Phase variation and microevolution at homopolymeric tracts in Bordetella pertussis

<p>Abstract</p> <p>Background</p> <p><it>Bordetella pertussis</it>, 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).</p> <p>Results</p> <p>The genomes of <it>B. pertussis </it>and the two closely related species, <it>B. bronchiseptica </it>and <it>B. parapertussis</it>, 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 <it>B. pertussis </it>strains using the polymerase chain reaction/ligase detection reaction (PCR/LDR) assay. Six HPTs were polymorphic in this collection of <it>B. pertussis </it>strains. Of note, one of these polymorphic HPTs was found in the <it>fimX </it>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 <it>fimX </it>was found to be 3.8-fold lower in strains carrying the longer allele. HPTs in three other genes, <it>tcfA</it>, <it>bapC</it>, and BP3651, varied widely in composition across the strain collection and displayed allelic polymorphism within single cultures.</p> <p>Conclusion</p> <p>Allelic polymorphism at homopolymeric tracts is common within the <it>B. pertussis </it>genome. Phase variability may be an important mechanism in <it>B. pertussis </it>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 <it>B. pertussis </it>loci.</p

Identification of genetic variants associated with Huntington's disease progression: a genome-wide association study

Background Huntington's disease is caused by a CAG repeat expansion in the huntingtin gene, HTT. Age at onset has been used as a quantitative phenotype in genetic analysis looking for Huntington's disease modifiers, but is hard to define and not always available. Therefore, we aimed to generate a novel measure of disease progression and to identify genetic markers associated with this progression measure. Methods We generated a progression score on the basis of principal component analysis of prospectively acquired longitudinal changes in motor, cognitive, and imaging measures in the 218 indivduals in the TRACK-HD cohort of Huntington's disease gene mutation carriers (data collected 2008–11). We generated a parallel progression score using data from 1773 previously genotyped participants from the European Huntington's Disease Network REGISTRY study of Huntington's disease mutation carriers (data collected 2003–13). We did a genome-wide association analyses in terms of progression for 216 TRACK-HD participants and 1773 REGISTRY participants, then a meta-analysis of these results was undertaken. Findings Longitudinal motor, cognitive, and imaging scores were correlated with each other in TRACK-HD participants, justifying use of a single, cross-domain measure of disease progression in both studies. The TRACK-HD and REGISTRY progression measures were correlated with each other (r=0·674), and with age at onset (TRACK-HD, r=0·315; REGISTRY, r=0·234). The meta-analysis of progression in TRACK-HD and REGISTRY gave a genome-wide significant signal (p=1·12 × 10−10) on chromosome 5 spanning three genes: MSH3, DHFR, and MTRNR2L2. The genes in this locus were associated with progression in TRACK-HD (MSH3 p=2·94 × 10−8 DHFR p=8·37 × 10−7 MTRNR2L2 p=2·15 × 10−9) and to a lesser extent in REGISTRY (MSH3 p=9·36 × 10−4 DHFR p=8·45 × 10−4 MTRNR2L2 p=1·20 × 10−3). The lead single nucleotide polymorphism (SNP) in TRACK-HD (rs557874766) was genome-wide significant in the meta-analysis (p=1·58 × 10−8), and encodes an aminoacid change (Pro67Ala) in MSH3. In TRACK-HD, each copy of the minor allele at this SNP was associated with a 0·4 units per year (95% CI 0·16–0·66) reduction in the rate of change of the Unified Huntington's Disease Rating Scale (UHDRS) Total Motor Score, and a reduction of 0·12 units per year (95% CI 0·06–0·18) in the rate of change of UHDRS Total Functional Capacity score. These associations remained significant after adjusting for age of onset. Interpretation The multidomain progression measure in TRACK-HD was associated with a functional variant that was genome-wide significant in our meta-analysis. The association in only 216 participants implies that the progression measure is a sensitive reflection of disease burden, that the effect size at this locus is large, or both. Knockout of Msh3 reduces somatic expansion in Huntington's disease mouse models, suggesting this mechanism as an area for future therapeutic investigation

