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Previous Article | Next Article 
Infection and Immunity, December 2003, p. 7170-7172, Vol. 71, No. 12
0019-9567/03/$08.00+0 DOI: 10.1128/IAI.71.12.7170-7172.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Tetsuyuki Yoshimatsu, Chiew Ko, Paul J. Converse, and William R. Bishai*
Department of Medicine, Center for Tuberculosis Research, Johns Hopkins School of Medicine, Baltimore, Maryland 21231
Received 9 June 2003/ Returned for modification 2 August 2003/ Accepted 8 August 2003
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ABSTRACT |
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TEXT |
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70 class of factors
(3,
8). SigA and SigB are the
principal sigma factors, and the other 11 are alternative sigma
factors, with all but SigF considered to be extracytoplasmic function
factors, mediating interactions of the bacterium with the
extracellular environment. The sigE gene is conserved in
mycobacteria but is not essential for growth in vitro
(18). Deletion of
sigE (
sigE) results in increased
susceptibility to heat shock and chemical stress
(6,
15,
18). Expression of
sigE increases after phagocytosis by human and mouse
macrophages (9,
10). In the absence of
sigE, the viability of M. tuberculosis in both
activated and unactivated macrophages is decreased
(15). Finally, deletion
of sigE has been shown by microarray analysis to result in the
downregulation of genes which encode transcriptional regulators, fatty
acid metabolism enzymes, and heat shock proteins
(15).
In our
laboratory we have characterized a number of sigma factors in terms of
their responses to stress, their effects on the expression of other
genes, and their phenotypes in mice
(2,
5,
11). We report here our
findings with C3H/HeJ mice infected by aerosol with an M.
tuberculosis CDC1551 sigE deletion mutant compared to
mice infected with wild-type or sigE-complemented M.
tuberculosis CDC1551
(7). Utilizing a
targeted-disruption strategy based on the vector pCK0686, which
contains a hygromycin resistance cassette flanked by multiple cloning
sites (MCS) (11), we
cloned a left-flank PCR product with 1,943 bp proximal to the
sigE gene (MT1259/Rv1221) and a nonoverlapping right-flank
product of 1,889 bp into the MCS, resulting in the deletion of the
central region of sigE from the targeting vector. Only 19 bp
of the 5' end and 392 bp of the 3' end of the
sigE gene remained in place, while a central 360 bp was
deleted. The resulting plasmid, pCK0699, was then used to transform
M. tuberculosis CDC1551 in a two-step selection process, with
Southern blotting used to confirm allelic exchange. A complemented
sigE mutant strain was created by cloning a
2,930-bp M. tuberculosis CDC1551 genomic fragment including
the coding region as well as the regulatory region of the sigE
gene and 215 bp downstream into an L5 phage-based
integration-proficient vector for mycobacteria, pMH94
(12), and then
transforming the M. tuberculosis sigE mutant
with this vector. Groups of inbred C3H/HeJ or BALB/c-severe combined
immunodeficiency (SCID) mice were infected by aerosol with matched
inocula of the wild-type, knockout, or complemented strain, which
routinely implant 
100 organisms in the lungs. For CFU organ
burden determinations, groups of five mice were sacrificed at day 1 and
at 4, 12, and 20 weeks and lung and spleen CFU counts were enumerated.
For time-to-death studies, groups of 10 mice were infected with matched
inocula and monitored daily for mortality.
The M.
tuberculosis sigE mutant showed a CFU organ
burden in C3H/HeJ mouse lungs that was similar to the pattern observed
with wild-type infection (Fig.
1). The complemented sigE mutant gave lung CFU counts
that closely matched those of the wild type and the
sigE mutant (log10 CFU counts at 12 and 16
weeks of 3.67 ± 0.29 and 4.06 ± 0.3, respectively).
