Tài liệu Báo cáo khoa học: Survival mechanisms of pathogenic Mycobacterium tuberculosis H37Rv ppt

REVIEW ARTICLE
Survival mechanisms of pathogenic Mycobacterium
tuberculosis H
37
Rv
Laxman S. Meena and Rajni
Institute of Genomics and Integrative Biology, Delhi, India
Introduction
Five decades of tuberculosis (TB) control programs
using potentially efficacious drugs have failed to reduce
prevalence of infection by the causative organism,
Mycobacterium tuberculosis, in most parts of the world
[1]. A large number of individuals (more than three
billion) have been vaccinated with Bacillus Calmette-
Gue
´
rin (BCG), but TB still kills more than 50 000
people every week and approximately one-third of the
world’s population is asymptomatically infected by
M. tuberculosis [2]. It is estimated that 200 million
people will display symptoms and that 35 million will
die of TB between 2000 and 2020 if control and pre-
ventive measures are not strengthened (World Health
Organization Annual Report, 2000). TB accounts for
32% of the deaths in HIV infected individuals [3]. The
situation is exacerbated by the emergence of multi-
drug-resistant TB [4] and the catastrophic nexus
between AIDS and TB [5,6]. A prerequisite for effec-
tive control is an understanding of the host–pathogen
interaction and its contribution to the development of
diseases. Our knowledge about how M. tuberculosis

enters the host cells is currently limited.
Mycobacterium tuberculosis has evolved several
mechanisms to circumvent the hostile environment of
the macrophage, its primary host cell (Figs 1 and 2).
Despite extensive research, our knowledge about the
virulence factor(s) of M. tuberculosis is quite inade-
quate. Understanding the molecular mechanisms of
M. tuberculosis pathogenesis will provide insights into
the development of target-specific drugs or effective
Keywords
dormancy; host cell; lysosome;
Mycobacterium; phagosome; signaling
transduction; tuberculosis; virulence factor
Correspondence
L. S. Meena, Institute of Genomics and
Integrative Biology, Mall Road, Delhi-
110007, India
Fax: +91 11 27667471
Tel: +91 11 27666156
E-mail: ;

(Received 2 February 2010, revised 12
March 2010, accepted 29 March 2010)
doi:10.1111/j.1742-4658.2010.07666.x
Mycobacterium tuberculosis H
37
Rv is a highly successful pathogen and its
success fully relies on its ability to utilize macrophages for its replication
and, more importantly, the macrophage should remain viable to host the
Mycobacterium. Despite the fact that these phagocytes are usually very

effective in internalizing and clearing most of the bacteria, M. tuberculosis
H
37
Rv has evolved a number of very effective survival strategies, including:
(a) the inhibition of phagosome–lysosome fusion; (b) the inhibition of
phagosome acidification; (c) the recruitment and retention of tryptophan-
aspartate containing coat protein on phagosomes to prevent their delivery
to lysosomes; and (d) the expression of members of the host-induced repeti-
tive glycine-rich protein family of proteins. However, the mechanisms by
which M. tuberculosis H
37
Rv enters the host cell, circumvents host defenses
and spreads to neighboring cell are not completely understood. Therefore,
a better understanding of host–pathogen interaction is essential if the glo-
bal tuberculosis pandemic is ever to be controlled. This review addresses
some of the pathogenic strategies of the M. tuberculosis H
37
Rv that aids in
its survival and pathogenicity.
Abbreviations
BCG, Bacillus Calmette-Gue
´
rin; LAM, lipoarabinomannan; PE-PGRS, a repetitive glycine-rich protein family; TACO, tryptophan-aspartate
containing coat protein; TB, tuberculosis.
2416 FEBS Journal 277 (2010) 2416–2427 ª 2010 The Authors Journal compilation ª 2010 FEBS
vaccine candidates for the treatment of the disease.
A variety of mechanisms have been suggested to con-
tribute towards the survival of Mycobacterium within
macrophages. These mechanisms are shown as a sche-
matic representation in Fig. 2. The present review aims

