Binding of the fibronectin-binding protein FnBPA fromStaphylococcus aureus to the human protein fibronectin has previously been implicated in the development of infective endocarditis, specifically in the processes of platelet activation and invasion of the endothelium. We recently proposed a model for binding of fibronectin to FnBPA in which the bacterial protein contains 11 potential binding sites (FnBPA-1 to FnBPA-11), each composed of motifs that bind to consecutive fibronectin type 1 modules in the N-terminal domain of fibronectin. Here we show that six of the 11 sites bind with dissociation constants in the nanomolar range; other sites bind more weakly. The high affinity binding sites include FnBPA-1, the sequence of which had previously been thought to be encompassed by the fibrinogen-binding A domain of FnBPA. Both the number and sequence conservation of the type-1 module binding motifs appears to be important for high affinity binding. The in vivo relevance of the in vitro binding studies is confirmed by the presence of antibodies in patients with S. aureus infections that specifically recognize complexes of these six high affinity repeats with fibronectin.Staphylococcus aureus is one of the most important bacterial pathogens to affect humans. Clinical manifestations of infection range from superficial skin infections (1) to life-threatening conditions, such as endocarditis (2, 3) and difficult to treat infections of the bones and joints (4, 5). The increasing virulence and antibiotic resistance exhibited by this major source of both community and hospital-acquired infection presents an urgent challenge. Furthering our understanding of the mechanism by which staphylococcal pathogenesis occurs is thus imperative for the development of novel therapeutic and preventative strategies.Since attachment to host tissue is a critical early step in infection, one particular group of targets for intervention is the microbial surface components recognizing adhesive matrix molecules (MSCRAMM) 4 family of surface-expressed adhesins (6, 7). These proteins exploit extracellular matrix proteins, such as fibronectin (Fn), using them as a bridge between the bacterial cell surface and host cell receptors that effect downstream signaling (8, 9). Although classically regarded as an exclusively extracellular pathogen, S. aureus has been shown to adhere to and invade several host cell types (10 -14), and the Fn-binding subfamily of MSCRAMMs (FnBPs) appears to be involved in this process (15). There is an emerging view that S. aureus can exist intracellularly, hijacking and invading host cells to establish persistence (16). Conceivably, this mechanism could facilitate rapid and effective bloodstream dissemination while allowing the bacterium to evade antibiotics and host immune surveillance, an apposite theory given the prevalence of bacterial metastasis (3) and infection relapse in staphylococcal disease (17).Fn is a large glycoprotein present in a soluble form in human plasma and other body fluids and in an insoluble form in...

No abstract

Bacterial fibronectin-binding proteins (FnBPs) contain a large intrinsically disordered region (IDR) that mediates adhesion of bacteria to host tissues, and invasion of host cells, through binding to fibronectin (Fn). These FnBP IDRs consist of Fn-binding repeats (FnBRs) that form a highly extended tandem ␤-zipper interaction on binding to the N-terminal domain of Fn. Several FnBR residues are highly conserved across bacterial species, and here we investigate their contribution to the interaction. Mutation of these residues to alanine in SfbI-5 (a disordered FnBR from the human pathogen Streptococcus pyogenes) reduced binding, but for each residue the change in free energy of binding was <2 kcal/mol. The structure of an SfbI-5 peptide in complex with the second and third F1 modules from Fn confirms that the conserved FnBR residues play equivalent functional roles across bacterial species. Thus, in SfbI-5, the binding energy for the tandem ␤-zipper interaction with Fn is distributed across the interface rather than concentrated in a small number of "hot spot" residues that are frequently observed in the interactions of folded proteins. We propose that this might be a common feature of the interactions of IDRs and is likely to pose a challenge for the development of small molecule inhibitors of FnBP-mediated adhesion to and invasion of host cells.

We recently provided the first description of a nuclear mechanism used by Protein Kinase C-theta (PKC-θ) to mediate T cell gene expression. In this mode, PKC-θ tethers to chromatin to form an active nuclear complex by interacting with proteins including RNA polymerase II, the histone kinase MSK-1, the demethylase LSD1, and the adaptor molecule 14-3-3ζ at regulatory regions of inducible immune response genes. Moreover, our genome-wide analysis identified many novel PKC-θ target genes and microRNAs implicated in T cell development, differentiation, apoptosis, and proliferation. We have expanded our ChIP-on-chip analysis and have now identified a transcription factor motif containing NF-κB binding sites that may facilitate recruitment of PKC-θ to chromatin at coding genes. Furthermore, NF-κB association with chromatin appears to be a prerequisite for the assembly of the PKC-θ active complex. In contrast, a distinct NF-κB-containing module appears to operate at PKC-θ targeted microRNA genes, and here NF-κB negatively regulates microRNA gene transcription. Our efforts are also focusing on distinguishing between the nuclear and cytoplasmic functions of PKCs to ascertain how these kinases may synergize their roles as both cytoplasmic signaling proteins and their functions on the chromatin template, together enabling rapid induction of eukaryotic genes. We have identified an alternative sequence within PKC-θ that appears to be important for nuclear translocation of this kinase. Understanding the molecular mechanisms used by signal transduction kinases to elicit specific and distinct transcriptional programs in T cells will enable scientists to refine current therapeutic strategies for autoimmune diseases and cancer.

1. The dihydropyridine receptor (DHPR) II-III loop is an intrinsically unstructured region made up of alpha-helical and beta-turn secondary structure elements with the N and C termini in close spatial proximity. 2. The DHPR II-III loop interacts in vitro with a ryanodine receptor (RyR) 1 SPRY domain through alpha-helical segments located in the A and B regions. Mutations within the A and B regions in the DHPR II-III loop alter the binding affinity to the SPRY2 domain. 3. The A and C peptides derived from DHPR II-III loop show negative cooperativity in binding to the SPRY2 domain. 4. The SPRY2 domain of the RyR1 (1085-1208) forms a beta-sheet sandwich structure flanked by variable loop regions. An acidic loop region of SPRY2 (1107-1121) forms part of a negatively charged cleft that is implicated in the binding of the DHPR II-III loop. 5. The mutant E1108A located in the negatively charged loop of SPRY2 reduces the binding affinity to the DHPR II-III loop.