Functional characterization of the n-acetylmuramyl-l-alanine amidase, ami1, from Mycobacterium abscessus

Article abstract of DOI:10.3390/cells9112410

Peptidoglycan (PG) is made of a polymer of disaccharides organized as a three-dimensional mesh-like network connected together by peptidic cross-links. PG is a dynamic structure that is essential for resistance to environmental stressors. Remodeling of PG occurs throughout the bacterial life cycle, particularly during bacterial division and separation into daughter cells. Numerous autolysins with various substrate specificities participate in PG remodeling. Expression of these enzymes must be tightly regulated, as an excess of hydrolytic activity can be detrimental for the bacteria. In non-tuberculous mycobacteria such as Mycobacterium abscessus, the function of PG-modifying enzymes has been poorly investigated. In this study, we characterized the function of the PG amidase, Ami1 from M. abscessus. An ami1 deletion mutant was generated and the phenotypes of the mutant were evaluated with respect to susceptibility to antibiotics and virulence in human macrophages and zebrafish. The capacity of purified Ami1 to hydrolyze muramyl-dipeptide was demonstrated in vitro. In addition, the screening of a 9200 compounds library led to the selection of three compounds inhibiting Ami1 in vitro. We also report the structural characterization of Ami1 which, combined with in silico docking studies, allows us to propose a mode of action for these inhibitors.

Full text of DOI:10.3390/cells9112410

cells
Article
Functional Characterization of the
N-Acetylmuramyl-l-Alanine Amidase,
Ami1, from Mycobacterium abscessus
Tanja Küssau 1, Niël Van Wyk 1,† , Matt D. Johansen 1,‡ , Husam M. A. B. Alsarraf 1,2
,
Aymeric Neyret 3, Claire Hamela 1, Kasper K. Sørensen 4, Mikkel B. Thygesen 4
,
Claire Beauvineau 5, Laurent Kremer 1,6,
*
and Mickaël Blaise 1,
*
1
Institut de Recherche en Infectiologie de Montpellier (IRIM), Université de Montpellier, CNRS UMR 9004,
CEDEX 5, 34293 Montpellier, France; tanja.kuessau@gmail.com (T.K.); nielvw@gmail.com (N.V.W.);
Matt.Johansen@uts.edu.au (M.D.J.); husam.alsarraf@irim.cnrs.fr (H.M.A.B.A.);
claire.hamela@irim.cnrs.fr (C.H.)
Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus C, Denmark
CEMIPAI CNRS UM UMS3725, CEDEX 5, 34293 Montpellier, France; aymeric.neyret@irim.cnrs.fr
Department of Chemistry, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40,
DK-1871 Frederiksberg C, Denmark; kso@chem.ku.dk (K.K.S.); mbth@chem.ku.dk (M.B.T.)
Chemical Library Institut Curie/CNRS, CNRS UMR9187, INSERM U1196 and CNRS UMR3666,
INSERM U1193, Université Paris-Saclay, F-91405 Orsay, France; Claire.Beauvineau@curie.fr
INSERM, IRIM, 34293 Montpellier, France
2
3
4
5
6
*
Correspondence: laurent.kremer@irim.cnrs.fr (L.K.); mickael.blaise@irim.cnrs.fr (M.B.);
Tel.: +33-(0)-434-359-447 (L.K. & M.B.)
†
‡
Present address: Institut für Mikrobiologie und Biochemie, Hochschule Geisenheim University,
65366 Geisenheim, Germany.
Present address: Centre for Inflammation, Faculty of Science, Centenary Institute and University of
Technology Sydney, Sydney, NSW 2006, Australia.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 6 October 2020; Accepted: 29 October 2020; Published: 4 November 2020
Abstract: Peptidoglycan (PG) is made of a polymer of disaccharides organized as a three-dimensional
mesh-like network connected together by peptidic cross-links. PG is a dynamic structure that
is essential for resistance to environmental stressors. Remodeling of PG occurs throughout
the bacterial life cycle, particularly during bacterial division and separation into daughter cells.
Numerous autolysins with various substrate specificities participate in PG remodeling. Expression of
these enzymes must be tightly regulated, as an excess of hydrolytic activity can be detrimental
for the bacteria. In non-tuberculous mycobacteria such as Mycobacterium abscessus, the function of
PG-modifying enzymes has been poorly investigated. In this study, we characterized the function of
the PG amidase, Ami1 from M. abscessus. An ami1 deletion mutant was generated and the phenotypes
of the mutant were evaluated with respect to susceptibility to antibiotics and virulence in human
macrophages and zebrafish. The capacity of purified Ami1 to hydrolyze muramyl-dipeptide was
demonstrated in vitro. In addition, the screening of a 9200 compounds library led to the selection
of three compounds inhibiting Ami1 in vitro. We also report the structural characterization of
Ami1 which, combined with in silico docking studies, allows us to propose a mode of action for
these inhibitors.
Keywords:
N-acetylmuramyl-l-alanine amidase;
peptidoglycan;
drug screening;
x-ray crystallography; Mycobacterium abscessus
Cells 2020, 9, 2410; doi:10.3390/cells9112410
www.mdpi.com/journal/cells

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1. Introduction
Peptidoglycan (PG), composed of glycan chains cross-linked by short peptides, surrounds most
bacteria and protects them against environmental and osmotic stress. PG consists of polymers
of the N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) disaccharide units.
The three-dimensional PG network is formed by the cross-linking of the peptide stems attached
to the MurNAc moiety [
a highly-dynamic structure participating in important physiological processes, such as bacterial
elongation and separation during cell division [ ]. During these dynamic phases, PG is remodeled
and the balance between PG synthesis and hydrolysis is tightly regulated [ ]. PG remodeling is
1]. Although contributing to the rigidity of the cell envelope, PG remains
2
3
performed by numerous autolysins acting on specific substrates and at different stages during bacterial
growth. While glycosidases, such as muraminidases and glucosaminidases act to hydrolyze the
glycosidic bonds between the carbohydrate units, peptidases, such as N-acetylmuramyl-l-alanine
amidases, l,d-endopeptidases, d,l-endopeptidases, l,d-carboxypeptidases, and dd-carboxypeptidases,
hydrolyze the amide bonds of the peptide stem at specific positions [4].
In mycobacteria, PG differs from Gram-positive or Gram-negative bacteria as it is covalently
linked to the atypical mycobacterial envelope [
peptidase superfamily, possessing an NlpC/P60 catalytic domain [
5
]. d,l-endopeptidases belonging to the papain-like
], have been widely studied in
6
mycobacteria. Among them, RipA has been shown to interact with the resuscitation-promoting factor
B (RpfB), a lytic transglycosylase, and the bifunctional PG-synthesizing enzyme, penicillin-binding
protein 1 (PBP1) [7]. Rpf-interacting protein A (RipA) activation also requires the Mycobacterium acid
resistance protease (MarP) during acid stress [8]. These studies emphasize the coordination of the PG
hydrolytic enzymes, which are tightly regulated during the mycobacterial life cycle.
Until recently the role of the N-acetylmuramyl-l-alanine amidases (amidases) has been poorly
documented in mycobacteria. The genomes of Mycobacterium smegmatis and Mycobacterium tuberculosis
encode four potential amidases. CwlM (Rv3915), also known as Ami2, is annotated as a PG amidase
but does not appear to be catalytically active [
the activity of the UDP-N-acetylglucosamine 1-carboxyvinyltransferase MurA, a key player of PG
biosynthesis [ ]. Rv3811/MSMEG_6406, designated Ami3, is not essential for mycobacterial growth,
9]. However, it plays an important role in modulating
9
but overexpression of Ami3, due to its inherent PG hydrolytic activity, is lethal for mycobacteria [10].
Ami3 activity is therefore tightly modulated by proteolysis mediated by the HtrA protease [10].
MSMEG_6281 (Ami1Msm), containing an amidase_3 domain, is dispensable for bacterial growth
but ami1 deletion mutants present an atypical lateral branching and a defect in cell separation and
septal PG turnover [11]. This mutant is also more susceptible to antibiotics due to increased cell wall
permeability; a phenotype subsequently confirmed in an independent study which also reported the
attenuation of the virulence the ami1 mutant in a mouse model of infection [12]. While cell division
was impaired in the ami1Msm knockout strain, this phenotype could be rescued by complementation
with the orthologue gene, Rv3717, from M. tuberculosis (ami1Mtb), suggesting a similar mode of
action in both species [13]. Furthermore, ami1Msm has also been shown to be important for biofilm
formation [14]. In addition, mycobacterial Ami1 has been biochemically and structurally characterized.
Purified Ami1Mtb hydrolyzes PG fragments in vitro in a zinc-dependent manner and its crystal
structure revealed a typical amidase_3 signature [15,16]. However, the functional role of Ami1 in
mycobacteria appears more complex than originally thought, for several reasons: (1) a single ami1
deletion mutant or multiple deletions of ami1 and ami3 or ami4 do not display any growth defect in
M. smegmatis [10], suggesting that amidases have redundant functions as reported in other bacteria,
such as in Escherichia coli [17–19] and (2) Ami1 shares overlapping functions with the endopeptidase
RipA, as Ami1 rescued RipA’s activity during cell division but not for persistence and chronic infection
in a mice infection model [20].
The PG remodeling machinery of M. abscessus, a rapidly growing non-tuberculous
mycobacterium, has been poorly explored. The M. abscessus complex [21] comprises three different
subspecies—M. abscessus sensu stricto (hereafter designated M. abscessus), M. abscessus subsp. bolletii,