to summarize the mechanisms that M. tuberculosis uses
to establish itself with the phagosomes of the host
macrophages, with an emphasis on recent advances in
the field of mycobacterial pathogenesis.
Survival strategies employed by
M. tuberculosis to survive in host cells
Cell wall virulence factors
When Mycobacteria are stained by Gram staining, they
cannot be decolorized by acid alcohol and are there-
fore classified as acid-fast bacilli. Acid fastness is
largely a result of the high content of mycolic acids,
Rough endoplasmic
reticulum
Golgi appartus
Nucleus
Mitochondria
Vesicles
Lipofuscin
Engulfed material
Phagocytosis
Phagosome
Lysosome
Phagosome
lysosome fusion
Killing of ingested
pathogen
Release of digested
material by exocytosis
Fig. 1. Detailed structure of a macrophage
showing a typical process of phagocytosis.

Phagosome
Lysosome
Inhibition of fusion of phagosome harbouring
Mycobacteria with lysosome
TACO protein on phagosome
harbouring mycobacteria
TACO
Proton ATPase-
Pump
Virulence
Proteins
Expression of virulence proteins of
PE-PGRS family
Inhibition of acidification of phagosome
harbouring Mycobacteria
Protection from reactive oxidative radicals
Fusion
H
+
O
2
–
.
OH.
H
2
O
2
NO.
AD

BE
C
Fig. 2. Key factors of the survival mecha-
nisms involved in the phagosome matura-
tion arrest of Mycobacterium tuberculosis
inside macrophages.
L. S. Meena and Rajni Survival strategies of mycobacteria in host
FEBS Journal 277 (2010) 2416–2427 ª 2010 The Authors Journal compilation ª 2010 FEBS 2417
long chain cross-linked fatty acids and other cell-wall
lipids in the cell wall [7]. Mycolic acid and other lipids
are linked to underlying arabinogalactan and peptido-
glycan [8]. A variety of unique lipids, such as lipoara-
binomannan (LAM), trehalose dimycolate and
phthiocerol dimycocerate, anchor noncovalently with
the cell membrane and appear to play an important
role in the virulence of M. tuberculosis [9]. Lipids such
as cord factor (surface glycolipid that is toxic to mam-
malian cells and is also an inhibitor of polymorphonu-
clear leukocyte migration) induce cytokine-
mediated events [10,11], which may also contribute to
virulence. Treatment of Mycobacterium avium by isoni-
azid disrupts mycolic acid biosynthesis, which is
responsible for the cording (serpentine cording)
phenomenon, and thereby renders the mycobacteria
hydrophobic [12]. In the case of Mycobacterium
smegmatis, enhanced permeability as a result of disrup-
tion of a mycolate ⁄ cording factor gene causes reduced
growth both in vitro and in vivo [13]. Disruption of the
gene involved in mycolic acid cyclopropanation was
shown to alter cording properties and reduce virulence

[14]. Using whole genome transpositional mutagenesis
techniques, 30 mutants of M. tuberculosis were selected
from a total screen of approximately 2500 mutants
that showed reduced growth. Seven of these mutants
had insertion within a locus involved in the synthesis
of phthiocerol dimycocerosate, an abundant compo-
nent of cell wall biosynthesis [15]. Phthiocerol dimyco-
cerosate was subsequently shown to help entry of
Mycobacterium leprae into peripheral nerve cells by
binding to nerve cell laminin protein [16]. The majority
of exported proteins and protective antigens of
M. tuberculosis are a triad of related gene products
called the antigen 85 complex, each having fibronectin
binding capacity and thus an important role in disease
pathogenesis [17].
LAM is also a major constituent of mycobacterial
cell wall and has been shown to induce tumor necrosis
factor release from the macrophages [18], which plays
a prominent role in bacterial killing. Studies have
shown that LAM acts at several levels and that it can
scavenge potentially cytotoxic oxygen free radicals,
inhibit protein kinase C activity and block the tran-
scriptional activation of gamma interferon inducible
genes in human macrophages such as cell lines, and
hence contribute to the persistence of mycobacteria
within mononuclear phagocytes [19].
Host cell surface receptors
M. tuberculosis appears to gain entry into macrophages
via cell surface molecules, including those of the inte-
grin family CR1 and CR3 complement receptors [20]

