Blackwell Science, LtdOxford, UKMMIMolecular Microbiology 1365-2958Blackwell Publishing Ltd, 2003503751762Original ArticleR. Singh et al.Role of mptpB in the virulence of mycobacteria
Molecular Microbiology (2003) 50(3), 751–762
doi:10.1046/j.1365-2958.2003.03712.x
Disruption of mptpB impairs the ability of
Mycobacterium tuberculosis to survive in guinea pigs
Ramandeep Singh,1 Vivek Rao,1 H. Shakila,2
Radhika Gupta,1 Aparna Khera,1 Neeraj Dhar,1
Amit Singh,1 Anil Koul,1,3 Yogendra Singh,3
M. Naseema,2 P. R. Narayanan,2 C. N. Paramasivan,2
V. D. Ramanathan2 and Anil K. Tyagi1*
1
Department of Biochemistry, University of Delhi South
campus, Benito Juarez Road, New Delhi-110021, India.
2
Tuberculosis Research Centre, Mayor V. R. Ramanathan
Road, Chetput, Chennai-600031, India.
3
Institute of Genomics and Integrative Biology, Mall Road,
Delhi-110007, India.
Summary
Protein tyrosine kinases and tyrosine phosphatases
from several bacterial pathogens have been shown to
act as virulence factors by modulating the phosphorylation and dephosphorylation of host proteins. The
identification and characterization of two tyrosine
phosphatases namely MptpA and MptpB from Mycobacterium tuberculosis has been reported earlier.
MptpB is secreted by M. tuberculosis into extracellular mileu and exhibits a pH optimum of 5.6, similar to
the pH of the lysosomal compartment of the cell. To
determine the role of MptpB in the pathogenesis of M.
tuberculosis, we constructed a mptpB mutant strain
by homologous recombination and compared the
ability of parent and the mutant strain to survive intracellularly. We show that disruption of the mptpB gene
impairs the ability of the mutant strain to survive in
activated macrophages and guinea pigs but not in
resting macrophages suggesting the importance of
its role in the host–pathogen interaction. Infection of
guinea pigs with the mutant strain resulted in a 70fold reduction in the bacillary load of spleens in
infected animals as compared with the bacillary load
in animals infected with the parental strain. Upon reintroduction of the mptpB gene into the mutant strain,
the complemented strain was able to establish infection and survive in guinea pigs at rates comparable
to the parental strain. These observations demonstrate a role of MptpB in the pathogenesis of
M. tuberculosis.
Accepted 7 July, 2003. *For correspondence. E-mail akt1000@
hotmail.com; Tel. (+91) 11 26888434; Fax (+91) 11 26885270.
© 2003 Blackwell Publishing Ltd
Introduction
Mycobacterium tuberculosis is the foremost cause of
human deaths by any single infectious agent (Dolin et al.,
1994). This intracellular pathogen can successfully survive inside the host macrophages in spite of the antimicrobicidal effector functions of the macrophages (Lowrie,
1983; Sibille and Reynolds, 1990; Sathish and Shinnick,
1994). A number of studies have shown that mycobacteria
are able to persist within phagocytes because phagosomes containing live organisms fail to fuse with lysosomes and are depleted in proton ATPases responsible for
acidification of phago-lysosomes (Armstrong and D’Arcy,
1971; Sturgill-Koszycki et al., 1994). Several M. tuberculosis genes viz. katG (catalase peroxidase), ahpC
(alkylhydroperoxide reductase), sodA, sodC (superoxide
dismutase) and noxR3 (nitric oxide reductase) have been
implicated in protection against reactive oxygen and nitrogen intermediates (Sherman et al., 1996; Manca et al.,
1999; Ruan et al., 1999; Dussurget et al., 2001). Recently,
it has also been shown that phagosomes containing live
mycobacteria recruit and retain a tryptophan-aspartatecontaining host protein, which prevents their delivery to
lysosomes (Ferrari et al., 1999).
In order to evade the host immune machinery, many
bacterial pathogens secrete effector molecules into host
cells allowing a pathogen to modify host proteins that
promote their survival in the host (Devinney et al., 2000).
Protein phosphorylation has been shown to play an important role in sensing of extracellular signals by a pathogen
and co-ordinating intracellular events (Kennelly and Potts,
1996). Protein tyrosine kinases and phosphatases control
the growth and differentiation of eukaryotic cells by modulating the tyrosine phosphorylation of regulatory proteins
(Stone and Dixon, 1994). Several protein phosphatases
and kinases from bacterial pathogens have been shown
to act as virulence factors (Guan and Dixon, 1990; Galyov
et al., 1993; Kaniga et al., 1996). Protein tyrosine phosphatases YopH and SptP from Yersinia pseudotuberculosis and Salmonella typhimurium, respectively, have been
well characterized. Protein tyrosine dephosphorylation of
macrophage proteins by YopH prevents phagocytosislinked signalling pathways (Bliska et al., 1991; Bliska and
Black, 1995; Fallman et al., 1995; Andersson et al., 1996;
Black and Bliska, 1997). Similarly, SptP of S. typhimurium
has been shown to play an important role in cytoskeletal
752 R. Singh et al.
rearrangements by interacting with Rac-1 and Cdc42
leading to internalization of bacteria into non-phagocytic
cells (Fu and Galan, 1999).
