Cooperative Gsx2-DNA binding requires DNA bending and a novel Gsx2 homeodomain interface.

The conserved Gsx homeodomain (HD) transcription factors specify neural cell fates in animals from flies to mammals. Like many HD proteins, Gsx factors bind A/T-rich DNA sequences prompting the following question: How do HD factors that bind similar DNA sequences in vitro regulate specific target genes in vivo? Prior studies revealed that Gsx factors bind DNA both as a monomer on individual A/T-rich sites and as a cooperative homodimer to two sites spaced precisely 7 bp apart. However, the mechanistic basis for Gsx-DNA binding and cooperativity is poorly understood. Here, we used biochemical, biophysical, structural and modeling approaches to (i) show that Gsx factors are monomers in solution and require DNA for cooperative complex formation, (ii) define the affinity and thermodynamic binding parameters of Gsx2/DNA interactions, (iii) solve a high-resolution monomer/DNA structure that reveals that Gsx2 induces a 20° bend in DNA, (iv) identify a Gsx2 protein-protein interface required for cooperative DNA binding and (v) determine that flexible spacer DNA sequences enhance Gsx2 cooperativity on dimer sites. Altogether, our results provide a mechanistic basis for understanding the protein and DNA structural determinants that underlie cooperative DNA binding by Gsx factors.

BASIS: BioAnalysis SEDFIT integrated software for cGMP analysis of SV-AUC data.

Sedimentation velocity analytical ultracentrifugation (SV-AUC) has long been an important method for characterization of antibody therapeutics. Recently, SV-AUC has experienced a wave of new interest and usage from the gene and cell therapy industry, where SV-AUC has proven itself to be the "gold standard" analytical approach for determining capsid loading ratios for adeno-associated virus (AAV) and other viral vectors. While other more common approaches have existed in the realm of cGMP-compliant techniques for years, SV-AUC has long been used strictly for characterization, but not for release testing. This manuscript describes the challenges faced in bringing SV-AUC to a cGMP environment and describes a new program, "BASIS", which allows for 21 CFR Part 11-compliant data handling and data analysis using the well-known and frequently cited SEDFIT analysis software.

Cooperative Gsx2-DNA Binding Requires DNA Bending and a Novel Gsx2 Homeodomain Interface.

The conserved Gsx homeodomain (HD) transcription factors specify neural cell fates in animals from flies to mammals. Like many HD proteins, Gsx factors bind A/T-rich DNA sequences prompting the question - how do HD factors that bind similar DNA sequences in vitro regulate specific target genes in vivo? Prior studies revealed that Gsx factors bind DNA both as a monomer on individual A/T-rich sites and as a cooperative homodimer to two sites spaced precisely seven base pairs apart. However, the mechanistic basis for Gsx DNA binding and cooperativity are poorly understood. Here, we used biochemical, biophysical, structural, and modeling approaches to (1) show that Gsx factors are monomers in solution and require DNA for cooperative complex formation; (2) define the affinity and thermodynamic binding parameters of Gsx2/DNA interactions; (3) solve a high-resolution monomer/DNA structure that reveals Gsx2 induces a 20° bend in DNA; (4) identify a Gsx2 protein-protein interface required for cooperative DNA binding; and (5) determine that flexible spacer DNA sequences enhance Gsx2 cooperativity on dimer sites. Altogether, our results provide a mechanistic basis for understanding the protein and DNA structural determinants that underlie cooperative DNA binding by Gsx factors, thereby providing a deeper understanding of HD specificity.

Convergent behavior of extended stalk regions from staphylococcal surface proteins with widely divergent sequence patterns.

