Describing the World Within Whole Cell Assays for Phagosome Contents

Until recently, research into phagosome biogenesis was largely limited to analyzing which compartmental marker molecules are present on phagosomes at which stage

Fluorescence and compartment markers

Hard X-ray micro probe

Transmission electron microscopy

- Immunofluorescence (qualitative, quantitative) ■ Elemental analysis

- Fluorescent fusion proteins

- Fluorescent lipids

- Fluorescent probes for small compounds

Fluorescence and compartment markers

Hard X-ray micro probe

Transmission electron microscopy

Host cell manipulation

Genetic knockout Genetic knock in RNA interference

Phagosome membrane present? DAMP (acidification) Lysosomal tracer colocalization UJtrastructural compartment characteristics

Host cell manipulation

Genetic knockout Genetic knock in RNA interference

Homogenate {post-nuclear supernatant)

Density gradient -Magnetic separation -Latex bead floatation -Organelle electrophoresis -

Host p rote o m ics

Purified phagosomes

Cytoskeleton-phagosome

assays

Host lipidomics

Homogenate {post-nuclear supernatant)

Purified phagosomes

Fluorescence-activated cell sorter (FACS)

- Attached vs. ingested particles

- Quantification marker distribution

- Phagosome acidification

- "Proximity assays"

- Microbial mutant phagosome analysis

- Phagosome sorting

Pathogen proteomics

Cell-free phagosome fusion assays

Pathogen proteomics

Cell-free phagosome fusion assays

Fluorescence-activated cell sorter (FACS)

- Attached vs. ingested particles

- Quantification marker distribution

- Phagosome acidification

- "Proximity assays"

- Microbial mutant phagosome analysis

- Phagosome sorting

Transcriptomics Figure 5.1 Various methods can be employed to study phagosome biogenesis, including microscopic (top), biochemical (bottom) and FACS-driven (right). Genetic host cell manipulation adds to the arsenal of methods that can be used to manipulate and understand phagosome biogenesis.

of maturation. In these studies the pathogen-containing phagosomes ("vacuoles") would be assigned certain compartment characteristics such as those of a sorting or late endosome. Knowing a pathogen's compartmentation allows the microbiologists to focus their virulence research on certain metabolic, environmental, ecological and physiological aspects of pathogen biology (e.g., a microorganism multiplying in the endoplasmic reticulum (ER) does not have to develop long-lasting acid stress resistance). Compartment identification also helps to focus on certain host aspects of infection (e.g., a pathogen living in a vacuole that resembles the early endosome will likely be able to access and probably requires some nutrients funneled into the endocytic system).

In the long run, however, in order to really understand the environment which a particle, pathogenic or not, encounters in the phagocyte, methods have to be developed that allow the quantification of intraphagosome parameters such as pH, oxygen pressure, concentrations of ions and micronutrients, presence of hydrolytic enzymes, or osmolarity. An added advantage would be the ability to monitor these parameters in nonfixed, that is, live cells in real time. Knowledge of the kinetics of the processes can then be compared with data obtained with microorganisms mutated in relevant metabolic or environmental response pathways, for example, oxygen resistance, glucose metabolism or ion scavenging. In the end, this information will provide important pieces in the reconstruction of the complete puzzle of "host cell-pathogen interplay."

Unfortunately, until recently few assays have been available that allow quantification of these variables. Applying some new methods, however, marker molecule distribution, intraphagosomal pH, lysosome content incorporation and hydrolytic activities in phagosomes can be quantified and rough estimates about their metal ion contents can be made.

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