Sequence analysis of the M. tuberculosis genome
revealed the presence of 11 serine/threonine kinases,
two tyrosine phosphatases and one serine/threonine
phosphatase (Cole et al., 1998). A few serine/threonine
kinases (PknA, PknB, PknD, PknF and PknG) and
tyrosine phosphatases (MptpA and MptpB) of M. tuberculosis have been studied for their biochemical properties (Peirs et al., 1997; Av-Gay et al., 1999; Koul et al.,
2000; 2001; Chaba et al., 2002). Both MptpA and MptpB
are secreted by M. tuberculosis into extracellular fluid
(Koul et al., 2000). Using Southern blot analysis, it was
earlier demonstrated that mptpB was present in the slowgrowing species such as M. tuberculosis H37Rv, M. tuberculosis H37Ra and M. bovis BCG but absent from the
fast-growing species such as M. smegmatis (Koul et al.,
2000). MptpB of M. tuberculosis was shown to dephosphorylate the phosphotyrosine residue of myelin basic
protein (Koul et al., 2000). Because phosphorylation and
dephosphorylation regulate several crucial processes in
eukaryotic cells, secreted tyrosine phosphatases may
serve as key molecules that modify host proteins to its
advantage and enable the tubercle bacilli to survive
within the host. By virtue of its in vitro tyrosine phosphatase activity, secretory nature and optimal pH of 5.6
for activity, we speculated that MptpB might interfere with
host signal transduction pathways facilitating the survival
of the pathogen in the host.
In order to evaluate the role of MptpB in the pathogenesis of M. tuberculosis, an mptpB mutant strain was constructed by homologous recombination. The mutant strain
lacking tyrosine phosphatase activity associated with
MptpB was employed to understand the role of this protein
in the pathogenesis of M. tuberculosis. Using the guinea
pig model of experimental tuberculosis, we report that the
mptpB mutant strain showed impaired ability to survive in
the host.
Results
Construction of the mptpB mutant and complemented
strain of M. tuberculosis
In order to establish whether MptpB plays a role in the
pathogenesis of M. tuberculosis, an mptpB mutant strain
of M. tuberculosis was constructed by using a non-replicative suicidal vector pBKDB (Fig. 1). The targeting vector,
pBKDB, carried the coding region of mptpB along with
1045 bp upstream and 1140 bp downstream flanking
sequences. A portion of the coding region (108 bp) of
MptpB was deleted and replaced with the gene conferring
resistance to hygromycin in pBKDB. The vector also car-
Fig. 1. Schematic representation of the mptpB locus in the genome
of parental (A) and mutant strain (C) of Mycobacterium tuberculosis.
Details of construction of pBKDB (B), a non-replicative suicide vector
used for disruption of mptpB is given in Experimental procedures.
HygR and KanR represent genes conferring resistance to hygromycin
and kanamycin respectively. ML and MR represent M. tuberculosis
DNA from the mptpB locus, oriT represents the origin of DNA replication and sacB represents the gene encoding levansucrase.
ried the gene conferring resistance to kanamycin in its
backbone as a second antibiotic selection marker for negative screening of allelic exchange events at the homologous site (Fig. 1).
Electroporation of M. tuberculosis with pBKDB and UVtreated pBKDB resulted in 22 and 3 hygromycin-resistant
transformants respectively. PCR analysis revealed that all
the transformants contained a hygromycin cassette indicating that these colonies were not spontaneous resistance mutants and arose from integration of the suicidal
vector into the mycobacterial genome (data not shown).
An allelic exchange event by homologous recombination
should result in the incorporation of a hygromycin resistance gene but not the vector backbone (having kanamycin resistance gene) into the mycobacterial genome.
Thus, transformants resistant to hygromycin but sensitive
to kanamycin were selected to score for a homologous
recombination event. All the transformants obtained on
electroporation of untreated DNA were kanamycin resistant while the three transformants obtained on electroporation of UV-pre-treated DNA were sensitive to
kanamycin. This suggested that an allelic exchange event
at the homologous site had taken place in the case of
these three transformants obtained upon electroporation
of UV-pre-treated DNA.
© 2003 Blackwell Publishing Ltd, Molecular Microbiology, 50, 751–762
Role of mptpB in the virulence of mycobacteria 753
mptpB gene disruption was assessed by hybridization
analysis of genomic DNA isolated from the parental M.
tuberculosis strain and three HygRKanS transformants. A
DNA fragment containing the entire coding region
(831 bp) of mptpB was used as a probe. Southern blot
analysis showed the presence of a 1.85 kb band in the
parental strain whereas a 3.8 kb band was observed in all
the three HygRKanS transformants as expected upon
replacement of 108 bp internal fragment of mptpB with a
hygromycin resistance gene cassette (Fig. 2A). These
results indicated that mptpB was disrupted in all three of
the HygRKanS transformants. Expression of MptpB was
analysed in the mutant strains using polyclonal sera
raised against MptpB in rabbit. Western blot analysis
showed absence of MptpB expression in all three of the
mutant strains (Fig. 2B). The complemented strain was
constructed by electroporation of pSD5-mptpB into electrocompetent cells of the mutant strain. The electropora-
tion of pSD5-mptpB restored the expression of MptpB in
the complemented strain (Fig. 2B).
