A Naturally-Occurring Point Mutation in a Hyaluronidase Gene (hysA1) of Staphylococcus aureus UAMS-1 Results in Reduced Enzymatic Activity

Hyaluronic acid is a high molecular weight polysaccharide that is widely distributed in animal tissues. Bacterial hyaluronidases degrade hyaluronic acid as secreted enzymes and have been shown to contribute to infection. <i>Staphylococcus aureus</i> UAMS-1 is a clinical isolate that codes for two hyaluronidases (<i>hysA1</i> and <i>hysA2</i>). Previous research has shown the presence of a full-length HysA1 protein from the <i>S. aureus</i> UAMS-1 strain with no evidence of enzymatic activity. A single base change resulting in an E480G amino acid change was identified in the <i>S. aureus</i> UAMS-1 <i>hysA1</i> gene when compared to the <i>S. aureus</i> Sanger 252 <i>hysA1</i> gene. A plasmid copy of the <i>S. aureus</i> Sanger 252 <i>hysA1 </i>gene transduced into a <i>hysA2 </i>deletion mutant strain of <i>S. aureus</i> UAMS-1 restored near wild type levels of enzymatic activity. Homology modeling and structural analysis suggested that Glu-480 in the HysA1 is critically responsible for maintaining the structural and functional ensemble of the catalytic and tunnel-forming residues, which are essential for enzyme activity. A high degree of relatedness among several Gram-positive bacterial hyaluronidases indicate the loss of enzymatic activity of HysA1 in the <i>S. aureus</i> UAMS-1 strain is most likely caused by the mutation identified in our study.

Genetic variant in 3’ untranslated region of the mouse pycard gene regulates inflammasome activity

Quantitative trait locus mapping for interleukin-1β release after inflammasome priming and activation was performed on bone-marrow-derived macrophages (BMDM) from an AKRxDBA/2 mouse strain intercross. The strongest associated locus mapped very close to the Pycard gene on chromosome 7, which codes for the inflammasome adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC). The DBA/2 and AKR Pycard genes only differ at a single-nucleotide polymorphism (SNP) in their 3’ untranslated region (UTR). DBA/2 vs. AKR BMDM had increased levels of Pycard mRNA expression and ASC protein, and increased inflammasome speck formation, which was associated with increased Pycard mRNA stability without an increased transcription rate. CRISPR/Cas9 gene editing was performed on DBA/2 embryonic stem cells to change the Pycard 3’UTR SNP from the DBA/2 to the AKR allele. This single base change significantly reduced Pycard expression and inflammasome activity after cells were differentiated into macrophages due to reduced Pycard mRNA stability.

A gain-of-function single nucleotide variant creates a new promoter which acts as an orientation-dependent enhancer-blocker

Many single nucleotide variants (SNVs) associated with human traits and genetic diseases are thought to alter the activity of existing regulatory elements. Some SNVs may also create entirely new regulatory elements which change gene expression, but the mechanism by which they do so is largely unknown. Here we show that a single base change in an otherwise unremarkable region of the human α-globin cluster creates an entirely new promoter and an associated unidirectional transcript. This SNV downregulates α-globin expression causing α-thalassaemia. Of note, the new promoter lying between the α-globin genes and their associated super-enhancer disrupts their interaction in an orientation-dependent manner. Together these observations show how both the order and orientation of the fundamental elements of the genome determine patterns of gene expression and support the concept that active genes may act to disrupt enhancer-promoter interactions in mammals as in Drosophila. Finally, these findings should prompt others to fully evaluate SNVs lying outside of known regulatory elements as causing changes in gene expression by creating new regulatory elements.

Genetic Variant in 3’ Untranslated Region of the Mouse Pycard Gene Regulates Inflammasome Activity

Quantitative trait locus mapping for interleukin-1β release after inflammasome priming and activation was performed on bone marrow-derived macrophages (BMDM) from an AKRxDBA/2 strain intercross. The strongest associated locus mapped very close to the Pycard gene on chromosome 7, which codes for the inflammasome adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC). The DBA/2 and AKR Pycard genes only differ at single nucleotide polymorphism (SNP) in their 3’ untranslated region (UTR). DBA/2 vs. AKR BMDM had increased levels of Pycard mRNA expression and ASC protein, and increased inflammasome speck formation, which was associated with increased Pycard mRNA stability without an increased transcription rate. CRIPSR/Cas9 gene editing was performed on DBA/2 embryonic stem cells to change the Pycard 3’UTR SNP from the DBA/2 to the AKR allele. This single base change significantly reduced Pycard expression and inflammasome activity after cells were differentiated into macrophages due to reduced Pycard mRNA stability.

Mutations in rpsL that confer streptomycin resistance show pleiotropic effects on virulence and the production of a carbapenem antibiotic in Erwinia carotovora

Spontaneous streptomycin-resistant derivatives of Erwinia carotovora subsp. carotovora strain ATTn10 were isolated. Sequencing of the rpsL locus (encoding the ribosomal protein S12) showed that each mutant was missense, with a single base change, resulting in the substitution of the wild-type lysine by arginine, threonine or asparagine at codon 43. Phenotypic analyses showed that the rpsL mutants could be segregated into two groups: K43R mutants showed reduced production of the β-lactam secondary metabolite 1-carbapen-2-em-3 carboxylic acid (Car), but little effect on exoenzyme production or virulence in potato tuber tests. By contrast, the K43N and K43T mutations were pleiotropic, resulting in reduced exoenzyme production and virulence, as well as diminished Car production. The effect on Car production was due to reduced transcription of the quorum-sensing-dependent car biosynthetic genes. The effects of K43N and K43T mutations on Car production were partially alleviated by provision of an excess of the quorum-sensing signalling molecule N-(3-oxohexanoyl)-l-homoserine lactone. Finally, a proteomic analysis of the K43T mutant indicated that the abundance of a subset of intracellular proteins was affected by this rpsL mutation.