Considerably divergent data have been published from attempts to model the E. coli vs. S. aureus specific immune reaction of the udder using primary cultures of bovine mammary epithelial cells from cows (pbMEC). Some groups reported a swift, strong and transient inflammatory response against challenges with E. coli and only a weak and retarded response against S. aureus, in agreement with the respective reaction of the udder. Others found almost the reverse. Presence or absence of fetal calf serum distinguished the experimental setting between both groups. We examined here if this causes the divergent reaction of the pbMEC towards both pathogen species. We challenged pbMEC with proteins from heat killed E. coli or S. aureus pathogens or purified TLR2 and TLR4 ligands. The stimuli were applied in normal growth medium with (SM10) or without (SM0) 10% fetal calf serum, or in the basal medium supplemented with 10 mg/ml milk proteins (SM Milk).

Listeria monocytogenes is a firmicute bacterium causing serious infections in humans upon consumption of contaminated food. Most of its virulence factors are secretory proteins either released to the medium or attached to the bacterial surface. L. monocytogenes encodes at least six different protein secretion pathways. Although great efforts have been made in the past to predict secretory proteins and their secretion routes using bioinformatics, experimental evidence is lacking for most secretion systems. Therefore, we constructed mutants in the main housekeeping protein secretion systems, which are the Sec-dependent transport, the YidC membrane insertases SpoIIIJ and YqjG, as well as the twin-arginine pathway, and analyzed their secretion and virulence defects. Our results demonstrate that Sec-dependent secretion and membrane insertion of proteins via YidC proteins are essential for viability of L. monocytogenes. Depletion of SecA or YidC activity severely affected protein secretion, whereas loss of the Tat-pathway was without any effect on secretion, viability, and virulence. Two-dimensional gel electrophoresis combined with protein identification by mass spectrometry revealed that secretion of many virulence factors and of enzymes synthesizing and degrading the cell wall depends on the SecA route. This finding was confirmed by SecA inhibition experiments using sodium azide. Analysis of secretion of substrates typically dependent on the accessory SecA2 ATPase in wild type and azide resistant mutants of L. monocytogenes revealed for the first time that SecA2-dependent protein secretion also requires the ATPase activity of the house-keeping SecA protein.