Evidence for Steric Regulation of Fibrinogen Binding to Staphylococcus aureus Fibronectin-binding Protein A (FnBPA)

The adjacent fibrinogen (Fg)- and fibronectin (Fn)-binding sites on Fn-binding protein A (FnBPA), a cell surface protein from Staphylococcus aureus, are implicated in the initiation and persistence of infection. FnBPA contains a single Fg-binding site (that also binds elastin) and multiple Fn-binding sites. Here, we solved the structure of the N2N3 domains containing the Fg-binding site of FnBPA in the apo form and in complex with a Fg peptide. The Fg binding mechanism is similar to that of homologous bacterial proteins but without the requirement for “latch” strand residues. We show that the Fg-binding sites and the most N-terminal Fn-binding sites are nonoverlapping but in close proximity. Although Fg and a subdomain of Fn can form a ternary complex on an FnBPA protein construct containing a Fg-binding site and single Fn-binding site, binding of intact Fn appears to inhibit Fg binding, suggesting steric regulation. Given the concentrations of Fn and Fg in the plasma, this mechanism might result in targeting of S. aureus to fibrin-rich thrombi or elastin-rich tissues.     

A novel fibronectin type III module binding motif identified on C-terminus of Leptospira immunoglobulin-like protein, LigB

Infection by pathogenic strains of Leptospira hinges on the pathogen’s ability to adhere to host cells via extracellular matrix such as fibronectin (Fn). Previously, the immunoglobulin-like domains of Leptospira Lig proteins were recognized as adhesins binding to N-terminal domain (NTD) and gelatin binding domain (GBD) of Fn. In this study, we identified another Fn-binding motif on the C-terminus of the Leptospira adhesin LigB (LigBCtv), residues 1708-1712 containing sequence LIPAD with a β-strand and nascent helical structure. This motif binds to 15th type III modules (15F₃) (K_D = 10.70 μM), and association (k_on = 600 M⁻¹ s⁻¹) and dissociation (k_off = 0.0129 s⁻¹) rate constants represents a slow binding kinetics in this interaction. Moreover, pretreatment of MDCK cells with LigB_1706-1716 blocked the binding of Leptospira by 39%, demonstrating a significant role of LigB_1706-1716 in cellular adhesion. These data indicate that the LIPAD residues (LigB_1708-1712) of the Leptospira interrogans LigB protein bind 15F₃ of Fn at a novel binding site, and this interaction contributes to adhesion to host cells.     

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.     

Multidomain Structure and Cellulosomal Localization of the Clostridium thermocellum Cellobiohydrolase CbhA

The nucleotide sequence of the Clostridium thermocellum F7 cbhA gene, coding for the cellobiohydrolase CbhA, has been determined. An open reading frame encoding a protein of 1,230 amino acids was identified. Removal of a putative signal peptide yields a mature protein of 1,203 amino acids with a molecular weight of 135,139. Sequence analysis of CbhA reveals a multidomain structure of unusual complexity consisting of an N-terminal cellulose binding domain (CBD) homologous to CBD family IV, an immunoglobulin-like β-barrel domain, a catalytic domain homologous to cellulase family E1, a duplicated domain similar to fibronectin type III (Fn3) modules, a CBD homologous to family III, a highly acidic linker region, and a C-terminal dockerin domain. The cellulosomal localization of CbhA was confirmed by Western blot analysis employing polyclonal antibodies raised against a truncated enzymatically active version of CbhA. CbhA was identified as cellulosomal subunit S3 by partial amino acid sequence analysis. Comparison of the multidomain structures indicates striking similarities between CbhA and a group of cellulases from actinomycetes. Average linkage cluster analysis suggests a coevolution of the N-terminal CBD and the catalytic domain and its spread by horizontal gene transfer among gram-positive cellulolytic bacteria.     

