Dataset Information

Profiling the heme-binding proteomes of bacteria using chemical proteomics

ABSTRACT: Heme is a cofactor with myriad roles and is essential to almost all living organisms. Accordingly, bacte-rial pathogens have developed numerous mechanisms to acquire heme from their hosts. Beyond classi-cal gas transport and catalytic functions, heme is increasingly appreciated as a tightly controlled signal-ling molecule regulating protein expression. However, heme acquisition, biosynthesis and regulation is poorly understood beyond a few model organisms, and the heme-binding proteome has yet to be fully characterised in bacteria. Yet as heme homeostasis is critical for bacterial survival, heme-binding pro-teins are promising drug targets. Herein we report a chemical proteomics method for global profiling of heme-binding proteins in live cells for the first time. Employing a panel of heme-based clickable and photoaffinity probes enabled the profiling of 32-54% of the known heme-binding proteomes in Gram-positive and Gram-negative bacteria, and revealed previously unknown potential heme interactors. This simple-to-implement profiling strategy could be interchangeably applied to different cell types and sys-tems and fuel future research into heme biology.

INSTRUMENT(S): Orbitrap Fusion, Q Exactive HF

ORGANISM(S): Enterococcus Faecalis (streptococcus Faecalis) Escherichia Coli

SUBMITTER: Isabel Wilkinson

LAB HEAD: Stephan A. Sieber

PROVIDER: PXD036133 | Pride | 2023-12-05

REPOSITORIES: Pride

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Publications

Profiling the Heme-Binding Proteomes of Bacteria Using Chemical Proteomics.

Wilkinson Isabel V L IVL Bottlinger Max M El Harraoui Yassmine Y Sieber Stephan A SA

Angewandte Chemie (International ed. in English) 20230123 9

Heme is a cofactor with myriad roles and essential to almost all living organisms. Beyond classical gas transport and catalytic functions, heme is increasingly appreciated as a tightly controlled signalling molecule regulating protein expression. However, heme acquisition, biosynthesis and regulation is poorly understood beyond a few model organisms, and the heme-binding proteome has not been fully characterised in bacteria. Yet as heme homeostasis is critical for bacterial survival, heme-bindin ...[more]

PMID: 36495310

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Project description:Plasmodium falciparum, the most pathogenic human malaria parasite, infects millions of human beings and causes a serious public health threat. Currently, the most potent anti-malarial drugs are artemisinin and its derivatives1,2. Artemisinin is a sesquiterpene lactone with an endoperoxide bridge3. The activation of artemisinin requires the cleavage of the endoperoxide bridge in the presence of iron sources4. Once activated, artemisinins are converted into highly reactive carbon-centered radicals5,6 that attack macromolecules through alkylation and propagate a series of damages, eventually leading to parasite death7,8. Even though several parasite proteins have been reported as the targets of artemisinin9,10, the exact mechanism of artemisinin action is still controversial and its high potency and specificity against the malaria parasite could not be fully accounted. Here, we have developed an unbiased chemical proteomics approach to directly probe the mechanism of action of artemisinin in P. falciparum in situ. An alkyne-tagged artemisinin analogue coupled with biotin enables selective purification and identification of 124 artemisinin covalent-binding protein targets, many of which are involved in essential biological processes of the parasite. In vitro assays confirm the specific artemisinin binding and inhibition of selected targets. Such a broad targeting spectrum disrupts the biochemical landscape of the parasite and causes its death. Furthermore, using the alkyne-tagged artemisinin coupled with a fluorescent dye to monitor its protein binding, we showed that heme, rather than free ferrous iron, is predominantly responsible for artemisinin activation. The extremely high level of heme released from the hemoglobin digestion by the parasite make artemisinin exceptionally potent against late-stage parasites (trophozoite and schizont stages) compared to parasites at early ring stage, which have low level of heme, mainly from endogenous synthesis. The ‘blood eating’ nature of the parasite with the release of large amounts of heme confers artemisinin with extremely high specificity against the parasites, with minimum side effects towards healthy red blood cells. Taken together, our results established a unifying model to explain the action and specificity of artemisinin in parasite killing. Our findings could facilitate the development of better alternative strategies to treat malaria in times of emerging artemisinin resistance11,12.

Project description:The response regulator HrrA belonging to the HrrSA two-component system (previously named CgtSR11) is known to be repressed by the global iron-dependent regulator DtxR in Corynebacterium glutamicum. Sequence analysis indicated an involvement of the HrrSA system in heme-dependent gene expression. Growth experiments revealed that the non-pathogenic soil bacterium C. glutamicum is able to use hemin or hemoglobin as sole iron source. In DNA microarray analyses a putative operon encoding the hemin-binding protein HtaA and the putative hemin ABC transporter HmuTUV showed a strong upregulation in heme-grown cells. Deletion of the hmu operon clearly affects heme utilization, but does not completely abolish growth on heme or hemoglobin. As a central part of this study, we investigated the regulon of the response regulator HrrA via comparative transcriptome analysis of a hrrA deletion mutant and C. glutamicum wild type in combination with DNA-protein interaction studies with purified HrrA protein. Our data provide evidence for a heme-dependent transcriptional activation of heme oxygenase (hmuO), an enzyme involved in the utilization of heme as iron source. Besides hmuO, HrrA was shown to activate the expression of heme-containing components of the respiratory chain, namely ctaD and the ctaE-qcrCAB operon encoding subunits I and III of cytochrome aa3 oxidase and three subunits of the cytochrome bc1 complex. Furthermore, HrrA represses almost all genes involved in heme biosynthesis, including glutamyl-tRNA reductase (hemA), uroporphyrinogen decarboxylase (hemE), and ferrochelatase (hemH). Thus, our data clearly emphasize a central role of the HrrSA system in the control of heme homeostasis in C. glutamicum. Three biological replicates of each experiment were performed. Experiment 1: Transcriptome comparison of wild type grown und FeSO4 or heme as iron source; Exp. 2: WT vs. hrrA deletion mutant grown on FeSO4; Exp. 3: WT vs. hrrA mutant grown on heme. For analysis via DNA microarraysose RNA was isolated from exponentially growing cells cultivated in CgXII medium containing glucose as carbon source and either 2.5 uM FeSO4 or 2.5 uM heme as iron source.

Dataset Information

Profiling the heme-binding proteomes of bacteria using chemical proteomics

Publications

Profiling the Heme-Binding Proteomes of Bacteria Using Chemical Proteomics.

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