For intracellular pathogens such as Salmonellae, Mycobacteriae and Brucellae, infection requires adaptation to the intracellular environment of the phagocytic cell. The transition from extracellular to intravacuolar environment has been expected to involve a global modulation of bacterial gene expression, but the precise events have been difficult to determine. We now report the complete transcriptional profile of intracellular Salmonella enterica sv. Typhimurium following macrophage infection. ...[more]
Project description:NADPH dependent phagocytic oxidase, by producing hydroxyradicals such as hydrogen peroxide, is essential for host defense against Salmonella infection. We used gene array analysis to identify Salmonella enterica serovar Typhimurium genes regulated by NADPH dependent phagocytic oxidase intracellularly in comparison to those expressed in vitro by hydrogen peroxide.
Project description:Streptococcus mutans was grown for 48 h in a biofilm in the absence (single species) and in the presence (dual species) of Veillonella parvula. In addition V. parvula single species 48 h biofilms were grown, to be used as a control. RNA was harvested from all types of biofilms and the transcript levels of the two types of biofilms containing S. mutans were compared with the use of S. mutans microarrays. V. parvula RNA was hybridized to S. mutans microarrays as a control for possible cross-hybridisation.
Project description:P388D1 murine macrophages were cultured in 85 mm tissue culture plates to about semi confluency. L. monocytogenes serotypes (1/2a EGD-e, 4a L99, 4b CLIP80459 and 4b F2365) were infected to the P388D1 cell monolayer at a MOI of 100 per eukaryotic cell. Infection was carried for 45 min and followed by addition of fresh medium containing 20 µg/ml gentamicin. The medium of the plates (containing 20 µg/ml gentamicin) infected with L. monocytogenes serotypes were replaced after 2 h post infection with fresh medium containing 50 µg/ml gentamicin. At each step the plates were washed extensively with 1x PBS. Incubation of the bacterial tissue culture plates was carried out in a humidified incubator for up to 4 h post infection.
Project description:The integration host factor, IHF, is a DNA-binding and -bending protein with roles in local DNA structural organization and transcriptional regulation in Gram-negative bacteria. This heterodimeric protein is composed of the two highly homologous subunits IHF alpha and IHF beta. DNA microarray analysis was used to define the regulon of genes subject to IHF control in Salmonella enterica serovar Typhimurium. The transcription profile of the wild type was compared with those of mutants deficient in IHF alpha, IHF beta, or both IHF alpha and IHF beta. Our data reveal a new connection between IHF and the expression of genes required by the bacterium to undergo the physiological changes associated with the transition from exponential growth to stationary phase. When a mutant lacking IHF entered stationary phase, it displayed downregulated expression of classic stationary-phase genes in the absence of any concomitant change in expression of the RpoS sigma factor. Purified IHF was found to bind to the regulatory regions of stationary-phase genes indicating an auxiliary and direct role for IHF in RpoS-dependent gene activation. Loss of IHF also had a profound influence on expression of the major virulence genes and epithelial cell invasion, indicating a role in co-ordinating regulation of the pathogenic traits with adaptation to stationary phase. Although the three mutants showed considerable overlaps in the genes affected by the ihf lesions, the observed patterns were not identical, showing that S. Typhimurium has not one but three overlapping IHF regulons.