Spleen counts for C3H/HeJ mice showed that all three strains were
absent in the spleens on day 1 following aerosol infection but then
disseminated from lungs to the spleen by 4 weeks at equivalent levels
(log10 spleen CFU counts of 3.54 ± 0.83, 2.08
± 1.9, and 2.33 ± 2.31 for the wild type,
sigE mutant, and complemented sigE
mutant, respectively). Thus CFU organ burden studies of immunocompetent
C3H/HeJ mice showed that the M. tuberculosis
sigE mutant and the complemented strain had virulence
equivalent to that of the wild-type parental strain, CDC1551.
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sigE mutant survived 291 days (Fig.
2A). Remarkably, all BALB/c-SCID mice infected with wild-type or
complemented bacilli died by day 39 or 47, whereas none of the mice
infected with the mutant died until day 70 (Fig.
2B). Kaplan-Meier analysis
showed that times to death for both immunocompetent C3H/HeJ and
immunocompromised BALB/c-SCID mice infected with the
sigE mutant were significantly prolonged compared to
those for mice infected with the wild-type or complemented strain
(P < 0.001). In immunocompetent mice, but not
immunocompromised mice, the complemented strain exhibited a phenotype
intermediate between the wild-type and the knockout phenotypes. The
difference in time to death between wild-type and complemented bacilli
was statistically significant, suggesting that, as measured in this
assay, complementation may have been only partial. Thus, despite
achieving a persistent, high-organ-burden infection in mouse lungs, the
M. tuberculosis sigE mutant was found to be
significantly attenuated by time-to-death analysis.
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sigH mutant, in which high organ
CFU counts in both mutant and wild-type bacilli were observed but
animals infected with the mutant showed a significant delay in time to
death (11). One
distinction from the sigH mutant is that there is a
significant delay in the time to death for SCID mice as well as for
immunologically intact mice after infection with the
sigE mutant bacilli. This may suggest that the
sigE tubercle bacilli are more susceptible to innate,
T-cell-independent killing than are the sigH
organisms. Interestingly, for the M. tuberculosis
sigH mutant, both CD4 and CD8 cell recruitment to the
lung was reduced in mouse tissues at 4 weeks; similarly, histological
sections showed a corresponding delay in disease progression in mouse
tissues on infection with the sigH mutant
(11). We have designated
this phenotype of bacterial persistence with reduced host mortality the
immunopathology (imp) defect. Although the immunopathology
phenotype has been observed upon mouse infection with both the M.
tuberculosis sigH and now the
sigE mutants, it is significant that functional
redundancy does not appear to account for the similar animal
phenotypes. Microarray analysis of the M. tuberculosis
sigH mutant suggests that the sigma factor governs a
redox stress response regulon, with several thioredoxins
and thioredoxin reductases showing relative underexpression in the
sigH mutant compared with expression in the wild type
(11). In contrast, the
M. tuberculosis sigE mutant sigma factor has
been reported to govern a more generalized stress response regulon
(15), and inspection of
the genes putatively governed by these sigma factors reveals few common
genes. Interestingly, the impaired survival of the M.
tuberculosis sigE mutant within macrophages
reported by Manganelli et al.
(15) does not appear to
extend to a survival defect in lung and spleen tissues in C3H/HeJ mice.
Recently, several non-sigma factor M. tuberculosis mutants,
including the M. tuberculosis RD1 mutant
(13) and
whiB3 mutant
(17), have been reported;
these mutants show mouse infection patterns similar to the
immunopathology phenotype observed in the M.
tuberculosis sigH and sigE
mutants. As both the RD1 region
(14) and the
whiB-like gene family
(4,
16) have been implicated
in gene regulation, one might speculate that the immunopathology
phenotype is an attenuation pattern associated with aberrant gene
regulation pathways and an inability to adapt to key stimuli during
growth in vivo in live animal hosts. While further research will be
important in order to understand the basis of the immunopathology
defect, this class of M. tuberculosis mutants has attractive
features as live, attenuated vaccines against tuberculosis in view of
their reduced lethality but significant persistence in the
host—factors which may be important in the maintenance of
protective immunity
(1).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Present
address: Division of Pulmonary Diseases, Department of Immunology and
Allergy, Oita Medical University, Oita, Japan.
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