EDITORIAL
crossm Classic Spotlight: Persistence Persists Victor J. DiRita Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, USA
KEYWORDS Classic Spotlight, intracellular growth, antibiotic resistance
F
red Neidhardt passed away in October 2016, leaving a tremendous footprint on the microbial sciences. Given his impact in so many areas (textbook author, monograph editor, department chair, and ASM president), we risk forgetting that he was also a prolific and creative experimentalist. His research centered on how cells balance their growth in response to the different conditions in which they find themselves. We already saw one of his papers highlighted in our Classic Spotlight series by Rick Gourse, who described Neidhardt’s important contribution to discovering the heat shock sigma factor sigma-32. This Classic Spotlight features another Neidhardt paper investigating Salmonella growth inside macrophages (1). Yes, the developer of “Neidhardt MOPS minimal medium,” described in a Journal of Bacteriology paper cited over 1,000 times by investigators studying bacterial growth in vitro (2), sought to understand how Salmonella grows within host cells. Other investigators had previously tackled this question, but definitive conclusions were not provided from those studies (3, 4). A built-in molecular clock established in earlier published work from Neidhardt’s lab was exploited for this work. The ribosomal protein L7 is the acetylated form of another ribosomal protein, L12. The conversion rate of L12 to L7 by acetylation does not keep up with the overall rate of accumulation of the absolute levels of the proteins, which varies directly with the growth rate, so changes in detectable L12 protein levels as a fraction of the total L12/L7 protein can be used to clock relative cellular growth rates in different conditions. Armed with this knowledge and using two-dimensional gel electrophoresis (an approach pioneered by Fred to study cellular physiology), Abshire and Neidhardt carried out a series of simple and deceptively informative experiments. They grew Salmonella enterica serovar Typhimurium in various growth media formulated for different steady-state doubling rates. Using imaging software to quantify the amount of L7 and L12 in these cultures on two-dimensional gels, they calculated the percentage of L7/L12 that was in the L12 (nonacetylated) form under different growth rates. From this they made a calibration curve of growth rate versus percent L12, which showed a range from nearly undetectable L12 (compared to L7) in acetate-MOPS (doubling rate ⫽ 245 min) to over 80% L12 in glucose-rich MOPS. They applied this calibration curve to determine the apparent growth rate of S. Typhimurium within the macrophage-like cell line U937 at early, mid-, and late growth in 4-h blocks over a 25-h period. The L7/L12 measurements from intracellular bacteria plotted on this curve suggested a relatively vigorous doubling rate of 33 to 39 min throughout the 25 h. This was not what they observed, however, when isolating bacteria from the U937 cells and plating for cell counts. Viable cell counts stayed stable and low (about 2 ⫻ 106) for the first 4 h—showing no evidence of growth—and then rose slightly for the next 4 h to about 1 ⫻ 107. The theoretical growth curve using the rate calculated with the L7/L12 clock would have shown a steady increase of cells over the 8 h, to roughly 1 ⫻ 1010. Abshire and Neidhardt termed this a “growth rate paradox.” February 2017 Volume 199 Issue 4 e00734-16
Journal of Bacteriology
Citation DiRita VJ. 2017. Classic spotlight: persistence persists. J Bacteriol 199:e00734-16. https://doi.org/10.1128/JB.00734-16. Copyright © 2017 American Society for Microbiology. All Rights Reserved. Address correspondence to [email protected]. The views expressed in this Editorial do not necessarily reflect the views of the journal or of ASM.
jb.asm.org 1
Editorial
Journal of Bacteriology
They developed three hypotheses to explain this paradox: (i) bacteria were indeed growing rapidly but were being killed (by the U937 cells) at the same or slightly less than the same rate over the first 8 h; (ii) a small number of cells were growing rapidly (these were the ones that the clock was measuring), but the remainder, while still viable, were not actually growing (so the clock did not record them); and (iii) the bacteria were in fact all growing very slowly, as the plate counting suggested, but the typically reliable ribosomal protein clock was malfunctioning in some biologically interesting way. Abshire and Neidhardt acknowledged that the third hypothesis was not easily testable, but to distinguish between the first two hypotheses, they repeated the U937 growth experiments in the presence of two different antibiotics: chloramphenicol, which is bacteriostatic, and ampicillin, which kills dividing cells. What they observed in the presence of antibiotics was revealing. For the first 2 h of intracellular growth, viable cell counts were sensitive to both antibiotics, with significant dropoff in viability compared to the control. This implied that early after cell infection (i) the bacteria were growing (and hence were susceptible to ampicillin) and (ii) the host cells were killing them (hence the loss of numbers after growth cessation by chloramphenicol). For the next 6 h, however, neither drug had much of an effect on the observed viability compared to the controls grown without antibiotics. To Abshire and Neidhardt, this suggested that most of the intracellular bacteria were not actually dividing (or else they would have been wiped out by the ampicillin) and that the U937 cells were not killing them (or else the viable counts would have dropped when bacterial growth was inhibited by chloramphenicol). Abshire and Neidhardt concluded that the growth rate paradox could be explained if there are two populations within the U937 macrophages: a large population of mostly nongrowing (but viable) cells and a smaller, but relatively fast-growing, population. They noted that their work supported an earlier, also now-classic, study by Nancy Buchmeier and Fred Heffron, published in another ASM journal, Infection and Immunity, which used electron microscopy to observe that Salmonella was parceled in two different macrophage compartments: some cells were in phagosomes and could be seen dividing, and others were in phagolysosomes and were not seen dividing (5). Epilogue. In 2014 Sophie Helaine, David Holden, and colleagues published a beautiful paper on Salmonella that examined the medically relevant, antibiotic-resistant persister cells that develop during real-life bacterial infections (6). Using obviously more sophisticated approaches, they reached a conclusion that was clearly foreshadowed by the findings of Abshire and Neidhardt: “the vacuolar environment induces phenotypic heterogeneity, leading to either bacterial replication or the formation of nonreplicating persisters that could provide a reservoir for relapsing infection.” Like the other towering contributions from Neidhardt’s life, the insights drawn from the experiments highlighted here have firmly endured the test of time. ACKNOWLEDGMENTS I thank David Friedman and Christopher Alteri for comments that helped improve the manuscript.
