The impact of pneumolysin on the macrophage response to Streptococcus pneumoniae is strain-dependent

Streptococcus pneumoniae is the world's leading cause of pneumonia, bacteremia, meningitis and otitis media. A major pneumococcal virulence factor is the cholesterol-dependent cytolysin, which has the defining property of forming pores in cholesterol-containing membranes. In recent times a clinically significant and internationally successful serotype 1 ST306 clone has been found to express a non-cytolytic variant of Ply (Ply306). However, while the pneumococcus is a naturally transformable organism, strains of the ST306 clonal group have to date been virtually impossible to transform, severely restricting efforts to understand the role of non-cytolytic Ply in the success of this clone. In this study isogenic Ply mutants were constructed in the D39 background and for the first time in the ST306 background (A0229467) to enable direct comparisons between Ply variants for their impact on the immune response in a macrophage-like cell line. Strains that expressed cytolytic Ply were found to induce a significant increase in IL-1β release from macrophage-like cells compared to the non-cytolytic and Ply-deficient strains in a background-independent manner, confirming the requirement for pore formation in the Ply-dependent activation of the NLRP3 inflammasome. However, cytolytic activity in the D39 background was found to induce increased expression of the genes encoding GM-CSF (CSF2), p19 subunit of IL-23 (IL23A) and IFNβ (IFNB1) compared to non-cytolytic and Ply-deficient D39 mutants, but had no effect in the A0229467 background. The impact of Ply on the immune response to the pneumococcus is highly dependent on the strain background, thus emphasising the importance of the interaction between specific virulence factors and other components of the genetic background of this organism

Isolation site influences virulence phenotype of serotype 14 Streptococcus pneumoniae strains belonging to multilocus sequence type 15

Streptococcus pneumoniae is a diverse species causing invasive as well as localized infections that result in massive global morbidity and mortality. Strains vary markedly in pathogenic potential, but the molecular basis is obscured by the diversity and plasticity of the pneumococcal genome. We have previously reported that S. pneumoniae serotype 3 isolates belonging to the same multilocus sequence type (MLST) differed markedly in in vitro and in vivo phenotypes, in accordance with the clinical site of isolation, suggesting stable niche adaptation within a clonal lineage. In the present study, we have extended our analysis to serotype 14 clinical isolates from cases of sepsis or otitis media that belong to the same MLST (ST15). In a murine intranasal challenge model, five ST15 isolates (three from blood and two from ears) colonized the nasopharynx to similar extents. However, blood and ear isolates exhibited significant differences in bacterial loads in other host niches (lungs, ear, and brain) at both 24 and 72 h postchallenge. In spite of these differences, blood and ear isolates were present in the lungs at similar levels at 6 h postchallenge, suggesting that early immune responses may underpin the distinct virulence phenotypes. Transcriptional analysis of lung tissue from mice infected for 6 h with blood isolates versus ear isolates revealed 8 differentially expressed genes. Two of these were exclusively expressed in response to infection with the ear isolate. These results suggest a link between the differential capacities to elicit early innate immune responses and the distinct virulence phenotypes of clonally related S. pneumoniae strains

The variable region of the pneumococcal pathogenicity island 1 is responsible for the unusually high virulence of a serotype 1 isolate

Streptococcus pneumoniae is the leading infectious cause of death in children in the world. However, the mechanisms that drive the progression from asymptomatic colonization to disease are poorly understood. Two virulence-associated genomic accessory regions (ARs) were deleted in a highly virulent serotype 1 clinical isolate (strain 4496) and examined for their contribution to pathogenesis. Deletion of a prophage encoding a platelet-binding protein (PblB) resulted in reduced adherence, biofilm formation, reduced initial infection within the lungs, and a reduction in the number of circulating platelets in infected mice. However, the region’s overall contribution to the survival of mice was not significant. In contrast, deletion of the variable region of pneumococcal pathogenicity island 1 (vPPI1) was also responsible for a reduction in adherence and biofilm formation but also reduced survival and invasion of the pleural cavity, blood, and lungs. While the 4496�PPI1 strain induced higher expression of the genes encoding interleukin-10 (IL-10) and CD11b in the lungs of challenged mice than the wild-type strain, very few other genes exhibited altered expression. Moreover, while the level of IL-10 protein was increased in the lungs of 4496�PPI1 mutant-infected mice compared to strain 4496-infected mice, the levels of gamma interferon (IFN-�), CXCL10, CCL2, and CCL4 were not different in the two groups. However, the 4496�PPI1 mutant was found to be more susceptible than the wild type to phagocytic killing by a macrophage-like cell line. Therefore, our data suggest that vPPI1 may be a major contributing factor to the heightened virulence of certain serotype 1 strains, possibly by influencing resistance to phagocytic killing

