Zakład Biochemii Analitycznej

BacSp222 is a recently discovered peptide bacteriocin produced by the opportunistic pet pathogen Staphylococcus pseudintermedius strain 222. This bacteriocin kills a wide range of Gram-positive bacteria, allowing producer cells to eliminate competing bacterial strains residing in the same physiological niche of the infected host organism. Research on BacSp222 to date has mainly focused on the molecular biology of the peptide, on the physicochemical mechanism of its action against bacterial membranes, and on determining the structure of the peptide using nuclear magnetic resonance (NMR) technique. Nevertheless, the most intriguing results proved that the described bacteriocin is not only capable of killing bacteria, but also shows significant activity against host cells. At higher doses, the peptide has a cytotoxic effect, while at very low concentrations (nanomoles/l) it acts as an immunomodulatory pro-inflammatory factor, inducing in model macrophage cell lines the activity of nitric oxide synthase (iNOs) and stimulating TNF secretion. A second intriguing finding revealed that the bacteriocin BacSp222 is produced and secreted along with several post-translationally modified isoforms, identified as succinic acid derivatives. Regarding these preliminary results, the presented project is devoted to extensive studies on the immunomodulatory effect of bacteriocin on eukaryotic cells, on the identification of cellular receptors and possible cytokines involved in this phenomenon, as well as on the mechanism of formation of the mentioned post-translational modifications, their influence on the biological activity of bacteriocin and the structure of its molecule. 

Comprehensive studies on immunomodulatory activity of BacSp222 bacteriocin will be conducted using model cell lines and primary cells. Native bacteriocin and bacteriocin after removal of N-terminal formyl-methionine will be used to verify the effect of formylation on recognition by specific receptors. Analysis of cytokines or growth factors produced by cells after exposure to bacteriocin will be carried out using the LUMINEX platform, studies on the induction of iNOs will be conducted by direct measurements of enzyme activity in cells, as well as based on measurements of enzyme gene expression by RT-PCR. Identification of cellular receptors involved in bacteriocin recognition will be performed using co-stimulation of cells with bacteriocin and receptor-specific antagonistic compounds, by measuring secretion of specific cytokines, by measuring intracellular mobilization of cAMP or Ca+2, and/or using modified cell lines exposing specific cellular recombinant receptors. Determinations of the effects of bacteriocin on transcription factors (e.g., AP-1, NFκB) will be performed using the electrophoretic EMSA technique. Effects of bacteriocin on cell morphology and verification of internalization of bacteriocin into cells will be studied by confocal microscopy, fluorescently labeled peptide and/or immunochemical techniques. Studies on the post-translational regulation of bacteriocin biological activity will be conducted using modified isoforms of the peptide isolated from culture medium. The effect of succinylation on the structure of the peptide in solution will be determined by NMR technique, the biological properties of the isoforms will be determined by bactericidal and cytotoxic activity assays, as well as by cell biochemistry research techniques described above. The mechanism of post-translational processing of bacteriocin and the influence of environmental factors on this phenomenon will be studied by chromatographic techniques.

Biologically, bacteriocins are defined as peptides or proteins produced by bacteria on ribosomes, capable of killing closely related strains living in the same physiological or ecological niche. These molecules also act as regulators of metabolism dependent on population density or the presence of other bacteria. It is estimated that 99% of bacterial strains produce at least one bacteriocin, so the biological and ecological importance of these factors is enormous. Moreover, bacteriocins or bacteriocin-producing microorganisms are widely used in the food industry, feed production, veterinary medicine and medicine. The realization of the research presented in this project will first of all allow to determine whether bacteriocins produced by opportunistic pathogens can also play a role of virulence factors, able to modulate the activity of immune cells of the infected organism. In addition, it will also allow to investigate a new post-translational mechanism of regulation of the of the biological activity of bacteriocins, based on polypeptide chain succinylation.

”Studies of the role of toxin-antitoxin systems in antibiotic resistance in staphylococci”

Staphylococci are bacteria which constitute the natural microflora of the skin and mucous membranes of humans and warm-blooded animals. On the other hand, they are dangerous opportunistic pathogens that can cause a number of diseases in their hosts. This threat is further strengthen by the increasing drug resistance of bacteria which causes serious difficulties in the treatment of staphylococcal infections.

