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Unlabelled: Bacterial communities are established through a combination of cooperative and antagonistic interactions between the inhabitants. Competitive interactions often involve the production of antimicrobial substances, including bacteriocins, which are small antimicrobial peptides that target other community members. Despite the nearly ubiquitous presence of bacteriocin-encoding loci, inhibitory activity has been attributed to only a small fraction of gene clusters. In this study, we characterized a novel locus (the pld locus) in the pathogen Streptococcus pneumoniae that drives the production of a bacteriocin called pneumolancidin, which has broad antimicrobial activity. The locus encodes an unusual tandem array of four inhibitory peptides, three of which are absolutely required for antibacterial activity. The three peptide sequences are similar but appear to play distinct roles in regulation and inhibition. A modification enzyme typically found in loci encoding a class of highly modified bacteriocins called lantibiotics was required for inhibitory activity. The production of pneumolancidin is controlled by a two-component regulatory system that is activated by the accumulation of modified peptides. The locus is located on a mobile element that has been found in many pneumococcal lineages, although not all elements carry the pld genes. Intriguingly, a minimal region containing only the genes required for pneumolancidin immunity was found in several Streptococcus mitis strains. The pneumolancidin-producing strain can inhibit nearly all pneumococci tested to date and provided a competitive advantage in vivo. These peptides not only represent a unique strategy for bacterial competition but also are an important resource to guide the development of new antimicrobials., Importance: Successful colonization of a polymicrobial host surface is a prerequisite for the subsequent development of disease for many bacterial pathogens. Bacterial factors that directly inhibit the growth of neighbors may provide an advantage during colonization if the inhibition of competitors outweighs the energy for production. In this work, we found that production of a potent antimicrobial called pneumolancidin conferred a competitive advantage to the pathogen Streptococcus pneumoniae. S. pneumoniae secreting pneumolancidin inhibits a wide array of Gram-positive organisms, including all but one tested pneumococcal strain. The pneumolancidin genetic locus is of particular interest because it encodes three similar modified peptides (lantibiotics), each of which has a distinct role in the function of the locus. Lantibiotics represent a relatively untapped resource for the development of clinically useful antibiotics which are desperately needed. The broad inhibitory activity of pneumolancidin makes it an ideal candidate for further characterization and development., (Copyright © 2016 Maricic et al.)