Effect of mptpB inactivation on the in vitro and intracellular
survival of M. tuberculosis
To investigate the effect of mptpB disruption on the growth
of M. tuberculosis in vitro, the morphology and growth
characteristics of the mutant strain of M. tuberculosis were
compared with that of the parental strain. The growth
pattern of the mutant strain was comparable to that of the
parental strain (data not shown). Similarly, morphometric
measurements (length, width and area) of the bacilli
revealed no differences between the mutant and the wildtype strains (Fig. 3A). To study the effect of disruption of
the mptpB gene on the intracellular survival of M. tuberculosis, resting and activated murine macrophage cells
were infected with either wild-type or the mptpB mutant
Fig. 2. A. Southern blot analysis of the wild-type and mptpB mutant strains of M. tuberculosis. Genomic DNAs (3 mg) from wild-type (WT) and
mutant strains (MT) of M. tuberculosis were digested with NotI, separated on a 1.2% agarose gel, transferred to nylon membrane and probed
with 32P-labelled mptpB DNA probe. The presence of 3.8 kb band in lanes MT1, MT2 and MT3 confirmed that allelic exchange had occurred at
the mptpB locus. The wild-type gene is indicated by a hybridization signal of 1.8 kb in lane WT.
B. Immunoblot analysis of the wild-type strain (WT), mptpB mutant strains (MT1, MT2, MT3) and complemented strain (CT) of M. tuberculosis:
analysis of the expression of MptpB in whole cell lysates of wild-type, mutant and complemented strains of M. tuberculosis by immunoblotting.
The strains were grown in MB7H9 media to mid- log phase as described in Experimental procedures.
© 2003 Blackwell Publishing Ltd, Molecular Microbiology, 50, 751–762
754 R. Singh et al.
Fig. 3. A. Morphology of M. tuberculosis and mptpB mutant strains: single cell suspensions of M. tuberculosis (WT) and mutant strains (MT)
were stained with auramine and viewed using Zeiss LSM 510 confocal microscope at 100¥ magnification.
B, C. Survival of wild-type and mptpB mutant strains of M. tuberculosis in macrophages: The mouse macrophage cell line J774A.1 was infected
separately with the wild-type and mutant strain of M. tuberculosis at an MOI of 1:10 (macrophage/bacilli). At different time points post-infection
(day 0, 2, 4, 6 and 8), macrophages were lysed and the number of intracellular mycobacteria was assessed by plating on MB7H10 plates (B: in
resting macrophages, C: in activated macrophages). The experiments were carried out twice in duplicates and data are depicted as mean of all
four values ±SE.
strain of M. tuberculosis. The number of surviving intracellular bacteria was determined on days 0, 2, 4, 6 and 8.
Both, the wild-type as well as the mutant strain, displayed
a similar pattern of intracellular growth in the resting macrophages at all time points of study (Fig. 3B). While at the
initial time point (day 0) bacillary counts were approximately 104 cfu well-1, the number of bacilli increased at
later time points attaining the peak value of ~105 cfu at
day 8 post-infection. These results show that the wild-type
as well as the mutant strain were comparable in their
ability to infect and survive in the resting macrophages.
However, the two strains showed differences in their ability
to survive in the activated macrophages (Fig. 3C). The
number of wild-type M. tuberculosis and mptpB mutant
was maximum and comparable at the initial time point
(~104 cfu well-1, at day 0). At later time points, a reduction
in the number of bacilli was observed in both cases. While
the wild-type M. tuberculosis was reduced to 50% and
28.6% at days 4 and 6 post-infection, respectively, a much
sharper decline was noted in the case of the mptpB
mutant which was reduced to 10% and 4% at days 4 and
6 post-infection respectively (Fig. 3C). These observa© 2003 Blackwell Publishing Ltd, Molecular Microbiology, 50, 751–762
Role of mptpB in the virulence of mycobacteria 755
Fig. 4. Total post-mortem scores for various
organs of guinea pigs. Guinea pigs were
infected with 5 ¥ 105 cfu of different strains of
M. tuberculosis subcutaneously. Infected
guinea pigs (eight animals per group at each
time point) were euthanized 3 weeks (A) and
6 weeks (B) post-infection. At the time of sacrifice, depending on the amount of pathological
damage in lung, liver, spleen and lymph nodes,
scores were assigned to each organ. Mean
total scores were calculated for all three groups
and are depicted as mean ± SE.
tions indicated that disruption of the mptpB gene had
impaired the ability of M. tuberculosis to survive in the
activated macrophages.
Virulence of the mptpB mutant strain of M. tuberculosis in
guinea pig
To determine whether the disruption of mptpB genes
would have any effect on the survival of M. tuberculosis
in vivo, guinea pigs in groups of eight animals were
infected subcutaneously with 5 ¥ 105 cfu of parental,
mutant or the complemented strain of M. tuberculosis.
