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Infection and Immunity, October 2005, p. 7027-7031, Vol. 73, No. 10
0019-9567/05/$08.00+0 doi:10.1128/IAI.73.10.7027-7031.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Cloning Vectors and Fluorescent Proteins Can Significantly Inhibit Salmonella enterica Virulence in Both Epithelial Cells and Macrophages: Implications for Bacterial Pathogenesis Studies
Leigh A. Knodler,1
Aaron Bestor,1
Caixia Ma,2
Imke Hansen-Wester,3
Michael Hensel,3
Bruce A. Vallance,2 and
Olivia Steele-Mortimer1*
Laboratory of Intracellular Parasites, NIAID, NIH, Rocky Mountain Laboratories, Hamilton, Montana,1
Division of Gastroenterology, British Columbia's Children's Hospital and the University of British Columbia, Vancouver, British Columbia, Canada,2
Institut für Klinische Mikrobiologie, Immunologie und Hygiene, Universität Erlangen-Nuernberg, Erlangen, Germany3
Received 31 March 2005/
Returned for modification 31 May 2005/
Accepted 13 June 2005

ABSTRACT
Plasmid vectors and fluorescent protein reporter systems are
commonly used in the study of bacterial pathogenesis. Here we
show that they can impair the ability of
Salmonella enterica serovar Typhimurium to productively infect either cultured mammalian
cells or mice. This has significant implications for studies
that rely on these systems.

TEXT
The facultative intracellular pathogen
Salmonella enterica causes
gastroenteritis and systemic infections. Pathogenesis is dependent
on the pathogen's ability to survive and/or replicate within
host cells and is mediated by two type III secretion systems,
encoded on
Salmonella pathogenicity islands 1 and 2 (SPI1 and
-2, respectively), which translocate bacterial effector proteins
into host cells (
11). Characterization of the function and regulation
of these virulence factors is essential to our understanding
of
Salmonella pathogenesis and has been the subject of intensive
study. Many of these studies rely on the use of plasmid vectors
for complementation analysis of protein function or for monitoring
gene expression. In particular, plasmid-borne genes encoding
green fluorescent protein (GFP) or related molecules have been
used as reporters for gene expression (
6,
9,
21,
27,
28) or
to localize bacteria inside host cells (
7,
13,
17,
18,
32).
However, there is an inherent fitness cost associated with maintaining
plasmids or high levels of fluorescent proteins, and even plasmids
that do not appear to have a metabolic cost under normal laboratory
growth conditions may significantly reduce the ability of a
bacterial pathogen to adapt to the stress of intracellular life
(
1,
4).
Salmonella can be internalized into host cells by several different mechanisms (Table 1). Active invasion of nonphagocytic and phagocytic cells occurs via a "trigger"-type process that involves extensive actin rearrangements and plasma membrane ruffles and is mediated by the SPI1-encoded type III secretion system (12). Non-SPI1-induced Salmonella is unable to invade nonphagocytic cells but is internalized, albeit relatively inefficiently, into phagocytic cells (26). Opsonization of Salmonella with complement or specific antibodies considerably enhances the efficiency of this phagocytic uptake (Table 1).
We hypothesized that the presence of plasmid vectors or the
production of fluorescent proteins could affect the ability
of
Salmonella to establish an intracellular niche and that this
might depend on the mechanism of entry. To investigate this
possibility, we examined the effect of several plasmids on the
ability of
Salmonella to establish successful interactions with
host cells. Five plasmids were selected for comparison using
three selection criteria: (i) previous use in complementation
studies of
Salmonella, (ii) low to medium copy number, and (iii)
the presence of different selectable markers (Table
2). The
plasmids were electroporated into
S. enterica serovar Typhimurium
SL1344 (
16) and maintained by the presence of antibiotics. These
plasmids had no detectable effect on the growth of serovar Typhimurium
in Luria-Bertani-Miller (LB-Miller) broth or on LB plates (data
not shown). We compared the effects of these vectors on invasion
and intracellular survival/replication in HeLa and RAW 264.7
cells, which have been widely used to study
Salmonella-host
cell interactions. The nonphagocytic epithelial-cell-like HeLa
cells (ATCC CCL2) are efficiently invaded by SPI1-induced serovar
Typhimurium (
25). HeLa cells grown in 24-well plates (5
x 10
4 cells/well) were infected with a high multiplicity of infection
(MOI) (

