Studies on the implications of PE class proteins of Mycobacterium tuberculosis as determinants of pathogenicity and Immuno-prophylaxis

1. Studies on the implications of PE class
proteins of Mycobacterium tuberculosis
as determinants of pathogenicity and
Immuno-prophylaxis
Sameer Tiwari
Division of Microbiology
L/O/G/O

2. M. tuberculosis
1882 Robert Koch
2011
2011
~9 million new cases
~2 million deaths worldwide
~2 million new cases in India

3. M. tuberculosis genome
4,000 genes
40% orphans
100 highly
homologous
PE/PPE genes
Cole et al. (1998)
Nature 393: 537-544

4. General Classification of M. tuberculosis genes

5. PE/PPE proteins
100 highly homologous genes with signature sequence of
Pro-Glu (PE) amino acid near amino terminus.

6. PE-PGRS
(polymorphic GC rich repetitive sequences)
1901 aa
550 aa
175 aa
Rv3508
Average
Rv0742
Larger proteins domain extremely rich in Gly (~ 40%) & Ala (~ 25%)
residues PGRS domain occur as repetitive sequences (repeated >30times
within domain)

7. Properties of PE proteins
Extensive functional redundancy
Close genomic & evolutionary association with ESX regions
Highly immunogenic

8. Classification of PE/PPE
protein families
Sequence variation between M.
tuberculosis H37Rv & M. bovis
BCG
Cole et al. (1998)
Nature 393: 537-544

10. Role of PE_PGRS
Homeostasis of
mycobacterial cell
Intracellular survival
Multiplication within its
chosen environment

11. Why to study?
Unique gene
family
No counterpart
in bacteria
Potential
source of
antigenic
variation
No strict
correlation with
pathogenic
strains
Little is known

12. Known facts…..
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
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

13. Objectives
Evaluation of complete cellular immune responses
incurred by these proteins
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.
.

14. What we Achieve?
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.
Identification of domains involved in binding to cell
surface or other intracellular components will help in
designing targets against these proteins.

15. Impact on antigen-presentation pathways
Ensuing host immune responses, and
Also provide a mechanism for generating antigenic diversity in
mycobacteria

16. Research Plan
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18. Conclusions
The 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.

19. 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

21. Thank you for your
patience….