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Max Planck Institute for Infection Biology, Charitéplatz 1, 10117 Berlin, Germany
5Department of Microbiology, Yonsei University, College of Medicine, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-752, South Korea
6Department of Computational Molecular Biology, Max Planck Institute for Molecular Genetics, Ihnestrasse 73, 14195 Berlin, Germany
7These authors contributed equally to this work.
*Correspondence: kaufmann@mpiib-berlin.mpg.de
DOI 10.1016/j.chom.2008.01.002
Cell Host & Microbe 3, 97–103, February 2008 ª2008 Elsevier Inc. 97
Cell Host & Microbe
A Point Mutation in the phoP Gene of H37Ra
attempted but may provide insights into the behavior of these identical to the corresponding loci in the published genomes of
strains under dormancy- or starvation-like conditions. M. tuberculosis CDC1551, a recent clinical isolate (Fleischmann
Major changes in gene expression profiles could result from et al., 2002), and M. bovis (Garnier et al., 2003). These 53 poly-
differences in transcriptional regulation, and consequently nu- morphisms could represent sequencing errors in the original se-
merous studies have addressed the regulatory machinery in M. quence. A potential sequencing error reported in the literature
tuberculosis with respect to virulence. An important class of reg- (Dubey et al., 2002), which would fuse the reading frames of
ulatory proteins is found in two-component systems in which Rv1180 (pks3) and Rv1181 (pks4), was not found in this H37Rv
several proteins have been implicated in mycobacterial virulence strain by resequencing. However, CDC1551, M. bovis, and
(Parish et al., 2003). Prominent and recently identified members H37Ra apparently code for the fusion protein. Twenty-two poly-
of these protein families in pathogenic mycobacteria include morphisms could be confirmed by resequencing. There were 19
DosR, which mediates dormancy, and PhoP, which positively cases in which the H37Rv sequence peculiarly diverges from the
regulates polyketide-derived lipid biosynthesis (Gonzalo et al., consensus built from M. bovis, CDC1551, and H37Ra; these
2006; Martin et al., 2006; Park et al., 2003; Perez et al., 2001). regions were therefore unlikely to be related to virulence. Only
Since these genes are of importance to virulence, microarray three polymorphisms, located in Rv0101, Rv0637, and phoP,
studies of knockout (KO) strains have been performed to identify were unique to H37Ra and are therefore most likely responsible
potential PhoP target genes (Walters et al., 2006). Interestingly, for its phenotype.
mycobacterial PhoP, although sharing the same name, is not for-
mally orthologous to PhoP from Salmonella, which is also essen- A Mutation in the Response Regulator PhoP of H37Ra
tial for virulence (see Figure S1 available online). Therefore, Among the three mutations the most significant effect on atten-
transfer of experimental information on PhoP proteins between uation of H37Ra was likely located in the phoP gene (Figure 1A),
species should be interpreted with great caution. which encodes the response regulator of the PhoP/PhoR two-
In this work, we extend upon previous genomic and transcrip- component system and is essential for virulence (Perez et al.,
tomic approaches by comparing the genomic sequence of 2001). The mutation in H37Ra described here leads to an amino
H37Ra with several virulent strains at the single nucleotide level, acid change located in the DNA-binding domain of PhoP replac-
discovering mutations in phoP (Rv0757), Rv0101, and Rv0637. ing a serine by a leucine (see Figure S2 for a structural model).
The mutation in PhoP compromised the functionality of the To investigate whether the mutation in the DNA-binding
protein, and expression analysis revealed a significant overlap domain interfered with its functionality, we performed gel shift
between genes deregulated in H37Ra and a PhoP KO strain. assays using overexpressed proteins (Figure 1B). The only
The introduction of the original phoP gene into H37Ra enhanced described interaction between mycobacterial PhoP and DNA is
the bacterial persistence in murine bone marrow-derived macro- the binding to its own promoter, where it interacts with a 9-mer
phages when compared to H37Ra. Our findings suggest that the consensus motif containing the sequence ACT/GT/GT/GYARC
phoP mutation in H37Ra resulted in the partial loss of regulatory (Gupta et al., 2006). The intensity of the shifted band was higher
function and that this contributed to reduced virulence of H37Ra. when the H37Rv version of the protein was incubated with DNA
containing the PhoP-binding motif. Therefore, we conclude that
RESULTS the PhoP from H37Ra has a reduced DNA-binding capability.
