A novel endogenous inhibitor of the secreted streptococcal NAD-glycohydrolase

The Streptococcus pyogenes NAD-glycohydrolase (SPN) is a toxic enzyme that is introduced into infected host cells by the cytolysin-mediated translocation pathway. However, how S. pyogenes protects itself from the self-toxicity of SPN had been unknown. In this report, we describe immunity factor for SPN (IFS), a novel endogenous inhibitor that is essential for SPN expression. A small protein of 161 amino acids, IFS is localized in the bacterial cytoplasmic compartment. IFS forms a stable complex with SPN at a 1:1 molar ratio and inhibits SPN's NAD-glycohydrolase activity by acting as a competitive inhibitor of its beta-NAD+ substrate. Mutational studies revealed that the gene for IFS is essential for viability in those S. pyogenes strains that express an NAD-glycohydrolase activity. However, numerous strains contain a truncated allele of ifs that is linked to an NAD-glycohydrolase-deficient variant allele of spn. Of practical concern, IFS allowed the normally toxic SPN to be produced in the heterologous host Escherichia coli to facilitate its purification. To our knowledge, IFS is the first molecularly characterized endogenous inhibitor of a bacterial beta-NAD(+)-consuming toxin and may contribute protective functions in the streptococci to afford SPN-mediated pathogenesis.     

Generation of novel conditional and hypomorphic alleles of the Smad2 gene

Smad2 is an intracellular mediator of the transforming growth factor beta signaling (TGFbeta) pathway. It has been previously shown that, in the mouse, ablation of functional Smad2 results in embryonic lethality due to gastrulation defects. To circumvent the early lethality and study the spatially and temporally specific functions of Smad2, we utilized the Cre-loxP system to generate a Smad2 conditional allele. Here we show that a conditional allele, Smad2(flox), was generated. In this allele, exons 9 and 10 are flanked by loxP sites and the gene is functionally wildtype. Cre-mediated recombination results in a deletion allele which phenocopies our previously reported Smad2(DeltaC) null mutation. To generate this conditional allele, we first made a targeted mutation which introduced a floxed neo cassette into intron 10. This allele (Smad2(3loxP)) functions hypomorphically when placed opposite a null allele, and unlike the other published Smad2 hypomorphic allele, can be maintained in the homozygous state.     

A novel mutant allele of Ncx1: a single amino acid substitution leads to cardiac dysfunction

The biological role and structure-function relationship of the Na(+)Ca(2+) exchanger NCX1 have been the subject of much investigation. Subtle mutagenesis to study the function of a protein seems only feasible in in vitro systems, but genetic forward screens have the potential to provide in vivo models to study single amino acid substitutions. In a genetic screen in mouse, we have isolated a mutant line carrying a novel mutant allele of the mouse Ncx1 gene. In this allele, a point mutation causes the substitution of a highly conserved asparagine residue (N874) with lysine. Accepted models for NCX1 structure propose that the affected amino acid is located in one of the reentrant membrane loops and experiments in vitro have identified N874 as critical for the ion transport function of NCX1. We found severe circulation defects and defective placentation in homozygous Ncx1(N87K4) mutant embryos, making the phenotype essentially indistinguishable from those of previously described null mutants. By ex vivo analysis, we demonstrated intrinsic functional abnormalities of cardiomyocytes. Western blot analysis and immunohistochemistry demonstrated normal levels and subcellular localization of the altered protein, ruling out the possibility that the abnormalities are a mere consequence of a major disturbance of protein structure. This study confirms and extends studies in vitro indicating the significance of amino acid N874 for the function of the NCX1 protein. It provides an in vivo model for this mutation and demonstrates the potential of forward genetic screens in a mammalian system.