scholarly journals An Electrostatic Interaction between BlpC and BlpH Dictates Pheromone Specificity in the Control of Bacteriocin Production and Immunity in Streptococcus pneumoniae

2015 ◽  
Vol 197 (7) ◽  
pp. 1236-1248 ◽  
Author(s):  
Marisa D. Pinchas ◽  
Nathan C. LaCross ◽  
Suzanne Dawid
Keyword(s):  
Streptococcus Pneumoniae ◽  
Quorum Sensing ◽  
Electrostatic Interaction ◽  
Transmembrane Domain ◽  
Allelic Variation ◽  
Single Species ◽  
Bacteriocin Production ◽  
Content Type ◽  
Naturally Occurring ◽  
Pheromone Specificity

ABSTRACTTheblplocus ofStreptococcus pneumoniaesecretes and regulates bacteriocins, which mediate both intra- and interspecific competition in the human nasopharynx. There are four major alleles of the geneblpH, which encodes the receptor responsible for activating theblplocus when bound to one of four distinct peptide pheromones (BlpC). The allelic variation ofblpHis 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 ofblpHin which recombination events appear to have joined two distinctblpHalleles together. Using these allelic variants, a series of laboratory-generated chimericblpHalleles, 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.IMPORTANCEBacteria 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. Theblplocus inStreptococcus pneumoniaethat 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.

scholarly journals Antibodies to Streptococcus pneumoniae Capsular Polysaccharide Enhance Pneumococcal Quorum Sensing

mBio ◽  
2011 ◽  
Vol 2 (5) ◽  
Author(s):  
Masahide Yano ◽  
Shruti Gohil ◽  
J. Robert Coleman ◽  
Catherine Manix ◽  
Liise-anne Pirofski
Keyword(s):  
Monoclonal Antibodies ◽  
Streptococcus Pneumoniae ◽  
Quorum Sensing ◽  
Direct Effect ◽  
Pneumococcal Disease ◽  
Effector Cell ◽  
Capsular Polysaccharide ◽  
Genetic Exchange ◽  
Content Type ◽  
Specific Antibodies

ABSTRACTThe use of pneumococcal capsular polysaccharide (PPS)-based vaccines has resulted in a substantial reduction in invasive pneumococcal disease. However, much remains to be learned about vaccine-mediated immunity, as seven-valent PPS-protein conjugate vaccine use in children has been associated with nonvaccine serotype replacement and 23-valent vaccine use in adults has not prevented pneumococcal pneumonia. In this report, we demonstrate that certain PPS-specific monoclonal antibodies (MAbs) enhance the transformation frequency of two differentStreptococcus pneumoniaeserotypes. This phenomenon was mediated by PPS-specific MAbs that agglutinate but do not promote opsonic effector cell killing of the homologous serotypeinvitro. Compared to the autoinducer, competence-stimulating peptide (CSP) alone, transcriptional profiling of pneumococcal gene expression after incubation with CSP and one such MAb to the PPS of serotype 3 revealed changes in the expression of competence (com)-related and bacteriocin-like peptide (blp) genes involved in pneumococcal quorum sensing. This MAb was also found to induce a nearly 2-fold increase in CSP2-mediated bacterial killing or fratricide. These observations reveal a novel, direct effect of PPS-binding MAbs on pneumococcal biology that has important implications for antibody immunity to pneumococcus in the pneumococcal vaccine era. Taken together, our data suggest heretofore unsuspected mechanisms by which PPS-specific antibodies could affect genetic exchange and bacterial viability in the absence of host cells.IMPORTANCECurrent thought holds that pneumococcal capsular polysaccharide (PPS)-binding antibodies protect against pneumococcus by inducing effector cell opsonic killing of the homologous serotype. While such antibodies are an important part of how pneumococcal vaccines protect against pneumococcal disease, PPS-specific antibodies that do not exhibit this activity but are highly protective against pneumococcus in mice have been identified. This article examines the effect of nonopsonic PPS-specific monoclonal antibodies (MAbs) on the biology ofStreptococcus pneumoniae. The results showed that in the presence of a competence-stimulating peptide (CSP), such MAbs increase the frequency of pneumococcal transformation. Further studies with one such MAb showed that it altered the expression of genes involved in quorum sensing and increased competence-induced killing or fratricide. These findings reveal a novel, previously unsuspected mechanism by which certain PPS-specific antibodies exert a direct effect on pneumococcal biology that has broad implications for bacterial clearance, genetic exchange, and antibody immunity to pneumococcus.

