An antimicrobial origin of organelle targeting peptides – ChloroMitoRAMP

This original project is based on the combination of bioinformatics and computational approaches to analyze the evolutionary history of OTP import and HA-RAMP resistance mechanisms with biochemical and genetic reductionist experimental approaches. These latter are based on the unique ability to routinely transform both the chloroplast and nuclear genomes of C. reinhardtii and on the power of the tools of bacterial microbiology. Placed in light of HA-RAMP bacterial resistance factors, our phylogenetic analyzes will infer plausible evolutionary scenarios for the origin of the translocation apparatus and help us understand the «why« through their history rather than the «how«. through their existing function. Likewise, we should also make major advances on the poorly understood mechanism of bacteria 'importing and destroying' resistance to HA-RAMP, much less studied than those based on the extrusion of AMP into the external medium, the export of peptidases or remodeling of the phospholipid content of bacterial membranes. Genetic and biochemical experiments will allow to test our working hypotheses.

We set-up a comparison short peptides that do not share a significant similarity by using a more stable linear regression instead partial least-squares discriminant analysis for the knowledge-based classification of the peptides. We confirmed the physico-chemical proximities between oTP and HA-RAMPs, apart from signal peptides targeting other subcellular compartments (Garrido et al., Cells, 2020). Based on this analysis, we proposed a functional and physico-chemical classification of HA-RAMP, classically classified so far based on the organism from which they were isolated.
We confirmed the cross-functionality between these peptides, by showing that chemically synthesized TPs can exhibit antimicrobial activity. Conversely, we showed that HA-RAMPs target a fluorescent reporter to either mitochondria or the chloroplast in the model alga Chlamydomonas reinhardtii. These results are presented in an article we published in 2020 (Garrido et al., Cells, 2020)
We also reconstructed the evolutionary history of the peptidases involved in the cleavage and post-cleavage degradation of oTPs (the Stromal Processing Peptidase, SPP, the Matrix Processing Peptidase, MMP, the Presequence Protease, PreP, and the Organellar OligoPeptidase, OOP) and estimated the phylogenetic gene trees for each peptidase. The obtained gene trees proved robust and this preliminary results show that at least one horizontal transfer event from a bacterial genome explains the origin of each of these peptidases, and that the bacterial homologs closest to these eukaryotic peptidases are derived from bacteria resistant to antimicrobial peptides, and bacteria whose ancestors gave birth to chloroplasts and mitochondria. These results therefore support the hypothesis of a common evolutionary origin between peptidases degrading HA-RAMP and peptidases degrading oTPs.

Our bioinformatic analysis allowed us to discriminate key physico-chemical properties between mTP and cTP, that we further exploited to decipher the molecular properties required for an HA-RAMPs to efficiently target either to chloroplast or mitochondria. Using over 200 chimeric HA-RAMP-oTP constructs controlling the subcellular localization of a fluorescent reporter in Chlamydomonas, we showed that organelle specificity determinants responsible for targeting to either the mitochondria or the chloroplast are pre-existing among naturally occurring HA-RAMPs. We also contributed to the ongoing effort to understand what these specificity determinants are, showing that chloroplast targeting requires longer, more disordered peptides that are predicted to interact with proteins through semi-conserved motifs. A paper detailing our findings is in preparation.
Protocols aimed at testing antimicrobial peptides toxicity were initiated using different media and conditions, that will allow to test phylogenomic based predictions. Antipeptides are likely to perturb cell envelope homeostasis and targeted bacteria are predicted to mount an adaptative response. CpxR a known cell envelope stress sensing regulator has been studied for its effect on membrane homeostasis and particularly phospholipid synthesis.

• Garrido C, Caspari OD, Choquet Y, Wollman F-A, Lafontaine I. 2020. “Evidence Supporting an Antimicrobial Origin of Targeting Peptides to Endosymbiotic Organelles”. Cells 9 (8): 1795.https://doi.org/10.3390/cells9081795.
• Caspari OD, Lafontaine I. (2021) The role of antimicrobial peptides in the evolution of endosymbiotic protein import. Plos Pathogen, in press
• Hassoun Y., Bartoli J., Wahl A., Viala J., Bouveret E. (2021) Dual regulation of phosphatidylserine decarboxylase expression by envelope stress responses. Minor revision

It is now widely accepted that mitochondria result from the endosymbiosis of an ?-proteobacterium with an archaea or a protoeucaryote, ~2 By ago, while plastids derive from a later endosymbiosis (~1.0-1.5 By ago) between a cyanobacterium and a phagotrophic protist. These endosymbiotic events resulted in a massive gene transfer from the organelle progenitors to the nucleus of the host cell. Most of the organelle proteome is now nuclear-encoded, translated in the cytosol and imported back in the organelle. Such import relies on organelle targeting peptides (oTPs, either targeting to mitochondria, mTPs, or to chloroplast, cTPs) at the N-terminus of most organelle-targeted proteins. These oTPs enable the translocation of the imported proteins across the envelope of the organelle.
How these imported proteins acquired an organelle targeting sequence and how the organelle progenitors acquired the corresponding import machinery remains an enigma.
To survive, the proto-endosymbiont had to be able to resist the innate immunity of the host cell, which includes the production of Helical Amphiphilic AntiMicrobial Peptides (HA-RAMPs), a primary defence mechanism of ubiquitous significance. Bacteria facing the action of HA-RAMPs produced by the host cell have evolved several resistance mechanisms, including the uptake of HA-RAMP through bacterial transporters followed by their degradation by bacterial proteases, thereby recapitulating the mechanism of organelle protein targeting. On such grounds, we proposed that the organelle protein import machinery originated through co-opting an ancient bacterial resistance mechanism against AMPs.
Our project aims at testing the predictions of the above hypothesis: We will i) look for evolutionary evidence for the proposed scenario by characterising the sequence and physico-chemical similarites of oTPs and HA-RAMP to challenge the HA-RAMP origin of oTPs and explore “pre-endosymbiotic”-like situations in the genomes of extant protists, ii) assess experimentally the ability of HA-RAMP to target proteins into organelles as well as the antimicrobial potential of oTPs, with a determination of the key residues involved in the evolutionary switch from one function to the other, iii) dissect the molecular mechanisms of uptake-based bacterial resistance against HA-RAMP, compared to those of protein import into organelles, while deciphering the evolutionary history of translocons and peptidases in extant organelles, in parallel with that of bacterial transporters and peptidases involved in HA-RAMP-resistance.
As to scientific impact, this ambitious, paradigm-breaking, project will explore a new view of protein import machineries, a prerequisite for the evolution from endosymbionts to fully integrated mitochondria and chloroplasts. It will be carried out by two research teams with complementary expertises, respectively leaders in organelle biology and bacterial physiology and bridges two research fields (the biogenesis of bioenergetic organelles and the biology of antimicrobial peptides) that remain so far poorly connected, even if the role of AMPs in extant endosymbioses is being increasingly recognised.
As to applications, transferring metabolic pathways of biotechnological interest to novel species often entails the transfer of dozens of genes from one organism to another. Tailoring the rational design of protein import to introduce novel properties through endosymbiosis can be regarded as a new frontier of synthetic biology. Our proposal has also a significant potential for biomedical applications: defects in organelle import and proteolytic clearance of targeting peptides leads to the accumulation of neurotoxic ?-amyloid peptides in neuron mitochondria, a major determinant of the progression of the Alzheimer’s disease. We will also address some poorly known mechanisms of antimicrobial resistance, one of the greatest challenges to global public health today.