Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Next-Generation Sequencing of sgRNA spacer sequences from a CRISPRi library screen of the Salmonella enterica serovar Typhimurium 14028s genome in response to repeated monoclonal IgA treatment

ABSTRACT: For this study, we implemented the anti-lipopolysaccharide monoclonal IgA Sal4 in a genome-wide CRISPR interference (CRISPRi) screen to identify genes that modulate the extent of agglutination of Salmonella enterica serovar Typhimurium (STm) in response to antibody exposure. We leveraged our recently-developed \"snow globe assay\" method to enrich for cells containing sgRNAs that silence the expression of genes that contribute to the process of antibody-mediated agglutination. A high density (~10^8 CFU/mL) culture of an STm strain containing a library of 36,651 sgRNA-encoded plasmids was treated with Sal4 IgA (15 μg/mL) in PBS for five hours at room temperature. The remaining portion of the culture present at the top of the supernatant after antibody treatment was passaged and grown overnight to repeat the enrichment the following day. We then purified the sgRNA-containing plasmids from this agglutinating-evading population using a DNA extraction kit, amplified the spacer sequence via PCR, purified the PCR products using AMPure magnetic beads, and pooled the resulting DNA for Next Generation Sequencing. The files included here represent the raw .fastq files obtained from the Illumina NextSeq platform. For more information of this study, please see https://doi.org/10.1101/2024.12.16.628777.

INSTRUMENT(S): NextSeq 500

ORGANISM(S): Salmonella enterica subsp. enterica serovar Typhimurium

SUBMITTER:

PROVIDER: E-MTAB-14834 | biostudies-arrayexpress |

SECONDARY ACCESSION(S): ERP169222

REPOSITORIES: biostudies-arrayexpress

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Project description:During the colonization of hosts, bacterial pathogens are presented with many challenges that must be overcome for colonization to successfully occur. This requires bacterial sensing of the surroundings and adaptation to the conditions encountered. One of the major impediments to pathogen colonization of the mammalian gastrointestinal tract is the antibacterial action of bile. Salmonella enterica serovar Typhimurium has specific mechanisms involved in resistance to bile. Besides being resistant to it, Salmonella can also successfully multiply in bile, using it as a source of nutrients. This accomplishment is highly relevant to pathogenesis, as Salmonella colonizes the gallbladder of hosts, where it can be carried asymptomatically and promote further host spread and transmission. In order to gain insights into the mechanisms used by Salmonella to grow in bile, we studied the changes elicited by Salmonella in the chemical composition of bile during growth in vitro and in vivo through a metabolomics approach. Our data suggest that phospholipids are an important source of carbon and energy for Salmonella during growth in the laboratory as well as during gallbladder infections of mice. Further studies in this area will generate a better understanding of how Salmonella exploits this generally hostile environment for its own benefit. For in vitro studies, bile was extracted from C57BL/6 mice and used immediately. An overnight culture of Salmonella enterica serovar Typhimurium SL1344 was used to inoculate 10 ?L of bile at an approximate density of 5x10^6 cells/mL. The experiment was performed in duplicate, and a total of 2 uninfected and 2 infected bile samples were studied. Samples were incubated for 24 hours at 37 oC with shaking. After incubation, samples were centrifuged to remove bacteria and the supernatant was saved for metabolomic analyses. For in vivo studies, C57BL/6 mice were infected with approximately 10^8 bacterial cells by oral gavage. Four groups of three to four mice each were either infected with Salmonella or kept uninfected (total of 11 mice per treatment group). Five days after infection, all mice were sacrificed and bile was collected. Samples were centrifuged and supernatants saved for metabolomic analyses. Samples were prepared by mixing equal volumes of bile from three to four mice, generating three samples per treatment (uninfected and infected), which were used in the subsequent steps. Seven microliters of each sample was evaporated and the residue resuspended in 50% acetonitrine. Extracts were infused into a 12-T Apex-Qe hybrid quadrupole-FT-ICR mass spectrometer equipped with an Apollo II electrospray ionization source, a quadrupole mass filter and a hexapole collision cell. Raw mass spectrometry data were processed as described elsewhere (Han et al. 2008. Metabolomics. 4:128-140). To identify differences in metabolite composition between different groups of samples, we filtered the list of masses for metabolites which were present on one set of samples but not the other. Additionally, we calculated the ratios between averaged intensities of metabolites from each group of mice. To assign possible metabolite identities to the masses selected as described above, the monoisotopic neutral masses of interest were queried against the Human Metabolome Database (HMDB, http://www.hmdb.ca), with a tolerance of 0.001 Da.

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Next-Generation Sequencing of sgRNA spacer sequences from a CRISPRi library screen of the Salmonella enterica serovar Typhimurium 14028s genome in response to repeated monoclonal IgA treatment

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