Salmonella-Induced Filament Formation Is a Dynamic Phenotype Induced by Rapidly Replicating Salmonella enterica Serovar Typhimurium in Epith
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感染与免疫杂志 2005年第2期
Infection, Immunity, Injury and Repair Program, Hospital for Sick Children
Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada
Departments of Microbiology Medicine, University of Washington, Seattle, Washington
ABSTRACT
Salmonella enterica serovar Typhimurium has the fascinating ability to form tubular structures known as Salmonella-induced filaments (Sifs) in host cells. Here, we show that the prevalence of the Sif phenotype in HeLa cells is affected by host cell density, growth, and the multiplicity of infection. Sif formation was observed in cells that displayed rapid intracellular bacterial replication and was found to be dynamic, being maximal 8 to 10 h postinfection and declining thereafter. The virulence factors SpvB and SseJ were found to negatively modulate Sif formation. Our findings demonstrate the complex and dynamic nature of the Sif phenotype.
TEXT
Salmonella enterica serovar Typhimurium is a gram-negative facultative intracellular pathogen that can cause a variety of diseases in different hosts (38). Both in vitro (4, 15, 18) and in vivo (23, 30, 32, 33) models have demonstrated the ability of serovar Typhimurium to inhabit and replicate within a membrane-bound compartment within host cells, the Salmonella-containing vacuole (SCV). From within this compartment, these bacteria can modulate trafficking of the SCV. For example, serovar Typhimurium is capable of blocking delivery of both the NADPH oxidase (13, 39, 40) and inducible nitric oxide synthase (iNOS) (9) to the SCV. Modulation of SCV trafficking is mediated in part by two different type III secretion systems (TTSS) encoded on the Salmonella chromosome. These specialized protein delivery systems mediate translocation of bacterial proteins called effectors into the host cell, where they modulate host cell machinery (16).
Late stages of SCV modification (6 to 8 h postinfection) include the formation of tubular membrane extensions known as Salmonella-induced filaments (Sifs). Sifs are thought to result from the fusion of late endocytic compartments with the SCV (6). Sif formation requires at least four effectors of the Salmonella pathogenicity island 2 (SPI-2)-encoded TTSS. SifA is essential for Sif formation and mutants lacking either this effector or regulators of its expression do not induce the Sif phenotype (28, 36). Deletion of sifA also leads to instability of the SCV, possibly through the actions of the putative glycerophospholipid-cholesterol acyltransferase SseJ, another SPI-2 effector (1, 31). The SPI-2 effectors SseF and SseG are also essential for Sif formation; sseF and sseG mutants form pseudo-Sif structures which do not acquire the late endosomal markers characteristic of Sifs (14, 20). Also, recent studies have shown that the SPI-2 effector SopD2 plays a role in promoting maximal Sif formation (16a). Regulation of the Sif phenotype by these and possibly other bacterial proteins is not yet understood.
An intriguing question is why only some infected cells make Sifs. Previous studies demonstrated Sif formation in only 50 to 80% of infected cells (1, 2, 14, 19, 28, 31, 36). This result is exemplified in Fig. 1A, which shows that not all infected cells make Sifs in vitro. Recent observations in our laboratory suggested that the density of the host cell monolayer prior to infection affected the presentation of the Sif phenotype. To address the effect of host cell density on Sif formation, HeLa epithelial cells were plated at various densities prior to infection with serovar Typhimurium (Fig. 1B). For these studies, we used a method previously optimized for invasion by serovar Typhimurium (35). Cells were briefly (10 min) exposed to a high multiplicity of infection (MOI) of late log phase serovar Typhimurium. The bacteria invaded cells by using the SPI-1-encoded TTSS during this pulse, and extracellular bacteria were then removed by extensive washing and the addition of gentamicin to the media (35). As shown in Fig. 1B, cell density had a major influence on presentation of the Sif phenotype. Sifs were observed in up to 80% of infected cells at the optimal cell density of 2.5 x 104 cells/ml, but they were much less prevalent at other densities.
