The catalytic domains of Streptococcus mutans glucosyltransferases: a structural analysis.

Streptococcus mutans, found in the human oral cavity, is a significant contributor to the pathogenesis of dental caries. This bacterium expresses three genetically distinct types of glucosyltransferases named GtfB (GTF-I), GtfC (GTF-SI) and GtfD (GTF-S) that play critical roles in the development of dental plaque. The catalytic domains of GtfB, GtfC and GtfD contain conserved active-site residues for the overall enzymatic activity that relate to hydrolytic glycosidic cleavage of sucrose to glucose and fructose, release of fructose and generation of a glycosyl-enzyme intermediate in the reducing end. In a subsequent transglycosylation step, the glucosyl moiety is transferred to the nonreducing end of an acceptor to form a growing glucan polymer chain made up of glucose molecules. It has been proposed that both sucrose breakdown and glucan synthesis occur in the same active site of the catalytic domain, although the active site does not appear to be large enough to accommodate both functions. These three enzymes belong to glycoside hydrolase family 70 (GH70), which shows homology to glycoside hydrolase family 13 (GH13). GtfC synthesizes both soluble and insoluble glucans (α-1,3 and α-1,6 glycosidic linkages), while GtfB and GtfD synthesize only insoluble or soluble glucans, respectively. Here, crystal structures of the catalytic domains of GtfB and GtfD are reported. These structures are compared with previously determined structures of the catalytic domain of GtfC. With this work, apo structures and inhibitor-complex structures with acarbose are now available for the catalytic domains of GtfC and GtfB. The structure of GtfC with maltose allows further identification and comparison of active-site residues. A model of sucrose binding to GtfB is also included. The new structure of the catalytic domain of GtfD affords a structural comparison of the three S. mutans glycosyltransferases. Unfortunately, the catalytic domain of GtfD is not complete since crystallization resulted in the structure of a truncated protein lacking approximately 200 N-terminal residues of domain IV.

Systemic Checkpoint Blockade by PD-L1 Single-Chain Antibody Confers Potent Antitumor Immunity and Long-term Survival.

Immune checkpoint inhibitors (ICI) are promising in adjuvant settings for solid tumors and hematologic malignancies. They are currently used in the treatment as mAbs in high concentrations, raising concerns of toxicity and adverse side effects. Among various checkpoint molecules, targeting the programmed cell death protein-1 (PD-1)-programmed death-ligand 1 (PD-L1) axis has garnered more clinical utility than others have. To develop a physiologically relevant and systemically stable level of ICIs from a one-time application by genetic antibody engineering, we endeavored using a nonpathogenic, replication-deficient recombinant adeno-associated vector (rAAV) expressing single-chain variable fragments (scFv) of PD-L1 antibody and tested in syngeneic mouse therapy models of MC38 colorectal and EMT6 breast tumors. Results of this study indicated a significant protection against PD-L1-mediated inhibition of CD8+ T-cell function, against the growth of primary and secondary tumors, and durable antitumor CTLs activity by adoptive CD8+ T-cell transfer. Stable maintenance of PD-L1 scFv in vivo resulted in an increase in PD-1- CD8+ T cells and a concomitant decrease in regulatory T cells, M2 macrophages, and myeloid-derived suppressor cells in the tumor microenvironment. Overall, these data demonstrate the potential of rAAV-PD-L1-scFv as an alternative to mAb targeting of PD-L1 for tumor therapy.

The Glycoprotein 340's Scavenger Receptor Cysteine-Rich Domain Promotes Adhesion of Staphylococcus aureus and Pseudomonas aeruginosa to Contact Lens Polymers.

Contact lenses are biomaterials worn on the eye to correct refractive errors. Bacterial adhesion and colonization of these lenses results in adverse events, such as microbial keratitis. The adsorption of tear proteins to contact lens materials enhances bacterial adhesion. Glycoprotein 340 (Gp340), a tear component, is known to promote microbial colonization in the oral cavity; however, it has not been investigated in any contact lens-related adverse event. Therefore, this study examined the adsorption of Gp340 and its recombinantly expressed scavenger receptor cysteine-rich (iSRCR1Gp340) domain on two common contact lens materials, etafilcon A and lotrafilcon B, and the concomitant effects on the adherence of clinical isolates of microbial keratitis causative agents, Pseudomonas aeruginosa (PA6206; PA6294), and Staphylococcus aureus (SA38; USA300). Across all strains and materials, iSRCR1Gp340 enhanced adherence of bacteria in a dose-dependent manner. However, iSRCR1Gp340 did not modulate the lysozyme's or lactoferrin's effects on bacterial adhesion to the contact lens. The Gp340 binding serine-rich surface protein (SraP) significantly enhanced the binding of USA300 to iSRCR1Gp340-coated lenses. In addition, iSRCR1Gp340-coated surfaces had significantly diminished biofilms with the SraP mutant (ΔSraP), and there was a further reduction in biofilms with the sortase A mutant (ΔSrtA), indicating the likely involvement of additional surface proteins. Finally, the binding affinities between iSRCR1Gp340 and SraP were determined using surface plasmon resonance (SPR), where the complete SraP binding region displayed nanomolar affinity, whereas its smaller fragments adhered with micromolar affinities. This study concludes that Gp340 and its SRCR domains play an important role in bacterial adhesion to the contact lens.

