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Detection of bioorthogonal groups by correlative light and electron microscopy allows imaging of degraded bacteria in phagocytes.

ABSTRACT: The interaction between parasites and phagocytic immune cells is a key inter-species interaction in biology. Normally, phagocytosis results in the killing of invaders, but obligate intracellular parasites hijack the pathway to ensure their survival and replication. The in situ study of these parasites in the phagocytic pathway is very difficult, as genetic modification is often complicated and, if successful, only allows the tracking of pathogen phagocytosis up until the degradation of the engineered reporter constructs. Here we combine bioorthogonal chemistry with correlative light-electron microscopy (CLEM) to follow bacterial processing in the phagolysosomal system. Labelled bacteria are produced using bioorthogonal non-canonical amino tagging (BONCAT), precluding the need for any genetic modification. The bacterial proteome - even during degradation - was then visualised using a novel CLEM-based approach. This allowed us to obtain high resolution information about the subcellular location of the degrading bacteria, even after the proteolytic degradation of reporter constructs. To further explore the potential of CLEM-based imaging of bioorthogonal functionalities, azide-labelled glycans were imaged by this same approach, as well as active-subpopulations of enzymes using a 2-step activity-based protein profiling strategy.

SUBMITTER: van Elsland DM

PROVIDER: S-EPMC5529995 | biostudies-literature |

REPOSITORIES: biostudies-literature

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