Joint genomic and proteomic analysis identifies meta-trait characteristics of virulent and non-virulent Staphylococcus aureus strains

Staphylococcus aureus is an opportunistic pathogen of humans and warm-blooded animals and presents a growing threat in terms of multi-drug resistance. Despite numerous studies, the basis of staphylococcal virulence and switching between commensal and pathogenic phenotypes is not fully understood. Using genomics, we show here that S. aureus strains exhibiting virulent (VIR) and non-virulent (NVIR) phenotypes in a chicken embryo infection model genetically fall into two separate groups, with the VIR group being much more cohesive than the NVIR group. Significantly, the genes encoding known staphylococcal virulence factors, such as clumping factors, are either found in different allelic variants in the genomes of NVIR strains (compared to VIR strains) or are inactive pseudogenes. Moreover, the pyruvate carboxylase and gamma-aminobutyrate permease genes, which were previously linked with virulence, are pseudogenized in NVIR strain ch22. Further, we use comprehensive proteomics tools to characterize strains that show opposing phenotypes in a chicken embryo virulence model. VIR strain CH21 had an elevated level of diapolycopene oxygenase involved in staphyloxanthin production (protection against free radicals) and expressed a higher level of immunoglobulin-binding protein Sbi on its surface compared to NVIR strain ch22. Furthermore, joint genomic and proteomic approaches linked the elevated production of superoxide dismutase and DNA-binding protein by NVIR strain ch22 with gene duplications

Assembly of the genome sequence of Staphylococcus aureus strain CH21

Poznanie sekwencji DNA a następnie analiza jej konkretnych elementów jest głównym celem badania genomów. Dzięki temu możliwa jest lokalizacja genów i innych interesujących fragmentów sekwencji. Samo sekwencjonowanie jest jednak bezwartościowe, do momentu uporządkowania jego wyników, polegającego na złożeniu sekwencji w kontigi, pogrupowaniu a następnie połączeniu w celu otrzymania całkowitego obrazu sekwencji chromosomu oraz plazmidów. Aby w pełni zrozumieć zawartość genomu oraz funkcje poszczególnych elementów konieczne jest połączenie analizy komputerowej i eksperymentalnej. W ciągu ostatnich kilkunastu lat, dzięki rozwojowi metod sekwencjonowania całego genomu, dokonano olbrzymich postępów w badaniach nad ewolucją patogenów bakteryjnych oraz nad ich mechanizmami działania. Jednymi z najbardziej interesujących bakterii, z punktu widzenia medycznego oraz badań naukowych są gronkowce, a szczególnie szczepy gatunku Staphylococcus aureus. Bakterie gronkowca złocistego są ważnym składnikiem mikroflory skóry ludzi i zwierząt, mimo to mogą stać się główną przyczyną, zagrażających życiu zakażeń. Wykazano, że szczepy ludzkie i zwierzęce są genetycznie odrębne, oraz ustalono, że S. aureus może się przemieszczać między gatunkami. Organizm ten produkuje wiele specyficznych czynników wirulencji, które mogą odpowiadać za ułatwienie kolonizacji gospodarza oraz wywołanie infekcji. Ponadto szczepy S. aureus wykazują rosnącą lekooporność. W oparciu o powyższe doniesienia w niniejszej pracy magisterskiej podstawę badań stanowił, wykazujący wirulencję w modelu zarodka kurzego, szczep S. aureus CH21 izolowany ze zmian chorobowych u drobiu. Podjęto próbę złożenia genomu wspomnianego szczepu w oparciu o odczyty sekwencji uzyskane z sekwencjonowania drugiej generacji (Illumina ®). Praca opiera się na analizie komputerowej otrzymanych z sekwencjonowania wyników oraz uzupełnianiu danych bioinformatycznych, danymi eksperymentalnymi. Podjęte działania laboratoryjne sprowadzają się do projektowania starterów a następnie wykonaniu z ich użyciem reakcji PCR i standardowego sekwencjonowania. W wyniku czego otrzymano sekwencję chromosomu S. aureus CH21 w postaci sześciu kontigów oraz otrzymano pełną sekwencję plazmidu pAvX.Understanding DNA sequences and then analysis of its specific elements is the main purpose of study genomes. This enables location of genes and other interesting sequence fragments. Sequencing, however, is worthless, until the arrangement of the results, consisting of submitting sequence contigs, grouping them and then combining to obtain a complete picture of the sequence of the chromosome and plasmids. To fully understand the contents of the genome and the functions of individual elements it is necessary to connect computer with experimental analysis. Over the past several years, thanks to the development of whole genome sequencing methods, great progress has been made in the study of the evolution of bacterial pathogens and on their mechanisms of action. From the point of medical and scientific research view, the most interesting bacteria among the others are staphylococci, especially strains of Staphylococcus aureus. The bacteria are a major component of humans and animals skin microflora -despite the fact it may become a major cause life-threatening infections. It was shown that human or animal strains are genetically distinct, and it was found that S. aureus can transfer between species. This organism produces a number of specific virulence factors that may be responsible for facilitating the colonization of the host and calling infections. In addition, S. aureus strains show increasing drug resistance.The above described reports were the basis for studying S. aureus strain CH21, isolated from lesions in poultry and exhibiting virulence in chicken embryo model . An attempt was made to obtain a genome sequence of mentioned strain based on data derived from next-generation sequencing (Illumina ®). The work is based on computer analysis of results obtained from sequencing and complement bioinformatics data, the experimental data. Undertaken laboratory activities come down to design primers in order to use them to PCR reaction and standard Sanger sequencing.As a result, the sequence of S. aureus CH21 chromosome, in a form of six contigs, and the full sequence of plasmid pAvX was received