The impact of the competence quorum sensing system on Streptococcus pneumoniae biofilms varies depending on the experimental model

Background: Different models for biofilm in Streptococcus pneumoniae have been described in literature. To permit comparison of experimental data, we characterised the impact of the pneumococcal quorum-sensing competence system on biofilm formation in three models. For this scope, we used two microtiter and one continuous culture biofilm system. Results: In both microtiter models the competence system influences stability and structure of biofilm in the late attachment phase and synthetic competence stimulating peptide (CSP) restored wild type phenotypes in the comC mutants unable to produce the peptide. Early attachment of single cells to well bottoms was found for both systems to be competence independent, while later phases, including microcolony formation correlated to an intact competence system. The continuous culture biofilm model was not affected by mutations in the competence locus, but deletion of capsule had a significant impact in this model. Conclusions: Since biofilm remains a largely uncharacterised multi-parameter phenotype it appears to be advisable to exploit more than one model in order to draw conclusion of possible relevance of specific genotypes on pneumococcal physiology.Claudia Trappetti, Luciana Gualdi, Lorenzo Di Meola, Prashant Jain, Cindy C Korir, Paul Edmonds, Francesco Iannelli, Susanna Ricci, Gianni Pozzi and Marco R Oggion

Identification of genes that contribute to the pathogenesis of invasive Pneumococcal Disease by In Vivo transcriptomic analysis

Streptococcus pneumoniae (the pneumococcus) continues to be responsible for a high level of global morbidity and mortality resulting from pneumonia, bacteremia, meningitis, and otitis media. Here we have used a novel technique involving niche-specific, genome-wide in vivo transcriptomic analyses to identify genes upregulated in distinct niches during pathogenesis after intranasal infection of mice with serotype 4 or 6A pneumococci. The analyses yielded 28 common, significantly upregulated genes in the lungs relative to those in the nasopharynx and 25 significantly upregulated genes in the blood relative to those in the lungs in both strains, some of which were previously unrecognized. The role of five upregulated genes from either the lungs or the blood in pneumococcal pathogenesis and virulence was then evaluated by targeted mutagenesis. One of the mutants (ΔmalX) was significantly attenuated for virulence in the lungs, two (ΔaliA and ΔilvH) were significantly attenuated for virulence in the blood relative to the wild type, and two others (ΔcbiO and ΔpiuA) were completely avirulent in a mouse intranasal challenge model. We also show that the products of aliA, malX, and piuA are promising candidates for incorporation into multicomponent protein-based pneumococcal vaccines currently under development. Importantly, we suggest that this new approach is a viable complement to existing strategies for the discovery of genes critical to the distinct stages of invasive pneumococcal disease and potentially has broad application for novel protein antigen discovery in other pathogens such as S. pyogenes, Haemophilus influenzae type b, and Neisseria meningitidis

