Membrane Proteases and Aminoglycoside Antibiotic Resistance

Abstract

We present genetic studies that help define the functional network underlying intrinsic aminoglycoside resistance in Pseudomonas aeruginosa. Our analysis shows that proteolysis, particularly that controlled by the membrane protease FtsH, is a major determinant of resistance. First, we examined the consequences of inactivating genes controlled by AmgRS, a two-component regulator required for intrinsic tobramycin resistance. Three of the gene products account for resistance: a modulator of FtsH protease (YccA), a membrane protease (HtpX), and a membrane protein of unknown function (PA5528). Second, we screened mutations inactivating 66 predicted proteases and related functions. Insertions inactivating two FtsH protease accessory factors (HflK and HflC) and a cytoplasmic protease (HslUV) increased tobramycin sensitivity. Finally, we generated an ftsH deletion mutation. The mutation dramatically increased aminoglycoside sensitivity. Many of the functions whose inactivation increased sensitivity appeared to act independently, since multiple mutations led to additive or synergistic effects. Up to 500-fold increases in tobramycin sensitivity were observed. Most of the mutations also were highly pleiotropic, increasing sensitivity to a membrane protein hybrid, several classes of antibiotics, alkaline pH, NaCl, and other compounds. We propose that the network of proteases provides robust protection from aminoglycosides and other substances through the elimination of membrane-disruptive mistranslation products.

Determinants of Intrinsic Aminoglycoside Resistance in Pseudomonas aeruginosa

“…HflKC regulates a protease, FtsH, which is responsible, in part, for turning over aberrant/misfolded membrane-associated polypeptides (41), suggesting that the increased susceptibility and membrane depolarization of the mutants result from increases in the levels of aminoglycoside-generated mistranslated polypeptides and, so, membrane perturbation. Consistent with this and with enhanced aminoglycoside-promoted membrane depolarization in P. aeruginosa being an indicator of enhanced CM perturbation by aminoglycoside-generated mistranslated polypeptides, an ftsH mutant of P. aeruginosa also exhibits enhanced susceptibility to aminoglycosides (37).…”

Primary mechanisms mediating aminoglycoside resistance in the multidrug-resistant Pseudomonas aeruginosa clinical isolate PA7

“…carbapenemase-encoding genes) (e.g. Doi et al, 2007 Struble & Gill, 2009;Kindrachuk et al, 2011;Hinz et al, 2011) in addition to previous ones (Poole, 2005). It might be interesting to examine the relationship between the MexXY pump and each determinant, or combinatorial determinants, in order to eradicate aminoglycoside resistance in P. aeruginosa.…”

Membrane Proteases in the Bacterial Protein Secretion and Quality Control Pathway

“…It is therefore of crucial importance that the cell is able to control the quality of essential membrane proteins and to remove mistranslated, damaged, misfolded, or aggregated membrane proteins (1,57).…”