Department of Biochemistry and Molecular Biology

Molecular Mechanisms of Bacterial Antibiotic Resistance and Pathogenesis

Principal investigators

Prof. Edita Sužiedėlienė
Dr. Julija Armalytė
Dr. Jūratė Skerniškytė

In collaboration (VU, Faculty of Physics): Dr. Irina Buchovec

Group members

Dr. Danutė Labeikytė
Dr. Arvydas Markuckas
Dr. Kęstutis Sužiedėlis
Ramutė Pagalytė

Phd students

Laurita Klimkaitė
Tomas Liveikis
Ignas Ragaišis

Research topics

Our research is focused towards understanding the molecular basis underlying the bacterial antibiotic resistance in clinic and in the environment with the emphasis on novel resistance mechanisms and on the bacterial cell features contributing to pathogenesi.

Genetic elements responsible for the spread of antimicrobial resistance in the clinic and the environment

Infections caused by the group of gram-negative bacteria that are resistant to nearly all currently available antibiotics is a serious concern in clinical settings worldwide. Bacteria, previously considered as non-pathogenic, due to their ability to acquire multidrug-resistance and virulence traits, are currently becoming the ones of the most important hospital infection agents. The opportunistic pathogen Acinetobacter baumannii causes a variety of difficult-to-treat nosocomial infections to critically ill patients. The characteristic features of A. baumannii are the ability to withstand prolonged periods of dryness, form biofilms on various surfaces including medical equipment, upregulate intrinsic resistance mechanisms and acquire new resistance genes through plasmids, as well as the ability to adhere and colonise the host cells. Another opportunistic pathogen Stenotrophomonas maltophilia is receiving increased attention in the clinic due to its innate multidrug-resistance causing difficult-to-treat infections, combined with a lacking knowledge about the molecular mechanisms causing the pathogenicity. Understanding the molecular basis of pathogenesis and evaluating the impact of the mobile genetic elements in bacterial adaptation may bring novel insights into pathogenicity and development of novel antibacterial strategies.

The interplay between opportunistic bacteria and the innate immunity

Our research has revealed two bacterial features as important factors in the pathogenesis of opportunistic bacteria: 1) capsular polysaccharides (CPS) and 2) outer membrane vesicles (OMV). We demonstrated that CPS efficiently protect A. baumannii from environmental stress and phagocytosis by immune cells. Additionally, A. baumannii produces OMV that encapsulate antibiotic-degrading enzymes, making OMV secretion a powerful strategy to inactivate antimicrobials at a distance. Our investigations has revealed that secreted OMV can also modulate inflammatory response in macrophages. Our goal is to understand how bacteria modulate the response of immune response through the production of CPS and OMV.

Alternative antibacterial treatment techniques

Our research focuses on antimicrobial photodynamic therapy (aPDT) against multidrug-resistant Gram-negative bacteria, such as A. baumannii and S. maltophilia. aPDT is a biophotonic technology that can be used as a complementary or alternative treatment to antibiotics. It is based on the interaction of a photosensitizer (PS), molecular oxygen, and low doses of light with a wavelength corresponding to the absorption peak of PS. Laboratory experiments have been carried out with the naturally occurring PS magnesium chlorophyllin (Chl), riboflavin (Rf), and 5-aminolevulinic acid (ALA).  An LED-based light source (402 nm and 440 nm) with temperature and light flux control is used for irradiation experiments. Our recent works demonstrate Chl-, Rf- and ALA-based aPDT efficacy against both pathogens biofilms. Studies on oxidative stress and reactive oxygen species (ROS) generation in the cells of the pathogens under study after exposure to aPDT have been successfully carried out. However, aPDTs can usually only partially inactivate bacteria in the biofilms they form. To overcome this problem, aPDT could be combined with antibiotics to neutralize the bacteria completely, thus preventing the recurrence of infection.

