Steered molecular dynamics study reveals insights into the function of the repetitive B region of collagen- and fibrinogen-binding MSCRAMMs

MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) are modular proteins covalently anchored in the bacterial cell wall of many Gram-positive bacteria. The N-terminal region of most MSCRAMMs carries the ligand-binding domains (A region) which specifically target the host extracellular matrix (ECM) proteins such as collagen, fibrinogen and fibronectin. In Staphylococcus aureus Cna, the prototype collagen-binding MSCRAMM, the A region is followed by a repetitive B region which is found to be conserved among many Gram-positive bacteria. This conservation signifies an important functional role for the B region which is made of repetitive domains. It was suggested that this region could act as a ‘stalk’ as well as a ‘spring’ to present the ligand-binding A region, away from the bacterial surface. But there is no clear functional implication of this region available till date. Each repetitive domain in the B region possesses a variant of the Ig fold called the CnaB fold. Additionally, the B repeats are also paired and the pairs are clustered together. To investigate if the B domains have a function similar to the Ig domains in the I-band region of the giant muscle protein, titin, steered molecular dynamics simulations of one, two and four B repeats of Cna were carried out. The results of the simulations suggest that the B region could provide mechanical stability, extensibility and elasticity to Cna due to the CnaB fold as well as the clustered arrangement of their domains. This study thus provided further insights into the biological underpinnings of adhesin–host interaction.     

Isopeptide bond formation in epidermis

Summary Proteins which are major substrates of epidermal transglutaminases can be identified in cultured keratinocytes of human, cow, and new-born rat.Cow and human keratinocytes both contain substrate proteins which are 30000 to 50000 daltons in size but dissociable in SDS to 12000 daltons or less. In both species these proteins correspond to in vivo synthesized proteins which are probable precursors of cornified envelope. Human keratinocytes synthesize a 125000 dalton protein which is also a precursor of cornified envelope both in cells and tissue. By SDS electrophoresis two 100000 dalton substrate proteins are seen in cow keratinocyte extracts and a 23000 dalton substrate protein is seen in rat keratinocyte extracts. Minor substrates of transglutaminase are seen in human keratinocytes, and one has been isolated by preparative electrophoresis. Major structural proteins of epidermis which are in vitro substrates of epidermal transglutaminase include the keratins and the stratum corneum basic protein.     

Role of fibronectin-binding MSCRAMMs in bacterial adherence and entry into mammalian cells

Most bacterial infections are initiated by the adherence of microorganisms to host tissues. This process involves the interaction of specific bacterial surface structures, called adhesins, with host components. In this review, we discuss a group of microbial adhesins known as Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMMs) which recognize and bind FN. The interaction of bacteria with FN is believed to contribute significantly to the virulence of a number of microorganisms, including staphylococci and streptococci. Several FN-binding MSCRAMMs of staphylococci and streptococci exhibit a similar structural organization and mechanism of ligand recognition. The ligand-binding domain consists of tandem repeats of a ∼45 amino acid long unit which bind to the 29-kDa N-terminal region of FN. The binding mechanism is unusual in that the repeat units are unstructured and appear to undergo a conformational change upon ligand binding. Apart from supporting bacterial adherence, FN is also involved in bacterial entry into non-phagocytic mammalian cells. A sandwich model has been proposed in which FN forms a molecular bridge between MSCRAMMs on the bacterial surface and integrins on the host cell. However, the precise mechanism of bacterial invasion and the roles of FN and integrins in this process have yet to be fully elucidated.     

A Novel Fibronectin Binding Motif in MSCRAMMs Targets F3 Modules

Background

BBK32 is a surface expressed lipoprotein and fibronectin (Fn)-binding microbial surface component recognizing adhesive matrix molecule (MSCRAMM) of Borrelia burgdorferi, the causative agent of Lyme disease. Previous studies from our group showed that BBK32 is a virulence factor in experimental Lyme disease and located the Fn-binding region to residues 21–205 of the lipoprotein.

