Mutational and biochemical analysis of the DNA-entry nuclease EndA from Streptococcus pneumoniae

EndA is a membrane-attached surface-exposed DNA-entry nuclease previously known to be required for genetic transformation of Streptococcus pneumoniae. More recent studies have shown that the enzyme also plays an important role during the establishment of invasive infections by degrading extracellular chromatin in the form of neutrophil extracellular traps (NETs), enabling streptococci to overcome the innate immune system in mammals. As a virulence factor, EndA has become an interesting target for future drug design. Here we present the first mutational and biochemical analysis of recombinant forms of EndA produced either in a cell-free expression system or in Escherichia coli. We identify His160 and Asn191 to be essential for catalysis and Asn182 to be required for stability of EndA. The role of His160 as the putative general base in the catalytic mechanism is supported by chemical rescue of the H160A variant of EndA with imidazole added in excess. Our study paves the way for the identification and development of protein or low-molecular-weight inhibitors for EndA in future high-throughput screening assays

Structural insights into catalytic and substrate binding mechanisms of the strategic EndA nuclease from Streptococcus pneumoniae

EndA is a sequence non-specific endonuclease that serves as a virulence factor during Streptococcus pneumoniae infection. Expression of EndA provides a strategy for evasion of the host's neutrophil extracellular traps, digesting the DNA scaffold structure and allowing further invasion by S. pneumoniae. To define mechanisms of catalysis and substrate binding, we solved the structure of EndA at 1.75 Å resolution. The EndA structure reveals a DRGH (Asp-Arg-Gly-His) motif-containing ββα-metal finger catalytic core augmented by an interesting ‘finger-loop’ interruption of the active site α-helix. Subsequently, we delineated DNA binding versus catalytic functionality using structure-based alanine substitution mutagenesis. Three mutants, H154A, Q186A and Q192A, exhibited decreased nuclease activity that appears to be independent of substrate binding. Glu205 was found to be crucial for catalysis, while residues Arg127/Lys128 and Arg209/Lys210 contribute to substrate binding. The results presented here provide the molecular foundation for development of specific antibiotic inhibitors for EndA

Prediction of new gene products and characterization of hypothetical proteins of Bifidobacterium breve DS15-17 In Silico / Predição de novos produtos gênicos e caracterização de proteínas hipotéticas de Bifidobacterium breve DS15-17 In Silico

The representation of the gene content of an organism is impacted by several factors, ranging from sampling to sequencing and then the genome assembly task. The genome assembly process can generate errors that are related to insufficient coverage in the data set, an inadequate assembly methodology, and finally, errors related to the limitation of the assembly software used. Thus, some genes remain unidentified both in complete and draft genomes, this incomplete gene knowledge impacts on several organisms, mainly of medical and industrial interest, such as Bifidobacterium breve, a Gram-positive bacterium, found in the gastrointestinal microbiota of mammals, including humans, and has beneficial probiotic activities. Therefore, the objective of this work is to identify the new gene products not represented in the genome of Bifidobacterium breve DS15-17 using the raw reads of this organism. The reads were produced from the sequencing with the Illumina MiSeq platform. PAN2HGENE software was used to identify new gene products. After the analysis, 44 new gene products were identified, 26 with described function and 18 hypothetical proteins. The hypothetical proteins identified were analyzed in the ProtoNet and Superfamily databases

The structural characterization of a prophage-encoded extracellular DNase from Streptococcus pyogenes

The pathogenic bacterium Group A Streptococcus pyogenes produces several extracellular DNases that have been shown to facilitate invasive infection by evading the human host immune system. DNases degrade the chromatin in neutrophil extracellular traps, enabling the bacterium to evade neutrophil capture. Spd1 is a type I, nonspecific ββα/metal-dependent nuclease from Streptococcus pyogenes, which is encoded by the SF370.1 prophage and is likely to be expressed as a result of prophage induction. We present here the X-ray structure of this DNase in the wild-type and Asn145Ala mutant form. Through structural and sequence alignments as well as mutagenesis studies, we have identified the key residues His121, Asn145 and Glu164, which are crucial for Spd1 nucleolytic activity and shown the active site constellation. Our wild-type structure alludes to the possibility of a catalytically blocked dimeric form of the protein. We have investigated the multimeric nature of Spd1 using size-exclusion chromatography with multi-angle light scattering (SEC-MALLS) in the presence and absence of the divalent metal ion Mg2+, which suggests that Spd1 exists in a monomeric form in solution