Unlabelled: The blp locus of Streptococcus pneumoniae secretes and regulates bacteriocins, which mediate both intra- and interspecific competition in the human nasopharynx. There are four major alleles of the gene blpH, which encodes the receptor responsible for activating the blp locus when bound to one of four distinct peptide pheromones (BlpC). The allelic variation of blpH is presumably explained by a need to restrict cross talk between competing strains. The BlpH protein sequences have polymorphisms distributed throughout the sequence, making identification of the peptide binding site difficult to predict. To identify the pheromone binding sites that dictate pheromone specificity, we have characterized the four major variants and two naturally occurring chimeric versions of blpH in which recombination events appear to have joined two distinct blpH alleles together. Using these allelic variants, a series of laboratory-generated chimeric blpH alleles, and site-directed mutants of both the receptor and peptide, we have demonstrated that BlpC binding to some BlpH types involves an electrostatic interaction between the oppositely charged residues of BlpC and the first transmembrane domain of BlpH. An additional recognition site was identified in the second extracellular loop. We identified naturally occurring BlpH types that have the capacity to respond to more than one BlpC type; however, this change in specificity results in a commensurate drop in overall sensitivity. These natural recombination events were presumably selected for to balance the need to sense bacteriocin-secreting neighbors with the need to turn on bacteriocin production at a low density., Importance: Bacteria use quorum sensing to optimize gene expression to accommodate for local bacterial density and diffusion rates. To prevent interception of quorum-sensing signals by neighboring strains, the genomes of single species often encode strain-specific signal/receptor pairs. The blp locus in Streptococcus pneumoniae that drives bacteriocin secretion is controlled by quorum sensing that involves the interaction of the signal/receptor pair BlpC/BlpH. We show that the pneumococcal population can be divided into several distinct BlpC/BlpH pairs; however, there are examples of naturally occurring chimeric receptors that can bind to more than one BlpC type. The trade-off for this broadened specificity is a loss of overall receptor sensitivity. This suggests that under certain conditions, the advantage of signal interception can trump the requirements for self-induction., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Nontypeable Haemophilus influenzae (NTHi) are Gram-negative coccobacilli that colonize the human pharynx, their only known natural reservoir. Adherence to the host epithelium facilitates NTHi colonization and marks one of the first steps in NTHi pathogenesis. Epithelial cell attachment is mediated, in part, by a pair of high molecular weight (HMW) adhesins that are highly immunogenic, antigenically diverse, and display a wide range of amino acid diversity both within and between isolates. In this study, the prevalence of hmwA, which encodes the HMW adhesin, was determined for a collection of 170 NTHi isolates recovered from the middle ears of children with otitis media (OM isolates) or throats or nasopharynges of healthy children (commensal isolates) from Finland, Israel, and the U.S. Overall, hmwA was detected in 61% of NTHi isolates and was significantly more prevalent (P=0.004) among OM isolates than among commensal isolates; the prevalence ratio comparing hmwA prevalence among ear isolates with that of commensal isolates was 1.47 (95% CI (1.12, 1.92)). Ninety-five percent (98/103) of the hmwA-positive NTHi isolates possessed two hmw loci. To advance our understanding of hmwA binding sequence diversity, we determined the DNA sequence of the hmwA binding region of 33 isolates from this collection. The average amino acid identity across all hmwA sequences was 62%. Phylogenetic analyses of the hmwA binding revealed four distinct sequence clusters, and the majority of hmwA sequences (83%) belonged to one of two dominant sequence clusters. hmwA sequences did not cluster by chromosomal location, geographic region, or disease status., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms. We have shown that bacterially produced antimicrobial peptides (bacteriocins) dictate the outcome of these competitive interactions. All fully-sequenced pneumococcal strains harbor a bacteriocin-like peptide (blp) locus. The blp locus encodes for a range of diverse bacteriocins and all of the highly conserved components needed for their regulation, processing, and secretion. The diversity of the bacteriocins found in the bacteriocin immunity region (BIR) of the locus is a major contributor of pneumococcal competition. Along with the bacteriocins, immunity genes are found in the BIR and are needed to protect the producer cell from the effects of its own bacteriocin. The overlay assay is a quick method for examining a large number of strains for competitive interactions mediated by bacteriocins. The overlay assay also allows for the characterization of bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. The assay is performed by pre-inoculating an agar plate with a strain to be tested for bacteriocin production followed by application of a soft agar overlay containing a strain to be tested for bacteriocin sensitivity. A zone of clearance surrounding the stab indicates that the overlay strain is sensitive to the bacteriocins produced by the pre-inoculated strain. If no zone of clearance is observed, either the overlay strain is immune to the bacteriocins being produced or the pre-inoculated strain does not produce bacteriocins. To determine if the blp locus is functional in a given strain, the overlay assay can be adapted to evaluate for peptide pheromone secretion by the pre-inoculated strain. In this case, a series of four lacZ-reporter strains with different pheromone specificity are used in the overlay.