EIN ALTKROATISCHES GRABERFELD AUF DEM BERGE SPAS BEI KNIN

Autor objavljuje rezultate istraživanja starohrvatske nekropole na brdu Spasu kod Knina (otkriveno ukupno 228 grobova). Na temelju prikupljenih podataka tijekom višegodišnjih arheoloških istraživanja i sprovedene analize grobne arhitekture, pogrebnih običaja i grobnih nalaza utvrđeno je vrijeme ukopavanja na toj nekropoli i uputilo na mogući prostorni smještaj naselja kojemu je to groblje pripadalo. Završna razmatranja objelodanjenih grobnih nalaza otkrivaju niz novih spoznaja o gospoda.stvenim mogućnostima stanovništva, njihov kulturni i duhovni život u 9, 10. i 11. stoljeću. Osim starohrvatskih grobova na Spasu su otkopani i temeljni ostaci arhitekture, za koje se utvrdilo da pripadaju rimskom naselju, vjerojatno ostacima stare Ninie, odnosno Curcuma. Sve to popraćeno je obilnom dokumentacijskom građom i katalogom grobova.Westlich der Stadt Knin erstreckt sich der Berg Spas. Auf seinem siidlichen Teil erhebt sich die mittelalterliche Festung Knin, und an den nord lichen Teil schlieBt sich das Hochplateau des Berges Spas an, das im horizontalen Querschnitt cinE\u27 birnentormige Form hat. Spas ist anfangs des 18. lahrhunderts durch einen kiinstlichen Durchstich von der Festung getrennt worden. Im nordlichen, breitesten Teil des Plateaus wurde ein altkroatisches Graberfeld entdeckt, mit insgesamt 228 Grabern. Das erforsci1te Gebiet umfaBt etwa 1200 m2 Oberflache. Es wurden neben Grabern auch Fundamente von Bauwerken aus vorktoatischer Zeit gefunden, was bezeugt, daB es sich hier um eine komplexe archaologische Fundstatte handelt. Im ersten Teil seiner Abhandlung beschreibt. der Verfasser die Lage des Fundortes, seine strategische Bedeutung, und den geschichtlichen Ablauf der Forschungen an diesem Ort. AuBerdem erwahnt er unter anderen bedeutenden Funden auch die Entdeckung von Fundamenten von Bauwerken , fUr die er aufgrund von Fragmenten steinernen Skulpturen, Ziegeln und Fragmenten von KeramikgefaBen festelIen konnte, daB sie einer romisehen Ansiedlung angehorten. Durch Suchgrabung wurde festgestelIt, daB die Reste dieser Bauwerke sich auch auf den iibrigen Teil des Plateaus erstrecken, der bis jetzt nicht erforscht worden ist. Diese Entdeckungen, zusammen mit einem groBen, schon friiher untersuchten Graberfeld am ostlichen FuBe dieses Berges, genannt »Greblje«, sind ein starkes Argumentfi.ir die Annahme jener Forscher, die der Meinung sind, daB die alte, antike Stadtsiedlung Ninia, die von der Schriftstellern der Antike erwahnt wird , sich an der SteIle der heutigen Stadt Knin befunden hatte, beziehungsweise an der Stelle ihrer Festung und der Wallburg Spas. Im siidostlichen Teil des ausgegrabenen Graberfeldes hat man ziemlich groBe Schuttablagerungen gefunden, unter denen sich auch Stiicke von Mortel mit Farbresten befanden. An dieser Stelle und in ihrer unmittelbaren Nahe wurden einige steinerne Fragmente mit vorromanischer F1echtwerkplastik gefunden , die zu Kircheneinrichtungsgegenstanden aus dem 9. oder 10. lh. gehorten. Die erwahnten Funde weisen mjt Sicherheit darauf hin, daB sich an eben dieser Stelle, wo man den Schutt gefunden hat, eine altkroatische Kirche befunden hatte, der die gefundenen Fragmente angehorten. Da sich von diesem Kirchenbau auch nicht der allergeringste Rest einer Mauer erhalten hat, kann man nur schlieBen, daB die Kirche bis zu den Grundmauern zerstort worden war, und daB ihre Steine als Baurnaterial fUr die Mauern der nahegelegenen Festung verwendet wurden. Der zwei te Teil dieser Abhandlung enthalt den Katalog der Grabarchitektur und ihres lnventars. Hier findet man kurz gefaBt die wichtigsten Angaben i.iber jedes cinzeIne Grab und die osteologischen Funde, sowie eine genaue Beschreibung der Grabbeigaben. Der dritte Teil behandelt die allgemeinen Merkmale des Graberfeldes. Wegen der ungi.instigen Bodenstruktur, sowie der Fundamente der Bauwerke, besitzt das Graberfeld nicht die Merkmale von Reihengrabern, Die Graber sind sehr verschiedenartig orientiert, und liegen in allen Richtungen; der groBte Teil der Graber war trotzdem richtjg orientiert, in der Richtung Osten-Westen (103 Graber) . In den meisten Grabern befand sich nur ein Verstorbener, manchmal jedoch auch mehrere. Die gefundenen osteologischen Reste bestatigen, daB das Graberfeld einer Siedlung zugehorig; mit einer gleich groBen Anzahl von bestatteten Mannern , Frauern und Kindern. Die Graber waren meistens aus behauener Rauhwacke , aus unregelmaBigen Steinplatten oder aus Bruchsteinen gebaut. Die behauene Rauhwacke ftir die Graber stammte wahrscheinlich von den eingesttirzten Bauwerken in der nahen Umgebung. Weiterhin analysiert der Verfasser die Grabfunde, meistens Schmuck, und zwar Ohrringe und Ringe. Indem er die typologischen Besonderheiten der einzelnen Gegenstande genau beurteilt, und sie mit analogem Material aus den tibrigen altkroatischen und anderen Graberfeldern vergleicht, kommt er zu Schltissen ber die Herkunft und\u27 Datierung der Funde. Mehr Aufmerksamkeit schenkt der Verfasser den Ohrringen vom Typ mit drei Beeren und zwar deshalb, weil in letzter Zeit versucht wurde, fast alle entwickelteren Varianten dieses Typs, die in Grabern auf dem Gebiet des altkroatisches Staates gefunden worden waren, von allen tibrigen Ohrringen die ebenfalls auf diesem Gebiet gefunden wurden, zu trennen. [ndem er auf das Problem der Datierung und Herkunft der Ohrringe mit drei Beeren eingeht, versuchte der Verfasser aufgrund einer Reihe sicherer Tatsachen und Hinweise darauf hinzuweisen, daB dieser Typ von Ohrringen, beziehungsweise die starker entwickelten Varianten , nicht von den tibrigen Typenvarianten altkroatischer Ohrringe isoliert werden dtirfen, die auf dem Gebiet des dalmatinischen Kroatiens gefunden worden waren , und die meistens in die Zeit vom Beginn des 9. bis zum Ende des 11. lahrhunderts datiert werden. Er machte ebenfalls darauf aufmerksam, daB samtliche Typen von altkroatischen Ohrringen, und so auch die entwickelteren Varianten von Ohrringen mit drei Beeren, die aus Grabern des Mutterlandes stammen, aus einheimischen Goldschmiedewerkstatten stammen , und daB sie als Ganzes eine altkroatische Kulturgruppe bilden. AuBer seinen Vermutungen tiber die Lage und GroBe der Ansiedlung, zu der das Graberfeld auf dem Berge Spas gehorte, ist der Verfasser im Ganzen der Meinun~, daB das Graberfeld auf Spas dem Zeitraum des frtihfeudalen kroatischen Staates angehorte, d. h., daB das Graberfeld sei t dem Anfang des 9. Jahrhunderts bestanden hatte und kontinuierlich bis zum Ende des ll. Jahrhunderts fortdauerte

