Glycoconjugate Journal (2018) 35:421–432

https://doi.org/10.1007/s10719-018-9842-7

MINI-REVIEW

Peptidoglycan in Mycobacteria: chemistry, biology and intervention

Tripti Raghavendra 1 & Saniya Patil 1 & Raju Mukherjee 1

Received: 12 June 2018 / Revised: 20 August 2018 / Accepted: 5 September 2018 / Published online: 19 September 2018
# Springer Science+Business Media, LLC, part of Springer Nature 2018

Abstract
Peptidoglycan, a major glycoconjugate in the mycobacterial cell envelope provides strength to resist osmotic stress and plays a
pivotal role in maintaining the cellular morphology. Several unique growth stage specific structural alterations occur in its
constituent monosaccharides and peptides that allow Mycobacterium to survive nutrient starvation and environmental stress.
Here, we discuss the enzymes involved in its intricate biosynthesis that are novel targets for therapeutic intervention and provide
an opportunity for potential antibiotic adjuvants. We also revisit the enzymatic steps which are critical for maintaining the
equilibrium between peptidoglycan synthesis and hydrolysis during cellular growth and division specifically focused on the
importance of cell wall remodelling during Bexit from dormancy^ in Mycobacterium, a phenomenon with tremendous physio-
logical and therapeutic importance for intervention in mycobacterial infections.

Keywords Tuberculosis . Cell envelope . Peptidoglycan . Biosynthesis . Remodeling

Introduction multi-layered cell wall provides the first layer of protection,

Organisms grouped under Mycobacteria represent a wide
range of genus ranging from free-living non-
pathogenic saprophytes to the opportunistic and the patho-
genic Mycobacterium tuberculosis (Mtb), which was the
cause of 6.3 million new cases of human tuberculosis in
2016 [1]. Mycobacterial pathogens have thrived and
evolved successfully through decades posing a huge chal-
lenge for creation of effective antimicrobials and vaccines.
An important reason behind becoming world’s most suc-
cessful pathogen is attributed to the presence of phenotypic
resistance towards antibiotics, which is largely contributed
by the persistent nature of its physiology and the presence
of a relatively impermeable cell envelope. The lipid content
of cell envelope accounts for upto 40% of the total cell mass
which is enormously high as compared to those of Gram
positive or Gram negative bacteria [2]. Presence of a lipid
rich cell envelope also explains the reason for the repeated
discovery of new antimicrobials which are highly hydro-
phobic and with targets in the cell wall [3]. While the
its role in uptake of nutrients and mediating cross-talks
during its interaction with host system is not completely
understood. The global architecture of the cell envelope
when viewed through an electron microscope suggests the
presence of several electron dense and electron transparent
regions [4]. The cytoplasm is protected by a bilayered inner
membrane which is surrounded by a complex layer of con-
jugated carbohydrates and lipids forming a Bcell wall core^,
that in turn is coated with an outer layer of capsular poly-
saccharide [5]. In this mini review we try to take a deeper
insight into the structure of this fascinating biopolymer,
focusing primarily on the unusual peptidoglycan which is
unique to this group of organisms. We also revisit its biosyn-
thetic pathway and particularly discuss the alteration in its
structure upon exposure to environmental stress and nutrient
starvation. The importance of peptidoglycan remodelling dur-
ing cell division and during reactivation from dormancy is
highlighted together with the potential biosynthetic steps that
are crucial for novel therapeutic intervention in mycobacterial
infections including antibiotic adjuvants.

