Harnessing Metabolic Regulation to Increase Hfq-Dependent Antibiotic Susceptibility in Pseudomonas aeruginosa

Abstract

The opportunistic human pathogen Pseudomonas aeruginosa is responsible for ~ 10% of hospital-acquired infections worldwide. It is notorious for its high level resistance toward many antibiotics, and the number of multi-drug resistant clinical isolates is steadily increasing. A better understanding of the molecular mechanisms underlying drug resistance is crucial for the development of novel antimicrobials and alternative strategies such as enhanced sensitization of bacteria to antibiotics in use. In P. aeruginosa several uptake channels for amino-acids and carbon sources can serve simultaneously as entry ports for antibiotics. The respective genes are often controlled by carbon catabolite repression (CCR). We have recently shown that Hfq in concert with Crc acts as a translational repressor during CCR. This function is counteracted by the regulatory RNA CrcZ, which functions as a decoy to abrogate Hfq-mediated translational repression of catabolic genes. Here, we report an increased susceptibility of P. aeruginosa hfq deletion strains to different classes of antibiotics. Transcriptome analyses indicated that Hfq impacts on different mechanisms known to be involved in antibiotic susceptibility, viz import and efflux, energy metabolism, cell wall and LPS composition as well as on the c-di-GMP levels. Furthermore, we show that sequestration of Hfq by CrcZ, which was over-produced or induced by non-preferred carbon-sources, enhances the sensitivity toward antibiotics. Thus, controlled synthesis of CrcZ could provide a means to (re)sensitize P. aeruginosa to different classes of antibiotics.

Distinctive Regulation of Carbapenem Susceptibility in Pseudomonas aeruginosa by Hfq

“…Moreover, we have provided evidence for Hfq/Crcmediated regulation of opdP. Hence, the increased susceptibility of P. aeruginosa hfq deletion mutants toward imipenem (Pusic et al, 2018) can be rationalized at the molecular level by these studies. However, we cannot exclude that hitherto unknown Hfq-mediated regulatory circuits additionally impact oprD and opdP regulation.…”

“…Recent studies revealed that P. aeruginosa hfq deletion mutants showed an increased susceptibility to imipenem (Ducret et al, 2016;Pusic et al, 2018), which coincided with higher oprD and opdP transcript levels in the hfq mutant strain when compared with the parental strain during growth in different media (Supplementary Figure S1 and Supplementary Table S2) (Ducret et al, 2016;Pusic et al, 2018). To verify the impact of Hfq on oprD and opdP translation, oprD::lacZ and opdP::lacZ translational gene fusions were constructed and the β-galactosidase activities conferred by the respective fusion proteins were determined in strains PAO1 and PAO1 hfq harboring plasmids pME6015P tac oprD::lacZ and pME6015P tac opdP::lacZ, respectively.…”

“…Taken these studies together with the observation that a PAO1hfq-deletion mutant was more susceptible to carbapenems when compared with the parental strain (Ducret et al, 2016;Pusic et al, 2018), it appeared safe to assume that Hfq is involved in regulation of both, oprD and opdP. In this study, we addressed the question whether post-transcriptional regulation of oprD and opdP occurs through riboregulation and/or through CCR, i.e., through Hfq/Crc repressive complexes.…”

Systems-level analysis of NalD mutation, a recurrent driver of rapid drug resistance in acute Pseudomonas aeruginosa infection

“…We predicted that metabolic changes in the membrane transport and metabolism of BCAA and AAA are directly connected to the development of aztreonam resistance. Previous studies have suggested that the carbon catabolite control system CbrAB/Crc regulates BCAA uptake and utilization [51] as well as antibiotic resistance in P. aeruginosa [52][53][54]. Since channels that actively uptake amino acids can also transport antibiotics with sufficient structural similarity (e.g., Escherichia coli glycine transport system can also uptake the antibiotic D-cycloserine [55]), aztreonam resistance can be possibly potentiated by decreasing drug uptake through BCAA transporters via the CbrAB/Crc system, in addition to the efflux provided by the upregulated MexAB-OprM.…”

TpiA is a Key Metabolic Enzyme That Affects Virulence and Resistance to Aminoglycoside Antibiotics through CrcZ in Pseudomonas aeruginosa

“…Crc represses the translation of lon, thus stabilizing the RhlI and promoting the rhl QS pathway (14). Hfq has also been shown to regulate biofilm formation and contribute to bacterial resistance to gentamicin, fosfomycin, and tetracycline (15,16). Recent studies demonstrated that carbon sources and cellular respiration influence bacterial susceptibility to antibiotics (17,18).…”