Journal of Bacteriology, November 1999, p. 6840-6843, Vol. 181, No. 21
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Identification and Disruption of lisRK,
a Genetic Locus Encoding a Two-Component Signal Transduction System
Involved in Stress Tolerance and Virulence in Listeria
monocytogenes
Paul D.
Cotter,1
Nathan
Emerson,1
Cormac G. M.
Gahan,1,2 and
Colin
Hill1,2,*
Department of
Microbiology1 and National Food
Biotechnology Centre,2 University College Cork,
Cork, Ireland
Received 12 July 1999/Accepted 25 August 1999
 |
ABSTRACT |
lisRK encodes a two-component regulatory system in the
food pathogen Listeria monocytogenes LO28. Following
identification of the operon in an acid-tolerant Tn917
mutant, a deletion in the histidine kinase component was shown to
result in a growth phase variation in acid tolerance, an ability to
grow in high ethanol concentrations, and a significant reduction in virulence.
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TEXT |
The success of Listeria
monocytogenes as a pathogen owes much to its ability to sense and
respond to its environment. A number of studies have demonstrated that
the ability of L. monocytogenes to respond to stress has
consequences in terms of the virulence of the organism (18, 20,
22). In many other bacteria, the link between variations in
environmental factors and enhanced or reduced virulence has been shown
to result from the sensing and regulatory activities of two-component
signal transduction systems (8, 11). A typical two-component
system consists of a membrane-associated histidine kinase, which
monitors a specific environmental parameter, and a cytoplasmic response
regulator, which enables the cell to respond, often via regulation of
gene expression, when this parameter varies (21, 27). In
this study our aim was to identify mutants of L. monocytogenes with altered abilities to respond to stress and to
investigate whether such alterations influence virulence. We describe
the identification of a two-component signal transduction system and
demonstrate that it functions in stress response and plays a role in in
vivo survival. This represents the only description, other than that of
the cheA-cheY system (7), of genes encoding a
two-component regulatory system in L. monocytogenes.
Isolation and characterization of a mutant of L. monocytogenes with enhanced stationary-phase acid tolerance.
An L. monocytogenes LO28 (serotype 1/2c) Tn917
bank was created by using the temperature-sensitive plasmid pTV1-OK
(12). Isolated transformants were grown overnight in tryptic
soy broth-yeast extract (TSB-YE) containing kanamycin (50 µg/ml) at
30°C, subcultured (1%) into TSB-YE containing erythromycin (0.04 µg/ml) at 42°C, and selected for kanamycin-sensitive
Tn917 integrants on tryptic soy agar-yeast extract
containing erythromycin (10 µg/ml). Overnight cultures of the
transposon bank were exposed to TSB-YE (adjusted to pH 4) for 36 h. While no survivors were recovered from the LO28 control, some 140 pinpoint and 3 regularly sized colonies were recovered from the bank.
Southern hybridization showed that Tn917 had integrated only
once and in different locations in each of the three isolates which had
given rise to the regular colonies (data not shown). Inverse PCR was
used to isolate chromosomal DNA from one isolate, LO28-M9; in this
method HpaI was used to generate upstream flanking DNA, and
XbaI and HindIII were used to generate
downstream DNA. The inverse-PCR products were cloned into pBluescript.
In total, 3,619 bp flanking the point of insertion was sequenced from
both strands. Analysis showed that this region contained two partial
and two complete open reading frames (ORFs) (Fig. 1A). The ORF into which the transposon
had inserted demonstrates a high degree of similarity to regulator
components of many bacterial two-component systems. The predicted
amino-terminal receiver domain of this ORF includes the invariant
aspartate and lysine residues conserved in this family of regulatory
proteins (Fig. 1B). Alignments indicated greatest similarity in both
the receiver and output domains to the group A Streptococcus
regulator of hyaluronic acid capsule synthesis CsrR (17),
the uncharacterized Bacillus subtilis putative regulator
YkoG (GenBank accession no. AJ002571), and a Synechocystis
sp. regulator (accession no. D64002), all of which are members of the
OmpR-PhoB subfamily of response regulators. The carboxy termini of
members of this subfamily are thought to have DNA-binding roles, and
several have been shown to target specific sites upstream from the
promoters that they regulate. It is thus probable that the putative
protein is also a transcriptional activator. The listerial regulator
gene was subsequently assigned the designation lisR. The
start codon for this gene can be predicted from the high degree of
similarity exhibited at the N termini of response regulators. A
putative ribosome-binding site (5'-AGAGG-3') was identified 9 bp
upstream from this location.
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FIG. 1.