Microbial diversity and spread of antibiotic resistance in the environment

Microorganisms are found in high abundance and play a leading role in countless natural processes in the environment. However, the biodiversity in the soil, water and other bacterial habitats can be challenged due to human activities and established natural processes affected. Apart from the soil or water body management being vital for agricultural purposes, they are also considered a favorable environment for the evolution and development of antimicrobial resistance, due to their high complexity and ongoing competition between the microorganisms. We aim to evaluate the microorganism variation that can be observed in different environmental conditions (e.g. intensive agricultural lands, aquafarming, polluted areas) concentrating on the possibility of the spread of clinically important antibiotic resistance genes (ARGs). The comparison of ARGs composition as well as their mobilization potential could show the impact of anthropogenic activities on the ecosystems as well as the possibility of transferring the ARGs to the human hosts.

Analysis of human airway microbiome

The human microbiomes, including the respiratory tract, are described and characterized in an increasing numbers of studies; however, the composition and effects of the healthy or disturbed microbiome on pulmonary health and its interaction with the host are only beginning to be elucidated. In our study we aimed to investigate the upper respiratory tract as a less invasive alternative for understanding lung health and to identify potential biomarkers linked to the disease. Microbial diversity of upper and lower airways was determined by full-length 16S rRNA gene amplicon sequencing using Oxford Nanopore technologies. While the composition of the upper respiratory tract microbiome did not directly associate with the composition of lower respiratory tract microbiome, a subtle influence was observed, indicating several species of microorganisms which should be looked into as potential biomarkers.

Projects

National research program “Investigation of capsular polysaccharides and outer membrane vesicles as virulence factors in pathogenesis of opportunistic bacteria” (2023-2026) funded by The Research Council of Lithuania (S-MIP-23-109). Principal investigator Dr. Jūratė Skerniškytė

Science promotion fund of Vilnius university project “Prevalence of genetic determinants of antibiotic resistance in opportunistic pathogens Acinetobacter baumannii and Stenotrophomonas maltophilia“, No. MSF-JM-12/2023. Principal investigator – Laurita Klimkaitė, Project participant – T. Liveikis (2023-2024).

Science promotion fund of Vilnius University project “Synergistic Antibiotic-Photodynamic Therapy Combination in Inactivation of Opportunistic Pathogen Stenotrophomonas maltophilia”, No. MSF-JM-3/2021. Project leader - dr. I. Buchovec, participant – L. Klimkaitė (2020-2021).

Programme for the European Union Funds Investments in Lithuania, Funding instrument - European Regional Development Fund, measure 01.2.2-LMT-K-718 „Targeted Research in Smart Specialisation Areas“, project „Analysis of Individualized Upper Respiratory Tract Microbiome – a novel tool for diagnostics and healthcare (YourAirwayMicrobiome)“, No. 01.2.2-LMT-K-718-03-0079. Project leader Innovative Medicine Center, project coordinator for Vilnius University dr. J. Armalytė (2020-2023).

National research program “Healthy ageing”, project “Development of virus-like particles-based vaccine against Acinetobacter baumannii“ (No. S-SEN20-1). Principal investigator dr. Julija Armalytė (2020–2021).

National research program “Sustainability of agro, forest and water ecosystems ”, project “The influence of intensive fish farming on aquatic microbiome and resistome“, No. S-SIT-20-6. Researcher dr. Julija Armalytė (2020-2021).

Developing students' skills by participating in scientific summer internships (LMT): “Inactivation of Acinetobacter baumannii biofilms by antimicrobial photodynamic therapy“; 2021.07-2021.08; Project leader – dr. Irina Buchovec.

National research program “Sustainability of agro, forest and water ecosystems” project “Influence of intensive farming on development, persistence and spread of bacteria resistant to antimicrobials and biocides in soil and water”. Project coordinator for Vilnius University dr. J. Armalytė (2015–2018).

