In this work, the suitability of imidazolium-based ionic liquid solvents is investigated for the dissolution and regeneration of silkworm (Bombyx mori) silk. Within an ionic liquid the anion plays a larger role in dictating the ultimate solubility of the silk. The dissolution of the silk in the ionic liquid is confirmed using wide-angle X-ray scattering. The dissolved silk is also processed into 100 mum-thick, two-dimensional films, and the structure of these films is examined. The rinse solvent, acetonitrile or methanol, has a profound impact on both the topography of the films and the secondary structure of the silk protein. The image depicts a silkworm cocoon dissolved in 1-butyl-3-methylimidazolium chloride and then regenerated as a film with birefringence.

Hospital-acquired bacterial infections are an increasingly important cause of morbidity and mortality worldwide. Staphylococcal species are responsible for the majority of hospital-acquired infections, which are often complicated by the ability of staphylococci to grow as biofilms. Biofilm formation by Staphylococcus epidermidis and Staphylococcus aureus requires cell-surface proteins (Aap and SasG) containing sequence repeats known as G5 domains; however, the precise role of these proteins in biofilm formation is unclear. We show here, using analytical ultracentrifugation (AUC) and circular dichroism (CD), that G5 domains from Aap are zinc (Zn 2؉ )-dependent adhesion modules analogous to mammalian cadherin domains. The G5 domain dimerizes in the presence of Zn 2؉ , incorporating 2-3 Zn 2؉ ions in the dimer interface. Tandem G5 domains associate in a modular fashion, suggesting a ''zinc zipper'' mechanism for G5 domain-based intercellular adhesion in staphylococcal biofilms. We demonstrate, using a biofilm plate assay, that Zn 2؉ chelation specifically prevents biofilm formation by S. epidermidis and methicillin-resistant S. aureus (MRSA). Furthermore, individual soluble G5 domains inhibit biofilm formation in a dose-dependent manner. Thus, the complex threedimensional architecture of staphylococcal biofilms results from the self-association of a single type of protein domain. Surface proteins with tandem G5 domains are also found in other bacterial species, suggesting that this mechanism for intercellular adhesion in biofilms may be conserved among staphylococci and other Gram-positive bacteria. Zn 2؉ chelation represents a potential therapeutic approach for combating biofilm growth in a wide range of bacterial biofilm-related infections.bacterial pathogenesis ͉ G5 domain ͉ Aap ͉ chelation ͉ Staphylococcus

Staphylococcal bacteria, including Staphylococcus epidermidis and Staphylococcus aureus, cause chronic biofilm-related infections. The homologous proteins Aap and SasG mediate biofilm formation in S. epidermidis and S. aureus, respectively. The self-association of these proteins in the presence of Zn 2+ leads to the formation of extensive adhesive contacts between cells. This study reports the crystal structure of a Zn 2+ -bound construct from the self-associating region of Aap. Several unusual structural features include elongated β-sheets that are solvent-exposed on both faces and the lack of a canonical hydrophobic core. Zn 2+ -dependent dimers are observed in three distinct crystal forms, formed via pleomorphic coordination of Zn 2+ in trans across the dimer interface. These structures illustrate how a long, flexible surface protein is able to form tight intercellular adhesion sites under adverse environmental conditions.H ealthcare-associated infections affect an estimated 1.5-2 million patients annually in the United States, with ∼99,000 annual fatalities (1). Staphylococci represent the most commonly isolated genus in healthcare-associated infections (2) and are the most common cause of infections on implanted devices (3). The key pathogenic mechanism in these infections is formation of a biofilm. A biofilm is a specialized bacterial colony with higherorder organization analogous to that of a tissue in multicellular organisms, in which there is concerted regulation of metabolic activity and gene expression (3). The entire colony is encased in an extensive extracellular matrix that can comprise polysaccharide, protein, nucleic acids, or combinations thereof (4). The extracellular matrix is important for mediating adhesion among neighboring bacteria as well as to diverse surfaces (3). Bacteria within a biofilm are resistant to antibiotics (5) and to host immune defenses (6), reducing the efficacy of available antimicrobials. Understanding the mechanisms of biofilm formation will allow us to combat the significant pathogenic advantages of biofilm-based infectious diseases.There are species-and strain-specific differences that affect staphylococcal biofilm formation (7), but certain key similarities have been identified. An important group of adhesive proteins includes the Staphylococcus epidermidis protein Aap and its Staphylococcus aureus homologs SasG and Pls. These are multidomain, multifunctional proteins with significant roles in biofilm formation. Exogenous expression of Aap or SasG in non-biofilmforming cocci is sufficient to mediate adhesion to host cells (8,9) and to initiate biofilm formation (10, 11). Aap knockout ablates biofilm formation (12).Aap, SasG, and Pls all have similar domain arrangements. In Aap, the N-terminal portion of the protein is comprised of an A-repeat region, with short (∼16-residue), imperfect sequence repeats, followed by a putative globular (α/β) domain with predicted α-helical and β-sheet content (Fig. 1A). This region of Aap mediates attachment to host cells via interact...