* Raju Mukherjee
raju.mukherjee@iisertirupati.ac.in
Ultrastructure of the cell envelope
1
Department of Biology, Indian Institute of Science Education and Before we discuss the role and importance of peptidoglycan in
Research, Tirupati, Karakambadi Road, Tirupati 517507, India mycobacterial cell envelope, it would be prudent to describe
422
its ultrastructure in detail. The Bcell wall core^ is composed of
a covalently connected peptidoglycan with arabinogalactan
and the mycolic acids to form the mAGP complex (mycolyl-
Arabinogalactan-Peptidoglycan) and contains proteins, free
lipids and lipoglycans including lipomannans and
lipoarabinomannans (Fig. 1a) [6]. The peptidoglycan consists
of an aminosugar backbone crosslinked by the tetrapeptide
side chains with unique modifications which will be discussed
in the later sections [7]. The second component of this cell
wall core is the highly branched heteropolymeric
arabinogalactan where C-5 of three of the 30 alternating β
(1 → 5) and β (1 → 6) linked galactofuranose residues are
decorated with extensively branched arabinofuranose residues
at positions 8, 10, and 12 to form mature arabinogalactan.
The arabinogalactan is further capped with long chain fatty
acids consisting of C70-C90 α-alkyl branched and β-
hydroxylated fatty acids known as mycolic acids esterified at
the non-reducing terminus of the pentaarabinosyl motifs of
arabinogalactan [8]. The mycobacterial outer membrane, ini-
tially thought to be composed of a tightly packed layer of
mycolic acids was found to consist of a less fluidic inner
leaflet, contributing towards low permeability of hydrophilic
molecules and a more fluidic outer leaflet composed of short
chain fatty acids and free lipids [9]. This hypothesis was val-
idated by reverse micellar extraction of membrane lipids that
revealed the presence of a true bilayer membrane [10]. The
Fig. 1 Global architecture of M.tuberculosis cell envelope. a The Bcell
wall core^ is composed of a covalently connected peptidoglycan with
arabinogalactan and the mycolic acids to form the mAGP complex
(mycolyl-Arabinogalactan-Peptidoglycan) and contains periplasmic
proteins and lipoglycans including lipomannans and
lipoarabinomannans. Short chain fatty acids, free glycolipids and outer
Glycoconj J (2018) 35:421–432
outer leaflet was found to contain non-covalently associated
glycolipids, importantly the sulpholipids, phthiocerol
dimycocerosate, diacyltrehalose and the trehalose mono and
dimycolates also known as the ‘cord factor’, all contributing
significantly towards pathogenicity [11]. The electron trans-
parent region between the inner and outer membrane, posited
as the pseudo-periplasm in mycobacteria, has been recently
observed to be a critical component in the cell envelope
harbouring many vulnerable membrane targets [12]. All of
these features have attracted scientists to target the essential
components of cell wall for therapeutic interventions particu-
larly the steps involved in their biogenesis which is evident
from the large number of new candidate drugs [13, 14] that
inhibit cell wall synthesis in addition to the successful drugs
including isoniazid, ethambutol, ethionamide, carbapenems
and D-cycloserine with targets in the cell envelope [15, 16].
Atypical Gram character of cell envelope
and permeability
Mycobacteria generally stain purple owing to the similarity of
the peptidoglycan layer with that of other Gram positives [17].
Based on the high sequence conservation shown by the im-
portant genes and their evolutionary distances from ancestral
sequences, they may be classified as Gram positives [18].
membrane proteins make up the outer most layer which together with the
mycolic acid layer forms the impermeable Bmycomembrane^. b Types of
peptide crosslinks in peptidoglycan observed during the different growth
phases. (Rpf-Resuscitation promoting factor; Ami-Amidase; RipA-Rpf
interacting protein A)
Glycoconj J (2018) 35:421–432
However, presence of an outer membrane and the specialized
proteins in the mycobacterial cell wall which are the typical
characteristics of Gram negatives strengthen the ambiguity
with respect to the ribosomal analysis and places
Mycobacteria phylogenetically closer to the Gram negatives
[19]. Thus, their Gram character still remains questionable
with little correlation to either the staining property or the
peptidoglycan content.
The cell envelope is well known for its contribution to-
wards drug tolerance which had been demonstrated early in
1970s [20]. The primary barrier to the permeation of small
hydrophilic molecules is attributed to the lipid rich outer mem-
brane whose permeability is close to a thousand times lower
than that of Gram negatives, restricting the entry of small
hydrophilic compounds [21]. Uptake of such molecules is
probably regulated by proteins present in the outer membrane
which are also known as β-barrel forming ‘porins’ [22]. On
the contrary, the observation that increased uptake of these
compounds occurs with increasing temperature or upon expo-
sure to cell wall permeabilizing agents such as Tween-80 or
sub-inhibitory concentrations of ethambutol which increases
membrane fluidity indicates a passive mode of transport of
nutrients [23, 24]. However, membrane dynamic studies using
fluorescent trehalose molecules during cell wall biogenesis
revealed lack of fluidity in the outer leaflet of the cell wall
with an extremely low rate of lateral diffusion of trehalose
glycolipids [24].
Unique structure of Mycobacterial
peptidoglycan
Contesting studies have attempted to model the architecture of
the peptidoglycan layer suggesting that it runs parallel with
the plasma membrane (Fig. 1a) while it was also suggested to
form a right handed coiled structure perpendicular to the lipid
bilayer by others and is still a topic of debate [25–27].
Nevertheless, it is becoming clearer that this glycoconjugate
plays a pivotal role in maintaining the typical cellular mor-
phology (bacillary or coccoid) together with providing
strength to resist osmotic stress [28]. Long polymers of alter-
nating units of aminosugars N-acetylglucosamine (GlcNAc)
and N-acetylmuramic acid (MurNAc) attached through a β
(1 → 4) glycosidic bond [7] form the glycan component. A
pentapeptide consisting L-alaninyl-D-isoglutaminyl-meso-
diaminopimelyl-D-alaninyl-D-alanine when added through
the lactyl group on the MurNAc helps maintain integrity of
the cell wall through inter-peptide crosslinks (Fig. 1a,b).
While the presence of D-amino acids ensures protection
against host proteases [28], modifications such as O-acetyla-
tion, and N-deacetylation of glycan strands in pathogenic bac-
terium were implicated in decreasing susceptibility to lyso-
zyme [29, 30]. Intriguingly, the glycan component of
423
peptidoglycan has always been found to be modified in most
bacteria although no modified precursors were ever detected,
thus, suggesting that all modifications took place post poly-
merization of the glycan strand. Classification of peptidogly-
can is based on the positions of crosslinking between two
pentapeptide subunits and subgroups were assigned if
interpeptide bridges were involved in the crosslinks [31].
Mycobacterial peptidoglycan primarily belongs to the A1γ
class, though certain unique structural modifications have
been reported in this species [25] including: (a) Oxidation of
few N-acetyl derivatives of MurNAc to N-glycolyl groups in
Mtb and M. smegmatis, giving rise to two types of muramic
acid residues [32]; (b) Alternate crosslinks between the
tetrapeptide chain includes the non classical 3 → 3 linkage
between two meso-diaminopimelic acid (DAP) residues in
addition to the regular 3 → 4 linkage between DAP and D-
ala, particularly under environmental stress and during station-
ary phase of growth (Fig. 1b) [33, 34]; (c) While cross-linking
is observed to be only 50% in E. coli, the proportion is 70–
80% in Mycobacterium [34]; (d) The arabinogalactan layer is
covalently linked to the MurNAc residues at its C6-OH
through a phosphodiester linkage by an α-L-
