Project description:This SuperSeries is composed of the following subset Series: GSE25545: Prediction, microarray and Northern blot analyses identify new intergenic small RNAs in Aliivibrio salmonicida (LB medium vs. iron-limited condition) GSE25546: Prediction, microarray and Northern blot analyses identify new intergenic small RNAs in Aliivibrio salmonicida (wild type vs. LitR mutant) Refer to individual Series
Project description:Iron is an essential micronutrient for all living organisms, and sequestering of iron and the virulence of pathogenic bacteria are believed to be correlated. As the defense mechanisms, potential hosts therefore keep the level of free iron inside the body to a minimum. The iron metabolism is well studied in general for several pathogens of humans and animals, but it is still mostly unclear how gene expression levels change in pathogens during the initial stages of infections. In this work, using Aliivibrio salmonicida we studied the immediate changes in transcription levels in response to a sudden decrease in iron levels. Microarray technology was used to monitor global changes in transcriptional levels. Cultures of A. salmonicida were grown to mid log phase before the iron chelator 2,2’-dipyridyl was added and samples were collected after 15 minutes of growth. Using our statistical cut-off values, we retrieved thirty-two differentially expressed genes where the most up-regulated genes belong to an operon encoding proteins responsible for producing the siderophore bisucaberin.
Project description:Aliivibrio salmonicida causes “cold-water vibriosis” (or “Hitra disease”) in fish, including marine-reared Atlantic salmon. During development of the disease the bacterium will encounter macrophages with antibacterial activities such as production of damaging reactive oxygen species (ROS). To defend itself the bacterium will presumably start producing detoxifying enzymes, reducing agents, and proteins involved in DNA and protein repair systems. Even though responses to oxidative stress are well studied for a few model bacteria, little work has been done in general to explain how important groups of pathogens, like members of the Vibrionaceae family, can survive at high levels of ROS. We have used bioinformatic tools and an –omics approach to study how A. salmonicida responds to hydrogen peroxide (H2O2). First, we used the recently published genome sequence to predict potential binding sites for OxyR (H2O2 response regulator). The computer-based search identified OxyR sites associated with 20 single genes and 8 operons, and these predictions were compared to experimental data from Northern blot analysis, microarray analysis and 2D gel electrophoresis. In general, OxyR binding site predictions and experimental results are in agreement. Up- and down-regulated genes are distributed among all functional gene categories, but a striking number of ≥2 fold up-regulated genes encode proteins involved in detoxification or DNA protection and repair, are part of reduction systems, or are involved in carbon metabolism and regeneration of NADH/NADPH. Our predictions and –omics data corroborates well with findings from other model bacteria, but also suggest species-specific gene regulation.
Project description:Iron is an essential micronutrient for all living organisms, and sequestering of iron and the virulence of pathogenic bacteria are believed to be correlated. As the defense mechanisms, potential hosts therefore keep the level of free iron inside the body to a minimum. The iron metabolism is well studied in general for several pathogens of humans and animals, but it is still mostly unclear how gene expression levels change in pathogens during the initial stages of infections. In this work, using Aliivibrio salmonicida we studied the immediate changes in transcription levels in response to a sudden decrease in iron levels. Microarray technology was used to monitor global changes in transcriptional levels. Cultures of A. salmonicida were grown to mid log phase before the iron chelator 2,2M-bM-^@M-^Y-dipyridyl was added and samples were collected after 15 minutes of growth. Using our statistical cut-off values, we retrieved thirty-two differentially expressed genes where the most up-regulated genes belong to an operon encoding proteins responsible for producing the siderophore bisucaberin. Six biological replicates of A. salmonicida wild type strain LFI1238 were grown in suspension in LB medium, at 8 M-BM-0C , 200 rpm and split in two halves at OD600nm 0.5, keeping one half of each replicate as control (still growing while the treated samples were growing with 2,2-dipyridyl treatment) while the other half were treated with 50M-BM-5M 2,2-dipyridyl for 15 minutes.
Project description:Aliivibrio (Vibrio) salmonicida is the causative agent of cold-water vibriosis in salmonids (Oncorhynchus mykiss and Salmo salar L.) and gadidae (Gadus morhua L.). Virulence-associated factors that are essential for the full spectrum of Al. salmonicida pathogenicity are largely unknown. Chitin-active lytic polysaccharide monooxygenases (LPMOs) have been indicated to play roles in both chitin degradation and virulence in a variety of pathogenic bacteria. In the present study we investigated the role of LPMOs in the pathogenicity of Al. salmonicida LFI238 in Atlantic salmon (Salmo salar L.). In vivo challenge experiments using isogenic deletion mutants of the two LPMOs encoding genes AsLPMO10A and AsLPMO10B, showed that both LPMOs, and in particular AsLPMO10B, were important in the invasive phase of cold-water vibriosis. Crystallographic analysis of the AsLPMO10B AA10 LPMO domain (to 1.4 Å resolution) revealed high structural similarity to viral fusolin, an LPMO known to enhance the virulence of insecticidal agents. Finally, exposure to Atlantic salmon serum resulted in substantial proteome re-organization of the Al. salmonicida LPMO deletion variants compared to the wild type strain, indicating the struggle of the bacterium to adapt to the host immune components.