and the mannose receptors [21]. By contrast, M. avium
enters macrophages via avb3, another receptor of inte-
grin family [22]. Unlike other bacteria, pathogenic
mycobacteria are opsonized with C3 peptides in an
entirely different way, involving the recruitment of the
complement fragment C2a to form a C3 convertase
and the generation of opsonically active C3b in the
absence of early activation components [23]. Individual
strains of M. tuberculosis can vary in their modes of
interaction with CR3, by interacting with distinct
domains of the receptor [24]. It has been shown that
mannose receptors bind the virulent Erdman and
H
37
Rv strains but not the avirulent M. tuberculosis
H
37
Ra strain. This difference in binding may arise
because strain H
37
Rv has ligands, such as LAM, that
bind to mannose receptors at different sites compared
to the M. tuberculosis H
37
Ra strain [25]. Furthermore,
it has been suggested that Fc receptor-mediated intake
of mycobacteria may involve distinct intracellular traf-
ficking for the virulent M. tuberculosis [26].
The relative contribution of various macrophage
receptors, such as complement receptors CR1, CR3

and CR4, mannose receptor, lung surfactant protein
receptors, CD14, scavenger receptors and Fc receptors,
in the intracellular fate and survival of M. tuberculosis
is still far from being understood [24]. Successful
pathogens (e.g. Salmonella typhi) appear to survive in
phagosomes by entering a receptor-mediated pathway
that is not coupled to the activation of macrophage
antimicrobial mechanisms, such as the production of
reactive oxygen or nitrogen intermediates [27]. How-
ever, to date, it is not yet clear how mycobacteria use
the advantage of selective receptor-mediated intracellu-
lar survival as a pathogenic strategy. It is possible that
the distinct routes of entry of M. tuberculosis result in
different cytokine secretion responses or different
downstream activation signals in the host macrophages,
leading to the differential survival of this pathogenic
bacteria.
Inhibition of phagosome–lysosome fusion
Both inhibition of growth and killing of intracellular
pathogens within the host cell of the mononuclear
phagocyte lineage are considered to be dependent on
phagosome–lysosome fusion [28]. Immediately after
engulfment by macrophages, most tubercle bacilli are
directed to phagolysosomes [29]. Subsequently, how-
ever, individual M. tuberculosis bud out from the fused
phagolysosomes into vacuoles that fail to fuse to the
secondary lysosomes and thus escape lysosomal killing.
Thus, temporary residence within a phagolysosome
Survival strategies of mycobacteria in host L. S. Meena and Rajni
2418 FEBS Journal 277 (2010) 2416–2427 ª 2010 The Authors Journal compilation ª 2010 FEBS

stimulates a response to the intracellular environment in
M. tuberculosis that facilitates its long-term survival and
reproduction. Sulfatides (anionic trehalose glycolipids)
of M. tuberculosis also have an antifusion effect [30].
M. tuberculosis can produce ammonia in abundance,
which is considered to be responsible for the inhibitory
effect of the culture supernatant of virulent mycobacte-
ria on phagolysosomal fusion [31]. Ammonium chloride
affects the movement of lysosomes by alkalizing the in-
tralysosomal compartment [32] and, as a result, it
diminishes the potency of intralysosomal enzymes via
alkalization. Live M. tuberculosis were shown to infect
human macrophages in the presence of low cytosolic
Ca
2+
, which is correlated with inhibition of phago-
some–lysosome fusion and intracellular viability. It
was suggested that defective activation of the Ca
2+
dependent effector proteins calmodulin and calmodulin-
dependent protein kinase 2 contributes to the intracellu-
lar pathogenesis of tuberculosis [33].
Inhibition of phagosomal acidification
The restricted fusogenicity of the mycobacterial vacuole
may extend beyond limiting the access of lysosomal
hydrolases to the bacilli. It has been reported that vacu-
oles containing M. avium are less acidic than neighbor-
ing lysosomes [31,34]. Within M. avium, the absence of
a vesicular proton-ATPase pump results in a lack of
acidification of phagosomes [35]. Recently, a role for