Methodology/Principal Findings

Studies aimed at identifying interacting sites between BBK32 and Fn revealed an interaction between the MSCRAMM and the Fn F3 modules. Further analysis of this interaction showed that BBK32 can cause the aggregation of human plasma Fn in a similar concentration-dependent manner to that of anastellin, the superfibronectin (sFn) inducing agent. The resulting Fn aggregates are conformationally distinct from plasma Fn as indicated by a change in available thermolysin cleavage sites. Recombinant BBK32 and anastellin affect the structure of Fn matrices formed by cultured fibroblasts and inhibit endothelial cell proliferation similarly. Within BBK32, we have located the sFn-forming activity to a region between residues 160 and 175 which contains two sequence motifs that are also found in anastellin. Synthetic peptides mimicking these motifs induce Fn aggregation, whereas a peptide with a scrambled sequence motif was inactive, suggesting that these motifs represent the sFn-inducing sequence.

Conclusions/Significance

We conclude that BBK32 induces the formation of Fn aggregates that are indistinguishable from those formed by anastellin. The results of this study provide evidence for how bacteria can target host proteins to manipulate host cell activities.     

Identification and biochemical characterization of two novel collagen binding MSCRAMMs of Bacillus anthracis

Cell wall-anchored proteins play critical roles in the pathogenesis of infections caused by Gram-positive bacteria. Through the analysis of the genome of Bacillus anthracis Ames strain, we identified two novel putative cell wall-anchored proteins, BA0871 and BA5258, which have sequence homology to CNA, a cell wall-anchored collagen adhesin of Staphylococcus aureus. The two proteins have similar domain organization to that of CNA, with typical signal peptide sequences, a non-repetitive A region followed by repeats, and a characteristic cell wall-anchoring region. They are expressed on the surface of B. anthracis. The A regions of the two proteins were predicted to adopt similar structural folds as CNA. Circular dichroism analysis of the recombinant A regions of the two proteins (rBA0871A and rBA5258A) indicate that their secondary structure compositions are similar to those of the A regions of CNA and other cell wall-anchored adhesins. We demonstrate through solid phase binding assays and surface plasmon resonance analyses that rBA0871A and rBA5258A specifically bound type I collagen in a dose-dependent and saturable manner. Their dissociation constants (KD) for collagen are 1.6-3.2 microm for rBA0871A and 0.6-0.9 microm for rBA5258A, respectively. We further demonstrate that BA0871 and BA5258 can mediate cell attachment to collagen when expressed on the surface of a heterologous host bacterium. To our knowledge these are the first two adhesins of B. anthracis described, which may have important implications for our understanding of the pathogenic mechanisms explored by this organism.     

Basal lamina formation by porcine thyroid cells grown in collagen- and laminin-deficient medium

Summary Porcine thyroid cells isolated by dispase treatment were cultured in either (a) Matrigel, (b) agarose with the addition of different combinations of basic fibroblast growth factor and laminin, or (c) on agarose-coated dishes. The formation of follicles and the presence of a basal lamina was investigated by routine electron microscopy of Araldite-embedded material and by light and electron microscopical immunocytochemical detection of the basal lamina components, laminin and collagen type IV. After 10 days of culture in Matrigel or agarose, a basal lamina-like structure surrounded most follicles. Follicles of cells growing in agarose and overlaid with a medium containing thyrotropin and fibroblast growth factor showed a fluorescent band at the basal side of the follicles after immunocytochemical staining with anti-laminin and anti-collagen antibodies. Routine electron microscopy showed that a basal lamina-like structure lined the outside of the follicle. This structure could be subdivided into a lamina lucida and a lamina densa. Electron microscopical immunogold labelling revealed that immunologically detectable laminin was confined to the lamina densa. These findings suggest that even in the absence of basal lamina components in the culture medium, thyroid cells are able to form follicles with a regular basal lamina when they are cultured in a three-dimensional environment.     