rhamnopyranose-(1 → 3)-α-D-GlcNAc (1 → P) linker, unlike
in other organisms [35]; (e) Additionally, a single glycine is
added to the ε-amino group of meso-DAP [36]; (f) The first
amino acid residue (L-ala) of the tetrapeptide can be replaced
with L-glycine [36]. A combinatorial diversity thus formed by
the different crosslinks formed between different peptide
chains render a greater distribution of the turgor pressure
across the crosslinks although they do not determine their
susceptibility to β-lactams. Importantly, it may provide a clue
to the relative importance of the specific stereochemistry that
may be exploited for therapeutic intervention. Muramyl di-
peptide, a minimum peptidoglycan component consisting of
MurNAc and two amino acids, D-Ala and D-isoglutamate
forms the pathogen-associated molecular pattern (PAMP) in
Mycobacteria and plays an critical role in host pathogen inter-
action by being recognized by the human nucleotide-binding
oligomerization domain 2 (NOD2) receptors [37].
Intricate synthesis
Although several peptidoglycan biosynthetic enzymes were
selected for target based drug discovery campaigns against
Mycobacteria, the biosynthetic pathway involved in its poly-
merization, believed to be very similar to that of E.coli and
Bacillus, still remains unsettled [38]. After the genome of Mtb
was mapped, comparative genomic approaches have helped in
annotation of the gene products and their genetic arrangement
in other Mycobacteria based on sequence homology.
Synthesis of peptidoglycan occurs in three different stages
and is topologically separated across the inner membrane.
424
While the activated nucleotide sugar-pentapeptides are syn-
thesized in the cytoplasm, they are assembled at the inner
side of the membrane and finally undergo polymerization
after being flipped to the periplasmic side by a membrane
transporter (Fig. 2) [54].
The pathway begins with synthesis of UDP-GlcNAc from
fructose-6-phosphate that requires the activities of three en-
zymes. An aminotransferase, GlmS, successively catalyzes its
conversion to glucosamine-6-phosphate, which is further con-
verted to glucosamine-1-phophate by a mutase, GlmM [55,
56]. UDP-GlcNAc is finally formed through acetylation and
uridylation carried out by the essential enzyme, GlmU [57].
The second stage involves the synthesis of UDP-MurNAc-
pentapeptide which is catalyzed sequentially by the MurA-F
ligase pathway. Generation of UDP-MurNAc is a two-step
process wherein MurA adds an enolpyruvyl moiety to UDP-
GlcNAc while MurB reduces the enolpyruvyl moiety to D-
lactoyl ether using NADPH as the electron donor, giving the
Fig. 2 Three stage synthesis of the mycobacterial peptidoglycan wherein
the precursors are produced in the cytoplasm (Stage 1), thereafter
monomeric units are assembled on the inner leaflet of the inner
membrane (Stage 2) and polymerization, crosslinking of tetrapeptide
side chains and attachment of peptidoglycan to arabinogalactan occurs
in the periplasm (Stage 3). Additional crosslinking that takes place during
Glycoconj J (2018) 35:421–432
final UDP-MurNAc [58]. In addition, a glycolyl modification
unique to Mycobacterium is brought about by NamH, a UDP-
MurNAc hydroxylase, giving rise to the two types of UDP-
muramyl substrates (MurNAc and MurNGly) [59].
Thereafter, the ATP-dependent steps catalyzed by MurC-F
incorporate the pentapeptide sequentially by a common reac-
tion mechanism [60]. Alanine racemase, Alr and glutamate
racemase, MurI produce the D-amino acids from their L
forms, respectively while meso-DAP is produced by a series
of reactions involving multiple enzymes [61–63]. An ATP-
dependent D-Ala:D-Ala ligase, Ddl catalyzes the formation
of the peptide bond between two D-alanine residues forming
the di-peptide substrate for the terminal ligase MurF [64].
The transfer of nucleotide sugar-pentapeptide to
decaprenyl phosphate is catalyzed by MurX/Y which results
in the formation of the first membrane-bound peptidoglycan
precursor (Lipid I) [65]. A β (1 → 4) bond is formed by MurG
between GlcNAc of UDP-GlcNAc and MurNAc/Gly of Lipid
stationary phase and under stress is depicted with a dotted box. Enzymes
whose molecular structures have been determined are shaded in colored
ovals. Rv numbers for the different enzymes have been obtained from
tuberculist.epfl.ch and correspond to that of M.tuberculosis H37Rv strain.
Inhibitors of the peptidoglycan biosynthetic enzymes are numbered
according to Table 1 and colored in red
Glycoconj J (2018) 35:421–432
I, forming the monomeric unit of the peptidoglycan (Lipid II)
[66]. Although the precise mechanism for translocation of
Lipid II across the membrane is not known, MurJ has been
identified as the potential flippase [54]. The final stage in PG
synthesis requires polymerization of Lipid II by the bifunc-
tional enzymes with transglycosylase and transpeptidase ac-
tivities [67]. Since transglycosylation precedes
transpeptidation, dividing cells might retain a small amount
of nascent uncrosslinked peptidoglycan that may be available
in the soluble form. The transglycosylase domain of the
penicillin-binding proteins PonA1 and PonA2 are responsible
for ligating the GlcNAc moiety with the muramyl moiety,
while the catalytic activity of a conserved serine in
transpeptidase domain is responsible for the (3 → 4)
crosslinking between meso-DAP and D-alanine. A new type
of (3 → 3) linkage is established by the conserved cysteine
containing L,D-transpeptidases LdtMt1 and LdtMt2 that pre-
dominates during nutrient limitations [68]. Importantly, pepti-
doglycan maturation and formation of mAGP core is not com-
plete without being ligated to the arabinogalactan and this step
is catalyzed by Lcp1 phosphotransferase, a LytR-CpsA-Psr
homologue in Mycobacteria [69].
Unlike in Mycobacterium which modifies the UDP-linked
precursors, multiple post-polymerization enzymatic modifica-
tion of the glycan strand has been observed in certain prokary-
otes. In Streptococcus pneumoniae, a peptidoglycan GlcNAc
N-deacetylase (PgdA) has been identified whose function has
been implicated in providing resistance to the muramidase
lysozyme by substrate alteration [30]. Similar protection is
also provided by specific O-acetylation of the C6-OH group
of MurNAc by an O-acetyltransferase (OatA) in
Staphylococcus aureus, however, the source of the acetyl
group has remained cryptic with reports proposing OatA to
perform the dual role of acetate transport and transfer [70]. As
an unique example of providing chemical resistance during
dormancy in Bacillus subtilis, half the MurNAc residues are
modified to form δ-lactam through the intra-molecular amide
bond formation between the amino group and carboxyl group
at positions 2 and 3, respectively. Notably, the absence of a
peptide chain at position 3 and an acetyl group at position 2 is
a precondition for this form of protection through a δ-lactam
formation [30].
Thus, it appears that peptidoglycan biogenesis is a highly
coordinated process which occurs concomitantly with cell
wall elongation and cell division, mediated by protein-
protein interactions. However, until recently the mechanisms
that coordinate the polymerization were not clearly under-
stood. Through whole genome interaction studies employing
transposon mutagenesis screen on mutants of ponA1, ponA2
and ldtMt2, it was observed that ponA1 and ponA2 have
unique genetic interactions thus suggesting that peptidoglycan
biogenesis may follow independent paths involving multi-
protein complexes which may be temporally and spatially
425
distinct near the septum and at the cell poles [71].
Mycobacterial peptidoglycan also undergoes extensive struc-
tural modifications to adapt and interact with the host environ-
ment. Several gene products involved in specific steps of bio-
synthesis were observed to be upregulated during stationary
phase, hypoxia and under in vivo growth conditions and these
include the precursor producing murE, the transpeptidases
(ldtMt1, ldtMt2) and the low molecular weight PBPs (dacB1
