Monitoraggio idrobiologico della sorgente carsica di Gologone (Sardegna): indagini preliminari

The analysis of biological water quality of the aquifer feeding Gologone springs was performed using the methodology proposed by Gaiter et al. (2004); researches on benthic fauna and organic component of the sediment in spring waters were carried out. The site was selected due to its location within the pristine area of Supramonte and the northen part of Gennargentu Mt. For this reason, Gologone springs, may be used as a reference site for assessing biological water quality of the Sardinian karstic springs, defining the structure of the macrobenthic assemblages and their relationships with the abiotic parameters.Questo lavoro ha permesso l’analisi della qualità biologica dell’acquifero che alimenta le sorgenti di Gologone, mediante la sperimentazione della metodologia proposta da Gaiter et al. (2004) consistente nell’analisi della fauna bentonica e della componente organica del sedimento in acque sorgive. Il sito è stato scelto in relazione all’integrità della sua area di alimentazione costituta dal Supramonte e dalla porzione settentrionale del massiccio del Gennargentu. In questo modo, è possibile fissare per le sorgenti carsiche sarde uno scenario ecologico di riferimento sul quale basare futuri studi sulla qualità biologica delle acque, definendo il popolamento macrobentonico dell’acquifero che alimenta le sorgenti e la sua interrelazione con i parametri abiotici

Accelerating the manufacture of glycoconjugate vaccines for pneumococcal disease

Streptococcus pneumoniae (S. pneumoniae) is one of the leading causes of invasive bacterial disease in children. In 2000 it contributed to 11% of deaths in children aged 1-59 months (1). Invasive pneumococcal disease can result in septicemia, meningitis and pneumonia. Two pneumococcal glycoconjugate vaccines were introduced in 2000 and 2010 in the USA. Deaths due to the serotypes included in these vaccines in children under 5 have fallen from 183 cases per 100,000 in 1998, to 11 per 100,000 in 2015. The current gold standard glycoconjugate vaccine for immunization is Prevenar 13. It is the leading global vaccine product generating over $6 billion dollars in revenue in 2016. The cost per dose in the USA is over$100 with a total of four doses required in young children. Due to the complex manufacturing process for the vaccine the expense of the dose is driven up. The process involves separate fermentations for the thirteen serotypes and for the carrier protein component. Subsequent to this are stripping, purification, activation and chemical conjugation steps to make the vaccine and another series of purifications to make the final formulation. The chemical conjugation step requires personnel with a high level of experience and intricate knowledge of the reaction and is a limiting factor for new low income country manufacturers in entering this market. The advent of Protein Glycan Coupling Technology (PGCT) has been an important development. This plasmid-based technology is able to produce glycoconjugate vaccines intracellularly in E. coli (2). As a result, there is no need for the chemical conjugation steps, meaning personnel do not need to have the niche skills currently required. Furthermore, the number of purification steps during the process are also reduced. E. coli cells have been engineered with PGCT to produce a glycoconjugate vaccine of Serotype 4 of S. pneumoniae. Using this cell line, transition of E. coli growth from shake flasks into small scale bioreactors has been performed. Results show cell biomass is increased in bioreactors and volumetric productivity of cells is improved. It has also become apparent that any changes to the system need to be carefully considered. For example, comparison of two different proteins in this system has found one is more amenable to glycosylation indicating that choice of protein will have an effect on glycoconjugate production. Overall these experiments have demonstrated the scalability of PGCT and has laid the foundation for future optimization of the system. The work presented here is using S. pneumoniae in a platform process for the production of pneumococcal conjugate vaccines. Here a new vaccine production technology is being optimized and scaled to increase product yields, and with a long term aim of reducing cost per dose of the vaccine. O’Brien, K.L. et al., 2009. Burden of disease caused by Streptococcus pneumoniae in children younger than 5 years: global estimates. The Lancet, 374(9693), pp.893–902. Cuccui J, Thomas RM, Moule MG, D’Elia R V, Laws TR, Mills DC, et al. Exploitation of bacterial N-linked glycosylation to develop a novel recombinant glycoconjugate vaccine against Francisella tularensis. Open Biol. England; 2013 May;3(5):130002

Towards a platform process for the manufacture of glycoconjugate vaccines for pneumococcal disease

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Virulence of the emerging pathogen, Burkholderia pseudomallei, depends upon the O-linked oligosaccharyltransferase, PglL.

