Structure of the host-recognition device of Staphylococcus aureus phage ϕ11.

Phages play key roles in the pathogenicity and adaptation of the human pathogen Staphylococcus aureus. However, little is known about the molecular recognition events that mediate phage adsorption to the surface of S. aureus. The lysogenic siphophage ϕ11 infects S. aureus SA113. It was shown previously that ϕ11 requires α- or ß-N-acetylglucosamine (GlcNAc) moieties on cell wall teichoic acid (WTA) for adsorption. Gp45 was identified as the receptor binding protein (RBP) involved in this process and GlcNAc residues on WTA were found to be the key component of the ϕ11 receptor. Here we report the crystal structure of the RBP of ϕ11, which assembles into a large, multidomain homotrimer. Each monomer contains a five-bladed propeller domain with a cavity that could accommodate a GlcNAc moiety. An electron microscopy reconstruction of the ϕ11 host adhesion component, the baseplate, reveals that six RBP trimers are assembled around the baseplate core. The Gp45 and baseplate structures provide insights into the overall organization and molecular recognition process of the phage ϕ11 tail. This assembly is conserved among most glycan-recognizing Siphoviridae, and the RBP orientation would allow host adhesion and infection without an activation step.

An essential role for the baseplate protein Gp45 in phage adsorption to Staphylococcus aureus.

Despite the importance of phages in driving horizontal gene transfer (HGT) among pathogenic bacteria, the underlying molecular mechanisms mediating phage adsorption to S. aureus are still unclear. Phage ϕ11 is a siphovirus with a high transducing efficiency. Here, we show that the tail protein Gp45 localized within the ϕ11 baseplate. Phage ϕ11 was efficiently neutralized by anti-Gp45 serum, and its adsorption to host cells was inhibited by recombinant Gp45 in a dose-dependent manner. Flow cytometry analysis demonstrated that biotin-labelled Gp45 efficiently stained the wild-type S. aureus cell but not the double knockout mutant ΔtarM/S, which lacks both α- and ß-O-GlcNAc residues on its wall teichoic acids (WTAs). Additionally, adsorption assays indicate that GlcNAc residues on WTAs and O-acetyl groups at the 6-position of muramic acid residues in peptidoglycan are essential components of the ϕ11 receptor. The elucidation of Gp45-involved molecular interactions not only broadens our understanding of siphovirus-mediated HGT, but also lays the groundwork for the development of sensitive affinity-based diagnostics and therapeutics for S. aureus infection.

Genetic engineering of untransformable coagulase-negative staphylococcal pathogens.

Coagulase-negative staphylococci (CoNS) are recognized as significant opportunistic pathogens. However, current knowledge of virulence mechanisms is very limited because a significant proportion of CoNS are refractory to available techniques for DNA transformation. We describe an efficient protocol for plasmid transfer using bacteriophage Φ187, which can transduce plasmid DNA to a wide range of CoNS from a unique, engineered Staphylococcus aureus strain. The use of a restriction-deficient, modification-proficient S. aureus PS187 mutant, which has a CoNS-type bacteriophage surface receptor, allows plasmid transfer to CoNS even when they are refractory to electroporation. Once the Φ187 titer reaches 10(9) plaque-forming units per milliliter, plasmid transfer can be accomplished within 1-2 d. Thus, our protocol is a major technical advance offering attractive opportunities for research on CoNS-mediated infections.

An accessory wall teichoic acid glycosyltransferase protects Staphylococcus aureus from the lytic activity of Podoviridae.

Many Staphylococcus aureus have lost a major genetic barrier against phage infection, termed clustered regularly interspaced palindromic repeats (CRISPR/cas). Hence, S. aureus strains frequently exchange genetic material via phage-mediated horizontal gene transfer events, but, in turn, are vulnerable in particular to lytic phages. Here, a novel strategy of S. aureus is described, which protects S. aureus against the lytic activity of Podoviridae, a unique family of staphylococcal lytic phages with short, non-contractile tails. Unlike most staphylococcal phages, Podoviridae require a precise wall teichoic acid (WTA) glycosylation pattern for infection. Notably, TarM-mediated WTA α-O-GlcNAcylation prevents infection of Podoviridae while TarS-mediated WTA ß-O-GlcNAcylation is required for S. aureus susceptibility to podoviruses. Tracking the evolution of TarM revealed an ancient origin in other staphylococci and vertical inheritance during S. aureus evolution. However, certain phylogenetic branches have lost tarM during evolution, which rendered them podovirus-susceptible. Accordingly, lack of tarM correlates with podovirus susceptibility and can be converted into a podovirus-resistant phenotype upon ectopic expression of tarM indicating that a "glyco-switch" of WTA O-GlcNAcylation can prevent the infection by certain staphylococcal phages. Since lytic staphylococcal phages are considered as anti-S. aureus agents, these data may help to establish valuable strategies for treatment of infections.

Wall Teichoic Acid Glycosylation Governs Staphylococcus aureus Nasal Colonization.