The experiments shown in Fig. 1B suggested that the ratio of invading bacteria to cell density affected Sif formation. To determine the effect of MOI on this phenotype, we plated cells at a constant density and infected serovar Typhimurium at various MOIs. As shown in Fig. 1C, an MOI of 325:1 led to optimal Sif formation, and lesser numbers of bacteria were unable to induce Sifs to the same extent. Surprisingly, higher MOIs also had an inhibitory effect on Sif formation. Together, these findings suggest that differences in host cell density and MOI are likely to explain the variability in results obtained by different investigators (1, 2, 14, 19, 31, 36).
Despite many attempts to optimize conditions for Sif formation, we were unable to induce this phenotype in more than 80% of infected cells. This result may reflect at least two possibilities: (i) a population of infecting bacteria are unable to initiate the Sif phenotype, or (ii) a population of infected host cells have alterations to their endosomal systems that preclude the endocytic fusion events leading to Sif formation. To address the first possibility, we infected cells with serovar Typhimurium and counted the numbers of intracellular bacteria. Only lysosome-associated membrane protein 1-positive (LAMP-1+) bacteria were enumerated in order to exclude cytosolic bacteria (7). The numbers of intracellular bacteria were grouped into four categories and plotted for each time point. As shown in Fig. 2A, the majority of infected cells contain low numbers of bacteria 4 h postinfection, with very few (15%) cells containing >10 bacteria per cell. As time progressed during the experiment, the bacteria replicated within the SCV. As such, the numbers of infected cells with one to three bacteria decreased, while the numbers of infected cells with >10 bacteria increased over time, culminating with 60% of infected cells containing >10 bacteria by 23 h postinfection.
The results shown in Fig. 2A represent the total population of intracellular bacteria in LAMP-1+ vacuoles. During the enumeration of intracellular bacteria, we also noted which cells contained Sifs and plotted them separately from those without Sifs (Fig. 2B). As shown, there was a dramatic difference between the two infected cell populations. Infected cells with Sifs displayed a rapid loss in the population of cells with one to three bacteria, concomitant with a rapid increase in the population of cells with higher bacterial numbers. By 23 h postinfection, nearly 90% of infected cells with Sifs had 10 or more bacteria, compared to 40% in those cells without Sifs. These findings suggest that the bacteria capable of inducing Sifs are undergoing rapid replication and that a static (or slow-growing) population of bacteria exists that is unable to induce the Sif phenotype. Therefore, Sifs appear to be associated with rapidly replicating serovar Typhimurium in vitro.
To address whether host cell growth affects Sif formation, HeLa cells were infected with wild-type serovar Typhimurium and then cultured in media containing different percentages of fetal bovine serum (FBS) (Fig. 3). Lowering serum concentrations is a commonly used method to slow cell growth and replication in culture. Normal culture conditions (10% FBS) showed maximal Sif formation 8 h postinfection, while suboptimal growth conditions exhibited a detrimental effect. This effect was also observed after treatment of cells with aphidicolin, a drug that blocks normal cell cycle progression by inhibiting DNA replication (34). As shown in Fig. 3, the addition of aphidicolin also reduced the numbers of Sifs formed by invading bacteria, although this drug did not inhibit bacterial replication on its own (data not shown). These results show that the growth rate of the host cell has an impact on Sif formation.
To further characterize the Sif phenotype, we analyzed the regulation of Sif formation. In Fig. 4A, chloramphenicol was added to stop bacterial protein synthesis at various times postinfection. In this way, bacterial effectors involved in Sif formation would no longer be synthesized. All cells were then fixed at 8 h to enumerate the extent of Sif formation. By 5 h postinfection, effectors were synthesized and Sif formation was maintained until the 8 h endpoint in 40% of the infected cells. By 6 h, the number of Sifs formed was equivalent to the control, in which no drug was added (Fig. 4A). These results suggest that the bacterial effectors necessary for Sif formation were translocated into host cells by 5 to 6 h postinfection and are consistent with previous studies demonstrating translocation of effector proteins (3, 5, 11, 12, 17, 19-21, 25-27).
