A hybrid-hierarchical genome assembly strategy to sequence the invasive golden mussel Limnoperna fortunei

Background: For more than 25 years, the golden mussel Limnoperna fortunei has aggressively invaded South American freshwaters, having travelled more than 5,000 km upstream across five countries. Along the way, the golden mussel has outcompeted native species and economically harmed aquaculture, hydroelectric powers, and ship transit. We have sequenced the complete genome of the golden mussel to understand the molecular basis of its invasiveness and search for ways to control it. Findings: We assembled the 1.6 Gb genome into 20548 scaffolds with an N50 length of 312 Kb using a hybrid and hierarchical assembly strategy from short and long DNA reads and transcriptomes. A total of 60717 coding genes were inferred from a customized transcriptome-trained AUGUSTUS run. We also compared predicted protein sets with those of complete molluscan genomes, revealing an exacerbation of protein-binding domains in L. fortunei. Conclusions: We built one of the best bivalve genome assemblies available using a cost-effective approach using Illumina pair-end, mate pair, and PacBio long reads. We expect that the continuous and careful annotation of L. fortunei’s genome will contribute to the investigation of bivalve genetics, evolution, and invasiveness, as well as to the development of biotechnological tools for aquatic pest control

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

Noisy neighbourhoods: quorum sensing in fungal-polymicrobial infections

Quorum sensing was once considered a way in which a species was able to sense its cell density and regulate gene expression accordingly. However, it is now becoming apparent that multiple microbes can sense particular quorum-sensing molecules, enabling them to sense and respond to other microbes in their neighbourhood. Such interactions are significant within the context of polymicrobial disease, in which the competition or cooperation of microbes can alter disease progression. Fungi comprise a small but important component of the human microbiome and are in constant contact with bacteria and viruses. The discovery of quorum-sensing pathways in fungi has led to the characterization of a number of interkingdom quorum-sensing interactions. Here, we review the recent developments in quorum sensing in medically important fungi, and the implications these interactions have on the host's innate immune response

Characterisation of the Thrombospondin-1 mediated adherence of Streptococcus pneumoniae to human host cells

