Two Group A Streptococcal Peptide Pheromones Act through Opposing Rgg Regulators to Control Biofilm Development

Streptococcus pyogenes (Group A Streptococcus, GAS) is an important human commensal that occasionally causes localized infections and less frequently causes severe invasive disease with high mortality rates. How GAS regulates expression of factors used to colonize the host and avoid immune responses remains poorly understood. Intercellular communication is an important means by which bacteria coordinate gene expression to defend against host assaults and competing bacteria, yet no conserved cell-to-cell signaling system has been elucidated in GAS. Encoded within the GAS genome are four rgg-like genes, two of which (rgg2 and rgg3) have no previously described function. We tested the hypothesis that rgg2 or rgg3 rely on extracellular peptides to control target-gene regulation. We found that Rgg2 and Rgg3 together tightly regulate two linked genes encoding new peptide pheromones. Rgg2 activates transcription of and is required for full induction of the pheromone genes, while Rgg3 plays an antagonistic role and represses pheromone expression. The active pheromone signals, termed SHP2 and SHP3, are short and hydrophobic (DI[I/L]IIVGG), and, though highly similar in sequence, their ability to disrupt Rgg3-DNA complexes were observed to be different, indicating that specificity and differential activation of promoters are characteristics of the Rgg2/3 regulatory circuit. SHP-pheromone signaling requires an intact oligopeptide permease (opp) and a metalloprotease (eep), supporting the model that pro-peptides are secreted, processed to the mature form, and subsequently imported to the cytoplasm to interact directly with the Rgg receptors. At least one consequence of pheromone stimulation of the Rgg2/3 pathway is increased biogenesis of biofilms, which counteracts negative regulation of biofilms by RopB (Rgg1). These data provide the first demonstration that Rgg-dependent quorum sensing functions in GAS and substantiate the role that Rggs play as peptide receptors across the Firmicute phylum

Modular Architecture and Unique Teichoic Acid Recognition Features of Choline-Binding Protein L (CbpL) Contributing to Pneumococcal Pathogenesis

The human pathogen Streptococcus pneumoniae is decorated with a special class of surface-proteins known as choline-binding proteins (CBPs) attached to phosphorylcholine (PCho) moieties from cell-wall teichoic acids. By a combination of X-ray crystallography, NMR, molecular dynamics techniques and in vivo virulence and phagocytosis studies, we provide structural information of choline-binding protein L (CbpL) and demonstrate its impact on pneumococcal pathogenesis and immune evasion. CbpL is a very elongated three-module protein composed of (i) an Excalibur Ca 2+ -binding domain -reported in this work for the very first time-, (ii) an unprecedented anchorage module showing alternate disposition of canonical and non-canonical choline-binding sites that allows vine-like binding of fully-PCho-substituted teichoic acids (with two choline moieties per unit), and (iii) a Ltp-Lipoprotein domain. Our structural and infection assays indicate an important role of the whole multimodular protein allowing both to locate CbpL at specific places on the cell wall and to interact with host components in order to facilitate pneumococcal lung infection and transmigration from nasopharynx to the lungs and blood. CbpL implication in both resistance against killing by phagocytes and pneumococcal pathogenesis further postulate this surface-protein as relevant among the pathogenic arsenal of the pneumococcus.We gratefully acknowledge Karsta Barnekow and Kristine Sievert-Giermann, for technical assistance and Lothar Petruschka for in silico analysis (all Dept. of Genetics, University of Greifswald). We are further grateful to the staff from SLS synchrotron beamline for help in data collection. This work was supported by grants from the Deutsche Forschungsgemeinschaft DFG GRK 1870 (to SH) and the Spanish Ministry of Economy and Competitiveness (BFU2014-59389-P to JAH, CTQ2014-52633-P to MB and SAF2012-39760-C02-02 to FG) and S2010/BMD- 2457 (Community of Madrid to JAH and FG).Peer Reviewe

Exclusion of an Exotic Top Quark with -4/3 Electric Charge Using Soft Lepton Tagging

