Pracownie badawcze

Badania

Najważniejsze osiągnięcia badawcze

• We developed a CRISPRi platform for analysis of gene functions in Pseudomonas aeruginosa and successfully used it in high-throughput analyses allowing identification of genes (including essential genes), which silencing alters P. aeruginosa susceptibility to the action of a spectrum of antibiotics.
• We showed that the overproduction of some drug/metabolite transporters causes significant inhibition of bacterial growth and influence motility and biofilm formation - important functions connected with P. aeruginosa virulence and survival.
• We identified diverse partners of the partitioning ParB protein in P. aeruginosa indicating its potential role in the cross-talk between chromosome segregation and other cellular processes.
• We revealed the ability of ParB to act as a nucleoid-associated protein that specifically binds to multiple, short sequences in the P. aeruginosa genome, indicating that ParB may play a role in genome topology.

Opis badań

Bacteria as single-cell organisms are fascinating subjects of investigation showing incredible survival capabilities and a huge repertoire of mechanisms helping them to last in very distinct environments including the human body. Pseudomonas aeruginosa is a bacterium commonly found in various niches and able to survive in very unfavorable conditions. Alongside other ESKAPE group pathogens, P. aeruginosa is a leading cause of nosocomial infections, posing particular risks to immunocompromised individuals and cystic fibrosis patients. Our studies focus on understanding of resistance and adaptation potential associated with multilayered regulation, including the components required for stable maintenance of genetic material and keeping cellular homeostasis of this bacterium.

Currently, our research focuses on: 1/ adaptation of the CRISPR interference (CRISPRi) method for functional analysis of P. aeruginosa genes and use it in studying mechanisms of bacterial resistance to antimicrobial compounds; 2/ characterization of genes encoding drug/metabolite transporters (DMTs) and chosen transcription regulators to shed light on their role in physiology, drug resistance and virulence of P. aeruginosa; 3/ further characterization of the mechanisms involved in P. aeruginosa chromosome segregation and their links with other processes in the cell.

Ad. 1/ We use the CRISPRi method in the study of antibiotic resistance and survival strategies of P. aeruginosa to identify the new targets for the development of effective antibacterial drugs.

Ad. 2/ We are working on creation of a genetic platform to investigate the functions of drug/metabolite transporters and efflux pumps in P. aeruginosa, to explore their functional interplay, and find potential inhibitors. We adapted a non-mammalian infection model Galleria mellonella to study bacterial pathogenesis (in collaboration with University of Warsaw), as some studied drug/metabolite transporters play a role in pathogenesis of P. aeruginosa. Moreover, we are working on adaptation of the proximity-dependent biotynylation method, allowing identification of protein-protein interactions in vivo, to characterize membrane interactome of drug/metabolite transporters. Our recent studies revealed a connection between drug metabolite transporter PA2576 and LPS biogenesis, as well as PA2577 (regulator of PA2576 gene) and type III secretion system (T3SS) regulatory cascade, pointing out the potential multitasking roles of the indicated proteins in bacterial cells. Our research addresses important biological questions on how bacteria control transport functions to modulate cellular processes, increase adaptability, and promote survivability, also during the pathogenesis process.

Ad. 3/ P. aeruginosa possesses a single, circular chromosome, which encodes an active partitioning system consisting of ParA (a Walker-type ATPase), ParB (a DNA binding protein) and parSs, centromere-like sequences recognized and bound by ParB. Numerous aspects of ParAB-parS functioning in P. aeruginosa were studied by the members of our group. P. aeruginosa mutants lacking par genes, or parS sites were constructed and the consequences of these changes for cell biology were analysed. Additionally, interactions of ParA and ParB with proteins involved in the cell cycle, cell division, metabolism, motility, antibiotic resistance, and virulence were also studied. Our recent analyses confirmed that MksBEF proteins become crucial for DNA segregation in cells lacking ParAB-parS system. ParB binding to genome was analysed using chromatin-immunoprecipitation and sequencing (ChIP-seq), what demonstrated ParB nucleoprotein formation around parS-sites but also showed for the first time, binding of ParB proteins to numerous half-parS sites, containing sequence motif representing one arm of the parS. Using transcriptome analyses we demonstrated the ability of ParB to directly repress the expression of certain operons and postulated a broad, but indirect role for ParB in gene regulation. Using chromosome conformation capture with sequencing (3C-seq), we showed multifaceted influence of the ParAB-parS system on spatial organization of P. aeruginosa chromosomes. ParB was critical for loading of SMC condensin on DNA, which function is to bring together opposite arms of the circular chromosome. The genome structure analyses also showed the presence of ParB-dependent loop bringing together the origin of replication (the region where the DNA replication starts, also including the parS sites) and the region containing the operon encoding the PA0124-PA0125 toxin-antitoxin system, critical for formation of antibiotic-tolerant subpopulation of cells called ‘perister’ cells. This finding suggests an intricate relationship between DNA organization and cellular processes crucial for bacterial survival.

