[32,33,34,35], such as sensing of environmental stimuli and the activation and inactivation of response regulators [36,37]. This is generally achieved through cascades of phosphorylation reactions in the host, which focuses a strong interest on the role of kinases, such as the serine/threonine kinase PknB (also known as StpK) in staphylococcal persistence. The PknB kinase is composed of three extracellular PASTA domains (penicillin binding domains), a central transmembrane domain and an intracellular kinase domain [16,38]. Interestingly, it was recently reported that PknB is not only involved in regulation of the central metabolism of S. aureus [15], but also determines staphylococcal infection of mouse kidneys in an abscess model [17]. The latter observation raises the question whether the kinase activity of PknB is directly or indirectly involved in the pathogenicity of S. aureus. A direct role of PknB in infection is conceivable since serine/threonine kinases play key roles in mammalian cell signalling, and at least two bacterial equivalents, YpkA of Yersinia and PknG of Mycobacterium tuberculosis, have been shown to be directly involved in the subversion of host cells during the respective infectious processes [39,40]. However the exact role played by PknB in pathogenesis or staphylococcal persistence has thus far remained unclear. Here we show that full-size soluble PknB is present in the medium of growing S. aureus cells. We therefore investigated whether PknB of S. aureus can recognize and phosphorylate known substrates of human serine/threonine kinases. For this purpose, we used peptide microarrays with known human phosphorylation sites. The phosphorylation profile and mass spectrometry results show that PknB is a proline-directed kinase, which can indeed phosphorylate specific human targets. The observed target specificity of PknB indicates possible roles for this enzyme in a wide range of host cell signalling processes during S. aureus infection. Results and Discussion Identification of Extracellular PknB It has previously been reported that different bacteria such as M. tuberculosis and Yersinia species can secrete their eukaryotic-like serine/threonine kinases directly into the host. This mechanism allows these bacteria to survive intracellularly [9,39], to disrupt the actin cytoskeleton [41], or to cause host cell apoptosis [40]. Since these bacterial ser/thr kinases need to be exported in order to impact on host cells, we investigated whether PknB might be detectable in the extracellular milieu of S. aureus. As shown by Western blotting using polyclonal antibodies against PknB, the full-size PknB was detectable both in the cellular and growth medium fractions of cells of S. aureus NCTC 8325 harvested at an OD600 of 2. As expected, PknB was neither detectable in cellular nor growth medium fractions of the DpknB mutant. The precise mechanism by which PknB is liberated from the wild-type cells remains to be elucidated. However, there is precedence for the release of membrane proteins, or fragments thereof, into the extracellular milieu of Gram-positive bacteria, such as S. aureus, by as yet unknown mechanisms [42]. One possibility is that these proteins are released by cell lysis, remaining stable in the medium due to an intrinsic resistance to extracytoplasmic proteolysis [43]. The idea that PknB is released by lysis would be consistent with the detection of relatively small amounts of the cytoplasmic marker protein thioredoxin A (TrxA) in the growth medium fractions (Fig. 1). TrxA is a cytoplasmic bacterial protein, which acts as an antioxidant by facilitating the reduction of cysteine disulfides in other cytoplasmic proteins. Since TrxA is normally a cytoplasmic protein, it will only be found in the extracellular milieu when PknB Phosphorylation Profile Figure 1. Release of PknB into the growth medium of S. aureus. The S. aureus strain NCTC 8325 (wt) or a DpknB derivative were propagated at 37uC in TSB and harvested at OD600 2. Crude extracts (ce) and supernatant (sup) fractions were isolated, corrected for OD and separated by NuPAGE electrophoresis (Invitrogen). Two-fold higher amounts of the supernatant fractions were used for PAGE as compared to the crude extracts. Immunoblotting was conducted using specific antibodies against PknB (upper panel) or TrxA (lower panel). The latter served as an indicator for cell lysis. The position of the specific PknB signal is marked with a black arrow. The band at ,60 kDA corresponds to an unidentified protein, which cross-reacts with the antibodies against PknB. The molecular weight of marker proteins is indicated on the left. doi:10.1371/journal.pone.0009057.g001 bacterial cells have lysed. Notably, compared to the cellular samples, we detected relatively more extracellular PknB than extracellular TrxA, which might suggest that the release of PknB is the consequence of a specific process rather than cell lysis. Irrespective of the mechanism by which PknB is released into the extracellular milieu of S. aureus, its release may impact on human host cell functions. This could be the case upon internalization of S. aureus. Although S. aureus is primarily an extracellular pathogen, there is strong evidence that it can be internalized by a wide range of human host cells. For example, S. aureus invades non-professional phagocytes by a mechanism which requires a specific interaction between the bacterial fibronectin- binding protein and the host cell [44,45,46]. This leads to host signal transduction, activation of tyrosine kinases, cytoskeletal rearrangement and endosome uptake. Bayles and Qazi reported that internalized S. aureus is able to escape from the host endosome, and this fact opens up the possibility of direct interactions of released PknB with proteins of the human host [47,48] Eukaryotic Phosphorylation Sites Recognized by PknB Peptide arrays (PepChips) have previously been used success- fully to profile the activity of kinases in eukaryotic cell lysates [49]. We therefore employed this array-based technology to investigate whether the staphylococcal kinase PknB has the ability to recognize and phosphorylate human phosphorylation sites. When the PepChips were incubated with purified and active PknB [16] and [33P-c] ATP, radioactivity was efficiently incorporated in a particular subset of the peptides on the chip. In contrast, little radioactivity was incorporated when the arrays were incubated with [33P-c] ATP in the absence of PknB. We identified 68 PLoS ONE | www.plosone.org 2 February 2010 | Volume 5 | Issue 2 | e9057