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An inhibitory mechanism of AasS, an exogenous fatty acid scavenger: Implications for re-sensitization of FAS II antimicrobials [1]

['Haomin Huang', 'Key Laboratory Of Multiple Organ Failure', 'Ministry Of Education', 'Departments Of Microbiology', 'General Intensive Care Unit Of The Second Affiliated Hospital', 'Zhejiang University School Of Medicine', 'Hangzhou', 'Zhejiang', 'Shenghai Chang', 'Center Of Cryo-Electron Microscopy']

Date: 2024-07

Antimicrobial resistance is an ongoing “one health” challenge of global concern. The acyl-ACP synthetase (termed AasS) of the zoonotic pathogen Vibrio harveyi recycles exogenous fatty acid (eFA), bypassing the requirement of type II fatty acid synthesis (FAS II), a druggable pathway. A growing body of bacterial AasS-type isoenzymes compromises the clinical efficacy of FAS II-directed antimicrobials, like cerulenin. Very recently, an acyl adenylate mimic, C10-AMS, was proposed as a lead compound against AasS activity. However, the underlying mechanism remains poorly understood. Here we present two high-resolution cryo-EM structures of AasS liganded with C10-AMS inhibitor (2.33 Å) and C10-AMP intermediate (2.19 Å) in addition to its apo form (2.53 Å). Apart from our measurements for C10-AMS’ Ki value of around 0.6 μM, structural and functional analyses explained how this inhibitor interacts with AasS enzyme. Unlike an open state of AasS, ready for C10-AMP formation, a closed conformation is trapped by the C10-AMS inhibitor. Tight binding of C10-AMS blocks fatty acyl substrate entry, and therefore inhibits AasS action. Additionally, this intermediate analog C10-AMS appears to be a mixed-type AasS inhibitor. In summary, our results provide proof of principle that inhibiting salvage of eFA by AasS reverses the FAS II bypass. This facilitates the development of next-generation of anti-bacterial therapeutics, esp. the dual therapy consisting of C10-AMS scaffold derivatives combined with certain FAS II inhibitors.

An AasS-aided eFA recycling compromises the clinical efficacy of FAS II-directed antimicrobials. The C10-AMS compound is an effective inhibitor for AasS with the Ki value of ~0.6 μM. Efficient binding of AasS by C10-AMS blocks the entry of fatty acyl substrates of various length. The C10-AMS inhibitor mimics an acyl-AMP adenylate to trap AasS in a closed conformation rather than an open state. Collectively, inhibiting a bacterial eFA scavenger AasS enables the re-sensitization of FAS II-targeted antimicrobials. This provides proof of concept that a combination of C10-AMS scaffold derivatives with appropriate FAS II inhibitors constitutes a next-generation anti-virulence biotherapy.

Data Availability: The obtained cryo-EM density maps and the data of the structures were deposited to the Electron Microscopy Data Bank (EMDB) and Protein Data Bank (PDB) with the assigned accession numbers: EMD-35008 and 8HSY for Apo AasS; EMD-36725 and 8JYL for AasS complexed with its C10-AMS inhibitor; EMD-36731 and 8JYU for AasS liganded with an adenylate intermediate, C10-AMP. All data needed to evaluate the conclusions in this paper are present in the paper and/or the Supplementary Materials .

The annual V. harveyi infection leads to a substantial economic loss in aquacultural production. Combined with certain FAS II antimicrobials, development of AasS inhibitors, opens perspectives of a dual-therapy against marine pathogens. Recently, the lead compound, 5’-O-(N-decanylsulfamoyl) adenosine (termed as C10-AMS) was found to efficiently impair the activities of AasS [ 59 ] and its paralogs like AasN/AasC ( Fig 1A ) [ 60 ]. Despite that it assumed to mimic the decanoyl-adenylate intermediate (abbreviated as C10-AMP), how the broad inhibitor C10-AMS antagonizes AasS-based eFA recycling is largely unclear. We solved two high-resolution cryo-EM structures of AasS liganded with C10-AMS inhibitor (2.33 Å) and C10-AMP intermediate (2.19 Å) in addition to its apo form (2.53 Å). Structural and biochemical comparison explains how the inhibition of AasS proceeds by the mixed-type inhibitor C10-AMS. Altogether, this study constitutes a proof of concept for inhibiting an eFA scavenger AasS (and/or its isoform AasN/AasC) to overcome bacterial bypass of anti-FAS II antimicrobials.

