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The Staphylococcus aureus FASII bypass escape route from FASII inhibitors
Accepted Manuscript The Staphylococcus aureus FASII bypass escape route from FASII inhibitors Claire Morvan, David Halpern, Gérald Kénanian, Amit Pathania, Jamila MondoloniAnba, Gilles Lamberet, Alexandra Gruss, Karine Gloux PII:
S0300-9084(17)30175-X
DOI:
10.1016/j.biochi.2017.07.004
Reference:
BIOCHI 5235
To appear in:
Biochimie
Received Date: 5 April 2017 Revised Date:
0300-9084 0300-9084
Accepted Date: 13 July 2017
Please cite this article as: C. Morvan, D. Halpern, G. Kénanian, A. Pathania, J. Mondoloni-Anba, G. Lamberet, A. Gruss, K. Gloux, The Staphylococcus aureus FASII bypass escape route from FASII inhibitors, Biochimie (2017), doi: 10.1016/j.biochi.2017.07.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Abstract
2 Antimicrobials targeting the fatty acid synthesis (FASII) pathway are being developed as
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alternative treatments for bacterial infections. Emergence of resistance to FASII inhibitors was
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mainly considered as a consequence of mutations in the FASII target genes. However, an
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alternative and efficient anti-FASII resistance strategy, called here FASII bypass, was
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uncovered. Bacteria that bypass FASII make use of exogenous fatty acids to build their
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membranes, and thus dispense with the need for FASII. This strategy is used by numerous
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Gram-positive low GC% bacteria, including streptococci, enterococci, and staphylococci. Some
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bacteria repress FASII genes once fatty acids are available, and “constitutively” shift to FASII
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bypass. Others, such as the major pathogen Staphylococcus aureus, may undergo high
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frequency mutations that favor FASII bypass. This capacity is particularly relevant during
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infection, as the host supplies the fatty acids needed for bacteria to bypass FASII and thus
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become resistant to FASII inhibitors. Screenings for anti-FASII resistance in the presence of
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exogenous fatty acids confirmed that FASII bypass, anti-FASII-resistant strains exist among
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clinical and veterinary isolates. Polymorphisms in S. aureus FASII initiation enzymes favor FASII
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bypass, possibly by increasing availability of acyl-carrier protein, a required intermediate. Here
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we review FASII bypass and consequences in light of proposed uses of anti-FASII to treat
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infections, with a focus on FASII bypass in S. aureus.
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The Staphylococcus aureus FASII bypass escape route from FASII
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inhibitors
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Claire Morvan1,2, David Halpern1, Gérald Kénanian1, Amit Pathania1, Jamila Mondoloni-
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Anba1, Gilles Lamberet1, Alexandra Gruss 1a, Karine Gloux 1a
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cedex, France. 2 Present address : Institute for Integrative Biology of the Cell (I2BC), CEA,
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CNRS, Université Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette, France.
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INRA –UMR1319- MICALIS AgroParisTech. Domaine de Vilvert, 78352 Jouy-en-Josas,
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[email protected]
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[email protected]
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corresponding authors:
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Abstract
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Antimicrobials targeting the fatty acid synthesis (FASII) pathway are being developed as
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alternative treatments for bacterial infections. Emergence of resistance to FASII inhibitors
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was mainly considered as a consequence of mutations in the FASII target genes. However,
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an alternative and efficient anti-FASII resistance strategy, called here FASII bypass, was
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uncovered. Bacteria that bypass FASII make use of exogenous fatty acids to build their
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membranes, and thus dispense with the need for FASII. This strategy is used by numerous
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Gram-positive low GC% bacteria, including streptococci, enterococci, and staphylococci.
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Some bacteria repress FASII genes once fatty acids are available, and “constitutively” shift to
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FASII bypass. Others, such as the major pathogen Staphylococcus aureus, may undergo
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high frequency mutations that favor FASII bypass. This capacity is particularly relevant
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during infection, as the host supplies the fatty acids needed for bacteria to bypass FASII and
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thus become resistant to FASII inhibitors. Screenings for anti-FASII resistance in the
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presence of exogenous fatty acids confirmed that FASII bypass, anti-FASII-resistant strains
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exist among clinical and veterinary isolates. Polymorphisms in S. aureus FASII initiation
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enzymes favor FASII bypass, possibly by increasing availability of acyl-carrier protein, a
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required intermediate. Here we review FASII bypass and consequences in light of proposed
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uses of anti-FASII to treat infections, with a focus on FASII bypass in S. aureus.
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Key words
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Triclosan, AFN-1252, fatty acid auxotrophy, clinical isolates, antibiotics, cross-resistance
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1. Numerous Gram-positive bacteria can bypass the FASII pathway by using
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exogenous fatty acids
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52 Steps of the FASII biosynthesis pathway have been extensively studied (see references [1-4]
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for in-depth reviews); “FASII bypass” is defined here as the capacity of numerous bacteria to
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use exogenous fatty acids, rather than de novo synthesis to constitute their membranes
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(schematized in Fig. 1). At one extreme, Lactobacillus bulgaricus lacks a complete FASII
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pathway, and relies on exogenous fatty acids for growth [6]. Numerous food-fermenting
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bacteria and opportunist pathogens (e.g., Lactobacillus plantarum and Streptococcus
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agalactiae respectively) efficiently incorporate fatty acids directly in membranes, and may
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thereby adapt their membrane phospholipid profiles to their lipid environment [5, 7].
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Interestingly, in S. agalactiae FASII gene expression is repressed in the presence of
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exogenous fatty acids and the profile of fatty acids incorporated into phospholipids mimics
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that of environmental fatty acids (Fig. 1; [5]). The existence of FASII bypass mechanisms
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makes sense: using full-length exogenous fatty acids directly for phospholipid synthesis
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economizes on energy expenditure during growth. Other pathogenic species, including
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enterococci, other streptococci, and staphylococci can use exogenous fatty acids to
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compensate a FASII block triggered by fatty acid repression or by a FASII inhibitor [5, 8-11].
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2. Could FASII inhibitors be efficient antibiotics despite FASII bypass in Gram-positive
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bacteria?
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Development of FASII inhibitors. A FASII inhibitor, isoniazid, has long been used to treat
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mycobacterial infections. Mycobacteria synthesize and require specific and very long chain
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fatty acids that cannot be supplied by the environment, explaining the effectiveness of such
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drugs [12]. The FASII pathway has more recently been a privileged “hot” target for
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antimicrobial development against the low GC percent Firmicutes. In various studies, strains
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of listeria, streptococci, staphylococci, and enterococci, were reported to be inhibited by 3
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various anti-FASII [13-22]. Most reported anti-FASII drugs (reviewed in Table1) target
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FabB/F and FabI, which catalyze essential steps in the iterative FASII elongation cycle. It is
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notable that bacteria affected by FASII inhibitors may be limited to those expressing the
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targeted FASII enzyme. For instance, streptococci encode an enoyl reductase distinct from
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FabI, making them naturally resistant to anti-FabI antimicrobials. This feature may be useful
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when developing narrow range antibiotics, and concomitantly sparing non-pathogens present
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in the same ecosystem.