Interaction of fibrinogen-binding tetrapeptides with fibrin oligomers and fine fibrin clots

The polymerization of fibrin, at pH 8.5 and ionic strength 0.45, and under conditions where the action of thrombin on fibrinogen was the rate-determining step, was interrupted by inactivating thrombin with p-nitrophenyl-p′-guanidinobenzoate (NPGB). Addition of the tetrapeptide Gly-Pro-Arg-Pro (GPRP) partially dissociated the fibrin oligomers as shown by subsequent ligation with Factor XIIIa and calcium ion followed by denaturation and gel electrophoresis; polyacrylamide gel electrophoresis with reduction showed a decrease in the proportion of γ-γ ligation compared with controls untreated by GPRP, and agarose gel electrophoresis showed a shift in the distribution of oligomer sizes. The dissociation was accomplished within 15 min and its extent was consistent with establishment of an equilibrium in which two molecules of GPRP react to sever an oligomer. When GPRP was introduced into fine unligated fibrin clots by diffusion, there was some dissociation as shown by differences in the degree of γ-γ ligation after treatment by Factor XIIIa; but the action of GPRP was much slower and less complete than on soluble oligomers. However, even a small amount of dissociation affected the mechanical properties of fine clots profoundly. The shear modulus (measured 25 s after application of stress) decreased progressively with increasing concentration of GPRP introduced by diffusion. The rate of shear creep under constant stress and the proportion of irrecoverable deformation also increased enormously. If the steadystate creep rate is interpreted in terms of an effective viscosity, the latter is decreased by up to three orders of magnitude by the presence of GPRP. In terms of transient network theories of viscoelasticity, the average lifetime of a network strand is greatly diminished. However, the total density of strands remains constant during creep and creep recovery as shown by constancy of the differential modulus or compliance. Removal of GPRP by diffusion only partially restores the original shear modulus and creep behavior of the original clot. Some limited data on the effect of the tetrapeptide Gly-His-Arg-Pro are also reported.     

Alpha-defensin enhances expression of HSP47 and collagen-1 in human lung fibroblasts

Neutrophils and lung fibroblasts are thought to play a role in the pathogenesis of pulmonary fibrosis. We reported previously that heat shock protein 47 (HSP47), a collagen-specific molecular chaperon, and collagen-1 synthesis were involved in pulmonary fibrosis, and that plasma levels of alpha-defensins (HNP; human neutrophil peptide), cationic proteins with antimicrobial and cytotoxic activity in neutrophils, were significantly higher in patients with idiopathic pulmonary fibrosis than in control subjects. Here, we investigated the direct effect of HNP-1 in vitro on the expression of HSP47 and collagen-1 in human lung fibroblasts (NHLF). HNP-1 at 5 microg/ml induced fibroblast proliferation but at concentrations >50 microg/ml, HNP-1 reduced cell viability. Incubation of NHLF with 10 to 25 microg/ml of HNP-1 for 24-h increased the expression of HSP47 and collagen-1 mRNAs (p<0.05). The levels of HSP47 protein also increased significantly at 50 microg/ml, and those of collagen-1 protein increased at 10 to 50 microg/ml of HNP-1 (p<0.05). The mitogen-activated protein kinase (MAPK) signaling pathway in NHLF was activated by HNP-1 stimulation, but inhibitor of MEK (PD98059) did not block HNP-1-induced HSP47 protein production. Our results suggest that alpha-defensin is a fibrogenic mediator that promotes collagen synthesis through the upregulation of HSP47 and collagen-1 in lung fibroblasts and participates in the pathogenesis of neutrophil-induced pulmonary fibrosis.     

Isopeptide bonds in chemotactic tripeptides. Synthesis and activity of lysine-containing fMLF analogs.

