Project description:Comparison of gene expression between the virulent Rickettsia rickettsii R strain and avirulent Rickettsia rickettsii Iowa. Keywords: virulent vs avirulent
Project description:Comparison of gene expression between the virulent Rickettsia rickettsii R strain and avirulent Rickettsia rickettsii Iowa. Keywords: virulent vs avirulent Virulent Rickettsia rickettsii R strain in triplicate was compared to avirulent Rickettsia rickettsii Iowa in triplicate
Project description:Rickettsia spp. can cause mild to severe human disease. These intracellular bacteria are associated with arthropods, nematodes and trematodes, and usually, are efficiently transmitted transovarially to the progeny of the invertebrate host. We recently demonstrated foreign gene acquisition by lateral gene transfer in Rickettsia genomes. The unexpected presence of laterally transferred toxin-antitoxin (TA) genetic elements (including vapBC) in several Rickettsia genomes has not been connected with the pathogenic process or the host-bacteria relationship. We suspect that vapBC are selfish genetic elements that addict eukaryotic hosts to Rickettsia. We identified a statistical link between the transovarial transmission of Rickettsia in invertebrate hosts and the presence of TA operons, specifically vapBC, in the Rickettsia genome. These TA are neighboring to type IV secretion genes. Tunel assays and whole-genome expression of infected cells showed that antibiotic eradication of TA-containing Rickettsia from the host in cell culture initiates a proapoptotic program. Rickettsia VapC toxins inhibit the growth of transformed Escherichia coli and Saccharomyces cerevisiae. Rickettsia toxin presents in vitro RNase activity. Annexin-V staining and time-lapse video showed that intracytoplasmic injections of VapC toxins in cells cause apoptosis. These data demonstrate that host cells may develop a dependence on Rickettsia spp. expressing the vapBC operon. This would constitute a new evolutionary M-bM-^@M-^\mafia strategyM-bM-^@M-^] of intracellular bacteria based on host addiction. Fresh cells from the human microvascular endothelial cell line (HMEC-1) [26] were infected with R. felis California-2 strain in the presence and absence of antibiotics, at a rate of 5 bacteria per eukaryotic cell. Then, we added or not antibiotics (chloramphenicol 50 M-BM-5g/ml or doxycycline to 40 M-BM-5g/ml) in both experimental (R.felis-infected) and control, mock-infected cells for 6 hours. The cells were harvested and RNA was extracted using the RNeasy Mini Kit (Qiagen). DNA contamination was removed using the Turbo DNA-free Kit (Ambion). RNA were labeled using the Quick Amp Labeling Kit One-color (Agilent) and hybridized onto a Whole Human Genome Microarray, 4x44K (Agilent) as recommended by the manufacturer. Arrays were scanned with DNA Microarray Scanner (Agilent), and data were extracted using Feature Extractor (Agilent).
Project description:Rickettsia spp. can cause mild to severe human disease. These intracellular bacteria are associated with arthropods, nematodes and trematodes, and usually, are efficiently transmitted transovarially to the progeny of the invertebrate host. We recently demonstrated foreign gene acquisition by lateral gene transfer in Rickettsia genomes. The unexpected presence of laterally transferred toxin-antitoxin (TA) genetic elements (including vapBC) in several Rickettsia genomes has not been connected with the pathogenic process or the host-bacteria relationship. We suspect that vapBC are selfish genetic elements that addict eukaryotic hosts to Rickettsia. We identified a statistical link between the transovarial transmission of Rickettsia in invertebrate hosts and the presence of TA operons, specifically vapBC, in the Rickettsia genome. These TA are neighboring to type IV secretion genes. Tunel assays and whole-genome expression of infected cells showed that antibiotic eradication of TA-containing Rickettsia from the host in cell culture initiates a proapoptotic program. Rickettsia VapC toxins inhibit the growth of transformed Escherichia coli and Saccharomyces cerevisiae. Rickettsia toxin presents in vitro RNase activity. Annexin-V staining and time-lapse video showed that intracytoplasmic injections of VapC toxins in cells cause apoptosis. These data demonstrate that host cells may develop a dependence on Rickettsia spp. expressing the vapBC operon. This would constitute a new evolutionary “mafia strategy” of intracellular bacteria based on host addiction.
Project description:The green rice leafhopper Nephotettix cincticeps have two mutualistic symbiotic bacteria (Candidatus Sulcia muelleri and Candidatus Nasuia deltocephalinicola) in its symbiont special organ bacteriome and are also infected to rickettsia. In order to determine immune challenge is induced or not by rickettsia infection in N. cincticeps, we investigated gene expression between rickettsia-infected and rifampicin treated uninfected N. cincticeps colonies.
Project description:Rickettsia amblyommatis, formerly named Rickettsia amblyommii and 'Candidatus Rickettsia amblyommii' is an intracellular bacterium belonging to the spotted fever group Rickettsia. It is highly prevalent in Amblyomma americanum and in other Amblyomma spp. throughout the Western Hemisphere. R. amblyommatis has been cultivated in chicken fibroblast, primary embryonated chicken eggs, Vero cells and arthropod-derived cells. Because of the affinity of rickettsiae to invade vascular endothelial cells, we tried to isolate R. amblyommatis from a nymph of Amblyomma cajennense s.l. collected in Saltillo (Coahulia, Mexico) using human umbilical vein endothelial cells (HUVEC). One tick half was analysed by ompA PCR and was found to be positive for R. amblyommatis. The other half was selected for in vitro culture of Rickettsia spp. It was triturated in 1 mL of endothelial cell growth medium with 1% antibiotic-antimycotic solution, and the homogenate was inoculated into a HUVEC line. Culture was maintained at 33°C in endothelial cell growth medium plus 2 mM l-glutamine and 2% fetal calf serum, with 5% CO2. The medium was changed weekly. Culture was checked by Gimenez stain for Rickettsia-like intracellular organisms. After 48 days of incubation, Rickettsia-like organisms were observed in HUVEC. PCR assays and sequencing of ompA gene in the culture suspension showed 100% identity with R. amblyommatis. This isolate was successfully established in HUVEC, and it has been deposited in the collection of the Center of Rickettsioses and Arthropod-Borne Diseases, Infectious Diseases Department, Hospital San Pedro-Center of Biomedical Research from La Rioja, Logroño, Spain. The HUVEC line is a useful tool for the isolation of R. amblyommatis.
Project description:Proteomics profiling of the secretome of HUVEC cells infected with Rickettsia; Proteomics pprofiling of the plasma proteome of mice infected with Rickettsia.