Structural and functional analysis of the C-terminal region of Streptococcus gordonii SspB.

Streptococcus gordonii is a member of the viridans streptococci and is an early colonizer of the tooth surface. Adherence to the tooth surface is enabled by proteins present on the S. gordonii cell surface, among which SspB belongs to one of the most well studied cell-wall-anchored adhesin families: the antigen I/II (AgI/II) family. The C-terminal region of SspB consists of three tandemly connected individual domains that display the DEv-IgG fold. These C-terminal domains contain a conserved Ca2+-binding site and isopeptide bonds, and they adhere to glycoprotein 340 (Gp340; also known as salivary agglutinin, SAG). Here, the structural and functional characterization of the C123SspB domain at 2.7 Šresolution is reported. Although the individual C-terminal domains of Streptococcus mutans AgI/II and S. gordonii SspB show a high degree of both sequence and structural homology, superposition of these structures highlights substantial differences in their electrostatic surface plots, and this can be attributed to the relative orientation of the individual domains (C1, C2 and C3) with respect to each other and could reflect their specificity in binding to extracellular matrix molecules. Studies further confirmed that affinity for Gp340 or its scavenger receptor cysteine-rich (SRCR) domains requires two of the three domains of C123SspB, namely C12 or C23, which is different from AgI/II. Using protein-protein docking studies, models for this observed functional difference between C123SspB and C123AgI/II in their binding to SRCR1 are presented.

Structure-Function Characterization of Streptococcus intermedius Surface Antigen Pas.

Streptococcus intermedius, an oral commensal bacterium, is found at various sites, including subgingival dental plaque, purulent infections, and cystic fibrosis lungs. Oral streptococci utilize proteins on their surface to adhere to tissues and/or surfaces localizing the bacteria, which subsequently leads to the development of biofilms, colonization, and infection. Among the 19 genomically annotated cell wall-attached surface proteins on S. intermedius, Pas is an adhesin that belongs to the antigen I/II (AgI/II) family. Here, we have structurally and functionally characterized Pas, particularly focusing on its microbial-host as well as microbial-microbial interactions. The crystal structures of VPas and C123Pas show high similarity with AgI/II of Streptococcus mutans. VPas hosts a conserved metal binding site, and likewise, the C123Pas structure retains its conserved metal binding sites and isopeptide bonds within its three DEv-IgG domains. Pas interacts with nanomolar affinity to lung alveolar glycoprotein 340 (Gp340), its scavenger receptor cysteine-rich domains (SRCRs), and with fibrinogen. Both Candida albicans and Pseudomonas aeruginosa, the opportunistic pathogens that cohabitate with S. intermedius in the lungs of CFTR patients were studied in dual-species biofilm studies. The Pas-deficient mutant (Δpas) displayed significant reduction in dual-biofilm formation with C. albicans. In similar studies with P. aeruginosa, Pas did not mediate the biofilm formation with either the acute isolate (PAO1) or the chronic isolate (FRD1). However, the sortase A-deficient mutant (ΔsrtA) displayed reduced biofilm formation with both C. albicans and P. aeruginosa FRD1. Taken together, our findings highlight the role of Pas in both microbial-host and interkingdom interactions and expose its potential role in disease outcomes. IMPORTANCE Streptococcus intermedius, an oral commensal bacterium, has been clinically observed in subgingival dental plaque, purulent infections, and cystic fibrosis lungs. In this study, we have (i) determined the crystal structure of the V and C regions of Pas; (ii) shown that its surface protein Pas adheres to fibrinogen, which could potentially ferry the microbe through the bloodstream from the oral cavity; (iii) characterized Pas's high-affinity adherence to lung alveolar protein Gp340 that could fixate the microbe on lung epithelial cells; and (iv) most importantly, shown that these surface proteins on the oral commensal S. intermedius enhance biofilms of known pathogens Candida albicans and Pseudomonas aeruginosa.

Glucan Binding Protein C of Streptococcus mutans Mediates both Sucrose-Independent and Sucrose-Dependent Adherence.

The high-resolution structure of glucan binding protein C (GbpC) at 1.14 Å, a sucrose-dependent virulence factor of the dental caries pathogen Streptococcus mutans, has been determined. GbpC shares not only structural similarities with the V regions of AgI/II and SspB but also functional adherence to salivary agglutinin (SAG) and its scavenger receptor cysteine-rich domains (SRCRs). This is not only a newly identified function for GbpC but also an additional fail-safe binding mechanism for S. mutans Despite the structural similarities with S. mutans antigen I/II (AgI/II) and SspB of Streptococcus gordonii, GbpC remains unique among these surface proteins in its propensity to adhere to dextran/glucans. The complex crystal structure of GbpC with dextrose (ß-d-glucose; Protein Data Bank ligand BGC) highlights exclusive structural features that facilitate this interaction with dextran. Targeted deletion mutant studies on GbpC's divergent loop region in the vicinity of a highly conserved calcium binding site confirm its role in biofilm formation. Finally, we present a model for adherence to dextran. The structure of GbpC highlights how artfully microbes have engineered the lectin-like folds to broaden their functional adherence repertoire.