RNA-seq Brings New Insights to the Intra-Macrophage Transcriptome of Salmonella Typhimurium.

Salmonella enterica serovar Typhimurium is arguably the world's best-understood bacterial pathogen. However, crucial details about the genetic programs used by the bacterium to survive and replicate in macrophages have remained obscure because of the challenge of studying gene expression of intracellular pathogens during infection. Here, we report the use of deep sequencing (RNA-seq) to reveal the transcriptional architecture and gene activity of Salmonella during infection of murine macrophages, providing new insights into the strategies used by the pathogen to survive in a bactericidal immune cell. We characterized 3583 transcriptional start sites that are active within macrophages, and highlight 11 of these as candidates for the delivery of heterologous antigens from Salmonella vaccine strains. A majority (88%) of the 280 S. Typhimurium sRNAs were expressed inside macrophages, and SPI13 and SPI2 were the most highly expressed pathogenicity islands. We identified 31 S. Typhimurium genes that were strongly up-regulated inside macrophages but expressed at very low levels during in vitro growth. The SalComMac online resource allows the visualisation of every transcript expressed during bacterial replication within mammalian cells. This primary transcriptome of intra-macrophage S.-Typhimurium describes the transcriptional start sites and the transcripts responsible for virulence traits, and catalogues the sRNAs that may play a role in the regulation of gene expression during infection

Investigating the transcriptional regulation of small RNA expression in Salmonella enterica serovar Typhimurium

THESIS 10499Salmonella enterica serovar Typhimurium (S. Typhimurium) is an important foodborne pathogen that causes self-limiting gastroenteritis, or more serious systemic infections in susceptible hosts. S. Typhimurium can infect a wide host range and encounters a series of stressful conditions within various host environments. S. Typhimurium expresses a Type Three Secretion System (TTSS) encoded on a pathogenicity island (SPI1), to mediate invasion of the host intestinal epithelium. Once internalised, S. Typhimurium survives and replicates within the Salmonella containing vacuole (SCV). S. Typhimurium expresses a second TTSS, encoded on a second pathogenicity island (SPI2), to survive the harsh intracellular environment and establish systemic infection

ProP Is Required for the Survival of Desiccated <i>Salmonella enterica</i> Serovar Typimurium Cells on a Stainless Steel Surface

Consumers trust commercial food production to be safe, and it is important to strive to improve food safety at every level. Several outbreaks of food-borne disease have been caused by Salmonella strains associated with dried food. Currently we do not know the mechanisms used by Salmonella enterica serovar Typhimurium to survive in desiccated environments. The aim of this study was to discover the responses of S. Typhimurium ST4/74 at the transcriptional level to desiccation on a stainless steel surface and to subsequent rehydration. Bacterial cells were dried onto the same steel surfaces used during the production of dry foods, and RNA was recovered for transcriptomic analysis. Subsequently, dried cells were rehydrated and were again used for transcriptomic analysis. A total of 266 genes were differentially expressed under desiccation stress compared with a static broth culture. The osmoprotectant transporters proP, proU, and osmU (STM1491 to STM1494) were highly upregulated by drying. Deletion of any one of these transport systems resulted in a reduction in the long-term viability of S. Typhimurium on a stainless steel food contact surface. The proP gene was critical for survival; proP deletion mutants could not survive desiccation for long periods and were undetectable after 4 weeks. Following rehydration, 138 genes were differentially expressed, with upregulation observed for genes such as proP, proU, and the phosphate transport genes (pstACS). In time, this knowledge should prove valuable for understanding the underlying mechanisms involved in pathogen survival and should lead to improved methods for control to ensure the safety of intermediate- and low-moisture foods

Proton resonance frequency shift thermometry: A review of modern clinical practices

Magnetic resonance imaging (MRI) has become a popular modality in guiding minimally invasive thermal therapies, due to its advanced, nonionizing, imaging capabilities and its ability to record changes in temperature. A variety of MR thermometry techniques have been developed over the years, and proton resonance frequency (PRF) shift thermometry is the current clinical gold standard to treat a variety of cancers. It is used extensively to guide hyperthermic thermal ablation techniques such as high-intensity focused ultrasound (HIFU) and laser-induced thermal therapy (LITT). Essential attributes of PRF shift thermometry include excellent linearity with temperature, good sensitivity, and independence from tissue type. This noninvasive temperature mapping method gives accurate quantitative measures of the temperature evolution inside biological tissues. In this review, the current status and new developments in the fields of MR-guided HIFU and LITT are presented with an emphasis on breast, prostate, bone, uterine, and brain treatments.Level of Evidence 5Technical Efficacy Stage 3The project was co-financed by the European Regional Development Fund (ERDF) under Ireland’s European Structural, Investment Funds Programme 2014–2020 and Enterprise Ireland; Grant agreement: CF-2017-0826-P, Irish Research Council postgraduate scholarship GOIPG/2018/82 and the NUI Galway College of Science.peer-reviewed2021-11-2

Confirmation of novel regulatory interactions that control five sRNAs.

<p>(A) STnc520, (B) FnrS, (C) STnc1330, (D) RyeF, (E) STnc1480. Each panel shows the visualisation of sequencing reads obtained from RNA-seq experiments using the IGB browser beside northern blots that validate the differential expression data using RNA extracted from biological replicates of wild-type and mutant strains grown under the same <i>in vitro</i> environmental condition, as indicated. 5S RNA was probed as a loading control.</p

Overlap between HilD and SsrB-controlled gene expression reveals 8 putative virulence factors.

<p>Absolute expression (A) and relative expression (B) of the 52 CDS genes which are differentially-expressed (>3-fold) in the absence of HilD and SsrB, compared to wild-type grown under ESP and InSPI2 conditions, respectively (putative co-regulated genes). (C) Presence of a transposon insertion in the 52 co-regulated genes leads to attenuation or amplification of fitness in chicken, pig or calf models [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006258#pgen.1006258.ref041" target="_blank">41</a>]. The scoring methods used by the authors of each publication were applied independently to each dataset (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006258#sec013" target="_blank">Materials and Methods</a>). A negative score indicates attenuation of fitness as a result of the transposon insertion (blue), while an increase in fitness is denoted by a positive fitness score (orange). If no output reads were identified for a particular insertion an arbitrary negative fitness score of -15 was assigned [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006258#pgen.1006258.ref041" target="_blank">41</a>]. Replacement of co-regulated genes with either a sense-oriented Kanamycin resistance cassette or an antisense-oriented chloramphenicol resistance cassette resulted in fitness attenuation in colonisation of either the spleen or liver of BALB/c mice following infection via the i.p. route [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006258#pgen.1006258.ref042" target="_blank">42</a>]. A negative fitness score indicates attenuation of fitness as a result of the gene deletion (blue), while an amplification of fitness is reflected by a positive fitness score (orange). Grey indicates that no virulence data were available for that gene. Eight genes that were co-regulated by HilD and SsrB have putative roles in virulence, and are indicated in blue.</p

Bacterial strains and plasmids.

<p>Bacterial strains and plasmids.</p

Panel of regulatory mutants and environmental conditions used for RNA-seq experiments.

<p>Panel of regulatory mutants and environmental conditions used for RNA-seq experiments.</p