Graphic illustration of the lisRK locus. (A)
Line drawing of the orientation of ORFs flanking the Tn917
insertion site. The striped regions within lisK indicate the
potential membrane-spanning regions, while the lollipops indicate
putative rho-independent terminators. The 498-bp region deleted in the
mutant LO28 lisK is also indicated. (B) Alignment of the
predicted receiver domains of both lisR and csrR
and the consensus sequence motif for the receiver element of response
regulators (27). (C) Alignment of the predicted transmitter
domain of both lisR and csrR and the consensus
sequence motif for the transmitter element of histidine kinase sensor
proteins (27). aa, amino acids.
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|
Immediately downstream from lisR, an ORF encoding a
histidine kinase was identified. The carboxy terminus of the predicted histidine kinase, designated LisK, most resembles LlkinA from Lactococcus lactis (19), CsrS (the partner of
CsrR) (17), and YkoH (the partner of YkoG)
all of which
have transmitters typical of the EnvZ-NarX family of histidine kinases.
An alignment (Fig. 1C) also demonstrates the presence of the H, N, G1,
and G2 boxes conserved among transmitter domains. In contrast to the carboxy termini, which are usually cytoplasmic in location, the amino
termini of histidine kinases are quite often membrane bound, containing
at least two membrane-spanning regions with a periplasmic loop. The
membrane-spanning regions, which can be identified as stretches of
hydrophobic amino acids, are also present in LisK (data not shown). It
is not surprising that a procedure designed to identify genes involved
in sensing and responding to acid stress should have identified a
two-component regulatory system, the most-characterized mechanism by
which environmental signals can be sensed in bacteria. There are
numerous examples of two-component systems which regulate virulence
factor production in response to specific environmental signals
(8, 11).
The amino acid sequence deduced from the incomplete upstream
orfA showed high identity with the Escherichia
coli enzyme 6-phosphogluconate dehydrogenase, while the downstream
orfD most closely resembled a variety of inner membrane
proteins found in E. coli. However, due to the presence of
putative rho-independent terminators (hairpin loops) both upstream of
lisR and immediately downstream of lisK, it
appears that lisRK constitutes a discrete transcriptional unit.
Creation and analysis of a lisK deletion mutant.
A
mutant, with a nonpolar 498-bp deletion in lisK from
nucleotide 1872 to 2369, was created to confirm that the
Tn917 insertion event was responsible for the enhanced
survival at low pHH displayed by LO28-M9. The SOE (splicing by overlap
extension) PCR procedure (13) was used to splice two 348-bp
PCR products from either side of the sequence to be deleted. This
hybrid was subsequently cloned into the temperature-sensitive shuttle
vector pKSV7 (25) and transformed into LO28. An allelic
exchange between the SOE product on pKSV7 and the intact gene resulted
in the removal of a 498-bp sequence encoding one of the hydrophobic
regions and the conserved histidine of the histidine kinase (Fig. 1A).
The mutation was confirmed by PCR analysis, and the mutant was
subsequently designated LO28lisK.
Carbohydrate utilization (as assayed by API-CH50), listeriolysin O
production on blood agar plates, and phospholipase production on egg
yolk emulsion plates (all at 37°C) were found to be unaffected in
LO28lisK (data not shown). As LO28-M9 was isolated as an
acid-tolerant mutant, the acid resistance of the deletion mutant was
tested. The growth phase acid sensitivities of LO28lisK
and the parent strain were tested by inoculating (2%) overnight
cultures into TSB-YE, removing aliquots at regular intervals, and
inoculating them into TSB-YE adjusted to pH 3.5 with 3 M lactic acid.
Survivors were counted after 45- and 140-min periods. With the longer
acid exposure time, a significant difference (0.01 < P < 0.02) was observed, in that the mutant was more resistant to
acid than the parent during stationary phase (Fig.
2A), thus confirming the M9 phenotype.
This mutant was used for further analysis. No difference could be
observed at lower cell densities because this treatment regimen reduced
bacterial numbers to below detectable levels. However, when the time of
exposure to low pH was reduced to 45 min, the mutant was shown to be
significantly more sensitive (0.01 < P < 0.02)
than the parent strain during early logarithmic growth (Fig. 2A). No
difference between stationary-phase cells could be observed due to the
reduced exposure to acidified media. These results indicate that the
relative resistance of the mutant is growth phase dependent. These
results also matched those obtained with the original LO28-M9 mutant,
confirming that transposon insertion in lisR was responsible
for the mutant's selection in acidified broth (data not shown). It is
tempting to speculate that a signal which would normally stimulate
LisRK to induce acid tolerance is missed during the early stages of
growth, leading to an extremely sensitive cell. However, this supposes
that a separate induction event which overcompensates for the lack of
LisRK by inducing even greater tolerance subsequently occurs. In
response to the altered acid resistance profile of
LO28lisK, the effects of several other stresses were
examined. Overnight cultures were inoculated (2%) into TSB adjusted to
pH 5.75 with lactic acid, with 7% NaCl, or with 5% ethanol. Growth
was determined with a Spectra max 340 spectrophotometer (Molecular
Devices) over a 24-h period. The mutant proved able to grow in the
presence of 5% ethanol, a concentration which is bacteriostatic for
the parent strain (Fig. 2B). This result was representative of a trend
that was also apparent for a number of other ethanol concentrations
(data not shown). Ethanol stress, and alcohol stress in general, is
thought to be exerted by a reduction of cytoplasmic membrane integrity
(15, 23). An increase in unsaturated-fatty-acid content has
been shown to aid growth and survival when the organisms are exposed to
ethanol (14). It may be at the membrane that the
two-component system ultimately exerts an influence, as it has been
shown that the composition of the cell membrane reflects the relative
acid tolerance of a bacterial strain (2, 4, 5).