REFERENCES 1. Abshire KZ, Neidhardt FC. 1993. Growth rate paradox of Salmonella typhimurium within host macrophages. J Bacteriol 175:3744–3748. 2. Neidhardt FC, Bloch PL, Smith DF. 1974. Culture medium for enterobacteria. J Bacteriol 119:736 –747. 3. Lowrie DB, Aber VR, Carrol ME. 1979. Division and death rates of Salmonella typhimurium inside macrophages: use of penicillin as a probe. J Gen Microbiol 110:409 – 419. 4. Jenkin C, Benacerraf B. 1960. In vitro studies on the interaction between
February 2017 Volume 199 Issue 4 e00734-16
mouse peritoneal macrophages and strains of Salmonella and Escherichia coli. J Exp Med 112:403– 417. 5. Buchmeier NA, Heffron F. 1991. Inhibition of macrophage phagosomelysosome fusion by Salmonella typhimurium. Infect Immun 59:2232–2238. 6. Helaine S, Cheverton AM, Watson KG, Faure LM, Matthews SA, Holden DW. 2014. Internalization of Salmonella by macrophages induces formation of nonreplicating persisters. Science 343:204 –208. https://doi.org/ 10.1126/science.1244705.
jb.asm.org 2
crossm Classic Spotlight: Persistence Persists Victor J. DiRita Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, USA
KEYWORDS Classic Spotlight, intracellular growth, antibiotic resistance
F
red Neidhardt passed away in October 2016, leaving a tremendous footprint on the microbial sciences. Given his impact in so many areas (textbook author, monograph editor, department chair, and ASM president), we risk forgetting that he was also a prolific and creative experimentalist. His research centered on how cells balance their growth in response to the different conditions in which they find themselves. We already saw one of his papers highlighted in our Classic Spotlight series by Rick Gourse, who described Neidhardt’s important contribution to discovering the heat shock sigma factor sigma-32. This Classic Spotlight features another Neidhardt paper investigating Salmonella growth inside macrophages (1). Yes, the developer of “Neidhardt MOPS minimal medium,” described in a Journal of Bacteriology paper cited over 1,000 times by investigators studying bacterial growth in vitro (2), sought to understand how Salmonella grows within host cells. Other investigators had previously tackled this question, but definitive conclusions were not provided from those studies (3, 4). A built-in molecular clock established in earlier published work from Neidhardt’s lab was exploited for this work. The ribosomal protein L7 is the acetylated form of another ribosomal protein, L12. The conversion rate of L12 to L7 by acetylation does not keep up with the overall rate of accumulation of the absolute levels of the proteins, which varies directly with the growth rate, so changes in detectable L12 protein levels as a fraction of the total L12/L7 protein can be used to clock relative cellular growth rates in different conditions. Armed with this knowledge and using two-dimensional gel electrophoresis (an approach pioneered by Fred to study cellular physiology), Abshire and Neidhardt carried out a series of simple and deceptively informative experiments. They grew Salmonella enterica serovar Typhimurium in various growth media formulated for different steady-state doubling rates. Using imaging software to quantify the amount of L7 and L12 in these cultures on two-dimensional gels, they calculated the percentage of L7/L12 that was in the L12 (nonacetylated) form under different growth rates. From this they made a calibration curve of growth rate versus percent L12, which showed a range from nearly undetectable L12 (compared to L7) in acetate-MOPS (doubling rate ⫽ 245 min) to over 80% L12 in glucose-rich MOPS. They applied this calibration curve to determine the apparent growth rate of S. Typhimurium within the macrophage-like cell line U937 at early, mid-, and late growth in 4-h blocks over a 25-h period. The L7/L12 measurements from intracellular bacteria plotted on this curve suggested a relatively vigorous doubling rate of 33 to 39 min throughout the 25 h. This was not what they observed, however, when isolating bacteria from the U937 cells and plating for cell counts. Viable cell counts stayed stable and low (about 2 ⫻ 106) for the first 4 h—showing no evidence of growth—and then rose slightly for the next 4 h to about 1 ⫻ 107. The theoretical growth curve using the rate calculated with the L7/L12 clock would have shown a steady increase of cells over the 8 h, to roughly 1 ⫻ 1010. Abshire and Neidhardt termed this a “growth rate paradox.” February 2017 Volume 199 Issue 4 e00734-16
Journal of Bacteriology
Citation DiRita VJ. 2017. Classic spotlight: persistence persists. J Bacteriol 199:e00734-16. https://doi.org/10.1128/JB.00734-16. Copyright © 2017 American Society for Microbiology. All Rights Reserved. Address correspondence to [email protected]. The views expressed in this Editorial do not necessarily reflect the views of the journal or of ASM.
