Differences in the aromatic domain of homologous streptococcal fibronectin-binding proteins trigger different cell invasion mechanisms and survival rates

Group A streptococci (GAS, Streptococcus pyogenes) and Group G streptococci (GGS, Streptococcus dysgalactiae ssp. equisimilis) adhere to and invade host cells by binding to fibronectin. The fibronectin-binding protein SfbI from GAS acts as an invasin by using a caveolae-mediated mechanism. In the present study we have identified a fibronectin-binding protein, GfbA, from GGS, which functions as an adhesin and invasin. Although there is a high degree of similarity in the C-terminal sequence of SfbI and GfbA, the invasion mechanisms are different. Unlike caveolae-mediated invasion by SfbI-expressing GAS, the GfbA-expressing GGS isolate trigger cytoskeleton rearrangements. Heterologous expression of GfbA on the surface of a commensal Streptococcus gordonii and purified recombinant protein also triggered actin rearrangements. Expression of a truncated GfbA (lacking the aromatic domain) and chimeric GfbA/SfbI protein (replacing the aromatic domain of SfbI with the GfbA aromatic domain) on S. gordonii or recombinant proteins alone showed that the aromatic domain of GfbA is responsible for different invasion mechanisms. This is the first evidence for a biological function of the aromatic domain of fibronectin-binding proteins. Furthermore, we show that streptococci invading via cytoskeleton rearrangements and intracellular trafficking along the classical endocytic pathway are less persistence than streptococci entering via caveolae.     

Comparison of the folding mechanism of highly homologous proteins in the lipid‐binding protein family

The folding mechanism of two closely related proteins in the intracellular lipid‐binding protein family, human bile acid‐binding protein (hBABP), and rat bile acid‐binding protein (rBABP) were examined. These proteins are 77% identical (93% similar) in sequence. Both of these single domain proteins fit well to a two‐state model for unfolding by fluorescence and circular dichroism at equilibrium. Three phases were observed during the unfolding of rBABP by fluorescence but only one phase was observed during the unfolding of hBABP, suggesting that at least two kinetic intermediates accumulate during the unfolding of rBABP that are not observed during the unfolding of hBABP. Fluorine NMR was used to examine the equilibrium unfolding behavior of the W49 side chain in 6‐fluorotryptophan‐labeled rBABP and hBABP. The structure of rBABP appears to be more dynamic than that of hBABP in the vicinity of W49 in the absence of denaturant, and urea has a greater effect on this dynamic behavior for rBABP than for hBABP. As such, the folding behavior of highly sequence related proteins in this family can be quite different. These differences imply that moderately sized proteins with high sequence and structural similarity can still populate quite different structures during folding. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Identification of β integrin‐like‐ and fibronectin‐like proteins in the bivalve mollusk Mytilus trossulus

Integrins play a key role in the intermediation and coordination between cells and extracellular matrix components. In this study, we first determined the presence of the β integrin‐like protein and its presumptive ligand, fibronectin‐like protein, during development and in some adult tissues of the bivalve mollusc Mytilus trossulus. We found that β integrin‐like protein expression correlated with the development and differentiation of the digestive system in larvae. Besides the presence of β integrin‐like protein in the digestive epithelial larval cells, this protein was detected in the hemocytes and some adult tissues of M. trossulus. The fibronectin‐like protein was detected firstly at the blastula stage and later, the FN‐LP‐immunoreactive cells were scattered in the trochophore larvae. The fibronectin‐like protein was not expressed in the β integrin‐positive cells of either the veliger stage larvae or the adult mussel tissues and the primary hemocyte cell culture. Despite the β integrin‐ and fibronectin‐like proteins being expressed in different cell types of mussel larvae, we do not exclude the possibility of direct interaction between these two proteins during M. trossulus development or in adult tissues.     