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and M. abscessus subsp. massiliense. Infections caused by this complex are increasing globally and are
notoriously difficult to treat, particularly due to the extreme resistance of M. abscessus to most antibiotic
classes [21]. The two M. abscessus morphotypes, (R) rough and (S) smooth, display similar antibiotic
susceptibility profiles but, largely differ in their virulence profile as emphasized in the embryonic
zebrafish model [22]. The S variant is considered as the environmental form that colonizes the airways
and that can transition to the more virulent R form [21]. The presence or absence of surface-exposed
glycopeptidolipids (GPL) in both the S and R morphotypes, respectively, has been shown to correlate
with virulence in several animal models as well as in humans [23–25].
Herein, we evaluated the impact of ami1Mab deletion with respect to growth and bacterial division
in both S and R morphotypes of M. abscessus and assayed their capacity to infect human macrophages
and zebrafish larvae. We characterized the hydrolytic properties of purified Ami1Mab and determined
its crystal structure at high resolution. We also report a thermal-shift assay screening approach that led
to the discovery of three Ami1Mab inhibitors.
2. Materials and Methods
2.1. Generation of Unmarked Deletion Mutant
Deletion of MAB_0318c (ami1Mab) in the wild-type strain M. abscessus CIP104536T was carried
out using the double homologous recombination unmarked strategy, reported previously [26].
The DNA sequences located upstream, left arm (LA), and downstream, right arm (RA), of the
ami1 gene were PCR-amplified and subsequently cloned after an overlapping PCR reaction
into the pUX1-katG using the following primers, Forward: Mab0318_pUX1katG_LA_F and
Reverse: Mab0318_pUX1katG_LA_R and Forward: Mab0318_pUX1katG_RA_F and Reverse:
Mab0318_pUX1katG_RA_R (Supplementary Table S1).
The resulting plasmid was electroporated in the smooth (S) and rough (R) variants of M. abscessus
and the red fluorescent, kanamycin (KAN)-resistant colonies which underwent the first homologous
recombination event were selected on Middlebrook 7H10 (Sigma-Aldrich, Saint-Quentin Fallavier,
France) agar supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC) in the presence of
200
µg/mL KAN. Cultures of selected colonies were then subjected to a counter-selection on 7H10 agar
plates supplemented with OADC containing 50
µg/mL isoniazid (INH) to promote a second homologous
recombination and removal of the KAN cassette. Non-fluorescent, INH-resistant colonies were picked
up and further genotyped for ami1Mab deletion following PCR using the Mab0318_pUX1katG_LA_F
and Mab0318_pUX1katG_RA_R primers (Supplementary Table S1). Proper deletion was further
confirmed by DNA sequencing of the genomic region surrounding the site of gene deletion.
For functional complementation of the ami1Mab deletion mutant, the genomic sequence of ami1Mab
was fused at its 30end in frame with the sequence encoding the Streptag-II, using the following
primers, Forward: Mab0318_pMV306hsp_EcoRI_F and Reverse: Mab0318_SalI_C-termStrepTag
(Supplementary Table S1) and subsequently cloned into the pMV306 plasmid possessing a
KAN-resistance cassette and resulting in the creation of pMV306::ami1Mab. The ami1Mab deletion
mutants were transformed with pMV306::ami1Mab and selected onto 7H10 agar supplemented with
OADC and KAN (200µg/mL).
2.2. Cloning for Recombinant Protein Expression
The genomic sequence encoding ami1Mab depleted from its secretion peptide signal
was PCR-amplified from M. abscessus genomic DNA with the following primers,
Forward: Mab0318_pET151_F and Reverse: Mab0318_pET151_R (Supplementary Table S1) and
cloned in a ligation-free independent manner into the directional Champion
™ pET151 TOPO™
(Thermo Fisher Scientific, Villebon-sur-Yvette, France) using manufacturer’s instructions.
The genomic sequence of ami1Msm without its peptide signal of secretion was PCR-amplified
from M. smegmatis mc2155 genomic DNA with the Forward and Reverse primers, Smeg6281_pET30_F

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and Smeg6281_pET30_R (Supplementary Table S1) and was cloned into the pET-30 ek/LIC plasmid
between the KpnI and EcoRI restriction sites in frame with the His- and S-tags followed by a Tobacco
etch virus (TEV) protease cleavage site.
The genomic sequence of ami1Mtb without its peptide signal of secretion was PCR-amplified
from M. tuberculosis genomic DNA with the Forward Mtb_3717_F and Reverse Mtb_3717_R primers
(Supplementary Table S1) and was cloned in a ligation-free independent manner into the pET-30
ek/LIC plasmid in frame with the His- and S-tags followed by a Tobacco etch virus (TEV) protease
cleavage site.
2.3. Mycobacterial Culture Conditions
The MIC were determined according to the Clinical & Laboratory Standards Institute (CLSI)
guidelines [27]. M. abscessus strains were grown in Middlebrook 7H9 broth (BD Difco) supplement◦ed
with 10% OADC and 0.025% tyloxapol (Sigma-Aldrich, Saint-Quentin Fallavier, France) at 37 C
under agitation (unless otherwise stated) in the presence of antibiotics, when required (750
µg/mL
hygromycin for all tdTomato-expressing strains, and 200 g/mL kanamycin for complemented strains).
µ
On plates, strains were grown on Middlebrook 7H10 agar (BD Difco) supplemented with 10% OADC
enrichment in the presence of antibiotics when required and grown at 37 ◦C.
2.4. Single-Cell Preparation for M. abscessus
The various strains expressing tdTomato were grown in 50 mL 7H9 supplemented with 10% OADC,
0.025% tyloxapol and appropriate antibiotics (750
complemented strains) for 3 days at 37 ◦C under agitation. After bacteria were centrifuged for 15 min
at 3000 g, the pellet was resuspended in 1 mL of 7H9 and transferred into 1.5 mL Eppendorf tubes.
µg/mL hygromycin and 200 µg/mL KAN for
×
To dissociate bacterial aggregates and generate single-cell bacteria, the samples were syringed through
a 26G needle 20 times followed by a 15 s sonication step, resuspended with 7H9 media and passed
through a 5
µ
m filter. Bacterial suspensions were centrifuged for 15 min at 3000
×
g and the pellet
resuspended in 1 mL of 7H. The OD was measured and 10
Eppendorf tubes and stored at −80 ◦C.
µL aliquots were prepared in 1.5 mL
2.5. Protein Expression and Purification
The E. coli BL21(DE3) strain resistant to phage T1 (New England Biolabs, Evry, France) containing
the pRARE2 plasmid was transformed with the pET-151::ami1Mab, pET-30::ami1Msm, or pET-30::ami1Mtb
constructs. Bacterial cultures were performed in Luria-Bertani broth (LB) medium containing 200
µ
g/mL
◦
ampicillin or 50
µg/mL KAN and 30
µg/mL chloramphenicol at 37 C. When an OD at 600 nm of
0.6 to 1 was reached, the cultures underwent a 30 min cold shock in icy water before adding 1 mM
isopropyl-
spun-down (8400
β
-d-1-thiogalactopyranoside (IPTG). Cultures were then grown for 20 h at 16 ◦C and then
◦
×
g, 4 C, 20 min). Bacterial pellets were resuspended in lysis buffer (50 mM
Tris-HCl pH 8, 400 mM NaCl, 20 mM imidazole, 1 mM benzamidine, and 5 mM β-mercaptoethanol)
and the cells were disrupted by sonication prior to clarification by an additional centrifugation step
(27,000× g, 4 ◦C, 45 min). Proteins were then purified by a first ion metal affinity chromatography
(IMAC) step (Ni-nitrilotriacetic acid sepharose, Cytiva, Europe, GmbH). To remove the N-terminal
tags, the eluted proteins were mixed in a 1:50 (w:w) ratio with TEV protease and dialyzed overnight
at 4 ◦C against 50 mM Tris-HCl pH 8, 200 mM NaCl, and 5 mM
β-mercaptoethanol. The dialyzed
proteins were loaded onto a second IMAC, the non-tagged proteins were eluted in the flow-through,
while tags, uncleaved protein, and TEV protease bound the column. Proteins were concentrated using
a 10 kDa cut-off centricon (Sartorius) and subjected to a final step of purification on size-exclusion
chromatography, Supderdex 75 10/300 GL using an elution buffer containing 50 mM Tris-HCl pH 8,
200 mM NaCl, and 5 mM β-mercaptoethanol for Ami1Mab and Ami1Mtb or the same buffer but at
pH 7.4 for Ami1Msm. The proteins prepared in these conditions were for crystallization purpose.
Ami1Mab and Ami1Mtb used for the thin-layer chromatography (TLC)-based activity assay were

