Project description:A major virulence factor in S. enterica is the pathogenicity island Spi2. The goal of the experiment was to determine the impact of the toll-like receptor 4 (TLR-4) on expression of Spi2 in S. enterica wild type (WT) and triple mutant strain (TMS) with deletions in SPI-1 TTSS, SPI-2 TTSS, flagella. The stain 14028 was used for the experiment. Cells were grown in a TLR-4 wildtype (plus) or deleted (minus) macrophage background. Total RNA was extracted and processed by qRT-PCR.
Project description:A major virulence factor in S. enterica is the pathogenicity island Spi2. The goal of the experiment was to determine the impact of the NRAMP-1 (natural resistance-associated macrophage protein-1) on expression of Spi2 in S. enterica wild type (WT) and triple mutant strain (TMS) with deletions in SPI-1 TTSS, SPI-2 TTSS, flagella. The stain 14028 was used for the experiment. Cells were grown in a NRAMP-1 wildtype (plus) or deleted (minus) macrophage background. Total RNA was extracted and processed by qRT-PCR.
Project description:The Salmonella enterica serovar Typhimurium (ST) mutant lacking the msbB gene (ΔmsbB) has been widely studied as a candidate for attenuated bacterial vectors in therapeutic applications. Deletion of msbB results in LPS with under-acylated lipid A, which lowers endotoxicity while maintaining structural integrity. This attenuation has traditionally been attributed to reduced TLR4 activation due to weaker interaction between the modified lipid A and TLR4. In our study, we confirmed that ΔmsbB ST was less lethal than wild-type (WT) ST in a mouse sepsis model. However, this difference persisted even in TLR4- and caspase-11-deficient mice, suggesting that LPS signaling is not the primary determinant of virulence. In vitro, bone marrow–derived macrophages (BMDMs) from TLR4- or caspase-11-deficient mice showed only modest reductions in ST-induced cell death and cytokine production. Importantly, ΔmsbB ST behaved similarly to WT ST in these assays, further indicating that LPS-mediated signaling is not central to the observed attenuation. Additionally, the mutant exhibited increased outer membrane permeability, likely contributing to its heightened antibiotic sensitivity—and reduced motility due to lower flagellin protein levels.
Project description:A major virulence factor in S. enterica is the pathogenicity island Spi2. The goal of the experiment was to determine the impact of the toll-like receptor 4 (TLR-4) on expression of Spi2 in S. enterica wild type (WT) and triple mutant strain (TMS) with deletions in SPI-1 TTSS, SPI-2 TTSS, flagella. The stain 14028 was used for the experiment. Cells were grown in a TLR-4 wildtype (plus) or deleted (minus) macrophage background. Total RNA was extracted and processed by qRT-PCR. Two strains (WT and TMS) grown under 2 conditions (TLR-4 wildtype (plus) or TLR-4 deleted (minus)) - total 4 samples were analyzed in the study. All PCRs were done in quadruplicates, located on the same PCR plate. For each quadruplicates the median value was calculated and used as a data point for further normalization. A set of controls was selected for all data sets using the following criteria: the data points for the controls should have Cp <40 and be present in all the samples.
Project description:A major virulence factor in S. enterica is the pathogenicity island Spi2. The goal of the experiment was to determine the impact of the NRAMP-1 (natural resistance-associated macrophage protein-1) on expression of Spi2 in S. enterica wild type (WT) and triple mutant strain (TMS) with deletions in SPI-1 TTSS, SPI-2 TTSS, flagella. The stain 14028 was used for the experiment. Cells were grown in a NRAMP-1 wildtype (plus) or deleted (minus) macrophage background. Total RNA was extracted and processed by qRT-PCR. Two strains ( WT and TMS) grown under 2 conditions (NRAMP-1 wildtype (plus) or NRAMP-1 deleted (minus)) - total 4 samples were analyzed in the study. All PCRs were done in duplicates, located on the same PCR plate. For each duplicate the mean value was calculated and used as a data point for further normalization. We averaged the Cp values for all the data points in a sample and then subtracted this sample average Cp from all the data points of the sample. Only 192 primer pairs from 288 on the platform GPL11111 were used.
