Bacterial Cell Cycle Group

Antimicrobial Drug Discovery and Development

Thomas Thyge Thomsen
We are interested in the discovery of new antimicrobials. Specifically we are currently involved in the development of novel antimicrobial peptides targeting highly drug-resistant Gram-negative species (E. coli, K. pneumonia, A. baumannii and P. aeruginosa). This includes description of mechanism of action (MOA), through analysis of antimicrobial effect on the synthesis of macromolecules (peptidoglycan, protein, DNA, RNA), resistance development and effect of cellular physiology. This work also includes routine testing of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and combinatorial testing for collateral sensitivity through synergy of antimicrobials. We are also involved in work on novel antimicrobial compounds of natural origin (plant/microbiological), this work is in collaboration with industry (Ktedogen srl/NAICONS) and researchers in Brazil. Our work also includes collaborations with Rigshospitalet in Copenhagen for work on in vivo efficacy access to clinical isolates.
Furthermore, we are especially interested in the mechanisms involved in resistance development towards antimicrobial peptides such as Colistin. These resistance mechanisms usually involve modification to the bacterial membrane, such as the addition of phosphoethanolamine to the Lippopolysaccharide layer (LPS), which changes the membrane charge. We apply genetic manipulation and genome engineering to elucidate how these changes influence antibacterial efficacy on a larger scale and how these alterations to the membrane may lead to collateral sensitivity, new target discovery and treatment possibilities.

Initiation of chromosome replication in Staphylococcus aureus

Thias Oberg Boesen
In this project we would like to identify and characterize molecular players involved in the regulation of initiation of DNA replication in Staphylococcus aureus. S. aureus is a clinically important Gram-positive pathogenic bacterium. As infections caused by resistant S. aureus (such as MRSA) might be hard to treat with currently available antibiotics, an increased understanding of parameters involved in controlling initiation of DNA replication in S. aureus may suggest new targets for treatment, possibly rendering already existing antibiotics effective.
In Escherichia coli the cell cycle is tightly regulated at the step of initiation of chromosome replication, ensuring that each origin of replication, oriC, is initiated once and only once per cell cycle. In brief, the initiator protein, DnaA, will be active when bound to ATP, DnaAATP, and inactive when bound to ADP, DnaA^ADP. DnaA^ATP accumulates throughout the cell cycle and when the ratio of DnaA^ATP/DnaA^ADP is high, this will trigger an open complex formation at the oriC region, leading to recruitment of the DNA polymerase III holoenzyme, which will start the replication process. Following initiation of replication, several mechanisms act in concert to prevent further re-initiations within the same cell cycle. These include an inactivation of the initiated oriC region for a given period of time, mediated by the SeqA and Dam methylase proteins, along with an inactivation of the DnaA initiator protein. Late in the cell cycle, the active form of DnaA will start to accumulate, getting ready for a new round of initiation in the next cell cycle.
In Gram-positives, such as Staphylococcus aureus and Bacillus subtilis, an alternative system for initiation control exists, but not much is known about the molecular details involved in cell cycle control at the level of initiation of DNA replication. Specifically, Gram-negatives contain the SeqA and Dam methylase proteins, but these proteins are absent in Gram-positive bacteria. For S. aureus, DnaA-mediated open complex formation at oriC in vitro is known to occur in an ATP-dependent manner, and several proteins, DnaB, DnaI and DnaC are believed to act during DNA replication. The exact functions, however, remain uncertain, and overall the understanding of the regulation of initiation of chromosome replication in Gram-positives is far behind of that in Gram-negatives, such as the well-studied E. coli.

