The Pneumococcal Serine-Rich Repeat Protein Is an Intra-Species Bacterial Adhesin That Promotes Bacterial Aggregation In Vivo and in Biofilms

The Pneumococcal serine-rich repeat protein (PsrP) is a pathogenicity island encoded adhesin that has been positively correlated with the ability of Streptococcus pneumoniae to cause invasive disease. Previous studies have shown that PsrP mediates bacterial attachment to Keratin 10 (K10) on the surface of lung cells through amino acids 273–341 located in the Basic Region (BR) domain. In this study we determined that the BR domain of PsrP also mediates an intra-species interaction that promotes the formation of large bacterial aggregates in the nasopharynx and lungs of infected mice as well as in continuous flow-through models of mature biofilms. Using numerous methods, including complementation of mutants with BR domain deficient constructs, fluorescent microscopy with Cy3-labeled recombinant (r)BR, Far Western blotting of bacterial lysates, co-immunoprecipitation with rBR, and growth of biofilms in the presence of antibodies and competitive peptides, we determined that the BR domain, in particular amino acids 122–166 of PsrP, promoted bacterial aggregation and that antibodies against the BR domain were neutralizing. Using similar methodologies, we also determined that SraP and GspB, the Serine-rich repeat proteins (SRRPs) of Staphylococcus aureus and Streptococcus gordonii, respectively, also promoted bacterial aggregation and that their Non-repeat domains bound to their respective SRRPs. This is the first report to show the presence of biofilm-like structures in the lungs of animals infected with S. pneumoniae and show that SRRPs have dual roles as host and bacterial adhesins. These studies suggest that recombinant Non-repeat domains of SRRPs (i.e. BR for S. pneumoniae) may be useful as vaccine antigens to protect against Gram-positive bacteria that cause infection.     

The basic keratin 10-binding domain of the virulence-associated pneumococcal serine-rich protein PsrP adopts a novel MSCRAMM fold

Streptococcus pneumoniae is a major human pathogen, and a leading cause of disease and death worldwide. Pneumococcal invasive disease is triggered by initial asymptomatic colonization of the human upper respiratory tract. The pneumococcal serine-rich repeat protein (PsrP) is a lung-specific virulence factor whose functional binding region (BR) binds to keratin-10 (KRT10) and promotes pneumococcal biofilm formation through self-oligomerization. We present the crystal structure of the KRT10-binding domain of PsrP (BR_187–385) determined to 2.0 Å resolution. BR_187–385 adopts a novel variant of the DEv-IgG fold, typical for microbial surface components recognizing adhesive matrix molecules adhesins, despite very low sequence identity. An extended β-sheet on one side of the compressed, two-sided barrel presents a basic groove that possibly binds to the acidic helical rod domain of KRT10. Our study also demonstrates the importance of the other side of the barrel, formed by extensive well-ordered loops and stabilized by short β-strands, for interaction with KRT10.     

Structure of a Novel O-Linked N-Acetyl-d-glucosamine (O-GlcNAc) Transferase,GtfA, Reveals Insights into the Glycosylation of Pneumococcal Serine-rich Repeat Adhesins

Protein glycosylation catalyzed by the O-GlcNAc transferase (OGT) plays a critical role in various biological processes. In Streptococcus pneumoniae, the core enzyme GtfA and co-activator GtfB form an OGT complex to glycosylate the serine-rich repeat (SRR) of adhesin PsrP (pneumococcal serine-rich repeat protein), which is involved in the infection and pathogenesis. Here we report the 2.0 Å crystal structure of GtfA, revealing a β-meander add-on domain beyond the catalytic domain. It represents a novel add-on domain, which is distinct from the all-α-tetratricopeptide repeats in the only two structure-known OGTs. Structural analyses combined with binding assays indicate that this add-on domain contributes to forming an active GtfA-GtfB complex and recognizing the acceptor protein. In addition, the in vitro glycosylation system enables us to map the O-linkages to the serine residues within the first SRR of PsrP. These findings suggest that fusion with an add-on domain might be a universal mechanism for diverse OGTs that recognize varying acceptor proteins/peptides.     

