Much attention has focused on the Mycobacterium tuberculosis molecular chaperone chaperonin (Cpn) 60.2 (Hsp 65) in the pathology of tuberculosis because of its immunogenicity and ability to directly activate human monocytes and vascular endothelial cells. However, M. tuberculosis is one of a small group of bacteria that contain multiple genes encoding Cpn 60 proteins. We have now cloned and expressed both M. tuberculosis proteins and report that the novel chaperonin 60, Cpn 60.1, is a more potent inducer of cytokine synthesis than is Cpn 60.2. This is in spite of 76% amino acid sequence similarity between the two mycobacterial chaperonins. The M. tuberculosis Cpn 60.2 protein activates human peripheral blood mononuclear cells by a CD14-independent mechanism, whereas Cpn 60.1 is partially CD14 dependent and contains a peptide sequence whose actions are blocked by anti-CD14 monoclonal antibodies. The cytokine-inducing activity of both chaperonins is extremely resistant to heat. Cpn 60.1 may be an important virulence factor in tuberculosis, able to activate cells by diverse receptor-driven mechanisms.

Staphylococcus epidermidis has been reported to bind to a number of host cell extracellular matrix proteins, including fibronectin. Here we report the identification of a fibronectin-binding protein from S. epidermidis. A phage display library of S. epidermidis genomic DNA was constructed and panned against immobilized fibronectin. A number of phagemid clones containing overlapping inserts were identified, and one of these clones, pSE109FN, contained a 1.4-kb insert. Phage pSE109FN was found to bind to fibronectin but not to collagen, fibrinogen, laminin, or vitronectin. However, pSE109FN also bound to heparin, hyaluronate, and plasminogen, although to a lesser extent than it bound to fibronectin. Staphylococcus epidermidis, a normal skin commensal, is an important opportunistic pathogen responsible for infections of implanted medical devices and infections at sites of surgery (24). Medical devices infected by S. epidermidis include cardiac pacemakers (1), cerebrospinal fluid shunts (23), continuous ambulatory peritoneal dialysis catheters (14), intravascular catheters (2), orthopedic devices (11), and prosthetic heart valves (13). The accepted paradigm for S. epidermidis adhesion to biomaterial surfaces is a two-step process involving a primary attachment step mediated by hydrophobic interactions, followed by biofilm formation and intercellular interactions (6). However, it is acknowledged that the primary attachment step is mediated not only by hydrophobic interactions but also by bacterial proteins.Since foreign bodies become rapidly coated with plasma proteins when they are implanted in the body, S. epidermidis may bind to implants through interactions with immobilized extracellular matrix proteins. In vitro studies have shown that S. epidermidis can bind to biomaterials coated with fibronectin and fibrinogen (3,5,9,17). The ability of Staphylococcus aureus to bind to extracellular matrix proteins through a family of proteins termed the microbial surface components recognizing adhesive matrix molecules is well documented (for a review see reference 4). However, comparatively little is known about how S. epidermidis interacts with matrix proteins. It has been established that S. epidermidis binds to fibrinogen through SdrG, a microbial surface component recognizing adhesive matrix molecules, which is also known as Fbe (7,16,18,19). The autolysin AtlE of S. epidermidis, which has been implicated as the mediator of primary attachment of this organism to polystyrene surfaces, has also been shown to bind vitronectin (8). Although it has been demonstrated that S. epidermidis binds to fibronectin in vitro (3,5,9), no fibronectin-binding protein has been identified yet. In fact, it has been suggested that teichoic acid may mediate binding of S. epidermidis to immobilized fibronectin (10). The aim of the work described in this paper was to use shotgun phage display cloning to identify S. epidermidis genes that code for fibronectin-binding proteins. MATERIALS AND METHODSBacterial strains and plasmids. The type strain ...

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There is evidence that the lipid A-associated proteins (LAPs) of enteric bacteria can induce the synthesis of interleukin 1 (IL-1) and therefore may be important virulence factors. Porphyromonas gingivdis is now recognized as a major pathogen in the chronic inflammatory periodontal diseases and it has previously been reported that a crude LAP fraction from this organism could induce IL-1 and interleukin 6 (IL-6) synthesis. In the present study the chemical and biological properties of the LAPs of this bacterium have been further characterized. Analysis by SDS-PAGE has shown that the LAPs comprise nine proteins of molecular masses 81,68,48,47,28,25,20,17 and 16 kDa. These LAPS, at concentrations as low as 100 ng mV, were shown to stimulate human gingival fibroblasts, human peripheral blood mononuclear cells and whole human blood t o produce the pro-inflammatory cytokine IL-6. The cytokineinducing activity of the LAPs was reduced after heat-inactivation and trypsinization, suggesting that the activity was not due to contaminating LPS. To establish which proteins in this mixture were the active cytokine inducers, the LAPs were separated by electrophoresis on polyacrylamide gels. The majority of the activity was associated with the 17 kDa LAP. N-terminal sequence analysis demonstrated that this protein was homologous t o an internal region of a conserved adhesin domain contained within a family of P. gingivdis extracellular proteins including the RI protease, Lys-gingipain, porphypain and haemagglutinin A. In addition to a role in adherence, the adhesin domain(s) of these proteins may also have cytokine-inducing properties. Furthermore, it has also been shown that the previously observed degradation of cytokines by P. gingivelis may be attributable to the catalytic domain of the RI protease. Thus, different domains within the same molecule appear t o have opposing actions on pro-inflammatory cytokine levels and the balance between these two activities may influence the cytokine status of the periodontiurn in patients with the common chronic inflammatory conditions known as the periodontal diseases.Keywords : Porphyromonas gingivalis RI protease, lipopolysaccharide, lipid A-associated protein, interleukin 1/3, interleukin 6Abbreviations: HGFs, human gingival fibroblasts; IL-1/3, interleukin 18; IL-6, interleukin 6; IAL, Limulus amoebocyte lysate; LAP, lipid A-associated protein; PBMCs, peripheral blood mononuclear cells; P-LPS, P. gingivalis LPS.The SWISS-PROT accession number for the sequence reported in this paper is P81411.