scholarly journals Functional Determinants of Metal Ion Transport and Selectivity in Paralogous Cation Diffusion Facilitator Transporters CzcD and MntE in Streptococcus pneumoniae

2016 ◽  
Vol 198 (7) ◽  
pp. 1066-1076 ◽  
Author(s):  
Julia E. Martin ◽  
David P. Giedroc
Keyword(s):  
Streptococcus Pneumoniae ◽  
Metal Ion ◽  
Transmembrane Domain ◽  
Divalent Metal ◽  
Cation Diffusion ◽  
Metal Specificity ◽  
Cation Diffusion Facilitator ◽  
Content Type ◽  
Metal Selectivity ◽  
A Site

ABSTRACTCation diffusion facilitators (CDFs) are a large family of divalent metal transporters that collectively possess broad metal specificity and contribute to intracellular metal homeostasis and virulence in bacterial pathogens.Streptococcus pneumoniaeexpresses two homologous CDF efflux transporters, MntE and CzcD. Cells lackingmntEorczcDare sensitive to manganese (Mn) or zinc (Zn) toxicity, respectively, and specifically accumulate Mn or Zn, respectively, thus suggesting that MntE selectively transports Mn, while CzcD transports Zn. Here, we probe the origin of this metal specificity using a phenotypic growth analysis of pneumococcal variants. Structural homology toEscherichia coliYiiP predicts that both MntE and CzcD are dimeric and each protomer harbors four pairs of conserved metal-binding sites, termed the A site, the B site, and the C1/C2 binuclear site. We find that single amino acid mutations within both the transmembrane domain A site and the B site in both CDFs result in a cellular metal sensitivity similar to that of the corresponding null mutants. However, multiple mutations in the predicted cytoplasmic C1/C2 cluster of MntE have no impact on cellular Mn resistance, in contrast to the analogous substitutions in CzcD, which do have on impact on cellular Zn resistance. Deletion of the MntE-specific C-terminal tail, present only in Mn-specific bacterial CDFs, resulted in only a modest growth phenotype. Further analysis of MntE-CzcD functional chimeric transporters showed that Asn and Asp in theND-DD A-site motif of MntE and the most N-terminal His in theHD-HD site A of CzcD (the specified amino acids are underlined) play key roles in transporter metal selectivity.IMPORTANCECation diffusion facilitator (CDF) proteins are divalent metal ion transporters that are conserved in organisms ranging from bacteria to humans and that play important roles in cellular physiology, from metal homeostasis and resistance to type I diabetes in vertebrates. The respiratory pathogenStreptococcus pneumoniaeexpresses two metal CDF transporters, CzcD and MntE. How CDFs achieve metal selectivity is unclear. We show here that CzcD and MntE are true paralogs, as CzcD transports zinc, while MntE selectively transports manganese. Through the use of an extensive collection of pneumococcal variants, we show that a primary determinant for metal selectivity is the A site within the transmembrane domain. This extends our understanding of how CDFs discriminate among transition metals.

scholarly journals The LuxS-Dependent Quorum-Sensing System Regulates Early Biofilm Formation by Streptococcus pneumoniae Strain D39

2011 ◽  
Vol 79 (10) ◽  
pp. 4050-4060 ◽  
Author(s):  
Jorge E. Vidal ◽  
Herbert P. Ludewick ◽  
Rebekah M. Kunkel ◽  
Dorothea Zähner ◽  
Keith P. Klugman
Keyword(s):  
Streptococcus Pneumoniae ◽  
Quorum Sensing ◽  
Biofilm Formation ◽  
Single Copy ◽  
Middle Ear Mucosa ◽  
Transcript Levels ◽  
Content Type ◽  
Sensing System ◽  
Quorum Sensing System

