Biology subjects

Herr, A. B.

Publications and source records attributed to Herr, A. B..

10 recordsLinked to original sources

bioRxiv2026

Enterococcus faecalis autolysin, EpaU, binds the enterococcal polysaccharide antigen via its teichoic acid-like repeats and modulates c-di-AMP signaling

The cell wall of the Gram-positive bacterium Enterococcus faecalis is decorated with the enterococcal polysaccharide antigen (EPA), consisting of a core rhamnan backbone linked covalently with a strain-variable teichoic acid-like (TA) polymer. Current models propose that the TA decoration is a repeating polymer composed of two alternating subunits, designated TAI and TAII, which are attached to the rhamnan core via a mild-acid labile phosphodiester bond from the initiating TAI subunit. In this study, we characterize the EpaU autolysin encoded within the EPA biosynthetic gene cluster. We demonstrate that the cell wall-binding domain of EpaU associates with the intact TA domains of EPA synthesized with the aid of the glycosyltransferases EpaR and EpaX. We further show that EpaU is a potent autolysin that binds generally over the E. faecalis cell surface, suggesting that it functions as a remodeling peptidoglycan hydrolase. The absence of EpaU leads to increased ampicillin resistance and elevated intracellular levels of the second messenger c-di-AMP. These data suggest that E. faecalis possesses a mechanism that senses the integrity of the peptidoglycan meshwork and employs c-di-AMP to regulate cell turgor, potentially altering the antibiotic resistance. ImportanceEnterococcus faecalis is an important opportunistic pathogen that can cause severe nosocomial infections. Knowledge of how bacteria remodel the cell wall is key to understanding many important cellular processes, such as antibiotic resistance, cell division, biofilm formation, and stress resistance. In this study, we shed new light on the structural details of the main cell wall polysaccharide, Enterococcal Polysaccharide Antigen (EPA), and its interaction with EpaU, an autolysin that cleaves peptidoglycan during cell wall remodeling. We also report a link between EpaU and the regulation of turgor pressure via cyclic dinucleotide signaling. This work contributes to a more complete picture of E. faecalis cell wall and may provide insight into the development of antimicrobial agents based on autolysins.

microbiology↗

bioRxiv2025

The secreted staphylococcal biofilm protein Sbp forms biomolecular condensates in the presence of DNA

Staphylococcus epidermidis is the leading cause of device-related infections, primarily due to its ability to form biofilms, surface-adherent bacterial communities that confer remarkable resistance to antibiotics and host defenses. Small basic protein, Sbp, is a 16- kDa protein expressed by S. epidermidis that has been shown to be crucial for biofilm formation, but little is known about its function. Sbp features a high proportion of basic residues as well as several predicted regions of intrinsic disorder. Consistent with its high positive charge density, Sbp is shown here to interact with double-stranded DNA, a ubiquitous component of the biofilm matrix, forming soluble complexes or large aggregates with short or longer DNA oligonucleotides, respectively. The observed multivalent interaction of Sbp with DNA along with its predicted disorder suggested that it might form biomolecular condensates with DNA. Confocal and differential interference contrast microscopy revealed that Sbp and dsDNA form phase-separated droplets and/or solid aggregates, depending on the concentration and stoichiometry of Sbp and DNA as well as the DNA oligonucleotide length. Fluorescence recovery after photobleaching experiments demonstrated that Sbp-DNA condensate droplets initially exhibit liquid-like behavior but gradually transition to a gel-like state. This work provides the first evidence that Sbp binds DNA and undergoes biomolecular condensation, revealing a previously unrecognized mechanism that may contribute to biofilm matrix organization in S. epidermidis.

biophysics↗

bioRxiv2023

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{degrees} 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.

biochemistry↗

bioRxiv2023

Staphylococcus aureus skin colonization is mediated by SasG lectin variation

Staphylococcus aureus causes the majority of skin and soft tissue infections, but this pathogen only transiently colonizes healthy skin. However, this transient skin exposure enables S. aureus to transition to infection. Initial adhesion of S. aureus to skin corneocytes is mediated by surface protein G (SasG). Here, phylogenetic analyses reveal the presence of two major divergent SasG alleles in S. aureus, SasG-I and SasG-II. Structural analyses of SasG-II identified a unique non-aromatic arginine in the binding pocket of the lectin subdomain that mediates adhesion to corneocytes. Atomic force microscopy and corneocyte adhesion assays indicated SasG-II can bind to a broader variety of ligands than SasG-I. Glycosidase treatment resulted in different binding profiles between SasG-I and SasG-II on skin cells. Additionally, SasG-mediated adhesion was recapitulated using differentiated N/TERT keratinocytes. Our findings indicate that SasG-II has evolved to adhere to multiple ligands, conferring a distinct advantage to S. aureus during skin colonization.

microbiology↗

bioRxiv2023

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.

microbiology↗

bioRxiv2023

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.

biophysics↗

bioRxiv2023

The assembly landscape of the complete B-repeat superdomain from Staphylococcus epidermidis strain 1457

