Intrinsic resistance mechanisms and CRISPR-Cas immunity of the opportunistic pathogen, Staphylococcus aureus

Intrinsic resistance mechanisms and CRISPR-Cas immunity of the opportunistic pathogen, Staphylococcus aureus

PhD Student: Kasper Mikkelsen, email:
Thesis defended: 23 April 2021

Staphylococcus aureus is a serious human pathogen with a remarkable ability to adapt to challenging conditions and cause life-threatening infections. The most important class of antimicrobials for the treatment of S. aureus infections is the β-lactams. By the acquisition of the staphylococcal chromosome cassette (SCCmec), some S. aureus isolates have evolved methicillin-resistance (MRSAs) that are resistant to nearly every β-lactam antibiotic, why additional measures are needed to combat this pathogen.


The purpose of this PhD project was to find novel targets suitable for future antimicrobial solutions, and gain insight into unknown resistance mechanisms of S. aureus. Additionally, we wanted to examine the prevalence of CRISPR-Cas systems in S. aureus, which protect the bacterium against phage attack.


During this PhD project, we identify and characterize two novel auxiliary factors, AuxA and AuxB, which are essential for MRSA to withstand β-lactam antimicrobials. AuxA and AuxB are membrane-associated proteins that interact with one another to form a hetero-multimeric complex. This complex further interacts with members of the peptidoglycan synthesis machinery, the divisome, and lipoteichoic acid and D-alanylation pathways. Mutants lacking either of the Aux proteins show attenuated autolytic activity and release wild type-length lipoteichoic acid polymers into the growth medium. This suggest that the Aux proteins carry a stabilizing role of the lipoteichoic acid polymer and might carry additional roles in division and peptidoglycan synthesis of the staphylococcal cell.

The SCCmec may not only be important for β-lactam resistance, but is likely to be involved in the spread of anti-phage immunity. The emerging Danish clone ST630 carry the SCCmec type V(5C2&5) in which the S. aureus CRISPR-Cas type III-A adaptive immune system is located. The S. aureus CRISPR-Cas system is active against phage attack and excise at high rates together with the type V(5C2&5) SCCmec. This could serve to spread antimicrobial and phage resistance simultaneously, endangering both antibiotic and phage therapy approaches.


We have characterized novel targets, that if inhibited, will re-sensitize MRSA to β-lactam antibiotics. Inhibitors of AuxA and/or AuxB can thereby serve in adjuvant therapy strategies to supplement β-lactam treatment to overcome MRSA infections.

Characterizing CRISPR-Cas systems in S. aureus is important to gain insight into whether potential phage therapies would work against this pathogen. In addition, CRISPR-Cas systems have been utilized for genetic manipulation, why full characterization could accompany new ideas for novel molecular biology tools.

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