Thrombospondin-1 (TSP1) ist ein matrizelluläres, Calcium-bindendes Glykoprotein, das an der Regulation verschiedener zellulärer Prozesse beteiligt ist. TSP1 wird von unterschiedlichen Zelltypen gebildet und ist vor allem in den α-Granula der Thrombozyten zu finden, aus denen es nach deren Aktivierung sekretiert wird. Streptococcus pneumoniae (Pneumokokken) sind Gram-positive humanpathogene Bakterien. Sie besiedeln asymptomatisch den menschlichen Respirationstrakt und können schwerwiegende lokale Infektionen und lebensbedrohliche Erkrankungen, wie z.B. Sepsis, bakterielle Meningitis oder invasive Pneumonien auslösen. Die Anheftung von S. pneumoniae an Wirtsstrukturen ist ein initialer Schritt für die Kolonisierung mukosaler Epitheloberflächen. In dieser Arbeit wird die Bedeutung des humanen TSP1 für die Pathogen-Wirt Interaktion analysiert und der Effekt für die Pathogenese demonstriert. Verschiedene Bindungsstudien und durchflusszytometrische Analysen zeigten eine Assoziation von S. pneumoniae an aktivierte Thrombozyten und an lösliches und immobilisiertes TSP1. In in vitro Infektionsversuchen konnte nachgewiesen werden, dass wirtszellgebundenes TSP1 die Adhärenz an und Invasion in Epithel- bzw. Endothelzellen vermittelt. TSP1 übernimmt die Funktion als Brückenmolekül zwischen S. pneumoniae und eukaryontischen Wirtszellen. Zur Charakterisierung des bakteriellen Adhäsins für TSP1 wurden die Pneumokokken mit dem proteolytischen Enzym Pronase E bzw. mit der Zucker oxidierenden Substanz Natriumperiodat inkubiert. Eine Behandlung mit Natriumperiodat reduzierte die TSP1 vermittelte Adhärenz der Pneumokokken an humane Wirtszellen. Im Gegensatz dazu hatte die Behandlung mit Pronase E keinen Einfluss auf die TSP1 vermittelte Anheftung von S. pneumoniae an eukaryontische Zellen. Diese Ergebnisse deuten an, dass es sich bei dem bakteriellen Adhäsin für TSP1 um eine oberflächenlokalisierte Glykostruktur der Pneumokokken handelt. Die TSP1 vermittelte bakterielle Adhärenz der Pneumokokken an Wirtszellen konnte durch Pneumokokken-spezifisches Phosphorylcholin bzw. durch Lipoteichonsäuren nicht reduziert werden. Im Gegensatz dazu wurde die TSP1 vermittelte Adhärenz von S. pneumoniae an Wirtszellen durch Zugabe von löslichem Peptidoglykan signifikant inhibiert. In verschiedenen Bindungsstudien wurde das Peptidoglykan als Pneumokokken-Adhäsin für TSP1 identifiziert. Weiterhin wurde herausgestellt, dass nicht nur S. pneumoniae, sondern auch andere Gram-positive pathogene Bakterien, wie Staphylococcus aureus, Streptococcus pyogenes, Listeria monocytogenes und verschiedene apathogene Bakterien mit TSP1 interagieren, im Gegensatz zu Gram-negativen Bakterien. Es konnte gezeigt werden, dass TSP1 das Peptidoglykan aller getesteten Gram-positiven Bakterien erkennt. Diese Beobachtung weist auf einen allgemeingültigen Mechanismus der Bakterien-Wirt Interaktion hin, der wahrscheinlich von großer Bedeutung für die Pathogenese Gram-positiver Bakterien ist. Als Rezeptoren für TSP1 auf der Wirtszellseite wurden Proteoglykane auf der Oberfläche von eukaryontischen Zellen identifiziert. Weiterhin konnte herausgestellt werden, dass eine Interaktion der Gram-positiven Bakterien mit TSP1 nicht nur eine Adhärenz an Wirtszellen vermittelt, sondern die Bakterien vor einer Phagozytose durch primäre Granulozyten schützt. Zusammenfassend beweisen diese Ergebnisse eine spezifische Interaktion von Gram-positiven Bakterien mit TSP1, die zur bakteriellen Kolonisierung des Wirtsgewebes beiträgt. Das Peptidoglykan übernimmt die Funktion eines bakteriellen Adhäsins für TSP1, so dass TSP1 als molekulare Brücke die Interaktion von Gram-positiven Bakterien und Wirtszell-Proteoglykanen vermittelt. Diese Untersuchungen tragen in bedeutender Weise zu einem besseren Verständnis der Pathogenese von Infektionen durch S. pneumoniae und anderen Gram-positiven Bakterien bei.Thrombospondin-1 (TSP1) is a matricellular glycoprotein that has key roles in interactions between human cells and components of the extracellular matrix. TSP1 is produced by different cell types and is mainly found in the α-granules of human thrombocytes and secreted upon their stimulation. Streptococcus pneumoniae are Gram-positive human bacteria which reside asymptomatically in the human respiratory tract, but can also cause local infections and life-threatening diseases such as sepsis, bacterial meningitis and pneumonia. A prerequisite for pneumococcal colonization of mucosal epithelial cells is the bacterial interaction with host cell structures. This study reports a novel role for human TSP1 in pathogen-host interactions. Binding assays and flow cytometric analysis demonstrate that S. pneumoniae specifically interacts with human TSP1. It is shown that S. pneumoniae binds to activated thrombocytes and recruits TSP1 from human plasma. Host-cell bound TSP1 promotes adherence of S. pneumoniae to human epithelial and endothelial cells, thereby acting as a molecular bridge between pneumococci and eukaryotic cells. To identify the bacterial adhesin for TSP1, pneumococci were incubated with the proteolytic enzyme pronase E and with sodium periodate, which oxidizes surface-exposed sugars. Pretreatment of the bacteria with sodium periodate, but not pronase E substantially reduced TSP1 mediated adherence to host cells, suggesting a glycoconjugate as the pneumococcal receptor for TSP1. Pneumococcal phosphorylcholine and lipoteichoic acids did not affect TSP1 mediated adherence of S. pneumoniae to host cells. In contrast, attachment of pneumococci to host cells via TSP1 was blocked by soluble peptidoglycan, indicating the recognition of bacterial peptidoglycan by TSP1. Further studies demonstrate that in addition to S. pneumoniae other Gram-positive bacteria including Staphylococcus aureus, Streptococcus pyogenes, Listeria monocytogenes and several apathogenic bacteria interact with TSP1. Gram-negative bacteria did not interact with TSP1. Further it is shown that TSP1 recognizes the peptidoglycan of all tested Gram-positive bacteria, suggesting a general mechanism of bacteria-host protein interaction which probably has a significant impact on the pathogenesis of Gram-positive bacteria. Host cell proteoglycans were identified as the cellular receptors for TSP1. It is demonstrated that the bacterial binding to TSP1 does not only support adhesion to host cells, but can also protect the bacteria from phagocytosis by granulocytes. In conclusion, the results demonstrate an exploitation of TSP1 by Gram-positive bacteria which supports bacterial colonization of host tissues. In this scenario peptidoglycan functions as adhesin and TSP1 acts as a molecular bridge, linking Gram-positive bacteria with proteoglycan receptors on the host cells. These studies contribute to a better understanding of infections by S. pneumoniae and other Gram-positive bacteria