An interesting observation made during these studies was that Sifs are a dynamic phenotype. Maximal Sif formation is observed 8 to 10 h postinfection, but it then declines abruptly to 20% by 21 h (Fig. 5B, wild-type data). While there are many possibilities for this decline, we wished to determine whether effectors of the SPI-2 TTSS were responsible. In order to analyze the breakdown and stability of Sifs, HeLa cells were infected with serovar Typhimurium and chloramphenicol was added at 8 h. Cells were then fixed at 12 and 23 h to determine the role of bacterial protein synthesis in Sif stability and/or breakdown. As shown in Fig. 4B, antibiotic addition had no dramatic effect on Sif downregulation, suggesting that any bacterial factors required for Sif downregulation are stable or that the decline of Sifs is not dependent upon bacterial protein synthesis.
A previous study by Ruiz-Albert et al. demonstrated that transfection of cells with the SPI-2 effector SseJ prior to infection inhibits Sif formation induced by wild-type serovar Typhimurium (31). To determine whether SseJ is involved in downregulating Sifs, we infected cells with mutants lacking this effector. For these experiments, the serovar Typhimurium CS401 wild-type strain was used. Optimization was carried out for this strain as it was for SL1344. An MOI of 325:1 was found to be optimal for CS401, as well, although Sif formation reached a maximum of only 30 to 50% by 8 h (data not shown). As illustrated in Fig. 5A, deletion of sseJ led to a dramatic increase in the amount of Sif tubules formed per infected cell. An increased percentage of infected cells had Sifs, as well (Fig. 5B). This increase was observed by 6 h postinfection and maintained at later times. However, Sif formation by this mutant still underwent a decline after peaking at 10 h postinfection. These findings demonstrate that SseJ antagonizes Sif formation during the first 8 to 10 h postinfection and that it does not mediate the downregulation of the Sif phenotype at later times postinfection.
Downregulation of the Sif phenotype correlated temporally with depolymerization of actin around the SCV, which was previously examined by us and others (3, 24, 25). Depolymerization of this actin is largely dependent upon the actions of SpvB, an ADP-ribosylating enzyme encoded on the virulence plasmid of serovar Typhimurium (10, 22, 25, 29, 37). The SPI-2 effectors SspH2 and SseI also contribute to remodeling of actin on the SCV, though their contribution is more subtle (25). To test whether actin depolymerization events are involved in the downregulation of Sifs, we infected cells with an isogenic mutant of serovar Typhimurium lacking spvB. This mutant displayed a slightly higher number of Sifs at 8 to 10 h; however, the breakdown of Sifs in the mutant was not affected compared to that in the wild type (Fig. 5B). Nonetheless, this is the first evidence that SpvB is involved in the Sif phenotype. We also infected cells with a double mutant lacking both sseJ and spvB. As expected, these bacteria induced Sifs in a larger fraction of infected cells at 10 h postinfection in a synergistic manner than the sseJ and spvB mutants alone (Fig. 5B).
Conclusions. We have shown that many conditions affect Sif formation in vitro, which may explain the variance of this phenotype among different investigators. Additionally, we report a number of novel observations relating to the Sif phenotype. First, Sif formation was found to correlate with cells infected with rapidly replicating bacteria. This observation is consistent with previous evidence suggesting that static (or dying) and rapidly growing populations of serovar Typhimurium exist in host cells (8). Second, bacterial numbers per infected cell do not appear to correlate directly with Sifs, as initial small numbers of intracellular bacteria (1-2) are sufficient to initiate Sif formation (see Fig. 2B). Also, the sseJ mutant, which is attenuated for intracellular replication (11, 31), can produce more Sifs than wild-type bacteria. Finally, we show that Sifs are a dynamic phenotype and are downregulated after 8 to 10 h in a manner that does not appear to require bacterial protein synthesis. While it remains unclear how the Sif phenotype benefits intracellular bacteria, this phenotype is tightly controlled by both positive (SifA, SseF, SseG, SopD2) (14, 36) and negative (SseJ and SpvB) (31) regulators. For future studies of the Sif phenotype, the double sseJ spvB mutant will provide a valuable reagent.
ACKNOWLEDGMENTS
This work was supported by grant funding and a New Investigator Award from the Canadian Institutes of Health Research to J.H.B. Infrastructure for the Brumell laboratory was provided by a New Opportunities Fund from the Canadian Foundation for Innovation and the Ontario Innovation Trust. C.L.B. is the recipient of a University of Toronto Open Fellowship Award and a studentship from the Natural Sciences and Engineering Research Council of Canada.
