Biochemistry

Background Alternative splicing, which generates multiple mRNA isoforms from single genes, is crucial for the regulation of eukaryotic gene expression. The flux through competing splicing pathways cannot be determined by traditional RNA-Seq, however, because different mRNA isoforms can have widely differing decay rates. Indeed, some mRNA isoforms with extremely short half-lives, such as those subject to translation-dependent nonsense-mediated decay (AS-NMD), may be completely overlooked in even the most extensive RNA-Seq analyses. Results RNA immunoprecipitation in tandem (RIPiT) of exon junction complex components allows for purification of post-splicing mRNA-protein particles (mRNPs) not yet subject to translation (pre-translational mRNPs) and, therefore, translation-dependent mRNA decay. Here we compare exon junction complex RIPiT-Seq to whole cell RNA-Seq data from HEK293 cells. Consistent with expectation, the flux through known AS-NMD pathways is substantially higher than that...

ABSTRACTAntivirulence agents inhibit the production of disease-causing virulence factors but are neither bacteriostatic nor bactericidal. Antivirulence agents against methicillin-resistantStaphylococcus aureus(MRSA) strain USA300, the most widespread community-associated MRSA strain in the United States, were discovered by virtual screening against the response regulator AgrA, which acts as a transcription factor for the expression of several of the most prominentS. aureustoxins and virulence factors involved in pathogenesis. Virtual screening was followed by similarity searches in the databases of commercial vendors. The small-molecule compounds discovered inhibit the production of the toxins alpha-hemolysin and phenol-soluble modulin α in a dose-dependent manner without inhibiting bacterial growth. These antivirulence agents are small-molecule biaryl compounds in which the aromatic rings either are fused or are separated by a short linker. One of these compounds is the FDA-approve...

Small-molecule antivirulence agents represent a promising alternative or adjuvant to antibiotics. These compounds disarm pathogens of disease-causing toxins without killing them, thereby diminishing survival pressure to develop resistance. Here we show that the small-molecule antivirulence agents F12 and F19 block staphylococcal transcription factor AgrA from binding to its promoter. Consequently, toxin expression is inhibited, thus preventing host cell damage by Gram-positive pathogens. Broad spectrum efficacy against Gram-positive pathogens is due to the existence of AgrA homologs in many Gram-positive bacteria. F12 is more efficacious in vitro and F19 works better in vivo. In a murine MRSA bacteremia/sepsis model, F19 treatment alone resulted in 100% survival while untreated animals had 70% mortality. Furthermore, F19 enhances antibiotic efficacy in vivo. Notably, in a murine MRSA wound infection model, combination of F19 with antibiotics resulted in bacterial load reduction. Thu...

The dwindling repertoire of antibiotics to treat Methicillin-resistant Staphylococcus aureus (MRSA) calls for novel treatment options. Quorum-quenching agents offer an alternative or an adjuvant to antibiotic therapy. Three biaryl hydroxyketone compounds discovered previously (F1, F12 and F19; Yu G., Kuo D., Shoham M., Viswanathan R. 2014. ACS Comb Sci 16:85-91) were tested for efficacy in MRSA-infected animal models. Topical therapy of compounds F1 and F12 in an MRSA murine wound infection model promotes wound healing compared to untreated control. Compounds F1, F12, and F19 afford significant survival benefits in an MRSA insect larvae model. Combination therapy of these quorum-quenching agents with cephalothin or nafcillin, antibiotics to which MRSA is resistant in monotherapy, revealed additional survival benefits. The quorum-quenching agents sensitize MRSA to the antibiotic by a synergistic mode of action that is also observed in vitro. An adjuvant of 1 μg/mL F1, F12 or F19 redu...

Many bacterial appendages have filamentous structures, often composed of repeating monomers assembled in a head-to-tail manner. The mechanisms of such linkages vary. We report here a novel protein oligomerization motif identified in the FadA adhesin from the ...

Dimerization represents a key regulatory step in the function of basic helix-loop-helix transcriptional factors. In many instances tissue-specific basic helix-loop-helix proteins, such as the hematopoietic factor SCL/tal or the myogenic factor MyoD, interact with ubiquitously expressed basic ...