We present a measurement of the electric charge of the top quark using \ppbar collisions corresponding to an integrated luminosity of 2.7~fb$^{-1}$ at the CDF II detector. We reconstruct \ttbar events in the lepton+jets final state and use kinematic information to determine which $b$-jet is associated with the leptonically- or hadronically-decaying $t$-quark. Soft lepton taggers are used to determine the $b$-jet flavor. Along with the charge of the $W$ boson decay lepton, this information permits the reconstruction of the top quark's electric charge. Out of 45 reconstructed events with $2.4\pm0.8$ expected background events, 29 are reconstructed as \ttbar with the standard model $+$2/3 charge, whereas 16 are reconstructed as \ttbar with an exotic $-4/3$ charge. This is consistent with the standard model and excludes the exotic scenario at 95\% confidence level. This is the strongest exclusion of the exotic charge scenario and the first to use soft leptons for this purpose.We present a measurement of the electric charge of the top quark using pp̅ collisions corresponding to an integrated luminosity of 2.7  fb-1 at the CDF II detector. We reconstruct tt̅ events in the lepton+jets final state. We use soft lepton taggers to determine the flavor of the b jets, which we use to reconstruct the top quark’s electric charge and exclude an exotic top quark with -4/3 charge at 95% confidence level. This is the strongest exclusion of the exotic charge scenario and the first to use soft leptons for this purpose.Peer reviewe

Elimination of Chromosomal Island SpyCIM1 from Streptococcus pyogenes Strain SF370 Reverses the Mutator Phenotype and Alters Global Transcription

This work was made possible by an Oklahoma Center for the Advancement of Science and Technology (OCAST) grant HR11-133 and by NIH Grant Number R15A1072718 to WMM and NIH Grant AI11822 to VAF.Streptococcus pyogenes chromosomal island M1 (SpyCIM1) integrates by site-specific recombination into the 5’ end of DNA mismatch repair (MMR) gene mutL in strain SF370SmR, blocking transcription of it and the downstream operon genes. During exponential growth, SpyCIM1 excises from the chromosome and replicates as an episome, restoring mutL transcription. This process is reversed in stationary phase with SpyCIM1 re-integrating into mutL, returning the cells to a mutator phenotype. Here we show that elimination of SpyCIM1 relieves this mutator phenotype. The downstream MMR operon genes, multidrug efflux pump lmrP, Holliday junction resolution helicase ruvA, and DNA base excision repair glycosylase tag, are also restored to constitutive expression by elimination of SpyCIM1. The presence of SpyCIM1 alters global transcription patterns in SF370SmR. RNA sequencing (RNA-Seq) demonstrated that loss of SpyCIM1 in the SpyCIM1 deletion mutant, CEM1Δ4, impacted the expression of over 100 genes involved in virulence and metabolism both in early exponential phase, when the SpyCIM1 is episomal, as well as at the onset of stationary phase, when SpyCIM1 has reintegrated into mutL. Among these changes, the up-regulation of the genes for the antiphagocytic M protein (emm1), streptolysin O (slo), capsule operon (hasABC), and streptococcal pyrogenic exotoxin (speB), are particularly notable. The expression pattern of the MMR operon confirmed our earlier observations that these genes are transcribed in early exponential phase but silenced as stationary phase is approached. Thus, the direct role of SpyCIM1 in causing the mutator phenotype is confirmed, and further, its influence upon the biology of S. pyogenes was found to impact multiple genes in addition to the MMR operon, which is a novel function for a mobile genetic element. We suggest that such chromosomal islands are a remarkable evolutionary adaptation to promote the survival of its S. pyogenes host cell in changing environments.Yeshttp://www.plosone.org/static/editorial#pee

The Relevance of Dissociations and the Irrelevance of Dissociationism: A Reply to Schwartz and Pritchard

This note examines Schwartz and Pritchard\u27s critique of Donchin and Lindsley\u27s study of the ERPs and retroactive masking. The critique is seen to be an example of the Dissociationist posture in which the failure to confirm a reported relationship between performance and ERPs is reported. The Dissociationist treats psychophysiology as an attempt to establish universally applicable correlations between performance measures and physiological measures. Such universality seems to be the necessary attribute of “physiological correlates” of psychological constructs. To demonstrate that ERPs are not correlates of perception, Schwartz and Pritchard try to show that the correlation reported by Donchin and Lindsley does not hold for U‐shaped masking functions. Donchin and Lindsley did not, however, attempt to establish a universal correlation. Rather, they attempted to utilize the parallelism between ERPs and two‐flash interactions to test models proposed to account for retroactive masking and enhancement. That the relationship is not universal, in that there are masking phenomena wherein the correlation fails, is quite irrelevant to the purpose. The methodology used by Schwartz and Pritchard is also questioned here. Specifically, it is noted that they err in combining in one curve, data obtained with a single stimulus with data obtained with two stimuli. They are thus led by focusing on a product, the d\u27, to the unwarranted assumption that the underlying processes are uniform