Post on 25-May-2015
description
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
M. tuberculosisM. tuberculosis
Robert Koch1882
~9 million new cases~2 million deaths worldwide2011
2011 ~2 million new cases in India
M. tuberculosis genomeM. tuberculosis genome
Cole et al. (1998) Nature 393: 537-544
4,000 genes 40% orphans
100 highly homologous
PE/PPE genes
General Classification of M. tuberculosis genesGeneral Classification of M. tuberculosis genes
CLINICAL MICROBIOLOGY REVIEWS, July 2003, p. 463–496
PE/PPE proteinsPE/PPE proteins
100 highly homologous genes with signature sequence of Pro-Glu (PE) amino acid near amino terminus.
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
Extensive functional redundancy
Close genomic & evolutionary association with ESX regions
Highly immunogenic
Properties of PE proteinsProperties of PE proteins
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
Features of PE-PGRS family
Role of PE_PGRSRole of PE_PGRS
Homeostasis of mycobacterial cell
Intracellular survival
Multiplication within its chosen environment
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
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
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
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.
Impact on antigen-presentation pathways Ensuing host immune responses, and Also provide a mechanism for generating antigenic diversity in mycobacteria
Research PlanResearch Plan
Cell culture1
2
3
4
Phagocytosis & intracellular surival
Cloning, expression & purification of PE3 & PE4
Immunological studies
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
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.
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
Thank you for your patience….Thank you for your patience….