50 to 100 CFU/cell) for a short time (10 min), after
which extracellular bacteria were removed by washing the cells
in Hanks balanced salt solution. After a short chase (10 min)
in growth media at 37°C, the remaining extracellular bacteria
were killed by the addition of gentamicin sulfate (100 µg/ml
for 1 h and then reduced to 10 µg/ml). Intracellular bacteria
were enumerated by solubilizing the cells in lysis buffer (1.0%
Triton X-100, 0.1% sodium dodecyl sulfate in phosphate-buffered
saline [PBS]) and plating on LB agar plates. Only one plasmid,
pACYC184, significantly decreased the invasion efficiency of
serovar Typhimurium under these conditions (Fig.
1). This plasmid
also reproducibly reduced intracellular replication in HeLa
cells, although without statistical significance (Fig.
1B).
Similar results were obtained when phagocytic macrophage-like
cells were infected with SPI1-induced serovar Typhimurium. Invasion
was carried out as described above, except that RAW 264.7 cells
(ATCC TIB-71) were seeded in six-well tissue culture plates
(1
x 10
6 cells/well), the MOI was

5 to 10 CFU/cell, and monolayers
were lysed for bacterial enumeration at 1 h postinfection (p.i.)
and 15 h p.i. The pACYC184-associated invasion defect was somewhat
enhanced in RAW 264.7 cells compared to HeLa cells (Fig.
1A).
Replication was also considerably reduced by the presence of
pACYC184 to approximately 30% of that seen for the wild-type
infections. We also observed decreased replication in RAW 264.7
cells of bacteria containing two other plasmids, pBR322 and
pWSK29 (Fig.
1B), although these plasmids had no detectable
effect on bacterial fitness in HeLa cells.
We next investigated whether these plasmids could affect the phagocytic uptake and/or subsequent intracellular replication of noninvasive bacteria (i.e., not SPI1 induced). Salmonella was inoculated into 10-ml LB-Miller broth with appropriate antibiotics for 16 to 18 h (stationary phase) and then opsonized by incubation in 14% normal human serum for 30 min or left untreated. Internalization was initiated (MOI of
10 to 20 CFU/cell) by centrifugation at 1,000 x g for 10 min at 25°C. After incubation at 37°C for 15 min, the remaining extracellular bacteria were killed by the addition of gentamicin sulfate. In contrast to what occurred with SPI1-mediated invasion, no plasmid had a detrimental effect on the ability of complement-opsonized bacteria to enter cells (Fig. 2A). However, entry of nonopsonized bacteria was compromised by pBR322 and pWSK29. As for SPI1-induced bacteria, pACYC184 reduced intracellular replication, although this was statistically significant only for nonopsonized bacteria (Fig. 2B).
GFP and its variants have been used as reporters of intracellular
bacterial gene expression with some success (
27-
30), although
a recent study proposed that GFP is costly for gastrointestinal
bacteria and could affect the ability of
Salmonella to interact
with host cells (
22). Other fluorescent reporter proteins are
likely to cause similar problems (
24). We compared the effects
of GFP (pFPV25.1) and DsRed (pRFP) on the ability of
Salmonella to invade, and survive within, host cells. These plasmids comprise
the same vector backbone, pFPV25, which has a promoterless
gfp gene and was developed for gene expression analysis with
Salmonella (
27) (Table
2). In pFPV25.1, the promoter region of
rpsM is
added upstream of
gfp, resulting in the constitutive synthesis
of GFP. In pRFP, the DsRed gene simply replaces the
gfp gene
(Table
2). To construct pRFP, primers RFP-SD-For-XbaI (5'-GATT
TCTAGATTTAAGAAGGAGATATACATATGAGGTCTTCCAAGAATG-3')
and RFP-Rev-SphI (5'-ACAT
GCATGCCTAAAGGAACAGATGGTGG-3') were
used to amplify the DsRed gene of vector pDsRed (Clontech) with
the Expand High Fidelity system (Roche). The forward primer
contained a ribosome-binding site identical to that of pFPV25.1
(
28). The product was digested by XbaI and SphI (restriction
sites are underlined) and cloned in XbaI/SphI-digested plasmid
pFPV25.1, thereby replacing the
gfp-mut3 gene. The construct
was confirmed by DNA sequencing. Neither pFPV25.1 nor pRFP had
any apparent effect on bacterial growth in LB-Miller broth (not
shown).
HeLa or RAW 264.7 cells were infected with SPI1-induced bacteria bearing either pFPV25 or one of the fluorescent-protein-producing pFPV25 derivatives, pFPV25.1 or pRFP. We found that the presence of either GFP or red fluorescent protein (RFP) significantly decreased bacterial invasion in both cell lines (Fig. 3). Furthermore, RFP- but not GFP-producing bacteria displayed a severe replication defect in HeLa cells (Fig. 3B). In contrast, fluorescent protein production did not appear to affect entry or replication when non-SPI1-induced bacteria were internalized into RAW 264.7 cells via phagocytosis (data not shown).
Our results suggested that the presence of certain cloning vectors
or the production of fluorescent proteins can significantly
impair the ability of serovar Typhimurium to invade and survive/replicate