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The first, and by far most widely used anti-FASII is triclosan, which has been
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extensively employed as a biocide, e.g., in hygiene, household, and hospital products [23,
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24]. At low concentrations, triclosan targets FabI [25]. However, at higher concentrations
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(e.g., 0.2 to 1% in soaps, creams, sutures), triclosan provokes membrane permeability and
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generalized non-specific membrane damage [26] and was linked to host-related damage
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(leading to its recent removal from hygiene and cosmetic products), and to widespread
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resistance (see 3.) [27, 28]. These unwanted properties stimulated the development of new
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FabI inhibitors with higher specificity, which eliminate the side effects observed with triclosan
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[16, 20, 29-31]. At least one such drug, AFN-1252 (renamed Debio 1452), is currently in
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clinical trials for treatment of S. aureus infections ([31] see 3.2.).
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Limitations of FASII inhibitors due to FASII bypass. Some bacteria use fatty acids,
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which are abundant in the host, to bypass a block in FASII synthesis. As a formal proof, a S.
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agalactiae strain deleted for its FASII biosynthesis genes was shown to retain full virulence
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[5]. As a second example, an E. faecalis fatty acid auxotroph was identified as the agent
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responsible for an umbilical cord infection [32]. These results essentially ruled out the use of
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anti-FASII to treat infections due to streptococci and enterococci, while the status of
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staphylococci was still debated.
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3. The case of S. aureus and FASII inhibitors
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Until recently, clinical S. aureus resistance to FASII inhibitors was mainly attributed to
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compensatory mutations in their targets or their precursor enzymes. A brief summary of this
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clinically significant class of anti-FASII resistance is given prior to consideration of the FASII
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bypass strategy.
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S. aureus anti-FASII resistance by modifications of the antimicrobial target.
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Documented cases of resistance to the widely used biocide triclosan mapped to point
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mutations in the fabI gene or its promoter [28, 29, 33]. FabI mutations were also selected in
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the presence of other FabI inhibitors, AFN-1252 and MUT056399 [16, 34]. Interestingly, S.
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aureus triclosan resistance was discovered in several cases to be associated with horizontal
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transfer of a fabI allele from S. haemolyticus (sh-fabI) [28, 33, 35]. Such isolates were
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subsequently found to be frequent among triclosan-resistant hospital screenings [28, 35].
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Moreover, resistance to FabF inhibitors was found to be associated with mutations in FabH,
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possibly by increasing malonyl-ACP availability, a substrate shared by FabF and FabH [36].
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S. aureus anti-FASII resistance by FASII bypass. The role of S. aureus FASII bypass in
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response to FASII inhibition has been a point of controversy [5, 8, 10, 14, 37-39].
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Biochemical studies led to the belief that the main fatty acid synthesized by S. aureus,
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anteiso C15:0 (ai15), was indispensable for phospholipid synthesis and membrane fluidity
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[10, 37, 38]. Moreover, PlsC, the enzyme that mediates fatty acid incorporation at position 2
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of the glycerophosphate backbone is reportedly highly specific [10, 29, 38]. The first S.
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aureus shown to be able to bypass FASII were fatty acid auxotroph mutants mapping to
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accD, encoding an acetyl-CoA carboxylase subunit involved in FASII initiation [10]. An acc
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mutant grew poorly in the absence of ai15, and was noninfectious in a mouse model, leading
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authors to suggest that emergence of FASII bypass variants during treatment would not
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compromise anti-FASII efficacy [38].
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However, the capacity of S. aureus to incorporate exogenous fatty acids as a means of
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adapting to a FASII block turned out to be more common than reported. Our studies revealed
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that fatty acid supplementation favored high frequency mutations leading to FASII bypass
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(mutation frequency leading to anti-FASII resistance of 10-6 compared to 10-8 in the absence
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of fatty acids) [40]. A robust class of FASII bypass mutants mapped to another initiation 5
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enzyme, FabD (malonyl-CoA:ACP transacylase). Some fabD mutants displayed a gain of
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function phenotype, contrasting to previously identified acc auxotrophs. The strains are
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mutated in the active site of FabD and are able to synthesize their own fatty acids, yet they
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also grow well by incorporating different combinations of exogenous fatty acids when FASII
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is blocked by either triclosan (Fig. 2) or AFN-1252 [40]. One fabD mutation may have
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resulted from replication slippage [41] in a run of “A” repeats, conferring triclosan resistance
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that might readily “slip back” to restore a wild type phenotype when selection is lifted (data
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not shown). A FASII bypass event can be also generated by anti-FabF treatment. Resistance
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to the FabF inhibitor maps to mutations in FabH, a third enzyme needed for FASII initiation.
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Interestingly, poor growth of these strains is rescued by exogenous fatty acid addition [36].
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Selective pressure by FASII inhibitors thus leads to resistant S. aureus that use
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different fatty acid combinations to comprise their membranes. Membrane composition is
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therefore not stringently controlled, an observation that also extends to clinical strains [35].
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3.1. A model for FASII bypass in S. aureus
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Recently elucidated steps of FASII bypass in S. aureus led to the following model
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(Fig. 3). The first step in exogenous fatty acid incorporation is phosphorylation by fatty acid
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kinase complex FakA FakB1 FakB2 (to acyl-PO4) [37]. Phosphorylated fatty acids are
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substrates for PlsY, which attaches an acyl group to position 1 of the glycerol-phosphate
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backbone [1]. A FASII block changes the balance of metabolic intermediates, including a
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pileup of malonyl-ACP. However, certain FabD variants produce less malonyl-ACP, leaving
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more free ACP available. This leads to a key step in the model, in which PlsX, which
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converts acyl-ACP to acyl-PO4, reverses its enzymatic activity. Acyl-ACPs may then be
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elongated by FASII (if it is not blocked) or used to complete phospholipid synthesis by PlsC,
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which transfers acyl-ACP to glycerol phosphate at position 2 to form phosphatidic acid [42].
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We provided direct evidence for this FASII bypass model in S. aureus isolates carrying
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FabD196 polymorphisms, in which phospholipids comprise exclusively exogenous fatty acids
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when FASII is blocked [40]. Differential regulation of FASII bypass in S. aureus and other
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Gram-positive bacteria (that directly utilize exogenous fatty acids in all tested conditions) is
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being investigated.
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3.2. Anti FASII efficiency on S. aureus in vivo: What’s the verdict?
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Difficulties in comparing in vivo models. Numerous animal experiments examined the
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efficacy of experimental FASII inhibitors (mainly anti-FabI) against S. aureus infections [16,
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30, 31, 43-47]. However, protocols varied between studies, e.g., infection regime, bacterial
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inocula, drug concentrations compared to MIC (minimal inhibitory concentration), drug
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treatment regimens post-infection, and evaluation of inhibitor efficiency (summarized in
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Supplementary Tables 1 and 2). The non-uniformity of test systems poses a main problem
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for gaining a real assessment of drug efficacies. Furthermore, current methods used to
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detect surviving S. aureus following anti-FASII treatments may fail to screen for fatty acid
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auxotrophs.
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The case in favor of anti-FASII inhibitors: i- Tests in animal models gave positive
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results on the efficacy of anti-FASII drugs in treating S. aureus infections [16, 19, 46]. ii-
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Clinical trials using AFN-1252 reported promising results in reducing lesion size of S. aureus
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skin infections (Clinicaltrials.gov identifier: NCT02426918) [31]. These reports concluded on
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the effectiveness of the tested FASII inhibitors. iii- Numerous bacteria (and also humans)
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lack a FabI enzyme. A narrow spectrum antimicrobial is a desired characteristic, which
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allows infections to be treated with minimal detrimental effects on the host and its harmless
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microbiomes. The anti-FabI Debio 1452 was shown to have low impact on the main intestinal
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bacterial populations, thus making the oral drug route a possible option [58].