jb.asm.org 1
Editorial
Journal of Bacteriology
They developed three hypotheses to explain this paradox: (i) bacteria were indeed growing rapidly but were being killed (by the U937 cells) at the same or slightly less than the same rate over the first 8 h; (ii) a small number of cells were growing rapidly (these were the ones that the clock was measuring), but the remainder, while still viable, were not actually growing (so the clock did not record them); and (iii) the bacteria were in fact all growing very slowly, as the plate counting suggested, but the typically reliable ribosomal protein clock was malfunctioning in some biologically interesting way. Abshire and Neidhardt acknowledged that the third hypothesis was not easily testable, but to distinguish between the first two hypotheses, they repeated the U937 growth experiments in the presence of two different antibiotics: chloramphenicol, which is bacteriostatic, and ampicillin, which kills dividing cells. What they observed in the presence of antibiotics was revealing. For the first 2 h of intracellular growth, viable cell counts were sensitive to both antibiotics, with significant dropoff in viability compared to the control. This implied that early after cell infection (i) the bacteria were growing (and hence were susceptible to ampicillin) and (ii) the host cells were killing them (hence the loss of numbers after growth cessation by chloramphenicol). For the next 6 h, however, neither drug had much of an effect on the observed viability compared to the controls grown without antibiotics. To Abshire and Neidhardt, this suggested that most of the intracellular bacteria were not actually dividing (or else they would have been wiped out by the ampicillin) and that the U937 cells were not killing them (or else the viable counts would have dropped when bacterial growth was inhibited by chloramphenicol). Abshire and Neidhardt concluded that the growth rate paradox could be explained if there are two populations within the U937 macrophages: a large population of mostly nongrowing (but viable) cells and a smaller, but relatively fast-growing, population. They noted that their work supported an earlier, also now-classic, study by Nancy Buchmeier and Fred Heffron, published in another ASM journal, Infection and Immunity, which used electron microscopy to observe that Salmonella was parceled in two different macrophage compartments: some cells were in phagosomes and could be seen dividing, and others were in phagolysosomes and were not seen dividing (5). Epilogue. In 2014 Sophie Helaine, David Holden, and colleagues published a beautiful paper on Salmonella that examined the medically relevant, antibiotic-resistant persister cells that develop during real-life bacterial infections (6). Using obviously more sophisticated approaches, they reached a conclusion that was clearly foreshadowed by the findings of Abshire and Neidhardt: “the vacuolar environment induces phenotypic heterogeneity, leading to either bacterial replication or the formation of nonreplicating persisters that could provide a reservoir for relapsing infection.” Like the other towering contributions from Neidhardt’s life, the insights drawn from the experiments highlighted here have firmly endured the test of time. ACKNOWLEDGMENTS I thank David Friedman and Christopher Alteri for comments that helped improve the manuscript.
REFERENCES 1. Abshire KZ, Neidhardt FC. 1993. Growth rate paradox of Salmonella typhimurium within host macrophages. J Bacteriol 175:3744–3748. 2. Neidhardt FC, Bloch PL, Smith DF. 1974. Culture medium for enterobacteria. J Bacteriol 119:736 –747. 3. Lowrie DB, Aber VR, Carrol ME. 1979. Division and death rates of Salmonella typhimurium inside macrophages: use of penicillin as a probe. J Gen Microbiol 110:409 – 419. 4. Jenkin C, Benacerraf B. 1960. In vitro studies on the interaction between
February 2017 Volume 199 Issue 4 e00734-16
mouse peritoneal macrophages and strains of Salmonella and Escherichia coli. J Exp Med 112:403– 417. 5. Buchmeier NA, Heffron F. 1991. Inhibition of macrophage phagosomelysosome fusion by Salmonella typhimurium. Infect Immun 59:2232–2238. 6. Helaine S, Cheverton AM, Watson KG, Faure LM, Matthews SA, Holden DW. 2014. Internalization of Salmonella by macrophages induces formation of nonreplicating persisters. Science 343:204 –208. https://doi.org/ 10.1126/science.1244705.
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