The primary mechanism of attenuation of bacillus Calmette-Guerin is a loss of secreted lytic function required for invasion of lung interstitial tissue.
ABSTRACT Tuberculosis remains a leading cause of death worldwide, despite the availability of effective chemotherapy and a vaccine. Bacillus Calmette-Guérin (BCG), the tuberculosis vaccine, is an attenuated mutant of Mycobacterium bovis that was isolated after serial subcultures, yet the functional basis for this attenuation has never been elucidated. A single region (RD1), which is absent in all BCG substrains, was deleted from virulent M. bovis and Mycobacterium tuberculosis strains, and the resulting DeltaRD1 mutants were significantly attenuated for virulence in both immunocompromised and immunocompetent mice. The M. tuberculosis DeltaRD1 mutants were also shown to protect mice against aerosol challenge, in a similar manner to BCG. Interestingly, the DeltaRD1 mutants failed to cause cytolysis of pneumocytes, a phenotype that had been previously used to distinguish virulent M. tuberculosis from BCG. A specific transposon mutation, which disrupts the Rv3874 Rv3875 (cfp-10 esat-6) operon of RD1, also caused loss of the cytolytic phenotype in both pneumocytes and macrophages. This mutation resulted in the attenuation of virulence in mice, as the result of reduced tissue invasiveness. Moreover, specific deletion of each transcriptional unit of RD1 revealed that three independent transcriptional units are required for virulence, two of which are involved in the secretion of ESAT-6 (6-kDa early secretory antigenic target). We conclude that the primary attenuating mechanism of bacillus Calmette-Guérin is the loss of cytolytic activity mediated by secreted ESAT-6, which results in reduced tissue invasiveness.
- [show abstract] [hide abstract]
ABSTRACT: Bacille Calmette-Guérin (BCG) vaccines are live attenuated strains of Mycobacterium bovis administered to prevent tuberculosis. To better understand the differences between M. tuberculosis, M. bovis, and the various BCG daughter strains, their genomic compositions were studied by performing comparative hybridization experiments on a DNA microarray. Regions deleted from BCG vaccines relative to the virulent M. tuberculosis H37Rv reference strain were confirmed by sequencing across the missing segment of the H37Rv genome. Eleven regions (encompassing 91 open reading frames) of H37Rv were found that were absent from one or more virulent strains of M. bovis. Five additional regions representing 38 open reading frames were present in M. bovis but absent from some or all BCG strains; this is evidence for the ongoing evolution of BCG strains since their original derivation. A precise understanding of the genetic differences between closely related Mycobacteria suggests rational approaches to the design of improved diagnostics and vaccines.Science 06/1999; 284(5419):1520-3. · 31.03 Impact Factor
Article: Genomics of Mycobacterium bovis.[show abstract] [hide abstract]
ABSTRACT: The imminent completion of the genome sequence of Mycobacterium bovis will reveal the genetic blueprint for this most successful pathogen. Comparative analysis with the genome sequences of M. tuberculosis and M. bovis BCG promises to expose the genetic basis for the phenotypic differences between the tubercle bacilli, offering unparalleled insight into the virulence factors of the M. tuberculosis complex. Initial analysis of the sequence data has already revealed a novel deletion from M. bovis, as well as identifying variation in members of the PPE family of proteins. As the study of bacterial pathogenicity enters the postgenomic phase, the genome sequence of M. bovis promises to serve as a cornerstone of mycobacterial genetics.Tuberculosis 02/2001; 81(1-2):157-63. · 3.03 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The mechanism(s) by which Mycobacterium tuberculosis crosses the alveolar wall to establish infection in the lung is not well known. In an attempt to better understand the mechanism of translocation and create a model to study the different stages of bacterial crossing through the alveolar wall, we established a two-layer transwell system. M. tuberculosis H37Rv was evaluated regarding the ability to cross and disrupt the membrane. M. tuberculosis invaded A549 type II alveolar cells with an efficiency of 2 to 3% of the initial inoculum, although it was not