Department of Immunology and Microbiology,
Wayne State University School of Medicine, Detroit,
Michigan,1 and the Public Health
Research Institute, New York, New York2
Reporter gene fusions were used to investigate the contributions of
PrfA DNA binding sites to Listeria monocytogenes virulence gene expression. Our results suggest that the DNA sequence of PrfA
binding sites determines the levels of expression of certain virulence
genes, such as hly and mpl. Other virulence
genes, such as actA and plcB, may depend upon
additional factors for full regulation of gene expression.
 |
TEXT |
Listeria monocytogenes is
a ubiquitous, gram-positive bacterial pathogen that can cause
relatively rare but serious infections in immunocompromised
individuals and pregnant women (11, 29). The bacterium
is a facultative intracellular parasite that invades a wide variety of
cell types and is capable of escaping from membrane-bound phagosomes or
endosomes to replicate within the host cell cytosol (24,
35). Subsequent to bacterial replication, L. monocytogenes can spread directly into adjacent cells by a
motility mechanism based on host cell actin polymerization (5, 8,
17, 24, 28, 34, 35).
Several gene products important for L. monocytogenes
intracellular growth and/or cell-to-cell spread have been identified. hly encodes listeriolysin O, a pore-forming hemolysin
required for efficient escape of the bacterium from host cell vacuoles; plcA encodes a phosphatidylinositol-specific phospholipase C
(PI-PLC) that aids in vacuolar escape; plcB encodes a
broad-specificity phospholipase C (PC-PLC) that is important for
cell-to-cell spread; mpl encodes a metalloprotease that
processes proPC-PLC to its active form; and actA encodes a
membrane protein essential for bacterial actin-based motility (reviewed
in references 4, 13, 25, 32, and
33). The expression of each of these genes is regulated by a transcriptional activator protein known as PrfA (21, 22). The prfA gene product is a key
regulatory component of L. monocytogenes pathogenesis;
bacterial strains that lack functional PrfA protein are avirulent in
mouse models of infection (9, 21, 22, 30).
PrfA is a 27-kDa protein with significant homology to members of the
cyclic AMP (cAMP) receptor protein (CRP)-FNR family of pleiotropic
transcription regulators (19, 20, 32). It recognizes and
binds to a 14-bp DNA palindromic sequence that is present in the 
40
region of target gene promoters (1, 6, 9, 31). Certain
PrfA-regulated promoters contain PrfA DNA binding sites that are
imperfect palindromes, and it has been postulated that PrfA binds with
a lower affinity to imperfect promoter palindromes (such as those
present upstream of mpl and actA) than to perfect ones (hly and plcA) (9). In support of
this hypothesis, it has been recently demonstrated, using in vitro DNA
footprinting studies, that PrfA binds to the
hly/plcA promoter with a higher affinity than to
the inlA promoter (6). Specific nucleotide substitutions within the PrfA binding site have also been shown to
abolish PrfA-dependent activation of gene expression (10).
Expression of PrfA-dependent gene products is differentially regulated.
Certain gene products, such as those encoded by hly or
plcA, are expressed at significant levels by bacteria grown in rich broth culture, whereas other PrfA-regulated gene products, such
as mpl and actA, are expressed at small to
undetectable amounts under standard broth culture conditions
(23). Upon entry of L. monocytogenes into the
environment of the host cell cytosol, hly expression
increases approximately 20-fold, whereas actA expression increases over 226-fold and ActA becomes one of the most abundant surface proteins expressed (2, 3, 23). A model has been proposed to describe the ability of PrfA to differentially regulate gene expression in an environment-dependent manner (9).
Outside of host cells or at the onset of infection, PrfA is present
within the bacterial cell at low to moderate levels and binds and
activates transcription from promoters with high-affinity PrfA binding
sites (such as hly and plcA). Upon entry into the
cytosol, PrfA protein synthesis increases to provide sufficient levels
of PrfA to occupy low-affinity promoter sites (such as those upstream
of mpl and actA). Recent evidence suggests that
PrfA may also require posttranslational modification or interaction
with additional factors for full activity, and a revised model
incorporating these observations has been proposed (36).
