Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Feb 5;10(1):e02056-18.
doi: 10.1128/mBio.02056-18.

Identification of Novel Acinetobacter baumannii Host Fatty Acid Stress Adaptation Strategies

Jhih-Hang Jiang  1 , Karl A Hassan  2 , Stephanie L Begg  3   4 , Thusitha W T Rupasinghe  5 , Varsha Naidu  6 , Victoria G Pederick  3 , Marjan Khorvash  3 , Jonathan J Whittall  3   7 , James C Paton  3 , Ian T Paulsen  6 , Christopher A McDevitt  4 , Anton Y Peleg  1   8 , Bart A Eijkelkamp  9
Affiliations

Identification of Novel Acinetobacter baumannii Host Fatty Acid Stress Adaptation Strategies

Jhih-Hang Jiang et al. mBio. .

Abstract

Free fatty acids hold important immune-modulatory roles during infection. However, the host's long-chain polyunsaturated fatty acids, not commonly found in the membranes of bacterial pathogens, also have significant broad-spectrum antibacterial potential. Of these, the omega-6 fatty acid arachidonic acid (AA) and the omega-3 fatty acid decosahexaenoic acid (DHA) are highly abundant; hence, we investigated their effects on the multidrug-resistant human pathogen Acinetobacter baumannii Our analyses reveal that AA and DHA incorporate into the A. baumannii bacterial membrane and impact bacterial fitness and membrane integrity, with DHA having a more pronounced effect. Through transcriptional profiling and mutant analyses, we show that the A. baumannii β-oxidation pathway plays a protective role against AA and DHA, by limiting their incorporation into the phospholipids of the bacterial membrane. Furthermore, our study identified a second bacterial membrane protection system mediated by the AdeIJK efflux system, which modulates the lipid content of the membrane via direct efflux of lipids other than AA and DHA, thereby providing a novel function for this major efflux system in A. baumannii This is the first study to examine the antimicrobial effects of host fatty acids on A. baumannii and highlights the potential of AA and DHA to protect against A. baumannii infections.IMPORTANCE A shift in the Western diet since the industrial revolution has resulted in a dramatic increase in the consumption of omega-6 fatty acids, with a concurrent decrease in the consumption of omega-3 fatty acids. This decrease in omega-3 fatty acid consumption has been associated with significant disease burden, including increased susceptibility to infectious diseases. Here we provide evidence that DHA, an omega-3 fatty acid, has superior antimicrobial effects upon the highly drug-resistant pathogen Acinetobacter baumannii, thereby providing insights into one of the potential health benefits of omega-3 fatty acids. The identification and characterization of two novel bacterial membrane protective mechanisms against host fatty acids provide important insights into A. baumannii adaptation during disease. Furthermore, we describe a novel role for the major multidrug efflux system AdeIJK in A. baumannii membrane maintenance and lipid transport. This core function, beyond drug efflux, increases the appeal of AdeIJK as a therapeutic target.

Keywords: AdeIJK; RND efflux; antimicrobial host lipids; free fatty acids; lipidomics; β-oxidation.

