Project description:In this work, we applied an RNA analysis method, Selective Capture of Transcribed Sequences (SCOTS), and cDNA hybridization-microarray technology to identify S. Paratyphi A transcripts expressed by bacteria in the blood of three patients in Bangladesh. In total, we detected 1798 S. Paratyphi A mRNAs expressed in the blood of infected humans (43.9% of the ORFeome). Of these, we identified 868 in at least two patients, and 315 in all three patients. S. Paratyphi A transcripts identified in at least two patients encode proteins involved in energy metabolism, nutrient and iron acquisition, vitamin biosynthesis, stress responses, oxidative stress resistance, and pathogenesis. A number of detected transcripts are expressed from PhoP and SlyA-regulated genes associated with intra-macrophage survival, genes contained within Salmonella Pathogenicity Islands (SPIs) 1-4, 6, 10, 13, and 16, as well as RpoS-regulated genes. The largest category of identified transcripts are those encoding proteins with unknown function. When comparing level of bacterial mRNA detection using in vivo samples collected from infected patients to samples from in vitro grown organisms, we found significant differences for 347, 391, and 456 S. Paratyphi A transcripts in each of three individual patients (approximately 9.7% of the ORFeome). Of these, expression of 194 transcripts (4.7% of ORFs) was concordant in two or more patients, and 41 in all patients. Genes encoding these transcripts are contained within SPI-1, 3, 6 and 10, are PhoP-regulated genes, are involved in energy metabolism, nutrient acquisition, drug resistance, or are uncharacterized genes. Using quantitative RT-PCR, we confirmed increased gene expression in vivo for a subset of genes identified in our analyses. We compared transcriptional profiles of S. Paratyphi A from the blood of infected humans to S. Paratyphi A grown in vitro. Replicates and dye-swaps were performed.
Project description:Comparative genomic analysis of a temporally and locally diverse set of S. enterica ssp I sv Paratyphi A isolates Keywords: ordered
Project description:Salmonella enterica subsp. enterica contains more than 2,600 serovars of which four are of major medical relevance for humans. While the typhoidal serovars (Typhi and Paratyphi A) are human-restricted and cause enteric fever, non-typhoidal Salmonella serovars (Typhimurium and Enteritidis) have a broad host range and predominantly cause gastroenteritis. In this study, we compared the core proteomes of Salmonella Typhi, Paratyphi A, Typhimurium and Enteritidis using contemporary proteomics. Five isolates, covering different geographical origins, and one reference strain per serovar were grown in vitro to the exponential phase. Protein levels of orthologous proteins between serovars were compared and subjected to gene ontology term enrichment and inferred regulatory interactions. Differential expression of the core proteomes of the typhoidal serovars appears mainly related to cell surface components and, for the non-typhoidal serovars, to pathogenicity. Our findings may guide future development of novel diagnostics and vaccines, and understanding of disease progression.
Project description:Comparative genomic analysis of a temporally and locally diverse set of S. enterica ssp I sv Paratyphi A isolates Keywords: ordered
Project description:Salmonella enterica causes serious global burden of morbidity and mortality and is a major cause of infant bacteremia in sub Saharan Africa. Diseases caused by Salmonella are treatable with antibiotics but successful antibiotic treatment has become difficult due to antimicrobial resistance. An effective vaccine together with public health effort may therefore be a better strategy to control these infections. Protective immunity against Salmonella depends primarily on T cell-mediated immune responses and therefore identifying relevant T cell antigens is necessary for Salmonella vaccine development. Our laboratory has used an immunoproteomics approach to identify Chlamydia T cell antigens that exhibited significant protection against Chlamydia infection in mice. In this study, we infected murine bone marrow derived dendritic cells from C57BL/6 mice with Salmonella enterica strain SL1344 followed by isolation of MHC class I and II- molecules and elution of bound peptides. The sequences of the peptides were then identified using tandem mass spectrometry. We identified 87 MHC class II and 23 MHC class I Salmonella derived peptides. Four of 12 peptides stimulated IFN-? production by CD4 T cells from the spleens of mice with persistent Salmonella infection. These antigens will be useful for Salmonella immunobiology research and are potential Salmonella vaccine candidates.
Project description:In this work, we applied an RNA analysis method, Selective Capture of Transcribed Sequences (SCOTS), and cDNA hybridization-microarray technology to identify S. Paratyphi A transcripts expressed by bacteria in the blood of three patients in Bangladesh. In total, we detected 1798 S. Paratyphi A mRNAs expressed in the blood of infected humans (43.9% of the ORFeome). Of these, we identified 868 in at least two patients, and 315 in all three patients. S. Paratyphi A transcripts identified in at least two patients encode proteins involved in energy metabolism, nutrient and iron acquisition, vitamin biosynthesis, stress responses, oxidative stress resistance, and pathogenesis. A number of detected transcripts are expressed from PhoP and SlyA-regulated genes associated with intra-macrophage survival, genes contained within Salmonella Pathogenicity Islands (SPIs) 1-4, 6, 10, 13, and 16, as well as RpoS-regulated genes. The largest category of identified transcripts are those encoding proteins with unknown function. When comparing level of bacterial mRNA detection using in vivo samples collected from infected patients to samples from in vitro grown organisms, we found significant differences for 347, 391, and 456 S. Paratyphi A transcripts in each of three individual patients (approximately 9.7% of the ORFeome). Of these, expression of 194 transcripts (4.7% of ORFs) was concordant in two or more patients, and 41 in all patients. Genes encoding these transcripts are contained within SPI-1, 3, 6 and 10, are PhoP-regulated genes, are involved in energy metabolism, nutrient acquisition, drug resistance, or are uncharacterized genes. Using quantitative RT-PCR, we confirmed increased gene expression in vivo for a subset of genes identified in our analyses.
Project description:In order to characterize pathogen specific T cell responses against Salmonella volunteers challenged with Salmonella enterica serovar Typhi (S. Typhi) or Salmonella Paratyphi A (S. Paratyphi). we used mass cytometry, to identify effector CD4+ T cells circulating during infection. We identified a population of CCR7-CD38+ cells accumulating during infection, and via unbiased single cell cloning and expansion we demonstrated that these CCR7-CD38+ cells are enriched in Salmonella specific T cells. In this experiment we performed TCR repertoire analysis of CCR7-CD38+ and CCR7-CD38- cells to determine the clonality of CCR7-CD38+ cells, the overlap between the repertoire of CCR7-CD38+ cells and of non-activated effector CCR7-CD38- cells, and to identify within CCR7-CD38+ and CCR7-CD38- cells the presence of the CDR3b TCR sequence of the pathogen specific T cell clones isolated from CCR7-CD38+ cells