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Interrogation of global mutagenesis data with a genome scale model of Neisseria meningitidis to assess gene fitness in vitro and in sera.

ABSTRACT: BACKGROUND: Neisseria meningitidis is an important human commensal and pathogen that causes several thousand deaths each year, mostly in young children. How the pathogen replicates and causes disease in the host is largely unknown, particularly the role of metabolism in colonization and disease. Completed genome sequences are available for several strains but our understanding of how these data relate to phenotype remains limited. RESULTS: To investigate the metabolism of N. meningitidis we generated and then selected a representative Tn5 library on rich medium, a minimal defined medium and in human serum to identify genes essential for growth under these conditions. To relate these data to a systems-wide understanding of the pathogen's biology we constructed a genome-scale metabolic network: Nmb_iTM560. This model was able to distinguish essential and non-essential genes as predicted by the global mutagenesis. These essentiality data, the library and the Nmb_iTM560 model are powerful and widely applicable resources for the study of meningococcal metabolism and physiology. We demonstrate the utility of these resources by predicting and demonstrating metabolic requirements on minimal medium, such as a requirement for phosphoenolpyruvate carboxylase, and by describing the nutritional and biochemical status of N. meningitidis when grown in serum, including a requirement for both the synthesis and transport of amino acids. CONCLUSIONS: This study describes the application of a genome scale transposon library combined with an experimentally validated genome-scale metabolic network of N. meningitidis to identify essential genes and provide novel insight into the pathogen's metabolism both in vitro and during infection.

SUBMITTER: Mendum TA

PROVIDER: S-EPMC3334622 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Interrogation of global mutagenesis data with a genome scale model of Neisseria meningitidis to assess gene fitness in vitro and in sera.

Mendum Tom A TA Newcombe Jane J Mannan Ahmad A AA Kierzek Andrzej M AM McFadden Johnjoe J

Genome biology 20111230 12

<h4>Background</h4>Neisseria meningitidis is an important human commensal and pathogen that causes several thousand deaths each year, mostly in young children. How the pathogen replicates and causes disease in the host is largely unknown, particularly the role of metabolism in colonization and disease. Completed genome sequences are available for several strains but our understanding of how these data relate to phenotype remains limited.<h4>Results</h4>To investigate the metabolism of N. meningi ...[more]

PMID: 22208880

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Project description:Neisseria meningitidis remains an important cause of septicemia and meningitis. One of its major virulence traits is its ability to avoid killing by human serum. In the present work, the effect of growth phase (exponential versus stationary) and growth medium (THB versus Catlin) on normal human serum (NHS) sensitivity was assessed on two N. meningitidis serogroup B strains (MC58 and M982). Both strains were found to be dramatically more resistant to 20% NHS killing at early exponential phase than at stationary phase (73-100% survival after 1 to 2 hours of growth compared to less than 10% survival after 7 hours of growth). This growth phase effect was detected only when a rich medium (THB) was used while no such effect was observed using Catlin medium. KDO (2-Keto-3-deoxyoctulosonic acid) and sialic acid content were measured on serum-resistant and serum-sensitive bacteria and were found comparable, indicating that differences in LPS and capsule expression were not at the origin of the observed phenotypes. Transcriptome profiling using a PCR-array was used to compare serum-resistant and serum-sensitive bacteria. A set of 255 genes was found to be differentially expressed with 159 genes up-regulated in serum-resistant bacteria, among them 12 genes involved in glucose catabolism and 22 are known virulence factors. Overall, this study shows that serum-resistant phenotype of N. meningitidis could be obtained through modulating growth conditions. The nature of growth media and growth phases have a major impact on the ability of N. meningitidis to resist to NHS killing. Consequently, the present work underlines the critical importance of carefully controlling those parameters in any studies aimed at investigating the mechanisms and factors involved in N. meningitidis serum-resistance. All experiments were performed in dye swap. These experiments were performed with 2 strains of N. meningitidis serogroup B. In one set of experiments, the gene expression profile of bacteria cultured in THB medium during 7h was compared with the corresponding profile of bacteria cultured during 1h in the same medium. In a second set of experiments, the gene expression profile of 1 strain cultured in THB medium during 7h was compared with the corresponding profile of the same strain cultured in Catlin medium during 7h. 3 biological replicates were performed for each comparison.

Dataset Information

Interrogation of global mutagenesis data with a genome scale model of Neisseria meningitidis to assess gene fitness in vitro and in sera.

Publications

Interrogation of global mutagenesis data with a genome scale model of Neisseria meningitidis to assess gene fitness in vitro and in sera.

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