Trypanosoma cruzi extracellular amastigotes trigger the protein kinase D1‐cortactin‐actin pathway during cell invasion

Trypanosoma cruzi extracellular amastigotes (EAs) display unique mechanisms for cell invasion that are highly dependent on host actin filaments. Protein kinase D1 (PKD1) phosphorylates and modulates the activity of cortactin, a key regulator of actin dynamics. We evaluated the role of host cortactin and PKD1 in actin filament dynamics during HeLa cell invasion by EAs. Host cortactin, PKD1 and actin are recruited by EAs based on experiments in fixed and live cells by time lapse confocal microscopy. EAs trigger PKD1 and extracellular signal‐regulated kinase 1/2 activation, but not Src family kinases, and selectively phosphorylate cortactin. Heat‐killed EAs and non‐infective epimastigotes both triggered distinct host responses and did not recruit the molecules studied herein. EA invasion was influenced by depletion or overexpression of host cortactin and PKD1, respectively, suggesting the involvement of both proteins in this event. Collectively, these results show new host cell mechanisms subverted during EA internalization into non‐phagocytic cells.     

Variation and association of fibronectin‐binding protein genes fnbA and fnbB in Staphylococcus aureus Japanese isolates

Fibronectin‐binding proteins A and B (FnBPA and FnBPB) mediate adhesion of Staphylococcus aureus to fibrinogen, elastin and fibronectin. FnBPA and FnBPB are encoded by two closely linked genes, fnbA and fnbB, respectively. With the exception of the N‐terminal regions, the amino acid sequences of FnBPA and FnBPB are highly conserved. To investigate the genetics and evolution of fnbA and fnbB, the most variable regions, which code for the 67th amino acids of the A through B regions (A67–B) of fnbA and fnbB, were focused upon. Eighty isolates of S. aureus in Japan were sequenced and 19 and 18 types in fnbA and fnbB, respectively, identified. Although the phylogeny of fnbA and fnbB were found to be quite different, each fnbA type connected with a specific fnbB type, indicating that fnbA and fnbB mutate independently, whereas the combination of both genes after recombination is stable. Hence those fnbA–fnbB combinations were defined as FnBP sequence types (FnSTs). Representative isolates of each FnST were assigned distinct STs by multilocus sequence typing, suggesting correspondence of FnST with genome lineage. Linkage disequilibrium (LD) analysis of the A67–B region revealed that subdomains N2, N3 and FnBR1 form a LD block in fnbA, whereas N2 and N3 form two independent LD blocks in fnbB. N2–N3 three‐dimensional structural models indicated that not only the variable amino acid residues, but also well‐conserved amino acid residues between FnBPA and FnBPB, are located on the surface of the protein. These results highlight a molecular process of the FnBP that has evolved by mingled mutation and recombination with retention of functions.     

Mapping the recognition domains of pneumococcal fibronectin‐binding proteins PavA and PavB demonstrates a common pattern of molecular interactions with fibronectin type III repeats

Colonization of mucosal respiratory surfaces is a prerequisite for the human pathobiont Streptococcus pneumoniae (the pneumococcus) to cause severe invasive infections. The arsenal of pneumococcal adhesins interacts with a multitude of extracellular matrix proteins. A paradigm for pneumococci is their interaction with the adhesive glycoprotein fibronectin, which facilitates bacterial adherence to host cells. Here, we deciphered the molecular interaction between fibronectin and pneumococcal fibronectin‐binding proteins (FnBPs) PavA and PavB respectively. We show in adherence and binding studies that the pneumococcal interaction with fibronectin is a non‐human specific trait. PavA and PavB target at least 13 out of 15 type III fibronectin domains as demonstrated in ligand overlay assays, surface plasmon resonance studies and SPOT peptide arrays. Strikingly, both pneumococcal FnBPs recognize similar peptides in targeted type III repeats. Structural comparisons revealed that the targeted type III repeat epitopes cluster on the inner strands of both β‐sheets forming the fibronectin domains. Importantly, synthetic peptides of FnIII¹, FnIII⁵ or FnIII¹⁵ bind directly to FnBPs PavA and PavB respectively. In conclusion, our study suggests a common pattern of molecular interactions between pneumococcal FnBPs and fibronectin. The specific epitopes recognized in this study can potentially be tested as antimicrobial targets in further scientific endeavours.     