and dacB2) which hydrolyze the terminal D-alanine residues
and maintain the 3 → 3 crosslinks [52]. Recent studies have
hinted that peptidoglycan synthesis plausibly occurs in spa-
tially segregated metabolically active membrane domains that
are distinct from the usual plasma membrane and are predom-
inantly located at the cell poles [72].
Peptidoglycan remodelling in cell division
Next most important feature of this glycoconjugate is its role
in cell division. The continuous macromolecular framework
of the peptidoglycan sacculus requires to be expanded uni-
formly during growth and cell division. Elongation of the
sacculus during cell division necessitates the requirement for
breakdown of existing peptidoglycan layer for allowing inser-
tion of the newly synthesized glycan [73]. Periplasmic hydro-
lases help in this process of carving the size, shape and thick-
ness of peptidoglycan wherein the D,D-carboxypeptidase and
L,D-carboxypeptidase sequentially trim the pentapeptide side
chain while the transglycosylases help maintain the length of
the glycan chain [52]. Mycobacterial genome also codes for
two amidases, Ami3 and Ami4 that cleave between the
MurNAc and L-ala in the stem peptide to release the entire
peptide stem from the glycan chain [74]. Multiple enzymes
organized into well-defined units known as ‘elongasome’ and
‘divisosome’ help to maintain the balance between hydrolysis
and synthesis of peptidoglycan to form a perfect cell wall [75,
76]. These remodelling proteins mainly belong to the five
classes: lytic transglycosylases, penicillin binding proteins
(PBPs), endopeptidases, L,D-transpeptidases and amidases.
The transglycosylases, RpfA-E (resuscitation promoting fac-
tors) and the endopeptidases, RipA and RipB (Rpf interacting
proteins) act in concert to hydrolyze the peptidoglycan and
were found to interact and coordinate with the biosynthetic
enzyme PonA1 (PBP1) in order to sustain continuous enlarge-
ment of the sacculus without any rupture [77]. However, the
monofunctional PbpA (PBP2) and PbpB were observed to be
involved only in septation during cell division [78]. In addi-
tion to the biosynthetic and remodelling enzymes, structural
proteins such as Wag31, FtsZ and FtsW form pivotal units of
this process in Mycobacteria [75, 79]. Super-resolution mi-
croscopy detected the presence of an active cell wall synthetic
complex at a region below the tip of the old poles, where
addition of new peptidoglycan units occurs during cell
426
elongation [80]. Following elongation, an asymmetric cell di-
vision takes place giving rise to non-identical daughter cells,
unlike other bacteria, resulting in a heterogeneity in cellular
morphology within a population and has been a primary rea-
son for differential susceptibility towards antimycobacterials
[81, 82].
Alteration in peptidoglycan during dormancy
and resuscitation
Dormancy in Mycobacteria is a state of temporary suspen-
sion of most metabolic activities as a protective response to
external unfavourable conditions which is reflected in a
range of morphological and physiological adaptations
[83]. In Mtb, entry into dormancy is accompanied by for-
mation of a thick cell wall and an accumulation of cell wall
metabolites for any future requirement for cell wall forma-
tion upon reactivation [84, 85]. Gram positive bacteria re-
lease huge quantities of peptidoglycan fragments during
cell division due to lack of a peptidoglycan recycling
mechanisms [86]. These peptidoglycan fragments, also re-
ferred to as muropeptides, have proved to be potent signal-
ing molecules for resuscitation of Bacillus subtilis spores
and similar behaviour was observed recently in case of
mycobacterial species [87, 88]. Cell wall receptors known
as eukaryotic like serine/threonine kinases (PrkC), con-
trolled by reversible protein phosphorylation, were pro-
posed to bind these muropeptides during the exit from dor-
mancy in Bacillus [89]. Interestingly, the PASTA
(Penicillin-binding protein and serine threonine
associated) domains found in these membrane kinases are
also present in the extracytoplasmic region of the kinase,
PknB in Mycobacteria [90]. PknB, which interacts and
interferes with several membrane associated proteins
through phosphotransfer reactions was also found to bind
synthetic muropeptides in vitro [91]. Furthermore, RpfB in
combination with its interacting partners RipA and RipB
was observed to play an important role in generating
muropeptides required for ligand-mediated kinase activa-
tion during cell wall remodelling [92, 93]. Therefore, on
the whole, peptidoglycan appears to hold a very significant
place in the overall process of dormancy and resuscitation.
Its importance was drawn from a recent study demonstrat-
ing an anticipatory behaviour of Mtb in accumulating tre-
halose derived from the outer membrane mycolates and
converting them into precursors required for peptidoglycan
synthesis during resuscitation, taking this aspect to a new
level for understanding the multifaceted roles of this
glycoconjugate [94].
Glycoconj J (2018) 35:421–432
Peptidoglycan as an attractive drug target
Since peptidoglycan biosynthesis plays a crucial role in over-
all cellular and structural integrity, the biosynthetic and re-
modelling enzymes become vulnerable candidates for drug
discovery. Several recent reports on the unique mechanism
of enzyme catalysis re-positions mycobacterial peptidoglycan
as an attractive drug target [15]. GlmU which catalyzes the
formation of UDP-GlcNAc contains both acetyltransferase
and uridyltransferase activities, however, structural homology
of the uridyltransferase domain with its human counterpart
leaves the acetyltransferase domain as a potential target [39,
95]. Synthetic inhibitors targeting GlmM and GlmU, particu-
larly a novel oxazolidine have shown promising results
against Mtb in vivo [40, 96]. D-cycloserine, which targets both
the alanine racemase and D-ala:D-ala ligase is used as an
effective second line therapy [45]. Encouragingly, an ana-
logue of the nucleoside-based natural product capuramycin,
SQ641 that inhibits the translocase MurX has shown efficacy
against the difficult to eliminate non-replicating form of
Mycobacterium [46]. Sansanmycin uridylpeptide analogues
strongly inhibited phospho-MurNAc-pentapeptide
translocase responsible for Lipid-I synthesis in vitro and
in ex vivo conditions [47].
In addition to biosynthetic enzymes, the non proteinaceous
peptidoglycan precursors can also be targeted by antibiotics
such as vancomycin which inhibits the polymerization of
tetrapeptide side chain by binding to the alanine moiety.
Ramoplanin and enduracidin were reported to inhibit the
transglycosylation step by blocking the MurG-catalyzed con-
version of Lipid I to Lipid II [50]. Recently, a new natural
product teixobactin was discovered from uncultured soil bac-
teria which inhibits peptidoglycan biosynthesis in Mtb by
binding to lipid II precursors [51]. Importantly, prolonged pe-
riods of exposure to the compound at low concentrations pro-
duced no resistant forms, thus indicating its promising future
in treating the MDR and XDR forms of Mtb [97] and reviving
the Achilles’ heel of Gram positives. Table 1 presents an up-
dated list of potential peptidoglycan targets and the corre-
sponding inhibitors having efficacy against Mycobacteria.
Due to the rapid rise in multidrug resistant forms of tuber-
culosis, alternate strategies employing lytic bacteriophage or
bacteriophage derived proteins can become an attractive op-
portunity. Bacteriophage based therapy has been very popular
in eastern European and former Soviet Union nations which
were incedentally also the centre for MDR TB outbreaks due
to armed conflicts, weak public health system and
unavailibility of new anti-tuberculosis drugs [98]. The virion
tail associated peptidoglycan hydrolases that are effective in
lysing the extracellular bacilli by targetting the peptidoglycans
may be considered as adjuvants in treating drug resistant
Glycoconj J (2018) 35:421–432 427

