Project description:The obligate intracellular bacterium, Ehrlichia ruminantium (ER) is the causal agent of Heartwater, a fatal disease in ruminants. It is transmitted by ticks of the genus Amblyomma. Here, we report the genomic comparative and the global transcriptional profile of 4 strains of ER, Gardel and Senegal, two distant virulent strains with their corresponding attenuated strains. Our results showed a higher metabolic activity in attenuated strains compared to virulent strains, suggesting a better adaptation in vitro of attenuated strains to the host cells. There was a strong modification of membrane protein encoding genes expression for the 4 strains. A major over-expression of map1-related genes was observed for virulent strains, whereas attenuated strains over-expressed genes encoding for hypothetical membrane proteins. This result suggests that in vivo, MAP-1 related proteins could induce non-protective immune responses for virulent strains. For the attenuated strains, the lack of expression of map1-related genes and over-expression of other membrane proteins encoding genes could be important in induction of efficient immune responses.The diminution of expression of many genes in attenuated Senegal was caused by severe mutation. One of them, the gene recO is involved in DNA repair and its mutation could explain the higher proportion of mutated genes in attenuated Senegal, inducing the faster attenuation of Senegal compared to Gardel.
Project description:The Rickettsiales Ehrlichia ruminantium (ER), the causal agent of the fatal tick-borne disease Heartwater, induces severe damage to the vascular endothelium in ruminants. Nevertheless, E. ruminantium-induced pathobiology remains largely unknown. Our work paves the way for understanding this phenomenon by using quantitative proteomic analyses (2D-DIGE-MS/MS, 1DE-nanoLC-MS/MS and biotin-nanoUPLC-MS/MS) of host bovine aorta endothelial cells (BAE) during the in vitro bacterium intracellular replication cycle. We detect 265 bacterial proteins (including virulence factors), at all time-points of the E. ruminantium replication cycle, highlighting a dynamic bacterium–host interaction. We show that E. ruminantium infection modulates the expression of 433 host proteins: 98 being over-expressed, 161 under-expressed, 140 detected only in infected BAE cells and 34 exclusively detected in non-infected cells. Cystoscape integrated data analysis shows that these proteins lead to major changes in host cell immune responses, host cell metabolism and vesicle trafficking, with a clear involvement of inflammation-related proteins in this process. Our findings led to the first model of E. ruminantium infection in host cells in vitro, and we highlight potential biomarkers of E. ruminantium infection in endothelial cells (such as ROCK1, TMEM16K, Albumin and PTPN1), which may be important to further combat Heartwater, namely by developing non-antibiotic-based strategies.
Project description:The inability to propagate obligate intracellular pathogens under axenic (host cell-free) culture conditions imposes severe experimental constraints that have negatively impacted progress in understanding pathogen virulence and disease mechanisms. Coxiella burnetii, the causative agent of human Q (Query) fever, is an obligate intracellular bacterial pathogen that replicates exclusively in an acidified, lysosome-like vacuole. To define conditions that support C. burnetii growth, we systematically evaluated the organismâ??s metabolic requirements using expression microarrays, genomic reconstruction, and metabolite typing. This led to development of a complex nutrient medium that supported substantial growth (~ 3 log10) of C. burnetii in a 2.5% oxygen environment. Importantly, axenically grown C. burnetii were highly infectious for Vero cells and exhibited developmental forms characteristic of in vivo grown organisms. Axenic cultivation of C. burnetii will facilitate studies of the organismâ??s pathogenesis and genetics, and aid development of Q fever preventatives such as an effective subunit vaccine. Furthermore, the systematic approach used here may be broadly applicable to development of axenic media that support growth of other medically important obligate intracellular pathogens. Host cell-free growth, Vero cell growth and carryover baseline of Coxiella burnetii