INFECTION AND IMMUNITY, Mar. 2003, p. 1134–1140 Vol. 71, No.

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0019-9567/03/$08.00⫹0 DOI: 10.1128/IAI.71.3.1134–1140.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Deletion of Two-Component Regulatory Systems Increases the

Virulence of Mycobacterium tuberculosis
Tanya Parish,† Debbie A. Smith, Sharon Kendall,‡ Nicola Casali,§
Gregory J. Bancroft, and Neil G. Stoker*
Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical
Medicine, London WC1E 7HT, United Kingdom
Received 9 July 2001/Returned for modification 21 August 2001/Accepted 2 December 2002

Two-component regulatory signal transduction systems are widely distributed among bacteria and enable
the organisms to make coordinated changes in gene expression in response to a variety of environmental
stimuli. The genome sequence of Mycobacterium tuberculosis contains 11 complete two-component systems, four
isolated homologous regulators, and three isolated homologous sensors. We have constructed defined muta-
tions in six of these genes and measured virulence in a SCID mouse model. Mice infected with four of the
mutants (deletions of devR, tcrXY, trcS, and kdpDE) died more rapidly than those infected with wild-type
bacteria. The other two mutants (narL and Rv3220c) showed no change compared to the wild-type H37Rv
strain. The most hypervirulent mutant (devR⌬) also grew more rapidly in the acute stage of infection in
immunocompetent mice and in gamma interferon-activated macrophages. These results define a novel class of
genes in this pathogen whose presence slows down its multiplication in vivo or increases its susceptibility to
host killing mechanisms. Thus, M. tuberculosis actively maintains a balance between its own survival and that
of the host.

Mycobacterium tuberculosis is an extraordinarily successful
pathogen that currently infects approximately one-third of the
global population and causes 8 million new cases of tubercu-
losis annually (41). The tubercle bacillus functions within sev-
eral hostile environments in order to survive within the human
host and cause disease. For instance, the bacteria must be able
to gain entry into macrophages, multiply intracellularly, survive
within the lung granuloma for years, and disperse to a new host
via aerosols (9). It is therefore likely that the expression of
different sets of genes by M. tuberculosis at various stages of
infection is crucial to its survival.
Two-component regulatory systems (2CRs) are widely dis-
tributed among bacteria and plants and enable the organisms
to respond to many different external stimuli (20, 37, 38).
These systems form a large family of related proteins that
consist of a membrane-bound sensor protein that activates an
effector protein, generally a transcriptional regulator, by phos-
phorylation. 2CRs have been shown to play a crucial role in the
controlled expression of virulence genes in other bacteria (11).
For example, in Salmonella enterica serovar Typhimurium, the
membrane-bound sensor protein PhoQ is activated by chang-
ing levels of magnesium, and this activates the PhoP response
regulator. PhoP is a DNA-binding protein that binds in a
* Corresponding author. Present address: Department of Pathology
and Infectious Diseases, Royal Veterinary College, Royal College St.,
London NW1 0TU, United Kingdom. Phone: 44 (0) 20 7468 5272. Fax:
44 (0) 20 7468 5306. E-mail: nstoker@rvc.ac.uk.
† Present address: Department of Medical Microbiology, Barts and
the London, Queen Mary’s School of Medicine and Dentistry, London
E1 2AD, United Kingdom.
‡ Present address: Department of Pathology and Infectious Dis-
eases, Royal Veterinary College, London NW1 0TU, United Kingdom.
§ Present address: School of Public Health, University of California
at Berkeley, Berkeley, CA 94720.
sequence-specific manner to selected promoters, thereby in-
ducing or repressing their transcription, and S. enterica serovar
Typhimurium phoP mutants are highly attenuated (14, 17).
The genome of M. tuberculosis contains 11 paired 2CRs, four
isolated regulators, and three isolated sensors (6). Mutants in
two of these, phoP and mprA are highly attenuated in a murine
model (31, 43), while a prrA mutant grows more slowly in mac-
rophages (12), indicating that these regulators control genes
which are important for successful infection by the pathogen.
In this work, we have investigated the role of other M. tuber-
culosis 2CRs in infection by systematically constructing defined
mutations in these systems and phenotypically characterizing
the resultant mutants in vivo and in vitro.
MATERIALS AND METHODS
Growth of bacteria and extraction of DNA. M. tuberculosis was grown at 37°C
in Middlebrook 7H9 medium (Difco) supplemented with 10% (vol/vol) oleic
acid-albumin-dextrose-catalase (Becton Dickinson) and 0.05% (wt/vol) Tween
80 or on Middlebrook 7H10 agar (Difco) supplemented with 10% (vol/vol) oleic
acid-albumin-dextrose-catalase. Hygromycin at 100 ␮g/ml, kanamycin at 20 ␮g/
ml, and X-Gal (5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside) at 50 ␮g/ml
were used where appropriate. DNA for Southern analysis was extracted as
described previously (2).
Mutant construction. Delivery vectors were constructed using the pNIL and
pGOAL series vectors (30). Mutants were constructed using a two-step strategy
as previously described (30). Briefly, 1 to 5 ␮g of vector DNA was pretreated with
UV to stimulate homologous recombination and used to electroporate M. tuber-
culosis (19, 30). Single-crossover strains were selected on agar containing hygro-
mycin, kanamycin, and X-Gal. An individual colony was streaked out onto agar
(without antibiotics) to allow the second crossover to occur. The cells were
resuspended in medium, and serial dilutions were plated onto X-Gal with 10%
(wt/vol) sucrose. Sucrose-resistant white colonies were tested for kanamycin
sensitivity and analyzed by PCR and Southern hybridization. Genomic DNA was
prepared according to the method of Belisle and Sonnenberg (2). Southern
hybrizidation was carried out using the AlkPhos Direct kit (Amersham) accord-
ing to the manufacturer’s instructions. In this way, individual strains carrying six
unmarked and one marked mutation were isolated (Fig. 1 and Table 1).

