Recycling of Peptidyl-tRNAs by Peptidyl-tRNA Hydrolase Counteracts Azithromycin-Mediated Effects on Pseudomonas aeruginosa

Abstract

Acute and chronic infections caused by the opportunistic pathogen Pseudomonas aeruginosa pose a serious threat to human health worldwide, and its increasing resistance to antibiotics requires alternative treatments that are more effective than available strategies. Clinical studies have clearly demonstrated that cystic fibrosis (CF) patients with chronic P. aeruginosa infections benefit from long-term low-dose azithromycin (AZM) treatment. Immunomodulating activity, the impact of AZM on the expression of quorum-sensing-dependent virulence factors, type three secretion, and motility in P. aeruginosa seem to contribute to the therapeutic response. However, to date, the molecular mechanisms underlying these AZM effects have remained elusive. Our data indicate that the AZM-mediated phenotype is caused by a depletion of the intracellular pools of tRNAs available for protein synthesis. Overexpression of the P. aeruginosa peptidyl-tRNA hydrolase, which recycles the tRNA from peptidyl-tRNA drop-off during translation, counteracted the effects of AZM on stationary-phase cell killing, cytotoxicity, and the production of rhamnolipids and partially restored swarming motility. Intriguingly, the exchange of a rare for a frequent codon in rhlR also explicitly diminished the AZM-mediated decreased production of rhamnolipids. These results indicate that depletion of the tRNA pools by AZM seems to affect the translation of genes that use rare aminoacyl-tRNA isoacceptors to a great extent and might explain the selective activity of AZM on the P. aeruginosa proteome and possibly also on the protein expression profiles of other bacterial pathogens. Pseudomonas aeruginosa is an opportunistic bacterial pathogen that causes both life-threatening acute and devastating chronic infections in the human host (1). In cystic fibrosis (CF) patients, the respiratory tract is especially prone to chronic infections caused by the most dominant bacterial pathogen, P. aeruginosa, and these infections largely determine the fate and prognosis of these patients (2, 3). Improved antimicrobial treatment strategies have greatly increased the life expectancy of CF patients in the last decades. Nevertheless, even aggressive antimicrobial therapy rarely eradicates established chronic P. aeruginosa infections (4, 5, 6). Hence, for the management of chronic infectious diseases, there is a strong need for alternative treatment strategies that amend classical antimicrobial therapy (7,8,9). Several clinical studies have demonstrated that CF patients and patients suffering from diffuse panbronchiolitis (DPB) who are chronically infected with P. aeruginosa benefit from treatment with the macrolide azithromycin (AZM), although the 14-and 15-C macrolides (erythromycin, azithromycin, and clarithromycin) do not inhibit the growth of P. aeruginosa at concentration levels below the breakpoint concentration for susceptibility to the macrolides (10,11,12,13,14,15).The nature of this beneficial effect of AZM is still unclear. Macrolides were shown to have immunomodulatory a...

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