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Previous Article | Next Article 
Journal of Bacteriology, October 2001, p. 6119-6125, Vol. 183, No. 20
Division of Infectious Diseases, Children's
Hospital, Harvard Medical School, Boston, Massachusetts
02115,1 and Howard Hughes Medical
Institute, Albert Einstein College of Medicine, Bronx, New York
104612
Received 11 May 2001/Accepted 30 July 2001
Mycobacterium tuberculosis is a specialized
intracellular pathogen that must regulate gene expression to overcome
stresses produced by host defenses during infection. SigH is an
alternative sigma factor that we have previously shown plays a role in
the response to stress of the saprophyte Mycobacterium
smegmatis. In this work we investigated the role of
sigH in the M.
tuberculosis response to heat and oxidative stress. We
determined that a M. tuberculosis sigH
mutant is more susceptible to oxidative stresses and that the inducible
expression of the thioredoxin reductase/thioredoxin genes
trxB2/trxC and a gene of unknown function,
Rv2466c, is regulated by sigH via expression from
promoters directly recognized by SigH. We also determined that the
sigH mutant is more susceptible to heat stress and that
inducible expression of the heat shock genes dnaK and
clpB is positively regulated by sigH. The
induction of these heat shock gene promoters but not of other
SigH-dependent promoters was markedly greater in response to heat
versus oxidative stress, consistent with their additional regulation by
a heat-labile repressor. To further understand the role of
sigH in the M.
tuberculosis stress response, we investigated the
regulation of the stress-responsive sigma factor genes
sigE and sigB. We determined that
inducible expression of sigE is regulated by
sigH and that basal and inducible expression of
sigB is dependent on sigE and
sigH. These data indicate that sigH plays
a central role in a network that regulates heat and oxidative-stress
responses that are likely to be important in M.
tuberculosis pathogenesis.
Tuberculosis remains a major cause
of human suffering, exacting an enormous toll of morbidity and
mortality in much of the world (11). The cause of
tuberculosis, the obligate pathogen Mycobacterium
tuberculosis, is highly adapted for survival in the host organism.
Following infection M. tuberculosis is ingested by macrophages and must persist in this environment in order to survive, either in a quiescent state or through active replication that
results in tissue destruction and the disease of tuberculosis. In
adapting to this intracellular environment, this bacterium must
regulate its physiology to survive a variety of stresses produced by
the macrophage, including reactive oxygen and reactive nitrogen species
produced by these cells (1, 4, 28). In addition
M. tuberculosis has been shown to alter the
physiology of the macrophage to modulate host defenses
(51). Although the sequencing of the M. tuberculosis genome and recent insights are beginning to
shed light on pathogenic mechanisms of this organism (7, 8, 27,
30), the means by which M. tuberculosis
adapts to survive and replicate in the host remain poorly understood.
Data from a number of laboratories have implicated several alternative
sigma factors of mycobacteria, including SigB, SigE, SigF, and SigH, in
the adaptation of the pathogen M. tuberculosis and the saprophyte Mycobacterium smegmatis to several
stresses (5, 9, 12, 20, 29, 53). M. smegmatis SigH has been shown to be important in surviving
organic peroxide stress and heat shock, and the transcription of
sigH in M. tuberculosis is induced in
response to a variety of stresses in vitro, including heat shock and
oxidative stress, and following uptake by macrophages (12, 16,
29). In Streptomyces coelicolor, a member of the actinomycete family that includes mycobacteria, SigR, a close homologue
of M. tuberculosis SigH, was recently shown to be
important in responding to redox stress through regulation of the
thioredoxin/thioredoxin reductase system (39).
The importance of heat stress and oxidative-stress responses in
M. tuberculosis pathogenesis and immunity has
been extensively investigated. Heat shock proteins, includng DnaK and
GroEL, are induced during macrophage infection and have been shown to
be major antigens recognized by the host immune system following infection by M. tuberculosis (2, 22, 25,
46). A recent report demonstrated enhanced protective immunity
in mice overexpressing dnaK (49). The induction
of heat shock proteins during infection and their function in
maintenance of protein structure suggest that these proteins play a
role protecting M. tuberculosis against oxidative
and other stresses generated by host macrophages during infection.
