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The Minimal Unit of Infection: in the Macrophage

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  • Authors: Brian C. VanderVen1, Lu Huang2, Kyle H. Rohde3, David G. Russell4
  • Editors: William R. Jacobs Jr.5, Helen McShane6, Valerie Mizrahi7, Ian M. Orme8
  • VIEW AFFILIATIONS HIDE AFFILIATIONS
    Affiliations: 1: Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853; 2: Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853; 3: Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL 32827; 4: Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853; 5: Howard Hughes Medical Institute, Albert Einstein School of Medicine, Bronx, NY 10461; 6: University of Oxford, Oxford OX3 7DQ, United Kingdom; 7: University of Cape Town, Rondebosch 7701, South Africa; 8: Colorado State University, Fort Collins, CO 80523
    • Source: microbiolspec December 2016 vol. 4 no. 6 doi:10.1128/microbiolspec.TBTB2-0025-2016
  • Received 28 July 2016 Accepted 16 August 2016 Published 09 December 2016
  • D. Russell, [email protected]
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  • Abstract:

    The interaction between and its host cell is highly complex and extremely intimate. Were it not for the disease, one might regard this interaction at the cellular level as an almost symbiotic one. The metabolic activity and physiology of both cells are shaped by this coexistence. We believe that where this appreciation has greatest significance is in the field of drug discovery. Evolution rewards efficiency, and recent data from many groups discussed in this review indicate that has evolved to utilize the environmental cues within its host to control large genetic programs or regulons. But these regulons may represent chinks in the bacterium’s armor because they include off-target effects, such as the constraint of the metabolic plasticity of . A prime example is how the presence of cholesterol within the host cell appears to limit the ability of to fully utilize or assimilate other carbon sources. And that is the reason for the title of this review. We believe firmly that, to understand the physiology of and to identify new drug targets, it is imperative that the bacterium be interrogated within the context of its host cell. The constraints induced by the environmental cues present within the host cell need to be preserved and exploited. The -infected macrophage truly is the “minimal unit of infection.”

  • Citation: VanderVen B, Huang L, Rohde K, Russell D. 2016. The Minimal Unit of Infection: in the Macrophage. Microbiol Spectrum 4(6):TBTB2-0025-2016. doi:10.1128/microbiolspec.TBTB2-0025-2016.

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/content/journal/microbiolspec/10.1128/microbiolspec.TBTB2-0025-2016
2016-12-09
2021-04-16

Abstract:

The interaction between and its host cell is highly complex and extremely intimate. Were it not for the disease, one might regard this interaction at the cellular level as an almost symbiotic one. The metabolic activity and physiology of both cells are shaped by this coexistence. We believe that where this appreciation has greatest significance is in the field of drug discovery. Evolution rewards efficiency, and recent data from many groups discussed in this review indicate that has evolved to utilize the environmental cues within its host to control large genetic programs or regulons. But these regulons may represent chinks in the bacterium’s armor because they include off-target effects, such as the constraint of the metabolic plasticity of . A prime example is how the presence of cholesterol within the host cell appears to limit the ability of to fully utilize or assimilate other carbon sources. And that is the reason for the title of this review. We believe firmly that, to understand the physiology of and to identify new drug targets, it is imperative that the bacterium be interrogated within the context of its host cell. The constraints induced by the environmental cues present within the host cell need to be preserved and exploited. The -infected macrophage truly is the “minimal unit of infection.”

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The Minimal Unit of Infection: in the Macrophage
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Citation: VanderVen B, Huang L, Rohde K, Russell D. 2016. The minimal unit of infection: in the macrophage. microbiolspec 4(6): doi:10.1128/microbiolspec.TBTB2-0025-2016
/content/microbiolspec/10.1128/microbiolspec.TBTB2-0025-2016.fig1
FIGURE 1
Life and death dynamics during long-term intracellular survival of . Survival assays. Resting murine bone marrow-derived macrophages were infected at low multiplicity of infection (∼1:1) with CDC1551. Viable CFU were quantified at day 0 and at 2-day intervals postinfection (p.i.) over a 14-day time course by lysis of monolayers, serial dilution, and plating on 7H10 medium. Error bars indicate standard error of the mean. Replication clock plasmid. The percentage of bacteria containing the pBP10 plasmid during growth in resting macrophages was determined by comparing CFU (mean ± standard deviation) recovered on kanamycin versus nonselective media (red). The cumulative bacterial burden (CBB) (black) was determined by mathematical modeling based on total viable CFU and plasmid frequency data. Data shown represent two independent experiments. The “bottleneck” response. Temporal expression profiles of genes differentially regulated at day 2 p.i., including genes from A that were upregulated (red) or downregulated (blue) >1.5-fold (shown as ratio of signal intensity relative to control). Note the maximal change in transcript levels at day 2 p.i. followed by majority trending back toward control levels. “Guilt by association” analysis. Genes regulated in sync with known virulence regulons. i.e., the DosR regulon, were identified by using a highly regulated member of this regulon, , in place of synthetic profiles. This figure is reproduced from Rohde et al. ( 24 ).
microbiolspec/4/6/TBTB2-0025-2016-fig1_thmb.gif
microbiolspec/4/6/TBTB2-0025-2016-fig1.gif
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FIGURE 1