Nontypeable Haemophilus influenzae (NTHi) is a bacterium that resides within the human pharynx. Because NTHi is human-restricted, its long-term survival is dependent upon its ability to successfully colonize new hosts. Adherence to host epithelium, mediated by bacterial adhesins, is one of the first steps in NTHi colonization. NTHi express several adhesins, including the high molecular weight (HMW) adhesins that mediate attachment to the respiratory epithelium where they interact with the host immune system to elicit a strong humoral response. hmwA, which encodes the HMW adhesin, undergoes phase variation mediated by 7-base pair tandem repeats located within its promoter region. Repeat number affects both hmwA transcription and HMW-adhesin production such that as the number of repeats increases, adhesin production decreases. Cells expressing large amounts of HMW adhesins may be critical for the establishment and maintenance of NTHi colonization, but they might also incur greater fitness costs when faced with an adhesin-specific antibody-mediated immune response. We hypothesized that the occurrence of large deletion events within the hmwA repeat region allows NTHi cells to maintain adherence in the presence of antibody-mediated immunity. To study this, we developed a mathematical model, incorporating hmwA phase variation and antibody-mediated immunity, to explore the trade-off between bacterial adherence and immune evasion. The model predicts that antibody levels and avidity, catastrophic loss rates, and population carrying capacity all significantly affected numbers of adherent NTHi cells within a host. These results suggest that the occurrence of large, yet rare, deletion events allows for stable maintenance of a small population of adherent cells in spite of HMW adhesin specific antibody-mediated immunity. These adherent subpopulations may be important for sustaining colonization and/or maintaining transmission., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

A significant fraction of seasonal and in particular pandemic influenza deaths are attributed to secondary bacterial infections. In animal models, influenza virus predisposes hosts to severe infection with both Streptococcus pneumoniae and Staphylococcus aureus. Despite its importance, the mechanistic nature of the interaction between influenza and pneumococci, its dependence on the timing and sequence of infections as well as the clinical and epidemiological consequences remain unclear. We explore an immune-mediated model of the viral-bacterial interaction that quantifies the timing and the intensity of the interaction. Taking advantage of the wealth of knowledge gained from animal models, and the quantitative understanding of the kinetics of pathogen-specific immunological dynamics, we formulate a mathematical model for immune-mediated interaction between influenza virus and S. pneumoniae in the lungs. We use the model to examine the pathogenic effect of inoculum size and timing of pneumococcal invasion relative to influenza infection, as well as the efficacy of antivirals in preventing severe pneumococcal disease. We find that our model is able to capture the key features of the interaction observed in animal experiments. The model predicts that introduction of pneumococcal bacteria during a 4-6 day window following influenza infection results in invasive pneumonia at significantly lower inoculum size than in hosts not infected with influenza. Furthermore, we find that antiviral treatment administered later than 4 days after influenza infection was not able to prevent invasive pneumococcal disease. This work provides a quantitative framework to study interactions between influenza and pneumococci and has the potential to accurately quantify the interactions. Such quantitative understanding can form a basis for effective clinical care, public health policies and pandemic preparedness.

All fully sequenced strains of Streptococcus pneumoniae (pneumococcus) contain a version of the blp locus which is responsible for the regulation and secretion of a variable repertoire of pneumococcal bacteriocins called pneumocins and their associated immunity proteins. Pneumocins mediate intra- and interspecies competition in vitro and have been shown to provide a competitive advantage in vivo. Pneumocin production is stimulated by extracellular accumulation of the peptide pheromone, BlpC. Both BlpC and the functional pneumocins are secreted out of the cell via the Blp transporter, BlpAB. The conserved surface-expressed serine protease, HtrA, has been shown to limit activation of the locus and secretion of functional pneumocins. In this work, we demonstrate that htrA mutants stimulate the blp locus at lower cell density and to a greater extent than strains expressing wild-type HtrA. This effect is not due to direct proteolytic degradation of secreted pheromone by the protease, but instead is a result of HtrA-mediated disruption of peptide processing and secretion. Because pneumocins are secreted through the same transporter as the pheromone, this finding explains why pheromone supplementation cannot completely restore pneumocin inhibition to strains expressing high levels of HtrA despite restoration of blp transcriptional activity. HtrA restricts pneumocin production to high cell density by limiting the rate of accumulation of BlpC in the environment. Importantly, HtrA does not interfere with the ability of a strain to sense environmental pheromones, which is necessary for the induction of protective immunity in the face of pneumocin-secreting competitors.