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The case against FASII inhibitors: i- Triclosan (anti-FabI) treatment was found to be
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associated with an increase in S. aureus colonization [59]. ii- In vitro-isolated fabD variants
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were shown to be infectious, and arose in response to different anti-FASII drugs [40]. iii- In 6
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cases, anti-FASII-resistance status of clinical isolates was correlated to the presence of fabD 7
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polymorphisms that favor FASII bypass [35, 40]. iv- A screening involving 700 clinical
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isolates revealed that 19% were triclosan-resistant, among which 1/3 were revealed only
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when fatty acids were present in screening medium [35]. v- Clinical fatty acid auxotrophs,
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which have a fatty acid growth requirement, were isolated in 2 out of 103 patient samples
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[35], and were also reported in a patient with septic arthritis [60].
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So what is the verdict on anti-FASII? There is evidence that these drugs are active
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against S. aureus in certain cases. Yet after anti-FASII treatment, resistant bacteria might
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persist and not be detected in the short term. The presence of FASII bypass variants in
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human and farm animal isolates raise questions on their efficient detection and on the long-
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term efficacy of FASII inhibitors in treating infections.
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3.3. Cross resistance of FASII bypass strains
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FASII bypass isolates are expectedly cross-resistant to all antimicrobials targeting FASII
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enzymes. Indeed, in S. aureus, triclosan-selected FASII bypass variants were resistant to
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AFN-1252, the FabI inhibitor in clinical trials, and platensimycin, a FabF inhibitor and
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antimicrobial candidate [21, 31, 40]. Both clinical and laboratory strains bearing the same
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FabD polymorphism (FabD196) were both triclosan and AFN-1252 resistant in medium
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containing fatty acids ([40], Fig. 4), and grew at concentrations of >500 fold the AFN-1252
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MIC [10] (data not shown). Thus, in addition to their frequent occurrence in clinical,
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veterinary, and laboratory settings, fatty acid auxotrophs and isolates that bypass FASII are
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all expectedly multi-anti-FASII-resistant.
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We screened S. aureus wild type and FASII bypass fabD mutants for altered
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sensitivities to antibiotics having targets unrelated to FASII (Table 2). In conditions where
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bacteria relied on exogenous fatty acids, fabD mutants showed increased tolerance to
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fosfomycin (inhibitor of cell wall synthesis enzyme MurA) and trimethoprim (inhibitor of
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nucleic acid synthesis enzyme dihydrofolate reductase). Changes in phospholipids due to
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altered fatty acids by FASII bypass may thus impact overall membrane composition and
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bacterial behavior including antibiotic resistance.
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Numerous Firmicutes including pathogens, incorporate exogenous fatty acids, which can
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compensate a FASII inhibition. The FASII pathway can be inhibited simply by exogenous
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fatty acid supplementation, or in some bacteria, with further addition of FASII inhibitors. In S.
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aureus, the essentiality of the major endogenous fatty acid ai15 has been in debate.
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However, the finding that mutants arising at high frequencies can replace endogenous fatty
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acid species with varied combinations of exogenous fatty acids casts doubts on this dogma.
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Thus staphylococci, like other Firmicutes, may have a higher membrane flexibility than
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previously recognized.
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S. aureus isolates that bypass FASII inhibition are already present among clinical and
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veterinary isolates, likely as a consequence of the widespread use of triclosan. We suggest
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that FASII bypass is one main strategy by which S. aureus overcomes FASII inhibitors in
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animals, environments with ubiquitous fatty acids. Experiments on animals and humans have
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supported the use of anti-FASII to treat S. aureus infections. However altered behavior of
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FASII bypass strains, such as small colony variant formation [60] and antibiotic resistance,
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may lead to cross antibiotic resistance, bacterial persistence and chronic disease.
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In view of these studies, we underline the importance of performing follow-up
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screenings prior to validating novel anti-FASII antimicrobials for treatment. Screenings for
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FASII bypass variants should be performed in conditions closer to those found in vivo, i.e.,
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that include fatty acids in assays [35]. FASII bypass variants that emerge after anti-FASII
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therapy may deserve further investigation to assess the risk of recurrence and chronic
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infection.
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Acknowledgements
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We thank all the members of the MicrobAdapt team for numerous stimulating discussions on
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FASII bypass. Research presented in this review received funding from the Agence
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Nationale de la Recherche (ANR) FattyBact project 525 ANR-132101. CM and GK were
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awarded stipends from the French Research Ministry. AP received a postdoctoral fellowship
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from the DIM-Malinf program, Conseil Régional Île de France (www.dim-malinf.org).
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59(5) (2015) 2583–2587.
443 444
inhibitor
with
potent
activity
against
S.
aureus
and
coagulase-negative
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ACCEPTED MANUSCRIPT selective antibiotic minimizes disturbance to the microbiome, Antimicrob. Agents
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Figure Legends
ACCEPTED MANUSCRIPT
460
Fig. 1. FASII pathway and FASII bypass. The FASII pathway comprises 3 main steps: i-
461
initiation, during which a priming molecule (short chain fatty acid) is synthesized; ii-
462
elongation, during which 4 enzymes elongate the fatty acid chain by two carbons per iterative
463
cycle; and iii- termination, in which a mature length acyl moiety is driven towards
464
phospholipid synthesis enzymes. The enzymes involved in each stage of FASII in S. aureus
465
are indicated; Int1-ACP to Int3-ACP represent reaction intermediates. FASII is blocked by
466
environmental fatty acids (FA; green dotted line), as occurs in the streptococci [5], and/or by
467
FASII inhibitors (red dotted line, see Table 1 for inhibitors thus far developed). The FASII
468
block favors the use of exogenous fatty acids (in green), leading to a change in the final
469
phospholipid product (called FASII bypass). Steps of FASII pathway have been extensively
470
characterized by numerous laboratories (see references [1-4] for in-depth reviews). The
471
schema presented here is based on Fig. S1 of reference 40.
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472
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Fig. 2. FASII bypass occurs with different fatty acid cocktails. Fatty acid profiles are
475
shown for the following strains and conditions: Top, S. aureus Newman strain grown in BHI
476
medium. The diagonal arrow indicates the position of ai15, the major fatty acid synthesized
477
by S. aureus. Center, a fabDm2 mutant corresponds to “Dep-5”, which depends on
478
exogenous fatty acids for normal growth [40]. It is grown in BHI medium containing triclosan
479
and three exogenous fatty acids, C14:0, C16:0, and C18:1. Bottom, the fabDm2 is grown in
480
3% liver extract as fatty acid source. The fatty acid profile of this mutant is equivalent to that
481
of the liver extract. Data in this figure correspond to gas chromatography results from
482
reference [40].
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Fig. 3. Model for FASII bypass in S. aureus fabD mutants and natural variants. FASII
485
and fatty acid incorporation kinase dependent pathways are as previously identified [1, 42].
