Abstract
Tuberculosis remains a scourge of humanity with a high mortality rate
and killing a couple of million people every year. Tuberculosis is caused by
Mycobacterium tuberculosis which is an incredibly successful pathogen. One
third of the world’s population has been infected with M. tuberculosis.
Although The World Health Organization has implemented several programs
to eradicate this dreaded disease, the number of new cases of tuberculosis
being reported every year is increasing at an alarming rate.
M. tuberculosis is transmitted in the form of aerosolized droplets
generated by the cough of an infected individual. These ingested bacilli are
engulfed by alveolar macrophages. Mycobacterium not only survives the
microbicidal techniques of the macrophages and evades the immune response
but also replicates during the early stages of infection. The site of infection is
characterized by the formation of granulomatous lesion or a tubercle. In spite
of the highly effective immune response against M. tuberculosis, the bacillus
persists within the tubercle, in a latent state, for a prolonged time interval and
is called latent tuberculosis. The reactivation of latent tuberculosis sometimes
occurs in these infected individuals due to compromised immune system. The
mechanism of latency and its subsequent reactivation is not fully
characterized and is a subject of active research. The elucidation of these
mechanisms will pave the way for new drugs and vaccines for the treatment of
tuberculosis.
The heat shock proteins have been implicated in the pathogenesis of M.
tuberculosis. The GroE and DnaK families are the major heat shock
chaperone machineries in M. tuberculosis. The genome of M. tuberculosis has
two paralogs of groEL, groEL1 (Rv3417c) and groEL2 (Rv0440). These
v
GroELs have been previously characterized to be present in uncanonical
oligomerisation states. Sequence analyses of more than 200 sequences have
revealed that both the M. tuberculosis GroELs have significantly diverged in
their functions during the course of evolution. Unlike E.coli GroEL,
M. tuberculosis GroEL1 has a histidine rich C- terminal tail which has been
demonstrated to be defective in biofilm formation in Mycobacterium
smegmatis. M. tuberculosis GroEL1 is a nonessential gene and mutant lacking
groEL-1 is defective in cytokine dependent granulomatous response. These
intriguing observations emphasize the necessity to further characterize the
heat shock machinery of M. tuberculosis.
The work embodied in this thesis aims to understand the structural
and functional aspects of M. tuberculosis transcription factors and stress
proteins, with primary focus being on the Hsp60 family of chaperones. This
thesis demonstrates the DNA binding property of un-canonical chaperone
GroEL1 for the first time. The role of GroEL1 as a nucleoid associated protein
is also elucidated. Furthermore, experiments have been performed to identify
the genome wide binding sites of GroEL1. The putative transcriptional
repressor protein of M. tuberculosis GroE system, HrcA has also been
characterized. The in depth characterization of these proteins have been
described systematically in six chapters. Chapter 1 of the thesis introduces
the topic of research, reviews the various aspects of the mode of action of
M. tuberculosis, the role of heat shock proteins in the pathogenesis and the
un- canonical nature of GroE system and its regulation. This chapter
describes the recent advancements in the field and also defines the main
objectives of the study.
The various techniques implemented during the course of this study
are described in Chapter 2. This study utilizes the recombinant DNA
vi
techniques and a spectrum of protein purification procedures. Several DNA
protein interaction protocols (Electrophoretic mobility shift assay, Chromatin
immuno-precipitation followed by Southern blot hybridization) are employed
to address the objective of the study has been described in detail. The
procedure for isolation of nucleoid from M. tuberculosis has been elaborately
described.
The regulation of stress response has an important role in pathogenesis
of M. tuberculosis. Chapter 3 focuses on the biochemical and biophysical
characterization
of
HrcA
(Heat
regulation
at
CIRCE),
a
putative
transcriptional regulator protein of the GroE operon. ANS fluorescence
studies demonstrates that the protein to have exposed hydrophobic residues
and Circular Dichroism studies characterize HrcA to be a predominantly
α- helical protein. These studies further demonstrate that GroEL1 and HrcA
do not interact to each other in the experimental conditions. The probability
of cross regulation of both the GroE and DnaK heat shock operons by HspR
has been experimentally disproved. The most striking observation of this
chapter is the observation of DNA binding ability of GroEL1 to CIRCE instead
of HrcA under identical experimental conditions. This chapter reveals the
uncanonical nature of stress response in M. tuberculosis, a feature which is
further studied in the following chapters.
The DNA binding ability of M. tuberculosis GroEL1 and its probable
physiological
functions
have
been
characterized
in
Chapter
4.
M. tuberculosis genome sequence shows the remarkable absence of many
nucleoid associated proteins. The nucleoids of M. tuberculosis have been
purified and interestingly discovered that a novel nucleoid associated protein
in M. tuberculosis is a sequence homolog of GroEL chaperonin. This chapter
reports that M. tuberculosis GroEL1 binds nucleic acid substrates without
vii
sequence specificity and plays a role in the condensation of DNA in nucleoid
formation. Chromatin immunoprecipitation followed by Southern blot
hybridization and immunofluorescence imaging of M. tuberculosis confirms
the in vivo GroEL-1 DNA interaction in M. tuberculosis. This chapter
therefore reveals that GroEL1 has evolved to be associated with the nucleoids.
The southern blot hybridization and immuno-fluorescence microscopy
reported in Chapter 4 suggested that GroEL1 has a preference for binding
some regions of the M. tuberculosis genome. To address this observation,
Chromatin immunoprecipitation followed by microarray analysis (ChIP-chip)
was performed to identify all the binding sites of GroEL1 across the entire
genome. The ChIP-chip experiment and its experimental validations have
been reported in Chapter 5. Surprisingly, genome-wide binding analyses
revealed preferential enrichment in the genes encoding PE-PGRS class of
family by ChIP-chip assay. A consensus motif for the GroEL1-DNA interaction
was also identified from the ChIP-chip interaction.
The implications of the present research work on the understanding of
the heat shock proteins of M. tuberculosis have been described in Chapter 6.
This is the first report to describe the DNA binding property of
M. tuberculosis GroEL1. The results presented in this thesis characterize this
novel property of the unique chaperonin GroEL1 of M. tuberculosis in detail.
The present results suggest several directions and opportunities for future
research work.
viii