Discoveries made during the 1918 influenza A pandemic and reports of severe disease associated with coinfection during the 2009 hemagglutinin type 1 and neuraminidase type 1 (commonly known as H1N1 or swine flu) pandemic have renewed interest in the role of coinfection in disease pathogenesis. The authors assessed how various timings of coinfection with influenza virus and pneumonia-causing bacteria could affect the severity of illness at multiple levels of interaction, including the biologic and population levels. Animal studies most strongly support a single pathway of coinfection with influenza inoculation occurring approximately 7 days before inoculation with Streptococcus pneumoniae, but less-examined pathways of infection also may be important for human disease. The authors discussed the implications of each pathway for disease prevention and what they would expect to see at the population level if there were sufficient data available. Lastly, the authors identified crucial gaps in the study of timing of coinfection and proposed related research questions.

Unlabelled: All fully sequenced strains of Streptococcus pneumoniae possess a version of the blp locus, which is responsible for bacteriocin production and immunity. Activation of the blp locus is stimulated by accumulation of the peptide pheromone, BlpC, following its secretion by the ABC transporter, BlpA. The blp locus is characterized by significant diversity in blpC type and in the region of the locus containing putative bacteriocin and immunity genes. In addition, the blpA gene can represent a single large open reading frame or be divided into several smaller fragments due to the presence of frameshift mutations. In this study, we use a collection of strains with blp-dependent inhibition and immunity to define the genetic changes that bring about phenotypic differences in bacteriocin production or immunity. We demonstrate that alterations in blpA, blpC, and bacteriocin/immunity content likely play an important role in competitive interactions between pneumococcal strains. Importantly, strains with a highly conserved frameshift mutation in blpA are unable to secrete bacteriocins or BlpC, but retain the ability to respond to exogenous peptide pheromone produced by cocolonizing strains, stimulating blp-mediated immunity. These "cheater" strains can only coexist with bacteriocin-producing strains that secrete their cognate BlpC and share the same immunity proteins. The variable outcome of these interactions helps to explain the heterogeneity of the blp pheromone, bacteriocin, and immunity protein content., Importance: Streptococcus pneumoniae resides in a polymicrobial environment and competes for limited resources by the elaboration of small antimicrobial peptides called bacteriocins. A conserved cluster of genes in the S. pneumoniae genome is involved in the production of bacteriocins and their associated protective immunity proteins through secretion of a signaling pheromone. In this study, we show that a significant number of strains have lost the ability to secrete bacteriocins and signaling pheromones due to a specific mutation in a dedicated transporter protein. Because the regulatory and immunity portion of the locus is retained, these "cheater" strains can survive in the face of invasion from a bacteriocin-producing strain without the cost of bacteriocin secretion. The outcome of such interactions depends on each strain's repertoire of pheromone, immunity protein, and bacteriocin genes, such that intrastrain competition drives the diversity in bacteriocin, immunity protein, and pheromone content.

We report on the comparative genomics and characterization of the virulence phenotypes of four S. pneumoniae strains that belong to the multidrug resistant clone PMEN1 (Spain(23F) ST81). Strains SV35-T23 and SV36-T3 were recovered in 1996 from the nasopharynx of patients at an AIDS hospice in New York. Strain SV36-T3 expressed capsule type 3 which is unusual for this clone and represents the product of an in vivo capsular switch event. A third PMEN1 isolate - PN4595-T23 - was recovered in 1996 from the nasopharynx of a child attending day care in Portugal, and a fourth strain - ATCC700669 - was originally isolated from a patient with pneumococcal disease in Spain in 1984. We compared the genomes among four PMEN1 strains and 47 previously sequenced pneumococcal isolates for gene possession differences and allelic variations within core genes. In contrast to the 47 strains - representing a variety of clonal types - the four PMEN1 strains grouped closely together, demonstrating high genomic conservation within this lineage relative to the rest of the species. In the four PMEN1 strains allelic and gene possession differences were clustered into 18 genomic regions including the capsule, the blp bacteriocins, erythromycin resistance, the MM1-2008 prophage and multiple cell wall anchored proteins. In spite of their genomic similarity, the high resolution chinchilla model was able to detect variations in virulence properties of the PMEN1 strains highlighting how small genic or allelic variation can lead to significant changes in pathogenicity and making this set of strains ideal for the identification of novel virulence determinants.

