21. The number of sorted cells can vary. For one experiment,
samples from 3 to 5 mice are typically pooled before cell sorting to obtain enough cells for analysis. Collecting
0.5–1 Â 10
4 cells of each population usually generates sufficient sample for imaging and quantification.
Acknowledgments
This work was supported by grants from the National Institutes of
Health (R21 AI137759 and R01 AI143768) to ST. DG was supported by a National Institutes of Health training grant T32
GM007310. LH was supported in part by a Centers of Biomedical
Research Excellence (COBRE) grant from the National Institutes
of Health through the Center for Microbial Pathogenesis and Host
Inflammatory Responses at the University of Arkansas for Medical
Sciences (P20 GM103625).
References
1. MacGilvary NJ, Tan S (2018) Fluorescent
Mycobacterium tuberculosis reporters: Illuminating host-pathogen interactions. Pathog Dis
76:fty017
2. Kentner D, Sourjik V (2010) Use of fluorescence microscopy to study intracellular signaling in bacteria. Annu Rev Microbiol
64:373–390
3. Campbell-Valois FX, Sansonetti PJ (2014)
Tracking bacterial pathogens with geneticallyencoded reporters. FEBS Lett 588:2428–2436
4. Rodriguez EA, Campbell RE, Lin JY et al
(2017) The growing and glowing toolbox of
fluorescent and photoactive proteins. Trends
Biochem Sci 42:111–129
5. Kremer L, Baulard A, Estaquier J et al (1995)
Green fluorescent protein as a new expression
marker in mycobacteria. Mol Microbiol
17:913–922
6. Tan S, Sukumar N, Abramovitch RB et al
(2013) Mycobacterium tuberculosis responds
to chloride and pH as synergistic cues to the
immune status of its host cell. PLoS Pathog 9:
e1003282
7. Sukumar N, Tan S, Aldridge BB et al (2014)
Exploitation of Mycobacterium tuberculosis
reporter strains to probe the impact of vaccination at sites of infection. PLoS Pathog 10:
e1004394
8. Nazarova EV, Montague CR, La T et al (2017)
Rv3723/LucA coordinates fatty acid and cholesterol uptake in Mycobacterium tuberculosis.
eLife 6:e26969
9. Joyce G, Williams KJ, Robb M et al (2012) Cell
division site placement and asymmetric growth
in mycobacteria. PLoS One 7:e44582
10. Stanley SA, Barczak AK, Silvis MR et al (2014)
Identification of host-targeted small molecules
that restrict intracellular Mycobacterium tuberculosis growth. PLoS Pathog 10:e1003946
11. Sorrentino F, Gonzalez del Rio R, Zheng X
et al (2015) Development of an intracellular
screen for new compounds able to inhibit
Mycobacterium tuberculosis growth in human
macrophages. Antimicrob Agents Chemother
60:640–645
12. VanderVen BC, Fahey RJ, Lee W et al (2015)
Novel inhibitors of cholesterol degradation in
Mycobacterium tuberculosis reveal how the bacterium’s metabolism is constrained by the
intracellular environment. PLoS Pathog 11:
e1004679
13. Rodrigues Felix C, Gupta R, Geden S et al
(2017) Selective killing of dormant Mycobacterium tuberculosis by marine natural products.
Antimicrob
Agents
Chemother
61:
e00743–e00717
14. Meniche X, Otten R, Siegrist MS et al (2014)
Subpolar addition of new cell wall is directed by
DivIVA in mycobacteria. Proc Natl Acad Sci U
S A 111:E3243–E3251
15. Rego EH, Audette RE, Rubin EJ (2017) Deletion of a mycobacterial divisome factor collapses single-cell phenotypic heterogeneity.
Nature 546:153–157
382
David Giacalone et al.
samples from 3 to 5 mice are typically pooled before cell sorting to obtain enough cells for analysis. Collecting
0.5–1 Â 10
4 cells of each population usually generates sufficient sample for imaging and quantification.
Acknowledgments
This work was supported by grants from the National Institutes of
Health (R21 AI137759 and R01 AI143768) to ST. DG was supported by a National Institutes of Health training grant T32
GM007310. LH was supported in part by a Centers of Biomedical
Research Excellence (COBRE) grant from the National Institutes
of Health through the Center for Microbial Pathogenesis and Host
Inflammatory Responses at the University of Arkansas for Medical
Sciences (P20 GM103625).
References
1. MacGilvary NJ, Tan S (2018) Fluorescent
Mycobacterium tuberculosis reporters: Illuminating host-pathogen interactions. Pathog Dis
76:fty017
2. Kentner D, Sourjik V (2010) Use of fluorescence microscopy to study intracellular signaling in bacteria. Annu Rev Microbiol
64:373–390
3. Campbell-Valois FX, Sansonetti PJ (2014)
Tracking bacterial pathogens with geneticallyencoded reporters. FEBS Lett 588:2428–2436
4. Rodriguez EA, Campbell RE, Lin JY et al
(2017) The growing and glowing toolbox of
fluorescent and photoactive proteins. Trends
Biochem Sci 42:111–129
5. Kremer L, Baulard A, Estaquier J et al (1995)
Green fluorescent protein as a new expression
marker in mycobacteria. Mol Microbiol
17:913–922
6. Tan S, Sukumar N, Abramovitch RB et al
(2013) Mycobacterium tuberculosis responds
to chloride and pH as synergistic cues to the
immune status of its host cell. PLoS Pathog 9:
e1003282
7. Sukumar N, Tan S, Aldridge BB et al (2014)
Exploitation of Mycobacterium tuberculosis
reporter strains to probe the impact of vaccination at sites of infection. PLoS Pathog 10:
e1004394
8. Nazarova EV, Montague CR, La T et al (2017)
Rv3723/LucA coordinates fatty acid and cholesterol uptake in Mycobacterium tuberculosis.
eLife 6:e26969
9. Joyce G, Williams KJ, Robb M et al (2012) Cell
division site placement and asymmetric growth
in mycobacteria. PLoS One 7:e44582
10. Stanley SA, Barczak AK, Silvis MR et al (2014)
Identification of host-targeted small molecules
that restrict intracellular Mycobacterium tuberculosis growth. PLoS Pathog 10:e1003946
11. Sorrentino F, Gonzalez del Rio R, Zheng X
et al (2015) Development of an intracellular
screen for new compounds able to inhibit
Mycobacterium tuberculosis growth in human
macrophages. Antimicrob Agents Chemother
60:640–645
12. VanderVen BC, Fahey RJ, Lee W et al (2015)
Novel inhibitors of cholesterol degradation in
Mycobacterium tuberculosis reveal how the bacterium’s metabolism is constrained by the
intracellular environment. PLoS Pathog 11:
e1004679
13. Rodrigues Felix C, Gupta R, Geden S et al
(2017) Selective killing of dormant Mycobacterium tuberculosis by marine natural products.
Antimicrob
Agents
Chemother
61:
e00743–e00717
14. Meniche X, Otten R, Siegrist MS et al (2014)
Subpolar addition of new cell wall is directed by
DivIVA in mycobacteria. Proc Natl Acad Sci U
S A 111:E3243–E3251
15. Rego EH, Audette RE, Rubin EJ (2017) Deletion of a mycobacterial divisome factor collapses single-cell phenotypic heterogeneity.
Nature 546:153–157
382
David Giacalone et al.