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purified under similar conditions, except the last step of purification on SEC was performed in
1 x Phosphate-Buffered-Saline (PBS) buffer.
2.6. Crystallization, Data Collection, and Structure Refinement
Crystals were grown in sitting drops in MR Crystallization Plates
by mixing 2 L of protein solution with 2 L of reservoir solution. Ami1Mab crystals were obtained
™
(Hampton Research) at 18 ◦C
µ
µ
at a protein concentration of 10 mg/mL and crystallized in 0.1 M Bis-Tris pH 5.5, 1.6 M ammonium
sulfate, and 1% (w/v) polyethylene glycol (PEG). Ami1Mab plus dipeptide crystals were obtained in
similar conditions and in presence of 5 mM of the ligand. Crystals were grown for several months and
cryo-protected by a brief soaking step into a solution made of 0.1 M Bis-Tris pH 5.5, 1.6 M ammonium
sulfate, 1% (w/v) PEG 3350, and 10–15% glycerol prior being cryo-cooled in liquid nitrogen.
Ami1Msm crystals were obtained at a concentration of 13 mg/mL in the presence of 5 mM
muramyl-dipeptide (MDP) and in a reservoir solution made of 0.1 M Bis-Tris pH 5.5, and 17% (w/v)
PEG. Crystals were cryo-protected by a brief soaking step into a solution made of 0.1 M Bis-Tris pH 5.5,
30% (w/v) PEG 3350, and 10% PEG 400 prior being cryo-cooled in liquid nitrogen.
X-ray diffraction data were collected at the Swiss-light source on the X06DA-PXIII beamline and
at the European Synchrotron Radiation Facility on the ID30B beamline.
The Ami1Mab structure was solved by molecular replacement using the crystal structure of the
Ami1Mtb structure (PDB: 4LQ6) as a search request. The Ami1Mab dipeptide and Ami1Msm structures
were solved similarly but using the Ami1Mab structure as a search model. Autobuild from the
Phenix (version 1.18.2) software suite [28] was used to automatically improve the models which
were then manually rebuilt and refined using the Coot (version 0.89) [29] and Phenix software suites.
The coordinates and structures factors have been deposited at the protein data bank under accession
numbers 7AGL for Ami1Mab, 7AGO for Ami1Mab plus dipeptide, and 7AGM for Ami1Msm
.
2.7. In Silico Docking
The three-dimensional structure of Ami1Mab bound to dipeptide was depleted from its ligand
and was used for in silico docking. The docking search was performed with the PyRx software
(version 0.9.4) [30] running AutoDock Vina [31]. All the amino acid side chains were kept as rigid.
Docking was carried out using the following grid parameters: center X = 0.59, Y = 16.38, and Z =
and dimensions (Å) X = 40, Y = 44, and Z =28 and an exhaustiveness of 8.
−
8.5
2.8. Drug Susceptibility Testing and MIC Determination
Minimum inhibitory concentrations (MICs) were determined in cation-adjusted Mueller–Hinton
broth (Sigma-Aldrich, Saint Quentin Fallavier, France) using the microdilution method. Briefly, 2 L of
the drug was added to 198 L bacterial suspension and 2 times serial dilutions were performed with a
final volume of 100 L per well (5
106 colony forming unit CFU/mL). The MIC was defined as the
µ
µ
µ
×
minimal drug concentration required to inhibit growth and was recorded by visual inspection after
4 days of incubation at 30 ◦C. All experiments were completed in technical replicates and performed in
three independent replicates.
2.9. Macrophage Experiments
THP-1 macrophages were grown in Roswell Park Memorial Institute (RPMI) medium (BD Gibco)
supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, Saint Quentin Fallavier, France)
(RPMIFBS) and incubated at 37 ◦C with 5% CO2. THP-1 monocytes were differentiated into macrophages
in the presence of 20 ng/mL phorbol myristate acetate (PMA) in 24-well flat-bottom tissue culture
microplates (105 cells/mL) and incubated for 48 h at 37 ◦C with 5% CO2. Macrophages were infected
with M. abscessus strains at a multiplicity of infection (MOI) of 2:1 (bacteria:macrophage) in 500 µL of
RPMIFBS for 2–4 h at 37 ◦C with 5% CO2. Following infection, the bacterial inoculum was removed and
cells were carefully washed three times with PBS and then incubated with fresh RPMIFBS supplemented

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with 250
times with PBS prior to the addition of 500
prevent extracellular growth. At various time points, macrophages were washed three times with
µ
g/mL amikacin for 2 h to kill extracellular bacteria. Macrophages were then washed three
µ
L RPMIFBS supplemented with 50
µg/mL amikacin to
PBS and lysed with 100
colony-forming units.
µL of 1% Triton X-100. Serial dilutions were plated on LB agar to count the
2.10. Synthesis of Peptidoglycan Dipeptide, l-Alanyl-d-Isoglutamine (DP)
The dipeptide was prepared by Fmoc solid-phase peptide synthesis and was performed
on a Biotage® Initiator+ AlstraTM microwave-assisted, automated peptide synthesizer from
Biotage AB, Uppsala, Sweden. Amino acids, reagents, and solvents were purchased from
Iris Biotech GmbH, Marktredwitz, Germany.
Peptide couplings were carried out using
5 equivalents of amino acid, 5 equivalents of 1-hydroxybenzotriazole (HOBt), 4.75 equivalents of
N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate
N-oxide (HBTU), and 9.75 equivalents of N,N-diisopropylethylamine (DIEA) in N-methylpyrrolidone
(NMP). A coupling time of 10 min at 75 ◦C, and subsequent deprotection with piperidine in
N,N-dimethylformamide (DMF) (2:3) for 3 min, followed by piperidine-DMF (1:4) for 10 min was used.
After each coupling and deprotection step, a washing procedure with 2x NMP, 1x dichloromethane,
then 2 x NMP was used.
The synthesis was carried out on a 0.1 mmol scale on 0.25 mmol/g Tenta Gel S Rink resins
from Rapp Polymere GmbH, Tuebingen, Germany. Peptide coupling of Fmoc-d-Glu(tBu)-OH
was followed by Fmoc-d-Ala-OH. The peptide was cleaved from the resin with trifluoroacetic
acid–water–triethylsilane 95:3:2 for two hours. Because of the high polarity of the product, purification
was performed in reversed-phase mode on an Isolera
™ system (Biotage AB, Uppsala, Sweden) with a
SNAP Bio C18 300 Å column (Biotage AB, Uppsala, Sweden), with water–acetonitrile containing
0.1% TFA, and using a gradient of acetonitrile from 5% to 30%. Peptide purity was evaluated by
analytical HPLC (Dionex Ultimate 3000 system from Thermo Scientific, Waltham, MA, USA) on an
analytical C18 column (Gemini NX 110 Å, 5
µ
m, 4.6
×
50 mm from Phenomenex, Torrance, CA, USA)
using a linear gradient flow from 2% to 25% acetonitrile, containing 0.1% formic acid, giving a purity
>98%, and identification was carried out by electrospray ionization mass spectrometry using an Impact
HD UHR-QTOF mass spectrometer from Bruker Corp., Billerica, MA, USA. MS (ESI): calculated for
C8H15N3O4: 217.1063; found: [M+H]+ 218.1132, and [M+Na]+ 240.0956.
2.11. Chemical Library
The Institut Curie-CNRS library (9200 compounds, 2013 version) was dissolved in anhydrous
dimethyl sulfoxide (DMSO, Carl Roth, Karlsruhe, Germany) and stored in a 10 mg/mL stock solution.
2.12. General Procedures Relative to the Chemistry of Ami1Mab Inhibitors
1
Proton (1H) NMR spectra were recorded on a Bruker Avance 300 (300 MHz for H), using
Tetramethylsilane as an internal standard. Deuterated DMSO-d6 was purchased from Euriso-Top
(Gif-Sur-Yvette, France). Chemical shifts are given in parts per million (ppm) (δ relative to the residual
solvent peak for 1H). The following abbreviations are used: singlet (s), doublet (d), triplet (t), and
multiplet (m). The instrument used was an Alliance Waters system [Alliance Waters 2695 (pump)
and Waters 2998 (Photodiode Array detector)] and the column was a Waters XBridge C-18, 3.5
particle size (3.0 mm 100 mm) (Waters, Saint-Quentin-en-Yvelines, France). Electrospray ionization
mass spectrometry was recorded on a micromass ZQ 2000 (Waters, Saint-Quentin-en-Yvelines, France).
Compounds to (purity >90% by liquid chromatography-mass spectrometry) came from the Institute
Curie/CNRS chemical library (Orsay, France).
µm
×
1
3

