In eukaryotic cell types, virtually all cellular processes are under control of proline-directed kinases and especially MAP kinases. Serine/threonine kinases in general were originally considered as a eukaryote-specific enzyme family. However, recent studies have revealed that orthologues of eukaryotic serine/threonine kinases exist in bacteria. Moreover, various pathogenic species, such as Yersinia and Mycobacterium, require serine/threonine kinases for successful invasion of human host cells. The substrates targeted by bacterial serine/threonine kinases have remained largely unknown. Here we report that the serine/threonine kinase PknB from the important pathogen Staphylococcus aureus is released into the external milieu, which opens up the possibility that PknB does not only phosphorylate bacterial proteins but also proteins of the human host. To identify possible human targets of purified PknB, we studied in vitro phosphorylation of peptide microarrays and detected 68 possible human targets for phosphorylation. These results show that PknB is a proline-directed kinase with MAP kinase-like enzymatic activity. As the potential cellular targets for PknB are involved in apoptosis, immune responses, transport, and metabolism, PknB secretion may help the bacterium to evade intracellular killing and facilitate its growth. In apparent agreement with this notion, phosphorylation of the host-cell response coordinating transcription factor ATF-2 by PknB was confirmed by mass spectrometry. Taken together, our results identify PknB as the first prokaryotic representative of the proline-directed kinase/MAP kinase family of enzymes.

Staphylococcus aureus is a dangerous opportunistic human pathogen that causes serious invasive diseases when it reaches the bloodstream. Recent studies have shown that S. aureus is highly resistant to killing by professional phagocytes and that such cells even provide a favorable environment for intracellular survival of S. aureus. Importantly, the reciprocal interactions between phagocytes and S. aureus have remained largely elusive. Here we have employed kinase profiling to define the nature and time resolution of the human THP-1 macrophage response toward S. aureus and proteomics to identify the response of S. aureus toward macrophages. The results of these studies reveal major macrophage signaling pathways triggered by S. aureus and proteomic signatures of the responses of S. aureus to macrophages. We also identify human proteins bound to S. aureus that have potential roles in bacterial killing and internalization. Most noticeably, our observations challenge the classical concept that macrophage responses are mainly mediated through Toll-like receptor 2 and NF-κB signaling and highlight the important role of the stress-activated MAP kinase signaling in orchestrating the host defense.

Comparison milk and blood lymphocyte blastogenic responses to the K1 antigen of Escherichia coli and lipopolysaccharide (LPS) from E. coli 0127,B8 were examined in 16 postpartum women by [2H]thymidine uptake. Rabbit hemolysincoated sheep erythrocyte monolayers were used to deplete macrophages from milk lymphocyte preparations and to enrich for T lymphocytes in order to make milk preparations more comparable to blood preparations. Response was defined as a stimulation index of -2.0. There was no evidence of selective response to K1 antigen by milk lymphocytes, since both blood and milk lymphocytes responded in four women and neither blood nor milk lymphocytes responded in nine. Milk lymphocytes alone responded to K1 in one woman, whereas blood lymphocytes alone responded in two women. Additional nonpaired milk or blood cultures were available from three women. None of these responded to K1 antigen. Corresponding lymphocyte cultures were stimulated with LPS. A positive K1 response was always accompanied by an LPS response, and the LPS response correlated with the K1 response in 17 of 19 women. Stool cultures examined with an antiserum agar showed no correlation between the presence of K1 E. coli in the stool and milk or blood lymphocyte response to K1 antigen. In the system used here, no selectivity of response of breast milk lymphocytes to K1 antigen was noted.Previous investigators (19)(20)(21) have proposed that a selective homing or tracking of lymphocyte populations to the human mammary gland results in a milk lymphocyte population different from the peripheral blood lymphocyte population. In vitro lymphocyte stimulation with several antigens has shown that breast milk lymphocyte responses do not always correspond with peripheral blood lymphocyte responses. The K1 antigen of Escherichia coli (a gut-associated antigen) is the only antigen studied that has been reported to stimulate breast milk lymphocytes but not peripheral blood lymphocytes. These data of Parmely et al. (20) support the concept of selective tracking of lymphocytes from the human gut to the human mammary gland.Homing of B lymphocytes from the gut to the mammary gland had been suggested before the studies of Parmely et al. by other investigators who used both animal and human subjects (1,5,13). In their studies of the gut-associated lymphoid systems in mice, Guy-Grand et al. (15) demonstrated tracking of T and B lymphoblasts to the lamina propria and intestinal epithelium from thoracic duct lymph and mesenteric lymph nodes. More recently, Roux et al. (25) have performed experiments in mice with radiola-beled mesenteric lymph node lymphoblasts which suggest possible homing of immunoglobulin A (IgA)-secreting plasma cells to the mammary gland late in pregnancy and during lactation, in addition to a homing back to the intestinal epithelium. The K1 data of Parmely et al. (20) and their subsequent hypothesis of selected T-cell populations in human milk are consistent with existing information from animal and human studies.A problem with comparison bl...