Among oxidative-stress response mechanisms, catalase-peroxidase activities were identified decades ago as important M. tuberculosis virulence factors that also play a role in
susceptibility to the first-line antitubercular isoniazid
(31, 32). More recent molecular investigation has linked
these phenotypes to the genes encoding catalase/peroxidase
(katG) and alkylhydroperoxidase (ahpC) in
M. tuberculosis (48, 52, 55, 56).
Strikingly, the gene encoding OxyR, a positive regulator of the
peroxide stress response in other bacteria and in most other
mycobacterial species, was found to be inactivated by multiple
mutations in M. tuberculosis, suggesting altered
regulation of the oxidative-stress response in this pathogen (10,
45).
Based on these observations, we sought to investigate the role of SigH
in M. tuberculosis in survival and regulation of
gene expression in response to oxidative and heat stresses. In this report we demonstrate that a sigH mutant of M. tuberculosis is impaired in its ability to survive different
types of oxidative stress as well as heat stress. We further
demonstrate that SigH, in response to these stresses, strongly induces
the transcription of genes that have been shown to be of central
importance in the response to these stresses. Among these are the genes
encoding the only thioredoxin reductase of M. tuberculosis TrxB2 and the heat shock proteins DnaK and
ClpB. In addition we demonstrate that SigH regulates the
stress-inducible expression of the genes encoding the stress-responsive
sigma factors SigE and SigB.
These data indicate that SigH plays an important role in regulating the
response to heat and oxidative stress in M. tuberculosis through the regulation of major effectors and
regulators of the response to these stresses. Together with published
data, our results suggest that SigH plays a central role in the
regulation of specific gene expression that allows the bacillus to
adapt to and survive following exposure to host-generated stresses that this organism is likely to encounter during infection.
Bacterial strains and culture conditions.
Escherichia
coli DH5 Construction of mutant strains of M.
tuberculosis.
The M. tuberculosis
sigH and sigE mutants were constructed by allele
exchange using the specialized transducing phage system as described
elsewhere (14). Briefly, the sigH gene was
disrupted by inserting a hygromycin resistance cassette into the coding region at codon 151, and the resulting disrupted sigH gene
and flanking DNA were cloned into the cos vector pYUB572.
The resulting plasmid was digested with PacI and ligated to
PacI-digested pHAE87 DNA. The ligation mix was packaged in
lambda in vitro packaging mix (Gigapack III; Stratagene), transduced
into E. coli, and plated on L
broth-hygromycin. Plasmid DNA was isolated from several colonies and restriction digested to verify the presence of the desired insert,
and this DNA was electroporated into M. smegmatis
strain mc2-155 (47) and plated for
plaques at 30°C. Plate stocks were made from a
temperature-sensitive phage, and M. tuberculosis H37Rv was infected, followed by plating at
37°C with hygromycin selection. Colonies were picked after 3 to 4 weeks of growth and were screened by PCR for disruption of
sigH. Candidate clones were confirmed by Southern blotting
to contain a single, disrupted copy of sigH, and one,
designated RH349, was used as the sigH mutant strain in
subsequent experiments. The M. tuberculosis sigE
mutant, designated RH374, was constructed utilizing a similar strategy,
except that the hygromycin resistance cassette was introduced into a
675-bp deletion (codons 1 to 225) in the sigE gene. The
sigH/sigE double mutant, designated RH375, was
generated by first disrupting sigH with a kanamycin
resistance gene, utilizing the temperature-sensitive/counterselection strategy of Pelicic et al. (40), followed by disruption of
sigE as described above.
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.20.6119-6125.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
The Alternative Sigma Factor SigH Regulates Major Components
of Oxidative and Heat Stress Responses in
Mycobacterium tuberculosis
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
(Life Technologies) and XL1Blue (Stratagene) were used
as host strains for cloning experiments. M. tuberculosis H37Rv was used as the wild-type strain in the
stress experiments and as the parental strain from which sigma factor
mutant strains were constructed. For some primer extension experiments,
RNA was isolated from Mycobacterium bovis BCG. E. coli was grown on L agar or in L broth. M. tuberculosis was grown in Middlebrook 7H9 broth supplemented
with oleic acid, albumin, and dextrose complex (7H9-OADC) (Difco) plus
0.05% Tween 80 or on Middlebrook 7H9-OADC agar plates. Ampicillin (100 µg/ml), apramycin (30 µg/ml), and hygromycin (50 µg/ml for
mycobacteria and 100 µg/ml for E. coli) were
added to culture media as indicated.