Laboratory of Nucleic Acids Biochemistry

Principal investigator

Prof. Saulius Serva

Group members

Dr. Aleksandras Konovalovas
Dr. Algirdas Mikalkėnas
Dr. Lina Aitmanaitė

PhD students

Enrika Celitan
Gerda Skinderytė

Research

Group is actively engaged in research focused on two main directions:

• Molecular mechanisms of innate yeast Saccharomyces viruses;
• Design and investigation of nucleoside- and nucleotide-based antivirals.

The innate viruses of Saccharomyces and closely related yeasts are being investigated to understand the relations with host in order to elucidate the evolutionary pathways and uncover the principles of dsRNA virus distribution within an ecosystem. We use the molecular biology techniques, involving advanced level manipulations on genomic material such as RNA cloning along with reverse genetics approach. Transcriptomic, proteomic and phenomic consequences of dsRNA viruses on the host cell are interpreted as model framework to establish a universal mechanisms behind any virus of interest. Viral capsids are purified and developed for the facilitated delivery of bioactive materials.
Nucleoside/nucleotide based antivirals constitute an essence of the modern high efficacy retroviral treatment. We rest on the fundamental principles of enzyme catalysis to design and develop antiviral compounds. The aim of our research is to develop the drugs active at the level of catalytic cycle of retroviral replication enzymes, linking the exclusive specificity and efficacy into the binding approach. These compounds are also beneficial for elucidation of the molecular mechanisms of reverse transcriptase inhibition.

Methods

The broad range of methods from classic to those recently developed are employed in a lab. We master microbiology, gene and genome engineering, next generation DNA and RNA sequencing (Oxford Nanopore), bioinformatics, enzymology, cell culture and other techniques. The obtained data are integrated by Systems Biology approach so creating a paradigm network for the virus-host interactions.

Projects (since 2017)

Ministry of Education, Science and Sports, Short-term research in health and education project “System for virus spread control and extreme situation management during COVID-19 epidemics”, Nr. S-DNR-20-2 (2021).

European Cooperation in Science and Technology (COST) activity CA17103: “Delivery of antisense RNA therapeutics (DARTER)” (P-COST-20-3). 2020-2022.

Vilnius University Science Advancement Fund Project „Investigation of Totiviridae family virus ScV-LA biogenesis in native environment“, Nr. MSF-JM-7 (2019-2020).

EU Horizon 2020 Research and Innovation Program Project “Sonic Drilling coupled with Automated Mineralogy and chemistry On-Line-On-Mine-Real-Time” (SOLSA) (2016-2020).

Baltisch-Deutsches Hochschulkontor, Project "Life cycle of yeast dsRNA viruses uncovered by advanced fluorescent microscopy" (2019).

Research Council of Lithuania, Project of National Research Program „Sustainability of agro-, forest and water ecosystems”: “Agroecosystems microbiota under climate change: structure and concordance mechanisms", SIT-07/2015 (2015-2018).

Molecular mechanisms of resistance to anticancer treatment

Principal investigator

Dr. Aušra Sasnauskienė

Group members

Dr. Violeta Jonušienė
Dr. Daiva Dabkevičienė
Vilmantė Žitkutė
Eglė Žalytė

Research topics

Research interests of our group:

• Investigation of molecular mechanisms of resistance to anticancer treatment;
• Functional studies of human primary cells.

Investigation of molecular mechanisms of resistance to anticancer treatment
Acquired drug resistance is a major limitation of cancer treatment. Resistance of cancer cells can emerge due to various factors: alterations in drug transport and metabolism, modification of drug targets, activation of DNA repair or changes in cell death induction. Deeper understanding of chemoresistant cell physiology, in particular cell death and survival signalling, suggests new possible targets to overcome cancer cell resistance.
We study molecular mechanisms that determine cancer cell susceptibility to agents of chemotherapy, targeted therapy or immune checkpoint inhibition. Our aim is to identify potential targets for anti-cancer therapy in colorectal and endometrial cancer cells.
In collaboration with researchers from Vilnius University Life Sciences Center Institute of Biochemistry, we investigate the use of nanotubes of bacteriophage origin as potential drug carriers. We evaluate the mechanisms of nanotube entry and transport in cancer cells.