Cellular senescence increases expression of bacterial ligands in the lungs and is positively correlated with increased susceptibility to pneumococcal pneumonia

Cellular senescence is an age-associated phenomenon that promotes tumor invasiveness owing to the secretion of proinflammatory cytokines, proteases, and growth factors. Herein we demonstrate that cellular senescence also potentially increases susceptibility to bacterial pneumonia caused by Streptococcus pneumoniae (the pneumococcus), the leading cause of infectious death in the elderly. Aged mice had increased lung inflammation as determined by cytokine analysis and histopathology of lung sections. Immunoblotting for p16, pRb, and mH2A showed that elderly humans and aged mice had increased levels of these senescence markers in their lungs vs. young controls. Keratin 10 (K10), laminin receptor (LR), and platelet-activating factor receptor (PAFr), host proteins known to be co-opted for bacterial adhesion, were also increased. Aged mice were found to be highly susceptible to pneumococcal challenge in a PsrP, the pneumococcal adhesin that binds K10, dependent manner. In vitro senescent A549 lung epithelial cells had elevated K10 and LR protein levels and were up to 5-fold more permissive for bacterial adhesion. Additionally, exposure of normal cells to conditioned media from senescent cells doubled PAFr levels and pneumococcal adherence. Genotoxic stress induced by bleomycin and oxidative stress enhanced susceptibility of young mice to pneumonia and was positively correlated with enhanced p16, inflammation, and LR levels. These findings suggest that cellular senescence facilitates bacterial adhesion to cells in the lungs and provides an additional molecular mechanism for the increased incidence of community-acquired pneumonia in the elderly. This study is the first to suggest a second negative consequence for the senescence-associated secretory phenotype.     

Changes in capsular serotype alter the surface exposure of pneumococcal adhesins and impact virulence

We examined the contribution of serotype on Streptococcus pneumoniae adhesion and virulence during respiratory tract infection using a panel of isogenic TIGR4 (serotype 4) mutants expressing the capsule types 6A (+6A), 7F (+7F) and 23F (+23F) as well as a deleted and restored serotype 4 (+4) control strain. Immunoblots, bacterial capture assays with immobilized antibody, and measurement of mean fluorescent intensity by flow cytometry following incubation of bacteria with antibody, all determined that the surface accessibility, but not total protein levels, of the virulence determinants Pneumococcal surface protein A (PspA), Choline binding protein A (CbpA), and Pneumococcal serine-rich repeat protein (PsrP) changed with serotype. In vitro, bacterial adhesion to Detroit 562 pharyngeal or A549 lung epithelial cells was modestly but significantly altered for +6A, +7F and +23F. In a mouse model of nasopharyngeal colonization, the number of +6A, +7F, and +23F pneumococci in the nasopharynx was reduced 10 to 100-fold versus +4; notably, only mice challenged with +4 developed bacteremia. Intratracheal challenge of mice confirmed that capsule switch strains were highly attenuated for virulence. Compared to +4, the +6A, +7F, and +23F strains were rapidly cleared from the lungs and were not detected in the blood. In mice challenged intraperitoneally, a marked reduction in bacterial blood titers was observed for those challenged with +6A and +7F versus +4 and +23F was undetectable. These findings show that serotype impacts the accessibility of surface adhesins and, in particular, affects virulence within the respiratory tract. They highlight the complex interplay between capsule and protein virulence determinants.     

Formation of a Disulfide Bond in the Immunoglobulin Domain of the Myelin P0 Protein Is Essential for Its Adhesion

Abstract: It is widely accepted, although never demonstrated, that the formation of a disulfide bond in the majority of immunoglobulin (Ig)-like domains stabilizes their final conformation and thus is essential to their functioning as adhesion/recognition molecules. The myelin P₀ protein, which has been shown directly to behave as a homophilic adhesion molecule, contains a single Ig-like domain, stabilized by a putative Cys²¹-Cys⁹⁸ disulfide bond. To test if this bond is indeed necessary to the adhesive function of P₀, the nucleotides in the P₀ cDNA coding for Cys²¹ were altered to code for an alanine. The mutated P₀ cDNA was transfected into Chinese hamster ovary cells, expression of the mutated P₀ protein was characterized, and the adhesiveness of Cys²¹-mutated P₀-expressing cells and that of cells expressing equivalent surface amounts of the unmutated protein were compared. It was found, as we previously reported, that incubation of a single cell suspension of the unmutated P₀-expressing cells resulted in the rapid formation of large aggregates. In contrast, after a similar incubation the cells expressing the Cys²¹-mutated P₀ were still mostly single cells, a result indistinguishable from that observed with the control transfected cells. This suggests that the P₀ protein, when mutated at Cys²¹, does not behave as a homophilic adhesion molecule, which in turn implies that the formation of an Ig domain disulfide bond is essential to the functioning of this molecule.     

