Cloning Vectors and Fluorescent Proteins Can Significantly Inhibit Salmonella enterica Virulence in Both Epithelial Cells and Macrophages: I
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感染与免疫杂志 2005年第10期
Laboratory of Intracellular Parasites, NIAID, NIH, Rocky Mountain Laboratories, Hamilton, Montana
Division of Gastroenterology, British Columbia's Children's Hospital and the University of British Columbia, Vancouver, British Columbia, Canada
Institut für Klinische Mikrobiologie, Immunologie und Hygiene, Universitt Erlangen-Nuernberg, Erlangen, Germany
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 104 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 106 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'-GATTTCTAGATTTAAGAAGGAGATATACATATGAGGTCTTCCAAGAATG-3') and RFP-Rev-SphI (5'-ACATGCATGCCTAAAGGAACAGATGGTGG-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 105 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.
REFERENCES
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2. Alpuche-Aranda, C. M., E. L. Racoosin, J. A. Swanson, and S. I. Miller. 1994. Salmonella stimulate macrophage macropinocytosis and persist within spacious phagosomes. J. Exp. Med. 179:601-608.
3. Bolivar, F., R. L. Rodriguez, P. J. Greene, M. C. Betlach, H. L. Heyneker, and H. W. Boyer. 1977. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2:95-113.
4. Bongaerts, R. J., I. Hautefort, J. M. Sidebotham, and J. C. Hinton. 2002. Green fluorescent protein as a marker for conditional gene expression in bacterial cells. Methods Enzymol. 358:43-66.
5. Buchmeier, N. A., and F. Heffron. 1989. Intracellular survival of wild-type Salmonella typhimurium and macrophage-sensitive mutants in diverse populations of macrophages. Infect. Immun. 57:1-7.
6. Bumann, D. 2002. Examination of Salmonella gene expression in an infected mammalian host using the green fluorescent protein and two-colour flow cytometry. Mol. Microbiol. 43:1269-1283.
7. Bumann, D. 2001. In vivo visualization of bacterial colonization, antigen expression, and specific T-cell induction following oral administration of live recombinant Salmonella enterica serovar Typhimurium. Infect. Immun. 69:4618-4626.
8. Chang, A. C. Y., and S. N. Cohen. 1978. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J. Bacteriol. 134:1141-1156.
9. Cirillo, D. M., R. H. Valdivia, D. M. Monack, and S. Falkow. 1998. Macrophage-dependent induction of the Salmonella pathogenicity island 2 type III secretion system and its role in intracellular survival. Mol. Microbiol. 30:175-188.
10. Fields, P. I., R. V. Swanson, C. G. Haidaris, and F. Heffron. 1986. Mutants of Salmonella typhimurium that cannot survive within the macrophage are avirulent. Proc. Natl. Acad. Sci. USA 83:5189-5193.
11. Galan, J. E. 2001. Salmonella interactions with host cells: type III secretion at work. Annu. Rev. Cell Dev. Biol. 17:53-86.
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13. Gandhi, M., S. Golding, S. Yaron, and K. R. Matthews. 2001. Use of green fluorescent protein expressing Salmonella Stanley to investigate survival, spatial location, and control on alfalfa sprouts. J. Food Prot. 64:1891-1898.
14. Garmory, H. S., K. F. Griffin, S. E. Leary, S. D. Perkins, K. A. Brown, and R. W. Titball. 2002. The effect of recombinant plasmids on in vivo colonisation of Salmonella enterica serovar Typhimurium strains is not reflected by in vitro cellular invasion assays. Vaccine 20:3239-3243.
15. Guzman, L.-M., D. Belin, M. J. Carson, and J. Beckwith. 1995. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol. 177:4121-4130.
16. Hoiseth, S. K., and B. A. Stocker. 1981. Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature 291:238-239.
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18. Johansson, C., and M. J. Wick. 2004. Liver dendritic cells present bacterial antigens and produce cytokines upon Salmonella encounter. J. Immunol. 172:2496-2503.
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Division of Gastroenterology, British Columbia's Children's Hospital and the University of British Columbia, Vancouver, British Columbia, Canada
Institut für Klinische Mikrobiologie, Immunologie und Hygiene, Universitt Erlangen-Nuernberg, Erlangen, Germany
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 104 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 106 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'-GATTTCTAGATTTAAGAAGGAGATATACATATGAGGTCTTCCAAGAATG-3') and RFP-Rev-SphI (5'-ACATGCATGCCTAAAGGAACAGATGGTGG-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 105 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.
REFERENCES
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