Protein F2, a novel fibronectin-binding protein from Streptococcus pyogenes, possesses two binding domains

Binding of the group A streptococcus (GAS) to respiratory epithelium is mediated by the fibronectin (Fn)-binding adhesin, protein F1. Previous studies have suggested that certain GAS strains express Fn-binding proteins that are different from protein F1. In this study, we have cloned, sequenced, and characterized a gene ( prtF2 ) from GAS strain 100076 encoding a novel Fn-binding protein, termed protein F2. Insertional inactivation of prtF2 in strain 100076 abolishes its high-affinity Fn binding. prtF2 -related genes exist in most GAS strains that lack prtF1 (encoding protein F1) but bind Fn with high affinity. These observations suggest that protein F2 is a major Fn-binding protein in GAS. Protein F2 is highly homologous to Fn-binding proteins from Streptococcus dysgalactiae and Strep-tococcus equisimilis , particularly in its carboxy-terminal portion. Two domains are responsible for Fn binding by protein F2. One domain (FBRD) consists of three consecutive repeats, whereas the other domain (UFBD) resides on a non-repeated stretch of approximately 100 amino acids and is located 100 amino acids amino-terminal of FBRD. Each of these domains is capable of binding Fn when expressed as a separate protein. In strain 100076, protein F2 activity is regulated in response to alterations in the concentration of atmospheric oxygen.     

Chitinase of Bacillus licheniformis from oyster shell as a probe to detect chitin in marine shells

Bacillus licheniformis CBFOS-03 is a chitinase producing bacteria isolated from oyster (Crassostrea gigas) shell waste. We have cloned and expressed the chi18B gene of B. licheniformis CBFOS-03, which encodes a glycohydrolase family 18 chitinase (GH18). Chi18B is a predicted 598 amino acid protein that consists of a catalytic domain (GH18), a fibronectin type III domain (Fn3), and a chitin binding domain (CBD). Purified Chi18B showed optimum chitinase activity at pH 9 and 55 °C, and activity was stimulated with 25 mM Mn²⁺. In kinetic analysis, Chi18B showed Km values of 9.07?±?0.65 μM and 129.27?±?0.38 μM with the substrates 4-methylumbelliferyl-N-N′-diacetylchitobiose and α-chitin, respectively. Studies of C-terminal deletion constructs revealed that the GH18 domain with one amino acid in C-terminal region was sufficient for chitinase activity; however, fusions of full length and CBD-deleted constructs to green florescent protein (GFP) and yellow florescent protein (YFP) suggest that the C-terminus is supposedly important in binding to shell powder. Full length Chi18B with GFP showed green fluorescence with oyster shell powder, but GH18+Fn3 with GFP did not. Similarly, full length Chi18B with YFP showed yellow fluorescence with clam (Chamelea gallina) shell and disk abalone (Haliotis discus) shell powder, but GH18+Fn3 with YFP construct did not. So, the CBD domain of Chi18B appears to play an important role in binding of oyster and other marine shells. It is likely to be used as a probe to identify the presence of chitin in marine shells like oyster shell, clam shell, and disk abalone shell using fusions of Chi18B with fluorescent proteins.     

A 28-kilodalton fibronectin-binding protein of group a streptococci

Lipoteichoic acid (LTA) has been implicated as a major adhesin of group A streptococci that interacts with fibronectin (Fn). It has been suggested that protein adhesins may also be involved in the binding of Fn to streptococci. We searched for such a protein by transblotting membrane preparations from M types 5, 19, and 24 group A streptococci to nitrocellulose and reacting the blot with¹²⁵I-Fn. The Fn reacted with a 28-kDa polypeptide from all three serotypes of streptococci. Using affinity-purified antibodies to the 28-kDa protein in immunoblots of membrane preparations from various streptococci, we demonstrated that the 28-kDa protein is present in all 17 strains tested. Affinity-purified antibodies to the 28-kDa protein also reacted in varying degrees with intact streptococci, demonstrating that the antigen is exposed on the surface of intact organisms. Our results suggest that, in addition to LTA, group A streptococci contain a common Fn-binding moiety that is expressed as a major component of membrane preparations and that is accessible on the surface of streptococci for interactions with Fn.     