The accumulation-associated protein (Aap) is the primary determinant of Staphylococcus epidermidis device-related infections. The B-repeat superdomain is responsible for intercellular adhesion that leads to the development of biofilms occurring in such infections. It was recently demonstrated that Zn-induced B-repeat assembly leads to formation of functional amyloid fibrils, which offer strength and stability to the biofilm. Rigorous biophysical studies of Aap B-repeats from S. epidermidis strain RP62A revealed Zn-induced assembly into a stable, reversible dimer and tetramer, prior to aggregation into amyloid fibrils. Genetic manipulation is not tractable for many S. epidermidis strains, including RP62A; instead, many genetic studies have used strain 1457. Therefore, to better connect findings from biophysical and structural studies of B-repeats to in vivo studies, the B-repeat superdomain from strain 1457 was examined. Differences between the B-repeats from strain RP62A and 1457 include the number of B-repeats, which has been shown to play a critical role in assembly into amyloid fibrils, as well as the distribution of consensus and variant B-repeat subtypes, which differ in assembly competency and thermal stability. Detailed investigation of the Zn-induced assembly of the full B-repeat supderdomain from strain 1457 was conducted using analytical ultracentrifugation. Whereas the previous construct from RP62A (Brpt5.5) formed a stable tetramer prior to aggregation, Brpt6.5 from 1457 forms extremely large stable species on the order of 28- and 32-mers, prior to aggregation into similar amyloid fibrils. Importantly, both assembly pathways proceed through the same mechanism of dimerization and tetramerization, and both conclude with the formation of amyloid-like fibrils. Theoretical discussions of the energy landscape of B-repeats from different strains and of different length are provided with considerations of biological implications. Statement of SignificanceStaphylococcus epidermidis is a major pathogen responsible for device-related infections. The primary factor responsible for such infections is the accumulation-associated protein, Aap, through its ability to mediate formation of dense surface-adherent communities of bacteria known as biofilms. Our lab recently demonstrated that the B-repeat superdomain of Aap from strain RP62A undergoes Zn-dependent assembly to form functional amyloid fibrils that improve the strength and resilience of biofilms in vitro. These amyloid fibrils may be responsible for the difficulty in treating device-related infections. However, strain 1457 is commonly used for genetic manipulation. In this manuscript, the Zn-dependent assembly of B-repeats from strain 1457 is shown to lead to the same outcome of amyloidogenesis, although it occurs through different intermediate oligomeric states.

biophysics↗

bioRxiv2022

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 lower 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.

biophysics↗

bioRxiv2020

Accelerating Drug Discovery and Repurposingby Combining Transcriptional Signature Connectivity with Docking

The development of targeted treatment options for precision medicine is hampered by a slow and costly process of drug screening. While small molecule docking simulations are often applied in conjunction with cheminformatic methods to reduce the number of candidate molecules to be tested experimentally, the current approaches suffer from high false positive rates and are computationally expensive. Here, we present a novel in silico approach for drug discovery and repurposing, dubbed connectivity enhanced Structure Activity Relationship (ceSAR) that improves on current methods by combining docking and virtual screening approaches with pharmacogenomics and transcriptional signature connectivity analysis. ceSAR builds on the landmark LINCS library of transcriptional signatures of over 20,000 drug-like molecules and ~5,000 gene knock-downs (KDs) to connect small molecules and their potential targets. For a set of candidate molecules and specific target gene, candidate molecules are first ranked by chemical similarity to their concordant LINCS analogs that share signature similarity with a knock-down of the target gene. An efficient method for chemical similarity search, optimized for sparse binary fingerprints of chemical moieties, is used to enable fast searches for large libraries of small molecules. A small subset of candidate compounds identified in the first step is then re-scored by combining signature connectivity with docking simulations. On a set of 20 DUD-E benchmark targets with LINCS KDs, the consensus approach reduces significantly false positive rates, improving the median precision 3-fold over docking methods at the extreme library reduction. We conclude that signature connectivity and docking provide complementary signals, offering an avenue to improve the accuracy of virtual screening while reducing run times by multiple orders of magnitude.

bioinformatics↗

bioRxiv2020

Modification of streptococcal cell wall polysaccharide spatially controls cell division

Bacterial cell division is driven by a tubulin homolog FtsZ, which assembles into the Z-ring structure leading to the recruitment of the cell division machinery. In ovoid-shaped Gram-positive bacteria, such as streptococci, MapZ guides Z-ring positioning at cell equators through an, as yet, unknown mechanism. The cell wall of the important dental pathogen Streptococcus mutans is composed of peptidoglycan decorated with Serotype c Carbohydrates (SCCs). Here, we show that an immature form of SCC, lacking the recently identified glycerol phosphate (GroP) modification, coordinates Z-ring positioning. Pulldown assays using S. mutans cell wall combined with binding affinity analysis identified the major cell separation autolysin, AtlA, as an SCC binding protein. Importantly, AtlA binding to mature SCC is attenuated due to GroP modification. Using fluorescently-labeled AtlA, we mapped SCC distribution on the streptococcal surface to reveal that GroP-deficient immature SCCs are exclusively present at the cell poles and equators. Moreover, the equatorial GroP-deficient SCCs co-localize with MapZ throughout the S. mutans cell cycle. Consequently, in GroP-deficient mutant bacteria, proper AtlA localization is abrogated resulting in dysregulated cellular autolysis. In addition, these mutants display morphological abnormalities associated with MapZ mislocalization leading to Z-ring misplacement. Altogether, our data support a model in which GroP-deficient immature SCCs spatially coordinate the localization of AtlA and MapZ. This mechanism ensures cell separation by AtlA at poles and Z-ring alignment with the cell equator.Competing Interest StatementThe authors have declared no competing interest.View Full Text

microbiology↗