1134
VOL. 71, 2003 M. TUBERCULOSIS TWO-COMPONENT REGULATORY MUTANTS 1135

optical density at 600 nm (OD600) of 0.05. The cultures were incubated standing
at 37°C. Serial dilutions were plated to determine colony counts at 2, 4, and 24 h.
(ii) Hydrogen peroxide. Cultures were diluted to a theoretical OD600 of 0.01 in
phosphate-buffered saline and treated with 5 mM H2O2 (final concentration) for
4 h at 37°C. Serial dilutions were plated to determine colony counts.
(iii) Long-term stationary-phase culture. Strains were inoculated into 10 ml of
medium and incubated standing at 37°C. Serial dilutions were plated to deter-
mine colony counts over several months.
(iv) Total starvation. Strains were inoculated into 10 ml of sterile water to a
theoretical OD600 of 0.05. The cultures were incubated standing at 37°C. Serial
dilutions were plated to determine colony counts over several weeks.
In vivo studies. Experimental infections of SCID and DBA/2 mice and tissue
analyses were carried out as described previously (36). For survival analysis,
groups of six mice were infected with 106 viable mycobacteria in 200 ␮l of normal
saline via a lateral tail vein. The level of inoculum was routinely checked by CFU
analysis. Where appropriate, infected mice were killed by cervical dislocation in
accordance with humane endpoint protocols under the Animals Scientific Pro-
cedures Act, 1986 (United Kingdom). Median survival times were calculated for
each group, and statistical analysis was performed using the log rank tests of
survival.
For tissue analysis, lungs, livers, and spleens were collected aseptically from
three mice per group into 10 ml of medium and passed through a 100-␮m-pore-
size sieve (Falcon) in 7H9 medium containing 0.05% Tween 80. Serial 10-fold
dilutions were plated in 100-␮l volumes, and CFU were counted after 4 weeks
and checked at 6 weeks to allow for any change in the growth rate for the mutants
ex vivo.
Macrophage infections. Bone marrow-derived macrophages from BALB/c
mice were isolated and infected in the absence of penicillin and streptomycin as
described previously (36). Macrophage monolayers were prestimulated with
gamma interferon (IFN-␥) (Gibco) at a concentration of 200 U/ml for 4 h prior
to infection. The cells were infected for 4 h, then washed six times in warm tissue
culture medium. The infection dose was assayed independently by plating the
inoculum. The number of viable mycobacteria was assessed by lysis of the mac-
rophage monolayer with 1 ml of sterile distilled water containing 0.1% Triton
X-100 per well, followed by plating the bacteria on Middlebrook 7H10 plates.

