THE NEGATIVE REGULATOR NfxB: A SHARED MECHANISM UNDERLYING BIOFILMS AND ANTIBIOTIC RESISTANCE IN PSEUDOMONAS AERUGINOSA

MARTINO RA; TOBARES RA; BUFFA L; COLQUE CA; LOPEZ VA; MOYANO AJ; SMANIA AM

Salta

Congreso; LV Annual SAIB, LIV PABMB 2019; 2019

Pseudomonas aeruginosa grows as biofilm communities in the airways of cystic fibrosis patients, where small colony variants (SCVs) are frequently observed. These biofilm-adapted SCVs show a great instability when grown outside biofilms, thus revealing the ability to switch between phenotypes. We have previously performed an evolutionary assay to explore the adaptive potential of P. aeruginosa by subjecting clones to alternating SCV conversion and reversion cycles, from which we further isolated SCVs and revertant clones. Comparative genomics analysis showed that all the evolved lines accumulated 1 mutation/round in genes of the wsp and yfi systems, suggesting strong evolutionary parallelism. Both systems are key regulators of the second messenger c-di-GMP, which governs the transition between planktonic and biofilm mode of growth. By round 4, SCV conversion in all evolutionary lines was dramatically constrained. However, one of these lines evolved up to ten conversion/reversion cycles. Interestingly, subsequent mutations to those observed in the wsp and yfi systems were localized in morA and the repressor gene nfxB. MorA is a dual enzyme with diguanylate cyclase and phosphodiesterase domains and is associated with motility regulation. Gene nfxB, located 243 bp upstream of morA, is a self-regulating gene, which regulates the expression of the MexCD-OprJ pump, associated with resistance to fluoroquinolones. Interestingly, SCV conversion in round 7 (R7-SCV) showed a mutation (A600C) that removed the stop codon of nfxB. Importantly, this mutation was also able to provide resistance to the fluoroquinolone ciprofloxacin. We next wondered if this mutation was able to affect the expression of morA. RT-PCR analysis showed that transcripts of morA were significantly increased in R7-SCV compared to the parental wild type strain and to its immediate predecessor from round six. Heterologous expression of a wild type copy of nfxB in R7-SCV was able to revert the SCV phenotype, restore sensitivity to ciprofloxacin, decrease biofilm formation and, to fully eliminate transcripts of morA. By performing site directed mutagenesis we further generated a nfxB-A600C mutant under a parental wild type context. Surprisingly, although this mutation per se provided resistance to ciprofloxacin and increased the transcript levels of morA, this mutant did not show a SCV phenotype nor an increased ability to form biofilms. These results suggest that previous mutations in wsp and yfi systems provided the genetic context in which mutations in nfxB resulted adaptive for biofilm formation by providing a SCV phenotype. Most importantly, under this genetic context, resistance to fluoroquinolones can be co-selected in biofilm environments in association with the SCV phenotype. Our results constitute the first report describing the role of NfxB in biofilm formation and provides a new mechanism of association between biofilms and antibiotic resistance.