natural resistance-associated macrophage protein 1
has been demonstrated [36] in directly promoting
H
+
-ATPase-dependent acidification of Mycobacterium
bovis BCG phagosomes in peritoneal macrophages.
Maturation of phagosomes
M. tuberculosis residing within host phagosomes modi-
fies the maturation of the phagosomal compartment
and enhances intracellular survival. This maturation
leads to the inhibition of phagolysosomal fusion.
Moreover, the aberrant expression of Rab5 on the
phagosomes containing M. tuberculosis causes the mat-
uration arrest of these phagosomes at the early
endosomal stage [37]. Phagosomes containing inert
particles or avirulent bacteria transiently display Rab5,
whereas phagosomes containing virulent M. tuberculosis
exhibit a persistent display of Rab5 [37].
Recruitment and retention of tryptophan-aspartate
containing coat protein (TACO) on phagosome wall
Recruitment and retention of the host protein TACO
to phagosomes harboring mycobacteria prevents
bacterial delivery to lysosomes [38]. TACO⁄ coronin-1
is an actin binding protein known to associate with
cholesterol within the plasma membrane [39]. Reten-
tion of TACO on the phagosomal wall allows the
mycobacteria to escape the bactericidal action of
macrophages [38]. Vitamin D
3
and retinoic acid down-

regulate TACO gene transcription in a dose-dependent
manner. This down-regulation occurs through the
modulation of this gene via the VDR ⁄ RXR response
sequence present in the promoter region of TACO
gene. Treatment with vitamin D
3
and retinoic acid
inhibits mycobacterial entry, as well as survival within
macrophages [40]. Moreover, TACO-mediated uptake
of mycobacteria depends on cholesterol [39].
Dormancy or persistence within the
host macrophages
M. tuberculosis has the ability to remain dormant
within host cells for years at the same time as retaining
the potential to be activated. The dormancy or latency
of M. tuberculosis allows the bacterium to escape the
activated immune system of the host. Persistence of
M. tuberculosis in mice is facilitated by isocitrate lyase,
a glyoxylate shunt enzyme that is essential for the
metabolism of fatty acids [41]. Disruption of the icl
gene attenuated bacterial persistence and virulence in
immune-competent mice without affecting bacterial
growth during the acute phase of infection.
Several genes were identified as being preferentially
expressed when Mycobacterium marinum resides in the
host granulomas and ⁄ or macrophages [42]. Two of the
genes were found to be homologs of genes for
M. tuberculosis PE ⁄ PE-PGRS, a family encoding
numerous repetitive glycine-rich proteins of unknown
function(s). The mutation of these two genes for

PE-PGRS produced M. marinum strains that were
incapable of replication in macrophages. The strains
exhibited decreased persistence in granulomas, thereby
suggesting a direct role for PE-PGRS proteins in
mycobacterial virulence. Hypoxia was also observed to
be a major factor in inducing the nonreplicating persis-
tence of tubercle bacilli [43].
Protection against oxidative radicals
The macrophages offer a hostile environment to intra-
cellular bacteria by producing a vast array of chemi-
cals such as reactive oxygen and nitrogen radicals.
However, the virulent Erdman strain of M. tuberculosis
overexpresses a protein that cyclopropanates mycolic
acid double bonds, resulting in a ten-fold lower suscep-
tibility to peroxide [44]. Also, the oxyR (i.e. a sensor
L. S. Meena and Rajni Survival strategies of mycobacteria in host
FEBS Journal 277 (2010) 2416–2427 ª 2010 The Authors Journal compilation ª 2010 FEBS 2419
of oxidative stress and a transcriptional activator that
induces the expression of detoxifying enzymes such
as catalase ⁄ hydroperoxidase) of M. tuberculosis has
numerous deletions and frameshift mutations giving
the appearance of a pseudogene [45]. Perhaps the pro-
tection afforded by cyclopropanated cell wall compo-
nents has rendered oxyR superfluous in pathogenic
mycobacteria. Superoxide dismutases play an impor-
tant role in protection against oxidative stress and so
contribute to the pathogenicity of many bacterial
species [46].
Virulence genes of M. tuberculosis
Initial efforts aimed at identifying the genes involved

