MicL, a new σE-dependent sRNA, combats envelope stress by repressing synthesis of Lpp, the major outer membrane lipoprotein.

In enteric bacteria, the transcription factor σ(E) maintains membrane homeostasis by inducing synthesis of proteins involved in membrane repair and two small regulatory RNAs (sRNAs) that down-regulate synthesis of abundant membrane porins. Here, we describe the discovery of a third σ(E)-dependent sRNA, MicL (mRNA-interfering complementary RNA regulator of Lpp), transcribed from a promoter located within the coding sequence of the cutC gene. MicL is synthesized as a 308-nucleotide (nt) primary transcript that is processed to an 80-nt form. Both forms possess features typical of Hfq-binding sRNAs but surprisingly target only a single mRNA, which encodes the outer membrane lipoprotein Lpp, the most abundant protein of the cell. We show that the copper sensitivity phenotype previously ascribed to inactivation of the cutC gene is actually derived from the loss of MicL and elevated Lpp levels. This observation raises the possibility that other phenotypes currently attributed to protein defects are due to deficiencies in unappreciated regulatory RNAs. We also report that σ(E) activity is sensitive to Lpp abundance and that MicL and Lpp comprise a new σ(E) regulatory loop that opposes membrane stress. Together MicA, RybB, and MicL allow σ(E) to repress the synthesis of all abundant outer membrane proteins in response to stress.

A selective peptide inhibitor of Frizzled 7 receptors disrupts intestinal stem cells.

Regeneration of the adult intestinal epithelium is mediated by a pool of cycling stem cells, which are located at the base of the crypt, that express leucine-rich-repeat-containing G-protein-coupled receptor 5 (LGR5). The Frizzled (FZD) 7 receptor (FZD7) is enriched in LGR5+ intestinal stem cells and plays a critical role in their self-renewal. Yet, drug discovery approaches and structural bases for targeting specific FZD isoforms remain poorly defined. FZD proteins interact with Wnt signaling proteins via, in part, a lipid-binding groove on the extracellular cysteine-rich domain (CRD) of the FZD receptor. Here we report the identification of a potent peptide that selectively binds to the FZD7 CRD at a previously uncharacterized site and alters the conformation of the CRD and the architecture of its lipid-binding groove. Treatment with the FZD7-binding peptide impaired Wnt signaling in cultured cells and stem cell function in intestinal organoids. Together, our data illustrate that targeting the lipid-binding groove holds promise as an approach for achieving isoform-selective FZD receptor inhibition.

Publisher Correction: A selective peptide inhibitor of Frizzled 7 receptors disrupts intestinal stem cells.

The version of this article originally published contained older versions of the Life Sciences Reporting Summary and the Supplementary Text and Figures. The error has been corrected in the HTML and PDF versions of the article.

Small RNAs endow a transcriptional activator with essential repressor functions for single-tier control of a global stress regulon.

The Escherichia coli σ(E) envelope stress response monitors and repairs the outer membrane, a function central to the life of Gram-negative bacteria. The σ(E) stress response was characterized as a single-tier activation network comprised of ~100 genes, including the MicA and RybB noncoding sRNAs. These highly expressed sRNAs were thought to carry out the specialized function of halting de novo synthesis of several abundant porins when envelope homeostasis was perturbed. Using a systematic target profiling and validation approach we discovered that MicA and RybB are each global mRNA repressors of both distinct and shared targets, and that the two sRNAs constitute a posttranscriptional repression arm whose regulatory scope rivals that of the protein-based σ(E) activation arm. Intriguingly, porin mRNAs constitute only ~1/3 of all targets and new nonporin targets predict roles for MicA and RybB in crosstalk with other regulatory responses. This work also provides an example of evolutionarily unrelated sRNAs that are coinduced and bind the same targets, but at different sites. Our finding that expression of either MicA or RybB sRNA protects the cell from the loss of viability experienced when σ(E) activity is inadequate illustrates the importance of the posttranscriptional repression arm of the response. σ(E) is a paradigm of a single-tier stress response with a clear division of labor in which highly expressed noncoding RNAs (MicA, RybB) endow a transcriptional factor intrinsically restricted to gene activation (σ(E)) with the opposite repressor function.

NF-κB inducing kinase is a therapeutic target for systemic lupus erythematosus.

NF-κB-inducing kinase (NIK) mediates non-canonical NF-κB signaling downstream of multiple TNF family members, including BAFF, TWEAK, CD40, and OX40, which are implicated in the pathogenesis of systemic lupus erythematosus (SLE). Here, we show that experimental lupus in NZB/W F1 mice can be treated with a highly selective and potent NIK small molecule inhibitor. Both in vitro as well as in vivo, NIK inhibition recapitulates the pharmacological effects of BAFF blockade, which is clinically efficacious in SLE. Furthermore, NIK inhibition also affects T cell parameters in the spleen and proinflammatory gene expression in the kidney, which may be attributable to inhibition of OX40 and TWEAK signaling, respectively. As a consequence, NIK inhibition results in improved survival, reduced renal pathology, and lower proteinuria scores. Collectively, our data suggest that NIK inhibition is a potential therapeutic approach for SLE.

Phase variation and microevolution at homopolymeric tracts in Bordetella pertussis.

BACKGROUND: Bordetella pertussis, the causative agent of whooping cough, is a highly clonal pathogen of the respiratory tract. Its lack of genetic diversity, relative to many bacterial pathogens, could limit its ability to adapt to a hostile and changing host environment. This limitation might be overcome by phase variation, as observed for other mucosal pathogens. One of the most common mechanisms of phase variation is reversible expansion or contraction of homopolymeric tracts (HPTs). RESULTS: The genomes of B. pertussis and the two closely related species, B. bronchiseptica and B. parapertussis, were screened for homopolymeric tracts longer than expected on the basis of chance, given their nucleotide compositions. Sixty-nine such HPTs were found in total among the three genomes, 74% of which were polymorphic among the three species. Nine HPTs were genotyped in a collection of 90 geographically and temporally diverse B. pertussis strains using the polymerase chain reaction/ligase detection reaction (PCR/LDR) assay. Six HPTs were polymorphic in this collection of B. pertussis strains. Of note, one of these polymorphic HPTs was found in the fimX promoter, where a single base insertion variant was present in seven strains, all of which were isolated prior to introduction of the pertussis vaccine. Transcript abundance of fimX was found to be 3.8-fold lower in strains carrying the longer allele. HPTs in three other genes, tcfA, bapC, and BP3651, varied widely in composition across the strain collection and displayed allelic polymorphism within single cultures. CONCLUSION: Allelic polymorphism at homopolymeric tracts is common within the B. pertussis genome. Phase variability may be an important mechanism in B. pertussis for evasion of the immune system and adaptation to different niches in the human host. High sensitivity and specificity make the PCR/LDR assay a powerful tool for investigating allelic variation at HPTs. Using this method, allelic diversity and phase variation were demonstrated at several B. pertussis loci.