Scl1-dependent internalization of group A Streptococcus via direct interactions with the alpha2beta(1) integrin enhances pathogen survival and re-emergence

The molecular pathogenesis of infections caused by group A Streptococcus (GAS) is not fully understood. We recently reported that a recombinant protein derived from the collagen-like surface protein, Scl1, bound to the human collagen receptor, integrin α₂β₁. Here, we investigate whether the same Scl1 variant expressed by GAS cells interacts with the integrin α₂β₁ and affects the biological outcome of host–pathogen interactions. We demonstrate that GAS adherence and internalization involve direct interactions between surface expressed Scl1 and the α₂β₁ integrin, because (i) both adherence and internalization of the scl1- inactivated mutant were significantly decreased, and were restored by in-trans complementation of Scl1 expression, (ii) GAS internalization was reduced by pre-treatment of HEp-2 cells with anti-α₂ integrin-subunit antibody and type I collagen, (iii) recombinant α₂-I domain bound the wild-type GAS cells and (iv) internalization of wild-type cells was significantly increased in C2C12 cells expressing the α₂β₁ integrin as the only collagen-binding integrin. Next, we determined that internalized GAS re-emerges from epithelial cells into the extracellular environment. Taken together, our data describe a new molecular mechanism used by GAS involving the direct interaction between Scl1 and integrins, which increases the overall capability of the pathogen to survive and re-emerge.     

A streptococcal collagen-like protein interacts with the alpha2beta1 integrin and induces intracellular signaling

The streptococcal collagen-like proteins Scl1 and Scl2 are prokaryotic members of a large protein family with domains containing the repeating amino acid sequence (Gly-Xaa-Yaa)(n) that form a collagen-like triple-helical structure. Here, we test the hypothesis that Scl variant might interact with mammalian collagen-binding integrins. We show that the recombinant Scl protein p176 promotes adhesion and spreading of human lung fibroblast cells through an alpha2beta1 integrin-mediated interaction as shown in cell adhesion inhibition assays using anti-alpha2beta1 and anti-beta1 integrins monoclonal antibodies. Accordingly, C2C12 cells stably expressing alpha2beta1 integrin as the only collagen-binding integrin show productive cell adhesion activities on p176 that can be blocked by an anti-alpha2beta1 integrin antibody. In addition, p176 promotes tyrosine phosphorylation of p125(FAK) of C2C12 cells expressing alpha2beta1 integrin, whereas parental cells do not. Furthermore, C2C12 adhesion of human lung fibroblast cells to p176 induces phosphorylation of p125FAK, p130CAS, and p68Paxillin proteins. In a domain swapping experiment, we show that integrin binds to the collagenous domain of the Scl protein. Moreover, the recombinant inserted domain of the alpha2 integrin interacts with p176 with a relatively high affinity (K(D) = 17 nm). Attempts to identify the integrin sites in p176 suggest that more than one site may be involved. These studies, for the first time, suggest that the collagen-like proteins of prokaryotes retained not only structural but also functional characteristics of their eukaryotic counterparts.     

Identification of Strychnine Binding Sites in the Rat Retina

Abstract: [³H]Strychnine specifically binds to membrane fractions isolated from rat retinae. The binding is saturable, with an apparent dissociation constant, K _D, of 14.3 × 10⁻⁹ M and 205 fmol bound/mg protein. Specific binding is time-dependent and proportional to protein concentration. Glycine and taurine are equally potent inhibitors of [³H]strychnine binding ( K _i= 4 × 10⁻⁵ M); no other amino acids endogenously present in the retina inhibited [³H]strychnine binding.     

Receptor-mediated transcytosis of botulinum neurotoxin A through intestinal cell monolayers

Botulism is mainly acquired by the oral route, and botulinum neurotoxin (BoNT) escapes the gastrointestinal tract by crossing the digestive epithelial barrier prior to gaining access to the nerve endings. Here, we show that biologically active BoNT/A crosses intestinal cell monolayers via a receptor-mediated transcytosis, including a transport inhibition at 4°C and a passage at 37°C in a saturable manner within 30–60 min. BoNT/A passage rate was about 10-fold more efficient through the intestinal crypt cell line m-IC_cl2, than through the carcinoma Caco-2 or T84 cells, and was not increased when BoNT/A was associated with the non-toxic proteins (botulinum complex). Like for neuronal cells, BoNT/A binding to intestinal cells was mediated by the half C-terminal domain as tested by fluorescence-activated cytometry and by transcytosis competition assay. A 'double receptor model' has been proposed in which BoNT/A interacts with gangliosides of GD_1b and GT_1b series as well as SV2 protein. Gangliosides of GD_1b and GT_1b series and recombinant intravesicular SV2-C domain partially impaired BoNT/A transcytosis, suggesting a putative role of gangliosides and SV2 or a related protein in BoNT/A transcytosis through Caco-2 and m-IC_cl2 cells.     

