L/O/G/O

Sameer Tiwari Division of Microbiology

Studies on the implications of PE class

proteins of Mycobacterium tuberculosis

as determinants of pathogenicity and

Immuno-prophylaxis

M. tuberculosis

Robert Koch 1882

~9 million new cases ~2 million deaths worldwide

2011

2011 ~2 million new cases in India

M. 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 genes

PE/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)

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 proteins

Classification of PE/PPE

protein families

Sequence variation between M.

tuberculosis H37Rv & M. bovis

BCG

Cole et al. (1998) Nature 393: 537-544

Role of PE_PGRS

Homeostasis of mycobacterial cell

Intracellular survival

Multiplication within its chosen environment

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…..

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

Objectives

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?

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 Plan

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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.

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….