Impact of recA on Levofloxacin Exposure-Related Resistance Development

Resistance to antibacterial agents is a major health concern that is spreading widely. Increasingly, common drugs like β-lactams and fluoroquinolones have been rendered ineffective in the clinic. Consequently, it is necessary that new therapeutic interventions be developed to combat resistance and meet the medical demands of treating infections due to pathogens resistant to available antibacterial agents. Resistance to antibacterial agents occurs frequently via genetic mutations or the acquisition of genetic material carrying resistance determinants. Evidence suggests that genetic mutations are one of the main mechanisms of defense employed by bacteria against many antibacterial agents (1, 2). Bacteria can escape the lethal effect of antimicrobial agents by acquiring mutations in target binding site, genes regulating efflux pumps, and genes encoding porin synthesis (3, 12, 13, 20). Needless to say, mutations are important for acquiring resistance, and finding ways to reduce mutation rates in bacteria might help us to combat resistance. Bacteria commonly develop spontaneous mutations as a consequence of error-prone DNA replication. Some of these random mutations may confer resistance to antibiotics. Lately, studies have suggested that bacteria also play an active role in inducing mutations in the event of exposure to certain classes of antibiotics or other environmental stresses (4, 16). One of the mechanisms by which these mutations are induced is the SOS response system (8). The SOS response system is a global response to DNA damage in which DNA repair and mutagenesis are induced. A central part of the SOS response is the derepression of various SOS genes, which are under the direct and indirect transcription control of a repressor, LexA. Another important protein involved in regulating the SOS response is RecA (the sensor of the system). RecA is a recombinase protein that has coprotease activity, functions in DNA strand exchange, and facilitates replicative bypass of DNA lesions. In a nontriggered state, LexA binds as a dimer in the promoter region of SOS genes and downregulates its own expression and that of other SOS genes. Formation of single-stranded DNA or a stalled replication fork resulting from DNA damage acts as a trigger for the SOS system and activates RecA. Upon activation, coprotease activity of RecA causes autocatalytic cleavage of the LexA dimer and leads to derepression of the SOS genes. The LexA regulon in most cases includes the recombination and repair genes recA, recN, and ruvAB, the nucleotide excision repair genes uvrAB and uvrD, the error-prone DNA polymerase (Pol) genes dinB (encoding Pol IV) and umuDC (encoding Pol V), and DNA polymerase II, in addition to other functions. Thus, induction of the SOS response trades long-term fidelity for short-term viability by mediating such processes as cell division inhibition, excision repair, upregulation of the tricarboxylic acid cycle, and error-prone replication. Overall, this process leads to a higher rate of mutant formation. Since spontaneous mutations are an integral part of the bacterial adaptation and do not provide an obvious means to target, the pathways that lead to mutations linked with antibiotic resistance (such as the SOS response) seems to be a logical and potentially lucrative target for pharmacological intervention. Hence, we attempted to investigate the effect of RecA (one of the key regulatory proteins involved in the SOS response and a potential pharmacological target) on resistance development. Two clinically important pathogens, Staphylococcus aureus and Escherichia coli, were selected as our test microorganisms. S. aureus is a Gram-positive bacterium that is known to cause pneumonia and skin infections. E. coli is a Gram-negative bacterium that is a common cause of intra-abdominal and urinary tract infections.