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11
Intracellular Localization of
Brucella abortus
and
Francisella tularensis
in Primary Murine Macrophages
Jean Celli
Summary
Intracellular bacterial pathogens have evolved sophisticated strategies to survive and
proliferate within cells of their hosts. Studying their intracellular life cycle is key to
understanding virulence and requires methodologies that can identify the compartments
in which they localize and characterize the replicative niche they generate. Here, we
describe immunofluorescence-based microscopy techniques applied to the intracellular
pathogens
Brucella abortus
and
Francisella tularensis
during their respective intracellular
cycles inside murine bone marrow-derived macrophages. Standard immunofluorescence
techniques are used to define the intracellular localization of the pathogens based on their
co-localization with specifically labeled macrophage organelles. In addition, we describe
an assay to assess the integrity of
Francisella
-containing phagosomes and bacterial release
into the macrophage cytoplasm, which is a hallmark of
Francisella
intracellular patho-
genesis.
Key Words:
Brucella
;
Francisella
; macrophages; intracellular cycle; endosomes,
endoplasmic reticulum; confocal immunofluorescence microscopy; digitonin.
1. Introduction
Survival and proliferation within mammalian cells are key virulence features
of bacterial pathogens with an intracellular life cycle
(1)
. Such bacteria have
evolved sophisticated mechanisms to avoid intracellular bactericidal functions
of host cells, many of which aim to modulate host functions to control their
intracellular trafficking and reach or generate a niche permissive for repli-
cation. An essential step in our understanding of the intracellular pathogenesis
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