486
Fatty acids (FA) are ubiquitous in host environments. The recently discovered that
487
exogenous fatty acids are substrate for the kinase complex FakA FakB1 FakB2 to produce
488
phosphorylated fatty acids (FA-PO4), which are substrates for PlsY; this drives incorporation
489
of an exogenous fatty acid into position 1 of the glycerolphosphate backbone [1, 42]. FASII
490
inhibitors that block elongation (in gray), lead to an acyl-ACP precursor pileup. However, less
491
efficient FabD activity of certain fabD variants leaves more ACP available [40], which can be
492
used by PlsX to convert exogenous fatty acids (turquoise) into phospholipids. This “reverse”
493
PlsX activity drives exogenous fatty acid incorporation into the glycerolphosphate backbone.
494
As a consequence exogenous fatty acids fill both positions of the glycerolphosphate
495
phospholipid backbone. The diagram is derived from [40].
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497
ACCEPTED MANUSCRIPT Fig. 4. Fatty acids favor Staphylococcus aureus resistance to anti-FASII AFN-1252.
499
Left, medium does not contain fatty acids; right, medium is supplemented with fatty acids.
500
Both plates are overlaid with clinical S. aureus strain SA40 [61], which harbors a natural fabD
501
variant correlating with resistance to FASII inhibitors [40]. AFN-1252 (1 µg) is deposited in
502
the center of each plate. The dark halo (delineated by arrows, left plate) indicates growth
503
inhibition by AFN-1252. This S. aureus isolate is AFN-1252- resistant when fatty acids are
504
present (right plate). This strain is also triclosan-resistant in fatty-acid-supplemented medium
505
[40]. Plates are photographed 36 hours post- incubation at 37°C.
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Table 1. Anti-FASII inhibitors. Target
ACCEPTED MANUSCRIPT
Enzyme function
Inhibitors
References
Fatty acid transfer AcpS
ACP synthase
Sch 538415, 4H-Oxazol-5-one, anthranilic acid
AccABCD
Acetyl-CoA Carboxylase
Pyrrolidinediones (Moiramide B), NCI 65828, andrimid, sulfonamidobenzamide (SABA) analogs
FabD
Malonyl-CoA:ACP transacylase Corytuberin
FabH
B-ketoacyl-ACP synthase III
[48]
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Initiation
[48] [51-53]
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HR12 (RWJ-3981), indole (SB418001) analogs, 1,2-Dithiole-3-ones, benzoylaminobenzoic acids, platensin, phomallenic acids, cyclic sulfones, alkyl-CoAdisulfides, pyrazin-triazole containing Schiff base
[48-51]
Elongation FabB/F
B-ketoacyl-ACP synthase I et II Cerulenin, thiolactomycin (and analogs), thiotetromycin, BABX, phomallenic acids, fasamycines, platensimycin (and analogs)
FabG
B-ketoacyl-ACP reductase
FabA
[17, 51]
[48]
B-hydroxydecanoyl-ACP dehydratase
Allenic acids, 3-Decynoyl-NAC
[48]
FabZ
B-hydroxyacyl-ACP dehydratase
NAS-91, NAS-21
[48]
FabI
Enoyl-ACP reductase I
Isoniazid , ethionamid , prothionamid , 2 triclosan , pyrrolyl benzohydrazides, eneamides with azetidines, diphenyl ether analogs, diazoborine, aminopyridines, Debio-1452, Debio-1450, MUT-056399, CG-400549, CG02390, complestatin, neuropectine A, aquastatin A, chloropeptin I, 1 and 4 substituted imidazols, naphtyridinones, thiopyridines (GGTC-004061), vinaxanthone, cephalochromin, kalimantacin, batumin, flavonoïds (e.g. EGCG), meleagrin
AC C
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Polyphenols
FabK
1
1
1
1
Aminopyridines, Atromentin, Leucomelone, AG205
Enoyl-ACP reductase II
2
[16, 45, 48, 51, 53-57]
[48]
used to treat M. tuberculosis infections; used as broad spectrum biocide; italics, molecules tested on animal models against S. aureus infections, of which Debio-1450, Debio-1452, and CG-400549 are in clinical trials. Completed from Zhang et al. 2006 [48] and Parsons et al. 2011 [51]. References not cited in those articles are presented at right.
ACCEPTED MANUSCRIPT
Table 2. MICs of fosfomycin and trimethoprim in S. aureus Newman wild type and FASII bypass mutant strains. Fosfomycin
Trimethoprim
Newman
BHI
FA
FA-T
BHI
12
2
-
4
m1 a
8
4
64
2
fabD
m2 b
-
128
96
-
FA
FA-T
1.7
-
1.5
>32
>32
>32
SC
fabD
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Strain
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Antibiotic minimal inhibitory concentrations (MICs) were determined using Etests (BioMerieux; expressed as µg antibiotic/ml). MIC tests ranges are 0.064-1024 µg/ml for fosfomycin and 0.002-32 µg/ml for trimethoprim. Strips were placed on BHI medium, or BHI medium containing fatty acids (FA, 0.5 mM), and fatty acids plus the FASII inhibitor triclosan (0.25 µg/ml, called FA-T). -, strain a m1 does not grow in test condition. fabD corresponds to “Cond-17” a mutant that can synthesize b m2 fatty acids, but that uses FASII bypass in the presence of FASII inhibitors; fabD requires exogenous fatty acids for growth [40].
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Initiation
Acetyl-CoA
AccBCDA
Elongation [N+2]
Int3-ACP
FabI
Int1-ACP
Malonyl CoA
SC
Acetyl-CoA or BC acyl-CoA
FabG
FabD
Malonyl-ACP
FabH
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FabZ
FabF acyl-ACP
Anti-FabI Triclosan AFN-1252
Environmental FAs
Fak
TE D
PlsX PlsY PlsC
ACP
AC C
EP
Termination
Fig. 1
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WT
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Exogenous fatty acids
fabDm2
Liver extract
TE D
Anti-FASII added
SC
No additions
20
25
30
AC C
EP
15
Fig. 2
ACCEPTED MANUSCRIPT
Malonyl-CoA
ACP
FabD
(Elongation)
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Initiation
Malonyl-ACP
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ACP
FA-PO4
PlsXPlsC
Fak
(environmental) FA
AC C
EP
TE D
PlsY
SC
Competition for ACP?
Fig. 3
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Fig. 4
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Highlights
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The fatty acid synthesis pathway (FASII) is a target for antimicrobial development (antiFASII). Numerous Gram-positive pathogens bypass anti-FASII by using host fatty acids. Staphylococcus aureus, long considered as anti-FASII-sensitive, can mutate in the presence of fatty acids, and bypass FASII. Numerous veterinary and clinical S. aureus isolates carry polymorphisms that favor FASII bypass. This review underlines the importance of screening for emergence of resistant variants before concluding on anti-FASII efficacy.