Extracellular matrix formation enhances the ability of streptococcus pneumoniae to cause invasive disease

Extent: 17p.During infection, pneumococci exist mainly in sessile biofilms rather than in planktonic form, except during sepsis. However, relatively little is known about how biofilms contribute to pneumococcal pathogenesis. Here, we carried out a biofilm assay on opaque and transparent variants of a clinical serotype 19F strain WCH159. After 4 days incubation, scanning electron microscopy revealed that opaque biofilm bacteria produced an extracellular matrix, whereas the transparent variant did not. The opaque biofilm-derived bacteria translocated from the nasopharynx to the lungs and brain of mice, and showed 100- fold greater in vitro adherence to A549 cells than transparent bacteria. Microarray analysis of planktonic and sessile bacteria from transparent and opaque variants showed differential gene expression in two operons: the lic operon, which is involved in choline uptake, and in the two-component system, ciaRH. Mutants of these genes did not form an extracellular matrix, could not translocate from the nasopharynx to the lungs or the brain, and adhered poorly to A549 cells. We conclude that only the opaque phenotype is able to form extracellular matrix, and that the lic operon and ciaRH contribute to this process. We propose that during infection, extracellular matrix formation enhances the ability of pneumococci to cause invasive disease.Claudia Trappetti, Abiodun D. Ogunniyi, Marco R. Oggioni and James C. Pato

The central role of arginine in Haemophilus influenzae survival in a polymicrobial environment with Streptococcus pneumoniae and Moraxella catarrhalis

Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis are bacterial species which frequently co-colonise the nasopharynx, but can also transit to the middle ear to cause otitis media. Chronic otitis media is often associated with a polymicrobial infection by these bacteria. However, despite being present in polymicrobial infections, the molecular interactions between these bacterial species remain poorly understood. We have previously reported competitive interactions driven by pH and growth phase between H. influenzae and S. pneumoniae. In this study, we have revealed competitive interactions between the three otopathogens, which resulted in reduction of H. influenzae viability in co-culture with S. pneumoniae and in triple-species culture. Transcriptomic analysis by mRNA sequencing identified a central role of arginine in mediating these interactions. Arginine supplementation was able to increase H. influenzae survival in a dual-species environment with S. pneumoniae, and in a triple-species environment. Arginine was used by H. influenzae for ATP production, and levels of ATP generated in dual- and triple-species co-culture at early stages of growth were significantly higher than the combined ATP levels of single-species cultures. These results indicate a central role for arginine-mediated ATP production by H. influenzae in the polymicrobial community

Antibacterial Activity of a Competence-Stimulating Peptide in Experimental Sepsis Caused by Streptococcus pneumoniae

Streptococcus pneumoniae, a major cause of human disease, produces a 17-mer autoinducer peptide pheromone (competence-stimulating peptide [CSP]) for the control of competence for genetic transformation. Due to previous work linking CSP to stress phenotypes, we set up an in vivo sepsis model to assay its effect on virulence. Our data demonstrate a significant increase in the rates of survival of mice, reductions of blood S. pneumoniae counts, and prolonged times to death for mice treated with CSP. In vitro the dose of CSP used in the animal model produced a transitory inhibition of growth. When a mutant with a mutation in the CSP sensor histidine kinase was assayed, no bacteriostatic phenotype was detected in vitro and no change in disease outcome was observed in vivo. The data demonstrate that CSP, which induces in vitro a temporary growth arrest through stimulation of its cognate histidine kinase receptor, is able to block systemic disease in mice. This therapeutic effect is novel, in that the drug-like effect is obtained by stimulation, rather than inhibition, of a bacterial drug target