PubMed Disclaimer

Figures

FIG 1
FIG 1
(A) The effects of long-chain polyunsaturated fatty acids (LC-PUFAs) on A. baumannii growth were determined by growing A. baumannii AB5075_UW in Luria-Bertani (LB) broth without LC-PUFA stress or with arachidonic acid (AA) or docosahexaenoic acid (DHA). The AA or DHA stress was quantified by comparing the 50% effective concentration (EC50), which was calculated using Prism 8 (GraphPad) from the optical density at 600 nm (OD600) measurements taken every 30 min, as per previous work (20). The EC50 is the time (minutes) when cultures have reached 50% of the maximum cell density, thereby representing mid-log growth. (B) The cell-associated omega-6 fatty acids (AA, GLA, and total n-6) and omega-3 fatty acids (DHA, EPA, and total n-3) were examined by gas chromatography following growth of strain AB5075_UW in the presence of 125 μM AA- or DHA-supplemented LB, respectively, using routine methods (19, 20). (C) The modification of exogenous fatty acids into phospholipids, phosphatidylethanolamine (PE), and phosphatidylglycerol (PG), was quantified in AB5075_UW cells using liquid chromatography-mass spectrometry (LC-MS) and underwent further species verification by tandem MS (MS/MS) following published protocols (19, 21). (D) The membrane permeability of AB5075_UW and adeJ121::T26 cells was examined by exposing the cells to 5 μM Sytox for 5 min, followed by extensive washing and analysis on a PHERAstar spectrophotometer at excitation 485/emission 520 (BMG Labtech). (E) The effects of 125 μM AA or DHA on A. baumannii AB5075_UW and the fadB and fadA Tn insertion mutant derivative fadB167::T26 and fadA140::T26 strains (14), respectively. The growth delay (in minutes) was calculated by comparing the EC50s. (F) The conversion of exogenous DHA into cell-associated PE or PG phospholipids in AB5075_UW or fadB167::T26 cells as determined by LC-MS. For all panels, the results are the mean ± standard error of the mean (SEM) from at least biological triplicates. Statistical analyses were performed using a one-way analysis of variance (ANOVA) (A, C, D, and E) or a Student's t test (B and F). ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
FIG 2
FIG 2
(A) Transcription levels of genes that encode putative A. baumannii resistance nodulation cell division (RND) efflux pumps in AB5075_UW cells grown in LB media with or without AA or DHA were examined by quantitative reverse transcription-PCR (qRT-PCR), using the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as a reference gene, and data were corrected against untreated cells as per previous analyses (19, 20). AB5075_UW cultures were grown to an optical density at 600 nm (OD600) of 0.5 and were exposed to 125 μM AA or DHA for 30 min prior to processing for RNA isolation. Oligonucleotide sequences can be found in Table S1 in the supplemental material. The results are the mean ± SEM from at least biological triplicates. Statistical analyses were performed using a one-sample t test (**, P < 0.01; ***, P < 0.001). (B) The effect of 125 μM AA or DHA on adeJ-inactivated A. baumannii AB5075_UW (adeJ121::T26) and A. baylyi ADP1 (ΔadeJ) mutants (14, 15). The growth delay (in minutes) was calculated by comparing the 50% effective concentration (EC50) (20). The results are the mean ± SEM from at least biological triplicates. Statistical analyses were performed using a one-way ANOVA (*, P < 0.05; ***, P < 0.001; ****, P < 0.0001). (C) Endogenous phosphatidylglycerol (PG) and cardiolipin (CL) species that were significantly affected by mutation of adeJ in strain AB5075_UW (adeJ121::T26). The results are the mean ± SEM from at least biological triplicates. Statistical analyses were performed using a two-way ANOVA on all PG or CL species identified by LC-MS, which can be found in Table S2 in the supplemental material (***, P < 0.001; ****, P < 0.0001). (D) The membrane permeability of AB5075_UW and adeJ121::T26 cells was examined by exposing the cells to 5 μM Sytox for 5 min, followed by extensive washing and analysis on a PHERAstar spectrophotometer at excitation 485/emission 520 (BMG Labtech). The results are the mean ± SEM from at least biological triplicates. Statistical analyses were performed using a Student's t test (*, P < 0.05). (E) The fatty acids in the media were analyzed by gas chromatography following growth of the AB5075_UW or adeJ121::T26 strains. The baseline represents media in which no bacteria were grown. Samples were first centrifuged at low speed (300 × g for 5 min), with the supernatant then passed through a 0.4 μm-pore filter and insoluble material subsequently removed by ultracentrifugation (100,000 × g for 1 h). The results are the mean ± SEM from at least biological triplicates. Statistical analyses were performed using a Student's t test (*, P < 0.05). (F) The ability of AB5075_UW or adeJ121::T26 cells to form biofilms was assessed after 24 h of static growth in a polystyrene 96-well plate. Cells were stained using 0.1% crystal violet, with the absorbance determined at 590 nm (21, 22). The results are the mean ± SEM from at least biological triplicates. Statistical analyses were performed using a Student's t test (*, P < 0.05). (G) Surface motility of AB5075_UW or adeJ121::T26 cells was examined by inoculating the center of a semisolid LB plate (0.25% agar) and incubating for 18 h (22). The aberrant motility phenotype was observed in all 3 independent experiments (replicates 1, 2, and 3).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Anes E, Jordao L. 2008. Trick-or-treat: dietary lipids and host resistance to infectious disease. Mini Rev Med Chem 8:1452–1458. - PubMed
    1. Simopoulos AP. 2011. Evolutionary aspects of diet: the omega-6/omega-3 ratio and the brain. Mol Neurobiol 44:203–215. doi:10.1007/s12035-010-8162-0. - DOI - PubMed
    1. Desbois AP, Smith VJ. 2010. Antibacterial free fatty acids: activities, mechanisms of action and biotechnological potential. Appl Microbiol Biotechnol 85:1629–1642. doi:10.1007/s00253-009-2355-3. - DOI - PubMed
    1. Churchward CP, Alany RG, Snyder LAS. 2018. Alternative antimicrobials: the properties of fatty acids and monoglycerides. Crit Rev Microbiol 44:561–570. doi:10.1080/1040841X.2018.1467875. - DOI - PubMed
    1. Harding CM, Hennon SW, Feldman MF. 2018. Uncovering the mechanisms of Acinetobacter baumannii virulence. Nat Rev Microbiol 16:91–102. doi:10.1038/nrmicro.2017.148. - DOI - PMC - PubMed

Publication types

MeSH terms

Substances