Fba, a novel fibronectin-binding protein from Streptococcus pyogenes, promotes bacterial entry into epithelial cells, and the fba gene is positively transcribed under the Mga regulator

In infection by Streptococcus pyogenes, fibronectin (Fn)-binding proteins play important roles as adhesins and invasins. Here, we present a novel Fn-binding protein of S. pyogenes that exhibits a low similarity to other Fn-binding proteins reported. After searching the Oklahoma Streptococcal Genome Sequencing Database for open reading frames (ORFs) with an LPXTG motif, nine ORFs were found among those recognized as putative surface proteins, and one of them was designated as Fba. The fba gene was found in M types 1, 2, 4, 22, 28 and 49 of S. pyogenes, but not in other serotypes or groups of streptococci. Fba, a 37.8 kDa protein, possesses three or four proline-rich repeat domains and exhibits a high homology to FnBPA, the Fn-binding protein of Staphylococcus aureus. Recombinant Fba exhibited a strong binding ability to Fn. In addition, Fba-deficient mutants showed diminished invasive capabilities to HEp-2 cells and low mortality in mice following skin infection. The fba gene was located downstream of the mga regulon and analysis using an mga-inactivated mutant revealed that it was transcribed under the control of the Mga regulator. These results indicate that Fba is a novel protein and one of the important virulence factors of S. pyogenes.     

The Streptococcal Binding Site in the Gelatin-binding Domain of Fibronectin Is Consistent with a Non-linear Arrangement of Modules

Fibronectin-binding proteins (FnBPs) of Staphylococcus aureus and Streptococcus pyogenes mediate invasion of human endothelial and epithelial cells in a process likely to aid the persistence and/or dissemination of infection. In addition to binding sites for the N-terminal domain (NTD) of fibronectin (Fn), a number of streptococcal FnBPs also contain an upstream region (UR) that is closely associated with an NTD-binding region; UR binds to the adjacent gelatin-binding domain (GBD) of Fn. Previously, UR was shown to be required for efficient streptococcal invasion of epithelial cells. Here we show, using a Streptococcus zooepidemicus FnBP, that the UR-binding site in GBD resides largely in the ⁸F1⁹F1 module pair. We also show that UR inhibits binding of a peptide from the α1 chain of type I collagen to ⁸F1⁹F1 and that UR binding to ⁸F1 is likely to occur through anti-parallel β-zipper formation. Thus, we propose that streptococcal proteins that contain adjacent NTD- and GBD-binding sites form a highly unusual extended tandem β-zipper that spans the two domains and mediates high affinity binding to Fn through a large intermolecular interface. The proximity of the UR- and NTD-binding sequences in streptococcal FnBPs is consistent with a non-linear arrangement of modules in the tertiary structure of the GBD of Fn.     

Crystal Structure of CBD2 from the Drosophila Na/Ca Exchanger: Diversity of Ca Regulation and Its Alternative Splicing Modification

Na⁺/Ca²⁺ exchangers (NCXs) promote the extrusion of intracellular Ca²⁺ to terminate numerous Ca²⁺-mediated signaling processes. Ca²⁺ interaction at two Ca²⁺ binding domains (CBDs; CBD1 and CBD2) is important for tight regulation of the exchange activity. Diverse Ca²⁺ regulatory properties have been reported with several NCX isoforms; whether the regulatory diversity of NCXs is related to structural differences of the pair of CBDs is presently unknown. Here, we reported the crystal structure of CBD2 from the Drosophila melanogaster exchanger CALX1.1. We show that the CALX1.1-CBD2 is an immunoglobulin-like structure, similar to mammalian NCX1-CBD2, but the predicted Ca²⁺ interaction region of CALX1.1-CBD2 is arranged in a manner that precludes Ca²⁺ binding. The carboxylate residues that coordinate two Ca²⁺ in the NCX1-CBD1 structure are neutralized by two Lys residues in CALX1.1-CBD2. This structural observation was further confirmed by isothermal titration calorimetry. The CALX1.1-CBD2 structure also clearly shows the alternative splicing region forming two adjacent helices perpendicular to CBD2. Our results provide structural evidence that the diversity of Ca²⁺ regulatory properties of NCX proteins can be achieved by (1) local structure rearrangement of Ca²⁺ binding site to change Ca²⁺ binding properties of CBD2 and (2) alternative splicing variation altering the protein domain-domain conformation to modulate the Ca²⁺ regulatory behavior.     