efficient in invading endothelial cells. However, bacteria that invaded A549 cells were subsequently able to be taken up by endothelial cells with an efficiency of 5 to 6% of the inoculum. When incubated with a bicellular transwell monolayer (epithelial and endothelial cells), M. tuberculosis translocated into the lower chamber with efficiency (3 to 4%). M. tuberculosis was also able to efficiently translocate across the bicellular layer when inside monocytes. Infected monocytes crossed the barrier with greater efficiency when A549 alveolar cells were infected with M. tuberculosis than when A549 cells were not infected. We identified two potential mechanisms by which M. tuberculosis gains access to deeper tissues, by translocating across epithelial cells and by traveling into the blood vessels within monocytes.Infection and Immunity 02/2002; 70(1):140-6. · 4.07 Impact Factor
The primary mechanism of attenuation of bacillus
Calmette–Gue ´rin is a loss of secreted lytic function
required for invasion of lung interstitial tissue
Tsungda Hsu*†‡, Suzanne M. Hingley-Wilson*†‡, Bing Chen*†, Mei Chen†, Annie Z. Dai†, Paul M. Morin*†,
Carolyn B. Marks§, Jeevan Padiyar*†, Celia Goulding¶, Mari Gingery¶, David Eisenberg¶, Robert G. Russell?,
Steven C. Derrick**, Frank M. Collins**, Sheldon L. Morris**, C. Harold King††, and William R. Jacobs, Jr.*†‡‡
*Howard Hughes Medical Institute, Departments of?Pathology and†Microbiology and Immunology, and§Analytic Imaging Facility, Albert Einstein College
of Medicine, Bronx, NY 10461;¶UCLA–DOE Institute of Genomics and Proteomics, University of California, Los Angeles, CA 90095-1570; **Center for
Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892; and††Division of Infectious Diseases,
Department of Medicine, Emory School of Medicine, Atlanta, GA 30303
Communicated by Barry R. Bloom, Harvard School of Public Health, Boston, MA, August 13, 2003 (received for review April 3, 2003)
Tuberculosis remains a leading cause of death worldwide, despite
the availability of effective chemotherapy and a vaccine. Bacillus
Calmette–Gue ´rin (BCG), the tuberculosis vaccine, is an attenuated
mutant of Mycobacterium bovis that was isolated after serial
subcultures, yet the functional basis for this attenuation has never
been elucidated. A single region (RD1), which is absent in all BCG
substrains, was deleted from virulent M. bovis and Mycobacterium
tuberculosis strains, and the resulting ?RD1 mutants were signif-
icantly attenuated for virulence in both immunocompromised and
immunocompetent mice. The M. tuberculosis ?RD1 mutants were
also shown to protect mice against aerosol challenge, in a similar
manner to BCG. Interestingly, the ?RD1 mutants failed to cause
cytolysis of pneumocytes, a phenotype that had been previously
used to distinguish virulent M. tuberculosis from BCG. A specific
transposon mutation, which disrupts the Rv3874 Rv3875 (cfp-10
esat-6) operon of RD1, also caused loss of the cytolytic phenotype
in both pneumocytes and macrophages. This mutation resulted in
the attenuation of virulence in mice, as the result of reduced tissue
invasiveness. Moreover, specific deletion of each transcriptional
unit of RD1 revealed that three independent transcriptional units
are required for virulence, two of which are involved in the
secretion of ESAT-6 (6-kDa early secretory antigenic target). We
conclude that the primary attenuating mechanism of bacillus
Calmette–Gue ´rin is the loss of cytolytic activity mediated by se-
creted ESAT-6, which results in reduced tissue invasiveness.
medium (1, 2), when Drs. Calmette and Gue ´rin set out to test the
hypothesis that a bovine tubercle bacillus could transmit pul-
monary tuberculosis after oral administration (1, 3, 4). However,
unexpectedly after the 39th passage, the strain was unable to kill
experimental animals (1, 2), and showed no reversion to viru-
lence even after the authors had performed over 200 passages
(3), which is consistent with the attenuating mutation being a
deletion mutation. In proceeding studies, BCG was determined
to be able to protect animals receiving a lethal challenge of
virulent tubercle bacilli (5), and in 1921 was first used as an
anti-tuberculous vaccine (6). Presently, an estimated 3 billion
doses have been used to vaccinate the human population against
tuberculosis, yet the mechanism that causes the attenuation of
BCG remains unknown.