Structural and functional characterization of solute binding proteins for aromatic compounds derived from lignin: p‐Coumaric acid and related aromatic acids

Lignin comprises 15–25% of plant biomass and represents a major environmental carbon source for utilization by soil microorganisms. Access to this energy resource requires the action of fungal and bacterial enzymes to break down the lignin polymer into a complex assortment of aromatic compounds that can be transported into the cells. To improve our understanding of the utilization of lignin by microorganisms, we characterized the molecular properties of solute binding proteins of ATP‐binding cassette transporter proteins that interact with these compounds. A combination of functional screens and structural studies characterized the binding specificity of the solute binding proteins for aromatic compounds derived from lignin such as p‐coumarate, 3‐phenylpropionic acid and compounds with more complex ring substitutions. A ligand screen based on thermal stabilization identified several binding protein clusters that exhibit preferences based on the size or number of aromatic ring substituents. Multiple X‐ray crystal structures of protein–ligand complexes for these clusters identified the molecular basis of the binding specificity for the lignin‐derived aromatic compounds. The screens and structural data provide new functional assignments for these solute‐binding proteins which can be used to infer their transport specificity. This knowledge of the functional roles and molecular binding specificity of these proteins will support the identification of the specific enzymes and regulatory proteins of peripheral pathways that funnel these compounds to central metabolic pathways and will improve the predictive power of sequence‐based functional annotation methods for this family of proteins.Proteins 2013; 81:1709–1726. © 2013 Wiley Periodicals, Inc.     

Noncollagenous region of the streptococcal collagen‐like protein is a trimerization domain that supports refolding of adjacent homologous and heterologous collagenous domains

Proper folding of the (Gly‐Xaa‐Yaa)_n sequence of animal collagens requires adjacent N‐ or C‐terminal noncollagenous trimerization domains which often contain coiled‐coil or beta sheet structure. Collagen‐like proteins have been found recently in a number of bacteria, but little is known about their folding mechanism. The Scl2 collagen‐like protein from Streptococcus pyogenes has an N‐terminal globular domain, designated V_sp, adjacent to its triple‐helix domain. The V_sp domain is required for proper refolding of the Scl2 protein in vitro. Here, recombinant V_sp domain alone is shown to form trimers with a significant α‐helix content and to have a thermal stability of T_m = 45°C. Examination of a new construct shows that the V_sp domain facilitates efficient in vitro refolding only when it is located N‐terminal to the triple‐helix domain but not when C‐terminal to the triple‐helix domain. Fusion of the V_sp domain N‐terminal to a heterologous (Gly‐Xaa‐Yaa)_n sequence from Clostridium perfringens led to correct folding and refolding of this triple‐helix, which was unable to fold into a triple‐helical, soluble protein on its own. These results suggest that placement of a functional trimerization module adjacent to a heterologous Gly‐Xaa‐Yaa repeating sequence can lead to proper folding in some cases but also shows specificity in the relative location of the trimerization and triple‐helix domains. This information about their modular nature can be used in the production of novel types of bacterial collagen for biomaterial applications.     

Lactobacillus fermentum BCS87 expresses mucus‐ and mucin‐binding proteins on the cell surface

Aims: To identify and characterize adhesion‐associated proteins in the potential probiotic Lactobacillus fermentum BCS87. Methods and Results: Protein suspensions obtained from the treatment of Lact. fermentum BCS87 with 1 mol 1^?1 LiCl were analysed by Western blotting using HRP‐labelled porcine mucus and mucin. Two adhesion‐associated proteins with relative molecular weight of 29 and 32 kDa were identified. The N‐terminal and internal peptides of the 32 kDa protein (32‐Mmubp) were sequenced, and the corresponding gene (32‐mmub) was found by inverse polymerase chain reaction. The complete nucleotide sequence of 32‐mmub revealed an open reading frame of 903 bp encoding a primary protein of 300 amino acids and a mature protein of 272 residues. A basic local alignment search showed 47–99% identity to solute‐binding components of ATP binding cassette transporter proteins in Lactobacillus, Streptococcus and Clostridium. An OpuAC‐conserved domain was identified and phylogenetic relationship analysis confirmed that 32‐Mmubp belongs to the OpuAC family. Conclusions: Adhesion of Lact. fermentum BCS87 appeared to be mediated by two surface‐associated proteins. 32‐Mmubp is a component of ABC transporter system that also functions as an adhesin. Significance and Impact of the Study: Characterization of 32‐Mmubp and 32‐mmub will contribute to understanding the host–bacteria interactions of Lact. fermentum with the intestinal tract of pigs.     