Genetic transfer of fusion proteins effectively inhibits VCAM-1-mediated cell adhesion and transmigration via inhibition of cytoskeletal anchorage

The adhesion of leukocytes to endothelium plays a central role in the development of atherosclerosis and thus represents an attractive therapeutic target for anti-atherosclerotic therapies. Vascular cell adhesion molecule-1 (VCAM-1) mediates both the initial tethering and the firm adhesion of leukocytes to endothelial cells. Our work evaluates the feasibility of using the cytoskeletal anchorage of VCAM-1 as a target for gene therapy. As a proof of concept, integrin α_IIbβ₃-mediated cell adhesion with clearly defined cytoskeletal anchorage was tested. We constructed fusion proteins containing the intracellular domain of β₃ placed at various distances to the cell membrane. Using cell adhesion assays and immunofluorescence, we established fusion constructs with competitive and dominant negative inhibition of cell adhesion. With the goal being the transfer of the dominant negative mechanism towards VCAM-1 inhibition, we constructed a fusion molecule containing the cytoplasmic domain of VCAM-1. Indeed, VCAM-1 mediated leukocyte adhesion can be inhibited via transfection of DNA encoding the designed VCAM-1 fusion protein. This is demonstrated in adhesion assays under static and flow conditions using CHO cells expressing recombinant VCAM-1 as well as activated endothelial cells. Thus, we are able to describe a novel approach for dominant negative inhibition of leukocyte adhesion to endothelial cells. This approach warrants further development as a novel gene therapeutic strategy that aims for a locally restricted effect at atherosclerotic areas of the vasculature.     

A 49-residue peptide from adhesin F1 of Streptococcus pyogenes inhibits fibronectin matrix assembly

F1 is an adhesin of Streptococcus pyogenes which binds the N-terminal 70-kDa region of fibronectin with high affinity. The fibronectin binding region of F1 is comprised of a 43-residue upstream domain and a repeat domain comprised of five tandem 37-residue sequences. We investigated the effects of these domains on the assembly of fibronectin matrix by human dermal fibroblasts, MG63 osteosarcoma cells, or fibroblasts derived from fibronectin-null stem cells. Subequimolar or equimolar concentrations of recombinant proteins containing both the upstream and repeat domains or just the repeat domain enhanced binding of fibronectin or its N-terminal 70-kDa fragment to cell layers; higher concentrations of these recombinant proteins inhibited binding. The enhanced binding did not result in greater matrix assembly and was caused by increased ligand binding to substratum. In contrast, recombinant or synthetic protein containing the 43 residues of the upstream domain and the first 6 residues from the repeat domain exhibited monophasic inhibition with an IC(50) of approximately 10 nm. Truncation of the 49-residue sequence at its N or C terminus caused loss of inhibitory activity. The 49-residue upstream sequence blocked incorporation of both endogenous cellular fibronectin and exogenous plasma fibronectin into extracellular matrix and inhibited binding of 70-kDa fragment to fibronectin-null cells in a fibronectin-free system. Inhibition of matrix assembly by the 49-mer had no effect on cell adhesion to substratum, cell growth, formation of focal contacts, or formation of stress fibers. These results indicate that the 49-residue upstream sequence of F1 binds in an inhibitory mode to N-terminal parts of exogenous and endogenous fibronectin which are critical for fibronectin fibrillogenesis.     

