PRELUDIUM BIS - four grants for UPWr researchers

• Prof. Krzysztof Grzymajło – research leading to improved methods of prevention and treatment of salmonellosis.
• Prof. Joanna Kolniak-Ostek – health-promoting formulas derived from coloured varieties of raspberries
• Prof. Zbigniew Lazar – use of Yarrowia yeast in biofuel production
• Prof. Ewa Walecka-Zacharska – reducing cases caused by Campylobacter jejuni bacteria

In the PRELUDIUM BIS 4 competition of the National Science Center, 58 projects were recognized this year. Four of them were written by UPWr scientists. All of them are attempts to find answers to contemporary problems. The implementation of the project of the team led by Prof. Krzysztof Grzymajła will help develop current knowledge of the pathogenesis of Salmonella, which in the future may lead to improved methods of prevention and treatment of salmonellosis. Prof. Ewa Walecka-Zacharska's project will contribute to increasing food safety and reducing foodborne illnesses caused by Campylobacter jejuni bacteria. Prof. Joanna Kolniak-Ostek's research, meanwhile, is expected to help develop formulas with enhanced health-promoting properties derived from colored raspberry varieties. And Prof. Zbigniew Lazar will look into the use of yeasts in biofuel production.

Improving methods of prevention and treatment of salmonellosis

Prof. Krzysztof Grzymajło of the UPWr's Department of Biochemistry and Molecular Biology will study the regulation of the expression of Salmonella virulence factors and their impact on the course of infection. He has received more than PLN 688,000 for his project. As the UPWr scientist explains, Salmonella bacilli, belonging to the Enterobacteriaceae family of gram-negative are among the most common causes of foodborne diseases, this infection is a major epidemiological problem worldwide. The most common source of it among humans is food of animal origin, hence diseases caused by Salmonella are referred to as zoonoses.

– The process of Salmonella infection is a multi-stage interaction between the bacterium and the infected host, focused on reaching the site of infection, adhering and finally entering the host cells. These processes are based on virulence factors: specific structures formed by Salmonella – explains Prof. Grzymajło, adding that two types of these structures – flagella and fimbriae responsible for motility and adhesion, respectively, determine the success of the pathogen during the first stages of infection. Flagella allow Salmonella to reach the site of infection, while fimbriae adhere to host cells and resist mechanical forces attempting to remove them from the intestinal lumen. – Consequently, both types of structures are responsible for the first stages of infection and determine the subsequent course of the infection, the scientist stresses.

Flagella are made up of three basic elements: the shaft, the hook and the filament. The filament is the most important from the point of view of Salmonella, built by two types of proteins that are formed in a coordinated manner involving a mechanism called phase variation. However, among the structures responsible for the attachment process, the most intensively studied are type 1 (T1F) fimbriae, long, filamentous structures with the FimH protein present at their top. This is directly responsible for the binding of structures to the host cell surface. – Despite numerous studies focusing on the role and regulation of Salmonella virulence factors, there is insufficient data on how flagella and T1F are regulated. We do not know, for example, when, at which stages, for how long and in what quantities they are produced during the course of Salmonella infection. Also, the role and significance of the interactions between these structures remains unknown, the scientist admits, but stresses that the UPWr team's preliminary research sheds some light on these interactions.

– We discovered that T1F production is dependent on Salmonella growth conditions and positively correlates with the level of host cell adhesion and invasion. Moreover, genetic elements involved in T1F production directly influence the level of production of flagellin-associated proteins, indicating that the modes of regulation of both types of structures are closely related. We want to analyse the interplay between the structures responsible for Salmonella adherence and motility during the first stages of infection, explains Prof. Grzymajło. The team under his leadership expects that the project will help develop current knowledge of Salmonella pathogenesis, which may lead to improved methods of prevention and treatment of salmonellosis in the future.

Health-promoting bioactive compounds from coloured raspberry varieties

Prof Joanna Kolniak-Ostek from the Department of Fruit, Vegetable and Plant Nutraceutical Technology at UPWr received nearly PLN 688,000 for research into the health-promoting properties of bioactive compounds from coloured raspberry varieties using  symbiotic bacterial and yeast cultures. As the UPWr scientist explains, in recent years, more and more attention has been paid to the impact of food on the human body, one of the reasons being that, both in Poland and other European countries, the main cause of death is chronic non-communicable diseases (NCDs), linked, among other things, to poor nutrition. The development of chronic diseases, autoimmune diseases, neurodegenerative diseases, cancer and cachexia are associated with so-called oxidative stress, caused by high levels of free oxygen radicals (ROS).

– Epidemiological studies are providing us with more and more evidence that consumption of foods rich in antioxidants reduces the risk of developing chronic diseases and oxidative stress, and the consumption of fruit, vegetables and their preserves, which are an excellent source of antioxidant and health-promoting substances, has a particularly beneficial effect on the human organism – says Professor Joanna Kolniak-Ostek. And she emphasises that the leaves of red, yellow and black raspberries, which are characterised by a high content of polyphenolic compounds with bioactive properties, can play a role in the prevention of civilisation diseases. The aim of the UPWr scientist's project is to analyse the profile and content of phytochemicals in the leaves of coloured raspberry varieties and the possibility of modulating their health-promoting properties through alcohol-acetic acid fermentation. Based on the results obtained, stable model preparations of bioactive compounds with enhanced health-promoting properties will be developed.