98 Cell Host & Microbe 3, 97–103, February 2008 ª2008 Elsevier Inc.
Cell Host & Microbe
A Point Mutation in the phoP Gene of H37Ra
Cell Host & Microbe 3, 97–103, February 2008 ª2008 Elsevier Inc. 99
Cell Host & Microbe
A Point Mutation in the phoP Gene of H37Ra
diminished in H37Ra, whereas additional repressive features of et al., 2006). The phoP mutant’s colony morphology differed dis-
this protein seem to remain intact. tinctly from the wild-type strain when cultured on 7H10 Middle-
brook agar plates. We observed similar morphological differ-
The Transcriptome under Hypoxic Conditions ences between H37Rv and H37Ra. Colonies were smaller in
and Starvation H37Ra than in H37Rv, and wrinkling on the colony surface was
Comparing H37Rv under hypoxic conditions in the stationary reduced in H37Ra as compared to H37Rv. As depicted in
phase with exponential growth, Voskuil et al. (2003, 2004) Figure 4A, these differences were reduced when H37Ra was
observed the expression of a unique set of genes referred to complemented with pSO5-expressing wild-type PhoP (H37Ra-
as the dormancy regulon in H37Rv. The two-component system phoP-Rv). Previously, Perez et al. (2001) found no significant
DosR/DosS (DevR/DevS, Rv3133c/Rv3132c) is pivotal for growth differences between wild-type and phoP KO strains when
the transcriptional gene activation in models of dormancy cultured in liquid medium. However, H37Ra took slightly longer
(Boon et al., 2001; Sherman et al., 2001; Voskuil et al., 2003, to reach the logarithmic and stationary phase. The phoP-com-
2004).We were interested in whether genes induced under hyp- plemented H37Ra strain revealed a growth pattern similar to
oxic conditions in H37Rv were similarly activated in H37Ra H37Rv (Figure S4).
because attenuation of H37Ra could be caused by expression Next, we were interested if the complementation of H37Ra
changes in the dormancy regulon of the mycobacterium. At the with the original phoP gene from H37Rv increased bacterial sur-
2-week time point, when conditions became hypoxic, we no- vival in immune cells, which is strongly diminished in H37Ra
ticed upregulation (1.8-fold, p value 3 3 10 14) of dosR tran- (McDonough et al., 1993). To this end, we infected murine
scripts in H37Ra. Other studies also documented the upregula- bone marrow-derived macrophages with equal multiplicities of
tion of dosR under nonreplicating persistence (NRP) conditions infection of H37Ra, H37Rv, and H37Ra-phoP-Rv (Figure 4B).
in H37Rv when expression was compared to exponentially This resulted in similar bacterial loads on day 0 for the three
growing bacteria (Voskuil et al., 2004). We directly compared ex- strains (Figure S5). At later time points, the bacterial burden of
pression patterns of H37Rv and H37Ra and found that dosR is macrophages infected with H37Ra was reduced to about 10%
induced even higher in the latter. Interestingly, this activation of the colony-forming unit (CFU) count of macrophages infected
also resulted in transcriptional induction of several genes of the with H37Rv. Cells challenged with H37Ra-phoP-Rv reached
dormancy regulon in the avirulent strain. Of the 116 transcripts a CFU count, which was between 24% and 35% of that of
identified as being upregulated (Voskuil et al., 2004), 10 genes H37Rv depending on the time point investigated. Overall CFU
were significantly activated in H37Ra after 2 weeks of culture of H37Ra-phoP-Rv were about three times higher than for the
as compared to gene expression in H37Rv (Figure 3C and Table noncomplemented H37Ra, showing that the intracellular bacte-
S2; p value < 0.001). rial persistence, and thus its virulence, was increased by the
Conditions of the 1-year time point likely resemble starvation. complementation.