We thank Michael Woodside for assistance with confocal microscopy and Brett Finlay, Nat Brown, Andrew Perrin, and members of the Brumell laboratory for helpful discussions and critical reading of the manuscript.
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Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada
Departments of Microbiology Medicine, University of Washington, Seattle, Washington
ABSTRACT
Salmonella enterica serovar Typhimurium has the fascinating ability to form tubular structures known as Salmonella-induced filaments (Sifs) in host cells. Here, we show that the prevalence of the Sif phenotype in HeLa cells is affected by host cell density, growth, and the multiplicity of infection. Sif formation was observed in cells that displayed rapid intracellular bacterial replication and was found to be dynamic, being maximal 8 to 10 h postinfection and declining thereafter. The virulence factors SpvB and SseJ were found to negatively modulate Sif formation. Our findings demonstrate the complex and dynamic nature of the Sif phenotype.
TEXT
Salmonella enterica serovar Typhimurium is a gram-negative facultative intracellular pathogen that can cause a variety of diseases in different hosts (38). Both in vitro (4, 15, 18) and in vivo (23, 30, 32, 33) models have demonstrated the ability of serovar Typhimurium to inhabit and replicate within a membrane-bound compartment within host cells, the Salmonella-containing vacuole (SCV). From within this compartment, these bacteria can modulate trafficking of the SCV. For example, serovar Typhimurium is capable of blocking delivery of both the NADPH oxidase (13, 39, 40) and inducible nitric oxide synthase (iNOS) (9) to the SCV. Modulation of SCV trafficking is mediated in part by two different type III secretion systems (TTSS) encoded on the Salmonella chromosome. These specialized protein delivery systems mediate translocation of bacterial proteins called effectors into the host cell, where they modulate host cell machinery (16).
Late stages of SCV modification (6 to 8 h postinfection) include the formation of tubular membrane extensions known as Salmonella-induced filaments (Sifs). Sifs are thought to result from the fusion of late endocytic compartments with the SCV (6). Sif formation requires at least four effectors of the Salmonella pathogenicity island 2 (SPI-2)-encoded TTSS. SifA is essential for Sif formation and mutants lacking either this effector or regulators of its expression do not induce the Sif phenotype (28, 36). Deletion of sifA also leads to instability of the SCV, possibly through the actions of the putative glycerophospholipid-cholesterol acyltransferase SseJ, another SPI-2 effector (1, 31). The SPI-2 effectors SseF and SseG are also essential for Sif formation; sseF and sseG mutants form pseudo-Sif structures which do not acquire the late endosomal markers characteristic of Sifs (14, 20). Also, recent studies have shown that the SPI-2 effector SopD2 plays a role in promoting maximal Sif formation (16a). Regulation of the Sif phenotype by these and possibly other bacterial proteins is not yet understood.
An intriguing question is why only some infected cells make Sifs. Previous studies demonstrated Sif formation in only 50 to 80% of infected cells (1, 2, 14, 19, 28, 31, 36). This result is exemplified in Fig. 1A, which shows that not all infected cells make Sifs in vitro. Recent observations in our laboratory suggested that the density of the host cell monolayer prior to infection affected the presentation of the Sif phenotype. To address the effect of host cell density on Sif formation, HeLa epithelial cells were plated at various densities prior to infection with serovar Typhimurium (Fig. 1B). For these studies, we used a method previously optimized for invasion by serovar Typhimurium (35). Cells were briefly (10 min) exposed to a high multiplicity of infection (MOI) of late log phase serovar Typhimurium. The bacteria invaded cells by using the SPI-1-encoded TTSS during this pulse, and extracellular bacteria were then removed by extensive washing and the addition of gentamicin to the media (35). As shown in Fig. 1B, cell density had a major influence on presentation of the Sif phenotype. Sifs were observed in up to 80% of infected cells at the optimal cell density of 2.5 x 104 cells/ml, but they were much less prevalent at other densities.
The experiments shown in Fig. 1B suggested that the ratio of invading bacteria to cell density affected Sif formation. To determine the effect of MOI on this phenotype, we plated cells at a constant density and infected serovar Typhimurium at various MOIs. As shown in Fig. 1C, an MOI of 325:1 led to optimal Sif formation, and lesser numbers of bacteria were unable to induce Sifs to the same extent. Surprisingly, higher MOIs also had an inhibitory effect on Sif formation. Together, these findings suggest that differences in host cell density and MOI are likely to explain the variability in results obtained by different investigators (1, 2, 14, 19, 31, 36).
