tuberculosis pathogenesis will provide insights into the development of target-specific drugs or effective Keywords dormancy; host cell; lysosome; Mycobacterium; phagosome; signaling tran
Trang 1Survival mechanisms of pathogenic Mycobacterium
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: meena@igib.res.in;
laxmansm72@yahoo.com
(Received 2 February 2010, revised 12
March 2010, accepted 29 March 2010)
doi:10.1111/j.1742-4658.2010.07666.x
Mycobacterium tuberculosis H37Rv 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
H37Rv 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 H37Rv 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 H37Rv 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.
Trang 2vaccine 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+
O2. OH.
H2O2 NO.
C
Fig 2 Key factors of the survival
mecha-nisms involved in the phagosome
matura-tion arrest of Mycobacterium tuberculosis
inside macrophages.
Trang 3long 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 tuberculosisappears 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
H37Rv strains but not the avirulent M tuberculosis
H37Ra strain This difference in binding may arise because strain H37Rv has ligands, such as LAM, that bind to mannose receptors at different sites compared
to the M tuberculosis H37Ra 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
Trang 4stimulates a response to the intracellular environment in
M tuberculosisthat facilitates its long-term survival and
reproduction Sulfatides (anionic trehalose glycolipids)
of M tuberculosis also have an antifusion effect [30]
M tuberculosiscan 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
Ca2+, which is correlated with inhibition of
phago-some–lysosome fusion and intracellular viability It
was suggested that defective activation of the Ca2+
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
bovisBCG phagosomes in peritoneal macrophages
Maturation of phagosomes
M tuberculosisresiding 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 D3and 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 D3 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 radichemi-cals 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
Trang 5of 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 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
Trang 6the Src family of protein tyrosine kinases These
kinas-es rkinas-esult 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 Phosphaphospha-tidic 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 elongatransla-tion 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.
mmaA4
Rv3392c ⁄ Rv0503c ⁄ Rv0642c
basic protein at serine residues ⁄ serine ⁄ threonine-protein kinase protein kinase G
[76]
NADH-ubiquinone oxidoreductase chain G
[61]
required for intracellular growth
[79]
signaling events or by direct cytotoxicity ⁄ phospholipase c 3 plcc
[80]
signaling events or by direct cytotoxicity ⁄ phospholipase c 4 (fragment) plcd.
[80]
lipids and shown to be required in the production of a sulfated glycolipid, sulfolipid-1
[81]
Trang 7Recent 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 27C 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 addiaddi-tional 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 moleculkinas-es 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
Trang 8A 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
References
1 Scheindlin S (2006) The fight against tuberculosis Molecular interventions 6, 124–130
2 Snider DE, Raviglione M & Kochi A (1994) Global burden of tuberculosis In Tuberculosis: Pathogenesis, Protection, and Control (Bloom BR ed), pp 2–11 American Society for Microbiology, Washington, DC
3 Raviglione MC, Snider DE & Kochi A (1995) Global epidemic of tuberculosis: morbidity and mortality of a world wide epidemic JAMA 273, 220–226