In order to explore the properties of chemotactic N-formylpeptides containing isopeptide bonds within their backbones, a group of lysine-containing analogs of the prototypical chemotactic tripeptide N-formylmethionyl-leucyl-phenylalanine (fMLF) was synthesized. The new analogs were designed by adding to the HCO-Met or Boc-Met residue a dipeptide fragment made up of Lys and Phe residues joined through Lys N alpha or N epsilon bonds, in all possible combinations. Thus, the following six pairs of tripeptides were synthesized and examined for their bioactivity: RCO-Met-Lys(Z)-Phe-OMe (2a, b), RCO-Met-Lys(Z-Phe)-OMe (3a, b), Z-Lys(RCO-Met)-Phe-OMe (4a, b), Z-Phe-Lys(RCO-Met)-OMe (5a, b), RCO-Met-Phe-Lys(Z)-OMe (6a, b) and Z-Lys(RCO-Met-Phe)-OMe (7a, b), with R=OC(CH3)(3 )and R=H for compounds a and b, respectively. All the new models were characterized fully and their activity (chemotaxis, superoxide anion production and lysozyme release) on human neutrophils determined as agonists (compounds b) and antagonists (compounds a). All N-formyl derivatives 2b-7b are less potent than fMLF-OMe as chemoattractants, but compound 7b exhibits selective activity as superoxide anion producer. Derivatives 2a-7a do not show antagonistic activity towards fMLF induced chemotaxis and O(2)(-) production, however, all these compounds except 4a antagonize lysozyme release by 60%.     

Expression of recombinant platelet glycoprotein IIbIIIa results in a functional fibrinogen-binding complex

The ability of cDNAs encoding the human platelet glycoprotein IIbIIIa to be expressed and assembled into a functional integrin receptor was assessed by transient transfection into a human cell line. Transfection of full length cDNAs resulted in synthesis of high levels of integrin subunits which appear to be stable within the cell for several days. Coexpression of both subunits resulted in a proteolytically processed form of GPIIb that associated with GPIIIa as a heterodimeric complex as the cell surface. Transport to the cell surface required association of these subunits with each other or with endogenous integrin subunits. When expressed alone, the GPIIb subunit remained intracellular, while the GPIIIa subunit was found to complex with endogenous proteins and was mobilized to the cell surface. The GPIIbIIIa receptor complex facilitated attachment of cells to known ligands for GPIIbIIIa: fibrinogen, vitronectin, and von Willebrand factor. This adhesion was sensitive to inhibition by the peptide GRGDV and the monoclonal antibody AP2, known inhibitors of platelet aggregation     

Platelet C1q receptor interactions with collagen- and C1q-coated surfaces

We recently described specific binding sites for C1q on human blood platelets. Structural similarities between the amino-terminal of C1q and collagen have suggested that receptors for both molecules on platelets might be the same. The present study thus compared the interaction of purified C1q receptors (C1qR) and whole platelets with collagen- and C1q-coated polystyrene surfaces. Surfaces coated with BSA or gelatin served as controls. Purified 125I-labeled C1qR recognized both C1q- and collagen-coated surfaces in a divalent, cation-independent manner. This adhesion was inhibited by polyclonal or monoclonal (II1/D1) anti-C1qR antibodies. Although C1qR adhered preferentially to C1q-coated surfaces, adhesion to bovine and human type I collagen, as well as to human type III and V collagen, was also noted. In parallel studies, 51Cr-labeled platelets bound equally well to collagen- or C1q-coated surfaces, albeit in a magnesium-dependent manner. Partial inhibition of platelet adhesion was observed in the presence of RGDS, despite the inability of RGDS to modify C1qR interaction with C1q or collagen. Moreover, anti C1qR antibodies selectively inhibited platelet adhesion to C1q-coated surfaces, whereas antibodies specific for the GPIa/IIa collagen receptor (6F1) preferentially inhibited platelet collagen interactions. These data support the presence of distinct platelet membrane C1qR, which may cross-react with collagen, and suggest that C1qR are necessary but not sufficient for platelet adhesion to C1q-coated surfaces. Additional divalent cation and/or RGD-sensitive binding sites may participate.     