A central premise of the PrfA regulatory models is that the affinity of
PrfA for its target promoter sites determines the levels and timing of
gene expression. We wished to determine if the sequence variations
present in the PrfA binding sites of different target promoters were
sufficient to account for the levels of gene expression observed under
extracellular growth conditions.
Use of a B. subtilis-based expression system to monitor
PrfA-dependent gene expression.
Previous experiments using
B. subtilis-based expression systems have demonstrated that
PrfA protein is capable of activating transcription from target
promoters in the absence of any additional L. monocytogenes
factors (10, 30). Activation of expression from certain
promoters, namely actA and mpl, was considerably weaker and occurred more slowly than that observed for the
hly and plcA promoters, suggesting that greater
quantities of PrfA were required for productive interaction
(30). We used the Bacillus subtilis-based
expression system referred to above (10) to measure the
ability of PrfA to activate expression from target promoters containing
specific nucleotide substitutions in the PrfA binding sites (Fig.
1). The relevant B. subtilis
strains are shown in Table 1. Mutations
within the hly, mpl, and actA
promoters were generated by PCR (12) with the primers
described in Table 2, and the products
were cloned upstream of a promoterless copy of lacZ
(14). The mutant promoter-lacZ transcriptional
fusions were then transduced with SP
phage (15) into the
chromosome of B. subtilis strains that contained an
isopropyl--D-thiogalactopyranoside (IPTG)-inducible
chromosomal copy of prfA as previously described (10) (Fig. 1). PrfA-dependent activation of target
promoter-lacZ fusions was monitored by measuring
-galactosidase activity following induction of prfA
expression (10, 37).
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FIG. 1.
Strategy for measuring the ability of PrfA to activate
target promoters containing PrfA box nucleotide substitutions.
Expression of prfA in B. subtilis was placed
under the control of the IPTG-inducible promoter Pspac by
insertion of a DNA fragment containing the prfA coding
sequence into the SPAC expression cassette of pAG58-phleo
(38). The expression cassette was then integrated by
recombination at the site of a phenotypically silent Tn917
insertion (14). The activity of PrfA-dependent promoter
constructs was monitored by using lacZ transcriptional
fusions, which were integrated by recombination into SP
prophages and then transduced into a specific site within the
chromosome of B. subtilis strains containing
Pspac-prfA (39).
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TABLE 2.
Primers designed to amplify and introduce nucleotide
substitutions into PrfA target promoter PCR products
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Substitution of the hly promoter palindrome with the
mpl promoter palindrome reduces hly
expression.
Previous work demonstrated that hly-lacZ
fusions are efficiently expressed following induction of
prfA in B. subtilis, whereas mpl-lacZ
and actA-lacZ fusions are both poorly expressed in
comparison and require longer periods of prfA induction
(30). The mpl and actA promoter
palindromes each have 3 base substitutions in comparison to the
hly/plcA palindrome; to determine if these base
differences were sufficient to account for the lower levels of
transcriptional activation observed, specific nucleotide substitutions
were introduced into the hly PrfA DNA binding site as shown
in Fig. 2A, and their effect on
PrfA-dependent activation of expression was determined. A single base
substitution (T12
A), designed to make the hly
palindrome more closely resemble that of mpl, was sufficient
to lower expression levels of hly by 90% (Fig. 2B). Similar
reductions in hly expression were observed for the
hly(T7A, T8A) and
hly(T7A, T8A,
T12A) promoter substitutions (Fig. 2B). These data
indicate that the sequence composition of the PrfA DNA binding site is
a major factor in determining levels of hly expression.
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FIG. 2.
Effects of PrfA-box mutations on hly-lacZ
expression. (A) Nucleotide substitutions were introduced into the
hly promoter palindrome to increase its similarity to the
mpl promoter palindrome. Substituted nucleotides are
designated by arrows. (B) -Galactosidase activity was measured at
the indicated time intervals in cultures of B. subtilis
strains containing the wild-type hly promoter or
hly promoter substitution mutant-lacZ fusions and
Pspac-prfA in the presence (open symbols) or absence (solid
symbols) of IPTG. Units of -galactosidase are as described by
Youngman (37). Individual time points were done in
duplicate, and the data shown are representative of at least three
independent experiments.  and  , hly palindrome;  and  , hly(T12A);  and  ,
hly(T7A, T8A);  and  ,
hly(T7A, T8A,
T12A).