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S0300-9084(17)30175-X
DOI:
10.1016/j.biochi.2017.07.004
Reference:
BIOCHI 5235
To appear in:
Biochimie
Received Date: 5 April 2017 Revised Date:
0300-9084 0300-9084
Accepted Date: 13 July 2017
Please cite this article as: C. Morvan, D. Halpern, G. Kénanian, A. Pathania, J. Mondoloni-Anba, G. Lamberet, A. Gruss, K. Gloux, The Staphylococcus aureus FASII bypass escape route from FASII inhibitors, Biochimie (2017), doi: 10.1016/j.biochi.2017.07.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
1
Abstract
2 Antimicrobials targeting the fatty acid synthesis (FASII) pathway are being developed as
4
alternative treatments for bacterial infections. Emergence of resistance to FASII inhibitors was
5
mainly considered as a consequence of mutations in the FASII target genes. However, an
6
alternative and efficient anti-FASII resistance strategy, called here FASII bypass, was
7
uncovered. Bacteria that bypass FASII make use of exogenous fatty acids to build their
8
membranes, and thus dispense with the need for FASII. This strategy is used by numerous
9
Gram-positive low GC% bacteria, including streptococci, enterococci, and staphylococci. Some
10
bacteria repress FASII genes once fatty acids are available, and “constitutively” shift to FASII
11
bypass. Others, such as the major pathogen Staphylococcus aureus, may undergo high
12
frequency mutations that favor FASII bypass. This capacity is particularly relevant during
13
infection, as the host supplies the fatty acids needed for bacteria to bypass FASII and thus
14
become resistant to FASII inhibitors. Screenings for anti-FASII resistance in the presence of
15
exogenous fatty acids confirmed that FASII bypass, anti-FASII-resistant strains exist among
16
clinical and veterinary isolates. Polymorphisms in S. aureus FASII initiation enzymes favor FASII
17
bypass, possibly by increasing availability of acyl-carrier protein, a required intermediate. Here
18
we review FASII bypass and consequences in light of proposed uses of anti-FASII to treat
19
infections, with a focus on FASII bypass in S. aureus.
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The Staphylococcus aureus FASII bypass escape route from FASII
7
inhibitors
9
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Claire Morvan1,2, David Halpern1, Gérald Kénanian1, Amit Pathania1, Jamila Mondoloni-
11
Anba1, Gilles Lamberet1, Alexandra Gruss 1a, Karine Gloux 1a
12
SC
13 14
1
15
cedex, France. 2 Present address : Institute for Integrative Biology of the Cell (I2BC), CEA,
16
CNRS, Université Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette, France.
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INRA –UMR1319- MICALIS AgroParisTech. Domaine de Vilvert, 78352 Jouy-en-Josas,
17 18 19
a
20
[email protected]
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Abstract
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Antimicrobials targeting the fatty acid synthesis (FASII) pathway are being developed as
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alternative treatments for bacterial infections. Emergence of resistance to FASII inhibitors
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was mainly considered as a consequence of mutations in the FASII target genes. However,
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an alternative and efficient anti-FASII resistance strategy, called here FASII bypass, was
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uncovered. Bacteria that bypass FASII make use of exogenous fatty acids to build their
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membranes, and thus dispense with the need for FASII. This strategy is used by numerous
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Gram-positive low GC% bacteria, including streptococci, enterococci, and staphylococci.
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Some bacteria repress FASII genes once fatty acids are available, and “constitutively” shift to
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FASII bypass. Others, such as the major pathogen Staphylococcus aureus, may undergo
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high frequency mutations that favor FASII bypass. This capacity is particularly relevant
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during infection, as the host supplies the fatty acids needed for bacteria to bypass FASII and
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thus become resistant to FASII inhibitors. Screenings for anti-FASII resistance in the
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presence of exogenous fatty acids confirmed that FASII bypass, anti-FASII-resistant strains
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exist among clinical and veterinary isolates. Polymorphisms in S. aureus FASII initiation
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enzymes favor FASII bypass, possibly by increasing availability of acyl-carrier protein, a
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required intermediate. Here we review FASII bypass and consequences in light of proposed
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uses of anti-FASII to treat infections, with a focus on FASII bypass in S. aureus.
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Key words
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Triclosan, AFN-1252, fatty acid auxotrophy, clinical isolates, antibiotics, cross-resistance
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1. Numerous Gram-positive bacteria can bypass the FASII pathway by using
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exogenous fatty acids
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52 Steps of the FASII biosynthesis pathway have been extensively studied (see references [1-4]
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for in-depth reviews); “FASII bypass” is defined here as the capacity of numerous bacteria to
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use exogenous fatty acids, rather than de novo synthesis to constitute their membranes
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(schematized in Fig. 1). At one extreme, Lactobacillus bulgaricus lacks a complete FASII
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pathway, and relies on exogenous fatty acids for growth [6]. Numerous food-fermenting
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bacteria and opportunist pathogens (e.g., Lactobacillus plantarum and Streptococcus
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agalactiae respectively) efficiently incorporate fatty acids directly in membranes, and may
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thereby adapt their membrane phospholipid profiles to their lipid environment [5, 7].
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Interestingly, in S. agalactiae FASII gene expression is repressed in the presence of
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exogenous fatty acids and the profile of fatty acids incorporated into phospholipids mimics
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that of environmental fatty acids (Fig. 1; [5]). The existence of FASII bypass mechanisms
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makes sense: using full-length exogenous fatty acids directly for phospholipid synthesis
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economizes on energy expenditure during growth. Other pathogenic species, including
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enterococci, other streptococci, and staphylococci can use exogenous fatty acids to
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compensate a FASII block triggered by fatty acid repression or by a FASII inhibitor [5, 8-11].
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2. Could FASII inhibitors be efficient antibiotics despite FASII bypass in Gram-positive
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bacteria?
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Development of FASII inhibitors. A FASII inhibitor, isoniazid, has long been used to treat
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mycobacterial infections. Mycobacteria synthesize and require specific and very long chain
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fatty acids that cannot be supplied by the environment, explaining the effectiveness of such
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drugs [12]. The FASII pathway has more recently been a privileged “hot” target for
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antimicrobial development against the low GC percent Firmicutes. In various studies, strains
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of listeria, streptococci, staphylococci, and enterococci, were reported to be inhibited by 3
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various anti-FASII [13-22]. Most reported anti-FASII drugs (reviewed in Table1) target
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FabB/F and FabI, which catalyze essential steps in the iterative FASII elongation cycle. It is
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notable that bacteria affected by FASII inhibitors may be limited to those expressing the
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targeted FASII enzyme. For instance, streptococci encode an enoyl reductase distinct from
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FabI, making them naturally resistant to anti-FabI antimicrobials. This feature may be useful
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when developing narrow range antibiotics, and concomitantly sparing non-pathogens present
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in the same ecosystem.
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The first, and by far most widely used anti-FASII is triclosan, which has been
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extensively employed as a biocide, e.g., in hygiene, household, and hospital products [23,
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24]. At low concentrations, triclosan targets FabI [25]. However, at higher concentrations
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(e.g., 0.2 to 1% in soaps, creams, sutures), triclosan provokes membrane permeability and
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generalized non-specific membrane damage [26] and was linked to host-related damage
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(leading to its recent removal from hygiene and cosmetic products), and to widespread
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resistance (see 3.) [27, 28]. These unwanted properties stimulated the development of new
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FabI inhibitors with higher specificity, which eliminate the side effects observed with triclosan
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[16, 20, 29-31]. At least one such drug, AFN-1252 (renamed Debio 1452), is currently in
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clinical trials for treatment of S. aureus infections ([31] see 3.2.).
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Limitations of FASII inhibitors due to FASII bypass. Some bacteria use fatty acids,
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which are abundant in the host, to bypass a block in FASII synthesis. As a formal proof, a S.