Animals were euthanized 3 and 6 weeks post-infection. At
both time points of euthanasia, spleens were homogenized and viable bacilli were enumerated (represented as
log10 cfu for each group).
It was observed that at 3 weeks post-infection, the
mean total score of the animals infected with the mutant
strain was 26, which was similar to the scores in the case
of animals infected with parental (28) and complemented
strain (30) (Fig. 4A). These results were commensurate
with the splenic cfu obtained for various groups on euthanization of animals at 3 weeks post-infection. The bacterial load in the spleen of animals infected with the mutant
strain was log10 3.71, which was comparable to the bacterial load in the spleens of animals infected with parental
(log10 3.73) and complemented strain (log10 3.68, Fig. 5A).
However, the total scores of the animals infected with the
mutant strain at the end of 6 weeks was significantly lower
(12) than the total score of animals infected with parental
(35, P < 0.02) and the complemented strain (33, P < 0.02;
Fig. 4B). The animals infected with the mutant strain
exhibited a significant reduction of bacillary load in the
spleen (log10 3.07) when compared with bacillary load in
the spleen of animals infected with the parental (log10
4.77, P < 0.002) and complemented strain (log10 4.45,
P < 0.003, Fig. 5B). Thus, an approximately threefold
reduction in total score and a 50- to 70-fold reduction in
the bacillary load in spleens was observed in animals
infected with the mutant strain in comparison to parental
Fig. 5. Survival of wild-type, mptpB mutant and
complemented strain of M. tuberculosis in
guinea pigs. Guinea pigs (eight animals per
group at each time point) were euthanized
3 weeks (A) and 6 weeks (B) post-infection.
Spleens were homogenized in 5 ml of water
and 10-fold serial dilutions of the spleen homogenates were plated in duplicate on LJ slopes.
Splenic bacillary load was determined, converted to log10 cfu and depicted as log10 ± SE.
© 2003 Blackwell Publishing Ltd, Molecular Microbiology, 50, 751–762
756 R. Singh et al.
or complemented strains. We have carried out this experiment three times obtaining similar results.
Histopathological analysis
Sections of liver and lung from animals in various groups
were subjected to histological analysis to determine morphology of the organs, the presence and extent of granuloma and the type and number of infiltrating cells. It was
observed that at 3 weeks there were no significant histological differences in liver and lung of animals infected with
the parental, mutant or complemented strain. At 3 weeks
post-infection, animals from all three groups showed no
difference in the extent or composition of granuloma. In
the case of liver, granuloma consisted mainly of epitheloid
cells and lymphocytes, while the lung granuloma comprised mainly of lymphocytes macrophages and a few
epitheloid cells (Fig. 6A and B). At 6 weeks post-infection,
in the case of liver, granuloma was observed in the case
of three and five guinea pigs infected with the parental
and complemented strains respectively. Among the animals infected with the mutant strain, only one animal
exhibited granuloma. In the case of lung, five animals
each infected with either the parental or complemented
strain exhibited granuloma. Among the animals infected
with the mutant strain, granuloma was observed in the
case of three animals. A representative section of liver
and lung of animals from various groups is shown in
Fig. 7A and B respectively. In the case of animals infected
with the wild-type and complemented strain, the liver sections showed multiple well-defined granuloma comprising
of epitheloid cells and lymphocytes. However, the liver
tissue from animals infected with the mptpB mutant strain
exhibited a distinct qualitative difference with respect to
the presence of epitheloid cells with only a few lymphocytes. In the case of lung tissues, the animals infected
with the wild-type and complemented strain showed
extensive granulomas comprising of lymphocytes and
macrophages. In contrast, the lung tissue from animals
infected with the mutant strain showed partly organized
granuloma mainly of lymphocytes.
Discussion
The molecular basis of the pathogenicity of M. tuberculosis is poorly understood. The entry of M. tuberculosis into
the host cell and their subsequent survival involves specific cross-talk of signals between the host and mycobac-
Fig. 6. Histopathology of liver and lung from guinea pigs at 3 week post-infection. Portions of liver and lungs were removed under aseptical
conditions and fixed in 10% formalin. Five micron sections of tissues were stained with haematoxylin and eosin and subjected to histopathological
analysis at a magnification of 10¥. Representative sections of liver (A) and lung (B) from various groups of animals are shown. Sections of liver
and lung from uninfected guinea pig were used as reference for normal tissue histology.