UNLABELLED: Nasal colonization by the human pathogen Staphylococcus aureus is a major risk factor for hospital- and community-acquired infections. A key factor required for nasal colonization is a cell surface-exposed zwitterionic glycopolymer, termed wall teichoic acid (WTA). However, the precise mechanisms that govern WTA-mediated nasal colonization have remained elusive. Here, we report that WTA GlcNAcylation is a pivotal requirement for WTA-dependent attachment of community-acquired methicillin-resistant S. aureus (MRSA) and emerging livestock-associated MRSA to human nasal epithelial cells, even under conditions simulating the nutrient composition and dynamic flow of nasal secretions. Depending on the S. aureus strain, WTA O-GlcNAcylation occurs in either α or ß configuration, which have similar capacities to mediate attachment to human nasal epithelial cells, suggesting that many S. aureus strains maintain redundant pathways to ensure appropriate WTA glycosylation. Strikingly, a lack of WTA glycosylation significantly abrogated the ability of MRSA to colonize cotton rat nares in vivo. These results indicate that WTA glycosylation modulates S. aureus nasal colonization and may help to develop new strategies for eradicating S. aureus nasal colonization in the future. IMPORTANCE: Nasal colonization by the major human pathogen Staphylococcus aureus is a risk factor for severe endogenous infections and contributes to the spread of this microbe in hospitals and the community. Here, we show that wall teichoic acid (WTA) O-GlcNAcylation is a key factor required for S. aureus nasal colonization. These data provide a mechanistic explanation for the capacity of WTA to modulate S. aureus nasal colonization and may stimulate research activities to establish valuable strategies to eradicate S. aureus nasal colonization in high-risk hospitalized patients and in the general community.

Transfer of plasmid DNA to clinical coagulase-negative staphylococcal pathogens by using a unique bacteriophage.

Genetic manipulation of emerging bacterial pathogens, such as coagulase-negative staphylococci (CoNS), is a major hurdle in clinical and basic microbiological research. Strong genetic barriers, such as restriction modification systems or clustered regularly interspaced short palindromic repeats (CRISPR), usually interfere with available techniques for DNA transformation and therefore complicate manipulation of CoNS or render it impossible. Thus, current knowledge of pathogenicity and virulence determinants of CoNS is very limited. Here, a rapid, efficient, and highly reliable technique is presented to transfer plasmid DNA essential for genetic engineering to important CoNS pathogens from a unique Staphylococcus aureus strain via a specific S. aureus bacteriophage, Φ187. Even strains refractory to electroporation can be transduced by this technique once donor and recipient strains share similar Φ187 receptor properties. As a proof of principle, this technique was used to delete the alternative transcription factor sigma B (SigB) via allelic replacement in nasal and clinical Staphylococcus epidermidis isolates at high efficiencies. The described approach will allow the genetic manipulation of a wide range of CoNS pathogens and might inspire research activities to manipulate other important pathogens in a similar fashion.

Role of the amino-terminal transmembrane domain of sulfonylurea receptor SUR2B for coupling to K(IR)6.2, ligand binding, and oligomerization.

ATP-sensitive K(+) (K(ATP)) channels consist of two types of subunits, K(IR)6.x that form the pore, and sulfonylurea receptors (SURs) that serve as regulatory subunits. SURs are ATP-binding cassette (ABC) proteins and contain, in addition to two nucleotide binding folds, the binding sites for channel openers such as diazoxide and P1075 and channel inhibitors such as glibenclamide (GBC) and repaglinide. Structurally, SURs differ from most eukaryotic ABC proteins by an additional amino-terminal transmembrane domain (TMD0); in case of SUR1, the subunit of the pancreatic K(ATP) channel, TMD0 serves as a major domain for association with K(IR). In this study we sought to elucidate the roles of TMD0 in SUR2B, the smooth muscle gating subunit, in the coupling between SUR2B and K(IR)6.2, in the self-association of SUR2B and in channel modulator binding to SUR2B. SUR2B has a weaker affinity for sulfonylureas thus SUR2B(Y1206S), with a higher affinity for GBC, but an equivalent opener binding was used. Association of SUR2B(YS)Δ, lacking TMD0, with K(IR)6.2 was shown by immunoprecipitation; however, no evidence for formation of functional channels was obtained. SUR2B(YS)Δ self-associates like SUR2B(YS) and binds GBC, repaglinide, and P1075 with slightly reduced affinities. The binding profile of the SUR2B(YS)Δ/K(IR)6.2 complex differs slightly but significantly from that of SUR2B(YS)Δ alone showing impaired allosteric coupling of binding sites. We conclude that TMD0 is not required for oligomerization of SUR2B, is of only minor importance in ligand binding, but is essential for both functional and allosteric coupling of SUR2B to K(IR)6.2.

Importance of the Kir6.2 N-terminus for the interaction of glibenclamide and repaglinide with the pancreatic K(ATP) channel.

