Inhibition of Inflammasome-Dependent Interleukin 1β Production by Streptococcal NAD-Glycohydrolase: Evidence for Extracellular Activity.

Group A (GAS) is a common human pathogen and the etiologic agent of a large number of diseases ranging from mild, self-limiting infections to invasive life-threatening conditions. Two prominent virulence factors of this bacterium are the genetically and functionally linked pore-forming toxin streptolysin O (SLO) and its cotoxin NAD-glycohydrolase (NADase). Overexpression of these toxins has been linked to increased bacterial virulence and is correlated with invasive GAS disease.

Streptolysin O and NAD-glycohydrolase prevent phagolysosome acidification and promote group A Streptococcus survival in macrophages.

Unlabelled: Group A Streptococcus (GAS, Streptococcus pyogenes) is an ongoing threat to human health as the agent of streptococcal pharyngitis, skin and soft tissue infections, and life-threatening conditions such as necrotizing fasciitis and streptococcal toxic shock syndrome. In animal models of infection, macrophages have been shown to contribute to host defense against GAS infection. However, as GAS can resist killing by macrophages in vitro and induce macrophage cell death, it has been suggested that GAS intracellular survival in macrophages may enable persistent infection.

Vitamin D and the human antimicrobial peptide LL-37 enhance group a streptococcus resistance to killing by human cells.

The CsrRS two-component regulatory system of group A Streptococcus (GAS; Streptococcus pyogenes) responds to subinhibitory concentrations of the human antimicrobial peptide LL-37. LL-37 signaling through CsrRS results in upregulation of genes that direct synthesis of virulence factors, including the hyaluronic acid capsule and streptolysin O (SLO). Here, we demonstrate that a consequence of this response is augmented GAS resistance to killing by human oropharyngeal keratinocytes, neutrophils, and macrophages.

Signal transduction through CsrRS confers an invasive phenotype in group A Streptococcus.

The CsrRS (or CovRS) two component system controls expression of up to 15% of the genome of group A Streptococcus (GAS). While some studies have suggested that the sensor histidine kinase CsrS responds to membrane perturbations as a result of various environmental stresses, other data have implicated the human antimicrobial peptide LL-37 and extracellular Mg(2+) as specific signals. We now report that Mg(2+) and LL-37 have opposite effects on expression of multiple genes that are activated or repressed by the transcriptional regulator CsrR.

Mn(II) binding by the anthracis repressor from Bacillus anthracis.

The anthracis repressor (AntR) is a manganese-activated transcriptional regulator from Bacillus anthracis and is a member of the diphtheria toxin repressor (DtxR) family of proteins. In this paper, we characterize the Mn(II) binding and protein dimerization state using a combination of continuous wave (cw) and pulsed EPR methods. Equilibrium metal binding experiments showed that AntR binds 2 equivalents of Mn(II) with positive cooperativity and apparent dissociation constants of 210 and 16.

Sequence of ligand binding and structure change in the diphtheria toxin repressor upon activation by divalent transition metals.

The diphtheria toxin repressor (DtxR) is an Fe(II)-activated transcriptional regulator of iron homeostatic and virulence genes in Corynebacterium diphtheriae. DtxR is a two-domain protein that contains two structurally and functionally distinct metal binding sites. Here, we investigate the molecular steps associated with activation by Ni(II)Cl(2) and Cd(II)Cl(2).

Prolylpeptide binding by the prokaryotic SH3-like domain of the diphtheria toxin repressor: a regulatory switch.

Diphtheria toxin repressor (DtxR) regulates the expression of iron-sensitive genes in Corynebacterium diphtheriae, including the diphtheria toxin gene. DtxR contains an N-terminal metal- and DNA-binding domain that is connected by a proline-rich flexible peptide segment (Pr) to a C-terminal src homology 3 (SH3)-like domain. We determined the solution structure of the intramolecular complex formed between the proline-rich segment and the SH3-like domain by use of NMR spectroscopy.

Genetic and biophysical studies of diphtheria toxin repressor (DtxR) and the hyperactive mutant DtxR(E175K) support a multistep model of activation.

The diphtheria toxin repressor (DtxR) from Corynebacterium diphtheriae is the prototypic member of a superfamily of transition metal ion-activated transcriptional regulators that have been isolated from Gram-positive prokaryotes. Upon binding divalent transition metal ions, the N-terminal domain of DtxR undergoes a dynamic structural organization leading to homodimerization and target DNA binding. We have used site-directed mutagenesis and NMR analysis to probe the mechanism by which apo-DtxR transits from an inactive to a fully active repressor upon metal ion binding.

The src homology 3-like domain of the diphtheria toxin repressor (DtxR) modulates repressor activation through interaction with the ancillary metal ion-binding site.

The diphtheria toxin repressor (DtxR) is a transition metal ion-activated repressor that acts as a global regulatory element in the control of iron-sensitive genes in Corynebacterium diphtheriae. We recently described (L. Sun, J.

Design and development of a novel genetic probe for the analysis of repressor-operator interactions.

While the native diphtheria tox promoter/operator (toxPO)-lacZ transcriptional fusion has allowed initial isolation and characterization of the diphtheria toxin repressor (DtxR), the low level of reporter gene expression has limited the detection and analysis of mutations affecting subtle changes in repressor-operator binding. In order to overcome this difficulty, we have constructed a novel hybrid promoter/operator-lacZ transcriptional fusion in which the "-35" and spacing of the tac promoter was fused to the "-10" and interrupted palindromic sequence of toxO. We show that the hybrid tacPtoxO is regulated by the transition metal ion-dependent DtxR and that lacZ expression is increased approximately 70-fold in the reporter strain Escherichia coli DH5alpha/lambdaRS45-tacPtoxO-lacZ relative to DH5alpha/lambdaRS45-toxPO-lacZ.