title: Characterization of an Acinetobacter baumannii lptD Deletion Strain: Permeability Defects and Response to Inhibition of Lipopolysaccharide and Fatty Acid Biosynthesis.
abstract: Lipid A on the Gram-negative outer membrane (OM) is synthesized in the cytoplasm by the Lpx pathway and translocated to the OM by the Lpt pathway. Some Acinetobacter baumannii strains can tolerate the complete loss of lipopolysaccharide (LPS) resulting from the inactivation of early LPS pathway genes such as lpxC. Here, we characterized a mutant deleted for lptD, which encodes an OM protein that mediates the final translocation of fully synthesized LPS to the OM. Cells lacking lptD had a growth defect comparable to that of an lpxC deletion mutant under the growth conditions tested but were more sensitive to hydrophobic antibiotics, revealing a more significant impact on cell permeability from impaired LPS translocation than from the loss of LPS synthesis. Consistent with this, ATP leakage and N-phenyl-1-naphthylamine (NPN) fluorescence assays indicated a more severe impact of lptD deletion than of lpxC deletion on inner and outer membrane permeability, respectively. Targeted liquid chromatography-mass spectrometry (LCMS) analysis of LPS intermediates from UDP-3-O-R-3-hydroxylauroyl-N-acetyl-α-d-glucosamine through lipid IV(A) showed that the loss of LptD caused an accumulation of lipid IV(A). This suggested that pathway intermediate accumulation or mislocalization caused by the blockage of later LPS pathway steps impacts envelope integrity. Supporting this notion, chemical inhibition of lipid A precursor enzymes, including LpxC and FabB/F, in the lptD deletion strain partially rescued growth and permeability defects.
  
  
  OBJECTIVE
  New antibiotics to treat Gram-negative bacterial infections are urgently needed. Inhibition of LPS biosynthesis is attractive because this would impact viability and cell permeability. Therefore, a better understanding of this pathway is important, especially in strains such as A. baumannii ATCC 19606, where LPS biosynthesis is not essential in vitro. We show that ATCC 19606 also survives the loss of the final translocation of LPS into the OM (lptD deletion). Intriguingly, this impaired cell envelope integrity more than the loss of LPS biosynthesis (lpxC deletion), presumably due to the accumulation of toxic intermediates. Supporting this, chemical inhibition of LPS biosynthesis partially reversed this permeability defect. This extends our understanding of the LPS machinery and provides insights into potential interrelationships of the target steps along this important pathway.
authors.affiliation: Infectious Diseases Area, Novartis Institutes for BioMedical Research, Emeryville, California, USA.
authors.forename: Jade
authors.initials: J
authors.lastname: Bojkovic
authors.affiliation: Infectious Diseases Area, Novartis Institutes for BioMedical Research, Emeryville, California, USA.
authors.forename: Daryl L
authors.initials: DL
authors.lastname: Richie
authors.affiliation: Infectious Diseases Area, Novartis Institutes for BioMedical Research, Emeryville, California, USA.
authors.forename: David A
authors.initials: DA
authors.lastname: Six
authors.affiliation: Infectious Diseases Area, Novartis Institutes for BioMedical Research, Emeryville, California, USA.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Rath
authors.affiliation: Infectious Diseases Area, Novartis Institutes for BioMedical Research, Emeryville, California, USA.
authors.forename: William S
authors.initials: WS
authors.lastname: Sawyer
authors.affiliation: Infectious Diseases Area, Novartis Institutes for BioMedical Research, Emeryville, California, USA.
authors.forename: Qijun
authors.initials: Q
authors.lastname: Hu
authors.affiliation: Infectious Diseases Area, Novartis Institutes for BioMedical Research, Emeryville, California, USA charlesr.dean@novartis.com.
authors.forename: Charles R
authors.initials: CR
authors.lastname: Dean
mesh_terms: D040981:Acinetobacter baumannii / Q000235:genetics* / Q000378:metabolism*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D005227:Fatty Acids / Q000096:biosynthesis*; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D008070:Lipopolysaccharides / Q000096:biosynthesis*; D010539:Permeability
chemical_list: D001425:Bacterial Outer Membrane Proteins; D005227:Fatty Acids; D008070:Lipopolysaccharides