Studies on the Anticancer and Antioxidant Activities of Resveratrol and Long-Chain Fatty Acid Esters

Resveratrol (RES) is gaining recognition as a natural bioactive compound. To expand the possible applications of RES with its enhanced bioactivity as well as to increase the health benefits of long-chain fatty acids, a lipophilization process of RES was performed using three fatty acids: palmitic acid (PA), oleic acid (OA), and conjugated linoleic acid (CLA). The obtained mono-, di-, and tri-esters of RES were evaluated for their anticancer and antioxidant properties against lung carcinoma (A549), colorectal adenocarcinoma (HT29), and pancreatic ductal adenocarcinoma (BxPC3) cell lines. Human fibroblast (BJ) cells were used as a control. Several parameters were investigated: cell viability and apoptosis, including the expression of major pro- and anti-apoptotic markers, as well as the expression of superoxide dismutase, a key enzyme of the body’s antioxidant barrier. Three of the obtained esters: mono-RES-OA, mono-RES-CLA, and tri-RES-PA, which significantly reduced the tumor cell viability up to 23%, at concentrations 25, 10, 50 μg/mL, respectively, turned out to be particularly interesting. The above-mentioned resveratrol derivatives similarly increased the tumor cells’ apoptosis by modifying their caspase activity of pro-apoptotic pathways (p21, p53, and Bax). Moreover, among the mentioned esters, mono-RES-OA induced apoptosis of the analyzed cell lines most strongly, reducing the number of viable cells up to 48% for HT29 cells versus 36% for pure RES. Furthermore, the selected esters exhibited antioxidant properties towards the normal BJ cell line by regulating the expression of major pro-antioxidant genes (superoxide dismutases—SOD1 and SOD2) without the effect on their expression in the tumor, and therefore reducing the defense of cancer cells against increased oxidative stress induced by high ROS accumulation. The obtained results indicate that the esters of RES and long-chain fatty acids allow enhancement of their biological activity. The RES derivatives have the potential for being applied in cancer prevention and treatment, as well as for oxidative stress suppression