Identification of a fibronectin-binding domain within the Campylobacter jejuni CadF protein

The binding of Campylobacter jejuni to fibronectin (Fn), a component of the extracellular matrix, is mediated by a 37 kDa outer membrane protein termed CadF for Campylobacter adhesion to Fn. Previous studies have indicated that C. jejuni binds to Fn on the basolateral surface of T84 human colonic cells. To further characterize the interaction of the CadF protein with Fn, enzyme-linked immunosorbent assays were performed to identify the Fn-binding domain (Fn-BD). Using overlapping 30-mer and 16-mer peptides derived from translated cadF nucleotide sequence, maximal Fn-binding activity was localized to four amino acids (AA 134-137) consisting of the residues phenylalanine-arginine-leucine-serine (FRLS). A mouse alpha-CadF peptide polyclonal antibody (M alpha-CadF peptide pAb) was generated using FRLS containing peptides and found to react with viable C. jejuni as judged by indirect fluorescent microscopy, suggesting that the FRLS residues are surface-exposed. Binding of CadF to purified Fn and INT 407 human epithelial cells was significantly inhibited with peptides containing the Fn-BD. Moreover, a CadF recombinant variant protein, in which the Phe-Arg-Leu residues (CadF AA 134-136) were altered to Ala-Ala-Gly, exhibited a 91% decrease in Fn-binding activity as compared with the wild-type CadF protein. Collectively, these data indicate that the FRLS residues (CadF AA 134-137) of the C. jejuni CadF protein possess Fn-binding activity.     

Crystal Structure of the ATP-Dependent Maturation Factor of Ni,Fe-Containing Carbon Monoxide Dehydrogenases

CooC proteins are ATPases involved in the incorporation of nickel into the complex active site ([Ni-4Fe-4S]) cluster of Ni,Fe-dependent carbon monoxide dehydrogenases. The genome of the carboxydotrophic bacterium Carboxydothermus hydrogenoformans encodes five carbon monoxide dehydrogenases and three CooC-type proteins, of which CooC1 was shown to be a nickel-binding ATPase. We determined the crystal structure of CooC1 in four different states: empty, ADP-bound, Zn²⁺/ADP-bound, and Zn²⁺-bound. The structure of CooC1 consists of two spatially separated functional modules: an ATPase module containing the deviant Walker A motif and a metal-binding module that confers the specific function of CooC1. The ATPase module is homologous to other members of the MinD family and, in analogy to the dimeric structure of ATP-bound Soj, is likely responsible for the ATP-dependent dimerization of CooC1. Its core topology classifies CooC1 as a member of the MinD family of SIMIBI (signal recognition particle, MinD and BioD)-class NTPases. The crystal structure of Zn²⁺-bound CooC1 reveals a conserved C-X-C motif as the metal-binding site responsible for metal-induced dimerization. The competitive binding of Ni²⁺ and Zn²⁺ to CooC1 in solution confirms that the conserved C-X-C motif is also responsible for the interaction with Ni²⁺. A comparison of the different CooC1 structures determined suggests a mutual dependence of metal-binding site and nucleotide-binding site.     

Structural and functional analysis of the tandem β-zipper interaction of a Streptococcal protein with human fibronectin

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 β-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 β-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.     

O-mannosylation of the Mycobacterium tuberculosis Adhesin Apa Is Crucial for T Cell Antigenicity during Infection but Is Expendable for Protection

Glycosylation is the most abundant post-translational polypeptide chain modification in nature. Although carbohydrate modification of protein antigens from many microbial pathogens constitutes important components of B cell epitopes, the role in T cell immunity is not completely understood. Here, using ELISPOT and polychromatic flow cytometry, we show that O-mannosylation of the adhesin, Apa, of Mycobacterium tuberculosis (Mtb) is crucial for its T cell antigenicity in humans and mice after infection. However, subunit vaccination with both mannosylated and non-mannosylated Apa induced a comparable magnitude and quality of T cell response and imparted similar levels of protection against Mtb challenge in mice. Both forms equally improved waning BCG vaccine-induced protection in elderly mice after subunit boosting. Thus, O-mannosylation of Apa is required for antigenicity but appears to be dispensable for its immunogenicity and protective efficacy in mice. These results have implications for the development of subunit vaccines using post-translationally modified proteins such as glycoproteins against infectious diseases like tuberculosis.     