Despite many attempts to optimize conditions for Sif formation, we were unable to induce this phenotype in more than 80% of infected cells. This result may reflect at least two possibilities: (i) a population of infecting bacteria are unable to initiate the Sif phenotype, or (ii) a population of infected host cells have alterations to their endosomal systems that preclude the endocytic fusion events leading to Sif formation. To address the first possibility, we infected cells with serovar Typhimurium and counted the numbers of intracellular bacteria. Only lysosome-associated membrane protein 1-positive (LAMP-1+) bacteria were enumerated in order to exclude cytosolic bacteria (7). The numbers of intracellular bacteria were grouped into four categories and plotted for each time point. As shown in Fig. 2A, the majority of infected cells contain low numbers of bacteria 4 h postinfection, with very few (15%) cells containing >10 bacteria per cell. As time progressed during the experiment, the bacteria replicated within the SCV. As such, the numbers of infected cells with one to three bacteria decreased, while the numbers of infected cells with >10 bacteria increased over time, culminating with 60% of infected cells containing >10 bacteria by 23 h postinfection.
The results shown in Fig. 2A represent the total population of intracellular bacteria in LAMP-1+ vacuoles. During the enumeration of intracellular bacteria, we also noted which cells contained Sifs and plotted them separately from those without Sifs (Fig. 2B). As shown, there was a dramatic difference between the two infected cell populations. Infected cells with Sifs displayed a rapid loss in the population of cells with one to three bacteria, concomitant with a rapid increase in the population of cells with higher bacterial numbers. By 23 h postinfection, nearly 90% of infected cells with Sifs had 10 or more bacteria, compared to 40% in those cells without Sifs. These findings suggest that the bacteria capable of inducing Sifs are undergoing rapid replication and that a static (or slow-growing) population of bacteria exists that is unable to induce the Sif phenotype. Therefore, Sifs appear to be associated with rapidly replicating serovar Typhimurium in vitro.
To address whether host cell growth affects Sif formation, HeLa cells were infected with wild-type serovar Typhimurium and then cultured in media containing different percentages of fetal bovine serum (FBS) (Fig. 3). Lowering serum concentrations is a commonly used method to slow cell growth and replication in culture. Normal culture conditions (10% FBS) showed maximal Sif formation 8 h postinfection, while suboptimal growth conditions exhibited a detrimental effect. This effect was also observed after treatment of cells with aphidicolin, a drug that blocks normal cell cycle progression by inhibiting DNA replication (34). As shown in Fig. 3, the addition of aphidicolin also reduced the numbers of Sifs formed by invading bacteria, although this drug did not inhibit bacterial replication on its own (data not shown). These results show that the growth rate of the host cell has an impact on Sif formation.
To further characterize the Sif phenotype, we analyzed the regulation of Sif formation. In Fig. 4A, chloramphenicol was added to stop bacterial protein synthesis at various times postinfection. In this way, bacterial effectors involved in Sif formation would no longer be synthesized. All cells were then fixed at 8 h to enumerate the extent of Sif formation. By 5 h postinfection, effectors were synthesized and Sif formation was maintained until the 8 h endpoint in 40% of the infected cells. By 6 h, the number of Sifs formed was equivalent to the control, in which no drug was added (Fig. 4A). These results suggest that the bacterial effectors necessary for Sif formation were translocated into host cells by 5 to 6 h postinfection and are consistent with previous studies demonstrating translocation of effector proteins (3, 5, 11, 12, 17, 19-21, 25-27).
An interesting observation made during these studies was that Sifs are a dynamic phenotype. Maximal Sif formation is observed 8 to 10 h postinfection, but it then declines abruptly to 20% by 21 h (Fig. 5B, wild-type data). While there are many possibilities for this decline, we wished to determine whether effectors of the SPI-2 TTSS were responsible. In order to analyze the breakdown and stability of Sifs, HeLa cells were infected with serovar Typhimurium and chloramphenicol was added at 8 h. Cells were then fixed at 12 and 23 h to determine the role of bacterial protein synthesis in Sif stability and/or breakdown. As shown in Fig. 4B, antibiotic addition had no dramatic effect on Sif downregulation, suggesting that any bacterial factors required for Sif downregulation are stable or that the decline of Sifs is not dependent upon bacterial protein synthesis.