Staphylococcus epidermidis and Staphylococcus aureus are highly problematic bacteria in hospital settings. A major challenge is their ability to form biofilms on abiotic or biotic surfaces. Biofilms are well-organized, multicellular bacterial aggregates that resist antibiotic treatment and often lead to recurrent infections. Bacterial cell wall-anchored (CWA) proteins are important players in biofilm formation and infection. Many have putative stalk-like regions or regions of low complexity near the cell wall-anchoring motif. Recent work demonstrated the strong propensity of the stalk region of S. epidermidis accumulation-associated protein (Aap) to remain highly extended under solution conditions that typically induce compaction. This behavior is consistent with the expected function of a stalk-like region that is covalently attached to the cell wall peptidoglycan and projects the adhesive domains of Aap away from the cell surface. In this study, we evaluate whether the ability to resist compaction is a common theme among stalk regions from various staphylococcal CWA proteins. Circular dichroism spectroscopy was used to examine secondary structure changes as a function of temperature and cosolvents along with sedimentation velocity analytical ultracentrifugation, size-exclusion chromatography, and SAXS to characterize structural characteristics in solution. All stalk regions tested are intrinsically disordered, lacking secondary structure beyond random coil and polyproline type II helix, and they all sample highly extended conformations. Remarkably, the Ser-Asp dipeptide repeat region of SdrC exhibited nearly identical behavior in solution when compared to the Aap Pro/Gly-rich region, despite highly divergent sequence patterns, indicating conservation of function by various distinct staphylococcal CWA protein stalk regions.

AAV analysis by sedimentation velocity analytical ultracentrifugation: beyond empty and full capsids.

The recent surge of therapeutic interest in recombinant adeno-associated viral (AAV) vectors for targeted DNA delivery has brought analytical ultracentrifugation (AUC) into the spotlight. A major concern during formulation of AAV therapeutics is purity of the active species (DNA-containing capsid, or "filled capsids"). Insertion of DNA into AAV is not a highly efficient process; thus, a significant amount of empty and partial/intermediate AAV molecules may exist. Recent guidance from the FDA includes limiting the presence of empty AAV capsids and other impurities to reduce immunotoxicity. While chromatographic techniques (SEC, SEC-MALS, AEX) are often used for empty and full capsid quantitation due to the ease of accessibility and familiarity among most biochemists, the resolution and sensitivity attained by sedimentation velocity (SV-AUC) in the formulation buffer and purification buffers is unmatched. Approaches for using SV-AUC to determine the empty-to-full capsid ratio have already been discussed by others; however, in this report, we focus on the importance of characterizing other impurities, such as free DNA, partially filled capsids, and aggregates that are recognized as species of concern for immunotoxicity. We also demonstrate the usefulness of applying multiple analyses (e.g., c(s), g(s*), WDA) in confirming the presence of and determining the hydrodynamic parameters of these various species.

Strong non-ideality effects at low protein concentrations: considerations for elongated proteins.

A recent investigation was aimed at obtaining structural information on a highly extended protein via SEC-MALS-SAXS. Significantly broadened elution peaks were observed, reminiscent of a phenomenon known as viscous fingering. This phenomenon is usually observed above 50 mg/mL for proteins like bovine serum albumin (BSA). Interestingly, the highly extended protein (Brpt5.5) showed viscous fingering at concentrations lower than 5 mg/mL. The current study explores this and other non-ideal behavior, emphasizing the presence of these effects at relatively low concentrations for extended proteins. BSA, Brpt5.5, and a truncated form of Brpt5.5 referred to as Brpt1.5 are studied systematically using size-exclusion chromatography (SEC), sedimentation velocity analytical ultracentrifugation (AUC), and viscosity. The viscous fingering effect is quantified using two approaches and is found to correlate well with the intrinsic viscosity of the proteins-Brpt5.5 exhibits the most severe effect and is the most extended protein tested in the study. By AUC, the hydrodynamic non-ideality was measured for each protein via global analysis of a concentration series. Compared to BSA, both Brpt1.5 and Brpt5.5 showed significant non-ideality that could be easily visualized at concentrations at or below 5 mg/mL and 1 mg/mL, respectively. A variety of relationships were examined for their ability to differentiate the proteins by shape using information from AUC and/or viscosity. Furthermore, these relationships were also tested in the context of hydrodynamic modeling. The importance of considering non-ideality when investigating the structure of extended macromolecules is discussed.

Mechanistic basis of staphylococcal interspecies competition for skin colonization.

