Impact of AmpC Derepression on Fitness and Virulence: the Mechanism or the Pathway?
Authors: Pérez-Gallego Marcelo , Torrens Gabriel , Castillo-Vera Jane , Moya Bartolomé , Zamorano Laura , Cabot Gabriel , Hultenby Kjell , Albertí Sebastián , Mellroth Peter , Henriques-Normark Birgitta , Normark Staffan , Oliver Antonio , Juan Carlos 
Published: October 2016

Understanding the interplay between antibiotic resistance and bacterial fitness and virulence is essential to guide individual treatments and improve global antibiotic policies. A paradigmatic example of a resistance mechanism is the intrinsic inducible chromosomal β-lactamase AmpC from multiple Gram-negative bacteria, including
Pseudomonas aeruginosa
, a major nosocomial pathogen. The regulation of
ampC
expression is intimately linked to peptidoglycan recycling, and AmpC-mediated β-lactam resistance is frequently mediated by inactivating mutations in
ampD
, encoding an
N
-acetyl-anhydromuramyl-
l
-alanine amidase, affecting the levels of
ampC
-activating muropeptides. Here we dissect the impact of the multiple pathways causing AmpC hyperproduction on
P. aeruginosa
fitness and virulence. Through a detailed analysis, we demonstrate that the lack of all three
P. aeruginosa
AmpD amidases causes a dramatic effect in fitness and pathogenicity, severely compromising growth rates, motility, and cytotoxicity; the latter effect is likely achieved by repressing key virulence factors, such as protease LasA, phospholipase C, or type III secretion system components. We also show that
ampC
overexpression is required but not sufficient to confer the growth-motility-cytotoxicity impaired phenotype and that alternative pathways leading to similar levels of
ampC
hyperexpression and resistance, such as those involving PBP4, had no fitness-virulence cost. Further analysis indicated that fitness-virulence impairment is caused by overexpressing
ampC
in the absence of cell wall recycling, as reproduced by expressing
ampC
from a plasmid in an AmpG (muropeptide permease)-deficient background. Thus, our findings represent a major step in the understanding of β-lactam resistance biology and its interplay with fitness and pathogenesis.

IMPORTANCE
Understanding the impact of antibiotic resistance mechanisms on bacterial pathogenesis is critical to curb the spread of antibiotic resistance. A particularly noteworthy antibiotic resistance mechanism is the β-lactamase AmpC, produced by
Pseudomonas aeruginosa
, a major pathogen causing hospital-acquired infections. The regulation of AmpC is linked to the cell wall recycling pathways, and frequently, resistance to β-lactams is caused by mutation of several of the components of the cell wall recycling pathways such as AmpD. Here we dissect the impact of the pathways for AmpC hyperproduction on virulence, showing that the lack of all three
P. aeruginosa
AmpD amidases causes a major effect in fitness and pathogenicity, compromising growth, motility, and cytotoxicity. Further analysis indicated that fitness-virulence impairment is specifically caused by the hyperproduction of AmpC in the absence of cell wall recycling. Our work provides valuable information for delineating future strategies for combating
P. aeruginosa
infections by simultaneously targeting virulence and antibiotic resistance.