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agalactiae strain deleted for its FASII biosynthesis genes was shown to retain full virulence
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[5]. As a second example, an E. faecalis fatty acid auxotroph was identified as the agent
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responsible for an umbilical cord infection [32]. These results essentially ruled out the use of
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anti-FASII to treat infections due to streptococci and enterococci, while the status of
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staphylococci was still debated.
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3. The case of S. aureus and FASII inhibitors
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Until recently, clinical S. aureus resistance to FASII inhibitors was mainly attributed to
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compensatory mutations in their targets or their precursor enzymes. A brief summary of this
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clinically significant class of anti-FASII resistance is given prior to consideration of the FASII
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bypass strategy.
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S. aureus anti-FASII resistance by modifications of the antimicrobial target.
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Documented cases of resistance to the widely used biocide triclosan mapped to point
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mutations in the fabI gene or its promoter [28, 29, 33]. FabI mutations were also selected in
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the presence of other FabI inhibitors, AFN-1252 and MUT056399 [16, 34]. Interestingly, S.
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aureus triclosan resistance was discovered in several cases to be associated with horizontal
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transfer of a fabI allele from S. haemolyticus (sh-fabI) [28, 33, 35]. Such isolates were
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subsequently found to be frequent among triclosan-resistant hospital screenings [28, 35].
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Moreover, resistance to FabF inhibitors was found to be associated with mutations in FabH,
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possibly by increasing malonyl-ACP availability, a substrate shared by FabF and FabH [36].
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S. aureus anti-FASII resistance by FASII bypass. The role of S. aureus FASII bypass in
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response to FASII inhibition has been a point of controversy [5, 8, 10, 14, 37-39].
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Biochemical studies led to the belief that the main fatty acid synthesized by S. aureus,
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anteiso C15:0 (ai15), was indispensable for phospholipid synthesis and membrane fluidity
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[10, 37, 38]. Moreover, PlsC, the enzyme that mediates fatty acid incorporation at position 2
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of the glycerophosphate backbone is reportedly highly specific [10, 29, 38]. The first S.
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aureus shown to be able to bypass FASII were fatty acid auxotroph mutants mapping to
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accD, encoding an acetyl-CoA carboxylase subunit involved in FASII initiation [10]. An acc
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mutant grew poorly in the absence of ai15, and was noninfectious in a mouse model, leading
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authors to suggest that emergence of FASII bypass variants during treatment would not
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compromise anti-FASII efficacy [38].
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However, the capacity of S. aureus to incorporate exogenous fatty acids as a means of
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adapting to a FASII block turned out to be more common than reported. Our studies revealed
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that fatty acid supplementation favored high frequency mutations leading to FASII bypass
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(mutation frequency leading to anti-FASII resistance of 10-6 compared to 10-8 in the absence
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of fatty acids) [40]. A robust class of FASII bypass mutants mapped to another initiation 5
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enzyme, FabD (malonyl-CoA:ACP transacylase). Some fabD mutants displayed a gain of
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function phenotype, contrasting to previously identified acc auxotrophs. The strains are
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mutated in the active site of FabD and are able to synthesize their own fatty acids, yet they
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also grow well by incorporating different combinations of exogenous fatty acids when FASII
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is blocked by either triclosan (Fig. 2) or AFN-1252 [40]. One fabD mutation may have
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resulted from replication slippage [41] in a run of “A” repeats, conferring triclosan resistance
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that might readily “slip back” to restore a wild type phenotype when selection is lifted (data
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not shown). A FASII bypass event can be also generated by anti-FabF treatment. Resistance
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to the FabF inhibitor maps to mutations in FabH, a third enzyme needed for FASII initiation.
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Interestingly, poor growth of these strains is rescued by exogenous fatty acid addition [36].
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Selective pressure by FASII inhibitors thus leads to resistant S. aureus that use
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different fatty acid combinations to comprise their membranes. Membrane composition is
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therefore not stringently controlled, an observation that also extends to clinical strains [35].
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3.1. A model for FASII bypass in S. aureus
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Recently elucidated steps of FASII bypass in S. aureus led to the following model
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(Fig. 3). The first step in exogenous fatty acid incorporation is phosphorylation by fatty acid
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kinase complex FakA FakB1 FakB2 (to acyl-PO4) [37]. Phosphorylated fatty acids are
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substrates for PlsY, which attaches an acyl group to position 1 of the glycerol-phosphate
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backbone [1]. A FASII block changes the balance of metabolic intermediates, including a
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pileup of malonyl-ACP. However, certain FabD variants produce less malonyl-ACP, leaving
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more free ACP available. This leads to a key step in the model, in which PlsX, which
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converts acyl-ACP to acyl-PO4, reverses its enzymatic activity. Acyl-ACPs may then be
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elongated by FASII (if it is not blocked) or used to complete phospholipid synthesis by PlsC,
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which transfers acyl-ACP to glycerol phosphate at position 2 to form phosphatidic acid [42].
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We provided direct evidence for this FASII bypass model in S. aureus isolates carrying
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FabD196 polymorphisms, in which phospholipids comprise exclusively exogenous fatty acids
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when FASII is blocked [40]. Differential regulation of FASII bypass in S. aureus and other
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Gram-positive bacteria (that directly utilize exogenous fatty acids in all tested conditions) is
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being investigated.
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3.2. Anti FASII efficiency on S. aureus in vivo: What’s the verdict?
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Difficulties in comparing in vivo models. Numerous animal experiments examined the
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efficacy of experimental FASII inhibitors (mainly anti-FabI) against S. aureus infections [16,
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30, 31, 43-47]. However, protocols varied between studies, e.g., infection regime, bacterial
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inocula, drug concentrations compared to MIC (minimal inhibitory concentration), drug
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treatment regimens post-infection, and evaluation of inhibitor efficiency (summarized in
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Supplementary Tables 1 and 2). The non-uniformity of test systems poses a main problem
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for gaining a real assessment of drug efficacies. Furthermore, current methods used to
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detect surviving S. aureus following anti-FASII treatments may fail to screen for fatty acid
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auxotrophs.
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The case in favor of anti-FASII inhibitors: i- Tests in animal models gave positive
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results on the efficacy of anti-FASII drugs in treating S. aureus infections [16, 19, 46]. ii-
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Clinical trials using AFN-1252 reported promising results in reducing lesion size of S. aureus
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skin infections (Clinicaltrials.gov identifier: NCT02426918) [31]. These reports concluded on
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the effectiveness of the tested FASII inhibitors. iii- Numerous bacteria (and also humans)
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lack a FabI enzyme. A narrow spectrum antimicrobial is a desired characteristic, which
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allows infections to be treated with minimal detrimental effects on the host and its harmless
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microbiomes. The anti-FabI Debio 1452 was shown to have low impact on the main intestinal
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bacterial populations, thus making the oral drug route a possible option [58].
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The case against FASII inhibitors: i- Triclosan (anti-FabI) treatment was found to be
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associated with an increase in S. aureus colonization [59]. ii- In vitro-isolated fabD variants
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were shown to be infectious, and arose in response to different anti-FASII drugs [40]. iii- In 6
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cases, anti-FASII-resistance status of clinical isolates was correlated to the presence of fabD 7
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polymorphisms that favor FASII bypass [35, 40]. iv- A screening involving 700 clinical
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isolates revealed that 19% were triclosan-resistant, among which 1/3 were revealed only
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when fatty acids were present in screening medium [35]. v- Clinical fatty acid auxotrophs,
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which have a fatty acid growth requirement, were isolated in 2 out of 103 patient samples
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[35], and were also reported in a patient with septic arthritis [60].