Toxin-antitoxin systems (TA) are ubiquitous in bacteria but not present in eukaryotes. They consist of ‘toxin’, an intracellular protein often having enzymatic properties, and ‘antitoxin’ which is its specific inhibitor. Activation of the system as the consequence of the release of toxin leads to the “poisoning” of the cell resulting in, among others, growth inhibition or even its death. Although the role of the TA systems in bacterial physiology is still the subject of a heated discussion, it is known that these systems are involved in the stabilization of genetic material and the response to stress caused among others by antibiotics. Preliminary studies have shown that TA systems’ genes in staphylococci often occur together with genes for antibiotic resistance.

In the frame of the project we plan to explore the correlation between the presence of TA systems in staphylococci and their antibiotic resistance. The research will seek for the explanation whether and how TA systems influence the acquisition and stabilization of genetic material coding for antibiotic resistance. In addition, we intend to examine how the activation leading to the release of the enzymatic activity of the toxin (RNase) controls the degree of susceptibility of Staphylococcus to selected antibiotics.

Implementation of the project requires a number of techniques from the field of bacteriology, molecular biology and biochemistry. Among others, databases will be searched with the use of specialized software. For the functional characterization of the newly discovered TA systems chromatographic and electrophoretic techniques will be applied. Using specialized equipment the impact of TA systems on the action of antibiotics towards bacteria will be examined as well.

The study of the effect of TA systems on drug resistance of staphylococci are in concordance with the latest trends in the research in the field of modern bacteriology. This is particularly important in the case of bacteria from Staphylococcus genus, opportunistic pathogens,as these bacteria are the cause of many serious infections in humans and animals leading to a number of diseases that pose a serious public health hazard, and entail substantial losses in animal farming. Studies of relations and mechanisms linking TA systems to resistance of bacteria to antibiotics undoubtedly expand our knowledge in the field of the modern bacteriology with future implications for medical practice and veterinary medicine. Moreover, the planned research addresses the burning issue of the growing drug resistance in bacteria, for solving of which national and international institutions and organizations call insistently.

“Toxin-antitoxin systems as regulators of gene expression in Staphylococcus”

Staphylococci are members of natural microflora of the skin and mucous membranes of humans and warm-blooded animals. On the other hand, these bacteria are dangerous opportunistic pathogens that can cause a number of diseases in their hosts. This threat is further heighten by the increasing drug resistance and virulence of staphylococcal strains associated with the transfer of genetic material encoding factors responsible for these properties.

Toxin-antitoxin (TA) systems are commonly found in bacteria. The system consists of 'toxin', which is often a sequence-specific endoribonuclease and 'antitoxin' which is dedicated inhibitor of 'toxin'. Analysis of the literature data, combined with our very interesting preliminary results on the characteristics of the TA system (PemIKSa) present in Staphylococcus aureus CH91 allows us to assume that the role of TA systems beyond the originally postulated plasmid maintenance, suggesting their involvement in the global regulation of gene expression, in particular encoding virulence factors and proteins determining bacteria-environment interaction. In frame of this project, we plan to find the correlation between TA systems in staphylococci and their virulence and ability to survive exposure to antibiotics. Moreover, the influence of TA systems activation on transcriptome and proteome will be investigated and linked with molecular mechanisms of the activation of PemIKSa.

The project requires a number of techniques in the field of molecular biology, microbiology and “omics” techniques. Studies TA distribution systems in staphylococcal genomes will be performed using PCR technique, optimally in the form of multiplex PCR. Supplementation of the strains with the additional genes of TA system will be performed using a series of pCN plasmids. Preparation of plasmids allowing for expression of particular toxins will be performed with standard molecular biology methods. For preparation of knock-out strains markerless plasmid pKOR will be used. Using the techniques of quantitative RT-PCR (qRT-PCR) and Northern blot the analysis of the level of the TA operon transcript in the different phases of bacteria growth and in various stress conditions will be performed. The ability to form a biofilm will be determined spectrophotometrically after staining the cells adsorbed to the polystyrene plates. Next generation sequencing (RNA-seq, Illumina) will be used for transcrptome analysis. For proteomics, gel-free techniques with isobaric labelling (iTRAQ) and mass spectrometry will be applied.

The project will expanded knowledge on the role of TA systems in the regulation of gene expression in staphylococci, reflected by changes on transcriptome and proteome level, and their impact on virulence and closely related to the ability to form biofilms and to avoid the bactericidal activity of antibiotics. Exploring the molecular mechanisms that can link TA systems with the virulence and antibiotic resistance of these bacteria undoubtedly expand our knowledge in the field of modern bacteriology and molecular biology.

