur as repetitive sequences (repeated >30times
within domain)
Rv3508 Average Rv0742
1901 aa 550 aa 175 aa
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Extensive functional redundancy
Close genomic & evolutionary association with ESX regions
Highly immunogenic
Properties of PE proteinsProperties of PE proteins
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Classification of PE/PPE protein familiesClassification of PE/PPE protein families
Sequence variation between M. tuberculosis H37Rv & M. bovis BCG
Sequence variation between M. tuberculosis H37Rv & M. bovis BCG
Cole et al. (1998) Nature 393: 537-544
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Features of PE-PGRS family
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Role of PE_PGRSRole of PE_PGRS
Homeostasis of mycobacterial cell
Intracellular survival
Multiplication within its chosen environment
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Why to study?Why to study?
Unique gene family
No counterpart in bacteria
Potential source of antigenic
variation
No strict correlation with
pathogenic strains
Little is known
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Role in virulence (Ramakrishnan et al.) pathogenic Mycobacterium marinum expresses two PE_PGRS genes in granulomas of infected frogs and that M. marinum mutants containing deletions in these genes replicate poorly in macrophages
Known facts…..Known facts…..
Several PE_PGRS proteins have been localized to the cell surface, and allelic diversity among M. tuberculosis isolates indicates that they could serve as a source of antigenic variation for this pathogen.
Microarray analysis has also suggested that variable expression of certain PE_PGRS genes occurs under conditions that mimic in vivo pathogenesis such as nutrient depletion, low pH or oxidative stress
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ObjectivesObjectives
Delineation of protective immune response of the proteins, upon challenge with MTB
Regulation of PE genes in mycobacteria in Culture grown, persistent, Hypoxic and from infected macrophages/ from infected lungs.
.
Evaluation of complete cellular immune responses incurred by these proteins
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What we Achieve?What we Achieve?
Identification of domains involved in binding to cell surface or other intracellular components will help in designing targets against these proteins.
Characterization of PE_PGRS gene expression
mechanisms of pathogenesis of mycobacteria in macrophages
Vaccine candidates as well as virulence factors.
Quantification of the amounts of CFP-10/ESAT-6
secreted into the macrophages during early stages of infection will give
an insight of their role in virulence.
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Impact on antigen-presentation pathways Ensuing host immune responses, and Also provide a mechanism for generating antigenic diversity in mycobacteria
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Research PlanResearch Plan
Cell culture1
2
3
4
Phagocytosis & intracellular surival
Cloning, expression & purification of PE3 & PE4
Immunological studies
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Quantitation of RNA during infection5
6
7
8
Generation of antisense PE3 & PE4 mycobacteria
Localization of PE3 & PE4
Studies of immune response incurred by MS
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ConclusionsConclusionsThe PE proteins/PPE functions to be the rich source of protective antigens involved in antigenic variation and immune-pathogenic importance.
PE/PPE genes involve in host-pathogen interactions, such as antigenic variability, virulence, and persistence of the bacillus. Their surface-exposed domains are also involved in the shaping of the bacterial cell structure. These proteins are capable of forming heterodimers which are secreted and thought to play a role in signal transduction.
The study will investigate the characteristics of PE3 (Rv0159c) & PE4 (Rv0160c) and their associations with ESAT-6 & CFP-10 and finally their interactions with macrophage.
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FacilitiesFacilities
Laboratory: Microbiology Division, CDRI, Lucknow
Equipments Bio-hood, CO2 Incubator, Refrigerated Centrifuge,
Microfuge, Fluorescent Microscope, Incubator Shaker, Water Bath,
Incubators, -860C Deep freezer, Power supplies, Gel apparatus,
Gradient Thermal Cycler, Gel Documentation System, Sonicator,
Protein Purification System, 2-D Gel Electrophoresis System, ABI Real
Time PCR.
Other resources
Animal House and BSL-III facility
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Thank you for your patience….Thank you for your patience….