A previous study by Ruiz-Albert et al. demonstrated that transfection of cells with the SPI-2 effector SseJ prior to infection inhibits Sif formation induced by wild-type serovar Typhimurium (31). To determine whether SseJ is involved in downregulating Sifs, we infected cells with mutants lacking this effector. For these experiments, the serovar Typhimurium CS401 wild-type strain was used. Optimization was carried out for this strain as it was for SL1344. An MOI of 325:1 was found to be optimal for CS401, as well, although Sif formation reached a maximum of only 30 to 50% by 8 h (data not shown). As illustrated in Fig. 5A, deletion of sseJ led to a dramatic increase in the amount of Sif tubules formed per infected cell. An increased percentage of infected cells had Sifs, as well (Fig. 5B). This increase was observed by 6 h postinfection and maintained at later times. However, Sif formation by this mutant still underwent a decline after peaking at 10 h postinfection. These findings demonstrate that SseJ antagonizes Sif formation during the first 8 to 10 h postinfection and that it does not mediate the downregulation of the Sif phenotype at later times postinfection.
Downregulation of the Sif phenotype correlated temporally with depolymerization of actin around the SCV, which was previously examined by us and others (3, 24, 25). Depolymerization of this actin is largely dependent upon the actions of SpvB, an ADP-ribosylating enzyme encoded on the virulence plasmid of serovar Typhimurium (10, 22, 25, 29, 37). The SPI-2 effectors SspH2 and SseI also contribute to remodeling of actin on the SCV, though their contribution is more subtle (25). To test whether actin depolymerization events are involved in the downregulation of Sifs, we infected cells with an isogenic mutant of serovar Typhimurium lacking spvB. This mutant displayed a slightly higher number of Sifs at 8 to 10 h; however, the breakdown of Sifs in the mutant was not affected compared to that in the wild type (Fig. 5B). Nonetheless, this is the first evidence that SpvB is involved in the Sif phenotype. We also infected cells with a double mutant lacking both sseJ and spvB. As expected, these bacteria induced Sifs in a larger fraction of infected cells at 10 h postinfection in a synergistic manner than the sseJ and spvB mutants alone (Fig. 5B).
Conclusions. We have shown that many conditions affect Sif formation in vitro, which may explain the variance of this phenotype among different investigators. Additionally, we report a number of novel observations relating to the Sif phenotype. First, Sif formation was found to correlate with cells infected with rapidly replicating bacteria. This observation is consistent with previous evidence suggesting that static (or dying) and rapidly growing populations of serovar Typhimurium exist in host cells (8). Second, bacterial numbers per infected cell do not appear to correlate directly with Sifs, as initial small numbers of intracellular bacteria (1-2) are sufficient to initiate Sif formation (see Fig. 2B). Also, the sseJ mutant, which is attenuated for intracellular replication (11, 31), can produce more Sifs than wild-type bacteria. Finally, we show that Sifs are a dynamic phenotype and are downregulated after 8 to 10 h in a manner that does not appear to require bacterial protein synthesis. While it remains unclear how the Sif phenotype benefits intracellular bacteria, this phenotype is tightly controlled by both positive (SifA, SseF, SseG, SopD2) (14, 36) and negative (SseJ and SpvB) (31) regulators. For future studies of the Sif phenotype, the double sseJ spvB mutant will provide a valuable reagent.
ACKNOWLEDGMENTS
This work was supported by grant funding and a New Investigator Award from the Canadian Institutes of Health Research to J.H.B. Infrastructure for the Brumell laboratory was provided by a New Opportunities Fund from the Canadian Foundation for Innovation and the Ontario Innovation Trust. C.L.B. is the recipient of a University of Toronto Open Fellowship Award and a studentship from the Natural Sciences and Engineering Research Council of Canada.
We thank Michael Woodside for assistance with confocal microscopy and Brett Finlay, Nat Brown, Andrew Perrin, and members of the Brumell laboratory for helpful discussions and critical reading of the manuscript.
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