Absence of Par proteins triggered significant expression changes of numerous genes encoding uncharacterized proteins with predicted role in gene expression regulation. An extensive analysis of five transcriptional regulators encoded by PA2121, PA2577, PA3973, PA3458 and PA3027 was conducted, in order to understand their biological function and possibly link with DNA segregation. The genes under their control were established, extensive phenotypic analyses including e.g. phenotype microarrays were performed. Strikingly, the analysed proteins were involved in diverse vital cellular processes, including the TCA cycle, glycerolipid and amino acid metabolism, and transport or osmoprotection functions.

The knowledge obtained from ongoing research facilitates our understanding of the physiology and survival strategies of pathogenic bacteria such as P. aeruginosa. Our long-term goal is to identify potential targets that, when inactivated, limit the survival of bacteria and could be used in antibacterial therapies.

Metodologia

In our research we adapted and use various genetic and molecular biology techniques e.g. cloning by Gibson and Golden Gate assembly, BACTH, protein purification, co-immunoprecipitation, Western blot, pull down, electrophoretic mobility shift assays, RT-qPCR, RNA-seq, ChIP-seq, 3C-seq, Tn-seq or CRISPRi-seq. A number of vectors and protocols were optimized for CRISPRi mediated gene silencing in P. aeruginosa. The performance of the system was determined by silencing of the reporter genes, essential genes, and genes involved in antibiotic resistance. Protocol for highly efficient preparation      of pooled P. aeruginosa CRISPRi libraries, and analysis of sequencing data were also established. As CRISPRi-seq offers a powerful platform tool for quantitative assessment of the significance of essential cellular processes we are expecting to uncover essential genes and pathways which inactivation e.g. enhances the killing by known antibiotics.

Our laboratory is equipped with a LightCycler480 (Roche) system for qPCR analysis, a Multiscan FC as well as a multimode VarioskanLUX (ThermoFisher Scientific) microplate readers, which allow various measurements of absorbance, fluorescence intensity, and luminescence.

Wybrane publikacje

• Kawalek A, Glabski K, Bartosik AA, Wozniak D, Kusiak M, Gawor J, Zuchniewicz K, Jagura-Burdzy G, Diverse Partners of the Partitioning ParB Protein in Pseudomonas aeruginosa. Microbiol Spectr. 2023; e0428922; doi: 10.1128/spectrum.04289-22.
• Kawalek A, Bartosik AA, Jagura-Burdzy G. Robust ParB Binding to Half-parS Sites in Pseudomonas aeruginosa-A Mechanism for Retaining ParB on the Nucleoid? Int J Mol Sci. 2023; 24(15):12517. doi: 10.3390/ijms241512517
• Kotecka K, Kawalek A, Modrzejewska-Balcerek M, Gawor J, Zuchniewicz K, Gromadka R, Bartosik AA, Functional Characterization of TetR-like Transcriptional Regulator PA3973 from Pseudomonas aeruginosa. Int J Mol Sci. 2022; 23(23):14584; doi: 10.3390/ijms232314584
• Modrzejewska M, Kawalek A, Bartosik AA, The LysR-Type Transcriptional Regulator BsrA (PA2121) Controls Vital Metabolic Pathways in Pseudomonas aeruginosa. mSystems. 2021; 6(4):e0001521; doi .org/10.1128/mSystems.00015-21
• Modrzejewska M, Kawalek A, Bartosik AA, The Lrp/AsnC-Type Regulator PA2577 Controls the EamA-like Transporter Gene PA2576 in Pseudomonas aeruginosa. Int J Mol. Sci. 2021, 22, 13340; doi.org/10.3390/ijms222413340

Współpraca

• Joanna Trylska , Centre of New Technologies, University of Warsaw, Warsaw, Poland
• Xue Liu, Department of Pathogen Biology, International Cancer Center, Shenzhen University Medical School, Shenzhen, Guangdong, China
• Hwan You Chang, Institute of Molecular Medicine, National Tsing Hua University, Taiwan, ROC
• Magdalena Modrzejewska-Balcerek, Faculty of Biology and Environmental Sciences, Institute of Biological Sciences, Cardinal Stefan Wyszynski University, Warsaw, Poland
• Marta Polańska, Department of Animal Physiology, Faculty of Biology, University of Warsaw, Poland
• Paweł Wawrzyniak, Department of Bacterial Genetics, Institute of Microbiology, Faculty of Biology, University of Warsaw, Poland
• Dariusz Bartosik, Department of Bacterial Genetics, Institute of Microbiology, Faculty of Biology, University of Warsaw, Poland
• Jolanta Mierzejewska, Chair of Drug and Cosmetics Biotechnology, Department of Chemistry, Warsaw University of Technology, Warsaw, Poland
• Małgorzata Adamczyk, Chair of Drug and Cosmetics Biotechnology, Department of Chemistry, Warsaw University of Technology, Warsaw, Poland

Nagrody i wyróżnienia

• Aneta Bartosik. PhD thesis award of the Prime Minister of Poland. 2005.