Heat shock and oxidative-stress experiments. Wild-type M. tuberculosis H37Rv, the sigH mutant strain RH349, and the sigH-complemented mutant strains RH377 and RH395 were compared for their ability to survive several stresses. Survival assays in liquid medium for chemical and heat stresses were performed as previously described (12, 53). Chemicals were added to log-phase cultures to a final concentration of 50 mM diamide-0.1 mM plumbagin. Heat shock was performed at 52°C. The number of viable cells was determined by plating serial dilutions on Middlebrook 7H9-OADC or by inoculating the contents of BACTEC 12B bottles and measuring the growth index (Becton Dickinson). Colonies on plates were counted after 3.5 to 4 weeks at 37°C for all strains except RH395, which requires 4.5 to 5 weeks to form colonies. For experiments using the BACTEC system, standard curves of CFU per milliliter (determined by plating serial dilutions) versus time to reach a growth index of 100 (T-100) were determined experimentally in the absence of stress for each strain. The growth index of 100 was then determined for each aliquot inoculated at the various time points in the stress experiments, and the number of bacilli inoculated was derived from the standard curve of that strain (13). For plating and BACTEC experiments, duplicate samples were assayed at each time point and each experiment was performed at least twice.
RNA isolation and primer extension. RNA was isolated from mid-log-phase cultures. For experiments examining stress induction of transcription, the culture was divided into two aliquots, one of which was treated with 1 mM diamide for 20 min or 50°C heat shock for 15 min prior to RNA isolation. The cells were chilled, pelleted, resuspended in RNeasy lysis buffer (Qiagen), and transferred to a 2-ml tube containing ceramic and silica beads. The bacteria were lysed by shaking in an FP120 machine (BIO101), and the lysate was then removed to a fresh tube and centrifuged to remove the cell debris. The supernatant then was processed using the RNeasy kit to isolate total RNA according to the manufacturer's protocol (Qiagen).
For primer extension analysis, 0.5 pmol of
-32P-labeled primer was mixed with 10 µg of
RNA in a 6-µl volume. The mixture was heated at 65°C for 5 min and
was cooled on ice. Reverse transcription mixture was prepared by mixing
2 µl of 5× first-strand synthesis buffer (Life Technologies), 1 µl
of 0.1 M dithiothreitol, 1 µl of 10 mM deoxynucleoside triphosphate
mix, and 10 U of Superscript II reverse transcriptase (Life
Technologies). The 4-µl reverse transcription mixture was added to
the primer-RNA mixture, and the reaction was incubated for 1 h at
42°C. The reaction was stopped by adding 5 µl of loading dye, and 5 µl of the reaction was loaded on a sequencing gel. Sequencing
reactions, performed with the same primer used for primer extension,
were run in adjacent lanes to determine the size of the transcripts.
Quantification of 32P-labeled transcripts was
performed using a PhosphorImager and ImageQuaNT software (Molecular
Dynamics). In each case where the absence of a SigH-dependent
transcript was observed in RNA isolated from the SigH mutant, the
quality of the RNA was confirmed by using the same batch of RNA in a
primer extension to determine the presence of a SigH-independent transcript.
Purification of M. tuberculosis SigH protein and in vitro transcription assays. The M. tuberculosis sigH gene was cloned in pTYB1 (New England Biolabs) to overexpress SigH as a fusion protein with the intein and chitin binding domain tag from the vector. Following purification of the fusion protein on a chitin column (New England Biolabs), native SigH protein was released from the chitin-bound intein tag by self-cleavage of the tag in the presence of dithiothreitol. SigH protein was further purified by ion-exchange chromatography on a POROS 50 HQ column (PerSeptive Biosystems) and gel permeation chromatography on a Superdex-100 column (Amersham Pharmacia Biotech).