The project has seven research steps, which will result in the qualitative and quantitative identification of the bioactive fraction of compounds from leaves using LC-MS techniques, determination of the bioactive properties of the polyphenolic fraction and their bioavailability during simulated in vitro digestion, the use of alcohol-acetic acid fermentation to enhance biological properties and, finally, the derivation of stable model preparations of bioactive compounds with enhanced health-promoting properties.

The research will be interdisciplinary involving chemistry, medicine and food technology.

Use of yeast in biofuel production

Prof Zbigniew Lazar from the Department of Food Biotechnology and Microbiology at UPWr received almost PLN 563 000 for research on Yarrowia yeast and the creation of a platform for the simultaneous biosynthesis of lipids and extracellular heterologous proteins. The UPWr scientist's project is of great importance at a time when the world is experiencing a growing demand for alternative energy sources such as biofuels, as oil and natural gas resources are depleting. One promising solution to this problem is the use of microorganisms in biofuel production, which are characterised by their high capacity to accumulate intracellular lipids.

– There are microorganisms that are able to accumulate up to 80 percent of lipids on a dry weight basis. These include, for example, the yeast Yarrowia lipolytica – explains Prof. Zbigniew Lazar, and emphasises that these amazing microorganisms belong to a broader group of microorganisms called the Yarrowia clade (currently formed by 15 different yeast species). – And among the species that make up this clade, there are some whose abilities to accumulate lipids or secrete polyols are significantly higher than those of Y. lipolytica. It has also been shown that these yeasts are capable of efficiently utilising waste compounds such as crude glycerol or volatile fatty acids (VFAs), which are found in high concentrations in the digestion mixtures of food industry waste or municipal waste, adds the scientist and notes that Yarrowia keelungensis or Yarrowia phangngaensis are characterised by a significantly better uptake of these substrates than Y. lipolytica.

The analysis of the transcriptome of these two microorganisms is to be the starting point for improving the utilisation of these substrates. This is at the same time a prelude to a very important part of the professor's proposal, which is the characterisation of glycerol and VFA metabolism, as well as the lipid biosynthetic pathway through the use of an automated colony manipulation system.

– The analysis of tens of thousands of transformants will allow the identification of new, hitherto uncharacterised enzymes that will allow the understanding of the functioning of metabolic pathways in selected species of the Yarrowia clade. Another important aspect is to exploit the enormous protein secretion capacity of this yeast, which in turn will be the starting point for their use in the production of heterologous proteins, especially those with surfactant activity. In fact, these proteins will be able to replace chemically synthesised surfactants used, for example, in mining, says the scientist. And he emphasises that surfactants are used in many forms and perform a variety of functions also in the production of products used in households, orchards and fields, in the automotive industry, construction, road building or the cosmetics and pharmaceutical industries. – Each of these industries is looking for alternative, biodegradable compounds, the properties of which would allow the development of environmentally friendly technologies. The solutions proposed in the project will certainly contribute to this – Professor Lazar concludes.

Research will contribute to increased food safety

Professor Ewa Wałecka-Zacharska from the Department of Food Hygiene and Consumer Health Protection at UPWr received nearly PLN 688,000 for research on identifying new genes related to biofilm formation by Campylobacter jejuni bacteria. This is another project in which understanding the mechanisms will contribute to food safety and reduce public health risks.

Campylobacter jejuni is currently the most common cause of foodborne illness in the European Union and the leading cause of bacterial watery diarrhoea in humans worldwide. The most common source of human infection is poultry and poultry products, and it occurs through the consumption of food contaminated with this pathogenic bacterium that has not undergone sufficient heat treatment. After consumption of products contaminated with C. jejuni bacilli, the pathogen colonises the small and large intestine of humans causing fever, abdominal pain and diarrhoea, although complications in the form of autoimmune reactions can also occur.

– A key factor in helping a pathogen survive in adverse environmental conditions is the ability to form a biofilm. This is a spatial structure containing microorganisms and the substances that bind their cells together. Bacteria residing in a biofilm are difficult to eliminate due to their strong adhesion to surfaces. They are also characterised by higher resistance to disinfectants and antibiotics – says prof. Ewa Walecka-Zacharska, and explains that although the process of biofilm formation is relatively well understood in bacteria such as Pseudomonas aeruginosa and Escherichia coli, little is known about this mechanism in the case of C. jejuni. Therefore, the aim of the UPWr scientist's project is to identify previously unknown genes associated with biofilm formation in C. jejuni by transposon-mediated mutagenesis.

– Transposons are mobile genetic elements that have the ability to incorporate themselves into different places in the genome. The incorporation of a transposon into a gene sequence usually triggers its inactivation, preventing the production of the protein encoded by the gene. In order to find previously unknown genes involved in biofilm formation by C. jejuni, we will construct a library of mutants of this bacterium, and then evaluate them for their ability to form biofilm – Prof. Wałecka-Zacharska enumerates.

The results of the project will allow us to understand the mechanism of biofilm formation by C. jejuni, which is a key factor necessary for the pathogen's survival in the environment. Knowledge of these mechanisms may be helpful in designing substances that inhibit biofilm formation, which will contribute to increased food safety and reduce public health risks.

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