Inhibitors of Streptococcus pneumoniae Surface Endonuclease EndA Discovered by High-Throughput Screening Using a PicoGreen Fluorescence Assay

The human commensal pathogen, Streptococcus pneumoniae, expresses a number of virulence factors that promote serious pneumococcal diseases, resulting in significant morbidity and mortality worldwide. These virulence factors may give S. pneumoniae the capacity to escape immune defenses, resist antimicrobial agents, or a combination of both. Virulence factors also present possible points of therapeutic intervention. The activities of the surface endonuclease, EndA, allow S. pneumoniae to establish invasive pneumococcal infection. EndA’s role in DNA uptake during transformation contributes to gene transfer and genetic diversitifcation. Moreover, EndA’s nuclease activity degrades the DNA backbone of neutrophil extracellular traps (NETs), allowing pneumococcus to escape host immune responses. Given its potential impact on pneumococcal pathogenicity, EndA is an attractive target for novel antimicrobial therapy. Herein, we describe the development of a high-throughput screening assay for the discovery of nuclease inhibitors. Nuclease-mediated digestion of double-stranded DNA was assessed using fluorescence intensity changes of the DNA dye ligand, PicoGreen. Under optimized conditions, the assay provided robust and reproducible activity data (Z'=0.87) and was used to screen 4727 small molecules against an imidazole-rescued variant of EndA. In total, 10 small molecules were confirmed as novel EndA inhibitors that may have utility as research tools for understanding pneumococcal pathogenesis, and ultimately drug discovery

Etude du recrutement de la nucléase EndA au sein du complexe de transport de l'ADN transformant chez Streptococcus pneumoniae

La transformation est un mécanisme largement répandu dans le monde bactérien permettant des échanges génétiques via la capture d'ADN exogène. Elle requiert généralement le développement d'un état physiologique transitoire, la compétence, au cours de laquelle sont néo-synthétisées les protéines essentielles à la capture de l'ADN du milieu extérieur, à sa traversée de la paroi cellulaire, à son internalisation dans le cytoplasme sous forme simple brin et à son intégration dans le génome par recombinaison homologue. Collectivement, ces protéines définissent l'appareil de transformation génétique, l'ADN Transformasome, dont la caractérisation est la plus avancée pour les bactéries modèles Streptococcus pneumoniae et Bacillus subtilis. Mon projet de thèse visait à caractériser la dynamique de mise en place de la partie du transformasome en charge du transport de l'ADN exogène au travers de la membrane cytoplasmique chez S. pneumoniae. Pour cela, j'ai principalement utilisé (et adapté au pneumocoque) des techniques de biologie cellulaire permettant de suivre par épifluorescence l'assemblage de ce pore d'entrée de l'ADN transformant dans des cellules vivantes. Ainsi, j'ai pu mettre en évidence un retard du processus de septation cellulaire lors du premier événement de division qui suit l'induction de la compétence. Pour localiser le pore d'entrée du transformasome, j'ai étudié la localisation de l'endonucléase membranaire EndA, en charge de la dégradation à l'extérieur de la cellule du brin complémentaire de l'ADN transformant internalisé. Contrairement à toutes les autres protéines du Transformasome, EndA est exprimée de manière constitutive. Mes résultats montrent que, d'une part, EndA localise de manière homogène dans la membrane des cellules non compétentes et, d'autre part, forme des foci dans les cellules compétentes. La formation des foci d'EndA est dépendante de la protéine membranaire ComEA du Transformasome, dont le rôle est de recevoir l'ADN double-brin exogène au niveau du pore pore d'entrée. Lorsque la capacité de transformation est maximale, EndA et ComEA sont préférentiellement localisées au niveau de la zone équatoriale des cellules. Par ailleurs, j'ai pu montrer que l'ADN transformant marqué par des fluorophores se concentre aussi principalement à la zone équatoriale des cellules compétentes. Dans l'ensemble, ce travail suggère que l'assemblage du pore d'entrée de l'ADN transformant chez S. pneumoniae se ferait à la zone équatoriale des cellules compétentes. Chez B. subtilis il a été montré que la fixation de l'ADN a lieu aux pôles des cellules compétentes. Ainsi, bien que les composantes des transformasomes de B. subtilis et de S. pneumoniae soient très conservées, il semble qu'une (ou des) caractéristique(s) propre(s) à ces deux espèces bactériennes déterminent le site d'assemblage du pore d'entrée de l'ADN transformasome dans la membrane.Natural genetic transformation is widely distributed in bacteria, allowing genetics exchange. Transformation requires a specialized membrane-associated complex which forms a DNA entry pore allowing internalization of exogenous single-stranded DNA (ssDNA). It also requires dedicated cytosolic proteins to integrate internalised ssDNA into the chromosome by homologous recombination. This complex, named DNA Transformasome, has been intensively studied in the human pathogen S. pneumoniae and the soil bacteria B. subtilis. The aim of my project was to characterize the membrane-associated complex in S. pneumoniae. I adapted two complementary cellular biology methods. To identify physical links between Transformasome components, I developed a method to purify membrane complexes in S. pneumoniae. Moreover, I performed fluorescence microscopy, which allows us to track and record individual bacterial cells of S. pneumoniae upon competence induction. Using GFP (Green Fluorescent Protein) fused to a key division protein FtsZ, I demonstrated a delayed septation process in competent cells. To visualize the integration of components of the DNA entry pore, I focused on EndA. EndA is a sequence non-specific endonuclease bound to the membrane which is responsible for processing double-stranded DNA into single-stranded fragments that in turn enter the microbial cell. This nuclease has been identified only in the streptococcal system. Remarkably, EndA is the only known component of the DNA entry pore that is constitutively expressed in non-competent cells and is not induced in competence. I showed that EndA concentrates into localized foci upon competence induction. This specific localization depends on the presence of the DNA-receptor, ComEA. Moreover, EndA and ComEA appear preferentially located at midcell in cultures exhibiting optimal transformation efficiency. Similarly, binding of extracellular DNA was localized to the cell centre. Together, our results are consistent with a model in which the active entry pore for DNA transformation is located at the septum in pneumococcal cells. In contrast, it appears that the entire process of DNA transformation localizes at the cell poles in B. subtilis. Although components of Transformasome are conserved between species, it appears that bacteria have adapted the transformation process according to their own physiology