The Aas members belong to a ubiquitous group of acyl-activating enzyme (AAE), also called adenylate-forming enzyme. The in vitro Aas activity is originally traced to the E. coli bifunctional 2-acyl-glycerolphosphoethnomine (2-acyl-GPE) acyltransferase/acyl-ACP synthetase [ 30 ]. The physiological role of the E. coli Aas enzyme denotes the ligation of an activated fatty acyl chain from acyl-ACP intermediate to the 1-position of Lyso-phospholipid (LysoPL), a byproduct of lipoprotein synthesis [ 32 , 43 ]. Because the resultant acyl-ACP intermediate is tightly bound by the Aas enzyme, it cannot access the membrane phospholipid/lipopolysaccharide (LPS)-lipid A pathway [ 12 , 24 ]. Indeed, the interfacial Aas of E. coli displays an in vitro ‘artifact’ salt-dependent activity of synthesizing acyl-ACP thioester [ 30 , 32 ]. In contrast, the marine bioluminescent bacterium V. harveyi AasS (VhAasS, thereafter called AasS) is a cytoplasmic version of acyl-ACP synthetase [ 44 – 46 ]. Notably, this soluble AasS channels a pool of eFA nutrients to build bacterial phospholipids as well as LPS-lipid A ( Fig 1A and 1B ) [ 47 , 48 ]. This paradigmatic VhAasS is featured by its substrate promiscuity and is leveraged as a versatile tool in synthetic biology [ 49 – 51 ]. So far, the toolbox of Aas enzymes contains four additional members capable of eFA assimilation. Namely, they include (i) SynAas of Cyanobacteria [ 52 , 53 ] that is partially equivalent to AAE15, a cousin of the plant Arabidopsis [ 54 ]; (ii) AasC of the sexually-transmitted, obligate intracellular parasite Chlamydia trachomatis [ 33 ]; (iii) AasN exclusively in the human pathogens Neisseria meningitis and N. gonorrhoeae [ 55 ]; and (iv) two isoforms (AfAas1 for C12:0 & AfAas2 for C18:1) arising from Alistipes finegoldii, a representative resident in human gut microbiomes [ 56 ]. Very recently, an extensive cryo-EM study revealed that unlike all the other AAE members with known structures (e.g., ttLC-FACS dimer [ 57 ]), AasS acts as ring-shape hexamer, and adopts a ‘conformational rearrangement’ pattern to execute its catalysis cycle [ 58 ]. The gating role of W230 is functionally defined in the context of AasS action, allowing bacterial salvage of eFA nutrients [ 58 ]. This represents the first structural landscape for acyl-ACP synthetase of versatility. It is an open question to ask if the other pathogen cousins of AasS (such as AasC [ 33 ] and AasN [ 55 ]) also feature a similar structural architecture.

A. eFA salvage coupled with FAS II pathway contributes to membrane phospholipid synthesis. Three mechanisms for eFA scavenging were included here, namely (i) FadD acyl-CoA ligase [ 29 ]; (ii) FakA/B system composed of the FakA kinase component and the FakB fatty acid-binding subunit [ 25 , 28 ]; (iii) Acyl-ACP synthetase, AasS [ 47 , 48 ]. Cerulenin denoted the FabF inhibitor targeting a FAS II pathway. B. The combination of PlsB/Y-PlsC and PlsX/Y-PlsC represented two alternative routes for the synthesis of membrane phospholipids. The pathway begins with G3P as a recipient, and extends using different primer substrates (acyl-CoA/acyl-ACP for PlsB/PlsC vs acyl-Pi for PlsY/X, and acyl-ACP for PlsC). Abbreviations: C10-AMS, 5’-O-(N-decanylsulfamoyl) adenosine; FadD, Acyl-CoA ligase; FakA/B, Fatty acid kinase A in complex with fatty acid-biding subunit B; AasS, acyl-ACP synthetase; AccABCD, Acetyl-CoA carboxylase composed of four subunits (namely (i) AccA, α-subunit of carboxyltransferase; (ii) AccB, biotin carboxyl carrier protein (BCCP); (iii) AccC, biotin carboxylase (BC); and (iv) AccD, β-subunit of carboxyltransferase); FAS II, Type II fatty acid synthesis pathway; FabI, Enoyl-ACP reductase; FabF, β-ketoacyl-ACP synthase II; FabG, Ketoacyl-ACP reductase; FabZ, 3-hydroxyacyl-ACP dehydratase; G3P, Glycerol-3-phosphate; LPA, Lyso-phosphatidic acid; PA, Phosphatidic acid; acyl-Pi, acyl phosphate; PlsB, G3P acyltransferase; PlsY, Acyl-Pi-dependent G3P acyltransferase; PlsX, Phosphate: acyl-ACP transacylase; PlsC, LPA acyltransferase; G — , Gram-negative bacterium; G + , Gram-positive bacterium.