Aim: We sought to characterize the contribution of the O-OTase, PglL, to virulence in two Burkholderia spp. by comparing isogenic mutants in Burkholderia pseudomallei with the related species, Burkholderia thailandensis. Materials & methods: We utilized an array of in vitro assays in addition to Galleria mellonella and murine in vivo models to assess virulence of the mutant and wild-type strains in each Burkholderia species. Results: We found that pglL contributes to biofilm and twitching motility in both species. PglL uniquely affected morphology; cell invasion; intracellular motility; plaque formation and intergenus competition in B. pseudomallei. This mutant was attenuated in the murine model, and extended survival in a vaccine-challenge experiment. Conclusion: Our data support a broad role for pglL in bacterial fitness and virulence, particularly in B. pseudomallei

Recent advances in the production of recombinant glycoconjugate vaccines.

Glycoconjugate vaccines against bacteria are one of the success stories of modern medicine and have led to a significant reduction in the global occurrence of bacterial meningitis and pneumonia. Glycoconjugate vaccines are produced by covalently linking a bacterial polysaccharide (usually capsule, or more recently O-antigen), to a carrier protein. Given the success of glycoconjugate vaccines, it is surprising that to date only vaccines against Haemophilus influenzae type b, Neisseria meningitis and Streptococcus pneumoniae have been fully licenced. This is set to change through the glycoengineering of recombinant vaccines in bacteria, such as Escherichia coli, that act as mini factories for the production of an inexhaustible and renewable supply of pure vaccine product. The recombinant process, termed Protein Glycan Coupling Technology (PGCT) or bioconjugation, offers a low-cost option for the production of pure glycoconjugate vaccines, with the in-built flexibility of adding different glycan/protein combinations for custom made vaccines. Numerous vaccine candidates have now been made using PGCT, which include those improving existing licenced vaccines (e.g., pneumococcal), entirely new vaccines for both Gram-positive and Gram-negative bacteria, and (because of the low production costs) veterinary pathogens. Given the continued threat of antimicrobial resistance and the potential peril of bioterrorist agents, the production of new glycoconjugate vaccines against old and new bacterial foes is particularly timely. In this review, we will outline the component parts of bacterial PGCT, including recent advances, the advantages and limitations of the technology, and future applications and perspectives

Genome-wide saturation mutagenesis of Burkholderia pseudomallei K96243 predicts essential genes and novel targets for antimicrobial development.

UNLABELLED: Burkholderia pseudomallei is the causative agent of melioidosis, an often fatal infectious disease for which there is no vaccine. B. pseudomallei is listed as a tier 1 select agent, and as current therapeutic options are limited due to its natural resistance to most antibiotics, the development of new antimicrobial therapies is imperative. To identify drug targets and better understand the complex B. pseudomallei genome, we sought a genome-wide approach to identify lethal gene targets. As B. pseudomallei has an unusually large genome spread over two chromosomes, an extensive screen was required to achieve a comprehensive analysis. Here we describe transposon-directed insertion site sequencing (TraDIS) of a library of over 10(6) transposon insertion mutants, which provides the level of genome saturation required to identify essential genes. Using this technique, we have identified a set of 505 genes that are predicted to be essential in B. pseudomallei K96243. To validate our screen, three genes predicted to be essential, pyrH, accA, and sodB, and a gene predicted to be nonessential, bpss0370, were independently investigated through the generation of conditional mutants. The conditional mutants confirmed the TraDIS predictions, showing that we have generated a list of genes predicted to be essential and demonstrating that this technique can be used to analyze complex genomes and thus be more widely applied. IMPORTANCE: Burkholderia pseudomallei is a lethal human pathogen that is considered a potential bioterrorism threat and has limited treatment options due to an unusually high natural resistance to most antibiotics. We have identified a set of genes that are required for bacterial growth and thus are excellent candidates against which to develop potential novel antibiotics. To validate our approach, we constructed four mutants in which gene expression can be turned on and off conditionally to confirm that these genes are required for the bacteria to survive

Evaluation of glycosylated FlpA and SodB as subunit vaccines against campylobacter jejuni colonisation in chickens

Campylobacter jejuni is the leading bacterial cause of human gastroenteritis worldwide and the handling or consumption of contaminated poultry meat is the key source of infection. C. jejuni proteins FlpA and SodB and glycoconjugates containing the C. jejuni N-glycan have been separately reported to be partially protective vaccines in chickens. In this study, two novel glycoproteins generated by protein glycan coupling technology-G-FlpA and G-SodB (with two and three N-glycosylation sites, respectively)-were evaluated for efficacy against intestinal colonisation of chickens by C. jejuni strain M1 relative to their unglycosylated variants. Two independent trials of the same design were performed with either a high challenge dose of 107 colony-forming units (CFU) or a minimum challenge dose of 102 CFU of C. jejuni M1. While antigen-specific serum IgY was detected in both trials, no reduction in caecal colonisation by C. jejuni M1 was observed and glycosylation of vaccine antigens had no effect on the outcome. Our data highlight inconsistencies in the outcome of C. jejuni vaccination trials that may reflect antigen-, challenge strain-, vaccine administration-, adjuvant- and chicken line-specific differences from previously published studies. Refinement of glycoconjugate vaccines by increasing glycosylation levels or using highly immunogenic protein carriers could improve their efficacy

Production and efficacy of a low-cost recombinant pneumococcal protein polysaccharide conjugate vaccine.