FIG. 1. Genomic contexts of 2CRs analyzed in this study. The
genomic contexts of the two-component system genes are shown. Re-
sponse regulators are shown as open arrows, sensors are shown as
hatched arrows, and other genes are shown as solid arrows. The open
boxes show the regions deleted in the mutants made, and the names of
the mutants are given alongside. All were unmarked deletions, except
for tcrXY, which contained a deletion and an additional insertion of a
hygromycin resistance gene. In the case of mtrB, the open box shows
the region that we attempted to delete.
Complementation of devR⌬. In order to complement the devR mutation in
Tame16, the integrating vector pSOUP86 was made by cloning a 1.7-kb region
containing 150 bp upstream, Rv3134c, and devR, but not devS, into the unique
NarI site of pUC-Gm-Int (24).
In vitro tests. Mutants were tested in a variety of in vitro situations, none of
which showed any differences from the wild type.
(i) Extreme pH. Liquid medium was adjusted to pH 2 with HCl or to pH 12
with NaOH. Strains were inoculated into 10 ml of liquid medium to a theoretical
RESULTS
We initially selected six paired 2CRs as targets for gene
disruption: devRS (Rv3132c/Rv3133c), kdpDE (Rv1027c/
Rv1028c), mtrAB (Rv3245c/3246c), narLS (Rv0844c/Rv0845),
tcrXY (Rv3764c/Rv3765c), and trcRS (Rv1032c/Rv1033c) (Fig.
1). We also selected one isolated sensor (Rv3220c). These gene
names have all been used in other publications, except narS
and tcrXY, for which we use our own nomenclature. All re-
sponse regulators belong to the class 2 DNA-binding regulator
family (29) typified by Escherichia coli OmpR, except for DevR
and NarL, which belong to the class 3 (LuxR) family typified by
E. coli NarL. Little is known about the functions of these
genes, and only the kdpDE genes show convincing homology to
a well-characterized system; in E. coli, this is an emergency
high-affinity potassium transport system synthesized only at
low potassium concentrations (40).
Six mutants were successfully constructed (trcS⌬, kdpDE⌬,

TABLE 1. M. tuberculosis H37Rv strains used

Name Genotype Description

H37Rv Wild-type laboratory strain (ATCC 25618)

Tame12 trcS⌬ 882-bp BalI deletion in sensor
Tame13 tcrXY::hyg 786-bp EcoRV-StuI deletion in sensor and regulator; adjacent hyg insertion
Tame14 kdpDE⌬ 1,691-bp SphI deletion in sensor and regulator
Tame16 devR⌬ 447-bp BalI deletion in regulator
Tame19 Rv3220⌬ 1,170-bp SphI deletion
Tame37 narL⌬ 317-bp PmlI-SmaI deletion in regulator
Devcomp5 Tame16::pSOUP86 Contains integrating plasmid carrying devR
1136 PARISH ET AL. INFECT. IMMUN.

FIG. 2. Southern blot analysis of mutants. DNA was extracted from wild-type (WT) and mutant (mut) strains and analyzed. The gels show
diagnostic digests that confirm their genotypes. devR, EcoRI/XhoI digest probed with 0.8-kb NotI fragment; trcS, XhoI digest probed with 2.3-kb
XhoI fragment; trcXY, EcoRI digest probed with 2.5-kb SmaI fragment; kdpDE, XhoI digest probed with 3.7-kb XhoI fragment; narL, BamHI digest
probed with 2.4-kb mutagenesis fragment; Rv3220c, XhoI digest probed with 1-kb mutagenesis fragment.

