Biomedical subjects

Barry K Hurlburt

Publications and source records attributed to Barry K Hurlburt.

5 recordsLinked to original sources

PubMed2004

Staphylococcus aureus AgrA binding to the RNAIII-agr regulatory region.

The control of virulence gene expression in the human pathogen Staphylococcus aureus is under the partial control of the two-component quorum-sensing system encoded by genes of the agr locus. The product of the agrA gene has been shown by amino acid sequence similarity to be the putative response regulator; however, binding of AgrA to promoters under its control has not yet been demonstrated. In this study, we isolated and purified soluble AgrA by expression under osmotic shock conditions and ion-exchange chromatography. Purified AgrA showed high-affinity binding to the RNAIII-agr intergenic region by electrophoretic mobility shift assays. Binding was localized by DNase I protection assays to a pair of direct repeats in the P2 and P3 promoter regions of the agr locus. We found that this binding was enhanced by the addition of the small phosphoryl donor, acetyl phosphate. The difference in binding affinity between these two promoters was found to result from a 2-bp difference between the downstream direct repeats of the P2 and P3 sites. Mutation of these base pairs in the P3 site to match those found in the P2 site increased the affinity of AgrA for the P3 site relative to that for the P2 site. These results are consistent with the function of AgrA as a response regulator with recognition sites in the promoter regions of RNAIII and the agr locus.

Bacterial Proteins↗

PubMed2003

Characterization of Staphylococcus aureus SarA binding sites.

The staphylococcal accessory regulator locus (sarA) encodes a DNA-binding protein (SarA) that modulates expression of over 100 genes. Whether this occurs via a direct interaction between SarA and cis elements associated with its target genes is unclear, partly because the definitive characteristics of a SarA binding site have not been identified. In this work, electrophoretic mobility shift assays (EMSAs) were used to identify a SarA binding site(s) upstream of the SarA-regulated gene cna. The results suggest the existence of multiple high-affinity binding sites within the cna promoter region. Using a SELEX (systematic evolution of ligands by exponential enrichment) procedure and purified, recombinant SarA, we also selected DNA targets that contain a high-affinity SarA binding site from a random pool of DNA fragments. These fragments were subsequently cloned and sequenced. Randomly chosen clones were also examined by EMSA. These DNA fragments bound SarA with affinities comparable to those of recognized SarA-regulated genes, including cna, fnbA, and sspA. The composition of SarA-selected DNAs was AT rich, which is consistent with the nucleotide composition of the Staphylococcus aureus genome. Alignment of selected DNAs revealed a 7-bp consensus (ATTTTAT) that was present with no more than one mismatch in 46 of 56 sequenced clones. By using the same criteria, consensus binding sites were also identified upstream of the S. aureus genes spa, fnbA, sspA, agr, hla, and cna. With the exception of cna, which has not been previously examined, this 7-bp motif was within the putative SarA binding site previously associated with each gene.

Adhesins, Bacterial↗

PubMed2002

Analysis of the DNA-binding properties of MyoD, myogenin, and E12 by fluorescence anisotropy.

MyoD and Myogenin are dominant myogenic regulatory factors (MRFs), which are involved in control of muscle-specific gene expression. The ubiquitously expressed E12 dimerizes with MyoD and Myogenin and has been shown to enhance their DNA-binding and transcriptional activities. In this study, fluorescence anisotropy assays have been used to determine the Gibb's free energy of dissociation (DeltaG) for MyoD, Myogenin, and E12 as homo- and heterodimers to the well-characterized myosin light chain enhancer (MLC), muscle creatine kinase (MCK) enhancer, and mutant thereof. The heterodimers of MyoD or Myogenin with E12 bound the MCK enhancer equally well (DeltaG = 21 kcal/mol). The homodimers varied dramatically in both MLC and MCK enhancer binding affinity. MyoD homodimer bound the MCK enhancer with the highest affinity (DeltaG = 19.6 kcal/mol) in comparison with the Myogenin homodimer-MCK interaction (DeltaG = 16.6 kcal/mol) and E12 homodimer-MCK interaction (DeltaG = 18.0 kcal/mol). The slope and shape of the binding isotherms revealed that with the exception of the E12 homodimer-MCK enhancer interaction, the other proteins bound with high levels of positive cooperativity. In contrast, the E12 homodimer-MCK enhancer interaction actually occurs with significant negative cooperativity. The binding of these proteins to MLC enhancer mimicked binding to the MCK enhancer, but with much lower affinities. These data support the hypothesis that DNA acts as an allosteric ligand facilitating the dimerization of these proteins. The combination of differential affinity and cooperativity explains why the heterodimers are the active species in transcriptional regulation.

Creatine Kinase↗

PubMed2002

Strain-dependent differences in the regulatory roles of sarA and agr in Staphylococcus aureus.

The accessory gene regulator (agr) and the staphylococcal accessory regulator (sar) are central regulatory elements that control the production of Staphylococcus aureus virulence factors. To date, the functions of these loci have been defined almost exclusively using RN6390, which is representative of the laboratory strain 8325-4. However, RN6390 was recently shown to have a mutation in rsbU that results in a phenotype resembling that of a sigB mutant (I. Kullik et al., J. Bacteriol. 180:4814-4820, 1998). For that reason, it remains unclear whether the regulatory events defined in RN6390 are representative of the events that take place in clinical isolates of S. aureus. To address this issue, we generated mutations in the sarA and agr loci of three laboratory strains (RN6390, Newman, and S6C) and four clinical isolates (UAMS-1, UAMS-601, DB, and SC-1). Mutation of sarA in the cna-positive strains UAMS-1 and UAMS-601 resulted in an increased capacity to bind collagen, while mutation of agr had little impact. Northern blot analysis confirmed that the increase in collagen binding was due to increased cna transcription. Without exception, mutation of sarA resulted in increased production of proteases and a decreased capacity to bind fibronectin. Mutation of agr had the opposite effect. Although mutation of sarA resulted in a slight reduction in fnbA transcription, changes in the ability to bind fibronectin appeared to be more directly correlated with changes in protease activity. Lipase production was reduced in both sarA and agr mutants. While mutation of sarA in RN6390 resulted in reduced hemolytic activity, it had the opposite effect in all other strains. There appeared to be reduced levels of the sarC transcript in RN6390, but there was no difference in the overall pattern of sar transcription or the production of SarA. Although mutation of sarA resulted in decreased RNAIII transcription, this effect was not evident under all growth conditions. Taken together, these results suggest that studies defining the regulatory roles of sarA and agr by using RN6390 are not always representative of the events that occur in clinical isolates of S. aureus.

Adhesins, Bacterial↗

PubMedDate not supplied

Structural and functional alterations in major peanut allergens caused by thermal processing.

The majority of foods that we eat are subjected to some type of processing either at home or by the manufacturer. The biochemical reactions that occur in foods as a result of thermal processing can be both beneficial and harmful. Here, we briefly review the effects of thermal processing and some of the effects of the Maillard reaction on the allergenicity of food proteins. Specifically, we focus on the known effects of roasting on the allergenic properties of peanut proteins and the contribution of Maillard reaction products or advanced glycation end products to these observed effects. The most thorough understanding of the effects of thermal processing on allergenicity involves the peanut proteins. Thermal processing alters specific biophysical and immunological properties of peanut proteins, such as structure, function, solubility, digestibility, immunoglobulin E (IgE) binding, and T-cell responses. A better understanding of the effects of thermal processing-induced biochemical and immunological alterations is of utmost importance for proper risk assessment of existing and newly introduced proteins in the food source, as well as development of effective diagnostic tools and therapeutic treatments for food allergy.

Allergens↗