ABSTRACTStreptococcus pneumoniaeis the leading cause of death in children worldwide and forms highly organized biofilms in the nasopharynx, lungs, and middle ear mucosa. TheluxS-controlled quorum-sensing (QS) system has recently been implicated in virulence and persistence in the nasopharynx, but its role in biofilms has not been studied. Here we show that this QS system plays a major role in the control ofS. pneumoniaebiofilm formation. Our results demonstrate that theluxSgene is contained by invasive isolates and normal-flora strains in a region that contains genes involved in division and cell wall biosynthesis. TheluxSgene was maximally transcribed, as a monocistronic message, in the early mid-log phase of growth, and this coincides with the appearance of early biofilms. Demonstrating the role of the LuxS system in regulatingS. pneumoniaebiofilms, at 24 h postinoculation, two different D39ΔluxSmutants produced ∼80% less biofilm biomass than wild-type (WT) strain D39 did. Complementation of these strains withluxS, either in a plasmid or integrated as a single copy in the genome, restored their biofilm level to that of the WT. Moreover, a soluble factor secreted by WT strain D39 or purified AI-2 restored the biofilm phenotype of D39ΔluxS. Our results also demonstrate that during the early mid-log phase of growth, LuxS regulates the transcript levels oflytA, which encodes an autolysin previously implicated in biofilms, and also the transcript levels ofply, which encodes the pneumococcal pneumolysin. In conclusion, theluxS-controlled QS system is a key regulator of early biofilm formation byS. pneumoniaestrain D39.

scholarly journals Conserved Mutations in the Pneumococcal Bacteriocin Transporter Gene, blpA, Result in a Complex Population Consisting of Producers and Cheaters

mBio ◽  
2011 ◽  
Vol 2 (5) ◽  
Author(s):  
Matthew R. Son ◽  
Mikhail Shchepetov ◽  
Peter V. Adrian ◽  
Shabir A. Madhi ◽  
Linda de Gouveia ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Bacteriocin Production ◽  
Genetic Changes ◽  
Immunity Protein ◽  
Content Type ◽  
Reading Frame ◽  
Transporter Protein ◽  
Peptide Pheromone ◽  
Complex Population ◽  
The Face

ABSTRACT 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.

scholarly journals Timing Is Everything: Impact of Naturally Occurring Staphylococcus aureus AgrC Cytoplasmic Domain Adaptive Mutations on Autoinduction

2019 ◽  
Vol 201 (20) ◽  
Author(s):  
Tim J. Sloan ◽  
Ewan Murray ◽  
Maho Yokoyama ◽  
Ruth C. Massey ◽  
Weng C. Chan ◽  
...  
Keyword(s):  
Molecular Structure ◽  
Staphylococcus Aureus ◽  
Quorum Sensing ◽  
Binding Site ◽  
Conformational Changes ◽  
Cytoplasmic Domain ◽  
Atp Binding ◽  
Sensor Kinase ◽  
Content Type ◽  
Naturally Occurring

ABSTRACT Mutations in the polymorphic Staphylococcus aureus agr locus responsible for quorum sensing (QS)-dependent virulence gene regulation occur frequently during host adaptation. In two genomically closely related S. aureus clinical isolates exhibiting marked differences in Panton-Valentine leukocidin production, a mutation conferring an N267I substitution was identified in the cytoplasmic domain of the QS sensor kinase, AgrC. This natural mutation delayed the onset and accumulation of autoinducing peptide (AIP) and showed reduced responsiveness to exogenous AIPs. Other S. aureus strains harboring naturally occurring AgrC cytoplasmic domain mutations were identified, including T247I, I311T, A343T, L245S, and F264C. These mutations were associated with reduced cytotoxicity, delayed/reduced AIP production, and impaired sensitivity to exogenous AIP. Molecular dynamics simulations were used to model the AgrC cytoplasmic domain conformational changes arising. Although mutations were localized in different parts of the C-terminal domain, their impact on molecular structure was manifested by twisting of the leading helical hairpin α1-α2, accompanied by repositioning of the H-box and G-box, along with closure of the flexible loop connecting the two and occlusion of the ATP-binding site. Such conformational rearrangements of key functional subdomains in these mutants highlight the cooperative response of molecular structure involving dimerization and ATP binding and phosphorylation, as well as the binding site for the downstream response element AgrA. These appear to increase the threshold for agr activation via AIP-dependent autoinduction, thus reducing virulence and maintaining S. aureus in an agr-downregulated “colonization” mode. IMPORTANCE Virulence factor expression in Staphylococcus aureus is regulated via autoinducing peptide (AIP)-dependent activation of the sensor kinase AgrC, which forms an integral part of the agr quorum sensing system. In response to bound AIP, the cytoplasmic domain of AgrC (AgrC-cyt) undergoes conformational changes resulting in dimerization, autophosphorylation, and phosphotransfer to the response regulator AgrA. Naturally occurring mutations in AgrC-cyt are consistent with repositioning of key functional domains, impairing dimerization and restricting access to the ATP-binding pocket. Strains harboring specific AgrC-cyt mutations exhibit reduced AIP autoinduction efficiency and a timing-dependent attenuation of cytotoxicity which may confer a survival advantage during established infection by promoting colonization while restricting unnecessary overproduction of exotoxins.