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C8H4ClNO3—Theoretical molecular weight M = 197.5753 g/mol
4-chlorofuro [3,2-c]pyridine-3-carboxylic acid (inhibitor 1) 1H NMR (300 MHz, DMSO-d6)
δ
8.81 (s, 1H),
8.38 (d, J = 5.7 Hz, 1H), 7.85 (d, J = 5.7 Hz, 1H), LRMS (ESI-MS) m/z = 198.0 + 200.0 [M+H]+.
C7H4ClNO4—Theoretical molecular weight, M= 201.5640 g/mol
2-chloropyridine-3,4-dicarboxylic acid (inhibitor 2) 1H NMR (300 MHz, DMSO-d6)
δ 14.07 (s, 2H),
8.62 (d, J = 5.0 Hz, 1H), 7.82 (d, J = 5.0 Hz, 1H). LRMS (ESI-MS) m/z = 202.0 + 204.0 [M+H]+.
C7H7N5—Theoretical molecular weight M = 161.1640 g/mol
2-(1H-tetrazol-5-yl)aniline (inhibitor 3) 1H NMR (300 MHz, DMSO-d6)
δ 9.09 (s, 3H), 7.84 (dd, J = 7.9,
1.2 Hz, 1H), 7.37 – 7.18 (m, 1H), 7.00 (d, J = 8.0 Hz, 1H), 6.80 (t, J = 7.2 Hz, 1H), LRMS (ESI-MS)
m/z = 162.2 [M+H]+.
2.13. Transmission Electron Microscopy
50 mL cultures were grown in 7H9 (supplemented with 10% OADC and 0.025% tyloxapol) and
appropriate antibiotics to an OD600 = 0. Bacteria were harvested by centrifugation at 4000 g for 15
×
min and washed twice with 1 mL sterile PBS. Cells were fixed in 2.5% glutaraldehyde in PHEM (60
mM Pipes, 25 mM Hepes, 10 mM EGTA, 2 mM MgCl2, pH 6.9) buffer for 1 h at room temperature,
post-fixed in OsO4 1%/K4Fe(CN)6 0.8% 1 h at room temperature then dehydrated in successive ethanol
bathes (50/70/90/100%). The samples were then infiltrated with propylene oxide/epon 812 mixes,
embedded in epon 812 and polymerized at 60 ◦C for 24 h. Then, 70 nm ultrathin sections were cut
with a PowerTome XL ultramicrotome (RMC, Tucson, AZ, USA). Cuts were stained with 0.2% OTE
(Oolong Tea Extract)/lead citrate and observed on a Tecnai G2 F20 (200 kV, FEG) TEM at the platform
Plateau de Microscopie Electronique COMET, INM, Platform Montpellier RIO Imaging.
2.14. Zebrafish Infection Experiments
Experiments were done with M. abscessus strains expressing tdTomato, obtained after
transformation with the replicative plasmid pTEC27 [22]. Embryos were dechorionated using pronase
(1 mg/mL final concentration) for 5 min at room temperature, followed by extensive washes in zebrafish
water. At 30 h post-fertilization, embryos were anesthetized with 160 µg/mL tricaine (Sigma-Aldrich,
Saint Quentin Fallavier, France) followed by caudal vein microinjection of 2–3 nL of bacterial suspension
(approximately 100 CFU/nL). The size of the inoculum was verified a posteriori by injecting 2 nL of
each bacterial suspension in sterile PBS and plated on 7H10 + OADC. To monitor embryo survival,
infected larvae were transferred into 24-well plates (three embryos per well) and incubated at 28.5 ◦C.

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Embryos were monitored daily and dead embryos were determined based on the absence of a heartbeat
and removed from the well.
2.15. Zebrafish Breeding and Ethics
Zebrafish experiments were completed in accordance with the guidelines defined by the European
Union for the use of laboratory animals. All animal experimentations were approved by the Direction
Sanitaire et Vétérinaire de l’Hérault et Comité d’Ethique pour l’Expérimentation Animale de la région
Languedoc Roussillon under the reference CEEA-LR-1145. Adult zebrafish were housed at the Centre
National de la Recherche Scientifique, Montpellier, France. Zebrafish embryos were obtained by
natural spawning and maintained at 28.5 ◦C in 60
µg/mL ocean salts. Zebrafish experiments were
performed using the golden mutant line [32].
2.16. Statistical Analyses
Statistical analyses were performed on Prism 5.0 (Graphpad, La Jolla, CA, USA) and detailed
in each figure legend. Macrophage experiments were analyzed using Student’s unpaired two-tailed
t-tests. For zebrafish embryo survival curve analysis, log-rank (Mantel–Cox) statistical tests were used.
2.17. Muramyl-Dipeptide Hydrolysis Assay
The reaction mixture was composed of 0.5
µL of Ami1 at different concentrations, 0.5
µL of
MDP (InvivoGen, Toulouse, France) at 10 mg/mL and 4
30 min at 37 C. The whole reaction volume was spotted at the bottom of a silica gel TLC plate
µL of 1X PBS, and was incubated for
◦
(10 × 10 or 20 × 20 cm) in 1
µL steps. Migration of the reaction products was done in a glass chamber
using butanol–ethanol–water (ratio 50:32:18, v/v/v) as a mobile phase. After approximately 1 h,
plates were removed, allowed to dry, sprayed with 2% ninhydrin solution (diluted in ethanol)
and charred. MDP, the dipeptide, and N-acetylmuramic acid (Sigma-Aldrich, Saint-Quentin Fallavier,
France) were used as internal standards.
2.18. Compounds Screening by Thermal-Shift Assay
The procedure was largely based on the protocol described by Niesen et al., [33]. Thirty-nine
microliters of purified Ami1Mab at a concentration of 80
One microliter of each compound at 10 mg/mL was added in each well. As a non-treated control and
to determine Ami1Mab melting temperature (Tm), 1 L of DMSO was added to one well with Ami1Mab
Thereafter, 40 L of the fluorescent dye, SYPRO orange (Sigma-Aldrich, Saint-Quentin Fallavier,
µM were dispensed in 96-well PCR plates.
µ
.
µ
France), at a concentration of 10X (stock solution 5000X in 100% DMSO) was added. Plates were sealed
and placed in a preheated qPCR machine (Agilent Mx3005) equipped with a Cy3 fi◦lter (excitation 545,
emission 568). The heat cycle program was as followed: a 10 min step at 25 C followed by an
incremental of 1◦/min step until the temperature reached 95 ◦C. After the run, the melting curves of the
protein in each well were plotted using Graphpad Prism software. Curves were fitted using Boltzmann
sigmoidal functions and the Tm was subsequently determined. A Tm value approximately of 2 ◦C to
5 ◦C higher than the DMSO control was considered as strong binding to the protein.
3. Results
3.1. Phenotypic Analysis of ami1 Deletion Mutant in the R and S Morphotypes
3.1.1. ami1Mab Is Dispensable for M. abscessus Growth in Vitro
To investigate the role of Ami1Mab, a
∆ami1Mab deletion mutant was generated based on the
development of a recent recombineering technique allowing the production of unmarked deletion
mutants in M. abscessus [26]. Gene deletion was generated in both the S and R backgrounds. Proper gene
inactivation was confirmed by PCR and sequencing analysis (Figure 1A).