Mahairas et al. (6) first compared the genomic sequences of
BCG and M. bovis, by using subtractive hybridization, and found
that there were three regions of difference (designated RD1,
RD2, and RD3) present in the genome of M. bovis, but missing
in BCG. Behr et al. (7), and others (8), later identified 16 large
deletions, including RD1-RD3, which were present in the Myco-
bacterium tuberculosis genome but absent in BCG. Eleven of
these 16 deletions were unique to M. bovis whereas the remain-
acillus Calmette–Gue ´rin (BCG) was first isolated from
Mycobacterium bovis after serial subculturing in ox bile
ing 5 deletions were unique to BCG. One of these 5 deletions,
designated RD1 (9,454 bp), was absent from all of the BCG
it was concluded that the deletion of RD1 occurred very early
during the development of BCG, probably before 1921 (9).
Therefore, it is reasonable to hypothesize that RD1 was the
primary attenuating mutation, which resulted in the generation
of BCG from M. bovis. An attempt to restore virulence to BCG
with an RD1-complementing clone was only partially successful
(10) although the deletion of the esat-6 gene from M. bovis
resulted in an attenuated phenotype in guinea pigs (11). A recent
study has also determined that deletion of RD1 from M. tuber-
no definitive study looking at the mechanism of attenuation or
at the function of each of the genes that comprise RD1.
Materials and Methods
Mice. BALB?c and C57BL?6 mice were purchased from Charles
River Breeding Laboratories. Severe combined immunodefi-
cient (SCID) (BALB?c background) mice were purchased from
the National Cancer Institute. Aerosol infections at a low dose
of 50 colony-forming units (cfu) per mouse and high dose of 500
cfu per mouse were carried out as described in ref. 13. I.v.
injections in the tail vein (at a dose of 2 ? 106cfu) and plating
were carried out as described in ref. 14. At least 10 mice were
used for survival studies, and at least 3 mice per time point for
all other studies. For pathology, half of the organ was plated and
half was fixed in either 10% formalin (for light microscopy) or
2.5% glutaraldehyde, in 0.1 M sodium cacodylate buffer (for
Construction of the RD1 Mutants. The unmarked deletion mutant
of M. tuberculosis H37Rv, mc24002, was generated by transfor-
mation using a sacB counter selection, as described in ref. 15.
Specifically, the plasmid pJH506 was created by cloning UFS
(upstream flanking sequence) and DFS (downstream flanking
sequence; see Fig. 1A) into pJH12 [a pMV261-derived Myco-
bacterial shuttle plasmid in which UFS and DFS flanked a green
fluorescent protein gene (GFPuv, CLONTECH) controlled by
the Mycobacterium leprae promoter of 18 kDa]. pJH508 was
created by cloning UFS-DFS-GFPuv (from pJH506) into
pyUB657. After transformation into mycobacteria, selection was
Abbreviations: ESAT-6, 6-kDa early secretory antigenic target; BCG, bacillus Calmette–
Gue ´rin; SCID, severe combined immunodeficient; UFS, upstream flanking sequence; DFS,
downstream flanking sequence; LDH, lactate dehydrogenase; CFP-10, culture filtrate pro-
‡T.H. and S.M.H.-W. contributed equally to this work.
‡‡To whom correspondence should be addressed. E-mail: email@example.com.
© 2003 by The National Academy of Sciences of the USA
October 14, 2003 ?
vol. 100 ?
carried out using hygromycin, followed by 3% sucrose. Southern
analysis (16), using UFS- or DFS-specific probes, was performed
to confirm the RD1 deletion. The mycobacteriophage-based
method of specialized transduction, which utilizes conditionally
replicating shuttle phasmids, was also used to construct RD1
mutants, by using UFS and DFS as above, along with the
individual gene constructs (Rv3871, Rv3872?3, Rv3874?5, and
Rv3876?7), as described in ref. 17. Again, Southern analysis was
used to confirm deletion.
Construction and Screening of the M. tuberculosis Erdman Transpo-
son-Generated Mutant Library. A transposon library of M. tuber-
culosis Erdman (of ?6,460 clones) was constructed as described
(18) by using the hygromycin-resistant Tn5370 transposon.
Screening of the library was carried out by infection of A549 cells
in 96-well plates (without the addition of gentamicin), which
were then screened for reduced lactate dehydrogenase (LDH)
release (described in the infection experiment methodology).
Identification of the transposon insertion sites was carried out by
sequencing, as described in ref. 18.
Complementation of Mutants. Complementation analyses were
performed with the cosmid 2F9, which contains the entire RD1
region (Rv3860–Rv3885c) in the integration proficient vector
pYUB412 (constructed by F. Bange, Medical School, Hannover,
Germany, identified and provided by S. T. Cole Institut Pasteur,
Paris). After transformation of the mutant strains with the
in vivo. (A) Survival experiment using SCID mice i.v. infected with 2 ? 106cfu
per mouse. Infection was carried out as described in Materials and Methods.