scholarly journals Competitive Dominance within Biofilm Consortia Regulates the Relative Distribution of Pneumococcal Nasopharyngeal Density

2017 ◽  
Vol 83 (16) ◽  
Author(s):  
Xueqing Wu ◽  
Nathan T. Jacobs ◽  
Catherine Bozio ◽  
Preston Palm ◽  
Santiago M. Lattar ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Quorum Sensing ◽  
Relative Density ◽  
Physical Contact ◽  
Relative Distribution ◽  
Upper Airways ◽  
Content Type ◽  
Ecological Requirements ◽  
Asymptomatic Children ◽  
Vaccine Type

ABSTRACT Streptococcus pneumoniae is a main cause of child mortality worldwide, but strains also asymptomatically colonize the upper airways of most children and form biofilms. Recent studies have demonstrated that ∼50% of colonized children carry at least two different serotypes (i.e., strains) in the nasopharynx; however, studies of how strains coexist are limited. In this work, we investigated the physiological, genetic, and ecological requirements for the relative distribution of densities, and spatial localization, of pneumococcal strains within biofilm consortia. Biofilm consortia were prepared with vaccine type strains (i.e., serotype 6B [S6B], S19F, or S23F) and strain TIGR4 (S4). Experiments first revealed that the relative densities of S6B and S23F were similar in biofilm consortia. The density of S19F strains, however, was reduced to ∼10% in biofilm consortia, including either S6B, S23F, or TIGR4, in comparison to S19F monostrain biofilms. Reduction of S19F density within biofilm consortia was also observed in a simulated nasopharyngeal environment. Reduction of relative density was not related to growth rates, since the Malthusian parameter demonstrated similar rates of change of density for most strains. To investigate whether quorum sensing (QS) regulates relative densities in biofilm consortia, two different mutants were prepared: a TIGR4ΔluxS mutant and a TIGR4ΔcomC mutant. The density of S19F strains, however, was similarly reduced when consortia included TIGR4, TIGR4ΔluxS, or TIGR4ΔcomC. Moreover, production of a different competence-stimulating peptide (CSP), CSP1 or CSP2, was not a factor that affected dominance. Finally, a mathematical model, confocal experiments, and experiments using Transwell devices demonstrated physical contact-mediated control of pneumococcal density within biofilm consortia. IMPORTANCE Streptococcus pneumoniae kills nearly half a million children every year, but it also produces nasopharyngeal biofilm consortia in a proportion of asymptomatic children, and these biofilms often contain two strains (i.e., serotypes). In our study, we investigated how strains coexist within pneumococcal consortia produced by vaccine serotypes S4, S6B, S19F, and S23F. Whereas S6B and S23F shared the biofilm consortium, our studies demonstrated reduction of the relative density of S19F strains, to ∼10% of what it would otherwise be if alone, in consortial biofilms formed with S4, S6B, or S23F. This dominance was not related to increased fitness when competing for nutrients, nor was it regulated by quorum-sensing LuxS/AI-2 or Com systems. It was demonstrated, however, to be enhanced by physical contact rather than by a product(s) secreted into the supernatant, as would naturally occur in the semidry nasopharyngeal environment. Competitive interactions within pneumococcal biofilm consortia regulate nasopharyngeal density, a risk factor for pneumococcal disease.

scholarly journals Induction of Plasmid-CarriedqnrS1in Escherichia coli by Naturally Occurring Quinolones and Quorum-Sensing Signal Molecules

2013 ◽  
Vol 57 (8) ◽  
pp. 4031-4034 ◽  
Author(s):  
Yee Gyung Kwak ◽  
George A. Jacoby ◽  
David C. Hooper
Keyword(s):  
Escherichia Coli ◽  
Quorum Sensing ◽  
Homoserine Lactone ◽  
Signal Molecules ◽  
Induction Effect ◽  
Vibrio Splendidus ◽  
Content Type ◽  
Naturally Occurring