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Figure 1. Generation and phenotypic analyses of
region around ami1Mab (MAB_0318c) in the parental (WT) and
abscessus. The size of the PCR amplicons used for genotyping the ami1Mab mutants is indicated. (
profile confirming the proper deletion of ami1Mab in the mutant strains. The PCR products of 1.6 kb were
amplified from ami1Mab genomic DNA from rough (R) and smooth (S) variants while a band at 2.4 kb
is expected for the parental (WT) strain. (
In vitro growth curves at 37 ◦C of the M. abscessus (Mab)
WT, ami1Mab, and complemented strains, in the R and S variants, respectively. Bacteria were grown in
7H9 broth supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC) and 0.025% tyloxapol.
The experiment is one representative of three replicates. ( ) Transmission electron microscopy (TEM)
of the M. abscessus WT, ami1Mab, and complemented strain in the S morphotype. Septa are indicated
by black arrows. ( ) Cell length measurements from the TEM data. Mean cell length = 1.27 m for Mab
S WT (n = 518); mean cell length = 1.34 m for ami1Mab (n = 557); mean cell length = 1.27 m from the
complemented strain (n = 576), the scale bar represents 500 nm. For statistical analysis, an unpaired
t-test with Welch’s correction was performed. p = 0.0035 for WT vs. ami1Mab and p = 0.0053 for
ami1Mab vs. ami1Mab complemented (compl.) n.s. stands non statistically significant and ** stands
for p < 0.01.
∆
ami1Mab
(A
) Schematic representation of the genomic
ami1Mab strains of Mycobacterium
) PCR
.
∆
∆
B
∆
C
)
∆
D
∆
E
µ
µ
∆
µ
∆
∆
∆

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The size of the amplicon
∆
ami1 mutants relative to the wild-type strain confirms the proper
deletion of the gene in both the S and R morphotypes (Figure 1B). Complemented deletion strains
were generated by cloning ami1Mab into the integrative pMV306 [34], yielding pMV306::ami1Mab and
allowing gene expression under the control of the strong hsp60 promoter.
Growth curves of the ∆ami1Mab and the corresponding complemented strains were similar to
those of their wild-type progenitors, suggesting that the inactivation of ami1 does not affect the growth
of M. abscessus in broth medium (Figure 1C). Overall, these results indicate that ami1Mab is dispensable
for M. abscessus growth in vitro.
3.1.2. ami1Mab Deletion Mutants Do Not Present Division Defects
It was previously reported that 20% of the M. smegmatis ami1 deletion mutants harbored an atypical
cell division [11], although this observation was neither confirmed in an independent study [10] nor
in a M. tuberculosis ami1 deletion mutant [20]. We, therefore, investigated the shape and morphology
of the M. abscessus S ∆ami1 mutants by transmission electron microscopy (TEM) (Figure 1D). The R
variant was not included in this analysis because of its high propensity to aggregate, making it
difficult to obtain homogeneous bacterial suspensions, therefore rendering TEM processing very
challenging. Analysis of approximately 500 individual WT,
cells failed to show morphological or cell division defects. Chain formation, typifying bacteria
unable to divide properly, was not observed in ami1Mab, thus differing from the chaining phenotype
∆ami1Mab, and ∆ami1Mab complemented
∆
observed in Helicobacter pylori [35] or E. coli [17] strains in which single or multiple amidase-encoding
genes were invalidated. Analysis of the size of a large number of single bacilli showed a slight but
statistically-significant average shorter cell length for the WT (1.27
strains (1.27 µm ± 0.017) as compared with the ∆ami1Mab mutant (1.34 µm ± 0.017) (Figure 1E).
µ
m
±
0.018) and complemented
3.1.3. ∆ami1Mab Is More Sensitive to β-Lactam Antibiotics and Detergent
We showed previously that inactivation of a dehydratase involved in cell wall biosynthesis was
associated with increased susceptibility to detergents in M. abscessus [36]. In this study, we assessed
whether ami1 deletion affects cell wall integrity and thus susceptibility to sodium dodecyl sulfate
(SDS) on solid medium. A moderate increase in sensitivity to SDS treatment at a concentration of
0.0025% was observed in ∆
ami1 as compared to the WT strain, particularly visible at the 10−2 dilution
(Supplementary Figure S1). The effect was more pronounced in the presence of 0.0037% SDS for the R
variant. This low detergent sensitivity was partially restored upon complementation with ami1Mab
(Supplementary Figure S1).
As the
inquired whether a similar antibiotic susceptibility profile is seen in
-lactam antibiotics (imipenem, cefuroxime, and cefamandole), known to target PG, were two times
∆
ami1 M. smegmatis and M. tuberculosis were more susceptible to antibiotics [11
,20], we next
∆
ami1Mab. The MIC of several
β
lower in the ∆ami1Mab strains compared to their respective parental progenitors (Table 1).
Table 1. MIC determination
M. abscessus S
∆ami1
M. abscessus S
∆ami1 Compl.
M. abscessus S WT
Imipenem
Cefoxitine
4
16
2
4
16
16
Cefuroxime
Cefamandole
Amikacin
187–375
2000
16
93
500–1000
16
187
2000
16
Clofazimine
Tigecycline
0.78
4
0.78
0.78
4
4
MIC in µg/mL of the different antibiotics were determined as described in the Materials and Methods, Section 2.8.

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The wild-type sensitivity to these drugs was restored upon complementation. In contrast, the MIC
of antibiotics that do not target PG (amikacin, clofazimine, and tigecycline) remained unaffected
(Table 1).
Taken together, these results suggest that although ∆ami1Mab mutants do not present major growth
and cell division defects, they display a slightly-increased susceptibility profile to SDS and to
antibiotics, suggestive of a cell wall alteration.
β-lactam
3.2. Analysis of the Virulence Phenotype of the M. abscessus ami1 Deletion Mutant
3.2.1. ∆ami1Mab Is Not Impaired in Uptake and Persistence in Human Macrophages
The recent study by Healy et al., emphasized the importance of Ami1 for M. tuberculosis
virulence, particularly for the successful establishment of chronic infection in mice [20].
Because immunocompetent mice rapidly clear the infection with M. abscessus, this model is not
permissive to the establishment of a chronic infection [23]. We thus analyzed the virulence of the
∆
ami1Mab mutants in the human THP-1 macrophages. Both S and R ∆ami1Mab strains were internalized
by macrophages similarly to the parental strains, as judged from the CFU determination at day 0
(Figure 2A).
The capacity of the mutants to replicate inside macrophages was also slightly modified as CFU
counts between day 1 and day 5 post-infection modestly increased as compared to the CFU of
the corresponding WT strains (Figure 2A). Unexpectedly, the complemented strains overexpressing
Ami1 showed an enhanced capacity to invade macrophages (up to 1-Log and 0.5-Log higher for
the R and S variants at day 0, respectively) as compared to corresponding WT and mutant strains.
However, the difference was less prominent at day 5 post-infection (Figure 2A).
These results suggest that ami1Mab is not required for macrophage internalization and intracellular
replication but that overexpression of Ami1Mab confers an intracellular survival advantage to the bacilli.

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Figure 2. Assessment of
THP-1 macrophages with amikacin maintained at 50
day 0 to day 5 for CFU enumeration. Bacteria were plated at day 0 (2–4 h phagocytosis followed
by a 2 h amikacin treatment at 250 g/mL) day 1, 3 and day 5. Histograms and error bars represent
∆
ami1Mab in human macrophage and zebrafish larvae. (
A) M. abscessus-infected
µg/mL in the RPMI medium were lysed from
µ
means and standard deviations calculated from three independent experiments. For statistical analysis,
the unpaired t-test with Welch’s correction was applied. *, **, ***, and **** stand for p < 0.1, p < 0.01,
p < 0.001, and p < 0.0001, respectively. Left and right panels are for M. abscessus S and R variants,
respectively. The M. abscessus S and R wild-type strains used as controls were not transformed with
pMV306. (B) Survival curves of zebrafish embryos infected with the different M. abscessus strains.
The data shown are corresponding to a pool of data from three independent experiments. For each
experiment, 20 embryos were injected with 200–300 CFUs, respectively. Left and right panels are
for M. abscessus S and R variants, respectively. The M. abscessus S and R wild-type strains used as
controls were transformed with pTEC27 but not transformed with pMV306. (C) Representative images
of infected zebrafish embryos at 5 days post-infection. Scale bar represents 1 mm. Infection foci are
displayed in red.