Life and death dynamics during long-term intracellular survival of . Survival assays. Resting murine bone marrow-derived macrophages were infected at low multiplicity of infection (∼1:1) with CDC1551. Viable CFU were quantified at day 0 and at 2-day intervals postinfection (p.i.) over a 14-day time course by lysis of monolayers, serial dilution, and plating on 7H10 medium. Error bars indicate standard error of the mean. Replication clock plasmid. The percentage of bacteria containing the pBP10 plasmid during growth in resting macrophages was determined by comparing CFU (mean ± standard deviation) recovered on kanamycin versus nonselective media (red). The cumulative bacterial burden (CBB) (black) was determined by mathematical modeling based on total viable CFU and plasmid frequency data. Data shown represent two independent experiments. The “bottleneck” response. Temporal expression profiles of genes differentially regulated at day 2 p.i., including genes from A that were upregulated (red) or downregulated (blue) >1.5-fold (shown as ratio of signal intensity relative to control). Note the maximal change in transcript levels at day 2 p.i. followed by majority trending back toward control levels. “Guilt by association” analysis. Genes regulated in sync with known virulence regulons. i.e., the DosR regulon, were identified by using a highly regulated member of this regulon, , in place of synthetic profiles. This figure is reproduced from Rohde et al. ( 24 ).

Source: microbiolspec December 2016 vol. 4 no. 6 doi:10.1128/microbiolspec.TBTB2-0025-2016
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FIGURE 2
Demonstrating the usefulness of the :: reporter strain in assessing and reporting on the localized induction of iNOS at the site of infection. Phosphate-buffered saline-immunized (naive) mice and mice vaccinated with heat-killed (vac) were infected with the ::GFP, ::mCherry Erdman reporter strain. Fluorescence induction of the promoter-dependent GFP is higher at 14 days in the vaccinated animals, as assessed by confocal microscopy of thick tissue sections that were scored subsequently by Volocity . The thick tissue sections were probed with antibodies against murine NOS2 (magenta), demonstrating the colocalization between GFP induction and NOS2 expression at the site(s) of infection. N.S., not significant. Data shown are detailed in Sukumar et al. ( 31 ).
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FIGURE 2

Demonstrating the usefulness of the :: reporter strain in assessing and reporting on the localized induction of iNOS at the site of infection. Phosphate-buffered saline-immunized (naive) mice and mice vaccinated with heat-killed (vac) were infected with the ::GFP, ::mCherry Erdman reporter strain. Fluorescence induction of the promoter-dependent GFP is higher at 14 days in the vaccinated animals, as assessed by confocal microscopy of thick tissue sections that were scored subsequently by Volocity . The thick tissue sections were probed with antibodies against murine NOS2 (magenta), demonstrating the colocalization between GFP induction and NOS2 expression at the site(s) of infection. N.S., not significant. Data shown are detailed in Sukumar et al. ( 31 ).

Source: microbiolspec December 2016 vol. 4 no. 6 doi:10.1128/microbiolspec.TBTB2-0025-2016
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FIGURE 3
Flow sorting of activated and resting -infected host cells demonstrates that the drug sensitivity of recovered from infection correlates inversely with the immune activation status of the host phagocyte. recovered from activated host cells were more tolerant to both INH and RIF than those recovered from resting host cells. C57BL/6J mice were infected with mCherry-expressing Erdman for 21 days, and -containing myeloid cells with different immune activation status isolated from lung tissue by using flow cytometry. CD11b mCherry CD80 cells (activated population) and CD11b mCherry CD80 cells (resting population) were sorted according to the depicted gating strategies. Isolated cells were established in culture and subjected to treatment with 1 μg/ml INH or RIF or an equivalent volume of dimethyl sulfoxide (DMSO). Following 24 h of drug treatment, bacterial survival was determined by CFU enumeration , and the percentage of surviving drug treatment was quantified by normalizing bacterial load in drug-treated samples against that in DMSO-treated samples . This figure is modified from the work of Liu et al. ( 34 ).
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FIGURE 3

Flow sorting of activated and resting -infected host cells demonstrates that the drug sensitivity of recovered from infection correlates inversely with the immune activation status of the host phagocyte. recovered from activated host cells were more tolerant to both INH and RIF than those recovered from resting host cells. C57BL/6J mice were infected with mCherry-expressing Erdman for 21 days, and -containing myeloid cells with different immune activation status isolated from lung tissue by using flow cytometry. CD11b mCherry CD80 cells (activated population) and CD11b mCherry CD80 cells (resting population) were sorted according to the depicted gating strategies. Isolated cells were established in culture and subjected to treatment with 1 μg/ml INH or RIF or an equivalent volume of dimethyl sulfoxide (DMSO). Following 24 h of drug treatment, bacterial survival was determined by CFU enumeration , and the percentage of surviving drug treatment was quantified by normalizing bacterial load in drug-treated samples against that in DMSO-treated samples . This figure is modified from the work of Liu et al. ( 34 ).