Staphylococci, whether beneficial commensals or pathogens, often colonize human skin, potentially leading to competition for the same niche. In this multidisciplinary study we investigate the structure, binding specificity, and mechanism of adhesion of the Aap lectin domain required for Staphylococcus epidermidis skin colonization and compare its characteristics to the lectin domain from the orthologous Staphylococcus aureus adhesin SasG. The Aap structure reveals a legume lectin-like fold with atypical architecture, showing specificity for N-acetyllactosamine and sialyllactosamine. Bacterial adhesion assays using human corneocytes confirmed the biological relevance of these Aap-glycan interactions. Single-cell force spectroscopy experiments measured individual binding events between Aap and corneocytes, revealing an extraordinarily tight adhesion force of nearly 900 nN and a high density of receptors at the corneocyte surface. The SasG lectin domain shares similar structural features, glycan specificity, and corneocyte adhesion behavior. We observe cross-inhibition of Aap-and SasG-mediated staphylococcal adhesion to corneocytes. Together, these data provide insights into staphylococcal interspecies competition for skin colonization and suggest potential avenues for inhibition of S. aureus colonization.

Convergent behavior of extended stalk regions from staphylococcal surface proteins with widely divergent sequence patterns.

Staphylococcus epidermidis and S. aureus are highly problematic bacteria in hospital settings. This stems, at least in part, from strong abilities to form biofilms on abiotic or biotic surfaces. Biofilms are well-organized multicellular aggregates of bacteria, which, when formed on indwelling medical devices, lead to infections that are difficult to treat. Cell wall-anchored (CWA) proteins are known to be important players in biofilm formation and infection. Many of these proteins have putative stalk-like regions or regions of low complexity near the cell wall-anchoring motif. Recent work demonstrated the strong propensity of the stalk region of the S. epidermidis accumulation-associated protein (Aap) to remain highly extended under solution conditions that typically induce compaction or other significant conformational changes. This behavior is consistent with the expected function of a stalk-like region that is covalently attached to the cell wall peptidoglycan and projects the adhesive domains of Aap away from the cell surface. In this study, we evaluate whether the ability to resist compaction is a common theme among stalk regions from various staphylococcal CWA proteins. Circular dichroism spectroscopy was used to examine secondary structure changes as a function of temperature and cosolvents along with sedimentation velocity analytical ultracentrifugation and SAXS to characterize structural characteristics in solution. All stalk regions tested are intrinsically disordered, lacking secondary structure beyond random coil and polyproline type II helix, and they all sample highly extended conformations. Remarkably, the Ser-Asp dipeptide repeat region of SdrC exhibited nearly identical behavior in solution when compared to the Aap Pro/Gly-rich region, despite highly divergent sequence patterns, indicating conservation of function by various distinct staphylococcal CWA protein stalk regions.

Follistatin Forms a Stable Complex With Inhibin A That Does Not Interfere With Activin A Antagonism.

Inhibins are transforming growth factor-ß family heterodimers that suppress follicle-stimulating hormone (FSH) secretion by antagonizing activin class ligands. Inhibins share a common ß chain with activin ligands. Follistatin is another activin antagonist, known to bind the common ß chain of both activins and inhibins. In this study, we characterized the antagonist-antagonist complex of inhibin A and follistatin to determine if their interaction impacted activin A antagonism. We isolated the inhibin A:follistatin 288 complex, showing that it forms in a 1:1 stoichiometric ratio, different from previously reported homodimeric ligand:follistatin complexes, which bind in a 1:2 ratio. Small angle X-ray scattering coupled with modeling provided a low-resolution structure of inhibin A in complex with follistatin 288. Inhibin binds follistatin via the shared activin ß chain, leaving the α chain free and flexible. The inhibin A:follistatin 288 complex was also shown to bind heparin with lower affinity than follistatin 288 alone or in complex with activin A. Characterizing the inhibin A:follistatin 288 complex in an activin-responsive luciferase assay and by surface plasmon resonance indicated that the inhibitor complex readily dissociated upon binding type II receptor activin receptor type IIb, allowing both antagonists to inhibit activin signaling. Additionally, injection of the complex in ovariectomized female mice did not alter inhibin A suppression of FSH. Taken together, this study shows that while follistatin binds to inhibin A with a substochiometric ratio relative to the activin homodimer, the complex can dissociate readily, allowing both proteins to effectively antagonize activin signaling.

Solution Structural Studies of Pre-amyloid Oligomer States of the Biofilm Protein Aap.

