ELUCIDATING DNA REPAIR PROCESSES CENTRAL TO FLUOROQUINOLONE PERSISTENCE IN NON-GROWING E. COLI POPULATIONS

Abstract

Chronic relapsing infections are a major cause of clinical concern, as antibiotics that seem to work initially ultimately fail at eradicating the infection. One of the main culprits of relapsing infections is bacterial persisters, which are enriched in non-growing and difficult-to-treat infections. Fluoroquinolone (FQ) persisters in non-growing populations, for example, tolerate lethal doses of antibiotics despite experiencing considerable DNA damage and repopulate sites of infection following treatment. This thesis aims to identify DNA repair proteins and repair mechanisms that enable FQ persisters in non-growing populations to recover from antibiotic treatment. Using time-lapse fluorescence microscopy, we obtained the first microscopic images of FQ persisters recovering from treatment and demonstrated that FQ persisters induce impressive SOS responses and show hallmark signs of DNA damage. Despite being scathed from treatment, whole genome sequencing verified that FQ persisters generally repair the damage correctly. Since homologous recombination, which requires undamaged sister DNA for accurate repair, is considered an error-proof DNA repair mechanism, we questioned whether harboring multiple chromosomes gives bacteria an advantage when challenged with FQs. While diploidy promoted increased survival, it did not guarantee survival, nor did the absence of a second chromosome preclude persistence. Instead, monoploid cells survived FQ treatment at a rate 10-fold greater than that of homologous-recombination deficient mutants. The survival of the monoploid population was dependent on the catalytic activity of exonuclease VII and likely also involved the actions of additional repair proteins. In considering that monoploid E. coli can survive FQ treatment in the absence of an error-proof repair mechanism, we questioned whether FQ treatment increases the likelihood of genetic mutations as measured by enhanced antibiotic resistance. Our results showed that persistence accelerates antibiotic resistance to multiple classes of antibiotics even after a single FQ treatment, a finding which could have major implications in clinical settings. The work presented herein reveals that multiple FQ persister subtypes exist within the same population and begins to elucidate repair mechanisms that these persisters use to survive. Understanding additional strategies employed by persisters could reveal novel drug targets that could be used to eradicate relapsing infections, thus improving medical care and saving lives.