Optimized APEX2 peroxidase-mediated proximity labeling in fast- and slow-growing mycobacteria.

Proximity labeling is a technology for tagging proteins and other biomolecules in living cells. These methods use enzymes that generate reactive species whose properties afford high spatial resolution for the localization of proteins to subcellular compartments and the identification of endogenous interaction partners. Here we present the adaptation of the engineered peroxidase APEX2 to proximity labeling in mycobacteria, including the human pathogen Mycobacterium tuberculosis. APEX2 is uniquely suited for general use in bacteria because unlike other proximity labeling enzymes, it does not depend on metabolites like ATP that are found in the cytoplasm, but are absent from the bacterial periplasm. Importantly, we found that in slow-growing mycobacteria like M. tuberculosis, codon usage optimization is required for APEX2 export into the periplasm via fusion to an N-terminal secretion signal. APEX2 expressed from codon-optimized genes affords robust, compartment-specific protein labeling in the cytoplasm and the periplasm of both fast- and slow-growing species. Here we detail these updated constructs and provide an optimized protocol for APEX2-mediated protein labeling in mycobacteria. We expect this approach to be broadly useful for determining the localization of specific proteins, cataloging subcellular proteomes, and identifying interaction partners of 'bait' proteins expressed as fusions to APEX2.

Characteristics of monolayer formation in vitro by the chytrid Batrachochytrium dendrobatidis.

Batrachochytrium dendrobatidis is a globally distributed generalist pathogen that has driven many amphibian populations to extinction. The life cycle of B. dendrobatidis has two main cell types, motile zoospores, and sessile reproductive sporangia. When grown in a nutrient-rich liquid medium, B. dendrobatidis forms aggregates of sporangia that transition into monolayers on surfaces and at the air-liquid interface. Pathogenic microorganisms use biofilms as mechanisms of group interactions to survive under harsh conditions in the absence of a suitable host. We used fluorescent and electron microscopy, crystal violet, transcriptomic, and gas chromatographic analyses to understand the characteristics of B. dendrobatidis monolayers. The cell-free monolayer fraction showed the presence of extracellular ribose, mannose, xylose, galactose, and glucose. Transcriptome analysis showed that 27%, 26%, and 4% of the genes were differentially expressed between sporangia/zoospores, monolayer/zoospores, and sporangia/monolayer pairs respectively. In pond water studies, zoospores developed into sporangia and formed floating aggregates at the air-water interface and attached film on the bottom of growth flasks. We propose that B. dendrobatidis can form surface-attached monolayers in nutrient-rich environments and aggregates of sporangia in nutrient-poor aquatic systems. These monolayers and aggregates may facilitate dispersal and survival of the fungus in the absence of a host. We provide evidence for using a combination of plant-based chemicals, allicin, gingerol, and curcumin as potential anti-chytrid drugs to mitigate chytridiomycosis.