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….