Seven genes that were upregulated in this time point were also in-
duced in cultures kept in phosphate-buffered saline (Betts et al.,
2002). Three of these are coded in the genomic region between DISCUSSION
Rv2660c and Rv2664 and belong to operons that are highly
induced during starvation. However, other starvation-associated We identified three point mutations in coding regions that are
genes (e.g., those involved in energy metabolism) were not likely to cause the attenuation of H37Ra. The additional confir-
differentially transcribed during growth in nutrient-poor media mation of sequencing errors in the original reference sequence
(Betts et al., 2002). for H37Rv underscores the accuracy of the high-throughput
analysis. However, it is important to realize that not all confirmed
Complementation of H37Ra with phoP from H37Rv differences necessarily represent sequencing errors, as it is dif-
Physiological features of phoP KO mutants differ from wild-type ficult to estimate the number of generations between individual
bacteria in that they develop smaller colonies on agar-based me- sequencing attempts. Only a large-scale study of H37Rv strains
dia and show different cording properties, a feature associated from different sources would shed light on this question as it has
with virulence (Glickman et al., 2000; Perez et al., 2001; Walters been performed recently for M. bovis BCG (Brosch et al., 2007).
100 Cell Host & Microbe 3, 97–103, February 2008 ª2008 Elsevier Inc.
Cell Host & Microbe
A Point Mutation in the phoP Gene of H37Ra
Only 19 of 22 loci differed in H37Rv as compared to CDC1551 to speculate that the attenuation of this strain is caused by
and H37Ra. The far more frequent use of H37Rv over H37Ra hyperdormancy.
could have led to the accumulation of gene alterations specific Previously, a particular correlation of H37Ra with dormancy has
for laboratory conditions. The small number of bona fide poly- not been observed, since gene expression patterns have only
morphisms between H37Ra and H37Rv can be explained satis- been investigated in exponentially growing bacteria. Recently,
factorily by the long generation times of mycobacteria as the constitutive upregulation of genes belonging to the dormancy
compared to other bacteria and is consistent with the relatively regulon has been found in the Beijing lineage of M. tuberculosis
few differences between H37Rv, the recent clinical isolate during the exponential growth phase (Reed et al., 2007). It was
CDC1551 (Fleischmann et al., 2002), and M. bovis (Garnier speculated that this induction confers a growth advantage to Bei-
et al., 2003). We consider the mutation in PhoP most critical for jing strains since they could be preadapted to the conditions inside
the phenotypic differences. Four pieces of evidence support macrophages during infection and that this might underlie the in-
this. (1) Of the three mutated genes that are different between creased virulence of this mycobacterial subfamily. The interplay
all virulent strains and H37Ra, PhoP is the only protein known between dormancy and virulence of mycobacteria is not yet re-
to be involved in virulence. (2) The mutation occurs in a region solved. Other avirulent strains such as M. smegmatis exhibit dor-
that interferes with the biological function of this transcription mancy-like phenotypes, and many virulence factors do not belong
factor as determined by gel shift assays. (3) We revealed a signif- to the dormancy regulon (Mayuri et al., 2002; Smith, 2003). In ad-
icant overlap between genes with altered expression levels be- dition, the targeted deletion of key dormancy genes, including
tween H37Ra and the phoP KO mutant of H37Rv. (4) Comple- dosR and Rv2031c, results in hypervirulent phenotypes (Hu
mentation of H37Ra with the phoP gene of H37Rv reverted the et al., 2006). If the higher expression of dormancy genes is the
colony shape phenotype and was more virulent than the original key to virulence as postulated, the deletion of these genes should
H37Ra in a mouse macrophage infection model. have generated hypovirulent mycobacteria. Therefore, it remains
However, we do not formally rule out the possibility that addi- unresolved why the constitutive overexpression of dosR in the Bei-
tional mutations observed here or mutations in gaps or regions of jing lineage should be associated with its hypervirulence. As a ca-
low sequencing quality, which were not pursued, contributed to veat it should be mentioned that deleting the dosR gene in a guinea
phenotypic differences between H37Rv and H37Ra. Several gap pig model (Malhotra et al., 2004) led to attenuation. Further studies
regions span PE and PPE genes, which have been implicated in vir- will be necessary to clarify the meaning of dormancy for virulence
ulence although mechanistic insight is lacking (Gordon et al., 1999). and persistence of mycobacteria.