Sequence and structural analysis of fibronectin‐binding protein reveals importance of multiple intrinsic disordered tandem repeats

The location of certain amino acid sequences like repeats along the polypeptide chain is very important in the context of forming the overall shape of the protein molecule which in fact determines its function. In gram‐positive bacteria, fibronectin‐binding protein (FnBP) is one such repeat containing protein, and it is a cell wall‐attached protein responsible for various acute infections in human. Several studies on sequence, structure, and function of fibronectin‐binding regions of FnBPs were reported; however, no detailed study was carried out on the full‐length protein sequence. In the present study, we have made a thorough sequence and structure analysis on FnBP_A of Staphylococcus aureus and explored the presence of dual ligand‐binding ability of fibrinogen (fg)‐binding region and its molecular recognition processes. Multiple sequence alignment and protein‐protein docking analysis reveal the regions which are likely involved in dual ligand binding. Further analysis of docking of FnBP_A fg‐binding region and fn N‐terminal modules suggests that if the latter binds to the fg‐binding region of FnBP_A, it would inhibit the subsequent binding of fg because of steric hindrance. The sequence analysis further suggests that the abundance of disorder promoting residue glutamic acid and dual personality (both order/disorder promoting) residue threonine in tandem repeats of FnBP_A and B proteins possibly would help the molecule to undergo a conformational change while binding with fn by β‐zipper mechanism. The segment‐based power spectral analysis was carried out which helps to understand the distribution of hydrophobic residues along the sequence particularly in intrinsic disordered tandem repeats. The results presented here will help to understand the role of internal repeats and intrinsic disorder in the molecular recognition process of a pathogenic cell surface protein.     

Identification of homologous binding proteins in porcine and bovine gametes

The purpose of this study was to determine if sperm and oocyte proteins that mediate plasma membrane interaction during mammalian fertilization are conserved among porcine and bovine gametes. We examined homologous and heterologous sperm and zona-free oocyte interactions to determine the extent of cross-reactivity between the gametes of these two ungulate species. First, the numbers of ejaculated porcine and bovine sperm bound to the oocyte plasma membrane of intact porcine and bovine oocytes were determined in vitro. There was no significant difference between the number of porcine or bovine sperm that bound to porcine or bovine oocytes (P > 0.25). Second, individual porcine and bovine sperm plasma membrane proteins were identified by binding of homologous or heterologous oocyte plasma membrane to whole sperm plasma membrane on Western ligand blots. The relative amount of labeled oocyte plasma membrane bound to individual sperm plasma membrane proteins was analyzed by laser densitometry. Eight porcine sperm plasma membrane proteins and seven bovine sperm plasma membrane proteins were bound by both porcine and bovine oocyte plasma membrane. A significantly greater relative amount of porcine oocyte plasma membrane than bovine oocyte plasma membrane was bound to the 14- and 10-kD porcine sperm plasma membrane proteins (P < 0.001 and P < 0.01, respectively). A 27-kD bovine sperm plasma membrane protein bound proportionally more bovine oocyte plasma membrane probe than porcine oocyte plasma membrane probe (P < 0.04). These results are consistent with conservation of similar receptor ligand interactions at the gamete plasma membrane among porcine and bovine gametes.     

Quantifying the mechanisms of domain gain in animal proteins

Background  

Protein domains are protein regions that are shared among different proteins and are frequently functionally and structurally independent from the rest of the protein. Novel domain combinations have a major role in evolutionary innovation. However, the relative contributions of the different molecular mechanisms that underlie domain gains in animals are still unknown. By using animal gene phylogenies we were able to identify a set of high confidence domain gain events and by looking at their coding DNA investigate the causative mechanisms.     