Inhibition of colon carcinogenesis by a standardized Cannabis sativa extract with high content of cannabidiol

PurposeColon cancer is a major public health problem. Cannabis-based medicines are useful adjunctive treatments in cancer patients. Here, we have investigated the effect of a standardized Cannabis sativa extract with high content of cannabidiol (CBD), here named CBD BDS, i.e. CBD botanical drug substance, on colorectal cancer cell proliferation and in experimental models of colon cancer in vivo.MethodsProliferation was evaluated in colorectal carcinoma (DLD-1 and HCT116) as well as in healthy colonic cells using the MTT assay. CBD BDS binding was evaluated by its ability to displace [³H]CP55940 from human cannabinoid CB₁ and CB₂ receptors. In vivo, the effect of CBD BDS was examined on the preneoplastic lesions (aberrant crypt foci), polyps and tumours induced by the carcinogenic agent azoxymethane (AOM) as well as in a xenograft model of colon cancer in mice.ResultsCBD BDS and CBD reduced cell proliferation in tumoral, but not in healthy, cells. The effect of CBD BDS was counteracted by selective CB₁ and CB₂ receptor antagonists. Pure CBD reduced cell proliferation in a CB₁-sensitive antagonist manner only. In binding assays, CBD BDS showed greater affinity than pure CBD for both CB₁ and CB₂ receptors, with pure CBD having very little affinity. In vivo, CBD BDS reduced AOM-induced preneoplastic lesions and polyps as well as tumour growth in the xenograft model of colon cancer.ConclusionsCBD BDS attenuates colon carcinogenesis and inhibits colorectal cancer cell proliferation via CB₁ and CB₂ receptor activation. The results may have some clinical relevance for the use of Cannabis-based medicines in cancer patients.     

Interaction of calcium-bound C-reactive protein with fibronectin is controlled by pH: in vivo implications

C-reactive protein (CRP) binds with high affinity to fibronectin (Fn), a major component of the extracellular matrix (ECM), but at physiological pH the binding is inhibited by calcium ions (Ca2+). Because CRP circulates in the blood in Ca2+ -bound form, the occurrence of CRP-Fn interactions in vivo has been doubtful. To define the basis of inhibition of CRP-Fn interaction by Ca2+ at pH 7.0, we hypothesized that Fn-binding site on CRP consisted of amino acids co-ordinating Ca2+. Site-directed mutagenesis of amino acids co-ordinating Ca2+ drastically decreased the binding of CRP to Fn, indicating that the Ca2+ -binding site indeed formed the Fn-binding site. To determine the requirements for possible interaction between Ca2+ -bound CRP and Fn, we investigated inhibition of CRP-Fn interaction by Ca2+ as a function of pH. Ca2+ did not inhibit binding of CRP to Fn at pH 6.5 and lower. The contrasting Fn binding properties of CRP at physiological and mildly acidic pH indicated that the interaction of Ca2+ -bound CRP with Fn was controlled by pH. We conclude that the inhibition of binding of CRP to Fn by Ca2+ at pH 7.0 is a mechanism to prevent CRP-Fn interactions under normal conditions. CRP, in its Ca2+ -bound state, is capable of binding Fn but only at the inflammatory sites and tumors with low pH. CRP, Fn, and the ECM all have been implicated in cancer. Taken together our data raise the possibility that CRP-Fn interactions may change the architecture of ECM to modify the development of tumors.     