© 2003 Blackwell Publishing Ltd, Molecular Microbiology, 50, 751–762
Role of mptpB in the virulence of mycobacteria 757
Fig. 7. Histopathology of liver and lung from guinea pigs at 6 week post-infection. Sections (5 mm) of liver and lung from animals infected with
wild-type, mutant and complemented strains of M. tuberculosis were fixed, processed, stained with haematoxylin and eosin and observed under
microscope at a magnification of 10¥. Representative sections, with an inset of high magnification (20¥), of liver (A) and lung (B) from various
groups of animals are shown. Sections of liver and lung from uninfected guinea pig were used as reference for normal tissue histology.
terium resulting in reprogramming of the host-signalling
network. Bacterial tyrosine phosphatases have been
demonstrated to modulate signal transduction pathways
of the eukaryotic host for the benefit of the pathogen
(Bliska and Black, 1995; Fallman et al., 1995; Andersson
et al., 1996; Black and Bliska, 1997; Fu and Galan,
1999). From M. tuberculosis, two tyrosine phosphatases,
MptpA and MptpB, have been identified and characterized (Koul et al., 2000). The characteristic features of
MptpB such as pH optima of 5.6 for activity and its secretory nature suggested that MptpB might play a role in the
survival of mycobacteria in the acidic environment of the
macrophages. To investigate the role of MptpB in the
pathogenesis of M. tuberculosis, the gene encoding
MptpB was disrupted in the M. tuberculosis genome by
homologous recombination using a non-replicative suicidal vector and disruption was confirmed by Southern
blot and immunoblot analysis. Disruption of mptpB had
no significant effect on the morphology and growth of M.
tuberculosis in defined liquid culture medium suggesting
that MptpB is not required for the growth of M. tuberculosis under in vitro conditions. Similar results were also
observed when the resting murine macrophage cell line
© 2003 Blackwell Publishing Ltd, Molecular Microbiology, 50, 751–762
was infected with the mptpB mutant and wild-type M.
tuberculosis. Both strains were comparable in their ability
to infect and survive in the mouse macrophage cell line
J774A. 1. However, because the activated macrophages
better mimic the conditions encountered by mycobacteria
in vivo, we compared the ability of the wild-type and the
mptpB mutant of M. tuberculosis to infect and survive in
the activated murine macrophages. An approximately
fivefold reduction in the number of intracellular mptpB
mutants (10%) was observed in comparison to the number of intracellular parental strains (50%) at 4 days postinfection. At 6 days post-infection, an approximately sevenfold reduction was observed in the number of intracellular mptpB mutant strains (4%) in comparison to the
number of internalized parental strains (28.6%) suggesting that the mptpB mutant strain was more sensitive to
killing as compared with the wild-type strain by activated
macrophages. More importantly, however, using the
guinea pig model of infection, we demonstrated a significant reduction in the ability of the mutant strain to survive
in the host. An approximately 70-fold (1.7 log) reduction
in bacillary load was observed in the spleen of the animals infected with the mutant strain as compared with
758 R. Singh et al.
the bacillary load from the animals infected with wild-type
strain at 6 week post-infection. This difference in the
splenic bacillary load in both groups of animals was not
observed earlier, at 3 weeks post-infection (log10 3.73 for
the wild-type strain and log10 3.71 for the mptpB mutant
strain) suggesting that the wild-type as well as the mutant
strain were capable of establishing the infection to a comparable extent. However, at 6 weeks post-infection the
bacillary load in the spleens of animals infected with
parental strain increased from log10 3.73 to log10 4.77. In
the case of animals infected with the mptpB mutant
strain, the splenic bacterial load decreased from log10
3.71 to log10 3.07 and this decrease was found to be
statistically significant. These observations suggest that
initially both strains (mutant and wild type) of M. tuberculosis are capable of establishing the infection to a similar
extent. However, the mutant strain displayed reduced
ability to sustain infection in guinea pigs.
That the loss of virulence of M. tuberculosis was a direct
consequence of disruption of mptpB, was shown by the
ability of the complemented strain to establish an infection
and survive in the host tissues at levels similar to those
observed in the case of wild-type M. tuberculosis. These
observations clearly suggest that MptpB plays an essential role in the virulence of M. tuberculosis.
It is well established that phosphatases not only antagonize kinases but can also act co-operatively to generate
antagonistic signals (Sun and Tonks, 1994). Thus, it is not
surprising that pathogenic microorganisms secrete
tyrosine phosphatases to modulate host antimicrobial
functions. YopH is required for full display of virulence of
Yersinia (Guan and Dixon, 1990). A strain of S. typhimurium carrying a null mutation in sptP is defective in its
ability to survive and establish an infection in BALB/c mice
suggesting the role of SptP in the pathogenesis of Salmonella (Kaniga et al., 1996). At present it is not clear how
MptpB exerts its function. A number of host functions such
as antigen presentation, macrophage activation and
membrane trafficking that influence the course of M.
tuberculosis pathogenesis are modulated by the levels of
tyrosine phosphorylation (Adams and Hamilton, 1984).
Because the mptpB mutant is impaired in its ability to
survive in activated macrophages, IFN-g-mediated signal
transduction pathways may represent a plausible target
for MptpB.