within host cells under certain conditions. To investigate whether
such defects are also observed in vivo, we carried out competitive
index studies with mice (
19,
20). Since our in vitro experiments
indicated that the pACYC184 vector and GFP and RFP production
had the greatest influence on host cell interactions, only bacteria
carrying pACYC184, pACYC177, pFPV25, pFPV25.1, or pRFP were
tested against wild-type SL1344. Animal protocols were in direct
accordance with guidelines drafted by the University of British
Columbia's Animal Care Committee and the Canadian Council on
the Use of Laboratory Animals. Bacteria were grown to the stationary
phase by shaking them overnight at 37°C in 10 ml of LB broth
containing the appropriate antibiotics. Plasmid-containing strains
were diluted in PBS and mixed with equal numbers of CFU of wild-type
SL1344 (no plasmids). Female BALB/c mice (6 to 8 weeks old;
Jackson Laboratories) were inoculated with a total of 1
x 10
5 bacteria in 300 µl by intraperitoneal injection. Mice
were euthanized 48 h postinoculation by cervical dislocation,
and the infected spleens were removed and homogenized in PBS.
Bacteria were enumerated by serial dilutions onto LB agar containing
streptomycin to enumerate total bacteria or onto selective media
to enumerate plasmid-containing bacteria. The competitive index
was calculated by dividing the ratio of the number of plasmid-carrying
bacteria in the output to the number of total bacteria in the
spleens (output CFU) by the ratio of the number of plasmid-carrying
CFU to the total number of CFU in the inoculum (input CFU).
Experiments were repeated at least twice, with a total of 6
to 10 mice being used per group. Neither pACYC177 nor pACYC184
affected the virulence of serovar Typhimurium in the mouse model
of infection, as measured by determining the competitive index
(data not shown). In contrast, the mean competitive indices
for pFPV25.1 and pRFP were 0.45 (
P = 0.0016) and 0.25 (
P <
0.0001), respectively, compared to 0.79 for the empty pFPV25
vector, indicating that production of the fluorescent proteins
significantly reduced the ability of bacteria to compete against
wild-type bacteria during a systemic infection. It remains possible
that the pFPV25.1 and pRFP plasmids were lost during the course
of infection due to the absence of antibiotic selection, which
would also result in a lower confidence interval, although we
consider this unlikely in these experiments (
6,
23,
28).
In conclusion, our data demonstrate that the outcome of Salmonella infection can be impaired by the presence of plasmids or the production of fluorescent proteins, and the mechanism by which Salmonella is internalized into tissue culture cells is a major determining factor. Bacterial fate is more compromised under conditions where bacterial fitness is requisite, i.e., SPI1-mediated (bacterium-driven) invasion versus host cell-driven phagocytosis. Three of the plasmids we tested, pACYC184, pACYC177, and pBAD30, have the same origin of replication, yet only pACYC184 significantly impaired the ability of serovar Typhimurium to interact with host cells. The most likely explanation for this is that these plasmids carry different antibiotic resistance markers (Table 2), and indeed the tet gene, present in pACYC184, has recently been shown to have deleterious effects on Salmonella survival in macrophages (1). In our experiments, the ability of Salmonella to colonize a murine host was reduced by fluorescent proteins but not significantly affected by the presence of either pACYC184 or pACYC177. The differences observed between in vitro and in vivo studies presumably reflect the different stresses experienced by bacteria in these infections and highlight the importance of including suitable controls when using plasmids in complementation studies (14). Furthermore, results from in vitro and in vivo experiments that rely exclusively on GFP- and RFP-expressing bacteria should be interpreted with some caution. While our conclusions serve as a cautionary note, these tools remain a powerful asset to the study of bacterium-host cell interactions.

ACKNOWLEDGMENTS
This research was supported by the Intramural Research Program
of NIH NIAID. B.A.V. receives grants from the Canadian Institutes
of Health Research (CIHR) and is the C.H.I.L.D. Foundation Research
Scholar, the Canada Research Chair in Pediatric Gastroenterology,
and a Michael Smith Foundation for Health Research Scholar.

FOOTNOTES
* Corresponding author. Mailing address: Rocky Mountain Laboratories, 903 South 4th Street, Hamilton, MT 59840. Phone: (406) 363-9292. Fax: (406) 363-9380. E-mail:
omortimer{at}niaid.nih.gov.

Editor: F. C. Fang

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Infection and Immunity, October 2005, p. 7027-7031, Vol. 73, No. 10
0019-9567/05/$08.00+0 doi:10.1128/IAI.73.10.7027-7031.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
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