The main objective of our research group is to exploit properties of Prokaryotes that thrive in extreme habitats for potential biotech applications. Enzymes being in our focus such as Enzymes such as DNA replication/transcription enzymes, DNA/RNA ligases, nucleases, SSB proteins, helicases, recombinases constitute primary targets to advance the knowledge on genome plasticity, maintenance and evolution. Other proteins being in our focus are thermophilic phages lytic enzymes that due to their antibacterial activity are perfectly suited as a scaffold for the development of antimicrobial agents (Plotka et al., 2019b).
Our recent efforts were focused on (i) improving specificity of a PCR-based DNA amplification; (ii) using thermophilic lytic enzymes (phage endolysins) as an alternative for antibiotics. In both areas proteins derived from thermophilic sources have proven to be extremely promising. We found that among proteins tested RecA of Thermus thermophilus phage Tt72 and RadA of archaeon Pyrococcus woesei improve substantially specificity of simplex and multiplex PCR assays by eliminating non-specific products (Stefanska et al., 2014, J. Biotech. 182-183:1-10; Stefanska et al., 2016, J. Appl. Genet. 57:239-249). The results obtained were so encouraging that we decided to use RadA protein as a crucial component of an allelic discrimination PCR assay of a human HADHA gene associated with long-chain 3-hydroxylacyl-CoA dehydrogenase deficiency that in infancy may lead to hypotonia, serious heart and liver problems and even sudden death. This is an example how proteins from extreme microbes can improve performance of diagnostic tests.
The second area that we explore currently covers lytic enzymes. Studies on them are important in the development of effective antimicrobial agents as an alternative to traditional antibiotics. Lytic enzymes (endolysins) discovered recently in our laboratories come from two thermophilic Thermus scotoductus phages: Ph2119 and Tsc2631 (Plotka et al., 2014, Appl. Environ. Microbial. 80:886-895; Plotka et al., 2015, Plos One 10:e0137374). Both of them while sharing high identity (74%) do not resemble any of the known thermophilic phage lytic enzymes. Instead, they have conserved a Zn2+ binding site characteristic for T3 and T7 lysozymes as well as eukaryotic peptidoglycan recognition proteins, which directly bind to, but also may destroy bacterial peptidoglycan. The enzymes show high lytic activity towards thermophiles, i.e. Thermus scotoductus; T. thermophilus; T. flavus and also, towards mesophilic Gram-negative bacteria i.e. E. coli, Serratia marcescens; Pseudomonas fluorescens, and Salmonella panama. The enzymes showed no activity against a number of Gram-positive bacteria analyzed with the exception of Deinococcus radiodurans and Bacillus cereus. Both lysins were found to be highly thermostable (optimum 50-78°C) : they retain approx. 87% of lytic activity after 6 h incubation at 95°C. Using structural and functional approach we found that the active site residues are necessary for lysis yet not crucial for peptidoglycan binding. Because Ts2631 endolysin exhibits intrinsic antibacterial activity and unusual robust thermal stability, it is perfectly suited as a scaffold for the development of antimicrobial agents (Plotka et al., 2019, Sci. Rep. 9:1261). We also demonstrated that bactericidal potential of Ts2631 endolysin can be exploited against the alarming multidrug-resistant clinical strains of Acinetobacter baumannii, Pseudomonas aeruginosa and other pathogens from the Enterobacteriaceae family. The EDTA/Ts2631 combination reduced A. baumannii, P. aeruginosa and all pathogens of the Enterobacteriaceae family, particularly multidrug-resistant Citrobacter braakii, to levels below the detection limit (>6 log); these results indicate that Ts2631 endolysin could be useful to combat Gram-negative pathogens. The investigation of A. baumannii cells treated with Ts2631 endolysin variants under transmission electron and fluorescence microscopy demonstrated that the intrinsic antibacterial activity of Ts2631 endolysin is dependent on the presence of its N-terminal extension (Plotka et al., 2019, Viruses 11:657).
molecular cloning, gene overexpression, protein purification, DNA/RNA sequencing,