Deborah G. Conrady

A zinc-dependent adhesion module is responsible for intercellular adhesion in staphylococcal biofilms

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 (Zn2+)-dependent adhesion modules analogous to mammalian cadherin domains. The G5 domain dimerizes in the presence of Zn2+, incorporating 2–3 Zn2+ 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 Zn2+ 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 three-dimensional 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. Zn2+ chelation represents a potential therapeutic approach for combating biofilm growth in a wide range of bacterial biofilm-related infections.

Structural basis for Zn2+-dependent intercellular adhesion in staphylococcal biofilms.

Significance Under adverse environmental conditions, bacteria can form specialized antibiotic-resistant colonies called “biofilms.” In Staphylococcus epidermidis biofilms, a protein, Aap, links bacterial cells together but does so only in the presence of zinc ions. We have determined the atomic structure of an adhesive portion of Aap bound to zinc. The protein adopts an elongated, flexible fold with zinc ions bridging two protein chains. The mode of assembly indicates that Aap is likely to form twisted rope-like structures between bacterial cells. These data provide clues about regions of the protein that could be targeted to prevent intercellular adhesion in the developing biofilm. 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 Zn2+ leads to the formation of extensive adhesive contacts between cells. This study reports the crystal structure of a Zn2+-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. Zn2+-dependent dimers are observed in three distinct crystal forms, formed via pleomorphic coordination of Zn2+ 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.

Shiga toxin binding to glycolipids and glycans.

Background Immunologically distinct forms of Shiga toxin (Stx1 and Stx2) display different potencies and disease outcomes, likely due to differences in host cell binding. The glycolipid globotriaosylceramide (Gb3) has been reported to be the receptor for both toxins. While there is considerable data to suggest that Gb3 can bind Stx1, binding of Stx2 to Gb3 is variable. Methodology We used isothermal titration calorimetry (ITC) and enzyme-linked immunosorbent assay (ELISA) to examine binding of Stx1 and Stx2 to various glycans, glycosphingolipids, and glycosphingolipid mixtures in the presence or absence of membrane components, phosphatidylcholine, and cholesterol. We have also assessed the ability of glycolipids mixtures to neutralize Stx-mediated inhibition of protein synthesis in Vero kidney cells. Results By ITC, Stx1 bound both Pk (the trisaccharide on Gb3) and P (the tetrasaccharide on globotetraosylceramide, Gb4), while Stx2 did not bind to either glycan. Binding to neutral glycolipids individually and in combination was assessed by ELISA. Stx1 bound to glycolipids Gb3 and Gb4, and Gb3 mixed with other neural glycolipids, while Stx2 only bound to Gb3 mixtures. In the presence of phosphatidylcholine and cholesterol, both Stx1 and Stx2 bound well to Gb3 or Gb4 alone or mixed with other neutral glycolipids. Pre-incubation with Gb3 in the presence of phosphatidylcholine and cholesterol neutralized Stx1, but not Stx2 toxicity to Vero cells. Conclusions Stx1 binds primarily to the glycan, but Stx2 binding is influenced by residues in the ceramide portion of Gb3 and the lipid environment. Nanomolar affinities were obtained for both toxins to immobilized glycolipids mixtures, while the effective dose for 50% inhibition (ED50) of protein synthesis was about 10−11 M. The failure of preincubation with Gb3 to protect cells from Stx2 suggests that in addition to glycolipid expression, other cellular components contribute to toxin potency.

FERM Domain Phosphoinositide Binding Targets Merlin to the Membrane and Is Essential for Its Growth-Suppressive Function

ABSTRACT The neurofibromatosis type 2 tumor suppressor protein, merlin, is related to the ERM (ezrin, radixin, and moesin) family of plasma membrane-actin cytoskeleton linkers. For ezrin, phosphatidylinositol 4,5-bisphosphate (PIP2) binding to the amino-terminal FERM domain is required for its conformational activation, proper subcellular localization, and function, but less is known about the role of phosphoinositide binding for merlin. Current evidence indicates that association with the membrane is important for merlin to function as a growth regulator; however, the mechanisms by which merlin localizes to the membrane are less clear. Here, we report that merlin binds phosphoinositides, including PIP2, via a conserved binding motif in its FERM domain. Abolition of FERM domain-mediated phosphoinositide binding of merlin displaces merlin from the membrane and releases it into the cytosol without altering the folding of merlin. Importantly, a merlin protein whose FERM domain cannot bind phosphoinositide is defective in growth suppression. Retargeting the mutant merlin into the membrane using a dual-acylated amino-terminal decapeptide from Fyn is sufficient to restore the growth-suppressive properties to the mutant merlin. Thus, FERM domain-mediated phosphoinositide binding and membrane association are critical for the growth-regulatory function of merlin.