Microbial Quorum-Sensing Molecules Induce Acrosome Loss and Cell Death in Human Spermatozoa▿

Infertility in men and women is frequently associated with genital contamination by various commensal or uropathogenic microbes. Since many microorganisms are known to release quorum-sensing signals in substantial amounts, we raised the question whether such molecules can directly affect human spermatozoa. Here we show that farnesol and 3-oxododecanoyl-l-homoserine lactone, employed by the opportunistic pathogenic yeast Candida albicans and the gram-negative bacterium Pseudomonas aeruginosa, respectively, induce multiple damage in spermatozoa. A reduction in the motility of spermatozoa coincided in a dose-dependent manner with apoptosis and necrosis at concentrations which were nondeleterious for dendritic cell-like immune cells. Moreover, sublethal doses of both signaling molecules induced premature loss of the acrosome, a cap-like structure of the sperm head which is essential for fertilization. Addressing their mechanism of action, we found that the bacterial molecule, but not the fungal molecule, actively induced the acrosome reaction via a calcium-dependent mechanism. This work uncovers a new facet in the interaction of microorganisms with human gametes and suggests a putative link between microbial communication systems and host infertility

Binding of Streptococcus pneumoniae endopeptidase O (PepO) to complement component C1q modulates the complement attack and promotes host cell adherence.

The Gram-positive species Streptococcus pneumoniae is a human pathogen causing severe local and life-threatening invasive diseases associated with high mortality rates and death. We demonstrated recently that pneumococcal endopeptidase O (PepO) is an ubiquitously expressed, multifunctional plasminogen and fibronectin binding protein facilitating host cell invasion and evasion of innate immunity. In this study we found that PepO interacts directly with the complement C1q protein, thereby attenuating the classical complement pathway and facilitating pneumococcal complement escape. PepO binds both free C1q and C1 complex in a dose-dependent manner based on ionic interactions. Our results indicate that recombinant PepO specifically inhibits the classical pathway of complement activation in both hemolytic and complement deposition assays. This inhibition is due to direct interaction of PepO with C1q, leading to a strong activation of the classical complement pathway and results in consumption of complement components. In addition, PepO binds the classical complement pathway inhibitor C4BP, thereby regulating downstream complement activation. Importantly, pneumococcal surface-exposed PepO-C1q interaction mediates bacterial adherence to host epithelial cells. Taken together, PepO facilitates C1q-mediated bacterial adherence, while its localized release consumes complement as a result of its activation following binding of C1q, thus representing an additional mechanism of human complement escape by this versatile pathogen