The blp locus of a type 6A strain of Streptococcus pneumoniae encodes a two-peptide bacteriocin, pneumocin MN, which mediates intraspecies competition during mouse nasopharyngeal colonization. This locus is regulated by a quorum-sensing mechanism consisting of a dedicated two-component regulatory system and a peptide pheromone. Like most clinical isolates, this type 6A strain can be separated into opaque and transparent colony variants, each playing a different role during pneumococcal infection. In this study, we show that the blp locus is differentially regulated at the posttranscriptional level in pneumococcal opacity variants. Transparent and opaque variants produce equivalent amounts of blpMNPO transcript when stimulated with a synthetic pheromone, but transparent variants have no pneumocin MN-mediated inhibitory activity while opaque variants produce large zones of inhibitory activity. The differential regulation in opacity variants is driven by the two-component regulatory system CiaRH via its regulation of the serine protease HtrA. Transparent mutants deficient in CiaH or HtrA show increased pneumocin MN-mediated inhibition. In addition, these mutants demonstrate alterations in their dose response to a synthetic peptide pheromone, suggesting that HtrA activity impacts pneumocin MN production at the level of signaling. This, in addition to its known effects on competence, suggests that HtrA is a pleiotropic regulator whose protease activity affects several important bacterial pathways. The complex regulation of pneumocins may allow the pneumococcus to reserve the secretion of active peptides for situations where the benefit of their inhibitory activity outweighs the cost of their production.

The introduction of the conjugate seven-valent pneumococcal vaccine has led to the replacement of vaccine serotypes with nonvaccine serotypes of Streptococcus pneumoniae. This observation implies that intraspecies competition between pneumococci occurs during nasopharyngeal colonization, allowing one strain or set of strains to predominate over others. We investigated the contribution of the blp locus, encoding putative bacteriocins and cognate immunity peptides, to intraspecies competition. We sequenced the relevant regions of the blp locus of a type 6A strain able to inhibit the growth of the type 4 strain, TIGR4, in vitro. Using deletional analysis, we confirmed that inhibitory activity is regulated by the function of the response regulator, BlpR, and requires the two putative bacteriocin genes blpM and blpN. Comparison of the TIGR4 BlpM and -N amino acid sequences demonstrated that only five amino acid differences were sufficient to target the heterologous strain. Analysis of a number of clinical isolates suggested that the BlpMN bacteriocins divide into two families. A mutant in the blpMN operon created in the clinically relevant type 19A background was deficient in both bacteriocin activity and immunity. This strain was unable to compete with both its parent strain and a serotype 4 isolate during cocolonization in the mouse nasopharynx, suggesting that the locus is functional in vivo and confirming its role in promoting intraspecies competition.

Nontypeable Haemophilus influenzae is an important cause of localized respiratory tract disease, which begins with colonization of the upper respiratory mucosa. In previous work we reported that the nontypeable H. influenzae HMW1 and HMW2 proteins are high-molecular-weight nonpilus adhesins responsible for attachment to human epithelial cells, an essential step in the process of colonization. Interestingly, although HMW1 and HMW2 share significant sequence similarity, they display distinct cellular binding specificities. In order to map the HMW1 and HMW2 binding domains, we generated a series of complementary HMW1-HMW2 chimeric proteins and examined the ability of these proteins to promote in vitro adherence by Escherichia coli DH5alpha. Using this approach, we localized the HMW1 and HMW2 binding domains to an approximately 360-amino-acid region near the N terminus of the mature HMW1 and HMW2 proteins. Experiments with maltose-binding protein fusion proteins containing segments of either HMW1 or HMW2 confirmed these results and suggested that the fully functional binding domains may be conformational structures that require relatively long stretches of sequence. Of note, the HMW1 and HMW2 binding domains correspond to areas of maximal sequence dissimilarity, suggesting that selective advantage associated with broader adhesive potential has been a major driving force during H. influenzae evolution. These findings should facilitate efforts to develop a subcomponent vaccine effective against nontypeable H. influenzae disease.

