Fmt Bypass in Pseudomonas aeruginosa Causes Induction of MexXY Efflux Pump Expression

Abstract

The intrinsic resistance of P. aeruginosa PAO1 to the peptide deformylase inhibitor (PDF-I) LBM415 was mediated by the MexAB-OprM and MexXY-OprM efflux pumps, the latter of which was strongly induced by LBM415. Single-step exposure of PAO1 deleted for mexAB-oprM (therefore lacking both MexAB-OprM and MexXY-OprM functions) to PDF-Is selected for nfxB mutants, which express the MexCD-OprJ efflux pump, indicating that these compounds are also substrates for this pump. Selection of resistant mutants by use of levels of LBM415 greater than that accommodated by efflux yielded two additional groups of mutations, in the methionyl-tRNA fmet formyltransferase (fmt) and folD genes. Both mechanisms are known to impose an in vitro growth deficit (also observed here), presumably due to impairment of protein synthesis. We surmised that this inherent impairment of protein synthesis would upregulate expression of mexXY in a fashion similar to upregulation by LBM415 or by ribosome inhibitory compounds. Transcriptional profiling and/or mexX::lux promoter fusion analysis revealed that fmt and folD mutants were strongly upregulated for mexXY and another gene known to be required for upregulation of the pump, PA5471. Complementation of the fmt mutation in trans reversed this constitutive expression. This supports the notion that MexXY has a natural physiological function responding to impairment of ribosome function or protein synthesis and that fmt mutation (Fmt bypass) and folD mutation generate the intracellular mexXY-inducing signal.

The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria

“…The weak promoter identified ahead of oprM within the mexAB-oprM operon (442) could possibly account for an excess of OprM molecules relative to MexAB, thus allowing the interaction of OprM with other extrusion systems without impacting the activity of MexAB-OprM by titration. MexXY(OprA)-overproducing mutants can be easily selected in vitro and in vivo by substrate antibiotics (393,439,443,444) or protein synthesis inhibitors (445), which is consistent with the increased prevalence, sometimes Ͼ80%, of such mutants in cystic fibrosis (436,(446)(447)(448)(449)(450)(451) and non-cystic fibrosis (384, 385, 414, 416, 417, 420-423, 429, 431, 452) patients worldwide. The abundance of reactive oxygen species in the cystic fibrosis lung environment might explain the high rates of resistant mutants with this pathology (453).…”

Oxidative Stress Induction of the MexXY Multidrug Efflux Genes and Promotion of Aminoglycoside Resistance Development in Pseudomonas aeruginosa

“…Still, only those agents known to target the ribosome promote mexXY expression (26,31,34), and this is compromised by so-called ribosome protection mechanisms (26), suggesting that the MexXY-OprM efflux system is recruited in response to ribosome disruption or defects in translation and not antibiotics per se. Consistent with this, mutations in fmt (encoding a methionyl-tRNA-formyltransferase) and folD (involved in folate biosynthesis and production of the formyl group added to initiator methionine), which are expected to negatively affect protein synthesis, increase expression of mexXY (6). Upregulation of mexXY by antimicrobials or fmt/ folD mutations is dependent upon a gene, PA5471, encoding a conserved hypothetical protein whose expression is also promoted by ribosome-disrupting antimicrobials (34) and by fmt/ folD mutations (6).…”

“…Consistent with this, mutations in fmt (encoding a methionyl-tRNA-formyltransferase) and folD (involved in folate biosynthesis and production of the formyl group added to initiator methionine), which are expected to negatively affect protein synthesis, increase expression of mexXY (6). Upregulation of mexXY by antimicrobials or fmt/ folD mutations is dependent upon a gene, PA5471, encoding a conserved hypothetical protein whose expression is also promoted by ribosome-disrupting antimicrobials (34) and by fmt/ folD mutations (6). Despite this primary link to translation disruption, the MexXY-OprM efflux system is a significant determinant of resistance to antimicrobials in clinical isolates, particularly aminoglycosides (19,40,52) but also ␤-lactams (3,19,23,37,51).…”

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

“…The presence of the oprA gene in P. aeruginosa might be more advantageous for its survival in the presence of aminoglycosides, other antimicrobials such as b-lactams (e.g. cefepime, ceftobiprole; Hocquet et al, 2006;Baum et al, 2009), tigecycline (Dean et al, 2003), LBM415 (peptide deformylase inhibitor; Caughlan et al, 2009) and/or some other sources of stress (e.g. oxidative stress; Fraud & Poole, 2011).…”