Evolution and Prevention of Antibiotic Resistance: Small Molecule Inhibitors of Bacterial Recombination Enzymes

Antibiotic resistant bacteria are an ever-increasing problem for the modern chemotherapy of bacterial infectious diseases. The loss of effective antibiotic therapies due to antibiotic resistance and the withering antibiotic pipeline are resulting in a reemergence in deaths from bacterial infections. New strategies are needed to combat pathogenic bacteria and in this context bacterial targets involved in the development of resistance are emerging an intriguing candidates for inhibition studies. Recent evidence suggests that bacterial stress response pathways (i.e., SOS and competence for transformation) are responsible for accelerated genetic changes that ultimately establish antibiotic resistance. Intervening in these pathways by small molecule inhibition of key recombination enzymes, RecA and EndA, would impact the DNA repair, SOS mutagenesis and recombination-based horizontal gene transfer activities of these enzymes and hinder the acquisition of antibiotic resistance. Bacteria having loss-of-function mutations in the recA gene are more sensitive to antibiotic treatment and develop resistance more slowly or not at all. In addition, endA-null strains of S. pneumoniae have diminished transformation efficiencies and are unable to acquire resistance-conferring DNA. Therefore, we believe chemotherapeutic agents that impart these bacterial phenotypes could act synergistically with currently prescribed antibiotics to prevent the accumulation of populations that are resistant to them. Towards this goal, we sought to identify properly designed inhibitors of RecA and EndA. High-throughput screening (HTS) is recognized as a powerful tool in drug discovery to identify target-specific lead compounds. We developed rational high-throughput screening programs to discover small-molecule inhibitors of RecA and EndA. Through these studies, we have identified novel chemical classes that specifically target RecA or EndA and demonstrate that these enzymes hold potential as novel targets in the treatment of bacterial infections.Doctor of Philosoph

Etude de la transformation plasmidique naturelle d'Escherichia coli et de ses relations éventuelles avec la compétence programmée pour la transformation génétique et la compétence dite nutritionnelle