narL⌬, devR⌬, tcrXY⌬::hyg, and Rv3220c⌬) (Fig. 2), but we
were unable to isolate an mtrAB mutant, although we tried to
generate both marked and unmarked deletions. No differences
were observed between the growth rates in axenic culture of
the mutants and the wild type. In addition, bacterial survival
under a variety of culture conditions, including extreme pH,
long-term stationary-phase culture, total starvation, and hydro-
gen peroxide, was not affected by any of the mutations (see
Materials and Methods) (data not shown).
In vivo assays. We proceeded to test the ability of each
mutant to cause disease in the SCID mouse model (Fig. 3A
and B). We had previously shown that this is a rapid and
sensitive model for assaying changes in mycobacterial viru-
lence, with mutants that are attenuated in these mice also
being attenuated in immunocompetent mice (16, 36). Infection
with two of the mutants (narL⌬ and Rv3220⌬) resulted in the
same time to death as with the wild-type strain. In contrast, the
other four mutants (devR⌬, tcrXY⌬, trcS⌬, and kdpDE⌬) all
showed an increase in virulence, with significantly shorter sur-
vival times (P ⬍ 0.001). This contrasts with all previous studies
of this pathogen, in which gene deletion has resulted in atten-
uation or no change in virulence (3, 5, 7, 12, 15, 21, 27, 31, 36).
The most hypervirulent mutant in the SCID model, devR⌬
(median survival time, 30.5 days compared to 40.5 days for the
wild-type bacteria) was studied further. First, the role of devR
in causing the increase in virulence was confirmed by comple-
mentation. An integrating plasmid carrying the Rv3134 and
devR genes expressed from the promoter upstream of Rv3134
was introduced into devR⌬, producing strain Devcomp5. The
SCID experiment was repeated and clearly showed that the
increased virulence shown by the devR⌬ mutant was reduced to
the level of the wild type in the complemented strain (Fig. 3C).
The phenotype of the devR⌬ mutant was then examined in
immunocompetent DBA mice. Bacterial loads in different or-
gans were counted 15, 30, and 60 days following infection (Fig.
4). The numbers of bacteria were elevated in the mice infected
with the devR⌬ mutant in the lung, liver, and spleen at all time
points. This was statistically significant in the lung at 15 days
and in all three tissues after 30 days. By day 60, the numbers of
devR⌬ bacilli recovered from all three organs were still higher
VOL. 71, 2003 M. TUBERCULOSIS TWO-COMPONENT REGULATORY MUTANTS
FIG. 3. Virulence of mutant M. tuberculosis strains in SCID mice.
The mice (six per group) were infected i.v. with 106 wild-type or mutant
bacteria. (A) Solid squares, wild-type H37Rv (median survival time
[MST], 40.5 day); solid triangles, devR⌬ (MST, 30.5 days; P ⬍ 0.0001);
open inverted triangles, tcrXY⌬ (MST, 33.5 days; P ⬍ 0.0001); crosses,
trcS⌬ (MST, 35 days; P ⬍ 0.001); diamonds, kdpE⌬ (MST, 35 days;
P ⬍ 0.001); circles, Rv3220⌬ (MST, 40 days; no significant difference
[NS]). The initial inocula were 4 ⫻ 106 (H37Rv), 5 ⫻ 106 (devR⌬),
2.5 ⫻ 106 (tcrXY⌬), 2 ⫻ 106 (trcS⌬), 2.5 ⫻ 106 (kdpE⌬), and 2 ⫻ 106
(Rv3220⌬) bacteria. (B) Solid squares, wild-type H37Rv (MST, 34
days); solid circles narL⌬ (MST, 35.5 days; NS). The initial inocula
were 8 ⫻ 105 and 8.9 ⫻ 105, respectively. (C) Complementation of the
M. tuberculosis devR mutant. Solid squares, wild-type H37Rv (MST, 34
days); solid triangles, devR⌬ (MST, 27 days); open triangles, comple-
mented devR⌬ (MST, 35.5 days). The initial inocula were 5.4 ⫻ 106,
7.2 ⫻ 106, and 8.1 ⫻ 106, respectively. The differences between the
mutants and the other strains were significant at a level of ⬍0.001. The
results for each mutant are representative of two or three individual
experiments.
1137
FIG. 4. Virulence of M. tuberculosis devR⌬ in DBA mice. Mice
(three per group) were infected i.v. with 106 wild-type or mutant
bacteria. The P values of bacterial loads that differ significantly from
those of the wild type are given above the relevant bars. The results
represent means plus standard errors for three mice per group, with
significance measured using Student’s t test. Solid bars, wild-type
H37Rv; open bars, devR⌬. *, P ⬍ 0.05; ***, P ⬍ 0.0001. The results
shown are representative of three individual experiments.
than in the controls, but significantly so only in the liver. The
increase in bacterial load was also observed by histological
analysis and was coincident with increased inflammatory re-
