A proteomics workflow for quantitative and time-resolved analysis of adaptation reactions of internalized bacteria.

The development of a mass spectrometric workflow for the sensitive identification and quantitation of the kinetics of changes in metaproteomes, or in particular bacterial pathogens after internalization by host cells, is described. This procedure employs three essential stages: (i) SILAC pulse-chase labeling and infection assay; (ii) isolation of bacteria by GFP-assisted cell sorting; (iii) mass spectrometry-based proteome analysis. This approach displays greater sensitivity than techniques relying on conventional cell sorting and protein separation, due to an efficient combination of a filtration-based purification and an on-membrane digestion. We exemplary describe the use of the workflow for the identification and quantitation of the proteome of 106 cells of Staphylococcus aureus after internalization by S9 human bronchial epithelial cells. With minor modifications, the workflow described can be applied for the characterization of other host-pathogen pairs, permitting identification and quantitation of hundreds of bacterial proteins over a time range of several hours post infection.

Experimental measles encephalitis in Lewis rats: dissemination of infected neuronal cell subtypes.

Acute measles may lead in rare instances to the chronic progressive central nervous system disease process subacute sclerosing panencephalitis (SSPE). SSPE results from a persistent measles virus (MV) infection with incomplete virus replication involving the entire human brain. The experimental encephalitis model in Lewis rats was used to define affected cell populations after infection with the neurotropic MV strain CAM/RB. Distribution patterns of MV were analysed by appropriate cell markers in the brain sections of infected animals employing multiple immunofluorescence labelling and confocal laser scanning microscopy. MV was detected in neurones but not in astrocytes, oligodendrocytes, microglia, and endothelial cells. GABAergic and glutamatergic neurons displayed MV antigen whereas cholinergic and catecholaminergic neurons appeared devoid of MV immunoreactivity. Mapping of the rat brain has revealed MV-infected neurones predominantly in motor, somatosensory, auditory, and visual cortices as well as in the basal ganglia and thalamic nuclei of infected rats. The results indicate that MV apparently disseminates via GABAergic and glutaminergic neurones and their processes. The tightly restricted viral distribution pattern is consistent with both inefficient immune clearance from infected neurones and with the observed disease symptoms.