in the pathogenesis of M. tuberculosis involved the
cloning and expression of random genomic DNA frag-
ments of pathogenic bacteria into surrogate hosts such
as Escherichia coli, followed by the analysis of survival
of recombinant E. coli in macrophage cell lines. To
identify the genes involved in the invasion of macro-
phages by M. tuberculosis, a gene fragment mce was
identified that encodes a 52 kDa protein conferring
E. coli with the ability to invade HeLa cells and sur-
vive within the host macrophages [47]. The intracellu-
lar survival of bacteria was impaired with the
spontaneous loss of DNA from the transformants.
Four copies of the mce gene have been identified in the
M. tuberculosis genome and have been designated as
mce1, mce2, mce3 and mce4 [48]. The exact function of
Mce1 is still unknown; it appears to serve as an effec-
tor molecule expressed on the surface of M. tuberculo-
sis that is capable of eliciting plasma membrane
perturbations in nonphagocytic mammalian cells [49].
In another study, the gene encoding Mce3 protein was
disrupted in the vaccine strain M. bovis BCG [50]. The
mutant strain exhibited a reduced ability to invade
nonphagocytic HeLa cell lines compared to the wild-
type BCG, supporting the idea that this gene is
involved in the invasion host tissues.
M. smegmatis has been used as a surrogate host for
cloning, expressing genes and constructing genomic
libraries of M. tuberculosis [51,52]. To identify the
genes essential for survival of mycobacteria within
macrophages, a plasmid library was constructed by

using genomic DNA from M. tuberculosis and electro-
porated into M. smegmatis [53]. The transformants
were used to infect the human macrophages cell line
U-937, and one transformant (eis) was isolated that
showed an enhanced survival over a period of 48 h
compared to the wild-type M. smegmatis [53]. The eis
gene, which encodes a 42 kDa protein, confers
M. smegmatis with the ability to resist killing by host
macrophages. The function of the Eis protein is still
unknown. It has been suggested that the secreted pro-
teins of mycobacteria have a profound influence on its
pathogenicity. It was found that the disruption of an
erp gene of M. tuberculosis encoding a secretory pro-
tein effects the survival of M. tuberculosis in host mac-
rophages [54].
In many Gram-negative bacteria, iron-regulated
genes are essential for the expression of full virulence
[55]. It is likely that the acquisition of iron by M. tuber-
culosis is also essential for growth and survival during
the course of infection. M. tuberculosis synthesizes two
distinct iron-regulated siderophores: the cell surface-
associated mycobactin and the excreted siderophore,
exochelin [56]. The mbtB gene, which is involved in the
biosynthesis of siderophores, was disrupted in
M. tuberculosis and the resulting mutant was observed
to have a restricted growth in iron-depleted conditions
[57]. The mutant also exhibited stunted growth pattern
in human monocyte cell line THP-1, suggesting a role
for siderophores in virulence.
M. tuberculosis and other mycobacterial species also

produce a number of iron-regulated membrane pro-
teins [56]. For example, iron-dependent regulatory pro-
tein (IdeR) of M. tuberculosis has been characterized
as a functional homolog of the diphtheria toxin repres-
sor from Corynebacterium diphtheriae [58,59]. The ideR
gene was shown to be necessary for high-level expres-
sion of the SodA and INH proteins that are involved
in the pathogenesis of mycobacteria [60].
It was revealed that the anti-apoptosis activity was a
result of the type-1 NADH- dehydrogenase of
M. tuberculosis and the main subunit of this multicom-
ponent complex is encoded by the gene for Nuo G.
Deletion of nuo G in
M. tuberculosis resulted in its
inability to inhibit macrophage apoptosis and signifi-
cantly reduced its virulence [61]. Another gene, named
fad D33, encoding an acyl-coenzyme A synthase, plays
an important role in M. tuberculosis virulence by sup-
porting growth in the liver [62]. Several other genes
demonstrated to be essential for the survival of myco-
bacteria in macrophages are shown in Table 1.
Modulating host signal network
A new perspective in the pathogenesis of M. tuberculo-
sis is the exploitation of host cell signaling pathways
by the pathogen. Upon infection, the phosphatases
and kinases of several pathogenic bacteria modify host
proteins and help in the establishment of the disease.
The uptake of M. tuberculosis by macrophages is
associated with a number of early signaling events,
such as the recruitment and activation of members of