Streptococcus pneumoniae is the leading cause of bacterial pneumonia. Although this is a vaccine preventable disease, S. pneumoniae still causes over 1 million deaths per year, mainly in children under the age of five. The biggest disease burden is in the developing world, which is mainly due to unavailability of vaccines due to their high costs. Protein polysaccharide conjugate vaccines are given routinely in the developed world to children to induce a protective antibody response against S. pneumoniae. One of these vaccines is Prevnar13, which targets 13 of the 95 known capsular types. Current vaccine production requires growth of large amounts of the 13 serotypes, and isolation of the capsular polysaccharide that is then chemically coupled to a protein, such as the diphtheria toxoid CRM197, in a multistep expensive procedure. In this study, we design, purify and produce novel recombinant pneumococcal protein polysaccharide conjugate vaccines in Escherichia coli, which act as mini factories for the low-cost production of conjugate vaccines. Recombinant vaccine efficacy was tested in a murine model of pneumococcal pneumonia; ability to protect against invasive disease was compared to that of Prevnar13. This study provides the first proof of principle that protein polysaccharide conjugate vaccines produced in E. coli can be used to prevent pneumococcal infection. Vaccines produced in this manner may provide a low-cost alternative to the current vaccine production methodology

Cytoplasmic glycoengineering of Apx toxin fragments in the development of Actinobacillus pleuropneumoniae glycoconjugate vaccines.

BACKGROUND: Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia and represents a major burden to the livestock industry. Virulence can largely be attributed to the secretion of a series of haemolytic toxins, which are highly immunogenic. A. pleuropneumoniae also encodes a cytoplasmic N-glycosylation system, which involves the modification of high molecular weight adhesins with glucose residues. Central to this process is the soluble N-glycosyl transferase, ngt, which is encoded in an operon with a subsequent glycosyl transferase, agt. Plasmid-borne recombinant expression of these genes in E. coli results in the production of a glucose polymer on peptides containing the appropriate acceptor sequon, NX(S/T). However to date, there is little evidence to suggest that such a glucose polymer is formed on its target peptides in A. pleuropneumoniae. Both the toxins and glycosylation system represent potential targets for the basis of a vaccine against A. pleuropneumoniae infection. RESULTS: In this study, we developed cytoplasmic glycoengineering to construct glycoconjugate vaccine candidates composed of soluble toxin fragments modified by glucose. We transferred ngt and agt to the chromosome of Escherichia coli in order to generate a native-like operon for glycoengineering. A single chromosomal copy of ngt and agt resulted in the glucosylation of toxin fragments by a short glycan, rather than a polymer. CONCLUSIONS: A vaccine candidate that combines toxin fragment with a conserved glycan offers a novel approach to generating epitopes important for both colonisation and disease progression

Global transcriptional profiling of Burkholderia pseudomallei under salt stress reveals differential effects on the Bsa type III secretion system

BACKGROUND: Burkholderia pseudomallei is the causative agent of melioidosis where the highest reported incidence world wide is in the Northeast of Thailand, where saline soil and water are prevalent. Moreover, recent reports indicate a potential pathogenic role for B. pseudomallei in cystic fibrosis lung disease, where an increased sodium chloride (NaCl) concentration in airway surface liquid has been proposed. These observations raise the possibility that high salinity may represent a favorable niche for B. pseudomallei. We therefore investigated the global transcriptional response of B. pseudomallei to increased salinity using microarray analysis. RESULTS: Transcriptome analysis of B. pseudomallei under salt stress revealed several genes significantly up-regulated in the presence of 320 mM NaCl including genes associated with the bsa-derived Type III secretion system (T3SS). Microarray data were verified by reverse transcriptase-polymerase chain reactions (RT-PCR). Western blot analysis confirmed the increased expression and secretion of the invasion-associated type III secreted proteins BipD and BopE in B. pseudomallei cultures at 170 and 320 mM NaCl relative to salt-free medium. Furthermore, salt-treated B. pseudomallei exhibited greater invasion efficiency into the lung epithelial cell line A549 in a manner partly dependent on a functional Bsa system. CONCLUSIONS: B. pseudomallei responds to salt stress by modulating the transcription of a relatively small set of genes, among which is the bsa locus associated with invasion and virulence. Expression and secretion of Bsa-secreted proteins was elevated in the presence of exogenous salt and the invasion efficiency was enhanced. Our data indicate that salinity has the potential to influence the virulence of B. pseudomallei