Unravelling signaling pathways involved in encystment of disease-causing amoebae – AmoCyst

The process of cyst formation is called «encystment«. Although widespread in microscopic organisms, little is known about encystment due to the multitude of processes involved and the lack of genetic data (or molecular tools) in the organisms concerned. Our project provides an atlas of the changes observed at the transcript, protein, and phospho-protein levels during the first hours of encystment of the amoeba A. castellanii. Our results confirm an observation already reported by Griffiths and Maureen Bowen 50 years ago: encystment induces a strong dephosphorylation activity within the amoeba. We have completed our phospho-proteome by an analysis of the transcriptome (set of transcripts) and the proteome (sum of proteins) which showed that dephosphorylation is followed by a strong expression of genes related to protein degradation or in the synthesis of carbohydrates such as cellulose which is essential for the formation of the cyst wall. Furthermore, we observed a decrease in the expression of genes involved in the metabolic activity of the amoebae.

The output related to this project is 2 research articles (Bernard C. et al., 2022, Nature Communications 13(1): 4104 and Rolland S. et al., 2020, Pathogens 9 (5), 321) and one research article in Pre-print (Rolland S. et al., 2021, BioRxiv doi.org/10.1101/2021.01.07.425711).

The publication of Bernard C. et al., 2022 perfectly answers the question initially asked. We provide both transcriptomic, proteomic and phosphoproteomic data of A. castellanii encystment that give a complete picture of the biological processes affected during encystment. The overall analysis of the three different omics approaches shows that, in contrast to proteins, transcript and phospho-motif levels were altered as early as one hour after the onset of encystment. The changes in protein levels are more gradual, and mainly observable after eight hours. Thus, it appears that the amoeba A. castellanii responds rapidly to nutrient deficiency through protein phosphorylation events and at the level of transcription. Such a rapid response is probably due to a high number of receptors with 35 G protein coupled receptors (GPCRs). We have deposited on the bioRxiv server (Rolland S. et al., 2021, BioRxiv) a research paper in which we show that overexpression of a gene encoding a G-protein coupled receptor (GPCR) affects the encystment of A. castellanii. Similarly, overexpression of a putative bacterial-acquired acetyltransferase slows down cyst formation (Rolland S. et al., 2020, Pathogens). These 3 scientific publications provide a better understanding of the initiation of encystation in A. castellanii.

The AMOCYST project aimed to shed light on the signaling pathways differentially regulated during the encystment of the amoeba Acanthamoeba castellanii. We have achieved our goal with the publication of the article «Bernard C. et al., 2022 Nature Communications«. This mass of data should probably help to understand the molecular choreography of other organisms able to encyst such as the pathogens responsible for amoebiasis (Entamoeba histolytica) or toxoplasmosis (Toxoplasma gondii). These results can be used to search for new anti-amoebic targets effective against cysts. For example, we found that the ACA1_112600 protein is highly expressed at the RNA and protein level during the first hours of cyst formation. This 72 amino acid protein represents an interesting target for inhibiting cyst formation because it has no sequence homology, nor functional domain, with known proteins. Before approaching development organizations and their representatives (CNRS Innovation, Agence Aliénor Transfert, etc.) for technological maturation, which will ultimately be addressed to agencies that analyze water quality and to industrialists whose activities provide an environment favorable to the development of amoebae, it is necessary to search for possible structural homologies, to identify the proteins likely to interact with this protein and to validate the importance of this protein during encystment. These prospects still need to be financed.
In the AMOCYST project, we also proposed to identify the mechanism by which the bacterium Parachlamydia acanthamoebae inhibits the encystment of amoebae. While we were able to collect transcriptomics, proteomics and phosphoproteomics data, we, unfortunately, did not have the time to analyze these data. The COVID-19 crisis exacerbated the delay we had in the proteomic analysis. The exploitation of the data also requires getting new funding.

The output is:
- 2 scientific research articles (Bernard C. et al., 2022, Nature Communications 13(1): 4104; Rolland S. et al., 2020, Pathogens 9 (5), 321) ;
- 2 review articles (Samba-Louaka A., 2021, Toxins 13(8):526; Samba-Louaka A. et al., 2019, FEMS Microbiology Reviews 1; 43(4): 415-434)
- and a pre-print (research article) (Rolland S. et al., 2021, BioRxiv).

Free-living amoebae (FLA) are pathogenic and opportunistic protozoa commonly found in natural and man-made water systems that feed on bacteria. As macrophages, FLA are phagocytic cells. Thus, bacteria that resist amoebae digestion would be able to resist immune cells phagocytosis. In addition to represent a training ground for microorganisms against macrophages, FLA provide nutrients to bacteria and increase their resistance to antimicrobials facilitating dispersion of bacteria. FLA protect bacteria from harsh conditions and they are considered as a melting pot that favors exchange of DNA between various intra-amoebal microorganisms and even viruses. Thus, FLA improve emergence and transmission of bacterial pathogens. Under stress conditions such as biocides treatment, FLA differentiate into resistant forms named cysts. This process, known as encystment or encystation, needs a better understanding in order to eliminate FLA. Indeed, signaling pathways involved in encystment are poorly understood. The goal of the project AmoCyst is to highlight encystment signaling pathways in order to identify putative targets for development of molecules against amoebae. The project is relevant in a global medical context as amoebae themselves or the pathogenic bacteria associated to FLA might infect humans and lead to diseases. To achieve our goal, proteomic analysis with high-resolution mass spectrometry will highlight changes in the levels and/or in the activities of proteins during encystment. Signaling pathways involved in encystment will be established using network alignment algorithms to identify conserved and amoeba-specific pathways or proteins. These hypothetical networks will be validated with protein interactions and functional tests. We will elucidate the contribution of proteins of interest in FLA resistance to current biocides. The small amount of data on encystment underscores the originality of this project which lies on our expertise in analysis of both eukaryotic signaling pathways and FLA encystment. Our results will be valorized through publications in peer-reviewed journals and in international congresses. Furthermore, we anticipate that these results of AmoCyst will be of interest to companies dealing with water, as human activities create conditions that favor development of both FLA and amoebae-associated bacteria and a solution to eliminate them is urgently needed. This study offers, for the first time, a global analysis combining proteomic, cell function and treatment sensitivity data that will enable a global comprehension of FLA encystment.