In vitro single-round runoff transcription analysis was performed using conditions modified from those described earlier by Kang et al. and Miyazaki et al. (23, 33). Purified SigH protein (0.5 µg) was incubated with 0.2 U of E. coli RNA polymerase core enzyme (Epicentre Technologies) at 37oC for 30 min in 30 µl of transcription buffer (50 mM Tris-HCl [pH 8.0], 10 mM MgCl2, 50 mM KCl, 0.1 mM EDTA, 250 µg of bovine serum albumin per ml, and 0.5 U of SUPERase-In [Ambion] per µl). To the reconstituted E
H holoenzyme, 0.09 pmol of DNA template was
added for 5 min at 37°C, followed by a mixture of
[-32P]UTP and three other nucleotides for 2 min at 37°C and then by a mixture of heparin and unlabeled UTP for 5 min at 37°C. Concentrations of nucleotides and heparin in 40 µl of
final reaction volume were 0.15 mM ATP, 0.15 mM GTP, 0.15 mM UTP, 0.15 mM CTP, 4 µCi of [-32P]UTP, and 200 µg
heparin per ml. Samples were electrophoresed in a 6% denaturing
polyacryamide gel containing 7 M urea and were analyzed by
autoradiography. DNA templates for in vitro transcription reactions
were prepared by PCR except for the sigH template, which was
a ClaI restriction fragment containing DNA 5' of the
sigH coding sequence.
Computer database searching. Searches of the M. tuberculosis H37Rv genome sequence for consensus promoter elements were performed utilizing the program Findpatterns in the GCG package of software and the "search pattern" program available on the TubercuList web site of the Pasteur Institute (http://genolist.pasteur.fr/TubercuList).
| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Growth and survival following oxidative and heat stresses.
M. tuberculosis H37Rv, the sigH mutant
strain RH349, and the mutant strain complemented with sigH
alone (RH377) or complemented with sigH plus rshA
(RH395) were assessed for in vitro growth and susceptibility to heat
and oxidative stresses. H37Rv and RH349 were found to have very similar
growth rates. In contrast, both RH377 and RH395 grew more slowly in
liquid medium. These strains were tested for susceptibility to diamide,
an agent that specifically oxidizes sulfhydryl groups
(24), and the redox cycling compound plumbagin, which
generates superoxide (19). The sigH mutant was
found to be more susceptible to both diamide and plumbagin (Fig.
1). This strain was also more susceptible
to 52°C heat stress (Fig. 1). For each stress tested, the
sigH mutant was more susceptible than H37Rv in both the
plating assay and in the BACTEC assay (not shown).
|
Regulation of stress response genes by SigH.
Two approaches
were used to identify SigH-dependent promoters. First, based on the
regulation of thioredoxin reductase by the SigH homologue SigR in
S. coelicolor, the role of SigH in regulating
transcription of this gene was examined. Second, based on the 
10 and
35 region sequences of the M. tuberculosis and M. smegmatis sigH promoters previously
demonstrated to be SigH dependent (12), we performed
searches of the M. tuberculosis genome sequence
for similar sequences positioned 5' to the start codon of annotated
open reading frames (7). Primer extension experiments,
using RNA isolated from M. tuberculosis H37Rv and RH349, were performed to determine whether these sequences corresponded to in vivo promoters and whether they were dependent on SigH.
|
|
Regulation of sigB transcription by SigH and
SigE.
Having found that SigH regulates the inducible expression of
sigE in response to heat shock and oxidative stress, we
sought to determine whether there was additional cross-regulation of other sigma factors by SigH. Based on data indicating that
sigB expression is increased in the response to several
stresses, including heat stress in M. tuberculosis (29), we examined the
transcription of this gene. A transcription start site, present in
unstressed log-phase cells and induced in response to diamide stress,
was observed in M. tuberculosis H37Rv (Fig
4). This transcription start site is 3 bases 5' from the sigB transcription start previously identified. This difference is likely the result of the use of a
heterologous sequence ladder generated from a non-GC-rich template, resulting in imprecise localization of the start site in the previous report, versus the use of a sigB sequence ladder to
determine the exact start site in this work (Fig. 4) (20).
In the sigH mutant strain, transcription from this promoter
in log-phase growth was slightly decreased from that seen in the wild
type. Induction of sigB transcription by diamide, however,
was substantially blunted in the sigH mutant.
|
Direct recognition of SigH-dependent promoters by SigH.