4 Culliton BJ (1992) Drug-resistant tuberculosis may bring epidemic Nature (London) 356, 473
5 Butler D (2000) New fronts in old war Nature 406, 670–672
6 Chaisson RE & Slutkin G (1989) Tuberculosis and human immunodeficiency virus infection J Infect Dis
159, 96–100
7 Daffe´ M & Draper P (1998) The envelope layers of mycobacteria with reference to their pathogenicity Adv Microb Physiol 39, 131–203
8 Ratledge C (1982) Nutrition, growth, and metabolism
In The Biology of Mycobacterial (Ratledge C & Stanford J eds) pp 186–212 Academic Press Inc, London
9 Glickman MS & Jacobs WR (2001) Microbial patho-genesis of Mycobacterium tuberculosis: dawn of a disci-pline Cell 104, 477–485
10 Indrigo J, Hunter RL & Actor JK (2003) Cord factor trehalose mediates trafficking events during mycobacte-rial 6, 69-dimycolate (TDM) infection of murine mac-rophages Microbiology 149, 2049–2059
11 Devergne O, Emilie D, Peuchmaur M, Crevon MC, DiAgay MF & Galanaud P (1992) Production of cyto-kines in sarcoid lymph nodes: preferential expression of interleukin-1-beta and interferon-gamma genes Hum Pathol 23, 317–323
12 Barry CE (2001) Mycobacterium smegmatis: an absurd model for tuberculosis? Trends Microbiol 9, 473–474
13 Liu J & Nikaido H (1999) A mutant of Mycobacte-rium smegmatisdefective in the biosynthesis in mycolic acid biosynthesis accumulates meromycolates Proc Natl Acad Sci USA 96, 4011–4016
Trang 914 Glickmann MS (2000) A novel mycolic acid
cyclopro-pane synthetase is required for coding, persistence and
virulence of Mycobacterium tuberculosis Mol Cell 5,
717–727
15 Cox JS, Chen B, McNeil M & Jacobs WR Jr (1999)
Complex lipids determines the tissue-specific replication
of Mycobacterium tuberculosis in mice Nature 402,
79–83
16 Ng V, Zanazzi G, Timpl R, Talts JF, Salzer JL,
Brennan PJ & Rambukkana A (2000) Role of the cell
wall phenolic glycolipid-1 in the peripheral nerve
predilection of Mycobacterium leprae Cell 103,
511–524
17 Belisle JT (1997) Role of the major antigen of
Mycobacterium tuberculosisin cell wall biogenesis
Science 276, 1420–1422
18 Chatterjee D (1992) Lipoarabinomannan
Multigly-cosylated form of the mycobacterial
mannosylphos-phatidylinositols J Biol Chem 267, 6228–6233
19 Chan J, Fan X, Hunter SW, Brennan PJ & Bloom BR
(1991) Lipoarabinomannan, a possible virulence factor
involved in persistence of Mycobacterium tuberculosis
within macrophages Infect Immun 59, 1755–1761
20 Ahearn JM & Fearon DT (1989) Structure and
func-tion of the complement receptors, CR1 (CD35) and
CR 2 (CD21) Adv Immunol 46, 183–219
21 Schlesinger LS (1993) Macrophage phagocytosis of
vir-ulent but not attenuated strains of
Mycobacte-rium tuberculosisis mediated by mannose receptors in
addition to complement receptors J Immunol 150,
2920–2930
22 Rao SP, Ogata K & Cantanzaro A (1993)
Mycobacte-rium avium –M intracellulare binds to the integrin
receptor v3 on human monocyte and monocyte derived
macrophages Infect Immun 61, 663–670
23 Schorey JS, Carroll MC & Brown EJ (1997) A
macro-phage invasion mechanism of pathogenic mycobacteria
Science 277, 1091–1093
24 Ernst JD (1998) Macrophages receptors for
Mycobacte-rium tuberculosis Infect Immun 68, 1277–1281
25 Schlesinger LS, Kaufmann TM, Iyer S, Hull SR &
Marchiando LK (1996) Differences in mannose
recep-tor mediated uptake of lipoarabinomannan from
virulent and attenuated strains of
Mycobacte-rium tuberculosisby human macrophages J Immunol
157, 4558–4575
26 Armstrong J & DiArcy HP (1971) Response of
cul-tured macrophages to Mycobacterium tuberculosis, with
observation on fusion of lysosomes with phagosomes
J Exp Med 134, 713–740
27 Ishibashi Y & Arai T (1990) Roles of the complement
receptors type 1 (CR 1) and type 3 (CR 3) on
