看護学部教官業績目録 ; 2003年1月～12月

Acinetobacter baumannii ATCC 19606 tolerates loss of lipopolysaccharide (LPS) caused by inactivation of early LPS pathway genes. However, mutations in pathway genes encoding steps downstream of LpxD have not been reported, implying that later biosynthetic steps may be essential for viability. Here, we determined if LpxH, the UDP-2,3-diacylglucosamine hydrolase that generates UMP-2,3-diacylglucosamine 1-phosphate (lipid X), was essential in A. baumannii ATCC 19606. Multiple attempts to disrupt lpxH on the genome were unsuccessful. When expression of LpxH was placed under control of an isopropyl β-D-1-thiogalactopyranoside (IPTG) inducible promoter, the cells failed to grow under standard laboratory conditions without IPTG induction. Growth under LpxH depletion conditions (-IPTG) was rescued by chemical inhibition of LpxC, upstream of LpxH, indicating that toxic accumulation of LPS pathway intermediates underlies LpxH essentiality. Consistent with this, the levels of LpxH substrate (product of LpxD) and a C14:0(3-OH) acyl variant of the LpxD substrate had accumulated in cells that were depleted of LpxH causing a growth defect. Intriguingly, under these partial depletion conditions, there was also a smaller but reproducible accumulation of the downstream pathway intermediates disaccharide 1-monophosphate and lipid IVA suggesting a complex downstream response to LpxH depletion

Characterization of an Acinetobacter baumannii lptD Deletion Strain; Permeability Defects and Response to Inhibition of Lipopolysaccharide and Fatty Acid Biosynthesis

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 complete loss of lipopolysaccharide (LPS) resulting from 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 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 loss of LPS synthesis. Consistent with this, ATP leakage and NPN fluorescence assays indicated a more severe impact of lptD deletion than lpxC deletion on inner and outer membrane permeability, respectively. Targeted LCMS analysis of LPS intermediates from UDP-3-O-[(3R)-3-hydroxylauroyl]-N-acetyl-α-D-glucosamine through lipid IVA, showed that loss of LptD caused an accumulation of lipid IVA. This suggested that pathway intermediate accumulations or mislocalizations caused by blockage of later LPS pathway steps impact envelope integrity. Supporting this notion, chemical inhibition of lipid A precursor enzymes including LpxC and Fab B/F in the ΔlptD strain partially rescued growth and permeability defects

Characterization of an A. baumannii lptD Deletion Strain; Permeability Defects and Response to Inhibition of Lipopolysaccharide and Fatty Acid Biosynthesis

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 complete loss of lipopolysaccharide (LPS) resulting from 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::KmR 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 loss of LPS synthesis. Consistent with this, adenosine triphosphate (ATP) leakage and N-phenyl-1-naphthylamine (NPN) fluorescence assays indicated a more severe impact of lptD deletion than 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 IVA, showed that loss of LptD caused an accumulation of lipid IVA. This suggested that pathway intermediate accumulation or mislocalization caused by blockage of later LPS pathway steps impact envelope integrity. Supporting this notion, chemical inhibition of lipid A precursor enzymes including LpxC and Fab B/F in the lptD::KmR strain partially rescued growth and permeability defects

Characterization of an Acinetobacter baumannii lptD deletion strain: Permeability defects and response to inhibition of lipopolysaccharide and fatty acid biosynthesis

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 anOMprotein 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-Dglucosamine through lipid IVA showed that the loss of LptD caused an accumulation of lipid IVA. 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

Depletion of LpxH causes accumulation of UDP-Diacyl-GlcN (LpxH substrate).