The crucial role of PhoP in M. tuberculosis virulence has been The 1-year sample of our time course study exhibited a differ-
well characterized in models of mycobacterial infections (Martin ent pattern of gene expression: almost all genes of the cluster
et al., 2006; Perez et al., 2001; Walters et al., 2006). Our data re- between Rv2660c and Rv2672, probably comprising four differ-
vealed a highly significant overlap of genes repressed in H37Ra ent operons, were strongly induced in H37Ra. The Rv2660c
and the phoP KO mutant of H37Rv. Interestingly, this correlation operon was also highly induced under starvation conditions in
was not observed for genes that are more actively transcribed in H37Rv (Betts et al., 2002). Thus—as under dormancy-like condi-
the phoP KO strain and H37Ra. The most likely reason for these tions—it needs to be determined whether the stronger expres-
differences is that PhoP is completely absent in the experiments sion of some genes involved in starvation are related to the
by Walters et al. (2006), whereas the H37Ra strain still comprises loss of virulence in H37Ra.
mutated PhoP. Since the mutation is located in the DNA-binding The complementation of H37Ra with phoP from H37Rv re-
domain, one would expect that activating and repressing functions verted the colony morphology and increased bacterial persis-
of a transcription factor are compromised to the same degree, tence within macrophages. Nevertheless, the complemented
which is apparently not the case. One hypothesis suggests that bacteria were still less virulent than H37Rv. It is conceivable
PhoP interacts with hitherto unknown additional proteins that act that other mutations in the genome of H37Ra might affect the
together as transcriptional repressors. A detailed characterization bacterial persistence in macrophages. Alternatively, the reduced
of the PhoP protein and its interaction partners will be necessary to virulence of the complemented strain could be caused by extra-
elucidate this issue. We have not determined whether PhoP is a di- chromosomal complementation of phoP, which does not mirror
rect regulator of the differentially expressed genes, and therefore, all aspects of its naturally occurring gene regulation, because
secondary effects should be considered as well: wild-type PhoP PhoP is autoregulatory (Gupta et al., 2006).
could induce the expression of transcriptional repressors; accord- Our combined approach demonstrates the feasibility of iden-
ingly, in the phoP KO mutant the relief of this repression would tifying minute but distinct differences between isogenic strains
cause indirect gene activation. Chromatin immunoprecipitation with respect to their genomes and transcriptomes. Perhaps
experiments could help to identify direct target genes of PhoP. more importantly, our experiments revealed paramount conse-
We also observed a significant similarity between gene ex- quences of single point mutations on the survival stratagem of
pression patterns of H37Ra and genes upregulated under dor- M. tuberculosis from which we conclude that the mutation in
mancy-like conditions at the 2-week time point. According to the phoP gene is of particular interest and at least partially
the work by Wayne and Hayes (1996) and Voskuil et al. (2003, responsible for the decreased virulence of H37Ra.
2004), mycobacteria have reached a state of nonreplicating per-
sistence in this stationary culture phase, which is characterized
EXPERIMENTAL PROCEDURES
by the upregulation of the dormancy regulon. Since this was ac-
companied by a transcriptional induction of dosR, the essential A detailed description of all procedures and protocols is available as Supple-
transcription factor mediating dormancy in H37Ra, it is tempting mental Data.
Cell Host & Microbe 3, 97–103, February 2008 ª2008 Elsevier Inc. 101
Cell Host & Microbe
A Point Mutation in the phoP Gene of H37Ra
102 Cell Host & Microbe 3, 97–103, February 2008 ª2008 Elsevier Inc.
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A Point Mutation in the phoP Gene of H37Ra
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