Experimental procedures
Bacterial strains and DNA manipulations
The bacterial strains and plasmids used in this study are
listed in Table 1. Plasmids were prepared in Escherichia
coli HB101 and DH5a strains using the Qiagen miniprep kit
according to the manufacturer’s recommendations (Qiagen
Inc.). All mycobacterial strains were grown in Middlebrook
(MB) 7H9 medium (Difco) supplemented with 0.5% glycerol, 0.2% Tween-80 and 1¥ ADC (Difco) or MB 7H10
medium (Difco) supplemented with 1¥ OADC (Difco). When
required, the following antibiotics were added at the specified concentration: ampicillin (50 mg ml-1 for E. coli ), kanamycin A sulphate (25 mg ml-1 for both E. coli and
mycobacteria) and hygromycin B (150 mg ml-1 for E. coli
and 50 mg ml-1 for mycobacteria). All restriction endonucleases, DNA-modifying enzymes such as T4 DNA ligase,
T4 polynucleotide kinase (New England Biolabs Inc.) and
Pfu DNA polymerase (Stratagene GmbH) were used
according to manufacturers’ recommendations. All recombinant DNA manipulations were carried out according to
standard protocols.
Table 1. Strains and plasmids used in this study.
Strain/plasmid
Escherichia coli HB101
E. coli DH5aF¢
Mycobacterium tuberculosis
Erdman
mptpB mutant strain
Complemented strain
pLitmus 38
pLit28 res-hyg-res
pSD5
pJQ200SK
pJQDB
pBKDB
pSD5-mptpB
Description
Reference
–
F D (gpt-proA) 62 leuB6 glnV44 ara-14 galK2 lacY1 D(mcrC-mrr)
rpsL20 (Strr ) xyl-5 mtl-1 recA13
F¢/endA1 hsdR17 (rk– mk+) glnV44 thi-1 recA1 gyrA (Nal r ) relA1 D
(lacIZYA-argF) U169 deoR [f80dlacD(lacZ) M15]
Virulent strain of M. tuberculosis
M. tuberculosis with its mptpB gene disrupted by the hygromycin
resistance cassette
mptpB mutant strain complemented with a wild-type copy of mptpB
Cloning vector with colE1 origin of DNA replication
pLitmus-28 derivative carrying an E. coli origin of DNA replication
and hygromycin resistance gene cassette
Mycobacteria – E. coli shuttle vector carrying the kanamycin
resistance gene
A non-replicative suicidal vector carrying genes for SacB and
gentamicin resistance
Derivative of pJQ200SK carrying mptpDB::hygr cloned in the XbaI site
Derivative of pJQDB carrying the kanamycin resistance gene cloned
in the vector backbone
Mycobacteria-E. coli shuttle vector pSD5 carrying the mptpB gene
Life Technologies
Life Technologies
Kindly provided by Dr Jaya S. Tyagi
AIIMS, New Delhi, India.
This study
This study
New England Biolabs
A kind gift from Dr Stoyan Bardarov,
Albert Einstein College of Medicine,
New York, USA
Dasgupta et al. (1998)
A kind gift from Dr Brigitte Gicquel,
Pasteur Institute, France
This study
This study
This study
© 2003 Blackwell Publishing Ltd, Molecular Microbiology, 50, 751–762
Role of mptpB in the virulence of mycobacteria 759
Construction of targeting vector pBKDB
For disruption of mptpB of M. tuberculosis, vector pBKDB
was constructed. For this, a DNA fragment containing
1045 bp upstream of the open reading frame (ORF) of
mptpB along with the initial 356 bp of the ORF of mptpB was
PCR amplified using M. tuberculosis Erdman DNA as template and primers ptpB.1 having a BspHI site (5¢-cca tca tga
ctg tgg aac cta ttc ctg tcg gcc-3¢) and ptpB.2 having a NdeI
site (5¢-ggg cat atg ggc tgg att cgc cgg act cgc cg-3¢). The
amplicon was end-repaired and cloned into EcoRV digested
pLitmus-38 resulting in pLitB1. Another DNA fragment containing 367 bp of the mptpB ORF corresponding to the Cterminal region of MptpB along with 1140 bp downstream of
the ORF of mptpB was PCR amplified using gene-specific
primers ptpB.3 carrying a NdeI site (5¢-ggg cat atg ggt gct
cac cca ctg ctt cgc ggg-3¢) and ptpB.4 having a BspHI site
(5¢-cca tca tga gtc ggt gac ccc cgt ata gcc cgg-3¢). The
amplicon was end-repaired and separately cloned into
EcoRV digested pLitmus-38 resulting in pLitB2. The vector
pLitB1 was digested with NdeI and ScaI and the larger DNA
fragment containing the initial 356 bp of the mptpB ORF
along with 1045 bp upstream of the ORF was gel purified
using a Qiagen gel extraction kit. Similarly, pLitB2 was
digested with NdeI and ScaI and the smaller DNA fragment
containing the 367 bp of the mptpB ORF corresponding to
the C-terminal region of MptpB along with 1140 bp downstream of the ORF was gel purified. The larger fragment
obtained by the digestion of pLitB1 and the smaller fragment
obtained by the digestion of pLitB2 were then ligated
together resulting into pLitDB. The mptpB-specific primers
ptpB.2 and ptpB.3 were non-overlapping; as a result, the
vector pLitDB contained the coding region of mptpB (with a
deletion of 108 bp from the central region of ORF) and
1045 bp of upstream and 1140 bp of downstream flanking
sequences and a unique NdeI site in the ORF of mptpB at
the deletion site for the cloning of the hygromycin resistance
gene cassette. The hygromycin resistance gene cassette
was excised out from pLit28res-hyg-res as a BamHI–XbaI
fragment, end-repaired and cloned into NdeI digested, endrepaired pLitDB resulting in pLitDBH. A 4.9 kb DNA fragment
containing mptpDB::hygr was excised out from pLitDBH as a
SpeI–NheI fragment and cloned into XbaI-digested
pJQ200SK (a non-replicative suicide vector, Pelicic et al.,
1996) yielding pJQDB. The gene conferring resistance to
kanamycin was excised out from pSD5 as a NheI–BstEII
fragment, end repaired and cloned into SmaI-digested
pJQDB resulting in pBKDB. The vector pBKDB provided 1.4
and 1.5 kb homologous regions upstream and downstream
of the hygromycin-resistant gene, respectively, for recombination to occur between targeting DNA and the mycobacterial genome.