Due to the importance of research for the scientific community and medical practice we intend to publish the results in international journals. Preference will be given to the journals with high citation index and free access to readers. In addition, it is planned to present the results at prestigious conferences, where there is an opportunity to established a collaboration with researchers of the leading centres of research in the field of studies. This is particularly important because the co-investigators of the project are also young scientists.

"Toxin-antitoxin systems as regulators of gene expression in Staphylococcus"

Staphylococci are members of natural microflora of the skin and mucous membranes of humans and warm-blooded animals. On the other hand, these bacteria are dangerous opportunistic pathogens that can cause a number of diseases in their hosts. This threat is further heighten by the increasing drug resistance and virulence of staphylococcal strains associated with the transfer of genetic material encoding factors responsible for these properties.

Toxin-antitoxin (TA) systems are commonly found in bacteria. The system consists of 'toxin', which is often a sequence-specific endoribonuclease and 'antitoxin' which is dedicated inhibitor of 'toxin'. Analysis of the literature data, combined with our preliminary results on the characteristics of the TA system (PemIKSa) present in Staphylococcus aureus CH91 allows us to assume that the role of TA systems beyond the originally postulated plasmid maintenance, suggesting their involvement in the global regulation of gene expression, in particular encoding virulence factors and proteins determining bacteria-environment interaction. In frame of this project, we plan to find the correlation between TA systems in staphylococci and their virulence and ability to survive exposure to antibiotics. Moreover, the influence of TA systems activation on transcriptome and proteome will be investigated and linked with molecular mechanisms of the activation of PemIKSa.

The project requires a number of techniques in the field of molecular biology, microbiology and "omics" techniques. Studies TA distribution systems in staphylococcal genomes will be performed using PCR technique, optimally in the form of multiplex PCR. Supplementation of the strains with the additional genes of TA system will be performed using a series of pCN plasmids. Preparation of plasmids allowing for expression of particular toxins will be performed with standard molecular biology methods. For preparation of knock-out strains markerless plasmid pKOR will be used. Using the techniques of quantitative RT-PCR (qRT-PCR) and Northern blot the analysis of the level of the TA operon transcript in the different phases of bacteria growth and in various stress conditions will be performed. The ability to form a biofilm will be determined spectrophotometrically after staining the cells adsorbed to the polystyrene plates. Next generation sequencing (RNA-seq, Illumina) will be used for transcrptome analysis. For proteomics, gel-free techniques with isobaric labelling (iTRAQ) and mass spectrometry will be applied.

"Identification and studies of factors involved in staphylococcal virulence using genomics and proteomics"

Interaction of pathogenic bacteria with the host organism is mediated by the so-called virulence factors, which may be located on the surface of cells of the microorganism or secreted to the environment. The genes coding for the virulence factors, their expression and its control is subjected to systems located inside the cell. Only coordinated action of these elements leads to the observed phenotype and results in a measurable biological effect.

As the aim of this project, we intend to examine the correlation between genotype, phenotype as well as proteome and virulence of carefully selected strains of staphylococci, in order to identify and characterize the factors affecting the observed biological effect (the ability to effectively colonize the host and develop an infection).

The subject of the research are carefully selected strains of S. aureus (commensal and from lesions) isolated from humans and animals (poultry). The project consists of two basic parts, genomic and proteomic. In the genomic part the genome sequence of selected S. aureus strains will be determined with the use of "next generation sequencing" techniques (platform Ilumina). The second part of the genomic studies will be bioinformatic analysis of the results. This high-throughput research will be carry out using freely-available software and databases. Moreover, we plan to create a dedicated scripting language Python facilitating the studies.

Studies of extracellular and cell surface-associated proteome (for selected S. aureus strains) are aimed to link genomic data with a pool of proteins that can potentially interact with the host during colonization and infection. These analyzes will run concurrently. In the first place we intent to compare extracellular proteomes of the selected strains in correlation with a high and low strain virulence in the chicken embryo model. This will be performed using 2D DIGE technique based on simultaneous analysis of two samples labeled with different fluorescent days. The second direction of proteomic studies will apply to proteins associated with the surface of bacterial cells (Surfome). To analyze this pool of proteins the cell shaving technique will be used.