Novel antimicrobial peptide discovery

Anna Elisabeth Ebbensgaard
In response to the increasing worldwide threat to human health posed by the emergence of bacterial resistance to currently used antibiotics, the overall aim of the recently established “Center for Peptide Antibiotics” (CEPAN) is to establish a discovery platform for peptide-based antibiotics against bacterial pathogens. In particular, we are interested in the discovery of new antimicrobial peptides and identification of novel targets for peptide and PNA (peptide nucleic acid) based peptides, as well as in the discovery of bacterial envelope permeabilizing peptides (EPPs). We used intracellular peptide display to identify novel hit peptides as well as novel targets for antibiotics. Combinatorial peptide libraries are generated, expressed intracellularly and screened for bioactivity. The SICLOPPS (split intein-mediated circular ligation of peptides and proteins) technology, a split-intein based method, enables the generation of genetically encoded cyclic peptide libraries in Escherichia coli. Cyclic peptides generated by the SICLOPPS technology that are interfering with bacterial growth are identified by deep sequencing and/or FACS based cell sorting and might as act as novel lead peptides for further drug development

Center for Peptide-Based Antibiotics: Peptide Antibiotics against Resistant Bacterial Infections

Agency: Novo Nordisk Challenge program
Main applicant: Prof. Peter E. Nielsen, University of Copenhagen, Denmark
Co-applicant: Anders Løbner-Olesens (shared) 
Expiration date: 2022
Link: https://cepan.ku.dk/

Re-inventing the bacterial membrane as a target for antimicrobial treatment of Multidrug-Resistant Gram- Negative Bacteria

Agency: Novo Nordisk Foundation – Tandem Programme
Main applicant: Prof. Niels Frimodt-Møller, Head of Department of Clinical Microbiology, Rigshospitalet 
Co-applicant: Anders Løbner-Olesens (shared) 
Expiration date: 2021

Engineering biological memory in bacteria

Agency: VILLUM Experiment Programme
Main applicant: Anders Løbner-Olesen (shared) 
Expiration date: 2022

Insane in the membrane; how to avoid crowding of the Eschericia coli inner membrane?

Agency: VILLUM Experiment Programme
Main applicant: Jakob Frimodt-Møller 
Expiration date: 2022 

ALO lab is involved in several different courses (both at bachelor-, master-, and Ph.D.-level) offered at the University of Copenhagen, Department of Biology, both as invited teachers but also as courses coordinators. More course information can be obtained at kurser.ku.dk.

Molecular Cell Biology (KU course code: NBIA07002U)
ALO lab share: Invited teacher (in charge of laboratory exercises).
Content: The main goal of the course is for the student to develop an understanding of how knowledge in molecular cell biology is obtained through experimental approaches; an aim particularly important and appropriate close to the point of the bachelor project.
Level: Bachelor
Credit: 15 ECTS points

General Molecular Biology (Molbiol) (KU course code: NBIA04033U)
ALO lab share: Invited teacher (in charge of laboratory exercises).
Content: The course aims at presenting the students with classical as well as modern molecular genetic terms and approaches. This course is a necessary for further enrollment in experiential-biological courses at the University of Copenhagen.
Level: Bachelor
Credit: 7.5 ECTS points

Biochemistry 1 (KU course code: LKEB10077U)
ALO lab share: Invited teacher.
Content: The course aims at presenting the students with the main topics within biochemistry, with emphasis on metabolism as well as the structure and function of important macro molecules.
Level: Bachelor
Credit: 7 ECTS points

Medical Bacteriology (KU course code: NBIK14035U)
ALO lab share: Course coordinator.
Content: To give students a thorough understanding of the field of Medical Bacteriology with emphasis on the specific traits that enables certain bacteria to cause disease. The course will cover specific bacteria, the diseases they cause and possible treatments. The course will rely heavily on genetics and molecular biology.
Level: Master
Credit: 7.5 ECTS points

Principal Subject in Molecular Genetics (KU course code: NBIA09016U)
ALO lab share: Invited teacher.
Content: The principal subject in molecular genetics deals with research that is based on phenotype/genotype relations in model organism and in humans, e.g. mutations that confer disease. Recent topics have been genome stability, cancer development, DNA replication and transcription regulation
Level: Master
Credit: 7.5 ECTS points

For public relations or our public outreach program please contact Postdoc Thomas Thyge Thomsen.