96-well growth restoration assay.

<p>A) Growth of JWK0013(pNOV044) was restored in the presence of LpxC inhibitors Compound 1 (32–128 μg/mL) and CHIR-090 (1–64 μg/mL) and fatty acid inhibitors pyridopyrimidine (8–32 μg/mL), andrimid (0.25–128 μg/mL), and cerulenin (2–64 μg/mL). Growth restoration was defined as an increase in fluorescence of at least 2-fold above background in consecutive wells. This was repeated at least three times in duplicate with similar results. In this representative example, SABA-1 displayed growth rescue at 0 μg/mL (DMSO control) due to revertants as evident by the lack of dose response, and growth was not observed in the replicate plates. B) Growth of JWK0013(pNOV044) was not observed under noninducing conditions (DMSO, 10 μL per disk); growth of JWK0013(pNOV044) was restored in the presence of IPTG (10 μL @ 1mM per disk); JWK0013(pNOV044) grew under noninducing conditions in the presence of pyridopyrimidine (10 μL @ 12.8 mg/ml per disk, ACC) and andrimid (10 μL @ 12.8 mg/ml per disk, ACC) but growth was not restored in the presence of SABA analogs that lack MIC values (10 μL @ 12.8 mg/mL per disk, SABA-1, 2).</p

A pathway-directed positive growth restoration assay to facilitate the discovery of lipid A and fatty acid biosynthesis inhibitors in <i>Acinetobacter baumannii</i>

<div><p><i>Acinetobacter baumannii</i> ATCC 19606 can grow without lipooligosaccharide (LOS). Lack of LOS can result from disruption of the early lipid A biosynthetic pathway genes <i>lpxA</i>, <i>lpxC</i> or <i>lpxD</i>. Although LOS itself is not essential for growth of <i>A</i>. <i>baumannii</i> ATCC 19606, it was previously shown that depletion of the lipid A biosynthetic enzyme LpxK in cells inhibited growth due to the toxic accumulation of lipid A pathway intermediates. Growth of LpxK-depleted cells was restored by chemical inhibition of LOS biosynthesis using CHIR-090 (LpxC) and fatty acid biosynthesis using cerulenin (FabB/F) and pyridopyrimidine (acetyl-CoA-carboxylase). Here, we expand on this by showing that inhibition of enoyl-acyl carrier protein reductase (FabI), responsible for converting trans-2-enoyl-ACP into acyl-ACP during the fatty acid elongation cycle also restored growth during LpxK depletion. Inhibition of fatty acid biosynthesis during LpxK depletion rescued growth at 37°C, but not at 30°C, whereas rescue by LpxC inhibition was temperature independent. We exploited these observations to demonstrate proof of concept for a targeted medium-throughput growth restoration screening assay to identify small molecule inhibitors of LOS and fatty acid biosynthesis. The differential temperature dependence of fatty acid and LpxC inhibition provides a simple means by which to separate growth stimulating compounds by pathway. Targeted cell-based screening platforms such as this are important for faster identification of compounds inhibiting pathways of interest in antibacterial discovery for clinically relevant Gram-negative pathogens.</p></div

Schematic of predicted lipid A and FASII biosynthetic pathway in <i>A</i>. <i>baumannii</i> ATCC 19606.

<p>Schematic of predicted lipid A and FASII biosynthetic pathway in <i>A</i>. <i>baumannii</i> ATCC 19606.</p

Inhibitors of fatty acid biosynthesis rescues growth of cells depleted for LpxK at 37°C but not 30°C.

<p>Growth of JWK0013(pNOV044) was not observed under noninducing conditions (DMSO, 10 μL per disk); growth of JWK0013(pNOV044) was restored in the presence of IPTG (10 μL @ 1 mM per disk) at 30 and 37°C; JWK0013(pNOV044) grew under noninducing conditions in the presence of CHIR-090 and Compound 1 (LpxC inhibitors, 10 μL @ 12.8 mg/mL) at 30 and 37°C; JWK0013(pNOV044) grew under noninducing conditions in the presence of fatty acid inhibitors at 37°C, but not 30°C, including pyridopyrimidine (10 μL @ 12.8 mg/mL per disk, ACC) and AFN-1252 (10 μL @ 3.2 mg/ml per disk, FabI) and cerulenin (10 μL @ 12.8 mg/mL per disk, FabB/F).</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

Depletion of LpxH causes accumulation of DSMP.

<p>(A) LCMS-MRM quantification of DSMP with three 12:0(3-OH) acyl groups and one 14:0(3-OH) acyl group and with two 12:0(3-OH) acyl groups and two 14:0(3-OH) acyl groups 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<0.05 and **, P<0.01) 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

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 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

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