Role of sRNAs in the sE Dependent Cell Envelope Stress Response in E.coli

The cell envelope in gram-negative bacteria is a specialized barrier that must be constantly remodeled to suit the bacteria's needs. To accomplish this end bacteria have numerous systems in place that monitor, repair, and reconfigure their cell envelope as needed. One of the most critical systems in E.coli that participates in this function is the σE dependent cell envelope stress response. Although much is known about the protein encoded components of this critical system, little was known about the components that encode for small regulatory RNAs (sRNAs).This work clearly establishes sRNAs as a central component of the σE dependent cell envelope stress response. We uncovered many new σE dependent sRNAs, including a novel sRNA, Reg26, which shares some characteristics with the other previously known σE dependent sRNAs MicA and RybB. Not only does this work fully characterize the regulatory breadth of MicA, RybB, and Reg26, it characterizes the importance of these sRNAs by assessing their contributions to cell survival. Overall this work demonstrates how these sRNAs, in concert with the protein encoded component of the response, enables σE to monitor and maintain a trait as complex as envelope homeostasis

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

Battling Btk Mutants With Noncovalent Inhibitors That Overcome Cys481 and Thr474 Mutations

The Bruton’s tyrosine kinase (Btk) inhibitor ibrutinib has shown impressive clinical efficacy in a range of B-cell malignancies. However, acquired resistance has emerged, and second generation therapies are now being sought. Ibrutinib is a covalent, irreversible inhibitor that modifies Cys481 in the ATP binding site of Btk and renders the enzyme inactive, thereby blocking B-cell receptor signal transduction. Not surprisingly, Cys481 is the most commonly mutated Btk residue in cases of acquired resistance to ibrutinib. Mutations at other sites, including Thr474, a gatekeeper residue, have also been detected. Herein, we describe noncovalent Btk inhibitors that differ from covalent inhibitors like ibrutinib in that they do not interact with Cys481, they potently inhibit the ibrutinib-resistant Btk C481S mutant <i>in vitro</i> and in cells, and they are exquisitely selective for Btk. Noncovalent inhibitors such as GNE-431 also show excellent potency against the C481R, T474I, and T474M mutants. X-ray crystallographic analysis of Btk provides insight into the unique mode of binding of these inhibitors that explains their high selectivity for Btk and their retained activity against mutant forms of Btk. This class of noncovalent Btk inhibitors may provide a treatment option to patients, especially those who have acquired resistance to ibrutinib by mutation of Cys481 or Thr474

Structure-Based Design of Tricyclic NF-κB Inducing Kinase (NIK) Inhibitors That Have High Selectivity over Phosphoinositide-3-kinase (PI3K)

We report here structure-guided optimization of a novel series of NF-κB inducing kinase (NIK) inhibitors. Starting from a modestly potent, low molecular weight lead, activity was improved by designing a type 11/2 binding mode that accessed a back pocket past the methionine-471 gatekeeper. Divergent binding modes in NIK and PI3K were exploited to dampen PI3K inhibition while maintaining NIK inhibition within these series. Potent compounds were discovered that selectively inhibit the nuclear translocation of NF-κB2 (p52/REL-B) but not canonical NF-κB1 (REL-A/p50)

Scaffold-Hopping Approach To Discover Potent, Selective, and Efficacious Inhibitors of NF-κB Inducing Kinase

NF-κB-inducing kinase (NIK) is a protein kinase central to the noncanonical NF-κB pathway downstream from multiple TNF receptor family members, including BAFF, which has been associated with B cell survival and maturation, dendritic cell activation, secondary lymphoid organ development, and bone metabolism. We report herein the discovery of lead chemical series of NIK inhibitors that were identified through a scaffold-hopping strategy using structure-based design. Electronic and steric properties of lead compounds were modified to address glutathione conjugation and amide hydrolysis. These highly potent compounds exhibited selective inhibition of LTβR-dependent p52 translocation and transcription of NF-κB2 related genes. Compound <b>4f</b> is shown to have a favorable pharmacokinetic profile across species and to inhibit BAFF-induced B cell survival in vitro and reduce splenic marginal zone B cells in vivo