The pancreatic K(ATP) channel, SUR1/Kir6.2, couples insulin secretion to the plasma glucose level. The channel is an octamer with four Kir6.2 subunits forming the pore and four sulphonylurea receptors (SUR1) regulating channel activity. SUR1 is an ABC protein with adenosine triphosphate (ATP)ase activity which activates the channel. It also contains the binding site for antidiabetic drugs like glibenclamide and repaglinide which close the channel by disrupting the stimulatory effect SUR-ATPase (MgATP-dependent) and by stabilising a long-lived closed channel state (MgATP-independent). In this study, we examined the effects of progressive truncation of the Kir6.2 N-terminus up to 20 amino acids on equilibrium binding and channel closure by glibenclamide and repaglinide, on the channel activating effect of the opener, 6-chloro-3-(1-methylcyclobutyl)amino-4H-thieno[3,2-e]-1,2,4thiadiazine 1,1-dioxide (NNC 55-0462), and on the binding kinetics of [(3)H]glibenclamide. Kir and SUR were transiently coexpressed in HEK cells and [(3)H]glibenclamide binding and patch-clamp experiments were performed in whole cells at 37°C and in isolated inside/out patches at 22°C. Truncation of the first 5 N-terminal amino acids abolished most of the affinity increase for glibenclamide and repaglinide that is produced by the association of Kir6.2 with SUR1. Progressive truncation continuously reduced the potency and efficacy of these drugs in closing the channel and impaired the ability to stabilise the closed state more than the ability to disrupt channel stimulation by SUR-ATPase. The effects of NNC 55-0462 were unchanged. Progressive truncation also speeded up dissociation of [(3)H]glibenclamide from the channel when dissociation was induced by an excess of (unlabelled) glibenclamide. This suggests the existence of a putative low affinity glibenclamide site on the channel whose affinity increases upon truncation. The data show that progressive truncation of the Kir6.2 N-terminus impairs the transduction of drug binding into channel closure more strongly than drug binding but leaves the effect of the opener NNC 55-0462 unchanged.

Substitution of the Walker A lysine by arginine in the nucleotide-binding domains of sulphonylurea receptor SUR2B: effects on ligand binding and channel activity.

Sulphonylurea receptors (SURs) serve as regulatory subunits of ATP-sensitive K(+) channels. SURs are members of the ATP-binding cassette (ABC) protein superfamily and contain two conserved nucleotide-binding domains (NBDs) which bind and hydrolyse MgATP; in addition, they carry the binding sites for the sulphonylureas like glibenclamide (GBC) which close the channel and for the K(ATP) channel openers such as P1075. Here we have exchanged the conserved Lys in the Walker A motif by Arg in both NBDs of SUR2B, the regulatory subunit of the vascular K(ATP) channel. Then the effect of the mutation on the ATPase-dependent binding of GBC and P1075 to SUR2B and on the activity of the recombinant vascular (Kir6.1/SUR2B) channel was assessed. Surprisingly, in the absence of MgATP, the mutation weakened binding of P1075 and the extent of allosteric inhibition of GBC binding by P1075. The mutation abolished most, but not all, of the MgATP effects on the binding of GBC and P1075 and prevented nucleotide-induced activation of the channel which relies on SUR reaching the posthydrolytic (MgADP-bound) state; the mutant channel was, however, opened by P1075 at higher concentrations. The data provide evidence that mutant SUR2B binds MgATP but that the posthydrolytic state is insufficiently populated. This suggests that the mutation locks SUR2B in an MgATP-binding prehydrolytic-like state; binding of P1075 may induce a posthydrolytic-like conformation to open the channel.

Testing the bipartite model of the sulfonylurea receptor binding site: binding of A-, B-, and A + B-site ligands.

ATP-sensitive K(+) (K(ATP)) channels are composed of pore-forming subunits (Kir6.x) and of regulatory subunits, the sulfonylurea receptors (SURx). Subtypes of K(ATP) channels are expressed in different organs. The sulfonylureas and glinides (insulinotropes) close the K(ATP) channel in pancreatic beta-cells and stimulate insulin secretion. The insulinotrope binding site of the pancreatic channel (Kir6.2/SUR1) consists of two overlapping (sub)-sites, site A, located on SUR1 and containing Ser1237 (which in SUR2 is replaced by Tyr1206), and site B, formed by SUR1 and Kir6.2. Insulinotropes bind to the A-, B-, or A + B-site(s) and are grouped accordingly. A-ligands are highly selective in closing the pancreatic channel, whereas B-ligands are nonselective and insensitive to the mutation S1237Y. We have examined the binding of insulinotropes representative of the three groups in [(3)H]glibenclamide competition experiments to determine the contribution of Kir6.x to binding affinity, the effect of the mutation Y1206S in site A of SUR2, and the subtype selectivity of the compounds. The results show that the bipartite nature of the SUR1 binding site applies also to SUR2. Kir6.2 as part of the B-site may interact directly or allosterically with structural elements common to all insulinotropes, i.e., the negative charge and/or the adjacent phenyl ring. The B-site confers a moderate subtype selectivity on B-ligands. The affinity of B-ligands is altered by the mutation SUR2(Y1206S), suggesting that the mutation affects the binding chamber of SUR2 as a whole or subsite A, including the region where the subsites overlap.