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Substitution of the mpl promoter palindrome with the
hly promoter palindrome increases mpl
expression.
Because a single base substitution within the
hly PrfA DNA binding site resulted in an hly
expression pattern comparable to that obtained for mpl
(compare Fig. 2B and 3B), we wanted to
determine if the converse situation would also prove true; would
specific nucleotide substitutions within the mpl palindrome,
designed to increase its similarity to the hly palindrome,
result in mpl expression levels that would be comparable to
those of hly? mpl-lacZ expression normally
exhibits an approximately threefold induction over background expression levels after 60 min of IPTG-mediated prfA
expression (Fig. 3B and C). The introduction of a single base change
(A12T) increased mpl-lacZ induction 7-fold,
and the mpl(A7T, A8T,
A12T) promoter substitution resulted in a 12-fold
induction of -galactosidase activity after 60 min (Fig. 3B and C).
In comparison, hly-lacZ exhibited a 36-fold induction over
background expression levels (Fig. 3B). Thus, nucleotide substitutions
designed to make the mpl palindrome more
"hly-like" resulted in significantly higher levels of
mpl expression. The substitution for the mpl
palindrome of the hly palindrome was, however, not
sufficient to raise levels of mpl expression to those
observed for hly, indicating that additional sequence
outside of the 14-bp palindrome may also contribute to optimize
activation.
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FIG. 3.
Effects of PrfA box mutations on mpl-lacZ
expression. (A) Nucleotide substitutions were introduced into the
mpl promoter palindrome to increase its similarity to the
hly promoter palindrome. Substituted nucleotides are
designated by arrows. (B) -Galactosidase activity was measured at
the indicated time intervals in cultures of B. subtilis
strains containing the wild-type mpl promoter or
mpl promoter substitution mutant-lacZ fusions and
Pspac-prfA in the presence (open symbols) or absence (solid
symbols) of IPTG. Individual time points were examined in duplicate,
and the data shown are representative of at least three independent
experiments. (C) Expanded view of the induction of -galactosidase
activity observed for the mpl promoter substitution mutants.
and , hly palindrome; and , mpl
palindrome; and , mpl(A12T); and
, mpl(A7T, A8T,
A12T).
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Substitution of the actA promoter palindrome with the
hly palindrome produces no effect on actA
expression.
The actA promoter palindrome, like
mpl, has several nucleotide substitutions within the PrfA
binding site that distinguish it from the hly palindrome. To
determine if actA expression levels could be increased by
nucleotide substitutions within the promoter palindrome that were
designed to make it more hly-like, two actA promoter-lacZ fusion constructs were generated
[actA(G14A) and actA(A7T, A8T,
G14A)] (Fig. 4A).
However, and in contrast to what was observed for mpl, the
introduction of a G14A change or the triple base change
(A7T, A8T, G14A) into the
actA promoter palindrome resulted in no increase in
actA expression levels (Fig. 4B). These results indicated
that altering the nucleotide composition of the actA
promoter palindrome was not sufficient to increase PrfA-directed
activation of expression.
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FIG. 4.
Effects of PrfA-box mutations on actA-lacZ
expression. (A) Nucleotide substitutions were introduced into the
actA promoter palindrome to increase its similarity to the
hly promoter palindrome. Substituted nucleotides are
designated by arrows. (B) -Galactosidase activity was measured at
the indicated time intervals in cultures of B. subtilis
strains containing the wild-type actA promoter or the
actA promoter substitution mutant-lacZ fusions
and Pspac-prfA in the presence (open symbols) or absence
(solid symbols) of IPTG. Individual time points were examined in
duplicate, and the data shown are representative of at least three
independent experiments. and , hly palindrome; and , actA palindrome; and ,
actA(G14A); and ,
actA(A7T, A8T,
G14A).