Crystal Structures of Progressive Ca2+ Binding States of the Ca2+ Sensor Ca2+ Binding Domain 1 (CBD1) from the CALX Na+/Ca2+ Exchanger Reveal Incremental Conformational Transitions

Na⁺/Ca²⁺ exchangers (NCX) constitute a major Ca²⁺ export system that facilitates the re-establishment of cytosolic Ca²⁺ levels in many tissues. Ca²⁺ interactions at its Ca²⁺ binding domains (CBD1 and CBD2) are essential for the allosteric regulation of Na⁺/Ca²⁺ exchange activity. The structure of the Ca²⁺-bound form of CBD1, the primary Ca²⁺ sensor from canine NCX1, but not the Ca²⁺-free form, has been reported, although the molecular mechanism of Ca²⁺ regulation remains unclear. Here, we report crystal structures for three distinct Ca²⁺ binding states of CBD1 from CALX, a Na⁺/Ca²⁺ exchanger found in Drosophila sensory neurons. The fully Ca²⁺-bound CALX-CBD1 structure shows that four Ca²⁺ atoms bind at identical Ca²⁺ binding sites as those found in NCX1 and that the partial Ca²⁺ occupancy and apoform structures exhibit progressive conformational transitions, indicating incremental regulation of CALX exchange by successive Ca²⁺ binding at CBD1. The structures also predict that the primary Ca²⁺ pair plays the main role in triggering functional conformational changes. Confirming this prediction, mutagenesis of Glu⁴⁵⁵, which coordinates the primary Ca²⁺ pair, produces dramatic reductions of the regulatory Ca²⁺ affinity for exchange current, whereas mutagenesis of Glu⁵²⁰, which coordinates the secondary Ca²⁺ pair, has much smaller effects. Furthermore, our structures indicate that Ca²⁺ binding only enhances the stability of the Ca²⁺ binding site of CBD1 near the hinge region while the overall structure of CBD1 remains largely unaffected, implying that the Ca²⁺ regulatory function of CBD1, and possibly that for the entire NCX family, is mediated through domain interactions between CBD1 and the adjacent CBD2 at this hinge.     

Fibronectin Binds to and Induces Conformational Change in a Disordered Region of Leptospiral Immunoglobulin-like Protein B