sponses (data not shown).
Macrophage studies. One of the most important early effec-
tor cells in controlling infection by M. tuberculosis is the mac-
rophage. Although M. tuberculosis is able to survive in resting
macrophages by inhibiting the maturation of phagosomes (32),
macrophages that are activated by IFN-␥ are able to kill the
bacteria using a variety of methods, including tumor necrosis
factor alpha and reactive oxygen and nitrogen intermediates
1138 PARISH ET AL.
FIG. 5. Virulence of M. tuberculosis devR⌬ in activated murine
macrophages. Squares, wild-type H37Rv; Solid triangles, devR⌬; open
triangles, complemented devR⌬; circles, narL⌬. The bacterial counts
after 24 h were significantly different in the devR⌬ and wild-type strains
using an unpaired t test (P ⬍ 0.001); this is an experiment represen-
tative of three, all of which showed devR⌬ to be hypervirulent at 24 h.
(1, 34). We therefore compared the abilities of activated mac-
rophages to control the growth of wild-type and devR⌬ bacilli.
Whereas wild-type bacteria were killed rapidly, there was a
reproducible rise in the numbers of devR⌬ bacteria in the first
24 h after infection (Fig. 5); we saw a 13% increase in the
growth of devR⌬ bacteria in 24 h compared to a reduction of
50% in wild-type bacteria. This effect was abrogated by
complementation of the mutation. As an additional control, we
showed that activated macrophages were able to kill narL⌬
bacilli with kinetics similar to those of wild-type bacteria, mir-
roring the in vivo results with this strain.
DISCUSSION
We chose 2CRs as targets for gene inactivation, as they have
been implicated in the virulence of many other pathogens. M.
tuberculosis has 11 2CRs, and we reasoned that they may play
an important role in the survival of the pathogen. We selected
seven systems (six 2CRs and one isolated sensor) as targets, of
which six were amenable to gene disruption, indicating they
were not essential for growth in culture. We were unable to
isolate a mutant lacking the mtrB gene (8, 39), suggesting that
the gene is essential. This is consistent with the studies of Zahrt
and Deretic (42), who were unable to isolate a mutant lacking
the mtrAB system. The reason for the essentiality of this system
is unknown, as are the genes which the system controls.
SCID mice infected with four of the six mutants (devR⌬,
trcS⌬, tcrXY⌬, and kdpDE) succumbed to infection more rap-
idly than those infected with wild-type M. tuberculosis. SCID
mice act as a model for assaying the innate immune response;
we have previously found that mutants that show altered re-
sponses in SCID mice show similar changes in immunocom-
petent mice. However, this preliminary screen will miss effects
that are due to the acquired immune response. Thus, the
increase in virulence in SCID mice was seen in the absence of
any T-cell-mediated response, although these mice retain the
INFECT. IMMUN.
capacity to generate organized inflammatory cell responses
surrounding foci of bacteria.
We carried out further experiments on the devR⌬ mutant,
which showed the greatest hypervirulence in the SCID model.
This gene was originally identified by its differential expression
in the M. tuberculosis strain H37Rv and its avirulent derivative
H37Ra (23). We saw increased bacillary load in the devR⌬
mutant-infected immunocompetent mice. Significantly greater
numbers of devR⌬ bacteria (⬃8 to 10-fold) than wild-type
bacteria were seen on day 15 in the lungs and on day 30 in the
lungs, liver, and spleen. By day 60, the wild-type bacteria in the
lungs had almost reached the levels seen with the mutant,
though the numbers of CFU in the liver were still significantly
higher. These early differences in CFU in immunocompetent
mice are consistent with the SCID mouse results, as T-cell-
specific controlling immunity only develops 21 to 28 days after
infection. As with the SCID data, the CFU differences ob-
served were not due to an intrinsic difference in the axenic
growth rate, as we described earlier. Sherman et al. (35) also
reported no difference in the in vitro exponential-phase growth
of a Mycobacterium bovis BCG strain lacking devR activity,
although stationary-phase survival was reduced.
In order to investigate the hypervirulent phenotype in a
more defined model, we investigated the growth of the devR⌬
mutant in activated macrophages. Again, the growth of the
bacteria was greater than that of the wild-type strain. This is