Strains used were M. tuberculosis H37Rv (F), M. tuberculosis ?RD1 (I), M.
tuberculosis ?RD1 (pYUB412::Rv3860–Rv3885c) (Œ), and M. bovis BCG (‚). (B)
(E), M. tuberculosis ?RD1 (‚), and M. bovis BCG (?), at a dose of 2 ? 106cfu.
(I), M. tuberculosis H37Rv (F), and M. tuberculosis H37Rv ?RD1 (‚). The
infecting dose per mouse was 2 ? 106cfu. Data represent the mean of cfus
from three mice per time point.
bovis. (A) Schematic of M. tuberculosis H37Rv RD1 region showing predicted
NcoI sites. Arrows at the top represent the genes in this region. UFSs and DFSs
used to generate the knockout are indicated as filled bars above the grid line.
Each increment in the grid line represents 1 kbp. The RD1 sequence deleted
from M. bovis BCG is represented by an open bar spanning from Rv3871 to
Rv3879c. The site of the insertion of transposon Tn5370 is also indicated. (B)
Southern analysis of the NcoI-digested genomic DNA isolated from the wild
tuberculosis and M. bovis. Lane 1, M. tuberculosis H37Rv; lane 2, M. tubercu-
losis H37Rv ?RD1; lane 3, M. tuberculosis Erdman; lane 4, M. tuberculosis
Erdman ?RD1; lane 5, M. tuberculosis CDC1551; lane 6, M. tuberculosis
CDC1551 ?RD1; lane 7, M. bovis Ravenel; and lane 8, M. bovis Ravenel ?RD1.
The probe used in the Southern analysis was either DFS (Left), demonstrating
the deletion of RD1, or IS6110-specific (Right). The IS6110 probe is used to
characterize the four different strains (described in ref. 31).
Generation of mutants in the RD1 region of M. tuberculosis and M.
Hsu et al.PNAS ?
October 14, 2003 ?
vol. 100 ?
no. 21 ?
constructs, as described in ref. 19, Southern analyses was carried
out to determine whether integration was successful.
Infection Experiments. A549 cells [an alveolar epithelial cell line,
American type Culture Collection (ATCC)] were infected at a
multiplicity of infection of 10:1, as described in detail in ref. 20.
Briefly, cells were seeded at 5 ? 105cells?ml, and, after infection
washed off and gentamicin (Sigma) was added to the medium.
LDH release was quantified at specified time points by using the
cytotoxicity detection kit (Roche Diagnostics), according to the
manufacturer’s instructions. The maximum and background
controls used were 0.1% Triton X-100-treated cells (maximum)
and uninfected cells (background). Values were calculated by
using the following equation: % LDH release ? (sample-
background)?(maximum-background). The levels of both apo-
ptosis and necrosis were determined by elucidating the concen-
tration of histone-associated DNA fragments (in the cell lysate
and supernatant, respectively), which were quantified by using
the cell death detection ELISA (Roche Diagnostics), again
according to the manufacturer’s instructions. The necrosis pos-
itive control was 0.1% Triton X-100, whereas 10 ?M campoth-
ecin (Sigma) was added for the apoptosis positive control. Five
mM exogenous glycine was added to the infected monolayers for
the glycine protection assay, as described in ref. 21. Bone
marrow-derived macrophages were obtained from the femurs
of BALB?c mice and were seeded at 2 ? 105cells?ml, after lysis
of red blood cells with 3% acetic acid?PBS and the removal of
Artificial Membrane Studies. Artificial planal bi-layers were con-
structed at room temperature by using the lipid di-
phytanoylphosphatidylcholine and the Mueller brush technique,
as described in ref. 22. Voltages of 20–50 mV were applied, and
the conductance across the bilayer was measured on the addition
of 1 ?g of purified protein.
Preparation of Culture Filtrates and Whole Cell Extracts. The culture
as described in ref. 16. Western blot analyses were performed by
using anti-ESAT-6 mouse monoclonal primary antibody
HYB76-8 at a 1:100 dilution and a sheep anti-mouse Ig second-
ary antibody (Amersham Pharmacia) at 1:10,000.
