Origins of glycan selectivity in streptococcal Siglec-like adhesins suggest mechanisms of receptor adaptation.

Bacterial binding to host receptors underlies both commensalism and pathogenesis. Many streptococci adhere to protein-attached carbohydrates expressed on cell surfaces using Siglec-like binding regions (SLBRs). The precise glycan repertoire recognized may dictate whether the organism is a strict commensal versus a pathogen. However, it is currently not clear what drives receptor selectivity. Here, we use five representative SLBRs and identify regions of the receptor binding site that are hypervariable in sequence and structure. We show that these regions control the identity of the preferred carbohydrate ligand using chimeragenesis and single amino acid substitutions. We further evaluate how the identity of the preferred ligand affects the interaction with glycoprotein receptors in human saliva and plasma samples. As point mutations can change the preferred human receptor, these studies suggest how streptococci may adapt to changes in the environmental glycan repertoire.

Tandem sialoglycan-binding modules in a Streptococcus sanguinis serine-rich repeat adhesin create target dependent avidity effects.

Sialic acid-binding immunoglobulin-like lectins (Siglec)-like domains of streptococcal serine-rich repeat (SRR) adhesins recognize sialylated glycans on human salivary, platelet, and plasma glycoproteins via a YTRY sequence motif. The SRR adhesin from Streptococcus sanguinis strain SK1 has tandem sialoglycan-binding domains and has previously been shown to bind sialoglycans with high affinity. However, both domains contain substitutions within the canonical YTRY motif, making it unclear how they interact with host receptors. To identify how the S. sanguinis strain SK1 SRR adhesin affects interactions with sialylated glycans and glycoproteins, we determined high-resolution crystal structures of the binding domains alone and with purified trisaccharides. These structural studies determined that the ligands still bind at the noncanonical binding motif, but with fewer hydrogen-bonding interactions to the protein than is observed in structures of other Siglec-like adhesins. Complementary biochemical studies identified that each of the two binding domains has a different selectivity profile. Interestingly, the binding of SK1 to platelets and plasma glycoproteins identified that the interaction to some host targets is dominated by the contribution of one binding domain, whereas the binding to other host receptors is mediated by both binding domains. These results provide insight into outstanding questions concerning the roles of tandem domains in targeting host receptors and suggest mechanisms for how pathogens can adapt to the availability of a range of related but nonidentical host receptors. They further suggest that the definition of the YTRY motif should be changed to ϕTRX, a more rigorous description of this sialic acid-recognition motif given recent findings.

An alternative N-terminal fold of the intestine-specific annexin A13a induces dimerization and regulates membrane-binding.

Annexin proteins function as Ca2+-dependent regulators of membrane trafficking and repair that may also modulate membrane curvature. Here, using high-resolution confocal imaging, we report that the intestine-specific annexin A13 (ANX A13) localizes to the tips of intestinal microvilli and determined the crystal structure of the ANX A13a isoform to 2.6 Å resolution. The structure revealed that the N terminus exhibits an alternative fold that converts the first two helices and the associated helix-loop-helix motif into a continuous α-helix, as stabilized by a domain-swapped dimer. We also found that the dimer is present in solution and partially occludes the membrane-binding surfaces of annexin, suggesting that dimerization may function as a means for regulating membrane binding. Accordingly, as revealed by in vitro binding and cellular localization assays, ANX A13a variants that favor a monomeric state exhibited increased membrane association relative to variants that favor the dimeric form. Together, our findings support a mechanism for how the association of the ANX A13a isoform with the membrane is regulated.

The Structure of the Bifunctional Everninomicin Biosynthetic Enzyme EvdMO1 Suggests Independent Activity of the Fused Methyltransferase-Oxidase Domains.

Members of the orthosomycin family of natural products are decorated polysaccharides with potent antibiotic activity and complex biosynthetic pathways. The defining feature of the orthosomycins is an orthoester linkage between carbohydrate moieties that is necessary for antibiotic activity and is likely formed by a family of conserved oxygenases. Everninomicins are octasaccharide orthosomycins produced by Micromonospora carbonacea that have two orthoester linkages and a methylenedioxy bridge, three features whose formation logically requires oxidative chemistry. Correspondingly, the evd gene cluster encoding everninomicin D encodes two monofunctional nonheme iron, α-ketoglutarate-dependent oxygenases and one bifunctional enzyme with an N-terminal methyltransferase domain and a C-terminal oxygenase domain. To investigate whether the activities of these domains are linked in the bifunctional enzyme EvdMO1, we determined the structure of the N-terminal methyltransferase domain to 1.1 Å and that of the full-length protein to 3.35 Å resolution. Both domains of EvdMO1 adopt the canonical folds of their respective superfamilies and are connected by a short linker. Each domain's active site is oriented such that it faces away from the other domain, and there is no evidence of a channel connecting the two. Our results support EvdMO1 working as a bifunctional enzyme with independent catalytic activities.

A quantitative method for analyzing establishing-efficiency of persistent viral infection.

A quantitative method for analyzing establishing-efficiency of persistent infection was devised. The efficiency of hPIV2 CA and SV5 T1 strains was found to be high, that is, 0.1 approximately 0.3 (an efficiency of 1.0 indicates that 100% of the virus-infected cells became persistently infected). The efficiency of the SV5 WR strain was also high, approximately 0.1, though the virus had no ability to immediately establish a steady state of persistent infection in whole cell-culture systems. At about 0.0007, the efficiency of SV41 was almost the same as that of the hPIV2 Toshiba strain. The establishing efficiencies of various rSeV were further analyzed in detail. The efficiencies of the rSeV(PA), rSeV(Ppi) and rSeV(HNpi) were below the limit of detection, while that of rSeV(Lpi) was nearly 1. Although the efficiency was around 0.001, the rSeV(Mpi) and the rSeV(Fpi) were unexpectedly found to be capable of forming persistently-infected cells, indicating that both the Fpi and Mpi proteins contribute to the establishing efficiency of persistent infection of SeVpi.

Failure of multinucleated giant cell formation in k562 cells infected with newcastle disease virus and human parainfluenza type 2 virus.

When K562 cells were infected with Newcastle disease virus (NDV) or human parainfluenza type 2 virus (hPIV-2), polykaryocyte formation could not be detected. Failure of multinucleated giant cell formation in K562 cells infected with either NDV or hPIV-2 is due to disturbance of the viral envelope-cell fusion step or to defect in the cell-cell fusion step, respectively. Especially, NDV completely replicated in K562 cells, and the hemagglutinin-neuraminidase and fusion proteins expressed on the cell surface of NDV-infected K562 cell were fully functional for fusion inducing activity. Therefore, the cell membranes of K562 cells are considered to be resistant to virus-induced cell fusion. Membrane fusion is regulated by many host factors including membrane fluidity, cytoskeletal systems, and fusion regulatory proteins system. An unknown regulatory mechanism of virus-induced cell fusion may function on the cell surface of K562 cells.