Table 1 Inhibitors of mycobacterial peptidoglycan biosynthesis and their targets

Sl. Inhibitor Class Target (Protein) Mechanism of inhibition References

No.

1, 2, 3 Glucosamine-6-phosphate analogues;
Thiazolidinone derivatives and related
scaffolds;
Aminoquinazoline compounds
4 3,5-dioxopyrazolidines
5 Benzylidino thiazolidinones
6 Substituted
5-Benzylidenethiazolidin-4-one
7 D-cycloserine
8 Muramycin: Nucleoside-peptide
antibiotics
9 Substituted prolines
10 Depsipeptide antibiotics
11 β-lactam with and without β-lactamase Penicillin binding protein (PonA1 and PonA2), Prevent cross linking of peptide chains [52, 53]
inhibitor
UDP-GlcNAc pyrophosphorylase: (GlmU)
UDP-N-acetylenolpyruvylglucosamine
reductase (MurB)
Mur Ligases (MurC/D/E/F)
D-alanine-D-alanine ligase (Ddl)
Phospho-N-acetylmuramoyl-pentapeptide-
transferase (MurX/MraY)
UDP-N-acetylglucosamine-N-
acetylmuramyl-(pentapeptide)
pyrophosphoryl-undecaprenol-N-
acetylglucosamine transferase (MurG)
Lipid II
L,D-transpeptidases (Ldt A, LdtB),
D,D-carboxipeptidase (DacB2)
Competitively inhibits the formation of [39–41]
UDP-GlcNAc
Competitively inhibits the formation of [42, 43]
UDP-MurNAc
Inhibits the formation of the [44]
peptapeptide chains.
Inhibits cell growth by reducing supply [45]
of substrates for the final peptide
ligation
Inhibits the synthesis of Lipid I [46–48]
Inhibits the synthesis of Lipid II [49]
Prevents the transglycosylation of the [50, 51]
translocated Lipid II by binding to it.
and β-lactamase inhibitor inhibits the
intrinsic activity of BlaC and
facilitates action of the penicillins.