Staphylococcus epidermidis is a commensal bacterium on human skin that is also the leading cause of medical device-related infections. The accumulation-associated protein (Aap) from S. epidermidis is a critical factor for infection via its ability to mediate biofilm formation. The B-repeat superdomain of Aap is composed of 5 to 17 Zn2+-binding B-repeats, which undergo rapid, reversible assembly to form dimer and tetramer species. The tetramer can then undergo a conformational change and nucleate highly stable functional amyloid fibrils. In this study, multiple techniques including analytical ultracentrifugation (AUC) and small-angle X-ray scattering (SAXS) are used to probe a panel of B-repeat mutant constructs that assemble to distinct oligomeric states to define the structural characteristics of B-repeat dimer and tetramer species. The B-repeat region from Aap forms an extremely elongated conformation that presents several challenges for standard SAXS analyses. Specialized approaches, such as cross-sectional analyses, allowed for in-depth interpretation of data, while explicit-solvent calculations via WAXSiS allowed for accurate evaluation of atomistic models. The resulting models suggest mechanisms by which Aap functional amyloid fibrils form, illuminating an important contributing factor to recurrent staphylococcal infections.

Pregnancy enables antibody protection against intracellular infection.

Adaptive immune components are thought to exert non-overlapping roles in antimicrobial host defence, with antibodies targeting pathogens in the extracellular environment and T cells eliminating infection inside cells1,2. Reliance on antibodies for vertically transferred immunity from mothers to babies may explain neonatal susceptibility to intracellular infections3,4. Here we show that pregnancy-induced post-translational antibody modification enables protection against the prototypical intracellular pathogen Listeria monocytogenes. Infection susceptibility was reversed in neonatal mice born to preconceptually primed mothers possessing L. monocytogenes-specific IgG or after passive transfer of antibodies from primed pregnant, but not virgin, mice. Although maternal B cells were essential for producing IgGs that mediate vertically transferred protection, they were dispensable for antibody acquisition of protective function, which instead required sialic acid acetyl esterase5 to deacetylate terminal sialic acid residues on IgG variable-region N-linked glycans. Deacetylated L. monocytogenes-specific IgG protected neonates through the sialic acid receptor CD226,7, which suppressed IL-10 production by B cells leading to antibody-mediated protection. Consideration of the maternal-fetal dyad as a joined immunological unit reveals protective roles for antibodies against intracellular infection and fine-tuned adaptations to enhance host defence during pregnancy and early life.

The molecular characterization of antibody binding to a superantigen-like protein from a commensal microbe.

Microorganisms have coevolved diverse mechanisms to impair host defenses. A major one, superantigens, can result in devastating effects on the immune system. While all known superantigens induce vast immune cell proliferation and come from opportunistic pathogens, recently, proteins with similar broad specificity to antibody variable (V) domain families were identified in a commensal microbiota. These proteins, identified in the human commensal Ruminococcus gnavus, are called immunoglobulin-binding protein (Ibp) A and B and have been shown to activate B cells in vitro expressing either human VH3 or murine VH5/6/7. Here, we provide molecular and functional studies revealing the basis of this Ibp/immunoglobulin (Ig) interaction. The crystal structure and biochemical assays of a truncated IbpA construct in complex with mouse VH5 antigen-binding fragment (Fab) shows a binding of Ig heavy chain framework residues to the Ibp Domain D and the C-terminal heavy chain binding domain (HCBD). We used targeted mutagenesis of contact residues and affinity measurements and performed studies of the Fab-IbpA complex to determine the stoichiometry between Ibp and VH domains, suggesting Ibp may serve to cluster full-length IgA antibodies in vivo. Furthermore, in vitro stimulation experiments indicate that binding of the Ibp HCBD alone is sufficient to activate responsive murine B cell receptors. The presence of these proteins in a commensal microbe suggest that binding a broad repertoire of immunoglobulins, particularly in the gut/microbiome environment, may provide an important function in the maintenance of host/microbiome homeostasis contrasting with the pathogenic role of structurally homologous superantigens expressed by pathogens.

Seroprevalence of SARS-CoV-2 infection in Cincinnati Ohio USA from August to December 2020.

