Project description:Transcriptional profiling of an El Tor biotype crp mutant The virulent V. cholerae El Tor Ogawa strain C7258 (Peru isolate 1991) and an isogenic deletion mutant (WL7258) lacking DNA sequences encoding the cAMP receptor protein were grown in LB medium to optical density at 600 nm of 1.5. The cultures were chilled in ice, cells quickly collected by centrifugation and total RNA imediately extracted. RNAwas extracted and purified using the Trizol plus RNA purification system (Invitrogen) followed RNEasy miniElute cleanup (Qiagen). RNA samples were conserved at - 80 C and used within a week. 4 replicates
Project description:This study assessed the development of disseminated candidiasis within Galleria mellonella larvae and characterised the proteomic responses of Candida albicans to larval hemolymph. Infection of larvae with an inoculum of 1 × 106 yeast cell reduced larval viability 24 (53.33 ± 3.33%), 48 (33.33 ± 3.33%) and 72 (6.66 ± 3.33%) hour post infection. C. albicans infection quickly disseminated from the site of inoculation and the presence of yeast and hyphal forms were found in nodules extracted from infected larvae at 6 and 24 hours. A range of proteins secreted during infection of G. mellonella were detected in larval hemolymph and these were enriched for biological processes such as interaction with host and pathogenesis. The candicidal activity of hemolymph after immediate incubation of yeast cells resulted in a decrease in yeast cell viability (0.23 ± 0.03 × 106, p < 0.05) as compared to control (0.99 ± 0.01 × 106). extracellular (in vivo) proteome of C. albicans in larval hemolymph were assessed. C. albicans responds to incubation in hemolymph ex vivo by the induction of an oxidative stress response, a decrease in proteins associated with protein synthesis and an increase in glycolytic proteins.
Project description:The transcription of the cldEFGC gene cluster of Bifidobacterium breve UCC2003 was shown to be induced upon growth on cellodextrins, implicating these genes in the metabolism of these sugars. Phenotypic analysis of a B. breve UCC2003::cldE insertion mutant confirmed that the cld gene cluster is exclusively required for cellodextrin utilization by this bacterium. HPAEC-PAD analysis of medium samples obtained during growth of B. breve UCC2003 on a mixture of cellodextrins revealed its ability to utilize cellobiose, cellotriose, cellotetraose and cellopentaose, with cellotriose representing the preferred substrate. The cldC gene of the cld operon of B. breve UCC2003 was shown to be the first described bifidobacterial β-glucosidase exhibiting hydrolytic activity towards various cellodextrins. In order to investigate differences in gene expression patterns of B. breve UCC2003 when grown on cellobiose or cellodextrins as compared to growth on glucose, DNA microarray experiments were performed. Total RNA was isolated from B. breve UCC2003 cultures grown on cellobiose, cellodextrins, or glucose (see Materials and Methods). The cultures were harvested at the time points that ensured that B. breve UCC2003 was metabolizing cellobiose or cellodextrins as opposed to the residual glucose present in the cellodextrin preparation. Analysis of the DNA microarray data was obtained from two independent biological replicates.
Project description:The differentiation of cells into distinct cell types, each of which is heritable for many generations, underlies many biological phenomena. White and opaque cells of the fungal pathogen Candida albicans are two such heritable cell types, each thought to be adapted to unique niches within their human host. To systematically investigate the differences between the two cell types, we performed strand-specific massively-parallel sequencing of RNA from C. albicans white and opaque cells. Combining the resulting data from both cell types, we first substantially re-annotated the C. albicans transcriptome, finding 1443 novel coding and non-coding transcriptionally active regions. Using the new annotation, we compared differences in transcript abundance between the two cell types with the genomic regions bound by the master regulator of the white-opaque switch (Wor1). We found that the revised transcriptional landscape considerably alters our understanding of the circuit governing differentiation. In particular, we can now resolve the poor concordance between binding of the master regulator and the differential expression of adjacent genes, a discrepancy observed in many other studies of cell differentiation. More than one third of the Wor1-bound differentially-expressed transcripts were previously unannotated, which explains the formerly puzzling presence of Wor1 at these positions along the genome. Indeed, many of these newly identified Wor1-regulated genes are non-coding and transcribed antisense to coding transcripts. We also found that 5' and 3' untranslated regions (UTRs) of mRNAs in the circuit are unusually long and that 5' UTRs often differ in length between white and opaque cells. These observations suggest that the use of alternative promoters is widespread in the circuit and that important regulatory information is carried in the long UTRs. Further analysis revealed that the revised Wor1 circuit bears several striking similarities to the Oct4 circuit that specifies the pluripotency of mammalian embryonic stem cells. Additional characteristics shared with the Oct4 circuit suggest a set of general hallmarks characteristic of heritable differentiation states in eukaryotes. RNA-Seq was applied to Candida albicans white and opaque cells to identify novel transcripts and UTRs that are differentially regulated between the two cell types. Two biological replicates each of white and opaque cell cultures. One of the white cell RNA samples was split just after isolation to allow a comparison of the poly(A)-selection and ribo-depletion sample preparation strategies.
Project description:Neutrophils are one of the first responders to infection and are a key component of the innate immune system through their ability to phagocytose and kill invading pathogens, secrete antimicrobial molecules and produce extracellular traps. Neutrophils are produced in the bone marrow, circulate within the blood and upon immune challenge migrate to the site of infection. We wanted to understand whether this transition shapes the mouse neutrophil protein landscape, how the mouse neutrophil proteome is impacted by systemic infection and perform a comparative analysis of human and mouse neutrophils. Using quantitative mass spectrometry we reveal tissue-specific, infection-induced and species-specific neutrophil protein signatures. We show a high degree of proteomic conservation between mouse bone marrow, blood and peritoneal neutrophils, but also identify key differences in the molecules that these cells express for sensing and responding to their environment. Systemic infection triggers a change in the bone marrow neutrophil population with considerable impact on the core machinery for protein synthesis and DNA replication along with environmental sensors. We also reveal profound differences in mouse and human blood neutrophils, particularly their granule contents and their receptor repertoires. Our proteomics data provides a valuable resource for understanding neutrophil function and phenotypes across species and model systems.