the first time to our knowledge that mycobacteria have been
found to increase in number in IFN-␥-activated macrophages.
The phenotype was consistently seen at 24 h (Fig. 5) but
seemed to be lost by 72 h (data not shown). Our results did not
show whether the increase in bacteria compared to the wild
type was due to increased growth, reduced killing, or a com-
bination of the two. The transient loss of susceptibility to
killing was not due to a deficiency in the production of nitric
oxide or tumor necrosis factor alpha, previously described as
potential key mechanisms of mycobacterial killing (1, 4). Both
of these were in fact elevated in supernatants collected at 24 h
from devR⌬ mutant-infected macrophages compared to those
infected with the wild-type strain (data not shown).
It has been reported that virulence in a trcS mutant is unal-
tered (12), which is inconsistent with our results. There are a
number of experimental differences which may explain this
discrepancy. We tested our trcS mutant using intravenous (i.v.)
inoculation of SCID mice and assayed survival, while Ewann et
al. used aerosol infection of C57Bl/6 mice and assayed CFU in
the lungs. In addition, the strains of M. tuberculosis were dif-
ferent, as we used H37Rv whereas they used strain Mt103.
Further work will be needed to clarify this; in particular, it
would be valuable to test our mutants using an aerosol model.
Our results, together with the previous reports on mprA,
phoP, and prrA, show that a large proportion of 2CR mutants
are attenuated or hypervirulent, demonstrating the critical
roles 2CRs play in determining the outcome of mycobacterial
infection. Virtually nothing is known about the mechanisms by
which this is achieved, either at the level of the signals they
respond to or the genes they modulate. It is difficult to predict
the signals to which 2CRs respond; infection and multiplica-
tion within a host requires the ability to adjust to multiple
stimuli, thereby exposing the bacteria to many different micro-
environments. Of the systems studied here, we know that tran-
VOL. 71, 2003 M. TUBERCULOSIS TWO-COMPONENT REGULATORY MUTANTS
scription of devR is induced directly or indirectly by hypoxia, a
signal that is associated with persistence (35). Our results sug-
gest either that hypoxia is relevant at more than one stage of
the infection or that M. tuberculosis embarks down the road to
a persistent state much earlier than has been previously sug-
gested. The trcR system has been reported to be induced in
early exponential phase (18), while the kdp system appears to
be orthologous to the E. coli system, which is a high-affinity
potassium transport system (40). This is induced during potas-
sium-limited growth and is also affected by oxidative stress
(33). The further use of a macrophage killing model with
combinations of bacterial and murine mutants may help us to
elucidate both signals and killing mechanisms.
It has been shown that there is a spectrum of virulence in
clinical isolates (22, 25, 28), which presumably is related to loss
or alteration of genes. This work represents the first report of
an increase in mycobacterial virulence following targeted gene
deletion. We propose that the four regulatory systems identi-
fied here, inactivation of which leads to hypervirulence, are
involved in suppressing the growth rate of M. tuberculosis at
early stages of infection. It would be expected that most gene
deletions will lead to pathogens that are less virulent. The few
precedents for gene knockout causing increased virulence are
found in regulatory genes, where mutations lead to derepres-
sion of virulence genes (10, 13). We could be observing the
same phenomenon, as we too have studied regulatory muta-
tions. However, it is remarkable that this phenotype was seen
in several of the mutants, while we have recently reported that
insertion of a transposon into a nonregulatory gene can also
lead to hypervirulence (26). This suggests that we are observ-
ing an important and previously undescribed aspect of the
biology of M. tuberculosis.
ACKNOWLEDGMENTS
Tanya Parish and Debbie A. Smith contributed equally to this work.
This work was funded by the GlaxoSmithKline Action TB project
and the European Union TB vaccine consortium (QLK2-CT-1999-
01093).
We thank Ruth McAdam, Ken Duncan, and Kirsten Jung for their
useful discussions and Heidi Alderton for excellent technical assis-
tance.
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