To
determine whether these promoters found to be SigH dependent in vivo
were directly recognized by SigH, single-round in vitro transcription
analysis was performed. Specific transcription requiring SigH,
resulting in transcripts of the size expected based on the location of
the promoter relative to the 3' end of each template, was observed for
all of the in vivo SigH-dependent promoters identified in this study
(Fig. 5). In the context of the in vivo
data, these results indicate that SigH regulates the transcription of
these genes directly, via the recognition of promoter sequences by RNA
polymerase holoenzyme containing SigH.
|
35 and 10 region sequences are
observed. The 8-base 35 region consensus includes 7 specific bases,
plus one position that allows a pyrimidine (cytosine or thymine). The
10 region has a 5-base consensus in which the central GTT is
conserved in all seven promoters. As seen in Fig. 5, differences in
promoter activity were observed in the in vitro transcription assay
using equal amounts of template under conditions where SigH was not
limiting, as determined by titration of SigH. These differences show
some correspondence to the extent of variation from consensus in the
35 region of these promoters, with trxB2, dnaK,
clpB, and Rv2466c forming a group of stronger promoters and
sigE, sigB, and sigH a group of weaker
promoters. Examination of the promoter regions of these genes shows
that the sigE, sigH, and sigB 35
sequences vary by at least 1 base from the consensus. Of interest,
clpB differs from the consensus in the first two positions
yet appears to be a strong promoter in vitro and in vivo.
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
In this work we demonstrate that a sigH mutant of M. tuberculosis is more susceptible to heat and oxidative stresses and that this sigma factor directly regulates the expression of major effectors of the response to these stresses. We further show that sigH regulates the stress-inducible expression of sigE and sigB, two sigma factors previously shown to play a role in the mycobacterial response to these and other stresses (29, 53). Together with previous data indicating that sigH is autoregulated (12), these results suggest a major role for this alternative sigma factor in the regulation of M. tuberculosis gene expression in response to stresses generated by the host during infection by this pathogen. The observation that sigH transcription is induced following infection of macrophages (16) and preliminary data indicating attenuation of the sigH mutant in a mouse model of infection (W. R. Jacobs and R. N. Husson, unpublished) support a role for SigH in the regulation of gene expression required for M. tuberculosis virulence. The regulation by SigH of both stress response effector proteins and additional transcriptional regulators creates a regulatory network with the potential for modulation of gene expression, with responses varying quantitatively and in the specific components of the response that are induced by different classes of stress.
The inconsistent complementation of the sigH mutant strain by sigH alone and the consistent complementation by sigH plus rshA suggest that RshA is required for effective function of SigH in vivo. RshA is highly similar to RsrA, a redox-regulated negative regulator of SigR, a S. coelicolor homologue of SigH (23, 39), and functions as a negative regulator of SigH (Song and Husson, unpublished). In Streptomyces, rsrA mutant strains frequently acquire inactivating mutations in sigR (38), while in M. smegmatis it has not been possible to generate a rshA mutant (X. Puyang and R. N. Husson, unpublished). These data indicate that unregulated SigH or SigR activity, a situation that would result in an uncontrolled positive-feedback loop, is poorly tolerated by the bacterium.
The coordinate regulation of the heat shock genes dnaK and
clpB makes physiologic sense in the context of recent
insights into the interaction of the DnaK-DnaJ-GrpE complex with ClpB
in the prevention of aggregate formation and insights into
disaggregation and refolding of denatured proteins (15, 34,
35). The positive regulation of these heat shock genes by an
alternative sigma factor, however, is a novel finding among the
high-G+C-content, gram-positive bacteria. In these organisms and in
most gram-positive bacteria, negative regulation by repressors, such as
HscR, which regulates GroES/GroEL, and HrpA (HspR), which regulates the
dnaK-dnaJ-grpE-hrpA and clpB operons
in Streptomyces species and other species, has been
described as the primary mechanism by which heat shock gene transcription is controlled (3, 17, 36, 44, 57). In contrast, in E. coli the alternative sigma
factors 32 (RpoH) and
24 (RpoE) play a major role in regulating the
heat shock response (18, 42-44).
