15. At the end of the immunofluorescence staining, wash the plate
with DW to remove any salt crystals that can interfere with the
microscopic observation.
16. Analyze the samples by quantifying the number of Leptospiracontaining phagosomes (LCPs) with LAMP-1 and cathepsin
D. LCPs recruit LAMP1 during phagosome maturation; LCPs
will further recruit cathepsin D when fused with the lysosomes.
As a control of normal phagolysosome fusion, a
non-pathogenic strain (e.g., L. biflexa Patoc I) or a heat-killed
pathogenic Leptospira can be used (Fig. 1).
Acknowledgments
This work was supported by JSPS KAKENHI grant JP21590484
(C.T.).
References
1. Stuart LM, Ezekowitz RA (2005) Phagocytosis:
elegant complexity. Immunity 22:539–550
2. Toma C, Murray GL, Nohara T, Mizuyama M,
Koizumi N, Adler B, Suzuki T (2014) Leptospiral outer membrane protein LMB216 is
involved in enhancement of phagocytic uptake
by macrophages. Cell Microbiol 16:1366–1377
3. Zhang L, Zhang C, Ojcius DM, Sun D, Zhao J,
Lin X, Li L, Li L, Yan J (2012) The mammalian
cell entry (Mce) protein of pathogenic Leptospira
species is responsible for RGD motif-dependent
infection of cells and animals. Mol Microbiol
83:1006–1023
4. Toma C, Okura N, Takayama C, Suzuki T
(2011) Characteristic features of intracellular
pathogenic Leptospira in infected murine
macrophages. Cell Microbiol 13:1783–1792
5. Eshghi A, Lourdault K, Murray GL, Bartpho T,
Sermswan RW, Picardeau M, Adler B, Snarr B,
Zuerner RL, Cameron CE (2012) Leptospira
interrogans catalase is required for resistance to
H 2 O 2 and for virulence. Infect Immun
80:3892–3899
6. Picardeau M (2017) Virulence of the zoonotic
agent of leptospirosis: still terra incognita? Nat
Rev Microbiol 15:297–307
7. Hu W-L, Dong H-Y, Li Y, Ojcius DM, Li S-J,
Yan J (2017) Bid-induced release of AIF/EndoG from mitochondria causes apoptosis of
macrophages during infection with Leptospira
interrogans. Front Cell Infect Microbiol 7:471
8. Du P, Li SJ, Ojcius DM, Li KX, Hu WL, Lin X,
Sun AH, Yan J (2018) A novel Fas-binding
outer membrane protein and lipopolysaccharide
of Leptospira interrogans induce macrophage
apoptosis through the Fas/FasL-caspase-8/À3
pathway. Emerg Microbes Infect 7:135
206
Claudia Toma and Toshihiko Suzuki
with DW to remove any salt crystals that can interfere with the
microscopic observation.
16. Analyze the samples by quantifying the number of Leptospiracontaining phagosomes (LCPs) with LAMP-1 and cathepsin
D. LCPs recruit LAMP1 during phagosome maturation; LCPs
will further recruit cathepsin D when fused with the lysosomes.
As a control of normal phagolysosome fusion, a
non-pathogenic strain (e.g., L. biflexa Patoc I) or a heat-killed
pathogenic Leptospira can be used (Fig. 1).
Acknowledgments
This work was supported by JSPS KAKENHI grant JP21590484
(C.T.).
References
1. Stuart LM, Ezekowitz RA (2005) Phagocytosis:
elegant complexity. Immunity 22:539–550
2. Toma C, Murray GL, Nohara T, Mizuyama M,
Koizumi N, Adler B, Suzuki T (2014) Leptospiral outer membrane protein LMB216 is
involved in enhancement of phagocytic uptake
by macrophages. Cell Microbiol 16:1366–1377
3. Zhang L, Zhang C, Ojcius DM, Sun D, Zhao J,
Lin X, Li L, Li L, Yan J (2012) The mammalian
cell entry (Mce) protein of pathogenic Leptospira
species is responsible for RGD motif-dependent
infection of cells and animals. Mol Microbiol
83:1006–1023
4. Toma C, Okura N, Takayama C, Suzuki T
(2011) Characteristic features of intracellular
pathogenic Leptospira in infected murine
macrophages. Cell Microbiol 13:1783–1792
5. Eshghi A, Lourdault K, Murray GL, Bartpho T,
Sermswan RW, Picardeau M, Adler B, Snarr B,
Zuerner RL, Cameron CE (2012) Leptospira
interrogans catalase is required for resistance to
H 2 O 2 and for virulence. Infect Immun
80:3892–3899
6. Picardeau M (2017) Virulence of the zoonotic
agent of leptospirosis: still terra incognita? Nat
Rev Microbiol 15:297–307
7. Hu W-L, Dong H-Y, Li Y, Ojcius DM, Li S-J,
Yan J (2017) Bid-induced release of AIF/EndoG from mitochondria causes apoptosis of
macrophages during infection with Leptospira
interrogans. Front Cell Infect Microbiol 7:471
8. Du P, Li SJ, Ojcius DM, Li KX, Hu WL, Lin X,
Sun AH, Yan J (2018) A novel Fas-binding
outer membrane protein and lipopolysaccharide
of Leptospira interrogans induce macrophage
apoptosis through the Fas/FasL-caspase-8/À3
pathway. Emerg Microbes Infect 7:135
206
Claudia Toma and Toshihiko Suzuki