phogocy-tosis and subsequent phagosome lysosome fusion in
salmonella infected murine macrophages FEMS
Microbiol Immunol 2, 89–96
28 Moulder JW (1985) Comparative biology of intracellu-lar parasitism Microbiol Rev 49, 298–337
29 McDonough KA, Kress Y & Bloom BR (1993) Patho-genesis of tuberculosis Interaction of Mycobacte-rium tuberculosiswith macrophages Infect Immun 61, 2763–2773
30 Goren MB (1977) Phagocyte lysosomes: interactions with infectious agents, phagosomes, and experimental perturbations in function Annu Rev Microbiol 31, 507– 533
31 Gorden AH, Hart PA & Young MR (1980) Ammonia inhibits phagosomes-lysosome fusion in macrophages Nature 286, 79–81
32 Hart D, Young MR, Jordan MW, Perkins WJ & Geisow MJ (1983) Chemical inhibitors of phagosome– lysosome fusion in cultured macrophages also inhibit salutary lysosomal movements A combined micro-scopic and computer study J Exp Med 158, 477–492
33 Malik ZA, Shankar SI & Kusner DJ (2001) Mycobac-terium tuberculosisphagosomes exhibit altered calmod-ulin-dependent signal transduction contribution to inhibition of phagosome–lysosome fusion and intracel-lular survival in human macrophages J Immunol 166, 3392–3401
34 Crowle AJ, Dahl R, Ross E & May MH (1991) Evidence that vesicles containing living, virulent, Mycobacterium aviumin cultured human macrophages are not acidic Infect Immun 59, 1823–1831
35 Sturgill-Koszycki S, Schlesinger PH, Chakraborty P, Haddix PL, Collins HL, Fok AK, Allen RD, Gluck SL, Heuser J & Russell DG (1994) Lack of acidification in Mycobacteriumphagosomes produced by exclusion of the vesicular proton-ATPase Science 263, 678–681
36 Hackam DJ, Roptstein OD, Zhang WJ, Cruenheid SP
& Grinstein S (1998) Host resistance to intracellular infection: mutation of natural resistance-associated macrophages protein 1 (Nramp 1) impairs phagosomal acidification J Exp Med 188, 351–364
37 Clemens DL, Lee B & Horwitz MA (2000) Deviant expression of Rab5 on phagosomes containing the intracellular pathogens Mycobacterium tuberculosis and Legionella pneumophilais associated with altered phag-osomal fate Infect Immun 68, 2671–2684
38 Ferrari G, Langen H, Naito M & Pieters J (1999) A coat protein on phagosomes involved in the intracellu-lar survival of mycobacteria Cell 97, 435–447
39 Gatfield J & Pieters J (2000) Essential role for choles-terol in entry of mycobacteria into macrophages Sci-ence 288, 1647–1650
40 Anand PK & Kaul D (2003) Vitamin D3-dependent pathway regulates TACO gene transcription Biochem Biophys Res Commun 310, 876–877
41 McKinney JD, Bentrup KHZ, Munoz-Elias EJ, Miczak A, Chen B, Chan W, Swenson D, Sacchettinl
JC, Jacobs WR & Russel DG (2000) Persistence of
Trang 10Mycobacterium tuberculosisin macrophages and mice
requires the glyoxylate shunt enzyme isocitrate lyase
Nature 406, 735–738
42 Ramakrishnan L, Federspiel NA & Falkow S (2000)
Granuloma-specific expression of Mycobacterium
viru-lence proteins from the glycine-rich PE-PGRS family
Science 288, 1436–1439
43 Wayne CD & Sohaskey (2001) Nonreplicating
persis-tence of Mycobacterium tuberculosis Annu Rev
Micro-biol 55, 139–163
44 Yuan Y, Lee RE, Besra GS, Belisle JT & Barry CE
(1995) Identification of a gene involved in the
biosynthesis of cyclopropanated mycolic acids in
Mycobacterium tuberculosis Proc Natl Acad Sci USA
92, 6630–6634
45 Sherman DR, Sabo PJ, Hickey MJ, Arain TM,
Mahatras GG, Yuan Y, Barry CE & Stover CK (1995)
Disparate response to oxidative stress in saprophytic
and pathogenic mycobacteria Proc Natl Acad Sci USA
92, 6625–6629
46 Dussurget O, Stewart G, Neyrolles O, Pescher P,
Young D & Marchal G (2001) Role of
Mycobacte-rium tuberculosiscopper-zinc superoxide dismutase
Infect Immun 69, 529–533
47 Arruda S, Bomfim G, Knights R, Huima-Byron T &
Riley LW (1993) Cloning of an M tuberculosis DNA