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FIG. 2.
(A) Percent survival of cultures of LO28 and
LO28lisK in TSB-YE (pH 3.5) after 45 min ( and  ,
respectively) and 120 min ( and  , respectively), at different
points during growth ( and  , respectively). Points below the
dotted line indicate that there were no survivors. (B) Growth of LO28
() and LO28lisK () in TSB-YE supplemented with 5%
ethanol at 37°C. Error bars represent the standard deviations of
triplicate experiments. OD600, optical density at 600 nm.
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|
However, relative growth rates were not affected under other stress
conditions, such as media adjusted to various pHs or containing various
concentrations of salt (data not shown). These results were similar to
those seen in LO28-M9. As it is likely that the Tn917
insertion in LO28-M9 resulted in a polar mutation in lisK, it is possible to attribute the phenotypic changes common in both mutants to lisK mutations.
LO28lisK is affected in virulence compared with
LO28.
A mouse model was used to measure any possible impact of the
lisK mutation on virulence. The number of bacteria in the
spleen was recorded, as this reflects the progress of the infection
(16). The data show that, regardless of whether a low
(1 × 105-CFU) or high (2 × 106-CFU)
dose is used, the number of LO28lisK cells in the spleen 1 day postinfection is much lower than that for wild-type-infected mice
(Fig. 3). This represents a significant
attenuation of the virulence of the bacteria. It is necessary to
understand the consequence of intraperitoneal inoculation to appreciate
how alterations in stress-induced responses can alter the virulence of
Listeria. Initially, the host immune system responds via the
arrival of infiltrating polymorphonuclear neutrophils and subsequently
of macrophages (9). Following phagocytosis,
Listeria produces hemolysin and phosphatidyl inositol
phospholipase C to enable its escape from the phagosome (1, 3,
10). It seems to be at this stage of escape that the sensing of
and response to stresses in vivo are most important: firstly, to adapt
to the presence of toxic radicals, acidification, and degradative
enzymes (24) and, secondly, to activate the production of
virulence factors, many of which have been shown to be regulated by
environmental conditions (6, 26). Thus, there is increasing
evidence that stress responses, possibly through a number of pathways,
can regulate the virulence of L. monocytogenes.
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FIG. 3.
Growth of LO28 () and LO28lisK () in
the spleens of BALB/c mice. Mice were inoculated with 1 × 105 (A) and 2 × 106 (B)
Listeria cells by the intraperitoneal route. The arrowheads
indicate the numbers inoculated into the peritoneal cavity on day 0. The cross indicates that one mouse died at that time point. Each datum
point represents the mean log10 number of
Listeria cells per spleen for four mice. Error bars
represent the standard deviations of triplicate experiments. (A)
Differences were significant on days 1 and 3 (0.1 < P < 0.5). (B) Differences were significant on days 1, 3, and 4 (0.01 < P < 0.02). In all cases, significance
was calculated by using the Student t test.
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|
It is thus significant that genes encoding LisRK, initially identified
as having a role in stress response, are also important in the
virulence of this pathogen. These results contribute to the increasing
evidence of the importance of functional stress-responsive mechanisms
for a successful infection.
Nucleotide sequence accession number.
The sequence of the
3,619-bp DNA determined in this study has been deposited in GenBank
under accession no. AF139908.
| |
ACKNOWLEDGMENTS |
We thank Kathryn J. Boor for providing plasmid pKSV7 and advice on
the SOE PCR technique.
This work has been funded by grant aid under the Food Sub-Programme
administered by the Department of Agriculture, Food and Forestry and is
supported by national and EU funds.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, University College Cork, Cork, Ireland. Phone:
353-21-902397. Fax: 353-21-903101. E-mail:
c.hill{at}ucc.ie.
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Journal of Bacteriology, November 1999, p. 6840-6843, Vol. 181, No. 21
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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