Project description:We developed a surface-displayed library of 117 human antimicrobial peptides (AMPs) and over 3000 human AMP variants in a laboratory E. coli strain expressing PhoP and PhoQ from Salmonella Typhimurium. We used sort-seq (fluorescence-activated cell sorting followed by next-generation sequencing) to characterize PhoPQ activation by these surface-displayed AMPs.
Project description:Transfer RNAs (tRNAs) maintain translational fidelity through strict charging by their cognate aminoacyl-tRNA synthetase and codon:anticodon base pairing with the mRNA at the ribosome. Mistranslation occurs when an amino acid not specified by the genetic code is incorporated into a protein. Since alanyl-tRNA synthetase uniquely recognizes a G3:U70 base pair in alanine tRNAs and the anticodon plays no role in charging, alanine tRNA variants with anticodon mutations have the potential to mistranslate alanine. Our goal was to quantify mis-incorporation of alanine into proteins in Saccharomyces cerevisiae strains expressing one of 57 different alanine tRNA anticodon variants. Using mass spectrometry, we observed mistranslation for 45 of the variants when expressed on single-copy plasmids.
Project description:Salmonella causes a range of diseases in different hosts, including enterocolitis and systemic infection. Lysine acetylation regulates many eukaryotic cellular processes, but its function in prokaryotes is largely unknown. Reversible acetylation in Salmonella Typhimurium depends on acetyltransferase Pat and nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase CobB. Here, we used cell and animal models to evaluate the virulence of pat and cobB deletion mutants in S. Typhimurium, and found that pat is essential for bacterial intestinal colonization, systemic infection and host inflammation response. Next, to understand the underlying mechanism, genome-wide transcriptome was analyzed. RNA-seq data showed the expression of Salmonella pathogenicity islands 1 (SPI-1) is partially dependent on pat. In addition, we found that HilD is a substrate of Pat, which is essential for maintaining HilD protein level. Taken together, these results suggested that protein acetylation system regulates SPI-1 expression by controlling HilD in a post-translational manner to mediate S. Typhimurium virulence. To use RNA-seq to analyze the transcriptome patterns of pat or cobB mutation in Salmonella Typhimurium 14028s.
Project description:Salmonella causes a range of diseases in different hosts, including enterocolitis and systemic infection. Lysine acetylation regulates many eukaryotic cellular processes, but its function in prokaryotes is largely unknown. Reversible acetylation in Salmonella Typhimurium depends on acetyltransferase Pat and nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase CobB. Here, we used cell and animal models to evaluate the virulence of pat and cobB deletion mutants in S. Typhimurium, and found that pat is essential for bacterial intestinal colonization, systemic infection and host inflammation response. Next, to understand the underlying mechanism, genome-wide transcriptome was analyzed. RNA-seq data showed the expression of Salmonella pathogenicity islands 1 (SPI-1) is partially dependent on pat. In addition, we found that HilD is a substrate of Pat, which is essential for maintaining HilD protein level. Taken together, these results suggested that protein acetylation system regulates SPI-1 expression by controlling HilD in a post-translational manner to mediate S. Typhimurium virulence.
Project description:The GeneChip Porcine Genome Array was used to identify the transcriptional response upon either Salmonella typhimurium (ST) or Salmonella choleraesuis (SC) infection in two porcine epithelial cell lines (IPEC-J2, from jejunum and IPI-2I, from ileum) during 2 and 4 hours post infection. The objectives in this study were first, to identify the different response between the epithelial cell lines from different gut regions; second, to study how the Salmonella serotypes used could elicit a different host response; and third, to determine the effect of the time-points on the differentially gene expression.
Project description:The GeneChip Porcine Genome Array was used to identify the transcriptional response upon either Salmonella typhimurium (ST) or Salmonella choleraesuis (SC) infection in two porcine epithelial cell lines (IPEC-J2, from jejunum and IPI-2I, from ileum) during 2 and 4 hours post infection. The objectives in this study were first, to identify the different response between the epithelial cell lines from different gut regions; second, to study how the Salmonella serotypes used could elicit a different host response; and third, to determine the effect of the time-points on the differentially gene expression.
Project description:Post-translational modification by ubiquitin and ubiquitin-like modifiers proteins regulate cellular processes at almost every levels. Ubiquitin itself is encoded by four different genes, either as single copy of ubiquitin fused to ribosomal proteins, or by polyubiquitin precursors3. Early studies identified several additional genes potentially coding for ubiquitin, but they were considered as pseudogenes due to differences in amino acids, or lack of apparent transcription4-6. Through analysis of large-scale proteomics and RNA sequencing experiments, we found evidence for expression at the mRNA and protein levels of several ubiquitin pseudogenes. Our results show that UBBP4, a pseudogene of the UBB subfamily, produces functional ubiquitin proteins with different amino acids composition compared to the canonical sequence. These ubiquitins variants are covalently conjugated to proteins that are different from ubiquitin, and proteins modified by UBBP4 are not targeted for proteasomal degradation. Furthermore, invalidation of UBBP4 results in slower cell division, and accumulation of lamin A within the nucleolus. This implies that a subset of proteins reported as ubiquitin targets could rather be through these variants arising from wrongly annotated pseudogenes, and that there is a specificity in the modification and differences in the functional consequence of proteins modified by these new ubiquitin variants. The identification of additional ubiquitins thus entails a new layer of complexity in protein ubiquitylation that has been unnoticed until now.