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3.2.2. Virulence of ∆ami1Mab Is Not Affected in the Zebrafish Model of Infection
Next, we assessed the virulence phenotype of
are particularly suitable to study the chronology of M. abscessus infection [22]. WT,
∆
ami1Mab strains in zebrafish embryos, which
ami1Mab
∆
,
and complemented strains were first transformed with pTEC27 encoding tdTomato, allowing infection
to be monitored in real-time by fluorescence microscopy. Bacterial suspensions (200–300 CFU) were
microinjected in the caudal vein of zebrafish embryos at 30 h post-fertilization [22]. As reported
previously, the S variant is predominantly avirulent in the embryos, with only 20% of embryo death
at 12 days post-infection [22,37]. The survival curves of the embryos infected with ∆ami1Mab and
the complemented strains were comparable to that of the WT (Figure 2B, left panel). In contrast,
the R strains appeared more virulent with 70% larval death at 12 dpi, in agreement with previous
reports [22] (Figure 2B, right panel). A slight but not statistically-significant killing delay was observed
for the ∆ami1Mab- complemented strain (Figure 2B, right panel). These findings were corroborated
with whole-embryo imaging, which failed to show infection foci in the embryos infected with the
different S-derivative strains (Figure 2C, left panel), while robust infection occurred in the embryos
challenged with the R derivative strains, typified by the presence of massive infection and important
bacterial burdens within the central nervous system (Figure 2C, right panel). Together, these results
suggest that Ami1Mab is not required for M. abscessus virulence in zebrafish.
3.3. Biochemical and Structural Characterization of Ami1Mab
3.3.1. Ami1Mab Is an Active PG Hydrolase
The biological function of Ami1 in M. smegmatis and M. tuberculosis underscores its requirement
for growth, cell division, and virulence. Ami1 appears, therefore, as a potential drug target to exploit
in mycobacteria. The macrophage infection results highlight the intracellular advantage conferred
by overexpression of Ami1Mab in M. abscessus. Comforted by these studies, we initiated biochemical
studies, searched for specific inhibitors, and characterized the structure of Ami1Mab
.
Ami1Mab was purified without its first 40 amino acids in N-terminus, predicted to correspond
to the signal peptide required for secretion. It was purified as a recombinant protein expressed in
E. coli following a three-step purification procedure, yielding a highly pure and homogeneous protein
preparation (Figure 3A).
The hydrolytic activity of pure Ami1Mab was next monitored by thin-layer chromatography
(TLC) and using the commercial N-acetylmuramyl-l-alanyl-d-isoglutamine substrate, also known as
muramyl-dipeptide (MDP). If the amidase is active on MDP, N-acetylmuramic acid and l-Ala-d-IsoGln
are expected to be released (Figure 3B). Substrate and reaction products could be visualized following
separation by TLC, spraying with ninhydrin, and charring. We first monitored the hydrolysis of MDP
using a range of protein concentrations (from 2 to 32 µM). MDP was rapidly hydrolyzed by Ami1Mab,
indicating that it is an active PG hydrolase that cleaves the amide bond between the N-acetylmuramic
acid and l-Ala (Figure 3C). Since Zn2+ is an essential divalent ion for PG amidases, we next assessed
the dependence of Ami1Mab for this metal ion. Incubating Ami1Mab with the chelating agent EDTA
prior to the reaction almost completely abolished the enzymatic activity (Figure 3D). These results
demonstrate that, like Ami1Mtb (Figure 3D), [15,16] Zn2+ is required for catalysis of Ami1Mab
.

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Figure 3.
Biochemical characterization of Ami1Mab
.
(A) Ami1Mab elution profile on
Superdex 75 10/300 GL size-exclusion chromatography column. The denaturating polyacrylamide
electrophoresis gel attests to the high protein purity after three chromatography steps. (B) Schematic of
the muramyl-dipeptide hydrolysis assay. Ami1Mab cleaves muramyl-dipeptide (MDP) to yield
N-acetylmuramic acid (MurNAc) and l-Ala-d-IsoGln (dipeptide). (C) The thin-layer chromatography
(TLC)-based activity assay revealed a correlation between the disappearance of MDP and the
concentration of Ami1Mab. Pure MDP, MurNAc, and dipeptide (DP) were loaded on the right part of
the TLC plate as migration controls. TLC was revealed by ninhydrin and charring. (D) The enzymatic
activity of Ami1 is Zn2+-dependent. Activity of both Ami1Mab and Ami1Mtb is inhibited after incubation
with EDTA.
3.3.2. Thermal-Shift-Based Drug Screening Identified Three Potent Inhibitors of Ami1Mab
Despite being a robust assay to monitor PG hydrolytic activity, the TLC-based assay is not
compatible with a large screening program to identify specific inhibitors of amidases. To overcome this
hurdle, we employed thermal-shift assay (TSA) [38,39] to screen a large chemical library. This technique

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is based on the principle that if a small molecule binds to the protein, the melting temperature (Tm) of
the protein is modified. The Tm can easily be followed by adding a fluorescent dye, SYPRO orange,
that binds to hydrophobic patches of a protein that are exposed after melting. Monitoring of the signal
can be performed by standard real time-PCR. It is noteworthy that this method does not necessarily
select for direct enzyme inhibitors but identifies molecules that can stabilize or destabilize the protein
of interest.
A TSA approach was developed in a 96-well plate format to screen the 9200 compounds of the
Curie-CNRS library. The entire screen consisted of 115 plates, each containing 80 chemicals as well
as a non-treated control and Ami1Mab plus 5% dimethyl sulfoxide (DMSO). Compound selection
was based on Tm values ranging from 2 to 5 ◦C higher than the Tm of Ami1Mab plus DMSO control.
Application of this threshold allowed the identification of 654 compounds. All selected compounds
were then assessed individually (at a concentration of 0.125 mg/mL) for their ability to inhibit Ami1Mab
activity using the TLC-based assay. This narrowed down our initial screen to 54 compounds. A third
round of selection was next applied, keeping only compounds inhibiting the enzyme activity at a
concentration below 1 mM. This led to the selection of only three potential hits.
3.3.3. Biochemical Characterization of Ami1Mab Inhibitors
Among these three hits, two belong to the pyridine chemical group while the third one
is a tetrazole-derived compound. Compound
acid while compound is 2-(1H-1,2,3,4-tetrazol-5-yl)aniline and compound
2-chloropyridine-3,4-dicarboxylic acid (Figure 4A).
1
is a 4-chlorofuro [3,2-c]pyridine-3-carboxylic
2
a
3
is a
The validity of these hits was subsequently reassessed by TSA with respect to their capacity to
increase the Tm of Ami1Mab. The three compounds increased the Tm of Ami1Mab by 5.5, 2.4, and 3.5 ◦C,
respectively, when compared with Ami1Mab incubated with DMSO (Tm= 45 ◦C) (Figure 4B).
The capacity of the three compounds to inhibit the MDP hydrolytic activity of Ami1Mab was
determined in a concentration-dependent manner using the TLC-based activity assay. Compound
displayed the highest inhibitory effect, starting at 150–200 M, followed by compound between
250–500 M, and compound , which showed a moderate inhibitory activity starting at 800
(Figure 4C).
1
µ
3
µ
2
µM
Since two of the inhibitors possess carboxylic groups, we addressed whether the inhibitory effect
was not due to a pH change during the reaction. The Ami1Mab activity was thus tested under various
acidic conditions, resulting in an active protein until pH 2 was reached. In parallel, products and
substrates of the reaction were separated by TLC under the same pH range to ensure that the observed
inhibition was not due to a TLC migration artifact dependent on the pH (Figure 4D).
These biochemical data indicate that all three compounds inhibit the Ami1Mab activity in vitro
.
To enlighten the mode of inhibition of these compounds, structural studies of Ami1Mab were undertaken.