Bien que la bactérie Escherichia coli ne soit pas connue pour être naturellement transformable, mon travail de thèse montre que l'on peut obtenir des transformants plasmidiques spontanément sur boîte. Cette transformation n'est pas induite par les cations divalents au contraire de la transformation ‘artificielle chimique’ (Sun et al., FEMS Microbiol. Lett. 2006. 265: 249–255). Les bactéries transformables utilisent une machinerie protéique transmembranaire, évolutivement conservée, pour internaliser l'ADN exogène sous forme simple brin. E.coli possède l'ensemble des gènes codant pour cette machinerie. J'ai inactivé les gènes clés de cette machinerie, dont hofQ (canal transmembranaire externe) et ycaI (canal transmembranaire interne), et observé qu'aucun de ces mutants n'est affecté pour la transformation sur boîte. L'ADN plasmidique ne pénètre donc pas via la machinerie de transformation, mais plutôt sous forme double brin ce que suggèrent les courbes de réponse à la concentration d'ADN (Sun et al., J. Bacteriol. 2009. 191: 713-719). Le troisième volet de ma thèse a consisté à tenter de mieux caractériser un phénomène appelé 'compétence nutritionnelle', appellation qui désigne la capacité d'utiliser l'ADN comme source de carbone. Pour établir si différents gènes de la machinerie de transformation étaient impliqués, j'ai cherché à reproduire les expériences publiées de croissance de la souche ZK126 sur milieu minimum M63 contenant de l'ADN. Malgré de nombreuses tentatives et contrôles, je n'ai pas pu reproduire ces expériences, ce qui m'a amené à clore mon mémoire de thèse par une discussion critique des données publiées relatives à la compétence nutritionnelle de E. coli.While Escherichia coli is not considered to belong to naturally transformable species, I established a transformation system allowing spontaneous plasmid transformation on plate (Sun et al., FEMS Microbiol. Lett. 2006. 265: 249–255). Transformation is not induced by divalent cations in contrast to chemically-induced 'artificial transformation' (Sun et al., J. Bacteriol. 2009. 191: 713-719). As DNA uptake in naturally transformable bacteria relies on a conserved multiprotein machinery and the E. coli genome contains all genes encoding this machinery, I investigated whether key genes are required for plasmid transformation. None of the mutants I constructed, including hofQ and ycaI which encode putative outer and inner membrane channel proteins were affected, indicating that plasmid DNA is not taken up via the transformation machinery. We proposed that plasmid DNA instead enters the cytoplasm as double stranded material as suggested by response curves to DNA concentration (Sun et al., J. Bacteriol. 2009. Ibid.). In the last part of my thesis, I reinvestigated so-called ‘nutritional competence’ of E. coli. Previously work reported that E. coli cells are able to use DNA as the sole carbon source. I wished to establish whether this phenomenon relies on the above-mentioned DNA uptake machinery. I therefore tried to reproduce the published growth experiments of E. coli ZK126 on M63 minimal medium with DNA. Despite numerous attempts and controls, I could not observe any growth. This failure to reproduce published observations led me to conclude my thesis by an in-depth discussion of the three articles dedicated to so-called nutritional competence of E. coli

Systematic classification of the His-Me finger superfamily

The His-Me finger endonucleases, also known as HNH or -metal endonucleases, form a large and diverse protein superfamily. The His-Me finger domain can be found in proteins that play an essential role in cells, including genome maintenance, intron homing, host defense and target offense. Its overall structural compactness and non-specificity make it a perfectly-tailored pathogenic module that participates on both sides of inter- and intra-organismal competition. An extremely low sequence similarity across the superfamily makes it difficult to identify and classify new His-Me fingers. Using state-of-theart distant homology detection methods, we provide an updated and systematic classification of His-Me finger proteins. In this work, we identified over 100 000 proteins and clustered them into 38 groups, of which three groups are new and cannot be found in any existing public domain database of protein families. Based on an analysis of sequences, structures, domain architectures, and genomic contexts, we provide a careful functional annotation of the poorly characterized members of this superfamily. Our results may inspire further experimental investigations that should address the predicted activity and clarify the potential substrates, to provide more detailed insights into the fundamental biological roles of these proteins