The differences that we observed in induction of expression of the SigH-dependent promoters of dnaK and clpB by heat versus oxidative stress suggested the presence of a heat-labile repressor. The role of HspR (HrpA) as a repressor of dnaK expression in M. tuberculosis has recently been demonstrated (49). The differential effect of heat versus oxidative stress on induction of expression from the SigH-dependent dnaK and clpB promoters, presumably mediated by the different susceptibility of HspR to these stresses, allows for modulation of the SigH-mediated response to different classes of stress.
The response of M. tuberculosis to oxidative stress has been the subject of active investigation. OxyR is a major positive regulator of the oxidative-stress response in many bacteria, where it regulates the expression of genes, including katG encoding catalase peroxidase and ahpC encoding alkylhydroperoxidase, in response to peroxide stress (6, 50). OxyR is present in most mycobacteria and has been shown in Mycobacterium marinum to positively regulate expression of ahpC but not of katG (37). A striking finding was that in M. tuberculosis, oxyR is inactivated by multiple mutations, resulting in minimal basal or inducible expression of ahpC in this organism (10, 45). In M. smegmatis and M. tuberculosis, katG was recently shown to be cotranscribed with and regulated by furA, which encodes a repressor similar to Fur proteins in many bacteria (41, 54). The regulation of other known genes that play a role in the oxidative-stress response, e.g., sodA and sodC encoding the superoxide dismutases of M. tuberculosis, remains to be determined.
In this context, the susceptibility of the M. tuberculosis sigH mutant to diamide, which generates a redox stress, and plumbagin, which generates superoxide, suggests that SigH regulates another arm of the oxidative-stress response in mycobacteria. While we have demonstrated the SigH-mediated induction of thioredoxin reductase and thioredoxin expression, the exact mechanisms by which sigH mediates the response to these stresses is not known. No SigH consensus promoter sequences are present 5' of sodA, sodC, or ahpC. The presence of a glutaredoxin motif in the protein encoded by Rv2466c, whose expression is SigH dependent, suggests that this gene may play a role in the SigH-regulated response to oxidative stress in M. tuberculosis.
A notable result of this work is the identification of a regulatory network that includes SigH, SigE, and SigB. Our findings indicate that the SigH-dependent sigE promoter is responsible for much of the increased transcription of sigE under high-heat and high-oxidative-stress conditions and that both SigE and SigH are required for maximal induction of sigB expression. The observation that transcription of sigH and sigE is induced following uptake of M. tuberculosis by macrophages (16) suggests that this SigH-dependent sigE promoter may be responsible for increased sigE transcription in the macrophage and that sigB transcription is also likely to be induced in this setting.
The consensus SigH-dependent promoter motifs identified in this report
are similar to those defined for SigR-dependent promoters in
S. coelicolor, with the potential expansion of
the 35 consensus to 8 nucleotides in M. tuberculosis and a single-base difference in the
6-nucleotide, overlapping consensus between these species (39). Three of the strongest M. tuberculosis promoters share the exact consensus 35
sequence, while the weaker sigB, sigE, and
sigH promoters vary by 1, 1, and 2 bases, respectively, from the consensus. The clpB promoter, despite varying from the
consensus at two positions, is a strong promoter in vivo and in vitro.
The relative strength of these promoters is consistent with the
regulatory roles of these sigma factors, whose effect on cell
physiology can be amplified through transcription of regulated genes,
in contrast with the direct effector roles of thioredoxin reductase, DnaK, and ClpB.
The regulation of a single sigB promoter by SigE and SigH
parallels the overlap in recognition of promoters by SigX and SigW in
Bacillus subtilis (21). Definition of the
importance of specific bases in the 10 and 35 regions through
targeted mutagenesis should allow clearer understanding of the promoter
elements involved in recognition by SigH and SigE. This insight,
together with functional characterization of regulated genes, will
allow further definition of the role of these sigma factors in the
regulation of M. tuberculosis gene expression in
response to stress and during infection.
| |
ACKNOWLEDGMENTS |
|---|
This work was supported by grants from the National Institutes of Health (AI37901 and AI27150) and by a grant from the Potts Memorial Foundation.
S.R. and T.S. contributed equally to this work.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Children's Hospital, Enders Rm. 609, 300 Longwood Ave., Boston, MA 02115. Phone: (617) 355-5151. Fax: (617) 355-8387. E-mail: robert.husson{at}tch.harvard.edu.
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Abstract
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Materials and Methods
Results
Discussion
References
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