Graphical Abstract Highlights d The crystal structure of EspB reveals a fused PE/PPE homology domain d EspB has a stabilized bipartite secretion signal that targets the EccCb1 ATPase d EspB oligomerizes to form a ring-shaped heptamer d A model of the heptamer was fit to EM density and crosslinking data In Brief Mycobacterium tuberculosis exports virulence factors to its surface using the ESX-1 secretion system, progressing the infection of human macrophages. Solomonson et al. show that one of these factors, EspB, adopts a PE/PPE-like fold and oligomerizes to form a barrel-shaped structure with heptameric symmetry. Accession Numbers 4WJ1 4WJ2 3J83 Solomonson et al., 2015, Structure 23, 1-13 March 3, SUMMARYMycobacterium tuberculosis (Mtb) uses the ESX-1 type VII secretion system to export virulence proteins across its lipid-rich cell wall, which helps permeabilize the host's macrophage phagosomal membrane, facilitating the escape and cell-to-cell spread of Mtb. ESX-1 membranolytic activity depends on a set of specialized secreted Esp proteins, the structure and specific roles of which are not currently understood. Here, we report the X-ray and electron microscopic structures of the ESX-1-secreted EspB. We demonstrate that EspB adopts a PE/PPE-like fold that mediates oligomerization with apparent heptameric symmetry, generating a barrel-shaped structure with a central pore that we propose contributes to the macrophage killing functions of EspB. Our structural data also reveal unexpected direct interactions between the EspB bipartite secretion signal sequence elements that form a unified aromatic surface. These findings provide insight into how specialized proteins encoded within the ESX-1 locus are targeted for secretion, and for the first time indicate an oligomerization-dependent role for Esp virulence factors.

Drug combinations are valuable tools for studying biological systems. Although much attention has been given to synergistic interactions in revealing connections between cellular processes, antagonistic interactions can also have tremendous value in elucidating genetic networks and mechanisms of drug action. Here, we exploit the power of antagonism in a high-throughput screen for molecules that suppress the activity of targocil, an inhibitor of the wall teichoic acid (WTA) flippase in Staphylococcus aureus. Well-characterized antagonism within the WTA biosynthetic pathway indicated that early steps would be sensitive to this screen; however, broader interactions with cell wall biogenesis components suggested that it might capture additional targets. A chemical screening effort using this approach identified clomiphene, a widely used fertility drug, as one such compound. Mechanistic characterization revealed the target was the undecaprenyl diphosphate synthase, an enzyme that catalyzes the synthesis of a polyisoprenoid essential for both peptidoglycan and WTA synthesis. The work sheds light on mechanisms contributing to the observed suppressive interactions of clomiphene and in turn reveals aspects of the biology that underlie cell wall synthesis in S. aureus. Further, this effort highlights the utility of antagonistic interactions both in high-throughput screening and in compound mode of action studies. Importantly, clomiphene represents a lead for antibacterial drug discovery.