infections [99]. BLytic cassette^ encoded endolysins, includ-
ing LysA and LysB, that specifically cleave most linkages
generally present in mycobacterial peptidoglycan has also
been promising in killing intracellular M.smegmatis when ad-
ministered ex vivo [100]. However, a major impediment in
using the above approaches could be a potential damage to
host tissues from release of mycobacterial toxins upon lysis.
Penicillin binding proteins and β-lactamases
β-lactam group of antibiotics consisting of the penicillins,
cephalosporins, monobactams and carbapenems which block
the cross-linking of peptidoglycan by transpeptidases are
among the most successful antimicrobial agents [101].
However, they have provided little success against the tuber-
cle bacilli except for the carbapenems, which resist the myco-
bacterial β-lactamase mediated hydrolysis of their lactam ring
and can still inhibit the non-classical L, D-transpeptidases [52,
102, 103]. Most β-lactamases fall under the category of
penicilloyl serine transferases along with PBPs which bind
penicillins at their active site and cleave the amide bond of
the lactam resulting in ring opening. In β-lactamases, the hy-
drolyzed substrate is released unlike in PBPs where the sub-
strate remains bound to the enzyme, distinguishing the two
functions which in fact forms the basis of evolution of β-
lactamases from PBPs [104]. The major β-lactamases of
Mtb are BlaA (in avirulent species) and BlaC (in virulent
species) [105] both belonging to the Ambler class-A. Their
deletion results in increased susceptibility of the organism
towards penicillins, cephalosporins and carbapenems [106].
The practical applicability of using a combinatorial therapy
of penicillin against Mtb was indirectly observed by Kasik
et al. in 1966 who administered dicloxallin, a penicillinase
resistant penicillin in combination with penicillin [107] but
went unobserved till the work by Hugonnet and Blanchard
reproduced it by using a β-lactamase analogue, clavulanate
[108]. A regimen was later formulated for treating TB patients
using a combination of amoxicillin and clavulanate which is
now sold as Augmentin®. Furthermore, drug combinations
such as the meropenem and imipenem in combination with
clavulanic acid, sulbactam and tazobactam showed clearance
of persistent cells in addition to the actively growing drug
resistant bacilli in vitro and ex vivo [108–111]. A new clinical
study reported that a combination of meropenem with
Augmentin® resulted in a synergistic effect against Mtb in
addition to improving the availability of the carbapenem
[112]. Novel β-lactam combinations and regimens are con-
stantly being investigated for improvized control strategies.
Recently, a study uncovered the complex mechanism of action
428
of Augmentin® involving the mycobacterial redox sensor,
WhiB4 which provides tolerance by suppressing the levels
of β-lactamases and reducing the intracellular concentration
of the reactive oxygen species thereby, underscoring the need
for including redox based adjuvants for potentiating efficacy
of β lactams [113]. Intriguingly, an intelligent computational
study on identifying association of repurposed drugs with
their targets predicted the lactams as the most populated drug
class, thus rekindling the importance of using lactams in a
combination therapy [114]. Furthermore, a recent report iden-
tified the presence of membrane microdomains, similar to
eukaryotic lipid rafts, in Staphylococcus that helps to recruit
and oligomerise membrane associated multimeric complexes
[115]. Intriguingly, Pbp2A, implicated in penicilin resistance,
was observed to oligomerize in these multi-protein platforms
before being compartmentalized for peptidoglycan biosynthe-
sis, thus opening yet another avenue for controlling drug
resistance.
Conclusion and future perspectives
Control of Mtb which took 1.3 million lives in 2016 is of
utmost priority in our quest for ending the global scourge of
tuberculosis by 2035 and this requires maximum understand-
ing of its unique cell envelope which provides multiple modes
of resistance in different manifestations. Our understanding of
the mycobacterial peptidoglycan though has steadily in-
creased with growing number of recent observations and con-
comitant new insights, it is apparent that the mechanistic
chemistry of the biosynthetic enzymes, regulation, interac-
tions and the spatio-temporal localization of the protein com-
plexes is still unclear and needs intense investigation.
Importantly, intelligent speculations based on comparative ge-
nomics have helped in identifying the peptidoglycan biosyn-
thetic enzymes while information on their molecular struc-
tures have tremendously helped in identifying new targets
and developing assays for screening antimycobacterials
[116]. Synthesis of advanced fluorescent probes coupled with
the development of super-resolution microscopy has also
helped to decipher the cardinal role of peptidoglycan during
cell elongation, division and re-entry from dormancy. These
new features can form future targets for chemotherapeutic
intervention with novel mechanism of action [51, 117].
Function of antibiotics go beyond the notion that their produc-
tion is primarily for protection against competitors [118] and
antimicrobial resistance in pathogens is ancient and wide-
spread in environment, with genomic evidences buried in
Pleistocene sediments [119]. This underpins the fact that rapid
emergence of resistance cannot be prevented even though we
are successful in developing candidate drugs at an unprece-
dented rate. However, disabling the features that provide in-
trinsic resistance may allow us to enhance the efficacy of
Glycoconj J (2018) 35:421–432
available drugs, which can be achieved by combining antibi-
otics with adjuvants, as testified by the success of
Augmentin®. Treatment shortening may not be a distant tar-
get with a regimen that can rapidly bring down the bacillary
load and is only possible by either developing new Bresis-
tance-proof^ molecules [120] or by introducing antibiotic ad-
juvants that can preserve the efficacy of the existing drugs.
Acknowledgements SP thanks IISER, Tirupati for research fellowship.
RM thanks DBT, Govt. of India and IISER, Tirupati for research support.
RM is a recipient of Early Career Research Award from SERB, Govt. of
India.
Compliance with ethical standards
Conflicts of interest The authors declare that they have no conflicts of
interest.
Ethical approval This article does not contain any studies with human
participants or animals performed by any of the authors.
References
1. WHO: Global tuberculosis report- 2017, vol. 2017, 2017 ed.
WHO, (2017)
2. Brennan, P.J.: Structure, function, and biogenesis of the cell
wall of Mycobacterium tuberculosis. Tuberculosis (Edinb)
83(1–3), 91–97 (2003). https://doi.org/10.1016/S1472-
9792(02)00089-6
3. Goldman, R.C.: Why are membrane targets discovered by pheno-
typic screens and genome sequencing in Mycobacterium tubercu-
losis? Tuberculosis (Edinb). 93(6), 569–588 (2013). https://doi.
org/10.1016/j.tube.2013.09.003
4. Hanks, J.H.: Significance of capsular components of
Mycobacterium leprae and other mycobacteria. Int. J. Lepr. 29,
74–83 (1961)
5. Daffe, M., Draper, P.: The envelope layers of mycobacteria with
reference to their pathogenicity. Adv. Microb. Physiol. 39, 131–
203 (1998)
6. Chatterjee, D., Bozic, C.M., McNeil, M., Brennan, P.J.: Structural
features of the arabinan component of the lipoarabinomannan of
Mycobacterium tuberculosis. J. Biol. Chem. 266(15), 9652–9660
(1991)
