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FRIMEDBIO-Fri prosj.st. med.,helse,biol

Disentangling penicillin resistance and compensatory adaption in pneumococci by combining genomics and molecular microbiology.

Alternative title: Å forene genomikk med molekylærbiologi for å forklare penicillinresistens og kompensatoriske tilpasninger hos pneumokokker.

Awarded: NOK 12.0 mill.

Pneumococci (Streptococcus pneumoniae) is a major human pathogen. This bacterium causes for example pneumonia, ear infection, bacteremia, and meningitis. Penicillin is the antibiotic of choice when treating pneumococcal infections. However, the quick rise of resistant isolates challenges healthcare systems, which are running out of treatment options. Increased penicillin resistance among pneumococci is also observed in Norwegian isolates. This bacterium is found on WHO’s list of species that must be prioritised in the efforts to develop new antibiotics and other antimicrobial strategies. We know that penicillin resistance comes with a fitness cost to the pneumococci, i.e. the bacteria often need to change some unknown genes in order to allow the functionality of the penicillin resistance genes. This project is a collaboration between the Norwegian University of Life Sciences (NMBU) and the Norwegian Institute of Public Health (NIPH) where one of the goals is to identify these unknown genes, whose change allow high penicillin resistance. When we have identified these genes, we can exploit this information to our advantage. For example, if one could find a compound that inhibits the function of a gene required for high level penicillin resistance, this compound could re-sensitize pneumococci to penicillins. By combining this new compound with existing penicillins, we could keep our penicillin antibiotics relevant as treatment options in the future. Another important goal of the project is to map the origin, spread and frequency of penicillin resistant pneumococci in Norway. We have hitherto sequenced all the genes in more than 1000 pneumococcal isolates collected by NIPH (“Norwegian isolates”). Several isolates are penicillin resistant and our bioinformatic analyses have identified mutations in genes with known functions as well as genes of unknown function. Among the known genes several encode so-called penicillin binding proteins (PBPs). Pneumococci have six PBPs, which are involved in building a cell wall that surrounds and protects bacteria. It is the PBPs that are attacked by penicillin. The latter “mimics” the function of the PBPs actual substrate used to build the cell wall. Penicillin binds stronger to the PBPs than the cell wall precursor, but cannot be used for construction of the cell wall. Thus, cell wall synthesis is blocked, and the bacterium stops dividing. It is well known that penicillin binds weaker to mutated PBPs leading to increased tolerance to penicillin, but how they at the same time have retained their ability to synthesise the cell wall is a mystery. The cell wall constructed by mutated PBPs is often structured differently and it seems that some of the mutated PBPs require a particular substrate variant in order to function. We are now studying which mutated PBP that are critical for high resistance and the link between mutated PBPs and an altered cell wall structure in resistant isolates. Furthermore, we have found mutations in genes of unknown functions. Experiments to characterize the function of these genes and their contribution to resistance is a main focus for the further progress of the project.

Pneumococci are major contributors to morbidity and mortality world-wide. The antibiotics of choice for treatment of pneumococcal infections are beta-lactams (including penicillin). However, their superior role as efficient drugs is fading in concert with the accelerating spread of beta-lactam resistance genes. Interestingly, beta-lactam resistant pneumococci must employ less efficient cell-wall synthesizing enzymes (low-affinity PBPs) which impose them a fitness cost that has great therapeutically potential. To become successful resistant clones, they acquire compensating mutations re-establishing their fitness. The proposed project will combine genome-wide association and epistasis analyses with molecular biology providing new knowledge on the mechanisms used by pneumococci to alleviate the fitness cost of being resistant to beta-lactam antibiotics. This will open the way for research aimed at generating more effective beta-lactams that are less prone to resistance development, keeping beta-lactams relevant as effective drugs in the future. By identifying specific pathways, and enzymatic functions required for relieving the fitness cost, it will be possible to screen for small molecules targeting these compensatory mechanisms that can be used in combination with existing beta-lactam antibiotics to sensitize already resistant strains. In our search for fitness cost mutations, we will genome sequence 1500 uncharacterized isolates from Norwegian patients and healthy carriers. This will also answer questions related to the spread and evolution of pneumococcal beta-lactam resistance in Norway, which is largely unknown. It will enhance our ability to trace resistant strains and give knowledge which can help implement strategies to slow the spread of resistant pneumococci in the population.

Funding scheme:

FRIMEDBIO-Fri prosj.st. med.,helse,biol

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