Survival strategies of mycobacteria in host L. S. Meena and Rajni
2420 FEBS Journal 277 (2010) 2416–2427 ª 2010 The Authors Journal compilation ª 2010 FEBS
the Src family of protein tyrosine kinases. These kinas-
es result in the increased tyrosine phosphorylation of
multiple macrophage proteins and the activation of
phospholipase D [82]. Activation of protein tyrosine
kinases appears to enhance stimulation of phospholi-
pase D activity and the associated increase in phospha-
tidic acid. Phosphatidic acid may trigger a number of
downstream processes that are necessary for membrane
remodeling during phagocytosis and the intracellular
survival of M. smegmatis in host cells [83]. Further-
more, LAM from the virulent species of M. tuberculo-
sis possesses the ability to modulate signaling
pathways linked to bacterial survival by phosphoryla-
tion of an apoptotic protein in the phosphatidylinositol
3-kinase-dependent pathway in THP-1 cells [84].
Many regulatory proteins or enzymes commonly
known as G-proteins play a vital role in cell signaling by
binding and hydrolyzing GTP to GDP [85]. Despite
their common biochemical function of GTP hydrolysis,
these proteins are associated with diverse biological
functions. In eukaryotes, G-proteins are classified into
three main groups: Ras and its homologs; the transla-
tion elongation factors [86], Tu and G; and the a
subunits of heterotrimeric G-proteins. All members of
this group share a common structural core, suggesting a
common evolutionary origin for these proteins. The
members of G-protein superfamily are known to play a
complex array of functions in eukaryotes, such as, hor-

mone action, visual transduction and protein synthesis.
By contrast to the eukaryotic counterparts, the
function of most of the universally conserved bacterial
GTPases is still poorly understood. In recent years,
there have been significant advances in the research
related to the GTP-binding protein in the prokaryotes.
Table 1. Genes involved in virulence of mycobacteria.
Serial number Gene name Gene number Function References
1 aceA Rv0467 Isocitrate lyase ⁄ dormancy [63]
2 mceD Rv0170 Cell invasion [64]
3 cmaA,
mmaA4
Rv3392c ⁄ Rv0503c ⁄
Rv0642c
Mycolic acid biosynthesis [65]
4 sigE ⁄ sigH Rv1221 ⁄ Rv3223c Sigma factors [66]
5 Acr Rv2031c Growth in macrophages [67]
6 drrC Rv2938 ABC transporter [68]
7 – Rv3718c PE-PGRS family [42]
8 erp Rv3810 Cell-wall associated surface proteins [54]
9 ideR Rv2711 Iron-dependent repressor [69]
10 glnA Rv2220 Nitrogen metabolism [70]
11 aphC Rv2428 Oxidative stress defense [71]
12 KatG Rv1908c Catalase ⁄ peroxidase [72]
13 fadD26 Rv2930 Lipid metabolism [73]
14 fadD28 Rv2941 Mycocerosis acid synthesis [74]
15 fbpa Rv3804c Mycolyl transferase [75]
16 PKnG Rv0410c Phosphorylates the peptide substrate myelin
basic protein at serine residues ⁄ serine ⁄
threonine-protein kinase protein kinase G

[76]
17 Pks2 Rv3825c Polyketide synthase PKS2 [77]
18 fadE28 Rv3544c Acyl-coenzyme A dehydrogenase [78]
19 nuoG Rv3151 NADH dehydrogenase I (chain G)
NADH-ubiquinone oxidoreductase chain G
[61]
20 phoP Rv0757 Positive regulator for the phosphate regulon,
required for intracellular growth
[79]
21 plcA Rv2351c Phospholipase c 1 plca (mtp40 antigen) [80]
22 plcB Rv2350c Membrane-associated phospholipase c 2 plcb [80]
23 plcC Rv2349c Intracellular survival, by the alteration of cell
signaling events or by direct cytotoxicity ⁄
phospholipase c 3 plcc
[80]
24 plcD Rv1755c Intracellular survival, by the alteration of cell
signaling events or by direct cytotoxicity ⁄
phospholipase c 4 (fragment) plcd.
[80]
25 mmpL8 Rv3823c Considered to be involved in the transport of
lipids and shown to be required in the
production of a sulfated glycolipid, sulfolipid-1
[81]
L. S. Meena and Rajni Survival strategies of mycobacteria in host
FEBS Journal 277 (2010) 2416–2427 ª 2010 The Authors Journal compilation ª 2010 FEBS 2421
Recent studies have shown that bacterial GTPases con-
trol vast arrays of function, such as the regulation of
ribosomal function and the cell cycle, the modulation
of DNA partitioning and DNA segregation [87]. The
best known prokaryotic small GTP-binding protein is

