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International audience; In Guadeloupe, a French overseas territory located in the Eastern Caribbean, infectious and non-infectious diseases, loss of biodiversity, natural disasters and global change threaten the health and well-being of animals, plants, and people. Implementing the “One Health” (OH) approach is crucial to reduce the archipelago's vulnerability to these health threats. However, OH remains underdeveloped in Guadeloupe, hampering efficient and effective intersectoral and transdisciplinary collaborations for disease surveillance and control. A multidisciplinary research group of volunteer researchers working in Guadeloupe, with collective expertise in infectious diseases, undertook a study to identify key attributes for OH operationalization by reviewing past and current local collaborative health initiatives and analyzing how much they mobilized the OH framework. The research group developed and applied an operational OH framework to assess critically collaborative initiatives addressing local health issues. Based on a literature review, a set of 13 opinion-based key criteria was defined. The criteria and associated scoring were measured through semi-directed interviews guided by a questionnaire to critically evaluate four initiatives in animal, human, plant, and environmental health research and epidemiological surveillance. Gaps, levers, and prospects were identified that will help health communities in Guadeloupe envision how to implement the OH approach to better address local health challenges. The methodology is simple, generic, and pragmatic and relies on existing resources. It can be transposed and adapted to other contexts to improve effectiveness and efficiency of OH initiatives, based on lessons-learned of local past or current multi-interdisciplinary and intersectoral initiatives.

International audience

International audience; Outer membrane proteins participate to pathogens adhesion to host cells and therefore often mediate cell infection. Such is the case for Ehrlichia ruminantium, an obligate intracellular bacterium that is transmitted by ticks and responsible for cowdriosis, a fatal disease of domestic and wild ruminants. Several experimental vaccines were developed, but the great genetic and presumably antigenic diversity of E. ruminantium make difficult to obtain an effective vaccine against all strains present in the field. In order to propose novel strategies to control cowdriosis, the interaction of E. ruminantium with its host cell, particularly the associated adhesion mechanisms must be first deciphered. A membrane protein of E. ruminantium, ERGA_CDS_01230, a probable iron transporter, initially identified by proteomics approaches in our group, was here studied for its role in host cell adhesion. The recombinant protein was expressed with post-translational glycosylation modifications and tagged GFP/Histidine in Leishmania tarentolae. Using cell biology approaches, we show that recProt01230 is able to adhere to bovine host cells and interacts with proteins from the cell lysate and the "membranes/ organelles” sub-fraction. Furthermore, recProt01230 does not adhere to heparan sulfate but other membrane polysaccharides seem to play a role in E. ruminantium's adhesion to the host cell. Indeed, preliminary experiments have shown that degrading dermatan sulfate and chondroitin sulfate at the cell surface is associated with a reduction of the number of bacteria in the host cells. Moreover, CDS ERGA_CDS_01230 is over expressed at early stages of infection when bacteria begin to attach to their host. So, our results show the implication of ERGA_CDS_01230 in the adhesion of E. ruminantium to host cells. ERGA_CDS_01230 also induces a humoral response in the vaccinated animals. In conclusion, ERGA_CDS_01230 could be a new promising target for vaccine or therapeutics development.

International audience; Bacterial pathogens have evolved numerous strategies to corrupt, hijack or mimic cellular processes in order to survive and proliferate. Among those strategies, Type IV effectors (T4Es) are proteins secreted by pathogenic bacteria to manipulate host cell processes during infection. They are delivered into eukaryotic cells in an ATP-dependent manner via the type IV secretion system, a specialized multiprotein complex. T4Es contain a wide spectrum of features including eukaryotic-like domains, localization signals or a C-terminal translocation signal. A combination of these features enables prediction of T4Es in a given bacterial genome. In this study, we developed a web-based comprehensive suite of tools with a user-friendly graphical interface. This version 2.0 of S4TE (Searching Algorithm for Type IV Effector Proteins; http://sate.cirad.fr) enables accurate prediction and comparison of T4Es. Search parameters and threshold can be customized by the user to work with any genome sequence, whether publicly available or not. Applications range from characterizing effector features and identifying potential T4Es to analyzing the effectors based on the genome G+C composition and local gene density. S4TE 2.0 allows the comparison of putative T4E repertoires of up to four bacterial strains at the same time. The software identifies T4E orthologs among strains and provides a Venn diagram and lists of genes for each intersection. New interactive features offer the best visualization of the location of candidate T4Es and hyperlinks to NCBI and Pfam databases. S4TE 2.0 is designed to evolve rapidly with the publication of new experimentally validated T4Es, which will reinforce the predictive power of the algorithm. The computational methodology can be used to identify a wide spectrum of candidate bacterial effectors that lack sequence conservation but have similar amino acid characteristics. This approach will provide very valuable information about bacterial host-specificity and virulence factors and help identify host targets for the development of new anti-bacterial molecules.