RD1 Deletions of M. bovis and M. tuberculosis Are Attenuated for
Virulence in Mice. To test whether RD1 (Fig. 1A) was essential for
virulence in M. bovis and M. tuberculosis, RD1 was deleted from
the M. tuberculosis strains H37Rv, Erdman, and CDC1551, and
from M. bovis Ravenel (Fig. 1B). The deletion of RD1 resulted
in the attenuation of virulence of M. tuberculosis H37Rv ?RD1
in a SCID mouse model of infection, which was restored on
complementation with RD1 (Fig. 2A). Similar attenuating phe-
notypes were observed for the RD1 deletion mutants made in M.
tuberculosis Erdman and CDC1551, and in M. bovis Ravenel
(data not shown). The M. tuberculosis H37Rv ?RD1 mutant was
also highly attenuated in immunocompetent BALB?c mice (Fig.
2B), but the pattern of in vivo growth remained similar to
wild-type, in contrast to BCG (Fig. 2C).
Protection of BCG and ?RD1 Against Challenge with Virulent M.
tuberculosis. One of the hallmark characteristics of BCG is its
ability to provide protection against aerosolized challenge with
virulent M. tuberculosis. At 3 and 8 months after vaccination with
either BCG or ?RD1 mutant, mice were challenged with 50–200
cfus of the acriflavin-resistant strain of M. tuberculosis Erdman,
by the aerosol route. Twenty-eight days after the challenge, the
mice were killed, and the bacterial burden in the lungs and
spleens was determined (see Table 1). In mice challenged after
8 months, both the BCG-vaccinated and M. tuberculosis ?RD1-
vaccinated mice exhibited ?1 log protection in the lung, with
similar cfu values. Protection was seen to a similar extent in mice
challenged 3 months after vaccination (see Table 1). The M.
tuberculosis ?RD1 mutant also protected against hematogenous
spread at both 3- and 8-month challenges (see Table 1). Thus, M.
tuberculosis ?RD1 exhibits long-term immunogenicity similar to
The cfp-10 esat-6 Operon of M. tuberculosis Is Required for Host Cell
Lysis. Because virulent M. tuberculosis, but not BCG, was previ-
ously reported to induced lysis of alveolar epithelial cells, we
screened for mutants that were defective in the ability to induce
cytolysis of lung epithelial cells (20, 23). Approximately 700
clones of a transposon-generated library of M. tuberculosis
Erdman mutants were screened for reduced cytolysis of lung
epithelial cells, by measuring LDH release. This screen resulted
in the identification of several mutants, one of which, mc24513,
mapped to the cfp-10 (Rv3874) gene, which is contained within
the RD1 region (Fig. 1). The mutant phenotype of reduced LDH
release was then confirmed in triplicate in A549 cells, along with
the parental and complemented strain controls (Fig. 3A). This
phenotype was also observed in primary macrophages, but at 7
days postinfection (Fig. 3B). It was also noted that both the broth
grown and intracellular growth rates of the mutant were not
significantly different to those of the parent strain (data not
shown). Previous studies have demonstrated that the cfp-10
(Rv3874) gene and the esat-6 (Rv3875) gene are coordinately
expressed as an operon (24). Therefore, a transposon insertion
in cfp-10 is likely to have a polar effect and prevent the
expression of both genes. Indeed, our studies showed that, as
expected, the cfp-10 mutant failed to express ESAT-6 (6-kDa
early secretory antigenic target), as evidenced by Western
analysis (data not shown).
To further elucidate the mode of cell death induced by the
wild-type strain and reduced in the mutant strain, the concen-
tration of histone-associated DNA fragments was determined,
both in the supernatant (associated with necrosis, in which the
cells lyse) and in the cell lysate (associated with apoptosis) in
lung epithelial cells. These results confirmed that necrosis was
reduced in the mutant when compared with the parental and
noted in the levels of apoptosis (data not shown). To further
Table 1. Vaccination with M. tuberculosis H37Rv ?RD1 protects as well as M. bovis BCG (day
3-month challenge (log10cfu) 8-month challenge (log10cfu)
Lung SpleenLung Spleen
4.77 ? 0.06
3.96 ? 0.20**
3.97 ? 0.39**
3.57 ? 0.21
2.18 ? 0.18***
2.12 ? 0.12***
6.61 ? 0.13
5.07 ? 0.10***
5.11 ? 0.14***
5.26 ? 0.11
3.85 ? 0.17**
4.00 ? 0.33**
**, P ? 0.01;***, P ? 0.001.
www.pnas.org?cgi?doi?10.1073?pnas.1635213100Hsu et al.
