Labeling strategies to track protozoan parasite proteome dynamics

Abstract

Intracellular protozoan parasites are responsible for wide-spread infectious diseases. These unicellular pathogens have complex, multi-host life cycles, which present challenges for investigating their basic biology and for discovering vulnerabilities that could be exploited for disease control. Throughout development, parasite proteomes are dynamic and support stage-specific functions, but detection of these proteins is often technically challenging and complicated by the abundance of host proteins. Thus, to elucidate key parasite processes and host-pathogen interactions, labeling strategies are required to track pathogen proteins during infection. Herein, we discuss the application of bioorthogonal non-canonical amino acid tagging and proximity-dependent labeling to broadly study protozoan parasites and include outlooks for future applications to study Plasmodium, the causative agent of malaria. We highlight the potential of these technologies to provide spatiotemporal labeling with selective parasite protein enrichment, which could enable previously unattainable insight into the biology of elusive developmental stages.

Keywords: Apicomplexan; Bioorthogonal non-canonical amino acid tagging; Plasmodium; Proximity-dependent labeling.

Figure 1.

Global methionine substitution for bioorthogonal non-canonical amino acid tagging (BONCAT). (A) The non-canonical amino acids (ncAA) L-azidohomoalanine (L-Aha) or L-homopropargylglycine (L-Hpg) are incorporated in place of L-methionine (L-Met) at initiator and internal codon sites via endogenous translation in both translationally active host and parasite cells. (B) After incorporation into proteins, ncAA can be conjugated through a click chemistry ligation reaction between the azide handle of L-Aha and an alkyne-containing tag, or, conversely, the alkyne handle of L-Hpg and an azide-containing tag. Conjugation to biotin enables affinity purification of labeled proteins, which can be identified and quantified by mass spectrometry. Alternatively, conjugation to a fluorophore enables visualization of labeled proteins by gel electrophoresis or microscopy. (C) BONCAT enables temporal control over protein labeling based on the time of ncAA addition. Adding ncAA concurrent to an experimental treatment followed by analysis enables interrogation of the nascent proteome, whereas adding ncAA prior to its replacement with L-Met enables labeling of the pre-existing proteome.

Figure 2.

Cell-specific incorporation of L-azidonorleucine (L-Anl). Parasite proteins can be specifically labeled with L-Anl by expression of a MetRS^NLL synthetase in the pathogen. In eukaryotes, MetRS^NLL charges initiator tRNA with L-Anl to facilitate its incorporation into proteins at the start codon. L-Met is incorporated at internal codons or in the absence of L-Anl. Since L-Anl is incompatible with endogenous translation, L-Met rather than L-Anl is also incorporated in host cells not expressing MetRS^NLL.The L-Anl azide moiety enables biotin/fluorophore conjugation and downstream analysis with the advantage of cell-specific (parasite not host) incorporation.

Figure 3.

Mechanisms and applications for enzyme-based proximity biotinylation. (A) BirA* and its derivatives fused to a bait protein can use exogenous biotin to covalently tag nearby interacting proteins (within 10 nm) at lysine residues. Peroxidases like APEX2 can be fused to a bait protein or to a signal sequence to enable trafficking to different subcellular localizations. APEX2 enzymes use hydrogen peroxide and biotin phenol to form a phenoxyl radical, which readily reacts with nearby proteins at electron rich residues up to 300 nm away. Labeling radius for biotin ligases and peroxidases indicated (dashed circles). (B) APEX2 (in blue) can be expressed in the host nucleus (top) or host cytosol (bottom) to biotinylate parasite effectors in cells.