Project description:In enteric bacteria, DNA supercoiling is highly responsive to environmental conditions. Host specific features of environment serve as cues for the expression of genes required for colonization of host niches via changing supercoiling [1]. It has been shown that substitution at position 87 of GyrA of Salmonella enterica str. SL1344 influences global supercoiling and results in an altered transcriptome with increased expression of stress response pathways [2]. Aminocoumarin antibiotics, such as novobiocin, can be used to relax supercoiling and alter the expression of supercoiling-sensitive genes. Meanwhile, Salmonella enterica demonstrates a significant resistance to this antibiotic and relatively small variability of supercoiling in response to the growth phase, osmotic pressure, and novobiocin treatment. Here we present for the first time transcriptome data of Salmonella enterica subsp. Enterica serovar Typhimurium str. 14028S grown in the presence of novobiocin. These data will help identify genes involved in novobiocin resistance and adaptation processes associated with torsion perturbations in S. enterica. Cleaned FASTQ files for the RNA-seq libraries are deposited in the NCBI Sequence Read Archive (SRA, Identifier: SRP239815) and have been assigned BioProject accession PRJNA599397.
Project description:The variable sigma (σ) subunit of the bacterial RNA polymerase (RNAP) holoenzyme, which is responsible for promoter specificity and open complex formation, plays a strategic role in the response to environmental changes. Salmonella enterica serovar Typhimurium utilizes the housekeeping σ70 and five alternative sigma factors, including σ54 The σ54-RNAP differs from other σ-RNAP holoenzymes in that it forms a stable closed complex with the promoter and requires ATP hydrolysis by an activated cognate bacterial enhancer binding protein (bEBP) to transition to an open complex and initiate transcription. In S. Typhimurium, σ54-dependent promoters normally respond to one of 13 different bEBPs, each of which is activated under a specific growth condition. Here, we utilized a constitutively active, promiscuous bEBP to perform a genome-wide identification of σ54-RNAP DNA binding sites and the transcriptome of the σ54 regulon of S. Typhimurium. The position and context of many of the identified σ54 RNAP DNA binding sites suggest regulatory roles for σ54-RNAP that connect the σ54 regulon to regulons of other σ factors to provide a dynamic response to rapidly changing environmental conditions.IMPORTANCE The alternative sigma factor σ54 (RpoN) is required for expression of genes involved in processes with significance in agriculture, bioenergy production, bioremediation, and host-microbe interactions. The characterization of the σ54 regulon of the versatile pathogen S. Typhimurium has expanded our understanding of the scope of the σ54 regulon and how it links to other σ regulons within the complex regulatory network for gene expression in bacteria.
Project description:Contamination of edible produce leaves with human bacterial pathogens has been associated with serious disease outbreaks and has become a major public health concern affecting all aspects of the market, from farmers to consumers. While pathogen populations residing on the surface of ready-to-eat produce can be potentially removed through thorough washing, there is no disinfection technology available that effectively eliminates internal bacterial populations. By screening 303 multi-gene deletion (MGD) mutants of Salmonella enterica serovar Typhimurium (STm) 14028s, we were able to identify ten genomic regions that play a role in opening the stomatal pore of lettuce leaves. The major metabolic functions of the deleted regions are associated with sensing the environment, bacterium movement, transport through the bacterial membrane, and biosynthesis of surface appendages. Interestingly, at 21 days post inoculation, seven of these mutants showed increased population titers inside the leaf, two mutants showed similar titers as the wild type bacterium, whereas one mutant with a large deletion that includes the Salmonella pathogenicity island 2 (SPI-2) showed significantly impaired persistence in the leaf apoplast. These findings suggest that not all the genomic regions required for initiation of leaf colonization (i.e., epiphytic behavior and tissue penetration) are essential for continuing bacterial survival as an endophyte. We also observed that mutants lacking either SPI-1 (Mut3) or SPI-2 (Mut9) induce callose deposition levels comparable to those of the wild type STm 14028s; therefore, these islands do not seem to affect this lettuce defense mechanism. However, the growth of Mut9, but not Mut3, was significantly impaired in the leaf apoplastic wash fluid (AWF) suggesting that the STm persistence in the apoplast may be linked to nutrient acquisition capabilities or overall bacterial fitness in this niche, which are dependent on the gene(s) deleted in the Mut9 strain. The genetic basis of STm colonization of leaves investigated in this study provides a foundation from which to develop mitigation tactics to enhance food safety.
Project description:Salmonella enterica is an ubiquitous pathogen throughout the world causing gastroenteritis in humans and animals. Survival of pathogenic bacteria in the external environment may be associated with the ability to overcome the stress caused by starvation. The bacterial response to starvation is well understood in laboratory cultures with a sufficiently high cell density. However, bacterial populations often have a small size when facing this challenge in natural biotopes. The aim of this work was to find out if there are differences in the transcriptomes of S. enterica depending on the factor of cell density during starvation. Here we present transcriptome data of Salmonella enterica subsp. enterica serovar Typhimurium str. 14028S grown in carbon rich or carbon deficient medium with high or low cell density. These data will help identify genes involved in adaptation of low-density bacterial populations to starvation conditions.