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So what is the verdict on anti-FASII? There is evidence that these drugs are active
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against S. aureus in certain cases. Yet after anti-FASII treatment, resistant bacteria might
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persist and not be detected in the short term. The presence of FASII bypass variants in
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human and farm animal isolates raise questions on their efficient detection and on the long-
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term efficacy of FASII inhibitors in treating infections.
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3.3. Cross resistance of FASII bypass strains
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FASII bypass isolates are expectedly cross-resistant to all antimicrobials targeting FASII
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enzymes. Indeed, in S. aureus, triclosan-selected FASII bypass variants were resistant to
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AFN-1252, the FabI inhibitor in clinical trials, and platensimycin, a FabF inhibitor and
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antimicrobial candidate [21, 31, 40]. Both clinical and laboratory strains bearing the same
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FabD polymorphism (FabD196) were both triclosan and AFN-1252 resistant in medium
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containing fatty acids ([40], Fig. 4), and grew at concentrations of >500 fold the AFN-1252
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MIC [10] (data not shown). Thus, in addition to their frequent occurrence in clinical,
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veterinary, and laboratory settings, fatty acid auxotrophs and isolates that bypass FASII are
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all expectedly multi-anti-FASII-resistant.
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We screened S. aureus wild type and FASII bypass fabD mutants for altered
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sensitivities to antibiotics having targets unrelated to FASII (Table 2). In conditions where
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bacteria relied on exogenous fatty acids, fabD mutants showed increased tolerance to
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fosfomycin (inhibitor of cell wall synthesis enzyme MurA) and trimethoprim (inhibitor of
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nucleic acid synthesis enzyme dihydrofolate reductase). Changes in phospholipids due to
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altered fatty acids by FASII bypass may thus impact overall membrane composition and
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bacterial behavior including antibiotic resistance.
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224 225 4. Conclusions
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Numerous Firmicutes including pathogens, incorporate exogenous fatty acids, which can
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compensate a FASII inhibition. The FASII pathway can be inhibited simply by exogenous
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fatty acid supplementation, or in some bacteria, with further addition of FASII inhibitors. In S.
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aureus, the essentiality of the major endogenous fatty acid ai15 has been in debate.
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However, the finding that mutants arising at high frequencies can replace endogenous fatty
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acid species with varied combinations of exogenous fatty acids casts doubts on this dogma.
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Thus staphylococci, like other Firmicutes, may have a higher membrane flexibility than
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previously recognized.
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S. aureus isolates that bypass FASII inhibition are already present among clinical and
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veterinary isolates, likely as a consequence of the widespread use of triclosan. We suggest
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that FASII bypass is one main strategy by which S. aureus overcomes FASII inhibitors in
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animals, environments with ubiquitous fatty acids. Experiments on animals and humans have
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supported the use of anti-FASII to treat S. aureus infections. However altered behavior of
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FASII bypass strains, such as small colony variant formation [60] and antibiotic resistance,
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may lead to cross antibiotic resistance, bacterial persistence and chronic disease.
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In view of these studies, we underline the importance of performing follow-up
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screenings prior to validating novel anti-FASII antimicrobials for treatment. Screenings for
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FASII bypass variants should be performed in conditions closer to those found in vivo, i.e.,
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that include fatty acids in assays [35]. FASII bypass variants that emerge after anti-FASII
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therapy may deserve further investigation to assess the risk of recurrence and chronic
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infection.
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Acknowledgements
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We thank all the members of the MicrobAdapt team for numerous stimulating discussions on
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FASII bypass. Research presented in this review received funding from the Agence
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Nationale de la Recherche (ANR) FattyBact project 525 ANR-132101. CM and GK were
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awarded stipends from the French Research Ministry. AP received a postdoctoral fellowship
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from the DIM-Malinf program, Conseil Régional Île de France (www.dim-malinf.org).
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C.C. Yuan, M.S. Head, D.J. Payne, S.F. Rittenhouse, T.D. Moore, S.C. Pearson, V. Berry,
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A.P. Fosberry, R. Greenwood, M.S. Head, D.A. Heerding, C.A. Janson, D.D. Jaworski,
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P.M. Keller, P.J. Manley, T.D. Moore, K.A. Newlander, S. Pearson, B.J. Polizzi, X. Qiu,
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S.F. Rittenhouse, C. Slater-Radosti, K.L. Salyers, M.A. Seefeld, M.G. Smyth, D.T. Takata,
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Mode of action, in vitro activity, and in vivo efficacy of AFN-1252, a selective
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ACCEPTED MANUSCRIPT antistaphylococcal FabI inhibitor, Antimicrob. Agents Chemother. 56 (2012) 5865-5874.
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enoyl-acyl carrier protein reductase inhibitor AFN-1252, Antimicrob. Agents Chemother.
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II inhibitors using a novel cellular bioluminescent reporter assay, Antimicrob. Agents
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Chemother. 59 (2015) 5775-5787.
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443 444
inhibitor
with
potent
activity
against
S.
aureus
and
coagulase-negative
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ACCEPTED MANUSCRIPT selective antibiotic minimizes disturbance to the microbiome, Antimicrob. Agents
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[59] A.K. Syed, S. Ghosh, N.G. Love, B.R. Boles, Triclosan promotes Staphylococcus aureus nasal colonization, mBio. http://doi: 10.1128/mBio.01015-13 (2014). [60] Y.T. Lin, J.C. Tsai, T. Yamamoto, H.J. Chen, W.C. Hung, P.R. Hsueh, L.J. Teng,
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Figure Legends
ACCEPTED MANUSCRIPT
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Fig. 1. FASII pathway and FASII bypass. The FASII pathway comprises 3 main steps: i-
461
initiation, during which a priming molecule (short chain fatty acid) is synthesized; ii-
462
elongation, during which 4 enzymes elongate the fatty acid chain by two carbons per iterative
463
cycle; and iii- termination, in which a mature length acyl moiety is driven towards
464
phospholipid synthesis enzymes. The enzymes involved in each stage of FASII in S. aureus
465
are indicated; Int1-ACP to Int3-ACP represent reaction intermediates. FASII is blocked by
466
environmental fatty acids (FA; green dotted line), as occurs in the streptococci [5], and/or by
467
FASII inhibitors (red dotted line, see Table 1 for inhibitors thus far developed). The FASII
468
block favors the use of exogenous fatty acids (in green), leading to a change in the final
469
phospholipid product (called FASII bypass). Steps of FASII pathway have been extensively
470
characterized by numerous laboratories (see references [1-4] for in-depth reviews). The
471
schema presented here is based on Fig. S1 of reference 40.
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Fig. 2. FASII bypass occurs with different fatty acid cocktails. Fatty acid profiles are
475
shown for the following strains and conditions: Top, S. aureus Newman strain grown in BHI
476
medium. The diagonal arrow indicates the position of ai15, the major fatty acid synthesized
477
by S. aureus. Center, a fabDm2 mutant corresponds to “Dep-5”, which depends on
478
exogenous fatty acids for normal growth [40]. It is grown in BHI medium containing triclosan
479
and three exogenous fatty acids, C14:0, C16:0, and C18:1. Bottom, the fabDm2 is grown in
480
3% liver extract as fatty acid source. The fatty acid profile of this mutant is equivalent to that
481
of the liver extract. Data in this figure correspond to gas chromatography results from
482
reference [40].