7. Lederer, E., Adam, A., Ciorbaru, R., Petit, J.F., Wietzerbin, J.: Cell
walls of mycobacteria and related organisms; chemistry and
immunostimulant properties. Mol. Cell. Biochem. 7(2), 87–104
(1975)
8. Jankute, M., Grover, S., Rana, A.K., Besra, G.S.: Arabinogalactan
and lipoarabinomannan biosynthesis: structure, biogenesis and
their potential as drug targets. Future Microbiol. 7(1), 129–147
(2012). https://doi.org/10.2217/fmb.11.123
9. Liu, J., Rosenberg, E.Y., Nikaido, H.: Fluidity of the lipid domain
of cell wall from Mycobacterium chelonae. Proc. Natl. Acad. Sci.
U. S. A. 92(24), 11254–11258 (1995)
10. Bansal-Mutalik, R., Nikaido, H.: Mycobacterial outer membrane
is a lipid bilayer and the inner membrane is unusually rich in diacyl
phosphatidylinositol dimannosides. Proc. Natl. Acad. Sci. U. S. A.
111(13), 4958–4963 (2014). https://doi.org/10.1073/pnas.
1403078111
Glycoconj J (2018) 35:421–432
11. Jackson, M.: The mycobacterial cell envelope-lipids. Cold Spring
Harb. Perspect. Med. 4(10), a021105 (2014). https://doi.org/10.
1101/cshperspect.a021105
12. Brecik, M., Centarova, I., Mukherjee, R., Kolly, G.S., Huszar, S.,
Bobovska, A., Kilacskova, E., Mokosova, V., Svetlikova, Z.,
Sarkan, M., Neres, J., Kordulakova, J., Cole, S.T., Mikusova, K.:
DprE1 is a vulnerable tuberculosis drug target due to its cell wall
localization. ACS Chem. Biol. 10(7), 1631–1636 (2015). https://
doi.org/10.1021/acschembio.5b00237
13. Makarov, V., Manina, G., Mikusova, K., Mollmann, U., Ryabova,
O., Saint-Joanis, B., Dhar, N., Pasca, M.R., Buroni, S., Lucarelli,
A.P., Milano, A., De Rossi, E., Belanova, M., Bobovska, A.,
Dianiskova, P., Kordulakova, J., Sala, C., Fullam, E., Schneider,
P., McKinney, J.D., Brodin, P., Christophe, T., Waddell, S.,
Butcher, P., Albrethsen, J., Rosenkrands, I., Brosch, R., Nandi,
V., Bharath, S., Gaonkar, S., Shandil, R.K., Balasubramanian, V.,
Balganesh, T., Tyagi, S., Grosset, J., Riccardi, G., Cole, S.T.:
Benzothiazinones kill Mycobacterium tuberculosis by blocking
arabinan synthesis. Science. 324(5928), 801–804 (2009). https://
doi.org/10.1126/science.1171583
14. Christophe, T., Jackson, M., Jeon, H.K., Fenistein, D., Contreras-
Dominguez, M., Kim, J., Genovesio, A., Carralot, J.P., Ewann, F.,
Kim, E.H., Lee, S.Y., Kang, S., Seo, M.J., Park, E.J., Skovierova,
H., Pham, H., Riccardi, G., Nam, J.Y., Marsollier, L., Kempf, M.,
Joly-Guillou, M.L., Oh, T., Shin, W.K., No, Z., Nehrbass, U.,
Brosch, R., Cole, S.T., Brodin, P.: High content screening iden-
tifies decaprenyl-phosphoribose 2′ epimerase as a target for intra-
cellular antimycobacterial inhibitors. PLoS Pathog. 5(10),
e1000645 (2009). https://doi.org/10.1371/journal.ppat.1000645
15. Abrahams, K.A., Besra, G.S.: Mycobacterial cell wall biosynthe-
sis: a multifaceted antibiotic target. Parasitology. 145(2), 116–133
(2018). https://doi.org/10.1017/S0031182016002377
16. Zumla, A., Nahid, P., Cole, S.T.: Advances in the development of
new tuberculosis drugs and treatment regimens. Nat. Rev. Drug
Discov. 12(5), 388–404 (2013). https://doi.org/10.1038/nrd4001
17. Beveridge, T.J.: Mechanism of gram variability in select bacteria.
J. Bacteriol. 172(3), 1609–1620 (1990)
18. Fu, L.M., Fu-Liu, C.S.: Is Mycobacterium tuberculosis a closer
relative to gram-positive or gram-negative bacterial pathogens?
Tuberculosis (Edinb). 82(2–3), 85–90 (2002)
19. Niederweis, M., Danilchanka, O., Huff, J., Hoffmann, C.,
Engelhardt, H.: Mycobacterial outer membranes: in search of pro-
teins. Trends Microbiol. 18(3), 109–116 (2010). https://doi.org/
10.1016/j.tim.2009.12.005
20. Hui, J., Gordon, N., Kajioka, R.: Permeability barrier to rifampin
in mycobacteria. Antimicrob. Agents Chemother. 11(5), 773–779
(1977)
21. Jarlier, V., Nikaido, H.: Permeability barrier to hydrophilic solutes
in Mycobacterium chelonei. J. Bacteriol. 172(3), 1418–1423
(1990)
22. Hancock, R.E.: Role of porins in outer membrane permeability. J.
Bacteriol. 169(3), 929–933 (1987)
23. Piddock, L.J., Williams, K.J., Ricci, V.: Accumulation of rifampi-
cin by Mycobacterium aurum, Mycobacterium smegmatis and
Mycobacterium tuberculosis. J. Antimicrob. Chemother. 45(2),
159–165 (2000)
24. Rodriguez-Rivera, F.P., Zhou, X., Theriot, J.A., Bertozzi, C.R.:
Visualization of mycobacterial membrane dynamics in live cells.
J. Am. Chem. Soc. 139(9), 3488–3495 (2017). https://doi.org/10.
1021/jacs.6b12541
25. Crick, D.C., Mahapatra, S., Brennan, P.J.: Biosynthesis of the
arabinogalactan-peptidoglycan complex of Mycobacterium tuber-
culosis. Glycobiology. 11(9), 107R–118R (2001)
26. Meroueh, S.O., Bencze, K.Z., Hesek, D., Lee, M., Fisher, J.F.,
Stemmler, T.L., Mobashery, S.: Three-dimensional structure of
the bacterial cell wall peptidoglycan. Proc. Natl. Acad. Sci. U. S.
429
A. 103(12), 4404–4409 (2006). https://doi.org/10.1073/pnas.
0510182103
27. Hayhurst, E.J., Kailas, L., Hobbs, J.K., Foster, S.J.: Cell wall pep-
tidoglycan architecture in Bacillus subtilis. Proc. Natl. Acad. Sci.
U. S. A. 105(38), 14603–14608 (2008). https://doi.org/10.1073/
pnas.0804138105
28. Cabeen, M.T., Jacobs-Wagner, C.: Bacterial cell shape. Nat. Rev.
Microbiol. 3(8), 601–610 (2005). https://doi.org/10.1038/nrmicro1205
29. Davis, K.M., Weiser, J.N.: Modifications to the peptidoglycan
backbone help bacteria to establish infection. Infect. Immun.
79(2), 562–570 (2011). https://doi.org/10.1128/IAI.00651-10
30. Vollmer, W.: Structural variation in the glycan strands of bacterial
peptidoglycan. FEMS Microbiol. Rev. 32(2), 287–306 (2008).
https://doi.org/10.1111/j.1574-6976.2007.00088.x
31. Schleifer, K.H., Kandler, O.: Peptidoglycan types of bacterial cell
walls and their taxonomic implications. Bacteriol. Rev. 36(4),
407–477 (1972)
32. Mahapatra, S., Scherman, H., Brennan, P.J., Crick, D.C.: N
Glycolylation of the nucleotide precursors of peptidoglycan bio-
synthesis of Mycobacterium spp. is altered by drug treatment. J
Bacteriol 187(7), 2341–2347 (2005). https://doi.org/10.1128/JB.
187.7.2341-2347.2005
33. Lavollay, M., Arthur, M., Fourgeaud, M., Dubost, L., Marie, A.,
Veziris, N., Blanot, D., Gutmann, L., Mainardi, J.L.: The peptido-
glycan of stationary-phase Mycobacterium tuberculosis predomi-
nantly contains cross-links generated by L,D-transpeptidation. J.
Bacteriol. 190(12), 4360–4366 (2008). https://doi.org/10.1128/JB.
00239-08
34. Matsuhashi, M.: Biosynthesis in the bacterial cell wall.
Tanpakushitsu Kakusan Koso. 11(10), 875–886 (1966)
35. McNeil, M., Daffe, M., Brennan, P.J.: Evidence for the nature of
the link between the arabinogalactan and peptidoglycan of myco-
bacterial cell walls. J. Biol. Chem. 265(30), 18200–18206 (1990)
36. Mahapatra, S., Crick, D.C., McNeil, M.R., Brennan, P.J.: Unique
structural features of the peptidoglycan of Mycobacterium leprae.
J. Bacteriol. 190(2), 655–661 (2008). https://doi.org/10.1128/JB.
00982-07
37. Schenk, M., Mahapatra, S., Le, P., Kim, H.J., Choi, A.W.,
Brennan, P.J., Belisle, J.T., Modlin, R.L.: Human NOD2 recog-
nizes structurally unique Muramyl dipeptides from
Mycobacterium leprae. Infect. Immun. 84(9), 2429–2438
(2016). https://doi.org/10.1128/IAI.00334-16
38. Glauner, B., Holtje, J.V., Schwarz, U.: The composition of the
murein of Escherichia coli. J. Biol. Chem. 263(21), 10088–
10095 (1988)
39. Tran, A.T., Wen, D., West, N.P., Baker, E.N., Britton, W.J., Payne,
R.J.: Inhibition studies on Mycobacterium tuberculosis N-
acetylglucosamine-1-phosphate uridyltransferase (GlmU). Org.
Biomol. Chem. 11(46), 8113–8126 (2013). https://doi.org/10.
1039/c3ob41896k
40. Li, Y., Zhou, Y., Ma, Y., Li, X.: Design and synthesis of novel cell
wall inhibitors of Mycobacterium tuberculosis GlmM and GlmU.