ABSTRACTNaturally occurring quinolone and quinolone-like compounds, such as quinine, 2-hydroxyquinoline, 4-hydroxyquinoline, and 2-heptyl-3-hydroxy-4(1H)-quinolone, increased expression ofqnrS1inEscherichia coli2.3- to 11.2-fold, similar to the synthetic quinolone ciprofloxacin. In contrast, chromosomalqnrVS1ofVibrio splendiduswas not induced by these compounds. Molecules associated with quorum sensing, such asN-3-hydroxybutyryl-homoserine lactone (HSL),N-hexanoyl-HSL, andN-3-(oxododecanoyl)-HSL, did not show an induction effect on eitherqnrS1orqnrVS1at the tested concentrations.

scholarly journals Quorum-Sensing Systems LuxS/Autoinducer 2 and Com Regulate Streptococcus pneumoniae Biofilms in a Bioreactor with Living Cultures of Human Respiratory Cells

2013 ◽  
Vol 81 (4) ◽  
pp. 1341-1353 ◽  
Author(s):  
Jorge E. Vidal ◽  
Kristen E. Howery ◽  
Herbert P. Ludewick ◽  
Porfirio Nava ◽  
Keith P. Klugman
Keyword(s):  
Streptococcus Pneumoniae ◽  
Quorum Sensing ◽  
Human Cells ◽  
Upper Respiratory Tract ◽  
Content Type ◽  
Autoinducer 2 ◽  
Sensing Systems ◽  
Abiotic Surfaces ◽  
Static Conditions ◽  
Respiratory Epithelial

ABSTRACTStreptococcus pneumoniaeforms organized biofilms in the human upper respiratory tract that may play an essential role in both persistence and acute respiratory infection. However, the production and regulation of biofilms on human cells is not yet fully understood. In this work, we developed a bioreactor with living cultures of human respiratory epithelial cells (HREC) and a continuous flow of nutrients, mimicking the microenvironment of the human respiratory epithelium, to study the production and regulation ofS. pneumoniaebiofilms (SPB). SPB were also produced under static conditions on immobilized HREC. Our experiments demonstrated that the biomass of SPB increased significantly when grown on HREC compared to the amount on abiotic surfaces. Additionally, pneumococcal strains produced more early biofilms on lung cells than on pharyngeal cells. Utilizing the bioreactor or immobilized human cells, the production of early SPB was found to be regulated by two quorum-sensing systems, Com and LuxS/AI-2, since a mutation in eithercomCorluxSrendered the pneumococcus unable to produce early biofilms on HREC. Interestingly, while LuxS/autoinducer 2 (AI-2) regulated biofilms on both HREC and abiotic surfaces, Com control was specific for those structures produced on HREC. The biofilm phenotypes of strain D39-derivative ΔcomCand ΔluxSQS mutants were reversed by genetic complementation. Of note, SPB formed on immobilized HREC and incubated under static conditions were completely lysed 24 h postinoculation. Biofilm lysis was also regulated by the Com and LuxS/AI-2 quorum-sensing systems.

scholarly journals New Antibody Weapons against an Old Foe

mBio ◽  
2011 ◽  
Vol 2 (5) ◽  
Author(s):  
Jorge L. Benach
Keyword(s):  
Immune Response ◽  
Streptococcus Pneumoniae ◽  
Capsular Polysaccharide ◽  
Single Species ◽  
Genetic Exchange ◽  
Content Type ◽  
Population Reduction ◽  
Diagnostic Capacity ◽  
Transformation Competence

ABSTRACT Antibodies have been used in a diagnostic capacity for many diseases and for identifying serotypes within single species of pathogens, notably between the multiple capsular polysaccharide serotypes ofStreptococcus pneumoniae. For many years, the functions of antibodies in infection were thought to be limited to the opsonization of microorganisms followed by phagocytosis and to the fixing of complement. The thought that antibodies could have other functions has emerged only recently. The study by Yano and coworkers from the laboratory of Liise-anne Pirofski published inmBio[M. Yano, S. Gohil, J. R. Coleman, C. Manix, and L.-A. Pirofski, mBio 2(5):e00176-11, 2011] identifies one mechanism whereby nonopsonic antibodies enhance the transformation competence of twoS. pneumoniaeserotypes, which leads to an increase in genetic exchange and bacterial variability with a resulting population reduction through fratricide. These new and revealing antibody functions will add another chapter to the burgeoning story of the diversity and versatility of the immune response to bacteria.

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