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Fig. 3. Model for FASII bypass in S. aureus fabD mutants and natural variants. FASII
485
and fatty acid incorporation kinase dependent pathways are as previously identified [1, 42].
486
Fatty acids (FA) are ubiquitous in host environments. The recently discovered that
487
exogenous fatty acids are substrate for the kinase complex FakA FakB1 FakB2 to produce
488
phosphorylated fatty acids (FA-PO4), which are substrates for PlsY; this drives incorporation
489
of an exogenous fatty acid into position 1 of the glycerolphosphate backbone [1, 42]. FASII
490
inhibitors that block elongation (in gray), lead to an acyl-ACP precursor pileup. However, less
491
efficient FabD activity of certain fabD variants leaves more ACP available [40], which can be
492
used by PlsX to convert exogenous fatty acids (turquoise) into phospholipids. This “reverse”
493
PlsX activity drives exogenous fatty acid incorporation into the glycerolphosphate backbone.
494
As a consequence exogenous fatty acids fill both positions of the glycerolphosphate
495
phospholipid backbone. The diagram is derived from [40].
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ACCEPTED MANUSCRIPT Fig. 4. Fatty acids favor Staphylococcus aureus resistance to anti-FASII AFN-1252.
499
Left, medium does not contain fatty acids; right, medium is supplemented with fatty acids.
500
Both plates are overlaid with clinical S. aureus strain SA40 [61], which harbors a natural fabD
501
variant correlating with resistance to FASII inhibitors [40]. AFN-1252 (1 µg) is deposited in
502
the center of each plate. The dark halo (delineated by arrows, left plate) indicates growth
503
inhibition by AFN-1252. This S. aureus isolate is AFN-1252- resistant when fatty acids are
504
present (right plate). This strain is also triclosan-resistant in fatty-acid-supplemented medium
505
[40]. Plates are photographed 36 hours post- incubation at 37°C.
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Table 1. Anti-FASII inhibitors. Target
ACCEPTED MANUSCRIPT
Enzyme function
Inhibitors
References
Fatty acid transfer AcpS
ACP synthase
Sch 538415, 4H-Oxazol-5-one, anthranilic acid
AccABCD
Acetyl-CoA Carboxylase
Pyrrolidinediones (Moiramide B), NCI 65828, andrimid, sulfonamidobenzamide (SABA) analogs
FabD
Malonyl-CoA:ACP transacylase Corytuberin
FabH
B-ketoacyl-ACP synthase III
[48]
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Initiation
[48] [51-53]
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HR12 (RWJ-3981), indole (SB418001) analogs, 1,2-Dithiole-3-ones, benzoylaminobenzoic acids, platensin, phomallenic acids, cyclic sulfones, alkyl-CoAdisulfides, pyrazin-triazole containing Schiff base
[48-51]
Elongation FabB/F
B-ketoacyl-ACP synthase I et II Cerulenin, thiolactomycin (and analogs), thiotetromycin, BABX, phomallenic acids, fasamycines, platensimycin (and analogs)
FabG
B-ketoacyl-ACP reductase
FabA
[17, 51]
[48]
B-hydroxydecanoyl-ACP dehydratase
Allenic acids, 3-Decynoyl-NAC
[48]
FabZ
B-hydroxyacyl-ACP dehydratase
NAS-91, NAS-21
[48]
FabI
Enoyl-ACP reductase I
Isoniazid , ethionamid , prothionamid , 2 triclosan , pyrrolyl benzohydrazides, eneamides with azetidines, diphenyl ether analogs, diazoborine, aminopyridines, Debio-1452, Debio-1450, MUT-056399, CG-400549, CG02390, complestatin, neuropectine A, aquastatin A, chloropeptin I, 1 and 4 substituted imidazols, naphtyridinones, thiopyridines (GGTC-004061), vinaxanthone, cephalochromin, kalimantacin, batumin, flavonoïds (e.g. EGCG), meleagrin
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Polyphenols
FabK
1
1
1
1
Aminopyridines, Atromentin, Leucomelone, AG205
Enoyl-ACP reductase II
2
[16, 45, 48, 51, 53-57]
[48]
used to treat M. tuberculosis infections; used as broad spectrum biocide; italics, molecules tested on animal models against S. aureus infections, of which Debio-1450, Debio-1452, and CG-400549 are in clinical trials. Completed from Zhang et al. 2006 [48] and Parsons et al. 2011 [51]. References not cited in those articles are presented at right.
ACCEPTED MANUSCRIPT
Table 2. MICs of fosfomycin and trimethoprim in S. aureus Newman wild type and FASII bypass mutant strains. Fosfomycin
Trimethoprim
Newman
BHI
FA
FA-T
BHI
12
2
-
4
m1 a
8
4
64
2
fabD
m2 b
-
128
96
-
FA
FA-T
1.7
-
1.5
>32
>32
>32
SC
fabD
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Strain
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Antibiotic minimal inhibitory concentrations (MICs) were determined using Etests (BioMerieux; expressed as µg antibiotic/ml). MIC tests ranges are 0.064-1024 µg/ml for fosfomycin and 0.002-32 µg/ml for trimethoprim. Strips were placed on BHI medium, or BHI medium containing fatty acids (FA, 0.5 mM), and fatty acids plus the FASII inhibitor triclosan (0.25 µg/ml, called FA-T). -, strain a m1 does not grow in test condition. fabD corresponds to “Cond-17” a mutant that can synthesize b m2 fatty acids, but that uses FASII bypass in the presence of FASII inhibitors; fabD requires exogenous fatty acids for growth [40].
RI PT
ACCEPTED MANUSCRIPT
Initiation
Acetyl-CoA
AccBCDA
Elongation [N+2]
Int3-ACP
FabI
Int1-ACP
Malonyl CoA
SC
Acetyl-CoA or BC acyl-CoA
FabG
FabD
Malonyl-ACP
FabH
M AN U
FabZ
FabF acyl-ACP
Anti-FabI Triclosan AFN-1252
Environmental FAs
Fak
TE D
PlsX PlsY PlsC
ACP
AC C
EP
Termination
Fig. 1
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WT
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Exogenous fatty acids
fabDm2
Liver extract
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Anti-FASII added
SC
No additions
20
25
30
AC C
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15
Fig. 2
ACCEPTED MANUSCRIPT
Malonyl-CoA
ACP
FabD
(Elongation)
RI PT
Initiation
Malonyl-ACP
M AN U
ACP
FA-PO4
PlsXPlsC
Fak
(environmental) FA
AC C
EP
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PlsY
SC
Competition for ACP?
Fig. 3
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Fig. 4
ACCEPTED MANUSCRIPT
Highlights
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The fatty acid synthesis pathway (FASII) is a target for antimicrobial development (antiFASII). Numerous Gram-positive pathogens bypass anti-FASII by using host fatty acids. Staphylococcus aureus, long considered as anti-FASII-sensitive, can mutate in the presence of fatty acids, and bypass FASII. Numerous veterinary and clinical S. aureus isolates carry polymorphisms that favor FASII bypass. This review underlines the importance of screening for emergence of resistant variants before concluding on anti-FASII efficacy.
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