<p>A) The LCMS-MRM quantification of lipid A precursor UDP-Diacyl-GlcN is shown for <i>A</i>. <i>baumannii</i> ATCC 19606 parent and NB48062-JWK0133 under inducing and non-inducing conditions. The <i>m/z</i> [M–H⁺]^- for UDP-Diacyl-GlcN containing 2 acyl groups, 12:0(3-OH), 12:0(3-OH) is 960.5 and for UDP-Diacyl-GlcN with 1 acyl group 12:0(3-OH) and 1 acyl group 14:0(3-OH) the <i>m/z</i> [M–H⁺]^- is 988.5. Experiments were performed in triplicate and bars show the mean value and SD (two-tailed student t-test, **, P<0.01) between NB46082-JWK0133 in the presence or absence of IPTG. Data shown were normalized to an internal standard (IS) as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160918#pone.0160918.ref040" target="_blank">40</a>]. B) Extracted ion chromatogram (EIC) of NB48062-JWK0133 in the presence or absence of IPTG for 2 acyl groups, 12:0(3-OH), 12:0(3-OH). C) Extracted ion chromatogram of NB48062-JWK0133 in the presence or absence of IPTG for 1 acyl group 12:0(3-OH) and 1 acyl group 14:0(3-OH).</p

The dependence of LpxH for growth is abrogated by inhibition of LpxC under standard laboratory conditions.

<p>(A) NB48062-JWK0133 was streaked on MHIIB agar supplemented with 1 mM IPTG and grown overnight at 37°C to induce <i>lpxH</i> expression. The following day, cells were washed repeatedly and resuspended to an OD₆₀₀ of 0.01, and 100 μL was plated on MHIIB plates without IPTG. Sterile filter discs containing IPTG, DMSO, or CHIR-090 were placed on the plates which were then incubated 37°C for 24 hours. Left panel; growth of NB48062-JWK0133 was not observed under non-inducing conditions (minus IPTG, DMSO). Center panel; growth of NB48062-JWK0133 is restored in the presence of IPTG. Right panel; NB48062-JWK0133 grew under non-inducing conditions in the presence of the LpxC inhibitor CHIR-090. (B) An overnight culture of NB48062-JWK0133 under inducing conditions (+ IPTG) was diluted to an OD₆₀₀ of 0.1 and then was diluted 100-fold into MHIIB containing 10% Alamar Blue. Next, 100 μL of the inoculum was added to the wells of a 96-well plate containing CHIR-090 to a final assay concentrations ranging from 0.25–64 μg/ml. The plate was incubated for 6 hours at 37°C before fluorescence reading (545ex (nm)– 590em (nm)) on the SpectraMax and analyzed with Softmax® Pro software v 5.4.1.</p

Depletion of LpxH causes accumulation of an alternative LpxC product containing a C14:0(3-OH) acyl chain.

<p>The LCMS-MRM quantification of UDP-3-<i>O</i>-[(<i>R</i>)-3-OH-C_12/14]-GlcN for acyl group 12:0(3-OH) and for acyl group 14:0(3-OH) is shown for <i>A</i>. <i>baumannii</i> ATCC 19606 parent and NB48062-JWK0133 under inducing and non-inducing conditions. The experiment was performed in triplicate and bars show the mean value and SD (two-tailed student t-test ***, P<0.001) between NB46082-JWK0133 in the presence or absence of IPTG. Data shown was normalized to an internal standard (IS) as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160918#pone.0160918.ref040" target="_blank">40</a>].</p

Transmission EM images of NB48062-JWK0133 plus or minus IPTG.

<p>LpxH depleted cells (-IPTG) display gross morphology changes. The experiment was repeated twice with similar results.</p

Schematic illustration of the <i>lpxH</i> regulated expression strain NB46082-JWK0133 and its dependence on IPTG induction for cell growth.

<p>(A) The chromosomal allele of <i>lpxH</i> is regulated by the P_tac promoter (inducible by IPTG). Plasmid pNOV108 provided extra copies of <i>lacI</i> to enhance repression of <i>lpxH</i> in the absence of IPTG. pNOV108 also contained <i>alaS</i> so that it could be maintained by complementation of an <i>alaS</i> deletion on the chromosome, eliminating the need for antibiotic selection. (B) Growth of NB48062-JWK0133 was IPTG dependent. C) Sub-culture growth curve of NB48062-JWK0133 plus or minus IPTG. Arrows indicate time points of sample collection for LCMS-MRM analysis, CFU determination, TEM images, RT-qPCR, and CHIR-090 rescue. D) A significant loss in viability (*, <i>P</i> ≤ 0.01) was observed at 1.5 OD₆₀₀ under LpxH depletion conditions (-IPTG) compared to inducing conditions (+IPTG).</p