Source: microbiolspec December 2016 vol. 4 no. 6 doi:10.1128/microbiolspec.TBTB2-0025-2016
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FIGURE 4
Examination of the bacterial stress reporter strain ::GFP, ::mCherry Erdman at the level of different host phagocyte populations in murine lung infection model. The flow cytometry gating strategy for the identification of -infected phagocyte subsets from infected mouse lung, showing preliminary identification of alveolar macrophages, recruited interstitial macrophages, and monocyte-derived dendritic cell populations. The level of expression of NO-driven GFP under regulation of the promoter, identifying those phagocytes that induce the highest level of bacterial stress, and how the stress intensifies from 14 to 28 days postinfection. The levels of induction of expression of GFP indicate that the most stressful host cells appear to be neutrophils and the least stressful appear to be alveolar macrophages. Labeling of the host cells with antibody against NOS2 demonstrates the direct correlation between expression levels of the host nitric oxide synthase, NOS2, and levels of expression of the bacterial stress response reporter ::GFP, shown in panel B. MFI, mean fluorescence intensity; PMN, polymorphonuclear leukocytes; IM, recruited interstitial macrophages; AM, alveolar macrophages.
microbiolspec/4/6/TBTB2-0025-2016-fig4_thmb.gif
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Image of FIGURE 4

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FIGURE 4

Examination of the bacterial stress reporter strain ::GFP, ::mCherry Erdman at the level of different host phagocyte populations in murine lung infection model. The flow cytometry gating strategy for the identification of -infected phagocyte subsets from infected mouse lung, showing preliminary identification of alveolar macrophages, recruited interstitial macrophages, and monocyte-derived dendritic cell populations. The level of expression of NO-driven GFP under regulation of the promoter, identifying those phagocytes that induce the highest level of bacterial stress, and how the stress intensifies from 14 to 28 days postinfection. The levels of induction of expression of GFP indicate that the most stressful host cells appear to be neutrophils and the least stressful appear to be alveolar macrophages. Labeling of the host cells with antibody against NOS2 demonstrates the direct correlation between expression levels of the host nitric oxide synthase, NOS2, and levels of expression of the bacterial stress response reporter ::GFP, shown in panel B. MFI, mean fluorescence intensity; PMN, polymorphonuclear leukocytes; IM, recruited interstitial macrophages; AM, alveolar macrophages.

Source: microbiolspec December 2016 vol. 4 no. 6 doi:10.1128/microbiolspec.TBTB2-0025-2016
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FIGURE 5
The ::GFP reporter strain responds to cholesterol during infection. GFP expression is induced with propionate and cholesterol. The GFP mean fluorescence intensity (MFI) was determined from 10,000 bacteria by flow cytometery. Resting bone marrow-derived macrophages were infected with the ::GFP reporter strain for 24 h. Within macrophages the ::GFP reporter is active. Treating macrophages infected with the ::GFP reporter strain with one of the inhibitors of cholesterol breakdown, identified in a recent drug screen ( 36 ), abolishes induction of the reporter signal. Nuclei are stained with DAPI, and the images are courtesy of Kaley Wilburn. DAPI, 4′,6-diamidino-2-phenylindole.
microbiolspec/4/6/TBTB2-0025-2016-fig5_thmb.gif
microbiolspec/4/6/TBTB2-0025-2016-fig5.gif
Image of FIGURE 5

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FIGURE 5

The ::GFP reporter strain responds to cholesterol during infection. GFP expression is induced with propionate and cholesterol. The GFP mean fluorescence intensity (MFI) was determined from 10,000 bacteria by flow cytometery. Resting bone marrow-derived macrophages were infected with the ::GFP reporter strain for 24 h. Within macrophages the ::GFP reporter is active. Treating macrophages infected with the ::GFP reporter strain with one of the inhibitors of cholesterol breakdown, identified in a recent drug screen ( 36 ), abolishes induction of the reporter signal. Nuclei are stained with DAPI, and the images are courtesy of Kaley Wilburn. DAPI, 4′,6-diamidino-2-phenylindole.

Source: microbiolspec December 2016 vol. 4 no. 6 doi:10.1128/microbiolspec.TBTB2-0025-2016
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