Construction of the mptpB mutant and
complementation strain
The targeting vector pBKDB was pre-treated with ultraviolet light (UV) as described (Hinds et al., 1999). In brief,
UV irradiation of DNA was carried out in an UV
stratalinker 1800 (Amersham Life Science) at 100 mJ cm-2.
UV-treated DNA as well as untreated DNA (3 mg each)
© 2003 Blackwell Publishing Ltd, Molecular Microbiology, 50, 751–762
was electroporated into electrocompetent cells of M. tuberculosis. The cells were revived in 1 ml of MB7H9 media
for 48 h, plated on MB7H10/OADC plates supplemented
with hygromycin (50 mg ml-1) and incubated at 37∞C for
3 week. Colonies were screened for hygromycin resistance
and kanamycin sensitivity. Disruption of the mptpB gene of
M. tuberculosis was confirmed by Southern blot and
immunoblot analysis.
For complementation studies, the complete mptpB gene
was PCR amplified using gene-specific primers and cloned
into pSD5 hsp60 (Dasgupta et al., 1998). The resulting plasmid pSD5-mptpB was electroporated into the mptpB mutant
strain of M. tuberculosis.
Southern blot analysis
Genomic DNA from mycobacteria was extracted as
described earlier. DNA (3 mg) was digested with NotI and
resolved on a 1.2% agarose gel. The gel was treated according to standard protocols. DNA fragments were transferred
to Hybond-N membrane (Amersham Pharmacia Biotech.),
cross-linked by UV irradiation and hybridized under standard
conditions (Southern, 1975) with 32P-labelled fragment containing the complete coding region of mptpB, in 50% formamide at 42∞C for 16 h. Blots were washed once with 2¥ SSC/
0.1% SDS at room temperature for 30 min followed by two
washes with 0.1¥ SSC/0.5% SDS at 65∞C for 30 min and
subjected to autoradiography.
Analysis of expression of MptpB in M. tuberculosis
To analyse the expression of MptpB in wild type, mutant and
complemented strains of M. tuberculosis, the bacteria was
grown in MB7H9 medium supplemented with 1¥ ADC enrichment, 0.5% glycerol and 0.2% Tween-80. All mycobacterial
cultures were grown at 37∞C to mid-log phase. The cells were
solubilized in SDS sample buffer and approximately 40 mg
whole cell lysate was electrophoresed on 10% SDS–PAGE
and transferred to nitrocellulose membrane (Amersham
Pharmacia Biotech.). The membrane was blocked for 2 h in
3% BSA and then incubated for 2 h with polyclonal sera
raised against MptpB (1:10 000) in rabbit. Thereafter the
membrane was washed thrice with PBS supplemented with
0.05% Tween-20 (PBST) and then incubated for 1 h with
HRP-conjugated goat anti-rabbit IgG (1:2500, Jackson
Immuno Research Laboratories, Inc.). The membrane was
then washed thrice with PBST and the expression was analysed using Di-amino Benzidene and H 2O2.
Morphological studies of bacilli
Smears prepared from single cell suspensions of the cultured wild-type and mutant strains were stained with
auramine (Blair et al., 1969). Confocal images of the bacilli
were obtained by scanning with a Zeiss 510 laser scanning
microscope using 100¥ oil immersion lens. The images
were then imported into Micro Image (Olympus Optical Co.,
Version 4.0) and morphometric measurements of at least
300 bacilli each of the mutant and wild-type strains were
made.
760 R. Singh et al.
Preparation of strains for infection studies
Table 2. Virulence scoring chart for infected organs.
Wild-type, mutant and complemented strains of M. tuberculosis were grown to mid-log phase in MB7H9 media at 37∞C.
The bacilli were harvested by centrifugation at 5000 g for
10 min, washed twice with 1¥ PBS, resuspended in 1/10th of
the culture volume and stored as 1 ml aliquots at -70∞C until
use. Viable count of bacteria was calculated by plating various dilutions in duplicates on MB7H10 and incubating the
plates at 37∞C for 3 weeks.