Promotion of phagocytosis of Streptococcus pyogenes in human blood by a fibrinogen-binding peptide

The binding of fibrinogen to M-related protein (Mrp) is known to contribute to the ability of Streptococcus pyogenes to evade phagocytosis by preventing the deposition of complement on the streptococcal surface. The objectives of this investigation were to map the common fibrinogen-binding domain of Mrp and to determine if this domain has a therapeutic potential to enhance phagocytosis of S. pyogenes in human blood. Using a series of recombinant, truncated proteins of Mrp, two fibrinogen-binding domains (FBD) were mapped. FBD1 was contained within amino acid residues 1-55 of Mrp and FBD2 within residues 81-138. FBD2 is found in all Mrp sequenced to date whereas FBD1 is not. Both FBD1 and FBD2 peptides but not a control peptide blocked the binding of fibrinogen to S. pyogenes and promoted phagocytosis of the streptococci in human blood. The data support the hypothesis that the binding of fibrinogen by S. pyogenes is centrally involved in their resistance to phagocytosis in human blood and suggest that treatments that interfere with the binding of fibrinogen to S. pyogenes may help in fighting infections by these organisms.     

Dietary soya lecithin decreases plasma triglyceride levels and inhibits collagen- and ADP-induced platelet aggregation

Elevated plasma lipid concentrations and increased platelet activation are risk factors in the development of atherosclerosis. Nine patients with type IIa hyperlipoproteinemia and nine patients with type IV hyperlipoproteinemia were given soya lecithin, 12 g/day, for 3 months. Plasma cholesterol and triglycerides were reduced by 15 and 23%, respectively, and HDL-cholesterol increased by 16% in the hypercholesterolemic patients. Platelet function was unchanged. In the hypertriglyceridemic patients, total cholesterol fell by 18%, triglycerides by 36%, and HDL-cholesterol increased by 14%. There was a 27% reduction in platelet aggregation (P less than 0.01). Seventeen hypertriglyceridemic patients then received increasing doses of soya lecithin for 1-month periods (6, 12, and 18 g/day). The optimal lipoprotein-lowering effect was achieved with a daily dose of 12 g soya lecithin per day. Both low-density lipoprotein and very-low-density lipoprotein levels were reduced, and HDL-cholesterol and apolipoprotein levels were reduced, and HDL-cholesterol and apolipoprotein A-I concentrations were increased. Platelet aggregation in response to collagen and ADP was significantly reduced, parallel with the reduction in triglyceride level. Soya lecithin supplementing the diet may be useful in the management of the hypertriglyceridemic patient.     

A role for the fibrinogen-binding regions of streptococcal M proteins in phagocytosis resistance

All virulent group A streptococcal isolates bind fibrinogen, a property that is closely linked to expression of type-specific antiphagocytic surface molecules designated M proteins. Here we show that although the M proteins from two different strains, M1 and M5, both bind fibrinogen with high affinity, they interact with different regions in the ligand. Moreover, mapping experiments demonstrated that the fibrinogen-binding regions in the M1 and M5 proteins are quite dissimilar at the amino acid sequence level and that they bind to different regions in the plasma protein. In spite of these differences, the fibrinogen-binding regions of M1 and M5 could both be shown to contribute to streptococcal survival in human blood, providing evidence for the distinct function of a plasma protein interaction in bacterial pathogenesis.     

Expression of clumping and fibrinogen-binding activities of Staphylococcus aureus at various growth stages

Clumping and fibrinogen-binding activities of 4 Staphylococcus aureus strains (Cowan I, Newman D2C, Wood 46 and NCTC 5655) were assayed with a semiquantitative clumping test and an enzyme-linked immunosorbent assay (ELISA), respectively. Distinct positive clumping was detected with whole cells of the 3 strains except Wood 46. Amounts of fibrinogen required for a definite clumping depended greatly on strains as well as on their growth phases. On the other hand, fibrinogen-binding activities were detected both in culture supernatants and in cell lysates of all the 4 strains, and the levels were rather comparable with one another and relatively steady through their growth cycles. No significant correlation was thus found among expression behavior of clumping and fibrinogen-binding activities.     