Extracellular DNA in head and neck biofilms

PhD ThesisExtracellular DNA (eDNA) is a ubiquitous component of the extracellular matrix of microbial biofilms. It has a number of functions that include a role as an adhesin during biofilm attachment, and facilitating matrix stability in mature biofilms. Increasingly, deoxyribonuclease (DNase) enzymes have been shown to reduce the colonisation of many microbial biofilms, both bacterial and fungal. Biofilms are estimated to be responsible for around 60% of bacterial infections, including many chronic diseases. The aim of this work was to determine the role of eDNA in chronic mixed-species biofilm infections, and to explore the potential of DNase enzymes for biofilm control. This included three major areas of research, focusing on chronic rhinosinusitis, tracheoesophageal speech valves (TESVs), and dental plaque. An important aspect was to test the efficacy of a novel bacterial nuclease, NucB, isolated from a seaweed-associated strain of Bacillus licheniformis, against microbial biofilms. The colonisation of speech valves by micro-organisms was studied using scanning electron microscopy (SEM). In keeping with previous observations, these biofilms were co-aggregations of fungal and bacterial species. Using confocal laser scanning microscopy, eDNA was observed in the biofilm matrix. Extracellular DNA was extracted and quantified from TESV biofilms. All six biofilms studied had detectable nucleic acids, as measured by NanoDrop spectrophotometry. The eDNA was apparently heavily degraded, and produced smears by agarose gel electrophoresis. Nevertheless, eDNA appeared to be providing biofilm stability since micro-organisms were liberated from the surface of the valves following treatment with NucB in over 60% of the TESVs tested. To assess the role of eDNA in biofilms associated with chronic rhinosinusitis, ‘obstructive mucin’ and sinus mucosa biopsy samples collected during functional endoscopic sinus surgery at the Freeman Hospital, Newcastle, were analysed for the presence of biofilms and biofilm-forming micro-organisms. An average of 3.75 bacterial species per patient were cultured from obstructive mucin. The most commonly isolated micro-organisms were Staphylococcus aureus, coagulase-negative staphylococci and α-haemolytic streptococci. Micro-organisms were not detected by transmission electron microscopy of the obstructive mucin and this material appeared to originate through a host inflammatory response. However, bacteria were visualised on the surface of sinus mucosa using a peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) universal bacteria probe. Twenty-four bacterial isolates were iv assessed for their ability to form biofilms in a microtitre plate model. All micro-organisms tested formed biofilms, and 14 of 22 were susceptible to NucB. In total, 15 of 24 microbial species produced eDNA that was detectable by agarose gel electrophoresis. By SEM, cellular colonisation was lower in treated samples and, in the case of Streptococcus constellatus FH20 stringy, matrix-like material was not present after DNase treatment. The role of eDNA in matrix stability and initial biofilm attachment was also studied in oral bacteria. Streptococcus gordonii DL1, Streptococcus mutans GS-5, Fusobacterium nucleatum 25586 and Actinomyces oris MG1 were examined using DNase treatment, CLSM, and eDNA extraction. Of these species, all except S. gordonii appeared to rely on high molecular weight (HMW) eDNA for biofilm attachment and biofilm stability. Although S. gordonii did not produce detectable HMW eDNA, nucleic acids were detectable by NanoDrop spectrophotometry. Furthermore, this species produces an extracellular nuclease which may degrade the HMW eDNA in the conditions used to culture biofilms. Interestingly, four S. mutans strains differed in their sensitivity to DNase treatment. Oral biofilms were also modelled in a BioFlux microfluidics device using flowing human saliva. Mixed-species biofilms and single species biofilms of S. mutans UA159 and S. gordonii DL1 were cultured using this technique, to determine whether this model would allow more realistic experiments for DNase testing. Finally, the extracellular nuclease, SsnA, of S. gordonii DL1 was characterised. A nuclease-deficient mutant did not produce extracellular nuclease activity on DNase Test agar or during a Forster Resonance Energy Transfer (FRET) assay. Nuclease activity was cell-wall-associated as predicted from the predicted amino acid sequence of SsnA. Allelic exchange mutagenesis determined that ssnA expression was regulated by CcpA in response to repressing sugars. However, in planktonic culture non-repressing carbon sources also inhibited enzyme activity during a FRET assay. Further experiments using acidic buffers replicated the inhibition of SsnA without the presence of sugars. SsnA was purified as a GST-tagged fusion protein in an Escherichia coli protein expression system, and had anti-biofilm activity against S. mutans GS-5. However, this species is strongly acidogenic and therefore it is hypothesised that although SsnA may be a competition biofilm factor, acid production by S. mutans may reduce its efficacy in vivo. In conclusion, this thesis has provided strong evidence for the role of eDNA in facilitating biofilm formation and mature biofilm stability of clinically relevant v biofilms. Nucleic acids were present in biofilms associated with a chronic infection, medical implant biofouling and dental plaque. A variety of DNase enzymes (NucB, DNase I, and SsnA) were capable of reducing biofilm colonisation. Given the adhesive function of eDNA in biofilm matrices it is proposed that DNase enzymes may be beneficial for controlling healthcare-related biofilms