The world is currently in a pandemic of COVID-19 (Coronavirus disease-2019) caused by a novel positive-sense, single-stranded RNA ß-coronavirus referred to as SARS-CoV-2. Here we investigated rates of SARS-CoV-2 infection in the greater Cincinnati, Ohio, USA metropolitan area from August 13 to December 8, 2020, just prior to initiation of the national vaccination program. Examination of 9,550 adult blood donor volunteers for serum IgG antibody positivity against the SARS-CoV-2 Spike protein showed an overall prevalence of 8.40%, measured as 7.56% in the first 58 days and 9.24% in the last 58 days, and 12.86% in December 2020, which we extrapolated to ~20% as of March, 2021. Males and females showed similar rates of past infection, and rates among Hispanic or Latinos, African Americans and Whites were also investigated. Donors under 30 years of age had the highest rates of past infection, while those over 60 had the lowest. Geographic analysis showed higher rates of infectivity on the West side of Cincinnati compared with the East side (split by I-75) and the lowest rates in the adjoining region of Kentucky (across the Ohio river). These results in regional seroprevalence will help inform efforts to best achieve herd immunity in conjunction with the national vaccination campaign.

Modification of cell wall polysaccharide guides cell division in Streptococcus mutans.

In ovoid-shaped, Gram-positive bacteria, MapZ guides FtsZ-ring positioning at cell equators. The cell wall of the ovococcus Streptococcus mutans contains peptidoglycan decorated with serotype c carbohydrates (SCCs). In the present study, we identify the major cell separation autolysin AtlA as an SCC-binding protein. AtlA binding to SCC is attenuated by the glycerol phosphate (GroP) modification. Using fluorescently labeled AtlA constructs, we mapped SCC distribution on the streptococcal surface, revealing enrichment of GroP-deficient immature SCCs at the cell poles and equators. The immature SCCs co-localize with MapZ at the equatorial rings throughout the cell cycle. In GroP-deficient mutants, AtlA is mislocalized, resulting in dysregulated cellular autolysis. These mutants display morphological abnormalities associated with MapZ mislocalization, leading to FtsZ-ring misplacement. Altogether, our data support a model in which maturation of a cell wall polysaccharide provides the molecular cues for the recruitment of cell division machinery, ensuring proper daughter cell separation and FtsZ-ring positioning.

Suppression of IgE-mediated anaphylaxis and food allergy with monovalent anti-FcεRIα mAbs.

BACKGROUND: Mast cell and basophil activation by antigen cross-linking of FcεRI-bound IgE is central to allergy pathogenesis. We previously demonstrated global suppression of this process by rapid desensitization with anti-FcεRIα mAbs. OBJECTIVES: We sought to determine whether use of monovalent (mv) anti-FcεRIα mAbs increases desensitization safety without loss of efficacy. METHODS: mv anti-human (hu) FcεRIα mAbs were produced with mouse-derived immunoglobulin variable regions and huIgG1 or huIgG4 C regions and were used to suppress murine IgE-mediated anaphylaxis and food allergy. mAbs were administered as a single dose or as serially increasing doses to mice that express hu instead of mouse FcεRIα; mice that additionally have an allergy-promoting IL-4Rα mutation; and hu cord blood-reconstituted immunodeficient, hu cytokine-secreting, mice that have large numbers of activated hu mast cells. Anaphylaxis susceptibility was sometimes increased by treatment with IL-4 or a ß-adrenergic receptor antagonist. RESULTS: mv anti-hu FcεRIα mAbs are considerably less able than divalent mAbs are to induce anaphylaxis and deplete mast cell and basophil IgE, but mv mAbs still strongly suppress IgE-mediated disease. The mv mAbs can be safely administered as a single large dose to mice with typical susceptibility to anaphylaxis, while a rapid desensitization approach safely suppresses disease in mice with increased susceptibility. Our huIgG4 variant of mv anti-huFcεRIα mAb is safer than our huIgG1 variant is, apparently because reduced interactions with FcεRs decrease ability to indirectly cross-link FcεRI. CONCLUSIONS: mv anti-FcεRIα mAbs more safely suppress IgE-mediated anaphylaxis and food allergy than divalent variants of the same mAbs do. These mv mAbs may be useful for suppression of huIgE-mediated disease.

The staphylococcal biofilm protein Aap forms a tetrameric species as a necessary intermediate before amyloidogenesis.