Leptospira interrogans is a pathogenic spirochete that causes disease in both humans and animals. LigB (Leptospiral immunoglobulin-like protein B) contributes to the binding of Leptospira to extracellular matrix proteins such as fibronectin (Fn), fibrinogen, laminin, and collagen. A high affinity Fn-binding region of LigB has been recently localized to LigBCen2, which contains the partial eleventh and full twelfth immunoglobulin-like repeats (LigBCen2R) and 47 amino acids of the non-repeat region (LigBCen2NR) of LigB. In this study, LigBCen2NR was shown to bind to the N-terminal domain (NTD) of Fn (K_D = 379 nm) by an enzyme-linked immunosorbent assay and isothermal titration calorimetry. Interestingly, this sequence was not observed to adopt secondary structure by far UV circular dichroism or by differential scanning calorimetry, in agreement with computer-based secondary structure predictions. A low partition coefficient (K_av) measured with gel permeation chromatography, a high hydrodynamic radius (R_h) measured with dynamic light scattering, and the insensitivity of the intrinsic viscosity to guanidine hydrochloride treatment all suggest that LigBCen2NR possesses an extended and disordered structure. Two-dimensional ¹⁵N-¹H HSQC NMR spectra of intact LigBCen2 in the absence and presence of NTD are consistent with these observations, suggesting the presence of both a β-rich region and an unstructured region in LigBCen2 and that the latter of these selectively interacts with NTD. Upon binding to NTD, LigBCen2NR was observed by CD to adopt a β-strand-rich structure, suggestive of the known β-zipper mode of NTD binding.Leptospira interrogans is a pathogenic spirochete that causes leptospirosis throughout the world, especially in developing countries but also in regions of the United States where it has reemerged (1). Weil''s syndrome, a severe form of this disease, is an acute febrile illness associated with multiorgan damage, including liver failure (jaundice), renal failure (nephritis), pulmonary hemorrhage, and meningitis (1), and has a 15% mortality rate if not treated (2). The molecular pathogenesis of leptospirosis is poorly understood, and the bacterial virulence factors involved are largely unknown. Recently, several potential Leptospira virulence factors have been described, including sphingomyelinases, serine proteases, zinc-dependent proteases, and collagenase (3); LipL32 (4); lipopolysaccharide (5); a novel factor H, laminin, and Fn-binding protein (Lsa24 or Len) (6–8); Loa 22 (9); and Lig (Leptospiral immunoglobulin-like) proteins (10–12).Lig proteins, including LigA, LigB, and LigC, contain multiple immunoglobulin-like repeat domains (13 in LigA, 12 in LigB and LigC) (10–12). Interestingly, the first 630 residues, from the N terminus to the first half of the seventh immunoglobulin-like domain, are conserved between LigA and LigB, but the rest of the immunoglobulin-like domains are variable (10–12) between the two proteins. Also, a non-immunoglobulin-like repeat region found on the C-terminal tail of LigB is not found in LigA (10–12). Lig proteins are categorized as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs)² due to their ability to bind to eukaryotic cells (13) through their interactions with extracellular matrix components, including fibronectin (Fn), laminin, collagens, elastin, and tropoelastin (13, 14, 45). Previously, a high affinity Fn-binding region was localized to LigBCen2, which includes the partial eleventh and complete twelfth immunoglobulin-like repeat region and the first 47 amino acids of the non-repeat regions of LigB (15). LigBCen2 was shown to bind to both the N-terminal domain (NTD) and the gelatin binding domain (GBD) of Fn. The addition of calcium induces a conformational change in LigBCen2 and enhances binding between LigBCen2 and the NTD of Fn (15).The first step in the process of bacterial infection is cellular adhesion, mediated by bacterial adhesins interacting with various components of the extracellular matrix (16). Known interaction modes between Fn and bacterial Fn-binding proteins include the β-zipper (17, 18) and the cationic cradle (19). It was recently discovered that the Fn-binding domains in certain Fn-binding proteins are disordered and extended but gain structure upon binding to the NTD of Fn (20–22).We have performed a fine-mapping study of the NTD-binding site on LigBCen2 and identified this site as LigBCen2NR, a portion of the non-repeat region (amino acids 1119–1165). The addition of NTD promotes the folding of LigBCen2NR from a disordered and extended structure to a folded structure. This finding is notable, since LigBCen2NR is located in the non-immunoglobulin-like region of LigB, as compared with other Fn-binding proteins, such as Staphylococcus aureus FnbpA and FnbpB (23), Streptococcus dysgalactiae FnBB (17), and Streptococcus pyogenes SfbI and SfbII (24). Thus, the binding mode appears to be similar to the known β-zipper mechanism but unique in sequence-specific interactions. This finding provides the fundamental groundwork for the development of a therapeutic agent to target this interaction in order to prevent or treat Leptospira infection.     

Effect of adsorbed fibronectin on the differential adhesion of osteoblast-like cells and Staphylococcus aureus with and without fibronectin-binding proteins

The influence of fibronectin (Fn) coated surfaces patterned with poly(ethylene glycol) microgels having inter-gel spacings between 0.5 and 3.0 μm on the adhesion of Staphylococcus aureus strains with and without Fn-binding proteins and cellular adhesion/spreading was investigated. Quantitative force measurements between a S. aureus cell and a patterned surface showed that the adhesion force between the bacterium and the patterned surface increased substantially after Fn adsorption, regardless of the strain used, but decreased with decreasing inter-gel spacing. In flow-chamber experiments, the Fn-binding strain adhered at a higher rate after Fn adsorption than the strain lacking Fn-binding proteins. In both cases, the adhesion rates decreased with decreasing inter-gel spacing. Osteoblast-like cells could bind to patterned surfaces despite the microgels, and adsorbed Fn substantially amplified this effect. Even under highly non-adhesive conditions associated with closely spaced microgels, adsorbed Fn preserves a window of inter-gel spacing around 1 μm where the adhesion of staphylococcal cells is hindered while cells can still adhere and spread.