Anaplasmataceae family includes obligate intracellular pathogenic Ehrlichia and endosymbiotic Wolbachia bacteria. A key factor of bacterial pathogenesis and symbiosis with eukaryotic cells is the ability to evade the innate immune system and hijack the host cellular pathways. Anaplasmataceae use effector proteins (T4Es) to manipulate cellular processes in order to survive and proliferate. It is still difficult to predict and study the repertoires of T4Es in Anaplasmataceae. Identifying such type IV effectomes is crucial to comprehend how the bacterium establishes symbiosis or pathogenesis. Deciphering bacterial interactions with mammalian or vector cells will foster development of alternative strategies to fight against the pathogen or prevent pathogen transmission by the vector. We propose a pipeline to identify T4 effectomes in Anaplasmataceae. We first use S4TE 2.0 software as a prediction tool for T4Es. The predicted effectors are confirmed using secretion assays in Legionella pneumophila and with cellular biology approaches. Then, we screen the effectome library for intracellular localization, for particular cellular phenotypes, and for protein partners or chromatin interactions. We then investigate for potential post-translational modifications of effectors after secretion (phosphorylation, truncation). Finally, we do phenotypic screening after ectopic expression in yeast. For each remarkable phenotype, the corresponding effector genes is silenced using PNA technology. This computational based- medium-throughput screening of Anaplasmataceae type IV effectors will accelerate the dissection of bacteria-host mutualistic or pathogenic interactions and will highlight the evolutionary history shared by these bacteria. These results will promote the development of novel strategies to prevent vector-borne transmission of pathogens and alternative therapeutics.

Outer membrane proteins participate to pathogens adhesion to host cells and therefore often mediate cell infection. Such is the case for Ehrlichia ruminantium, an obligate intracellular bacterium that is transmitted by ticks and responsible for cowdriosis, a fatal disease of domestic and wild ruminants. Several experimental vaccines were developed, but the great genetic and presumably antigenic diversity of E. ruminantium make difficult to obtain an effective vaccine against all strains present in the field. In order to propose novel strategies to control cowdriosis, the interaction of E. ruminantium with its host cell, particularly the associated adhesion mechanisms must be first deciphered. A membrane protein of E. ruminantium, ERGA_CDS_01230, a probable iron transporter, initially identified by proteomics approaches in our group, was here studied for its role in host cell adhesion. The recombinant protein was expressed with post-translational glycosylation modifications and tagged GFP/Histidine in Leishmania tarentolae. Using cell biology approaches, we show that recProt01230 is able to adhere to bovine host cells and interacts with proteins from the cell lysate and the "membranes/ organelles” sub-fraction. Furthermore, recProt01230 does not adhere to heparan sulfate but other membrane polysaccharides seem to play a role in E. ruminantium's adhesion to the host cell. Indeed, preliminary experiments have shown that degrading dermatan sulfate and chondroitin sulfate at the cell surface is associated with a reduction of the number of bacteria in the host cells. Moreover, CDS ERGA_CDS_01230 is over expressed at early stages of infection when bacteria begin to attach to their host. So, our results show the implication of ERGA_CDS_01230 in the adhesion of E. ruminantium to host cells. ERGA_CDS_01230 also induces a humoral response in the vaccinated animals. In conclusion, ERGA_CDS_01230 could be a new promising target for vaccine or therapeutics development.

Permits in silico screening of proteobacteria genomes. S4TE predicts type IV effectors (T4Es) based on the combined use of more than 10 features. It analyses the genome architecture and its hit content through the visualization of the length and distribution of intergenic regions and the distribution of the hits according to local gene density. The tool is based on characteristics of known T4Es from different bacterial species, genera and classes.