define the mechanism of wild-type-induced cytolysis, glycine was
added to the supernatant of the infected monolayer. The addi-
tion of exogenous glycine has been reported to protect the cells
against lethal ion fluxes across the plasma membrane (21) and
was observed to reduce the amount of cytolysis from ?87% to
?13% (Fig. 3D). This finding suggests that wild-type-induced
cytolysis is mediated by lethal ion fluxes across the plasma
membrane. That the effector protein causing lysis and death of
the cells was ESAT-6 was demonstrated by experiments using an
artificial planal bi-layer, constructed by using the lipid di-
phytanoylphosphatidylcholine, as described in ref. 22 (Fig. 3E).
In these studies it was determined that the ESAT-6 protein,
either alone or in combination with culture filtrate protein 10
(CFP-10), resulted in major disruptions in conductance, even-
tually resulting in total destruction of the artificial membrane. It
is also interesting to note that the purified ESAT-6 protein, again
either alone or in combination with CFP-10, formed structures
similar to those seen in amyloidogenic protein solutions before
amyloid fibrils are formed (data not shown), and that some
amyloidogenic proteins exhibit pore-forming properties similar
to those of bacterial toxins (25).
The cfp-10 esat-6 Operon of RD1 Is Required for Virulence. The
disruption of the cfp-10 esat-6 operon in M. tuberculosis also
resulted in severe attenuation of virulence in an i.v. SCID mouse
model of infection, which was comparable to that observed with
the RD1 deletion mutants (Fig. 4A). Three weeks after aerosol
challenge of immunocompetent BALB?c mice, the mutant-
infected mice exhibited rare mild foci composed of very low
numbers of mononuclear inflammatory cells, with no lesions
detected in some lobes of the lung. Low numbers of infiltrating
macrophages were present, predominantly in the alveolar spaces
of lungs from mice infected with the mutant strain. In compar-
ison, the wild-type-infected mice showed scattered foci of mild
pneumonitis, with focal infiltration of low numbers of macro-
phages, accompanied by infiltration of epitheloid cells. The cfu
counts in the lung, for both the mutant and wild-type-infected
mice, were similar (data not shown). Electron microscopy re-
vealed that the mutant, Rv3874::Tn5370, bacilli were located
intracellularly within intact macrophages in the alveolar spaces,
with some macrophages containing large numbers of bacilli (Fig.
4 B1 and B2). In direct contrast, the wild-type bacilli were
typically located in macrophages in the interstitium of the
interalveolar wall (Fig. 4 B3 and B4), with some macrophages
showing evidence of lysis (Fig. 4B4). Furthermore, ultrastruc-
tural studies of the M. tuberculosis cfp-10 esat-6 mutant and
wild-type-infected human alveolar epithelial cells (in vitro) re-
vealed that fewer cells were infected in the case of the mutant
bacilli (?20% more cells were infected by wild-type bacilli), and
that there were approximately ten times higher numbers of
bacilli per cell in the mutant treatment, compared with wild-type
(see Table 2). Together, these data suggest that the lack of the
cfp-10 esat-6 operon in the mutant results in intracellular con-
tainment of the organism and is associated with a lack of cell lysis
and tissue invasiveness, and with the attenuation of lung damage.
cell lysis. (A) LDH release assay of infected lung epithelial cells, at 72 h
postinfection. Cells were infected at a multiplicity of infection of 10:1, and
supernatants were analyzed for LDH release. wt, M. tuberculosis Erdman ?
pYUB412 (empty vector); mt, mc24513 ? cfp10::Tn5370 ? pYUB412; c,
mc24513 ? pYUB412::Rv3860–Rv3885c; DRD1, M. tuberculosis Erdman ?RD1.
Values are means ? SD of triplicate measurements. (B) LDH release assay of
c are as in A. Values are means ? SD of triplicate measurements. (C) Necrosis-
determining assay, measuring histone-associated DNA fragments in the
supernatant. Labels wt, mt, and c are as in A and B. (D) The M. tuberculosis
necrosis phenotype is inhibited by 5 mM exogenous glycine (?g), compared
with the control, in which there is no exogenous glycine (?g). Values are
means ? SD of triplicate measurements. (E) ESAT-6, but not CFP-10, is suffi-
cient to induce disruption, leading to total destruction (see arrowhead) of an
artificial lipid bilayer. Artificial planal bilayers were constructed by using the
lipid diphytanoylphosphatidylcholine as described in ref. 28. Voltages of
on the addition of 1 ?g of each protein. The addition of ESAT-6 and CFP-10 in
carried out in triplicate. For further details, see Materials and Methods.
