NEONATAL SOCIETY ABSTRACTS
Group B Streptococcus gene expression in nutrient limited environments
Presented at the Neonatal Society 2004 Summer Meeting (programme).
Herbert M, Beveridge C, Synder L, Saunders N
Neonatal Unit, Department of Paediatrics, University of Oxford, John Radcliffe Hospital, Oxford, UK
Purpose: We aimed to study gene expression in Group B Streptococcus (GBS) when grown in nutritionally deplete environments, such as amniotic fluid. Our long-term goal is to identify key regulators of virulence that can be targeted to develop novel means of preventing Early-Onset Sepsis (EOS).
Experimental design: We developed a 384-probe sub-microarray containing probes to all the known GBS regulator, virulence and transporter genes, and genes involved in adaptation to reactive oxygen intermediate stress. RNA was extracted from GBS grown in amniotic fluid and rich media (Todd Hewitt Broth; THB). RNA profiles were stabilised with RNAlater. RNA was converted to cDNA, indirectly labelled with Cy3 and Cy5 dyes, hybridised to the sub-array in Genetix hybridisation solution at 65°C for 8 hours, and washed with Genetix wash solutions. The hybridised arrays were read on a Genepix 4000B microarray scanner.
Results: The gene expression of GBS grown in THB was compared at mid- and late-logarithmic and early-stationary phases (figure). Comparisons between genes expressed in THB and amniotic fluid is on going.
Figure: Gene expression at 3 compared with 5 hours growth in THB. Blank spots represent no detectable gene expression, bright yellow spots indicate genes that are strongly expressed under both conditions, and red or green spots indicate genes up or down regulated, respectively, at 3 compared with 5 hours.
Identifying the genes that are essential for growth during nutritional depletion (such as late logarithmic phase growth in THB and in amniotic fluid) may reveal an Achilles heel that can be targeted to inhibit GBS growth during the initiation of EOS. Three regulators of virulence, encoded by mga, rofA and nra, were shown to be up or down regulated according to bacterial density and nutrient availability. Also as GBS reaches late logarithmic growth virulence genes encoding C5a peptidase, sialic acid synthesis genes and hemolysin are up regulated, as are peptide, sugar and phosphate transporters, and enzymes excreted to damage host cells in the immediate environment and thus release nutrients. Parallel experiments in amniotic fluid are underway.
Conclusions: We have identified regulators of virulence of GBS that are expressed during nutritional depletion in vitro. If these same genes are expressed during growth in amniotic fluid, then these can be targeted for the development of inhibitors to prevent GBS EOS.