Project description:Neospora caninum has been recognised as the main cause of transmissible abortion in cattle worldwide. Although the pathogenesis of this disease remains largely unknown, different factors associated to both the host and the parasite have influence in the dynamics of the disease. Intraspecific variation in virulence have been widely shown among N. caninum isolates. However, the molecular basis that govern such variability and the outcome of the infection have not been well-established yet. This study aims to improve the knowledge on the factors governing intra-specific variation in virulence in N. caninum. Quantitative label free LC-MS/MS was used to investigate proteome differences between the high virulent isolate Nc-Spain7 and the low virulent isolate Nc-Spain1H through tachyzoite lytic cycle: at the end of invasion (12 hours), during exponential growth (36 hours) and at early egress (56 hours). The results showed the higher differences in abundance of proteins at invasion and egress, 77 and 62 proteins, respectively. At 36 h, during active parasite replication only 19 proteins were found differentially abundant between isolates. Microneme protein repertoire, involved in parasite invasion and egress, was more abundant in Nc-Spain1H isolate, which displays a lower invasion rate. Soluble effectors related to Toxoplasma gondii virulence (ROP and GRA), proteins of carbohydrate and fatty acid metabolism and stress responses also showed different abundance between isolates. Differential expression analysis by RNAseq technology during tachyzoite egress from the host-cell (56 hours) was also performed showing an expression profile of genes associated with the bradyzoite stage the low virulent and low growing NcSpain1H isolate. The differences in proteome and RNA expression profiles between these two isolates reveal interesting insights into likely mechanisms involved in specific phenotypic traits and virulence in N. caninum.
Project description:Despite the importance of bovine neosporosis, important knowledge gaps remain concerning pathogenic mechanisms of N. caninum. The infection of the placenta is a crucial event in the pathogenesis of the disease, however, very little is known about the relation of the parasite with this target organ. Recent studies have shown that isolates with important variations in virulence also show different interactions with the bovine trophoblast cell line F3, involving proliferative capacity and transcriptome host cell modulation. Herein, we use the same model of infection to study the interaction of Neospora with these target cells at the proteomic level using LC-MS/MS over the course of the parasite lytic cycle. We also analyzed the proteome differences between high (Nc-Spain7)- and low (Nc-Spain1H)- virulence isolates. The results showed mitochondrial processes and metabolism as the main point of Neospora-host interactions. Interestingly, Nc-Spain1H infection showed the highest level of influence on host cell proteome in contrast to Nc-Spain7 infection. These results confirm Nc-Spain7 capacities to evade host cell response and establish infection more effectively.
Project description:Recent advances in high throughput sequencing methodologies allow the opportunity to probe in depth the transcriptomes of organisms including N. caninum. In this project, we are using Illumina sequencing technology to analyze the transcriptome (RNA-Seq) of experimentally accessible stages (e.g. tachyzoites at different times points) of N. caninum NCLiv. The aim is to make transcriptional landscape maps at different time points at different life cycle stages of N. caninum and compare it with equivalent datasets from the closely related parasite Toxoplasma gondii
Project description:Six isolates of PT21/28 and six of PT32 were analysed by CGH using UBECarray3 microarrays (containing probes for E. coli K-12 str. MG1655 and O157:H7 str. EDL933 and Sakai) to define genotypic differences between phage types. gDNA from E.coli O157 str. Sakai was hybridised to all arrays to provide a universal control channel on all arrays.
Project description:Six isolates of PT21/28 and six of PT32 were analysed by CGH using UBECarray3 microarrays (containing probes for E. coli K-12 str. MG1655 and O157:H7 str. EDL933 and Sakai) to define genotypic differences between phage types. gDNA from E.coli O157 str. Sakai was hybridised to all arrays to provide a universal control channel on all arrays. gDNA from 12 PT 21/28 & 32 isolates were labelled with Cy5 and control gDNA from str. Sakai was labelled with Cy3. Test and control gDNA was hybridised to UBECarray3 microarrays. The LOWESS normalised relative signal to the Sakai control channel was used to compare between samples.
Project description:Here, using ChIP-seq, we define the NF-κB cistrome which is comprised of 31,070 cis-acting binding sites responsive to LPS-induced signaling. In addition, we demonstrate that the transcriptional repressor B-cell lymphoma 6 (Bcl-6) regulates nearly a third of the Tlr4-regulated transcriptome and that 90% of the Bcl-6 cistrome is collapsed following Tlr4 activation. Bcl-6 deficient macrophages are acutely hypersensitive to lipopolysaccharide (LPS) and, using comparative ChIP-seq analyses, we find that the Bcl-6 and NF-κB cistromes intersect, within nucleosomal distance, at nearly half of Bcl-6 binding sites in stimulated macrophages to promote opposing epigenetic modifications of the local chromatin. These results reveal a genomic strategy for controlling the innate immune response in which repressive and inductive cistromes establish a dynamic balance between macrophage quiescence and activation via epigenetically marked cis-regulatory elements. keywords: Genome-wide location analysis Identification of BCL6 and NFkB binding sites in unstimulated and LPS-stimulated primary bone-marrow derived macrophages.