Genetic synergy between Acinetobacter baumannii undecaprenyl phosphate biosynthesis and the Mla system impacts cell envelope and antimicrobial resistance

ABSTRACT Acinetobacter baumannii is a Gram-negative bacterial pathogen that poses a major health concern due to increasing multidrug resistance. The Gram-negative cell envelope is a key barrier to antimicrobial entry and includes an inner and outer membrane. The maintenance of lipid asymmetry (Mla) system is the main homeostatic mechanism by which Gram-negative bacteria maintain outer membrane asymmetry. Loss of the Mla system in A. baumannii results in attenuated virulence and increased susceptibility to membrane stressors and some antibiotics. We recently reported two strain variants of the A. baumannii type strain ATCC 17978: 17978VU and 17978UN. Here, ∆mlaF mutants in the two ATCC 17978 strains display different phenotypes for membrane stress resistance, antibiotic resistance, and pathogenicity in a murine pneumonia model. Although allele differences in obgE were previously reported to synergize with ∆mlaF to affect growth and stringent response, obgE alleles do not affect membrane stress resistance. Instead, a single-nucleotide polymorphism (SNP) in the essential gene encoding undecaprenyl pyrophosphate (Und-PP) synthase, uppS, results in decreased enzymatic rate and decrease in total Und-P levels in 17978UN compared to 17978VU. The UppSUN variant synergizes with ∆mlaF to reduce capsule and lipooligosaccharide (LOS) levels, increase susceptibility to membrane stress and antibiotics, and reduce persistence in a mouse lung infection. Und-P is a lipid glycan carrier required for the biosynthesis of A. baumannii capsule, cell wall, and glycoproteins. These findings uncover synergy between Und-P and the Mla system in maintaining the A. baumannii cell envelope and antibiotic resistance. IMPORTANCE Acinetobacter baumannii is a critical threat to global public health due to its multidrug resistance and persistence in hospital settings. Therefore, novel therapeutic approaches are urgently needed. We report that a defective undecaprenyl pyrophosphate synthase (UppS) paired with a perturbed Mla system leads to synthetically sick cells that are more susceptible to clinically relevant antibiotics and show reduced virulence in a lung infection model. These results suggest that targeting UppS or undecaprenyl species and the Mla system may resensitize A. baumannii to antibiotics in combination therapies. This work uncovers a previously unknown synergistic relationship in cellular envelope homeostasis that could be leveraged for use in combination therapy against A. baumannii.


Figure S1 .
Figure S1.Dilution spotting of 17978UN and 17978VU wild-type and DmlaF strains 17978VU strains with obgE, lptD, and uppS alleles from 17978UN, and PCR on the ATCC17978 stock.(A) Overnight cultures were serially diluted in PBS before plating on an LB agar plate.Image is representative of 5 biological replicates from 2 independent experiments.(B) Overnight cultures were serially diluted in PBS before plating on an LB agar plate with 0.01% SDS + 0.25 mM EDTA.Image is representative of 5 biological replicates from 2 independent experiments.(C) Colony PCR on single colonies from ATCC 17978 (2021) stock using primers that distinguish the uppS allele between 17978VU and 17978UN.n = 46 with 43 isolates positive for uppS UN , 2 isolates positive for uppS VU , and one indeterminant.

Figure S2 .
Figure S2.Comparison of UppS VU and UppS UN protein secondary structure and transcript abundance (A-B) Purified UppS VU and UppS UN were subjected to circular dichroism analysis with a range of 190 nm to 210 nm.N=1.(C) Transcript abundance of uppS in 17978VU wild-type compared to 17978UN wild-type.Data are mean ± SEM, n=3.Not significant by one sample T test.

Figure S3 .
Figure S3.Dilution spotting of 17978UN wild-type, DmlaF, and DmlaF suppressor strains.Overnight cultures were serially diluted in PBS before plating on an LB agar plate with or without 0.01% SDS + 0.125 mM EDTA for overnight incubation.Experiments were repeated two independent times (total n=4) with similar results.Figure S4.17978VU and 17978UN capsule, peptidoglycan, and LOS staining.(A) The peptidoglycan of 17978VU and 17978UN wild-type, DmlaF, and DmlaF with the opposite uppS allele was stained with NADA-green (3-[(7-Nitro-2,1,3-benzoxadiazol-4yl)amino]-D-alanine hydrochloride).Scale bar = 5 µm.Images are representative of two biological replicates from one experiment.(B) 17978VU and 17978UN wild-type and DmlaF strains were subjected to Maneval's capsule stain.Scale bar = 5 µm.Experiments were repeated three independent times (total n=5) with similar results.(C) Raw image of Alcian blue stained capsular polysaccharide.Experiments were repeated three independent times (total n=7) with similar results.(D) Raw image of silver-stained LOS gel.Experiments were repeated three independent times (total n=7) with similar results.

Figure S4 .
Figure S3.Dilution spotting of 17978UN wild-type, DmlaF, and DmlaF suppressor strains.Overnight cultures were serially diluted in PBS before plating on an LB agar plate with or without 0.01% SDS + 0.125 mM EDTA for overnight incubation.Experiments were repeated two independent times (total n=4) with similar results.Figure S4.17978VU and 17978UN capsule, peptidoglycan, and LOS staining.(A) The peptidoglycan of 17978VU and 17978UN wild-type, DmlaF, and DmlaF with the opposite uppS allele was stained with NADA-green (3-[(7-Nitro-2,1,3-benzoxadiazol-4yl)amino]-D-alanine hydrochloride).Scale bar = 5 µm.Images are representative of two biological replicates from one experiment.(B) 17978VU and 17978UN wild-type and DmlaF strains were subjected to Maneval's capsule stain.Scale bar = 5 µm.Experiments were repeated three independent times (total n=5) with similar results.(C) Raw image of Alcian blue stained capsular polysaccharide.Experiments were repeated three independent times (total n=7) with similar results.(D) Raw image of silver-stained LOS gel.Experiments were repeated three independent times (total n=7) with similar results.

Figure S5 .
Figure S5.Antibiotic susceptibility, lysozyme sensitivity, and bacterial burdens from a lung infection model.(A-B) Antimicrobial susceptibility of A. baumannii ATCC 17978VU and 17978UN wild-type and mutant strains was determined by a disk-diffusion assay and measuring the zone of clearance.Data are representative of 3 experiments.n=3, data are mean ± SEM.Significance is by oneway ANOVA with Tukey's multiple comparisons.(C) Growth curve in LB with and without lysozyme at 1 mg/mL.n=3, data are means +/-SEM.(D-G) Bacterial CFU were enumerated 48 h after intranasal inoculation.n=5, medians are shown, and significance was determined by a Mann-Whitney test, * p < 0.05