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Figure 4. Characterization of Ami1Mab inhibitors. (
selected by thermal-shift assay. Chloride atom is in green, carbon in black, oxygen in red and nitrogen
in blue ( ) Melting curves of Ami1Mab in the presence of compound (blue) at 244 M, compound
(red) at 879 M, and compound (blue) at 845 M as compared with protein incubated with 5% DMSO
(black). ( ) Assessment of Ami1Mab inhibition by compounds 1, 2, and 3 by TLC. ( ) Evaluation of the
Ami1Mab activity over an acidic pH range.
A) Chemical structure of the three compounds
B
1
µ
2
µ
3
µ
C
D

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3.3.4. Biochemical Characterization of Ami1Mab Inhibitors
The crystal structure of Ami1Mab was solved at a high resolution in its free form and also bound
to l-Ala-d-IsoGln, one of the end-product of the reaction (Table 2).
In addition, to have a complete structural view and to compare the Ami1 structures from
M. tuberculosis, M. smegmatis, and M. abscessus, we have also solved the crystal structure of Ami1Msm
at a high resolution (Table 2).
Ami1Mab and Ami1Msm displayed an overall globular structure comprising a central
β-sheet
(β1, 4, 5, 6, 7, 8) surrounded by strands
310 helix (η1) (Figure 5A).
β2 and β3 forming an antiparallel β-sheet, six α-helices, and one
The catalytic zinc is coordinated by the highly-conserved triad which constitutes the amidase_3
motif signature, namely H25, E60, and H115 in Ami1Mab and H51, E86, and H141 in Ami1Msm
(Figure 5A, left and middle panels). Ami1Mab shares 68% and 65% primary sequence identity with
Ami1Msm and Ami1Mtb, respectively, while Ami1Msm and Ami1Mtb possess 79% identity. All three
structures of the apo forms are very similar since the superposition of Ami1Mab with Ami1Msm and
Ami1Mtb (PDB id: 4LQ6) leads to r.m.s.d. values of 0.46Å and 0.55Å, respectively, while Ami1Msm and
AmiMtb display a r.m.s.d. value of 0.51Å.
The co-crystal structure of Ami1Mab bound to l-Ala-d-IsoGln was also solved at a high resolution.
The Fo-Fc simulated-annealed OMIT map attests to the presence of the dipeptide in the active site
of the protein (Figure 5B). This structure represents the enzyme state after the hydrolysis of MDP.
The dipeptide binds close to the Zn2+ ion (Figure 5C). The l-Ala residue of the dipeptide is tightly
bound through water-mediated H-bonding established with the side chains of Asp117 and Arg173,
itself stabilized by the side chain of Asp175. Glu190 interacts with the dipeptide ligand and this residue
was shown to be critical for the in vitro and in vivo activity of Ami1Mtb [15,16,20]. The carboxylic
group of d-IsoGln is contacted by a salt-bridge interaction with Lys45 as well as by the main chain
of Ala177 and Gly178. The side chain of Gln48 finalizes the interaction with the amide group of
d-IsoGln. All these interactions are conserved in the dipeptide-bound Ami1Mtb structure (PDB id:
4M6G) (Figure 5D) and presumably also in Ami1Msm since all the residues interacting with the ligand in
Ami1Mab and Ami1Mtb are conserved in Ami1Msm (Figure 5E). We noticed, however, a slight difference
in ligand binding in the structures of Ami1Mab and Ami1Mtb bound to l-Ala-d-IsoGln. In the latter,
the NH2 group of the Ala residue of the dipeptide is close to Zn2+ and only interacting with Glu200
(Figure 5C,D). In Ami1Mab, the amine group is interacting with the side chains of Asp117, Glu190 and,
Arg173 (Figure 5C).
Overall, these findings demonstrate that Ami1Mab is a functional PG hydrolase that shares a very
strong structural conservation with Ami1Mab and Ami1Mtb
.

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Table 2. Crystallographic data collection and refinement statistics.
Ami1Mab
Ami1Mab
Ami1Msm
l-Ala-d-IsoGln
PDB accession
7AGL
ESRF-ID30B
0.979
7AGO
SLS-PXIII-X06DA
1
7AGM
SLS-PXIII-S06DA
0.979
Beamlime
Wavelength
Resolution range
Space group
38.4–1.6 (1.65–1.6)
P 41 21 2
42.8–1.7 (1.76–1.7)
P 41 21 2
46.7–1.35 (1.39–1.35)
P 1 21 1
85.92 85.92 73.66
90 90 90
85.74 85.74 75.63
90 90 90
41.85 68.84 63.72
90 91.866 90
Unit cell
Total reflections
Multiplicity
228,475 (22,850)
6.4 (6.3)
824,584 (80,940)
26.1 (26.1)
99.8 (100)
18.8 (2.4)
526,861 (52,052)
6.7 (6.6)
Completeness (%)
Mean I/sigma(I)
Wilson B-factor
R-meas
96.7 (99.2)
11.1 (1.3)
22.4
99.9 (99.9)
16.0 (1.3)
15.4
20.9
0.094 (1.25)
0.99 (0.48)
0.131 (1.29)
0.99 (0.85)
0.069 (1.47)
0.99 (0.61)
CC1/2
Reflections used in
refinement
35,744 (3601)
31,542 (3107)
79,191 (7884)
Reflections used for R-free
2000 (201)
0.168 (0.294)
0.195 (0.329)
2000 (197)
0.162 (0.256)
0.186 (0.307)
3960 (394)
0.155 (0.285)
0.180 (0.319)
R-work
R-free
Number of non-hydrogen
atoms
1831
1857
3672
Macromolecules
Ligands
1598
1
1628
15
3218
2
Solvent
232
242
452
Protein residues
RMS (bonds, Å)
RMS (angles, ◦)
Ramachandran favored (%)
Ramachandran allowed (%)
Ramachandran outliers (%)
Rotamer outliers (%)
Clashscore
212
214
434
0.007
0.89
97.14
2.86
0.00
1.18
0.63
27.6
25.8
20.4
40.1
0.006
0.79
97.17
2.83
0.00
1.18
1.55
22.6
20.5
33.4
35.8
0.005
0.77
97.91
2.09
0.00
0.30
1.72
23.2
21.6
18.3
34.3
Average B-factor (Å2)
Macromolecules
Ligands
Solvent
Values in parentheses are for the last resolution shell.

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Figure 5. Structural comparisons of the Ami1Mab, Ami1Mtb, and Ami1Msm. (
of Ami1Mab (left), Ami1Msm (middle) and Ami1Mtb (PDB id: 4LQ6) (right). Each structure is shown as a
cartoon representation and is colored differently and according to its secondary structures. , and
indicate alpha-helices, beta-strands, and 310 helices, respectively. The catalytic zinc ion (Zn2+) appears
as a wheat-colored sphere. ( ) Fo-Fc simulated-annealed OMIT map. The electron density map as
seen in the Ami1Mab:dipeptide co-crystal structure and surrounding l-Ala-d-IsoGln (yellow sticks) is
displayed as a grey mesh and contoured at 3 level. ( ) Close up of the active site of Ami1Mab as
A) Overall crystal structure
α
,
β
η
B
σ
C
seen in the Ami1Mab: dipeptide co-crystal structure. l-Ala-d-IsoGln (yellow sticks) tightly interacts by
H-bonding and salt-bridge as illustrated by the dashed lines. Water molecules appear as green spheres.
(
D) The figure shows the molecular interactions seen in the co-crystal structure of Ami1Mtb bound to
dipeptide (PDB id: 4M6G). (E) Active site of the Ami1Msm
.

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3.3.5. In Silico Docking Allows Predicting That Ami1Mab Inhibitors May Compete with
Substrate Binding
We did not succeed in solving the co-crystal structure of Ami1Mab bound to any of the three
selected inhibitors. The relatively-low potency of the inhibitors is in favor of a low affinity of these
inhibitors for the amidase and might explain our vain attempt at obtaining the co-structures. As an
alternative, in silico docking was performed to gain insight into their potential mechanism of inhibition.
To facilitate the search for the potential inhibitor’s binding-site, the Ami1Mab crystal structure was
probed with the CASTp server to identify cavities in the protein structure [40]. This analysis led to the
identification of a single cavity of a volume of 72 Å2 comprising the active site of Ami1Mab. In silico
docking by defining a search region in this area was performed with the AutoDock Vina software [31].
The three inhibitors could be docked into the Ami1Mab active site with similar binding energy (
(Figure 6).
∆
G◦)
Figure 6. In silico docking of the three inhibitors. The figure depicts the best docking poses of the three
compounds. All three inhibitors appear able to bind in the Ami1Mab active site with similar binding
energies. Inhibitors are displayed in stick representation. Nitrogen atoms are in blue, oxygen in red,
hydrogen in white and carbon atoms in yellow for the ligand and grey for the protein.
Inhibitor 1 (
∆
G◦ =
6.5 kcal/mol) appears to be able to occupy the active site by interacting with
−
the main chain of Ala177 and Gly178 through its carboxylic group and a weaker H-bonding with the
Lys45 side chain. The nitrogen of the pyridine moiety is interacting with the side chain of Asp22, His25,
Gln48, and His115 interact as well by Van der Waals interactions. Inhibitor 2 (
∆
G◦ =
6.0 kcal/mol)
−
interacts by H-bonding with Lys45 and Gln48 as well as with His25 and His115 by weaker contacts.
Inhibitor 3 could be docked with a ∆G◦ = −6.4 kcal/mol, where multiple interactions involve the two
carboxylic groups. The carboxylate in position 3 interacts with the side chains of Gln48, Arg99, and the
main chain of His25, while the carboxylate in position 4 is able to contact the side chains of Asp22,
Ser113, and Lys186. Ile96 side chain is in close vicinity with the chloride in position 2.
The proposed docking poses strongly suggest that the three inhibitors are able to occupy the
active site in a manner similar to the dipeptide seen in the crystal structure. The carboxylate group
of inhibitor 1 is interacting with the same residues Lys45, Ala177, and Gly178 involved in binding
of the d-IsoGln carboxylate. The docking study supports the view that these inhibitors might act by
competing with the binding of the PG peptide stem.
4. Discussion
The PG biosynthesis pathway has long been overlooked as an interesting drug target for
subsequent drug developments against mycobacterial diseases, largely due to the fact that most
β
-lactam antibiotics inhibiting transpeptidation show poor activity against mycobacteria. This is
explained by the presence of specific secreted -lactamases, which neutralize these antibiotics.
However, targeting PG biosynthesis in mycobacteria has recently regained interest after the discovery
β