Era (named for ‘E. coli Ras-like protein’). Era is essen-
tial for the growth of E. coli, Salmonella typhimurium
and Streptococcus mutans because mutants of Era
reveal pleiotropic phenotypes, including alterations in
the regulation of carbon metabolism, the stringent
response and cell division [88–92]. In E. coli, depletion
of Era at 27 °C was shown to cause cell filamentation
[92] and a mutation in the GTP-binding domain sup-
presses temperature-sensitive chromosome partitioning
mutations, indicating that Era is a cell-cycle check-
point regulator [89,90].
Interestingly, similar to Era, homologs of Obg (a new
subfamily of small GTP-binding protein) are also
present both in prokaryotes and eukaryotes [93].
Several bacterial homologs of the Obg subfamily have
been characterized, and examples include Obg proteins
from Bacillus subtilis, Streptomyces griseus, Streptomy-
ces coelicolor, CgtA (CgtA is also called ObgE)
proteins from Caulobacter crescentus, E. coli, Vibrio
harveyi and YhbZ from Haemophilus influenzae
[93–99]. Obg proteins of B. subtilis and Streptomyces
species are essential for vegetative growth and the
initiation of sporulation [93,94,96,100,101]. The Obg
homologue (CgtA) in C. crescentus was shown to be
indispensable for growth [97]. Similarly, E. coli homo-
log YhbZ (renamed ObgE) has also been reported to
comprise an essential gene involved in the chromosome
partitioning [95]. Besides these roles for Era and Obg,
this category of protein has also been shown to be
necessary for the stress-dependent activation of

transcription factors. Homologs of the Ras family of
GTP-binding proteins have also been shown to
contribute to morphology and virulence in several
pathogenic fungi [101].
LepA is another member of GTP-binding protein
family; however, its exact function is still not clear.
Helicobacter pylori resides in the gastric mucus layer,
where the pH is in the range 4.5–5.0; therefore, to per-
sist in the hostile acidic environment of the stomach, it
must survive acid shock and grow at acidic pH. Inacti-
vation of an ortholog of the E. coli LepA in H. pylori
resulted in the inability of mutant to grow at pH 4.8,
suggesting that LepA is essential for the growth of
H. pylori under acidic conditions and that it might
play a critical role in infection by this pathogen [103].
Microbial pathogens such as mycobacteria have sus-
tained a long lasting association with their host
because they have evolved sophisticated mechanisms to
interfere with the macrophage signaling process and
eventually affect the overall phagocytosis process.
Keeping in view the importance of G-proteins, one
approach to help understand this mechanism would
involve looking for the presence of such G-proteins in
M. tuberculosis, which might interfere with the cell sig-
naling and might be specifically expressed under
growth of bacteria in macrophage.
Keeping in mind the importance of members of
G-proteins in diverse functions such as bacterial
growth, survival, stress management and virulence, we
investigated the complete genome sequence of