Non-typable Haemophilus influenzae is a common commensal organism in the human upper respiratory tract and an important cause of localized respiratory tract disease. The pathogenesis of disease begins with bacterial colonization of the nasopharynx, a process that involves establishment on the mucosal surface and evasion of local immune mechanisms. Under the proper circumstances, the organism spreads contiguously to the middle ear, the sinuses, or the lungs, and then stimulates a brisk inflammatory response, producing symptomatic infection. In this review, we summarize our present understanding of the molecular determinants of this sequence of events. Continued investigation of the molecular mechanism of non-typable H. influenzae pathogenicity should facilitate development of novel approaches to the treatment and prevention of H. influenzae disease.

Expression of a number of eukaryotic genes is regulated by long stretches of tandem repeats located within the 5' untranslated region of the particular gene. In this study, we describe a regulatory system in Haemophilus influenzae with striking similarities to those found in eukaryotes. We show that expression of the HMW1 and HMW2 adhesins varies based on the number of 7-bp tandem repeats in the hmw1A and hmw2A promoters. The repeats lie between two separate transcription initiation sites and exert a repressive effect, such that increases in repeat number result in step-wise decreases in levels of specific mRNA and protein production and vice versa. The range of expression of HMW1 and HMW2 varies between very weak and very strong, with a series of gradations in between. Variation in the number of repeats in the hmw1A and hmw2A promoters occurs in individual colonies passaged in vitro, in an animal model of infection, and during natural infection in humans. This system of regulation is unique in prokaryotes and likely enhances the pathogenicity of the organism by increasing adaptive potential.

The excitability of the visual system was studied in ten adult chronic cats. Visual evoked potentials were recorded, using decreasing interstimulus intervals. A decrease of the excitability of the visual system is observed when interstimulus intervals are less than 800 milliseconds. Clinical applications with regard to visual evoked potential recording on comatose patients are suggested.

The axes: amplitude, frequency, time, define a trihedral where the most usual representations occur in time domain (plane defined by the axes amplitude-time). The frequency of an action with respect to time is represented in the plane defined by the axes frequency-time in the so called "Modulation Domain", it is being considered very useful, expressive and easy to construct in the automatic treatment of signals.

Research of the alcohol action mechanism on the SNC in acute alcoholic intoxication (AAI) has been dealt in various ways. On one side the alcohol action--apparently most unspecific--on cellular membranes has been studied. Other authors, instead, have studied more specific alcohol effects on three types of neurotransmitters: opioid peptides, GABA and catecholamines. The effect of alcohol on cellular membranes seems to be beyond any doubt. Alcohol action on specific neurotransmitters is the object of controversy, especially in the case of endogenous opioids. There are data which strongly support the participation of the GABA receptors in the AAI. Modifications produced in the cellular membrane by alcohol action can modify the structure of the function of the membrane receptors. On the other hand, distinct receptors may be localized in the same neuron, while the existence of interactions between different neurotransmitters is well known. Therefore, the various hypotheses previously stated are not mutually exclusive.

The effect of an i.v. bolus injection of nicotine 50 micrograms/kg-1, was compared in 6 awake dogs with the effect of nicotine injection after naloxone, 400 micrograms/kg-1. Respiratory inhibition was produced immediately after beginning the nicotine injection, followed by hyperpnea. Naloxone produced a significant decrease of the respiratory inhibition followed by hyperpnea increase compared to the trial with only nicotine.