Characteristics
In vitro infection of the mouse macrophage cell line by
wild-type and mutant M. tuberculosis strains
J774A.1 mouse macrophage cell line resting or activated with
rIFN-g (50Uml-1, 16 h) was seeded in a six-well plate (Nalge
Nunc International) at a density of 2 ¥ 105 per well. Before
infection, the cells were washed once with 1¥ Hanks’ balanced salt solution (HBSS, GibcoTM Invitrogen Corporation)
and medium was replaced with Dulbecco’s modified Eagle’s
medium (DMEM, GibcoTM Invitrogen Corporation) supplemented with 10% heat inactivated fetal calf serum (FCS,
GibcoTM Invitrogen Corporation). The bacterial strains were
washed twice with DMEM and resuspended in DMEM
supplemented with 5% FCS. The cells were infected with
wild-type and mutant strain at a MOI of 1:10 (macrophage/
bacteria). The cells were incubated at 37∞C in a 5% CO2
atmosphere. After 6 h of infection, cells were washed twice
with 1¥ HBSS and then with 2 ml of DMEM supplemented
with FCS (10%), Antibiotic-antimycotic (1%, GibcoTM Invitrogen Corporation) and amikacin (20 mg ml-1). On days 0, 2, 4,
6 and 8, infected cells were lysed in 1 ml of 0.1% Triton X100 for 15 min. The number of bacilli at different time points
was determined by plating 10-fold serial dilutions in duplicates on MB 7H10 medium and incubating the plates at 37∞C
for 3 weeks.
Virulence studies in guinea pigs
Guinea pigs (Hartley strain) weighing 200–400 g were purchased from National Center for Laboratory Animal Science
(NCLAS), Hyderabad, India. Guinea pigs in groups of eight
animals were infected with 5 ¥ 105 cfu of either the wild-type
or the mptpB mutant strain or the complemented strain of M.
tuberculosis subcutaneously. The animals (eight animals per
group at each time point) were euthanized at 3 or 6 weeks
post-infection. The visible lesions in liver, spleen, lung and
lymph glands were scored according to Mitchison’s scoring
system shown in Table 2 (Mitchison, 1964). To estimate the
viable bacilli count, the spleens were homogenized in 5 ml of
sterile water. Bacteria in the infected spleens were enumerated by plating 10-fold serial dilutions of the homogenates in
duplicate on Lowenstein–Jensen (LJ) slants and incubating
the slants at 37∞C for 4 weeks. The total organ score and cfu
data are expressed as the mean ± standard error obtained in
a group of eight animals.
For histopathological analysis, at each time point of sacrifice, portions of liver and lung were removed aseptically and
fixed in 10% formalin. The tissue was embedded in paraffin
wax and cut into five micron sections using a microtome. The
Spleen
Heavy involvement with numerous large tubercles and
areas of necrosis
Moderate involvement with numerous small tubercles
or markedly enlarged spleen plus numerous small
tubercles
Scanty involvement with few large tubercles or numerous small but easily visible tubercles
Minimal involvement with one or two large tubercles or
moderate number of just visible tubercles
Liver
Heavy involvement with numerous large tubercles and
areas of necrosis
Moderate involvement with moderate number of large
tubercles or numerous small tubercles, no areas of
necrosis
Scanty involvement with scanty tubercles, easily visible
Minimal involvement with just visible tubercles
Lung
Heavy involvement with numerous large tubercles
measuring 3–5 mm in diameter
Moderate involvement with occasional large tubercles
(3–5 mm) or more numerous small tubercles
(1–2 mm)
Scanty involvement up to four large tubercles or number
of small tubercles
Minimal involvement with small scanty tubercles
Secondary lymph glands
Abscess at the site of injection plus enlargement plus
caseation of inguinal gland and sublumbar/coelic
Abscess at the site of injection plus enlargement plus
caseation of inguinal gland, no sublumbar or coelic
involvement
Abscess at the site of injection plus enlargement but no
caseation
Or
No abscess at the site of injection but caseation of
inguinal gland
Abscess at the site of injection, but no enlargement or
caseation
Enlargement but no caseation or no abscess at the site
of injection
Score
40
30
20
10
30
23
15
8
20
15
10
5
10
8
6
5
2
The above virulence scoring chart is based on Mitchison’s scoring
system (Mitchison, 1964).
sections were stained with haematoxylin and eosin and subjected to histopathological analysis. In infected tissues, the
percentage of granuloma and cellular composition of a granuloma were microscopically accessed as described earlier
(Jayashankar and Ramanathan, 1999).
Statistical analysis
The Student’s unpaired t-test was applied for statistical evaluation of the data assuming normal distribution. A P-value of
<0.05 was considered as a statistically significant observation for any parameter.
Acknowledgements
This work was supported by a grant from the Indian Council
of Medical Research, India. R.S., V.R. and N.D. are thankful
© 2003 Blackwell Publishing Ltd, Molecular Microbiology, 50, 751–762
Role of mptpB in the virulence of mycobacteria 761
to the University Grants Commission, India and A.K., A.S.
are thankful to the Council of Scientific and Industrial
Research, India for research fellowships. We thank Dr J. S.
Tyagi for critically reading the manuscript. Bindu Nair is
acknowledged for technical assistance. Rajiv Chawla is
acknowledged for efficient preparation of the manuscript. We
thank Jaya Gopinath, S. Nambirajan, K. Chandran and M.
Asokan for their technical assistance.
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