title: Impact of AmpC Derepression on Fitness and Virulence: the Mechanism or the Pathway?
abstract: 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.
  
  
  
  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.
authors: Servicio de Microbiología and Unidad de Investigación, Hospital Son Espases, Instituto de Investigación Sanitaria de Palma (IdISPa), Palma de Mallorca, Spain.
authors: Marcelo
authors: M
authors: Pérez-Gallego
authors: Servicio de Microbiología and Unidad de Investigación, Hospital Son Espases, Instituto de Investigación Sanitaria de Palma (IdISPa), Palma de Mallorca, Spain.
authors: Gabriel
authors: G
authors: Torrens
authors: Servicio de Microbiología and Unidad de Investigación, Hospital Son Espases, Instituto de Investigación Sanitaria de Palma (IdISPa), Palma de Mallorca, Spain.
authors: Jane
authors: J
authors: Castillo-Vera
authors: Servicio de Microbiología and Unidad de Investigación, Hospital Son Espases, Instituto de Investigación Sanitaria de Palma (IdISPa), Palma de Mallorca, Spain.
authors: Bartolomé
authors: B
authors: Moya
authors: Servicio de Microbiología and Unidad de Investigación, Hospital Son Espases, Instituto de Investigación Sanitaria de Palma (IdISPa), Palma de Mallorca, Spain.
authors: Laura
authors: L
authors: Zamorano
authors: Servicio de Microbiología and Unidad de Investigación, Hospital Son Espases, Instituto de Investigación Sanitaria de Palma (IdISPa), Palma de Mallorca, Spain.
authors: Gabriel
authors: 0000-0002-6452-9945
authors: G
authors: Cabot
authors: Department of Laboratory Medicine, Karolinska Institutet and University Hospital, Huddinge, Sweden.
authors: Kjell
authors: K
authors: Hultenby
authors: Instituto Universitario de Investigación en Ciencias de la Salud, Universidad de las Islas Baleares, Palma de Mallorca, Spain.
authors: Sebastián
authors: S
authors: Albertí
authors: Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.
authors: Peter
authors: P
authors: Mellroth
authors: Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.|Department of Clinical Microbiology, Karolinska University Hospital, Stockholm, Sweden.
authors: Birgitta
authors: B
authors: Henriques-Normark
authors: Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.
authors: Staffan
authors: S
authors: Normark
authors: Servicio de Microbiología and Unidad de Investigación, Hospital Son Espases, Instituto de Investigación Sanitaria de Palma (IdISPa), Palma de Mallorca, Spain antonio.oliver@ssib.es carlos.juan@ssib.es.
authors: Antonio
authors: A
authors: Oliver
authors: Servicio de Microbiología and Unidad de Investigación, Hospital Son Espases, Instituto de Investigación Sanitaria de Palma (IdISPa), Palma de Mallorca, Spain antonio.oliver@ssib.es carlos.juan@ssib.es.
authors: Carlos
authors: C
authors: Juan
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D002473:Cell Wall / Q000378:metabolism*; D017353:Gene Deletion; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000235:genetics / Q000378:metabolism; D010457:Peptidoglycan / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000254:growth & development / Q000502:physiology*; D014774:Virulence; D001618:beta-Lactamases / Q000096:biosynthesis* / Q000235:genetics
chemical_list: C084284:AmpG protein, Bacteria; D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D010457:Peptidoglycan; C062460:AmpD protein, Bacteria; D009238:N-Acetylmuramoyl-L-alanine Amidase; C048795:AmpC beta-lactamases; D001618:beta-Lactamases