Hsu et al. PNAS ?
October 14, 2003 ?
vol. 100 ?
no. 21 ?
Rv3871 and Rv3876?Rv3877 Are Required for Virulence and for the
Secretion of ESAT-6. To test whether Rv3874 and Rv3875 were the
only genes of RD1 required for virulence, we generated deletions
and observed that the deletion of Rv3871 and Rv3876?77, as well as
or Rv3874::Tn5370 mutants in the SCID mouse model (Fig. 5A).
Based on homology to other protein translocation systems (26, 27),
we hypothesized that both Rv3871 and Rv3876?77 were involved in
secretion of CFP-10 and ESAT-6. Western analyses of the whole
cell extracts and culture filtrate proteins (Fig. 5B) clearly demon-
strate that both the ?Rv3871 and ?Rv3876?77 mutants can syn-
thesize ESAT-6, but that they are defective in their abilities to
secrete ESAT-6 to the culture filtrate. Thus, all of the attenuated
mutants of RD1 (?Rv3871, ?Rv8874?5, and ?Rv3876?77) are
defective in the synthesis or secretion of the effector protein
virulence. (A) Survival in SCID mice i.v. infected with 2 ? 106cfu of M.
tuberculosis Erdman ? pYUB412 (wt), mc24513 ? cfp10::Tn5370 ? pYUB412
(mt), and mc24513 ? pYUB412::Rv3860–Rv3885c (c). (B) Electron micrographs
of early lesions after high-dose aerosol infection of BALB?c mice with M.
tuberculosis Erdman wild type ? pYUB412, compared with mc24513 ?
pYUB412, at 3 weeks postinfection (?5,000). (B1) Mutant-infected macro-
phage in the alveolar airspace. This result was not observed in the wild-type-
infected mice at this time point. (B2) A macrophage in the lumen of the
airspace with large numbers of intracellular mutant bacilli. (B3) Wild-type
bacillus residing within a macrophage, located interstitially. This result was
not observed in mutant-infected mice at this time point. (B4) Cell within the
I pneumocyte. Arrows denote mycobacteria.
The loss of cfp-10?esat-6 (Rv3874?Rv3875) confers attenuation of
Table 2. Ultrastructural studies of the behaviour of the
M. tuberculosis cfp-10 esat-6 mutant in alveolar epithelial cell
culture (each value is representative of 62 4-grid squares)
mc24513 Wild type
% cells infected (24 h)
% cells infected (72 h)
% intracellular bacteria
% extracellular bacteria
Average number of bacilli per infected cell
42 ? 17***
4 ? 4***
***, P ? 0.001.
virulence and for the secretion of ESAT-6. (A) Survival time of SCID mice
infected i.v. with 2 ? 106cfu of M. tuberculosis H37Rv (I), ?Rv3871 (F),
whole cell extracts and culture filtrates. Arrow indicates the ESAT-6-specific
band. (B1) Culture filtrates probed with both anti-ESAT-6 primary antibody
HYB76-8 and goat anti-mouse Ig secondary antibody. (B2) Culture filtrates
probed only with secondary antibody. (B3) Whole-cell lysates probed with
both primary and secondary antibodies. In B1–B3, lanes are as follows: lane 1,
purified ESAT-6 protein (50 ng); lane 2, ?Rv3871; lane 3, ?Rv3872?3; lane 4,
M. tuberculosis H37Rv, H37Rv ?RD1, and complemented strain H37Rv ?RD1
(pYUB412::Rv3860–Rv3885c), reacted with both primary and secondary anti-
bodies. Lane 1, purified ESAT-6 protein (50 ng); lane 2, H37Rv; lane 3, H37Rv
?RD1; and lane 4, H37Rv ?RD1 (pYUB412::Rv3860–Rv3885c).
The genes Rv3871 and Rv3876?Rv3877 of RD1 are required for
www.pnas.org?cgi?doi?10.1073?pnas.1635213100Hsu et al.