Fatty acids (FA) are a group of energetically-expensive building blocks for membrane phospholipid synthesis in the tree of life. Unlike eukaryotic cells with Type I Fatty Acid Synthesis (FAS I) machinery, a giant multienzyme complex, diverse bacterial species exploit the type Il FAS system (FAS II), consisting of multiple discrete subunits [ 11 ]. The essentiality of the two FAS II metabolites [i.e., β-hydroxyl FA in Gram-negative bacterium [ 12 ], and pentadecanoic acid, a branched-chain FA in Gram-positive pathogen [ 13 ]] enables the possibility of the FAS II machinery as a druggable pathway. As expected, FAS II-directed mining of natural products led to a repertoire of attractive lead compounds. Namely, they include, but not limited to (i) cerulenin [ 14 – 16 ] and platensimycin [ 17 ], two selective FabB/F inhibitor ( Fig 1A ); (ii) platencin, a natural product with dual targets FabH and FabB/F [ 18 ]; and (iii) an arsenal of FabI-targeted antimicrobials (i.e., triclosan, a widely-used biocide [ 19 – 21 ]; isoniazid, the front-line anti-TB drug [ 22 ]; and Staphylococcus-specific AFN1252 [ 23 ]). Apart from the FAS II pathway, the majority of bacterial pathogens develop diverse mechanisms to recycle environment/exogenous FA (eFA) [ 12 , 24 ]. Unlike Gram-positive pathogens (Staphylococcus [ 25 , 26 ], and Streptococcus [ 27 , 28 ]) having fatty acid kinase FakAB systems, Gram-negative bacterium relies on either acyl-CoA ligase FadD [ 29 ] or acyl-ACP synthetase (Aas) exemplified with the E. coli bifunctional Aas [ 30 – 32 ] and its relic AasC of Chlamydia [ 33 ]. This raises the possibility that an eFA salvage compromises the effectivity of FAS II inhibitors by replacing de novo synthesized FAs ( Fig 1 ) [ 34 – 36 ]. In fact, S. aureus liberates host FAs from abundant low-density lipoproteins (LDL) [ 37 ], and the assimilated eFA favors staphylococcal anti-FAS II adaptation at the infection site [ 13 , 38 ]. In addition, staphylococcal FAS II bypass is in part, if not all, traced to the polymorphism of fabD [ 39 ] and acc [ 40 ], two essential genes that initiate a FAS II pathway [ 41 ]. A growing body of evidence explains the failure of FAS II antibiotic-based anti-MRSA therapy in a mouse bacteremia model [ 38 – 40 ]. Combined with (p)ppGpp inducer of stringent response, FAS II antimicrobials can block MRSA outgrowth, offering a synergistic bi-therapy strategy [ 42 ]. Because staphylococcal anti-FAS II bypass is engendered via an eFA scavenging by the FakAB system [ 37 , 38 ], it is in rational to formulate an alternative bi-therapeutics by using the anti-FAS II drug AFN1252 mixed with some FakAB inhibitor. Finally, we are eager to find out whether or not an Aas enzyme mimicking FakAB machinery exclusively in G-positive pathogens can confer G-negative bacterial adaptation to FAS II-directed antimicrobials.

Antimicrobial resistance (AMR) is recognized by the World Health Organization (WHO) as a top 10 challenge to global health and sustainable development, of which the main driver refers to misuse and/or overuse of antimicrobials. WHO declared that AMR-caused global deaths are estimated to rise from ~0.7 million in 2014 [ 1 ], to ~1.27 million, in 2019 [ 2 ]. Thereby, Jim O’Neil predicted that annual deaths might reach 10 million by 2050 [ 1 , 3 ]. Because of limited efforts to contain AMR spread during the COVID-19 period (from 2019 to 2022), it is expected to worsen partially, which forms a cross-cutting, silent pandemic approaching alarming proportions [ 4 ]. As the top priority member of ‘ESKAPE’ pathogens, the resistant Escherichia coli (E. coli) primarily causes life-threatening infections that might pose extensive health/economic impacts over the next decade [ 5 ]. The close relative of E. coli, Vibrio harveyi (V. harveyi) is an opportunistic pathogen of shrimps and invertebrates in marine aquacultures [ 6 ]. Multiple drug resistances observed for certain V. harveyi isolates are due to an ever-increasing number of acquired AMR determinants, like tetB and qnrA [ 7 – 9 ]. To tackle AMR crisis, a unified ‘one health’ approach is prioritized, comprising multiple sectors of humans, domestic/wild animals, plants and the wild environments (i.e., ecosystems) [ 10 ].

Results and discussion

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[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1012376

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