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Proteomic characterisation of surfomes, secretomes and membrane vesicles in Gram-positive bacteria
Bacterial surface proteins play a fundamental role in the interaction between the cell and its environment. They constitute a diverse group of molecules with important functions. They are also potential targets for drugs or vaccines. The post-genomic era offers new and exciting opportunities for vaccine research, which are expected to shorten the time to discovery. The new “omics” technologies allow the search for new targets on a large scale, but the large number of identified candidates can often exceed the required in vivo validation process, which questions the ultimate impact of these strategies to accelerate the discovery of new vaccines.
Infections caused by bacteria of the genus Streptococcus have a major impact on global public health, both in humans and animals. The pneumococcus (S. pneumoniae) is the leading cause of pneumonia in children and adolescents, accounting for more than one million deaths per year in children under five. Infections caused by this bacterium have increased in frequency and severity over the past two decades. Although a polysaccharide conjugate vaccine is available, it is of limited effectiveness, and its price is high. Recombinant protein-based vaccines would offer greater coverage at a more affordable price, which is crucial for developing countries. On the other hand, S. suis is a swine pathogen that causes significant economic losses in the livestock sector, but given its zoonotic nature it has led to outbreaks in humans that have caused dozens of deaths.
In this line of research, we characterized the “surfome” (i.e. the set of surface proteins) of collections of clinical isolates of S. pneumoniae and S. suis, as well as other Gram-positive species, using the strategy of “shaving” live bacteria (Rodríguez-Ortega et al, Nat Biotechnol 2006, 24(2): 191-197). This strategy consists of the digestion of the surface of living cells with proteases, the recovery ,liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). This allows rapid identification of highly expressed and exposed proteins. The identified proteins are validated to monitor their in vivo accessibility to antibodies, and then protective assays are performed on animal models to test their efficacy as vaccines. In addition, we characterise the “secretome”, defined as the set of proteins secreted by the bacteria, as well as the extracellular membrane vesicles produced by the bacterial cells, which have been shown to have a high immunogenicity and protective capacity against infection.
Keywords: Streptococcus, proteomics, surface proteins, secretome, membrane vesicles, vaccines.
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Development of diagnostic serological tools against pneumococcal infection
Pneumococcal infections are one of the leading causes of morbidity and mortality in children worldwide, with a particular incidence in developing countries. Epidemiological surveillance is crucial in order to elucidate changes in the patterns of infection by the microorganism at the population level and the effectiveness of preventive measures. Serological techniques offer a wide range of possibilities for such monitoring, and also provide an early diagnostic tool that can help make better decisions about the treatment of patients and guide the measures to be taken at population level. In this line, we have developed a serological tool for detecting pneumococcal infection in patients, which allows us to distinguish between infected people and healthy carriers. To do this, we use 95 recombinant pneumococcal proteins that we have in our laboratory as antigens, and which we have identified experimentally using the proteomic strategy of “shaving” live cells. With these recombinant proteins, we have built two protein arrays on two different platforms: printed on a glass surface, and immobilized in Luminex microspheres. This technique would facilitate epidemiological studies of vaccine effectiveness due to its ease of use and possibility of use for serodiagnostics in hospitals and primary care settings. Additionally, a longitudinal study of children will investigate the development of natural immunity to pneumococcal surface proteins and their relationship with colonization in populations in our environment and in another in Southeast Asia with early and very frequent colonization. These results will help in the selection of future vaccine candidates based on surface proteins.
Keywords: pneumococcus, proteomics, protein arrays, Luminex, serodiagnostics, epidemiology.
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Multiomic analysis of the effect of antimicrobial substances from plants, essential oils and their main bioactive compounds
Antibiotics are molecules produced by living beings that are used to treat infections caused by bacteria. However, their use and abuse has led to the emergence of resistances in infectious microorganisms. The emergence of antibiotic-resistant strains in pathogenic bacteria is a global public health concern, as prophylactic measures, such as vaccines, are hardly available to combat most infections caused by bacteria. If today’s antibiotics were to become ineffective, we would be defenceless against bacterial infections, which could result in terrible epidemics and deaths as in past eras. It is therefore a priority to carry out research into the discovery and mechanisms of action of new substances with antimicrobial capacity, which will provide us with tools to fight infections, should the main antibiotics in use cease to be effective.
To this end, plants have always been a source of remedies for diseases. The antimicrobial activity of many plants and their extracts, particularly essential oils of aromatic plants, has been widely described. In this line, we study the effect at proteomic level after exposing Gram-positive bacteria to various molecules, essential oils and their main bioactive compounds. The substances studied include rhodomyrtone, a molecule isolated from a South-East Asian plant, as well as essential oils of thyme, oregano and cinnamon, and their main bioactive compounds: thymol, carvacrol and cinnamaldehyde.
Keywords: antimicrobials, antibiotic resistance, Gram-positive bacteria, proteomics, essential oils, plants.
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Proteomics for the identification of bioactive peptides in kefir and other dairy products
Kefir is a drink resulting from the fermentation of milk by a symbiosis of microorganisms. Originating from the Caucasian mountains, it has been widely consumed in Eastern European countries, Russia and South-West Asia for at least 2000 years. Its consumption is increasing in Western countries, although in Mediterranean countries it is considerably less consumed than in the above-mentioned countries or in others such as Scandinavia. It has acquired the “status” of a probiotic food, being designated as “the yoghurt of the 21st century”. Kefir differs from other dairy ferments, such as yoghurts, in that it is not produced by one or a few microorganisms (bacteria), but by an association of bacterial species (Lactobacillus and other species) and yeasts (Saccharomyces and others), which gives it special characteristics. Thus, the fermentation produced is lactic-alcoholic, unlike yogurt, which is only lactic. This gives it a characteristic taste, and a sensation of effervescence in the mouth, due to the CO2 produced. The main products of fermentation, apart from CO2, are lactic acid and ethanol (the latter generally around 0.5%), as well as other minority components such as diacetyl, acetaldehyde, amino acids and peptides, which contribute to the product’s particular organoleptic characteristics.
This fermented beverage has the added value of being a probiotic food, which means potential access to a wide range of consumers looking for this type of product. In addition to the tradition that attributes to it the responsibility for the longevity of the inhabitants of the Caucasus area from which it comes from, numerous studies report many health benefits, including antimicrobial, antitumour, anti-carcinogenic and immune system enhancing activity, as well as its contribution to the reduction of cholesterolaemia. It is also suitable for people who are lactose intolerant, because lactose is practically absent from the product, and because it contains the enzyme β- galactosidase.
Although it is a food that arouses great interest, its study at biochemical and molecular level is minimal. Thus, very little is known about the enzymes that are secreted into the environment and which can contribute to the transformation of milk into kefir. Likewise, very few peptides have been characterised, some of which are bioactive. This line of research aims to deepen our knowledge of these two aspects, so that we can contribute to describing more of the beneficial properties of kefir and to finding, if possible, new bioactive molecules that can be isolated and used on their own or added to functional foods.
Keywords: kefir, fermented dairy products, bioactive peptides, proteomics, metabolomics, functional foods
