Production of Norspermidine Contributes to Aminoglycoside Resistance in pmrAB Mutants of Pseudomonas aeruginosa

Abstract

Emergence of resistance to polymyxins in Pseudomonas aeruginosa is mainly due to mutations in two-component systems that promote the addition of 4-amino-4-deoxy-l-arabinose to the lipopolysaccharide (LPS) through upregulation of operon arnBCADTEF-ugd (arn) expression. Here, we demonstrate that mutations occurring in different domains of histidine kinase PmrB or in response regulator PmrA result in coresistance to aminoglycosides and colistin. All seventeen clinical strains tested exhibiting such a cross-resistance phenotype were found to be pmrAB mutants. As shown by gene deletion experiments, the decreased susceptibility of the mutants to aminoglycosides was independent from operon arn but required the efflux system MexXY-OprM and the products of three genes, PA4773-PA4774-PA4775, that are cotranscribed and activated with genes pmrAB Gene PA4773 (annotated as speD2 in the PAO1 genome) and PA4774 (speE2) are predicted to encode enzymes involved in biosynthesis of polyamines. Comparative analysis of cell surface extracts of an in vitro selected pmrAB mutant, called AB16.2, and derivatives lacking PA4773, PA4774, and PA4775 revealed that these genes were needed for norspermidine production via a pathway that likely uses 1,3-diaminopropane, a precursor of polyamines. Altogether, our results suggest that norspermidine decreases the self-promoted uptake pathway of aminoglycosides across the outer membrane and, thereby, potentiates the activity of efflux pump MexXY-OprM.

Keywords: PmrAB; Pseudomonas aeruginosa; aminoglycosides; colistin; norspermidine; polyamines.

FIG 1

Schematic representation of histidine kinase (HK) PmrB. The mutations responsible for cross-resistance to aminoglycosides and colistin in in vitro-selected mutants and clinical strains are marked with asterisks. The first and second transmembrane domains of PmrB are colored in gray (from amino acid position 15 to 37 and from 161 to 183, respectively). Also represented are the periplasmic domain (from 38 to 160), the HAMP linker domain (from 186 to 238), the dimerization/phosphoacceptor (HisKA) domain (from 239 to 304, colored in black) that contains the conserved active site histidine 249, and the histidine kinase-like ATPase (HATPase) domain (from 348 to 459). The domains are available from the SMART protein database (http://smart.embl-heidelberg.de).

FIG 2

Contribution of genes PA4773, PA4774, and PA4775 to amikacin (A) and colistin (B) resistance. Deletion mutants AB16.2ΔPA4773, AB16.2ΔPA4774, and AB16.2ΔPA4775 were transcomplemented with a DNA fragment carrying the wild-type genes PA4773-PA4774-PA4775 inserted in chromosomal site attB (yielding constructs AB16.2ΔPA4773::CTX73-75, AB16.2ΔPA4774::CTX73-75, and AB16.2ΔPA4775::CTX73-75, respectively). The data presented are representative of 3 independent MIC determinations.

FIG 3

Amounts of spermidine (A), norspermidine (B), and 1,3-diaminopropane (C) in cell surface extracts of strain PAO1 and derived mutants. The histograms represent the area under the peak values of each compound as determined by LC-ESI-MS. The data correspond to means of normalized values (log scale) ± the standard deviation (SD) of three independent experiments. Tukey’s test results are indicated as follows: *, P < 0.05; **, P < 0.01.

FIG 4

Effects of gene PA4773, PA4774, and PA4775 overexpression on bacterial growth. Growth of strains PAO1 (circles, solid line), AB16.2 (squares, solid line), AB16.2ΔPA4773 (crosses, dashed line), AB16.2ΔPA4774 (open triangles, dotted line), and AB16.2ΔPA4775 (triangles, dashed line) in Mueller-Hinton broth at 37°C was measured spectrophotometrically at A₆₀₀. Error bars indicate SD of three biological replicates.

FIG 5

Norspermidine biosynthesis. The putative pathway of norspermidine synthesis in P. aeruginosa and the previously identified pathway of norspermidine biosynthesis in Vibrionales are represented with black and gray lines respectively.