Functional analysis of human primary cells
In collaboration with the Institute of Biomedical Sciences, Faculty of Medicine, Vilnius University, we analyze primary cells of patients having rare genetic diseases. We aim to characterize functional changes of primary cells and correlate them with alterations in genome and transcriptome.

Methods

Culturing of cell cultures and primary cells in 2D and 3D systems; immunoenzymatic assays: Western blot, ELISA; quantitative PCR; subcellular localization studies using confocal microscopy; flow cytometry; functional analysis of gene expression using siRNA and CRISPR-Cas techniques.

Projects (since 2017)

Science promotion fund of Vilnius University project “Application of CRISPR-Cas13 technology in studying mechanisms of chemoresistance”, No. MSF-JM-2/2021. Project leader V. Žitkutė (2021-2022).

Research Council of Lithuania National programme “Healthy ageing” project “Self-assembling Phage Proteins for Targeted Nanomedicine”, No. P-SEN-20-4. Participants: A. Sasnauskienė, V. Žitkutė (2020-2021).

Science promotion fund of Vilnius University project “Genome and transcriptome analysis in pathogenesis studies of rare inherited diseases”, No. MSF-JM-2/2020. Participants: A. Sasnauskienė, V. Žitkutė (2020).

Research Council of Lithuania. Project “Redox Chemistry, Biochemistry and Cytotoxicity of Aromatic Nitrocompounds and N-oxides: a New Look”, No. DOTSUT-34/09.33-LMT-K712-01-0058. Participant V. Jonušienė (2018–2021).

Research Council of Lithuania, National programme “Healthy ageing” project “Novel biomarkers for individualized therapy of colon cancer: proteomics, microRNomics and clinics”, No. SEN-17/2015. Participants V. Jonušienė, A. Sasnauskienė (2015-2018).

Molecular mechanisms of intracellular trafficking

Principal investigator

Prof. Vytautė Starkuvienė Erfle

Research topics

Intracellular trafficking distributes newly synthesized and endocytosed material to diverse cellular destinations and, by doing so, ensures cellular homeostasis. Deregulation of cargo trafficking leads to ever-increasing list of diseases such as cancer or cardio-vascular. Intracellular trafficking strongly contributes to cell differentiation and aging processes. Trafficking can be divided into several pathways: secretory mechanisms that transport cargo from the endoplasmic reticulum to the Golgi complex, and from there on – to the plasma membrane, lysosomes or cell outside. Endocytosis is responsible for cellular entry of various ligands, growth factors or viruses. Both, secretory and endocytic pathways are closely related to degradation, autophagy, cell death and division, transcription and translation. Our aim is to analyse how endocytosis machinery respond and adapt to changes in cell outside. The projects are running at Vilnius and Heidelberg (Germany) Universities.

Methods

To dissect the complexity of trafficking pathways, we use cell biology, molecular genetics and biochemistry techniques. The methodological focus in the group lies in high-throughput and high-resolution fluorescence microscopy. We work with cancer and healthy primary cells in 2D and 3D environment; and modify gene, transcript and protein expression and function by gene editing, RNAi, drug and antibody-mediated approaches, respectively. We also develop techniques to perform these experiments with little side effects and on varying biological scales..

Complexity of endocytosis defines the efficiency of cell modification

Cargo intracellular delivery depends on effectiveness of endocytosis, which differs in individual cells. GFP protein enters some cells efficiently and localizes to cytoplasm. In contrast, some cells fail to internalize this cargo. Part of cells trap GFP in their endosomes after internalization.

Courtesy: Dr S. Liechocki, Heidelberg University