fragment associated with entry and survival inside cells
Science 261, 1454–1457
48 Cole ST, Brosch R, Parkhill J, Garnier T, Churcher C,
Harris D, Gordon SV, Eiglmeier K, Gas S, Barry CE
et al.(1998) Deciphering the biology of
Mycobacte-rium tuberculosisfrom the complete genome sequence
Nature 393, 537–544
49 Chitale S, Ehrt S, Kawamura I, Fujimura T, Shimono
N, Anand N, Lu S, Cohen-Gould L & Riley LW
(2001) Recombinant Mycobacterium tuberculosis
pro-tein associated with mammalian cell entry Cell
Micro-biol 3, 247–254
50 Flesselles B, Anand NN, Remani J, Loosmore SM &
Klein HM (1999) Disruption of mycobacterial cell
entry gene of Mycobacterium bovis BCG results in a
mutant that exhibits a reduced invasiveness for
epithe-lial cells FEMS Microbiol Lett 177, 237–242
51 Wieles B, Ottenhoff TH, Steenwijk TM, Franken KL,
de Vries RR & Langermans JA (1997) Increased
intra-cellular survival of Mycobacterium smegmatis
contain-ing the Mycobacterium leprae thioredoxin-thioredoxin
reductase gene Infect Immun 65, 2537–2541
52 Garbe T, Harris D, Vordermeier M, Lathigra R,
Ivanyi J & Young D (1993) Expression of the
Mycobacterium tuberculosis19-kilodalton antigen in
Mycobacterium smegmatis: immunological analysis and
evidence of glycosylation Infect Immun 61, 260–267
53 Wei J, Dahl JL, Moulder JW, Roberts EA, OiGarra P,
Young DB & Freidmann V (1999) Identification of a
Mycobacterium tuberculosisgene that enhances mycobacterial survival in macrophages J Bacteriol
182, 377–384
54 Berthet FX, Lagranderie M, Gounon P, Laurent-Win-ter C, Ensergueix D, Chavaror P, Thouron F, Mara-nghi E, Pelicic V, Portnoi D et al (1998) Attenuation
of virulence by disruption of the Mycobacterium tuber-culosiserp gene Science 282, 759–762
55 Litwin CM & Calderwood SB (1993) Role of iron in regulation of virulence genes Clin Microbiol Rev 6, 137–149
56 Wheeler PR & Ratledge C (1994) Metabolism of Mycobacterium tuberculosis In Tuberculosis: Patho-genesis, Protection and Control (Bloom B ed),
pp 353–385 American Society for Microbiology, Washington, DC
57 Voss D, Rutter JK, Schroeder BG, Su H, Zhu Y & Barry CE (2000) The salicylate derived mycobactin sid-erophores of Mycobacterium tuberculosis are essential for growth in macrophages Proc Natl Acad Sci USA
97, 1252–1257
58 Schmitt MP, Predich M, Doukhan L, Smith I & Holmes RK (1995) Characterization of an Iron-depen-dent regulatory protein (IdeR) of Mycobacterium tuber-culosisas a functional homolog of the diphtheria toxin repressor (Dtx R) from Corynebacterium diptheriae Infect Immun 63, 4284–4289
59 Manabe YC, Saviola BJ, Sun L, Murphy JR & Bishai
WR (1999) Attenuation of virulence in Mycobacte-rium tuberculosisexpressing a constitutively active iron repressor Proc Natl Acad Sci USA 96, 12844–
12848
60 Dussurgett O, Rodriguez M & Smith I (1998) Protec-tive role of Mycobacterium smegmatis IdeR against reactive oxygen species and isoniazid toxicity Tuberc Lung Dis 79, 99–106
61 Velmurugan K, Chen B, Miller JL, Azogue S, Gurses
S, Hsu T, Glickman M, Jacobs WR Jr, Porcelli SA & Briken V (2007) Mycobacterium tuberculosis nuoG is a virulence gene that inhibits apoptosis of infected host cells PLoS Pathog 3, e110 doi:10.1371/journal.ppat 0030110
62 Rindi L (2002) Involvement of the fadD33 gene in the growth of Mycobacterium tuberculosis in the liver of BALB⁄ c mice Microbiology 148, 3873–3880
63 Honer ZU, Bentrup K, Miczak A, Swenson DL & Russell DG (1999) Characterization of activity and expression of isocitrate lyase in Mycobacterium avium and Mycobacterium tuberculosis J Bacteriol 181, 7161– 7167
64 Graham JE & Clark-Curtiss JE (1999) Identification of Mycobacterium tuberculosisRNAs synthesized in response to phagocytosis by human macrophages by selective capture of transcribed sequences (SCOTS) Proc Natl Acad Sci USA 96, 11554–11559