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Domann et al. (7) reported the presence of a putative
transcriptional terminator downstream of mpl in the promoter
region of actA. The 5' end of the 64-bp stem-loop structure
identified by Domann et al. (7) overlaps the site of
actA transcript initiation, and it seemed plausible that the
presence of such a stem-loop structure might influence or inhibit
PrfA-directed activation of actA expression. An
actA promoter-lacZ reporter gene construct was
generated that contained the hly promoter palindrome and a deletion of the majority of the actA putative stem-loop
structure, but retained the actA transcript initiation site
(from +3 relative to the start site of transcription to +51
a deletion
of 48 bp; the GTG translational start codon of actA is at
position +150). This promoter fusion was introduced into the B. subtilis expression system, and -galactosidase activity was
measured as a function of PrfA-directed activation of actA
expression. Deletion of the potential stem-loop structure did not
result in increased actA expression (J. R. Williams and
N. E. Freitag, unpublished data).
In summary, the experiments described in this study indicate that
although the sequence composition of the PrfA DNA binding site is a
major determinant of promoter activity in certain promoters, such as
hly and mpl, other promoters, such as
actA, may require additional factors or activation events
for optimal expression. Our study did not assess the binding affinity
of PrfA for different target promoters or the efficiency of
transcriptional activation. Instead, we demonstrated that the
substitution of the PrfA binding site from a highly expressed promoter
(hly) for that of a poorly expressed promoter binding site
(mpl or actA) was sufficient in the case of the
mpl promoter to increase expression, but had no effect on
the actA expression levels. DNA footprinting studies have
identified an approximately 26-bp region, beginning 10 bp upstream of
the 14-bp palindrome and ending 2 bp downstream, that is protected from
DNase I digestion in the presence of PrfA protein (6). Based
on these studies, it is likely that additional promoter sequences
flanking the 14-bp palindrome contribute to PrfA binding and
recognition. Nevertheless, it was surprising to see absolutely no
affect on PrfA-dependent activation of actA expression
following substitution of the 14-bp actA palindrome for that
of hly. Our results suggest that there may exist additional
events besides the binding of PrfA to the actA promoter that
are involved in the regulation of actA expression.
The use of the B. subtilis assay system allows measurement
of PrfA-dependent promoter activation in the absence of any additional L. monocytogenes factors. PrfA shares significant homology
with the CRP-FNR family of prokaryotic transcription factors (19, 20, 32), and recent experimental evidence suggests that PrfA, like CRP, requires the presence of a cofactor for full activity (26, 27, 36). It has been demonstrated that a number of L. monocytogenes strains that constitutively overexpress
PrfA-regulated gene products contain a mutant prfA allele
(prfA*) with a single amino acid substitution (Gly145Ser)
(27). These prfA* mutants resemble
crp* mutants which contain an analogous mutation in a region
of PrfA-CRP homology and which are active in the absence of the
cofactor cAMP (16, 18). Preliminary experiments suggest that
cAMP does not influence PrfA activity (36; L. M. Shetron-Rama and N. E. Freitag, unpublished data). Vega et al.
(36) have demonstrated that PrfA* has an increased binding
affinity for target DNA. It is possible that the activated form of PrfA
is required for full expression of certain target promoters and that one of these promoters is that of actA. If so, this would
suggest that PrfA activation does not occur in the B. subtilis system, and it accentuates the difference that exists
between hly, mpl, and actA expression.
The results presented in this study indicate that sequence variations
within the PrfA DNA binding sites of L. monocytogenes virulence gene promoters can produce a wide range of effects on gene
expression. It is necessary, however, to confirm that the behavior
observed for promoter fusions in the heterologous B. subtilis expression system reflects what is actually occurring in
L. monocytogenes. Preliminary experiments indicate that the behavior observed for substitutions within the actA promoter
in B. subtilis closely corresponds to what is observed when
the same substitutions are introduced into L. monocytogenes
(L. M. Shetran-Rama and N. E. Freitag, unpublished data). We
are in the process of using both heterologous systems and defined
mutations within L. monocytogenes to better define the
activation steps and cofactors required for optimal expression of
PrfA-dependent virulence genes.
We thank Kathleen Jacobs for technical assistance in the
construction of plasmids for this study. We thank David Dubnau for very
helpful discussions and the reviewers of the manuscript for insightful
criticism and valuable suggestions.
This work was supported by Public Health Service grant AI41816 from the
National Institutes of Health. N.E.F. thanks the Public Health Research
Institute for initial support and encouragement.
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