Chapter 18
Evaluation of Intracellular Trafficking in Macrophages
Claudia Toma and Toshihiko Suzuki
Abstract
Macrophages are phagocytic cells that constitute the primary barrier against pathogens. After phagocytosis
a single-membraned vesicle that contains the pathogen is formed. This phagosome undergoes a maturation
process to acquire an increasingly antimicrobial environment. Leptospiral uptake by macrophages induces
the formation of a Leptospira-containing phagosome (LCP). The kinetics of lysosomal marker recruitment
by the LCP is correlated with virulence. This chapter presents a protocol to study the intracellular trafficking
of Leptospira spp. within macrophages by fluorescent labeling bacteria and different markers of the
phagocytic pathway. We also describe a method to evaluate the bacterial survival within macrophages.
Key words Macrophage, Leptospira, Intracellular trafficking, Phagosome maturation, Immunofluorescence, Survival
1 Introduction
Pathogens internalized by macrophages are sequestered in phagosomes, which sequentially acquire their microbicidal properties
through a complex maturation process leading to the formation
of phagolysosomes. Thus, the intracellular trafficking of phagocytosed bacteria can be characterized by the acquisition of different
markers such as the early endosomal antigen (EEA1), the late
endosomal antigen (LAMP1), or cathepsin D [1].
Macrophage uptake of pathogenic leptospires is enhanced by
outer membrane proteins [2, 3]. After phagocytosis, L. interrogans
can evade host defense mechanisms by delaying phagosome maturation [4] and resisting to reactive oxygen species [5]. These strategies allow the successful dissemination of the pathogen to the
target organs [6]. Although L. interrogans serovar Manilae does
not induce macrophage cell death, several studies have reported
that macrophages undergo cell death by L. interrogans infection
when using serovar Lai or serovar Icterohaemorrhagiae, suggesting
that strain- and/or serovar-specific differences exist during Leptospira-macrophage interactions [7, 8].
Nobuo Koizumi and Mathieu Picardeau (eds.), Leptospira spp.: Methods and Protocols, Methods in Molecular Biology, vol. 2134,
https://doi.org/10.1007/978-1-0716-0459-5_18, © Springer Science+Business Media, LLC, part of Springer Nature 2020
199
Evaluation of Intracellular Trafficking in Macrophages
Claudia Toma and Toshihiko Suzuki
Abstract
Macrophages are phagocytic cells that constitute the primary barrier against pathogens. After phagocytosis
a single-membraned vesicle that contains the pathogen is formed. This phagosome undergoes a maturation
process to acquire an increasingly antimicrobial environment. Leptospiral uptake by macrophages induces
the formation of a Leptospira-containing phagosome (LCP). The kinetics of lysosomal marker recruitment
by the LCP is correlated with virulence. This chapter presents a protocol to study the intracellular trafficking
of Leptospira spp. within macrophages by fluorescent labeling bacteria and different markers of the
phagocytic pathway. We also describe a method to evaluate the bacterial survival within macrophages.
Key words Macrophage, Leptospira, Intracellular trafficking, Phagosome maturation, Immunofluorescence, Survival
1 Introduction
Pathogens internalized by macrophages are sequestered in phagosomes, which sequentially acquire their microbicidal properties
through a complex maturation process leading to the formation
of phagolysosomes. Thus, the intracellular trafficking of phagocytosed bacteria can be characterized by the acquisition of different
markers such as the early endosomal antigen (EEA1), the late
endosomal antigen (LAMP1), or cathepsin D [1].
Macrophage uptake of pathogenic leptospires is enhanced by
outer membrane proteins [2, 3]. After phagocytosis, L. interrogans
can evade host defense mechanisms by delaying phagosome maturation [4] and resisting to reactive oxygen species [5]. These strategies allow the successful dissemination of the pathogen to the
target organs [6]. Although L. interrogans serovar Manilae does
not induce macrophage cell death, several studies have reported
that macrophages undergo cell death by L. interrogans infection
when using serovar Lai or serovar Icterohaemorrhagiae, suggesting
that strain- and/or serovar-specific differences exist during Leptospira-macrophage interactions [7, 8].
Nobuo Koizumi and Mathieu Picardeau (eds.), Leptospira spp.: Methods and Protocols, Methods in Molecular Biology, vol. 2134,
https://doi.org/10.1007/978-1-0716-0459-5_18, © Springer Science+Business Media, LLC, part of Springer Nature 2020
199