Molecular basis of differential B-pentamer stability of Shiga toxins 1 and 2.

Escherichia coli strain O157:H7 is a major cause of food poisoning that can result in severe diarrhea and, in some cases, renal failure. The pathogenesis of E. coli O157:H7 is in large part due to the production of Shiga toxin (Stx), an AB5 toxin that consists of a ribosomal RNA-cleaving A-subunit surrounded by a pentamer of receptor-binding B subunits. There are two major isoforms, Stx1 and Stx2, which differ dramatically in potency despite having 57% sequence identity. Animal studies and epidemiological studies show Stx2 is associated with more severe disease. Although the molecular basis of this difference is unknown, data suggest it is associated with the B-subunit. Mass spectrometry studies have suggested differential B-pentamer stability between Stx1 and Stx2. We have examined the relative stability of the B-pentamers in solution. Analytical ultracentrifugation using purified B-subunits demonstrates that Stx2B, the more deadly isoform, shows decreased pentamer stability compared to Stx1B (EC50 = 2.3 µM vs. EC50 = 0.043 µM for Stx1B). X-ray crystal structures of Stx1B and Stx2B identified a glutamine in Stx2 (versus leucine in Stx1) within the otherwise strongly hydrophobic interface between B-subunits. Interchanging these residues switches the stability phenotype of the B-pentamers of Stx1 and Stx2, as demonstrated by analytical ultracentrifugation and circular dichroism. These studies demonstrate a profound difference in stability of the B-pentamers in Stx1 and Stx2, illustrate the mechanistic basis for this differential stability, and provide novel reagents to test the basis for differential pathogenicity of these toxins.

A human cancer-predisposing polymorphism in Cdc25A is embryonic lethal in the mouse and promotes ASK-1 mediated apoptosis.

BackgroundFailure to regulate the levels of Cdc25A phosphatase during the cell cycle or during a checkpoint response causes bypass of DNA damage and replication checkpoints resulting in genomic instability and cancer. During G1 and S and in cellular response to DNA damage, Cdc25A is targeted for degradation through the Skp1-cullin-β-TrCP (SCFβ-TrCP) complex. This complex binds to the Cdc25A DSG motif which contains serine residues at positions 82 and 88. Phosphorylation of one or both residues is necessary for the binding and degradation to occur.ResultsWe now show that mutation of serine 88 to phenylalanine, which is a cancer-predisposing polymorphic variant in humans, leads to early embryonic lethality in mice. The mutant protein retains its phosphatase activity both in vitro and in cultured cells. It fails to interact with the apoptosis signal-regulating kinase 1 (ASK1), however, and therefore does not suppress ASK1-mediated apoptosis.ConclusionsThese data suggest that the DSG motif, in addition to its function in Cdc25A-mediated degradation, plays a role in cell survival during early embyogenesis through suppression of ASK1-mediated apoptosis.

Thermodynamic Analysis of Metal Ion-Induced Protein Assembly

A large number of biological systems are regulated by metal ion-induced protein assembly. This phenomenon can play a critical role in governing protein function and triggering downstream biological responses. We discuss the basic thermodynamic principles of linked equilibria that pertain to metal ion-induced dimerization and describe experimental approaches useful for studying such systems. The most informative techniques for studying these systems are sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation, although a wide range of other spectroscopic, chromatographic, or qualitative approaches can provide a wealth of useful information. These experimental procedures are illustrated with examples from two systems currently under study: zinc-induced assembly of a staphylococcal protein responsible for intercellular adhesion in bacterial biofilms and calcium-induced dimerization of a human nucleotidase.