The accumulation-associated protein (Aap) from Staphylococcus epidermidis is a biofilm-related protein that was found to be a critical factor for infection using a rat catheter model. The B-repeat superdomain of Aap, composed of 5-17 B-repeats, each containing a Zn2+-binding G5 and a spacer subdomain, is responsible for Zn2+-dependent assembly leading to accumulation of bacteria during biofilm formation. We previously demonstrated that a minimal B-repeat construct (Brpt1.5) forms an antiparallel dimer in the presence of 2-3 Zn2+ ions. More recently, we have reported the presence of functional amyloid-like fibrils composed of Aap within S. epidermidis biofilms and demonstrated that a biologically relevant construct containing five and a half B-repeats (Brpt5.5) forms amyloid-like fibrils similar to those observed in the biofilm. In this study, we analyze the initial assembly events of the Brpt5.5 construct. Analytical ultracentrifugation was utilized to determine hydrodynamic parameters of reversibly associating species and to perform linked equilibrium studies. Linkage studies indicated a mechanism of Zn2+-induced dimerization similar to smaller constructs; however, Brpt5.5 dimers could then undergo further Zn2+-induced assembly into a previously uncharacterized tetramer. This led us to search for potential Zn2+-binding sites outside of the dimer interface. We developed a Brpt5.5 mutant that was unable to form the tetramer and was concordantly incapable of amyloidogenesis. CD and dynamic light scattering indicate that a conformational transition in the tetramer species is a critical step preceding amyloidogenesis. This mechanistic model for B-repeat assembly and amyloidogenesis provides new avenues for potential therapeutic targeting of staphylococcal biofilms.

The biofilm adhesion protein Aap from Staphylococcus epidermidis forms zinc-dependent amyloid fibers.

The skin-colonizing commensal bacterium Staphylococcus epidermidis is a leading cause of hospital-acquired and device-related infections. Its pathogenicity in humans is largely due to its propensity to form biofilms, surface-adherent bacterial accumulations that are remarkably resistant to chemical and physical stresses. Accumulation-associated protein (Aap) from S. epidermidis has been shown to be necessary and sufficient for mature biofilm formation and catheter infection. Aap contains up to 17 tandem B-repeat domains, capable of zinc-dependent assembly into twisted, rope-like intercellular filaments in the biofilm. Using microscopic and biophysical techniques, we show here that Aap B-repeat constructs assemble further into zinc-dependent functional amyloid fibers. We observed such amyloid fibers by confocal microscopy during both early and late stages of S. epidermidis biofilm formation, and we confirmed that extracellular fibrils from these biofilms contain Aap. Unlike what has been observed for amyloidogenic biofilm proteins from other bacteria, which typically use chaperones or initiator proteins to initiate amyloid assembly, our findings indicate that Aap from S. epidermidis requires Zn2+ as a catalyst that drives amyloid fiber formation, similar to many mammalian amyloid-forming proteins that require metals for assembly. This work provides detailed insights into S. epidermidis biofilm formation and architecture that improve our understanding of persistent staphylococcal infections.

The Proline/Glycine-Rich Region of the Biofilm Adhesion Protein Aap Forms an Extended Stalk that Resists Compaction.

Staphylococcus epidermidis is one of the primary bacterial species responsible for healthcare-associated infections. The most significant virulence factor for S. epidermidis is its ability to form a biofilm, which renders the bacteria highly resistant to host immune responses and antibiotic action. Intercellular adhesion within the biofilm is mediated by the accumulation-associated protein (Aap), a cell wall-anchored protein that self-assembles in a zinc-dependent manner. The C-terminal portion of Aap contains a 135-aa-long, proline/glycine-rich region (PGR) that has not yet been characterized. The region contains a set of 18 nearly identical AEPGKP repeats. Analysis of the PGR using biophysical techniques demonstrated the region is a highly extended, intrinsically disordered polypeptide with unusually high polyproline type II helix propensity. In contrast to many intrinsically disordered polypeptides, there was a minimal temperature dependence of the global conformational state of PGR in solution as measured by analytical ultracentrifugation and dynamic light scattering. Furthermore, PGR was resistant to conformational collapse or α-helix formation upon the addition of the osmolyte trimethylamine N-oxide or the cosolvent 2,2,2-trifluoroethanol. Collectively, these results suggest PGR functions as a resilient, extended stalk that projects the rest of Aap outward from the bacterial cell wall, promoting intercellular adhesion between cells in the biofilm. This work sheds light on regions of low complexity often found near the attachment point of bacterial cell wall-anchored proteins.