Small proteins and small RNAs in antibiotic susceptibility

Multi-drug resistant bacterial pathogens are the leading cause of healthcare associated infections (HAI). The opportunistic pathogen Pseudomonas aeruginosa (Pae) is responsible for ~10% of HAI worldwide and particularly devastating for immunocompromised individuals or patients with cystic fibrosis. The pathogenicity of Pae is accompanied by increased resistance towards many medically used antibiotics. In fact, Pae has rendered most antibiotics inactive, leaving polymyxins and aminoglycosides as a last resort. Although several resistance mechanisms toward aminoglycosides and polymyxins are known, a refined understanding of the molecular regulatory circuits that contribute to susceptibility and persister cell formation is key to develop new strategiesools to combat Pae. We have identified small regulatory RNA- and small protein candidates that affected the susceptibility towards these antibiotics. The major goal of these studies is to achieve a mechanistic understanding of these potential regulators. The studies with the small RNA candidates may either contribute to a refined understanding of the regulation of known antibiotic resistance determinants or even reveal unknown molecular circuits contributing to the susceptibility towards colistinobramycin. The studies with the small protein are anticipated to reveal new actors/mechanisms involved in the susceptibility towards these antibiotics.

Background: Multi-drug resistant bacterial pathogens are the leading cause of healthcare associated infections (HAI). Pseudomonas aeruginosa (Pae) is responsible for ~10% of HAI worldwide. The pathogenicity of Pae is accompanied by increased resistance towards many antibiotics. In fact, Pae has rendered most antibiotics inactive, leaving polymyxins and aminoglycosides as a last resort. Although several resistance mechanisms toward aminoglycosides and polymyxins are known, a refined understanding of the molecular regulatory circuits that contribute to susceptibility and persister cell formation is key to develop new strategies/tools to combat Pae. We have identified small regulatory RNA- and small protein candidates that affected the susceptibility towards these antibiotics. The major goal of these studies was to achieve a mechanistic understanding of these potential "regulators". Results: We initially identified two sRNAs termed IGR4759 and IGR4737, whose expression is induced upon colistin and tobramycin treatment, respectively. Deletion of IGR4759 resulted in a higher susceptibility to colistin, whereas the IGR4737 deletion mutant was impaired in persister cell formation upon exposure to lethal doses of tobramycin. Comparative transcriptome analyses revealed that IGR4759 stimulates the synthesis of functions that counteract reactive oxygen species that are known to be caused by colistin, while IGR4737 down-regulates function involved assembly of pili and flagella, which are involved in uptake of antibiotics. In this way IGR4737 could affect tobramycin uptake. In Pae, the 37 amino acids long and highly conserved small protein PhrS-P is encoded by the dual-function sRNA PhrS. Pae strains lacking PhrS-P exhibited significantly reduced fitness when exposed to sub-inhibitory concentrations of tobramycin and other aminoglycosides. Therefore, we performed in vivo co-purification studies coupled to mass spectrometry, which identified the kinase AmgS of the AmgRS two-component system as an interacting partner of PhrS-P. AmgRS is involved in response to envelope perturbations induced by aminoglycoside-generated mistranslated polypeptides. These aberrant polypeptides disrupt the inner membrane activating AmgS, which activates AmgR to induce, among others, the transcription of the membrane protease genes htpX and PA5528. The activities of the HtpX and PA5528 proteins in turn stimulate the expression of the mexXY efflux pump operon. The MexXY-OprM efflux pump is capable of extruding aminoglycosides from the cell, thus increasing the aminoglycoside resistance of Pae. We were able to shown that the absence of PhrS-P synthesis leads to a two-fold decrease in the steady state mRNA levels of the htpX and PA5528 transcripts. These findings are supported by RNA-seq based transcriptome analyses, which confirmed that multiple genes of the proposed AmgR/S regulon are differentially expressed depending on the presence or absence of PhrS-P. In summary, our studies revealed that PhrS-P affects the susceptibility to tobramycin by stimulating the activity of AmgS, and thus a new mechanism contributing to resistance toward aminoglycoside antibiotics.