Measurement of adhesive forces between individual Staphylococcus aureus MSCRAMMs and protein-coated surfaces by use of optical tweezers

The force required to rupture bonds between individual Staphylococcus aureus MSCRAMMs and surfaces coated with extracellular matrix molecules has been quantified by using optical tweezers. The observed binding forces between fibrinogen or fibronectin and S. aureus MSCRAMMs occurred as an approximate integer multiple of 20 or 25 pN, respectively.     

A secreted collagen- and fibronectin-binding streptococcal protein modulates cell-mediated collagen gel contraction and interstitial fluid pressure

Fibroblast-mediated collagen gel contraction depends on collagen-binding beta1 integrins. Perturbation of these integrins reveals an alternative contraction process that is integrin alphaVbeta3-dependent and platelet-derived growth factor (PDGF) BB-stimulated. Connective tissue cells actively control interstitial fluid pressure (IFP), and inflammation-induced lowering of IFP provides a driving force for edema formation. PDGF-BB normalizes a lowered IFP by an alphaVbeta3-dependent process. A potential modulation of IFP by extracellular matrix-binding bacterial proteins has previously not been addressed. The fibronectin (FN)-binding protein FNE is specifically secreted by the highly virulent Streptococcus equi subspecies equi. FNE bound FN and native collagen type I with K(d) values of approximately 20 and approximately 50 nm determined by solid-phase binding assays. Rotary shadowing revealed a single FNE binding site located at on average 122 nm from the C terminus of procollagen type I. FNE induced alphaVbeta3-mediated contraction by C2C12 cells in a concentration-dependent manner having a maximal effect at approximately 100 nm. This activity of FNE required cellular FN, and FNE acted synergistically to added plasma FN or PDGF-BB. FNE enhanced binding of soluble FN to immobilized collagen, and conversely the binding of collagen to immobilized FN. Marked bell-shaped concentration dependences for these interactions suggest that FNE forms a bridge between FN and collagen. Finally, FNE normalized dermal IFP lowered by anaphylaxis. Our data suggest that secreted FNE normalized lowering of IFP by stimulating connective tissue cell contraction.     

Effect of dietary lipid on collagen- and adenosine diphosphate-induced platelet aggregation and thromboxane A2 synthesis in the rat

Dietary lipids containing different proportions of long-chain polyunsaturated fatty acids can affect platelet thromboxane A(2) formation and aggregation. In the present work, the effects of dietary lipid, from animal and vegetable sources, on collagen- and adenosine diphosphate (ADP)-induced thromboxane A(2) (measured as thromboxane B(2)) production and aggregation in washed rat platelets were studied. In addition, plasma thromboxane B(2) levels in rats fed different dietary lipids were measured. Animals were fed 10% fat by weight as lard (LRD), corn oil, soy bean oil, canola oil (CAN), or cod liver oil (CLO) for a period of 7 weeks. Circulating thromboxane B(2) levels detected in platelet-poor plasma of the CLO-fed animals were significantly lower than those of rats fed all other dietary lipids. The platelets of CLO-fed animals synthesized significantly less thromboxane A(2) compared with those from other dietary groups following ex vivo stimulation of platelets with agonists such as collagen and ADP, with the exception of platelets from the LRD-fed animals. Ex vivo stimulation of platelets obtained from this group with collagen resulted in the synthesis of significantly greater levels of thromboxane A(2) compared with all other groups. However, aggregation responses to collagen and ADP were not significantly affected by dietary treatment, although relatively the lowest responses to these agonists were apparent in the CLO-fed and CAN-fed groups, respectively.