Nucleotides sequestered at different subsite loci within DNA‐binding pockets of two OB‐fold single‐stranded DNA‐binding proteins are unstacked to different extents

The gene 5 protein (g5p) encoded by the Ff strains of Escherichia coli bacteriophages is a dimeric single‐stranded DNA‐binding protein (SSB) that consists of two identical OB‐fold (oligonucleotide/oligosaccharide‐binding) motifs. Ultrafast time‐resolved fluorescence measurements were carried out to investigate the effect of g5p binding on the conformation of 2‐aminopurine (2AP) labels positioned between adenines or cytosines in the 16‐nucleotide antiparallel tails of DNA hairpins. The measurements revealed significant changes in the conformational heterogeneity of the 2AP labels caused by g5p binding. The extent of the changes was dependent on sub‐binding‐site location, but generally resulted in base unstacking. When bound by g5p, the unstacked 2AP population increased from ～22% to 59–67% in C‐2AP‐C segments and from 39% to 77% in an A‐2AP‐A segment. The OB‐fold RPA70A domain of the human replication protein A also caused a significant amount of base unstacking at various locations within the DNA binding site as evidenced by steady‐state fluorescence titration measurements using 2AP‐labeled 5‐mer DNAs. These solution studies support the concept that base unstacking at most of a protein's multiple sub‐binding‐site loci may be a feature that allows non‐sequence specific OB‐fold proteins to bind to single‐stranded DNAs (ssDNAs) with minimal preference for particular sequences. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 484–496, 2013.     

Differences in osteoblast miRNA induced by cell binding domain of collagen and silicate-based synthetic bone

Summary PerioGlas (PG) is an silicate-based (i.e. anorganic) material used for grafting periodontal osseous defects since the ninety whereas P-15 is an analog of the cell binding domain of collagen (i.e. organic material) that is successfully used in clinical trial to promote bone formation. However, how PG (i.e anorganic material) and P-15 (i.e. collagen) differentially alter osteoblast activity to promote bone formation is unknown. We therefore attempted to get more insight by using microRNA microarray techniques to investigate the translation process in osteoblasts differentially exposed to PG and P-15. We identified 3 up-regulated miRNA (i.e. mir-30b, mir-26a, mir-92) and 8 down-regulated miRNA (i.e. mir-337, mir-377, mir-25, mir-200b, mir-129, mir-373, mir-133b, mir-489). The data reported are, to our knowledge, the first study on translation regulation in osteoblatsts differentially exposed to cell binding domain of collagen and to silicate-based material. Both enhance the translation of several miRNA belonging to osteogenetic genes, but P-15 acts preferentially on homeobox genes.     

Chi hotspots trigger a conformational change in the helicase-like domain of AddAB to activate homologous recombination

In bacteria, the repair of double-stranded DNA breaks is modulated by Chi sequences. These are recognised by helicase-nuclease complexes that process DNA ends for homologous recombination. Chi activates recombination by changing the biochemical properties of the helicase-nuclease, transforming it from a destructive exonuclease into a recombination-promoting repair enzyme. This transition is thought to be controlled by the Chi-dependent opening of a molecular latch, which enables part of the DNA substrate to evade degradation beyond Chi. Here, we show that disruption of the latch improves Chi recognition efficiency and stabilizes the interaction of AddAB with Chi, even in mutants that are impaired for Chi binding. Chi recognition elicits a structural change in AddAB that maps to a region of AddB which resembles a helicase domain, and which harbours both the Chi recognition locus and the latch. Mutation of the latch potentiates the change and moderately reduces the duration of a translocation pause at Chi. However, this mutant displays properties of Chi-modified AddAB even in the complete absence of bona fide hotspot sequences. The results are used to develop a model for AddAB regulation in which allosteric communication between Chi binding and latch opening ensures quality control during recombination hotspot recognition.