International audience; The obligate intracellular pathogenic bacterium, Ehrlichia rurninantiurn, is the causal agent of heartwater, a fatal disease in ruminants transmitted by Amblyomrna ticks. So far, three strains have been attenuated by successive passages in mammalian cells. The attenuated strains have improved capacity for growth in vitro, whereas they induced limited clinical signs in vivo and conferred strong protection against homologous challenge. However, the mechanisms of pathogenesis and attenuation remain unknown. In order to improve knowledge of E. ruminantium pathogenesis, we performed a comparative transcriptomic analysis of two distant strains of E ruminantium, Gardel and Senegal, and their corresponding attenuated strains. Overall, our results showed an upregulation of gene expression encoding for the metabolism pathway in the attenuated strains compared to the virulent strains, which can probably be associated with higher in vitro replicative activity and a better fitness to the host cells. We also observed a significant differential expression of membrane protein-encoding genes between the virulent and attenuated strains. A major downregulation of map1-related genes was observed for the two attenuated strains, whereas upregulation of genes encoding for hypothetical membrane proteins was observed for the four strains. Moreover, CDS_05140, which encodes for a putative porin, displays the highest gene expression in both attenuated strains. For the attenuated strains, the significant downregulation of map1-related gene expression and upregulation of genes encoding other membrane proteins could be important in the implementation of efficient immune responses after vaccination with attenuated vaccines. Moreover, this study revealed an upregulation of gene expression for 8 genes encoding components of Type IV secretion system and 3 potential effectors, mainly in the virulent Gardel strain. Our transcriptomic study, supported by previous proteomic studies, provides and also confirms new information regarding the characterization of genes involved in E. ruminantium virulence and attenuation mechanisms.

International audience; Ehrlichia ruminantium is an obligatory intracellular bacterium that causes heartwater, a fatal disease in ruminants. Due to its intracellular nature, E. ruminantium requires a set of specific virulence factors, such as the type IV secretion system (T4SS), and outer membrane proteins (Map proteins) in order to avoid and subvert the host's immune response. Several studies have been conducted to understand the regulation of the T4SS or outer membrane proteins, in Ehrlichia, but no integrated approach has been used to understand the regulation of Ehrlichia pathogenicity determinants in response to environmental cues. Iron is known to be a key nutrient for bacterial growth both in the environment and within hosts. In this study, we experimentally demonstrated the regulation of virB, map1, and tr1 genes by the newly identified master regulator ErxR (for Ehrlichia ruminantium expression regulator). We also analyzed the effect of iron depletion on the expression of erxR gene, tr1 transcription factor, T4SS and map1 genes clusters in E. ruminantium. We show that exposure of E. ruminantium to iron starvation induces erxR and subsequently tr1, virB, and map1 genes. Our results reveal tight co-regulation of T4SS and map1 genes via the ErxR regulatory protein at the transcriptional level, and, for the first time link map genes to the virulence function sensu stricto, thereby advancing our understanding of Ehrlichia's infection process. These results suggest that Ehrlichia is able to sense changes in iron concentrations in the environment and to regulate the expression of virulence factors accordingly.

During infection, some intracellular pathogenic bacteria use a dedicated multiprotein complex known as the type IV secretion system to deliver type IV effector (T4E) proteins inside the host cell. These T4Es allow the bacteria to evade host defenses and to subvert host cell processes to their own advantage. Ehrlichia chaffeensis is a tick-transmitted obligate intracellular pathogenic bacterium, which causes human monocytic ehrlichiosis. Using comparative whole genome analysis, we identified the relationship between eight available E. chaffeensis genomes isolated from humans and show that these genomes are highly conserved. We identified the candidate core type IV effectome of E. chaffeensis and some conserved intracellular adaptive strategies. We assigned the West Paces strain to genetic group II and predicted the repertoires of T4Es encoded by E. chaffeensis genomes, as well as some putative host cell targets. We demonstrated that predicted T4Es are preferentially distributed in gene sparse regions of the genome. In addition to the identification of the two known type IV effectors of Anaplasmataceae, we identified two novel candidates T4Es, ECHLIB_RS02720 and ECHLIB_RS04640, which are not present in all E. chaffeensis strains and could explain some variations in inter-strain virulence. We also identified another novel candidate T4E, ECHLIB_RS02720, a hypothetical protein exhibiting EPIYA, and NLS domains as well as a classical type IV secretion signal, suggesting an important role inside the host cell. Overall, our results agree with current knowledge of Ehrlichia molecular pathogenesis, and reveal novel candidate T4Es that require experimental validation. This work demonstrates that comparative effectomics enables identification of important host pathways targeted by the bacterial pathogen. Our study, which focuses on the type IV effector repertoires among several strains of E. chaffeensis species, is an original approach and provides rational putative targets for the design of alternative therapeutics against intracellular pathogens. The collection of putative effectors of E. chaffeensis described in our paper could serve as a roadmap for future studies of the function and evolution of effectors.