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of
β-lactamases inhibitors which, together with
β-lactam antibiotics, can successfully inhibit growth of
M. tuberculosis [41] and M. abscessus [42,43].
PG remodeling in mycobacteria and particularly PG hydrolytic enzymes has been more thoroughly
investigated in recent years [44]. The NlpC/P60 endopeptidases RipA, RipB, RipC, and RipD have
been widely scrutinized [7,20,45–50]. The functional redundancy and the non-essential individual
roles of RipA and RipB hydrolyzing the PG peptide stem between the 2nd and 3rd amino acid were
established both in M. smegmatis [50] and in M. tuberculosis [20]. Redundancy of PG amidases has
also been proposed since single or multiple deletion of amide-encoding genes (ami2, ami3, and ami4)
in M. smegmatis is not detrimental for growth [10]. The major septal PG hydrolase RipA can sustain
the amidase Ami1 PG hydrolytic activity in M. tuberculosis [20]. However, despite the fact that
mycobacterial amidases share redundant functions and that ami1 is not essential for growth or cell
division of M. tuberculosis, Ami1 is required for the persistence of the bacilli within the host [20].
This contrasts with studies reporting that Ami1 is essential for cell separation and that inactivation of
the corresponding gene is associated with atypical cell division phenotypes [11,13].
Apart from M. smegmatis, little is known about PG-hydrolyzing enzymes in other non-tuberculous
mycobacteria. To fill this gap, we investigated the role of ami1 in the human pathogen, M. abscessus.
We showed that ami1Mab, as reported in M. smegmatis and M. tuberculosis, is dispensable for cell growth
in the R and S variants. Consistent with observations in M. tuberculosis, ami1Mab is also not essential
for cell division in M. abscessus. We also showed that ami1Mab is neither required for invasion nor
for persistence in macrophages while overexpression of ami1Mab partially increased the uptake and
intracellular survival of the bacilli in THP-1 macrophages. In M. smegmatis, deletion of ami1 increases
type 3-3 crosslinks as compared to the WT strain [11]. In M. abscessus, changes in PG cross-link ratio and
type of crosslinking is correlated with the growing phase. PG of exponentially-growing M. abscessus
has more cross-links characterized by an increasing ratio of 3-3 cross-links [51]. As the deletion of ami1
in M. smegmatis increases the 3-3 crosslinks ratio [11] one could propose that overexpression of ami1Mab
might trigger a decrease in 3-3 and consequently an increase in 4-3-type cross-links, which may confer,
for yet unidentified reasons, intracellular advantage. This, however, remains to be further investigated
in future studies. The evidence of ami1Mab being involved in PG remodeling was also supported by a
slight increase in the susceptibility to
β-lactam antibiotics in ∆ami1Mab. MICs for non-targeting PG
antibiotics were not modified, suggesting that ami1 deletion triggers specific cell wall modification in
PG rather than modifications of the permeability of the cell wall. The cell wall impairment in
is also supported by a modest increased sensitivity to detergent.
∆ami1Mab
Infections studies in zebrafish embryos and macrophages suggest that ∆ami1Mab is as virulent
as its WT progenitor. In light of the recent studies related to Ami1 from M. tuberculosis and M.
smegmatis, this work on Ami1Mab emphasizes functional differences regarding the contribution of Ami1
from the three species regarding growth, invasion, and persistence. These findings are particularly
unexpected considering that all three mycobacteria possess the four amidase-encoding genes and
that the corresponding orthologues share a highly-conserved primary sequence, suggesting that these
functional differences are unlikely connected to their enzymatic activity per se. Super-resolution
microscopy analyses unraveled differences in the spatiotemporal synthesis of PG in M. smegmatis
and M. tuberculosis [52], which may explain the functional divergence of Ami1 between the different
mycobacterial species.
Growth conditions and bacterial environment also appear crucial in modulating PG remodeling
enzymes as reported for ripA deletion mutant which is only impaired in cell division under acid stress
conditions (8, 37). A possible explanation for the difference of Ami1 contribution may arise from the
respective lifestyles of the environmental M. abscessus as opposed to the human-specific M. tuberculosis
pathogen. In this context, it is noteworthy that the obligate intracellular Mycobacterium leprae, carrying a
reduced genome, encodes for only two amidases, namely Ami1 (ML2131) and Ami2 (CwlM, ML2704).
This supports the view that Ami1 and Ami2 are required for the intracellular mycobacterial lifestyle and
were conserved in M. leprae due to the selection pressure. Additionally, transcriptomic analyses showed

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up- and down-regulation of multiple genes in M. abscessus subspecies massiliense when comparing
planktonic, intra-macrophage, and intra-amoebal growth [53]. These included genes from the PG
metabolic pathway found to be down-regulated in macrophages as compared with planktonic growth
conditions [53].
Biochemical and structural characterizations of Ami1Mab unambiguously demonstrate that it is an
active PG amidase, very closely related to Ami1Msm and Ami1Mtb. A noticeable difference in dipeptide
binding was observed between Ami1Mab and Ami1Mtb. This could be explained by the fact that the
Ami1Mab structure was co-crystallized with the dipeptide while the structure of Ami1Mtb which was
generated by crystallization with MDP, likely hydrolyzed during the crystallization process and only
the dipeptide part was visible in the structure. In the Ami1Mab co-structure, the peptide is further away
from the catalytic Zn2+. This state might, therefore, represent a post-hydrolysis state i.e., a state where
the product is about to be released from the enzyme.
This study describes the first inhibitors of this class of enzymes, which may represent a first step
towards the design of more potent inhibitors. Further work through structure–activity relationship
approaches is expected to improve the properties of these inhibitors.
In summary, this work represents the first study describing a PG hydrolytic enzyme in M. abscessus.
Further work is needed to (i) explore the role of Ami1 in M. abscessus and its intricate relationships
with other amidases/endopeptidases such as RipA and (ii) study its regulation under various
stress conditions.
Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4409/9/11/2410/s1,
Table S1: Primers sequences; Figure S1: Detergent susceptibility assay.
Author Contributions: Conceptualization, T.K., N.V.W., M.D.J., K.K.S., M.B.T., C.B., L.K., and M.B.; methodology,
T.K., N.V.W., M.D.J., K.K.S., M.B.T., C.B., H.M.A.B.A., L.K., and M.B.; validation, T.K., N.V.W., M.D.J., K.K.S., M.B.T.,
C.B., L.K., and M.B.; formal analysis, T.K., N.V.W., M.D.J., K.K.S., M.B.T., C.B., L.K., and M.B.; investigation, T.K.,
N.V.W., M.D.J., K.K.S., M.B.T., C.B., A.N., H.M.A.B.A., C.H., and M.B.; writing—original draft preparation, M.B.;
writing—review and editing, N.V.W., M.D.J., L.K., and M.B.; supervision, L.K. and M.B.; project administration,
L.K. and M.B.; funding acquisition, L.K. and M.B. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by the Fondation pour la Recherche M
é
dicale (FRM) (grant number
DEQ20150331719 to L.K.). T.K. was supported by a Ph.D. fellowship from Minist
è
re de l’Enseignement Sup rieur,
é
de la Recherche et de l’Innovation (MESRI). N.V.W. was supported by a postdoctoral fellowship from the
Infectiopôle Sud.
Acknowledgments: We thank the staff at the Swiss Light Source and European Synchrotron Radiation Facility
beamlines for support during data collection, W. Daher for preparing THP-1 macrophage cultures, A. Viljoen for
fruitful discussions, and F. Roquet-Banères for technical support and help with MIC determination.
Conflicts of Interest: The authors declare no conflict of interest.
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