Intracellular Localization of Brucella abortus and Francisella tularensis in Primary Murine Macrophages - Bacterial Pathogenesis: Methods and Protocols - page 127

of these bacteria is to define their intracellular trafficking, which is key to
comprehending their mechanisms of survival. Brucella abortus and Francisella
tularensis are the causative agents of brucellosis and tularemia, respectively.
Upon infection of their host, both bacteria encounter phagocytic cells, of
which macrophages constitute a major target for survival and replication (2,3) .
B. abortus ensures its intracellular survival by inhibiting the fusion of its
vacuole, the Brucella -containing vacuole (BCV), with degradative lysosomes
(4,5) . In addition to this early survival mechanism, Brucella uses a type IV
secretion system (VirB) (6) to control interactions and fusion of its vacuole
with the macrophage endoplasmic reticulum (ER) and generates an ER-derived
organelle that segregates Brucella from the degradative endocytic pathway
and allows unrestricted multiplication (4,7,8) . A different survival strategy
is used by F. tularensis , which is capable of rapid physical escape from its
initial phagosome following phagocytic uptake (9,10) . Once in the macrophage
cytoplasm, intracellular Francisella undergoes replication. As exemplified by
Brucella and Francisella , such pathogens traffic through, and interact with,
various intracellular compartments to complete their cycle. The ability to
localize these bacteria within the host cell at various stages of the infection
cycle is a major asset toward understanding pathogenesis. Here, we detail
fluorescence microscopy-based methods to localize B. abortus or F. tularensis
in various compartments of primary macrophages.
2. Materials
2.1. Bacterial Cultures
1. Tryptic soy broth (TSB) or agar (TSA) (Sigma, St Louis, MO).
2. Cystine heart agar blood (CHAB) made of cystine heart agar (CHA; Difco,
Beckton Dickinson, Sparks, MD, USA) supplemented with 9% sheep blood. The
sheep blood is chocolatized by adding it to autoclaved CHA before it cools
below 75 °C.
3. Kanamycin solution, 50 μg/mL (K0254; Sigma).
2.2. Culture and Infection of Primary Macrophages
1. C57BL/6J (or BALB/c) 6- to 10-week-old female mice for bone marrow
harvesting.
2. For bone marrow harvesting, sterile dissection tweezers, scissors, scalpels, and
1-mL syringe fitted with a 25G 5/8 -gauge needle.
3. Dulbecco's modified Eagle's medium (DMEM) containing 1 g/L d-glucose
(Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS;
Invitrogen) and 2 m M l-glutamine (Invitrogen).
4. Gentamicin solution, 10 μg/mL (G1272; Sigma).