M. tuberculosis, aiming to identify the presence of
genes encoding the GTP-binding proteins. The genome
sequence of M. tuberculosis demonstrated that, in addi-
tion to homologs of Obg and Era, the additional
family member LepA is also present.
In our earlier study, three G-proteins, Era, Obg and
LepA of M. tuberculosis, were cloned and expressed in
E. coli. Purified proteins showed GTP-binding and
hydrolyzing activities [104]. A point mutation in the
conserved GTP-binding motif, AspXXGly (Asp to Ala),
in Era (Asp-258) and Obg (Asp-212) proteins resulted in
the loss of the associated activities, confirming that
known key residues in well-established G-proteins are
also conserved in mycobacterial homologs. This study
confirms that M. tuberculosis harbors functional Era,
Obg and LepA proteins. Mycobacterium tuberculosis is
an intracellular pathogen and has evolved strategies to
survive in the acidic environment of macrophages.
Therefore, it would be interesting to determine whether
functional Era, Obg and LepA proteins of M. tuberculo-
sis, similar to their counterparts in other bacteria, play a
crucial role in its survival ⁄ pathogenesis.
Protein kinases have been found to coordinate the
stress response, the developmental process and patho-
genicity in several microorganisms [105]. The presence
of functional Ser ⁄ Thr kinases [106] in mycobacteria
was reported prior to the release of the complete gen-
ome sequence of M. tuberculosis, and the genomic
sequence then suggested the presence of eleven putative
protein kinases [48]. The serine ⁄ threonine kinases of

M. tuberculosis are likely to mediate specific signal
transduction events with host pathways. Protein kinas-
es G and F may comprise key molecules that change
the phosphorylation pattern of host proteins upon
infection, thereby promoting bacterial survival [107].
Inhibitors of protein kinases have also been shown to
prevent the uptake of M. leprae by peritoneal macro-
phages of mice [108]. This suggests that the protein
kinases of M. tuberculosis may be involved in modify-
ing the host phosphorylation pattern to promote their
establishment and survival within the host cells.
Survival strategies of mycobacteria in host L. S. Meena and Rajni
2422 FEBS Journal 277 (2010) 2416–2427 ª 2010 The Authors Journal compilation ª 2010 FEBS
A major anti-phosphotyrosine reactive protein is
present only in strains belonging to M. tuberculosis
complex [109]. Thus, protein phosphorylation may
play an important role in the pathogenesis of myco-
bacteria. It has been shown that M. tuberculosis has
two functional tyrosine phosphatases that are secreted
into the culture supernatant, and that they may inter-
fere within the host cells [110].
Recently, a new transporter family (mmpL) was
shown to transport lipid molecules into host cells [9],
where they may interact with specific host cellular tar-
gets and serve to modulate the host-signaling network.
Mycobacterial lipids can be found in host cytoplasm
without a mycobacterial presence within the host cells
[111]. Stress-induced p38 mitogen-activated protein
kinase is a component of M. tuberculosis phagosome
arrest. The uptake of Mycobacterium stimulates p38

phosphorylation in the macrophage. EEA1 (i.e. early
endosomal autoantigen) plays an essential role in
phagosome maturation. EEA1 is recruited to mem-
brane by Rab 5 and by PI3P [112]. It was proposed
that the PKnH kinase of M. tuberculosis mediates a
host signal and triggers events that are responsible for
the intracellular survival of the bacterium, thus leading
to chronic infection [113].
Conclusions
M. tuberculosis, the causative agent of tuberculosis is
still a major burden to human health. M. tuberculosis
is very unusual among the bacterial pathogens with
respect to its ability to persist in the face of host
immune responses. The ability of M. tuberculosis to
persist within macrophages is well known, although
the molecular mechanisms behind this resistance have
not been resolved so far. However, the release of the
complete genome sequence of M. tuberculosis, as well
as recent advances in functional genomics tools (e.g.
microarrays and proteomics), in combination with
modern approaches, has facilitated a more rational
and directional approach towards the understanding of
these mechanisms. Therefore, it is clear that a better
understanding of these mechanisms and the host–path-
ogen interaction will be essential not only to control
this pandemic, but also to elucidate the novel features
of macrophage defenses and host immune responses.
The success of M. tuberculosis during the parasitization
of macrophages involves a modulation of the normal
progression of the phagosome into an acidic and

hydrolytically active phagolysosome, and also avoids
the development of localized, productive immune
responses against M. tuberculosis in the host.
Acknowledgements
We thank Rajesh S. Gokhale for making this work
possible. We also thank Hemant Khanna (University
of Michigan, Flint, MI, USA) for providing valuable
suggestions. The authors acknowledge financial sup-
port from GAP0050 of the Department of Science and
Technology and Council of Scientific & Industrial
Research.
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