The bundlin pilin protein of enteropathogenic Escherichia coli is an N-acetyllactosamine-specific lectin

Synthetic N -acetyllactosamine (LacNAc) glycoside sequences coupled to BSA competitively inhibit enteropathogenic Escherichia coli (EPEC) localized adherence (LA) to human intestinal biopsy specimens and tissue culture cell monolayers. The LacNAc-specific adhesin appears to be associated with the bundle-forming pili (BFP) expressed by EPEC during the early stages of colonization. Herein, we report that recombinant bundlin inhibits EPEC LA to HEp-2 cells and binds to HEp-2 cells. Recombinant bundlin also binds, with millimolar association constants ( K _assoc), to synthetic LacNAc-Benzene and LacNAc-O(CH₂)₈CONH₂ glycosides as assessed in the gas phase by nanoelectrospray ionization mass spectrometry. Furthermore, LacNAc-BSA inhibits LA only of EPEC strains that express α bundlin alleles, suggesting putative locations for the LacNAc-binding pocket in the α bundlin monomer. Collectively, these results suggest that α bundlin possesses lectin-like properties that are responsible for LacNAc-specific initial adherence of α bundlin-expressing EPEC strains to host intestinal epithelial cells.     

The serine-rich domain from Crk-associated substrate (p130cas) is a four-helix bundle

p130(cas) (Crk-associated substrate) is a docking protein that is involved in assembly of focal adhesions and concomitant cellular signaling. It plays a role in physiological regulation of cell adhesion, migration, survival, and proliferation, as well as in oncogenic transformation. The molecule consists of multiple protein-protein interaction motifs, including a serine-rich region that is positioned between Crk and Src-binding sites. This study reports the first structure of a functional domain of Cas. The solution structure of the serine-rich region has been determined by NMR spectroscopy, demonstrating that this is a stable domain that folds as a four-helix bundle, a protein-interaction motif. The serine-rich region bears strong structural similarity to four-helix bundles found in other adhesion components like focal adhesion kinase, alpha-catenin, or vinculin. Potential sites for phosphorylation and interaction with the 14-3-3 family of cellular regulators are identified in the domain and characterized by site-directed mutagenesis and binding assays. Mapping the degree of amino acid conservation onto the molecular surface reveals a patch of invariant residues near the C terminus of the bundle, which may represent a previously unidentified site for protein interaction.     

The GRIP domain is a specific targeting sequence for a population of trans-Golgi network derived tubulo-vesicular carriers

Vesicular carriers for intracellular transport associate with unique sets of accessory molecules that dictate budding and docking on specific membrane domains. Although many of these accessory molecules are peripheral membrane proteins, in most cases the targeting sequences responsible for their membrane recruitment have yet to be identified. We have previously defined a novel Golgi targeting domain (GRIP) shared by a family of coiled-coil peripheral membrane Golgi proteins implicated in membrane trafficking. We show here that the docking site for the GRIP motif of p230 is a specific domain of Golgi membranes. By immuno-electron microscopy of HeLa cells stably expressing a green fluorescent protein (GFP)-p230_GRIP fusion protein, we show binding specifically to a subset of membranes of the trans -Golgi network (TGN). Real-time imaging of live HeLa cells revealed that the GFP-p230_GRIP was associated with highly dynamic tubular extensions of the TGN, which have the appearance and behaviour of transport carriers. To further define the nature of the GRIP membrane binding site, in vitro budding assays were performed using purified rat liver Golgi membranes and cytosol from GFP-p230_GRIP-transfected cells. Analysis of Golgi-derived vesicles by sucrose gradient fractionation demonstrated that GFP-p230_GRIP binds to a specific population of vesicles distinct from those labelled for β-COP or γ-adaptin. The GFP-p230_GRIP fusion protein is recruited to the same vesicle population as full-length p230, demonstrating that the GRIP domain is solely proficient as a targeting signal for membrane binding of the native molecule. Therefore, p230 GRIP is a targeting signal for recruitment to a highly selective membrane attachment site on a specific population of trans -Golgi network tubulo-vesicular carriers.     

Effects of Copper on Survival of Prion Protein Knockout Neurons and Glia

Abstract: The N-terminal region of the prion protein (PrP) contains an octameric repeat region suggested to bind copper. A 32-amino acid peptide (PrPOcta) based on this region in the protein was tested for its effects on cultured cerebellar cells. Cerebellar cells from mice deficient in cellular PrP (Prnp^0/0 mice) are more sensitive to copper toxicity and oxidative stress. PrPOcta selectively promotes the survival of Prnp^0/0 cerebellar cells. However, PrPOcta also reduces the toxicity of CuSO₄ on cerebellar cells and abolishes the difference in increased sensitivity of Prnp^0/0 cells to both copper toxicity and also oxidative stress from xanthine oxidase. PrPOcta does not promote the survival or proliferation of astrocytes or microglia. The survival-promoting effects of PrPOcta on neurons may be due to its ability to effectively chelate copper. The octameric repeat region of PrP may represent a functional domain of the native protein.