Carbohydr. Res. 346(13), 1714–1720 (2011). https://doi.org/10.
1016/j.carres.2011.05.024
41. Rani, C., Mehra, R., Sharma, R., Chib, R., Wazir, P., Nargotra, A.,
Khan, I.A.: High-throughput screen identifies small molecule in-
hibitors targeting acetyltransferase activity of Mycobacterium tu-
berculosis GlmU. Tuberculosis (Edinb). 95(6), 664–677 (2015).
https://doi.org/10.1016/j.tube.2015.06.003
42. Kumar, V., Saravanan, P., Arvind, A., Mohan, C.G.: Identification
of hotspot regions of MurB oxidoreductase enzyme using homol-
ogy modeling, molecular dynamics and molecular docking tech-
niques. J. Mol. Model. 17(5), 939–953 (2011). https://doi.org/10.
1007/s00894-010-0788-3
43. Rana, A.M., Trivedi, P., Desai, K.R., Jauhari, S.: Novel S-triazine
accommodated 5-benzylidino-4-thiazolidinones: synthesis and
430
in vitro biological evaluations. Med. Chem. Res. 23(10), 4320–
4336 (2014). https://doi.org/10.1007/s00044-014-0995-z
44. Tomasic, T., Zidar, N., Kovac, A., Turk, S., Simcic, M., Blanot,
D., Muller-Premru, M., Filipic, M., Grdadolnik, S.G., Zega, A.,
Anderluh, M., Gobec, S., Kikelj, D., Peterlin Masic, L.: 5-
Benzylidenethiazolidin-4-ones as multitarget inhibitors of bacteri-
al Mur ligases. ChemMedChem. 5(2), 286–295 (2010). https://
doi.org/10.1002/cmdc.200900449
45. Prosser, G.A., de Carvalho, L.P.: Kinetic mechanism and inhibi-
tion of Mycobacterium tuberculosis D-alanine:D-alanine ligase by
the antibiotic D-cycloserine. FEBS J. 280(4), 1150–1166 (2013).
https://doi.org/10.1111/febs.12108
46. Siricilla, S., Mitachi, K., Wan, B., Franzblau, S.G., Kurosu, M.:
Discovery of a capuramycin analog that kills nonreplicating
Mycobacterium tuberculosis and its synergistic effects with
translocase I inhibitors. J. Antibiot. (Tokyo). 68(4), 271–278
(2015). https://doi.org/10.1038/ja.2014.133
47. Tran, A.T., Watson, E.E., Pujari, V., Conroy, T., Dowman, L.J.,
Giltrap, A.M., Pang, A., Wong, W.R., Linington, R.G.,
Mahapatra, S., Saunders, J., Charman, S.A., West, N.P., Bugg,
T.D., Tod, J., Dowson, C.G., Roper, D.I., Crick, D.C., Britton,
W.J., Payne, R.J.: Sansanmycin natural product analogues as po-
tent and selective anti-mycobacterials that inhibit lipid I biosyn-
thesis. Nat. Commun. 8, 14414 (2017). https://doi.org/10.1038/
ncomms14414
48. Wiegmann, D., Koppermann, S., Wirth, M., Niro, G., Leyerer, K.,
Ducho, C.: Muraymycin nucleoside-peptide antibiotics: uridine-
derived natural products as lead structures for the development of
novel antibacterial agents. Beilstein J. Org. Chem. 12, 769–795
(2016). https://doi.org/10.3762/bjoc.12.77
49. Trunkfield, A.E., Gurcha, S.S., Besra, G.S., Bugg, T.D.: Inhibition
of Escherichia coli glycosyltransferase MurG and Mycobacterium
tuberculosis Gal transferase by uridine-linked transition state
mimics. Bioorg. Med. Chem. 18(7), 2651–2663 (2010). https://
doi.org/10.1016/j.bmc.2010.02.026
50. Fang, X., Tiyanont, K., Zhang, Y., Wanner, J., Boger, D., Walker,
S.: The mechanism of action of ramoplanin and enduracidin. Mol.
BioSyst. 2(1), 69–76 (2006). https://doi.org/10.1039/b515328j
51. Ling, L.L., Schneider, T., Peoples, A.J., Spoering, A.L., Engels, I.,
Conlon, B.P., Mueller, A., Schaberle, T.F., Hughes, D.E., Epstein,
S., Jones, M., Lazarides, L., Steadman, V.A., Cohen, D.R., Felix,
C.R., Fetterman, K.A., Millett, W.P., Nitti, A.G., Zullo, A.M.,
Chen, C., Lewis, K.: A new antibiotic kills pathogens without
detectable resistance. Nature. 517(7535), 455–459 (2015).
https://doi.org/10.1038/nature14098
52. Kumar, P., Arora, K., Lloyd, J.R., Lee, I.Y., Nair, V., Fischer, E.,
Boshoff, H.I., Barry 3rd, C.E.: Meropenem inhibits D,D-carboxy-
peptidase activity in Mycobacterium tuberculosis. Mol. Microbiol.
86(2), 367–381 (2012). https://doi.org/10.1111/j.1365-2958.2012.
08199.x
53. Rullas, J., Dhar, N., McKinney, J.D., Garcia-Perez, A., Lelievre,
J., Diacon, A.H., Hugonnet, J.E., Arthur, M., Angulo-Barturen, I.,
Barros-Aguirre, D., Ballell, L.: Combinations of beta-lactam anti-
biotics currently in clinical trials are efficacious in a DHP-I-
deficient mouse model of tuberculosis infection. Antimicrob.
Agents Chemother. 59(8), 4997–4999 (2015). https://doi.org/10.
1128/AAC.01063-15
54. Sham, L.T., Butler, E.K., Lebar, M.D., Kahne, D., Bernhardt, T.G.,
Ruiz, N.: Bacterial cell wall. MurJ is the flippase of lipid-linked
precursors for peptidoglycan biogenesis. Science. 345(6193),
220–222 (2014). https://doi.org/10.1126/science.1254522
55. Li, S., Kang, J., Yu, W., Zhou, Y., Zhang, W., Xin, Y., Ma, Y.:
Identification of M. tuberculosis Rv3441c and M. smegmatis
MSMEG_1556 and essentiality of M. smegmatis
MSMEG_1556. PLoS One. 7(8), e42769 (2012). https://doi.org/
10.1371/journal.pone.0042769
Glycoconj J (2018) 35:421–432
56. Durand, P., Golinelli-Pimpaneau, B., Mouilleron, S., Badet, B.,
Badet-Denisot, M.A.: Highlights of glucosamine-6P synthase ca-
talysis. Arch. Biochem. Biophys. 474(2), 302–317 (2008). https://
doi.org/10.1016/j.abb.2008.01.026
57. Zhang, Z., Bulloch, E.M., Bunker, R.D., Baker, E.N., Squire, C.J.:
Structure and function of GlmU from Mycobacterium tuberculo-
sis. Acta Crystallogr. D Biol. Crystallogr. 65(Pt 3), 275–283
(2009). https://doi.org/10.1107/S0907444909001036
58. Jankute, M., Cox, J.A., Harrison, J., Besra, G.S.: Assembly of the
mycobacterial cell wall. Annu. Rev. Microbiol. 69, 405–423
(2015). https://doi.org/10.1146/annurev-micro-091014-104121
59. Raymond, J.B., Mahapatra, S., Crick, D.C., Pavelka Jr., M.S.:
Identification of the namH gene, encoding the hydroxylase re-
sponsible for the N-glycolylation of the mycobacterial peptidogly-
can. J. Biol. Chem. 280(1), 326–333 (2005). https://doi.org/10.
1074/jbc.M411006200
60. Barreteau, H., Kovac, A., Boniface, A., Sova, M., Gobec, S.,
Blanot, D.: Cytoplasmic steps of peptidoglycan biosynthesis.
FEMS Microbiol. Rev. 32(2), 168–207 (2008). https://doi.org/
10.1111/j.1574-6976.2008.00104.x
61. Feng, Z., Barletta, R.G.: Roles of Mycobacterium smegmatis D-
alanine:D-alanine ligase and D-alanine racemase in the mecha-
nisms of action of and resistance to the peptidoglycan inhibitor
D-cycloserine. Antimicrob. Agents Chemother. 47(1), 283–291
(2003)
62. Li, Y., Mortuza, R., Milligan, D.L., Tran, S.L., Strych, U., Cook,
G.M., Krause, K.L.: Investigation of the essentiality of glutamate
racemase in Mycobacterium smegmatis. J. Bacteriol. 196(24),
4239–4244 (2014). https://doi.org/10.1128/JB.02090-14
63. Usha, V., Lloyd, A.J., Lovering, A.L., Besra, G.S.: Structure and
function of Mycobacterium tuberculosis meso-diaminopimelic ac-
id (DAP) biosynthetic enzymes. FEMS Microbiol. Lett. 330(1),
10–16 (2012). https://doi.org/10.1111/j.1574-6968.2012.02527.x
64. Prosser, G.A., de Carvalho, L.P.: Reinterpreting the mechanism of
inhibition of Mycobacterium tuberculosis D-alanine:D-alanine li-
gase by D-cycloserine. Biochemistry. 52(40), 7145–7149 (2013).
https://doi.org/10.1021/bi400839f
65. Kurosu, M., Mahapatra, S., Narayanasamy, P., Crick, D.C.:
Chemoenzymatic synthesis of Park’s nucleotide: toward the de-
velopment of high-throughput screening for MraY inhibitors.
Tetrahedron Lett. 48(5), 799–803 (2007). https://doi.org/10.
1016/j.tetlet.2006.11.160
66. Jha, R.K., Katagihallimath, N., Hota, S.K., Das, K.S., de Sousa,
S.M.: An assay for exogenous sources of purified MurG, enabled
by the complementation of Escherichia coli murG(Ts) by the
Mycobacterium tuberculosis homologue. FEMS Microbiol. Lett.
326(2), 161–167 (2012). https://doi.org/10.1111/j.1574-6968.
2011.02446.x
67. Hett, E.C., Chao, M.C., Rubin, E.J.: Interaction and modulation of
two antagonistic cell wall enzymes of mycobacteria. PLoS Pathog.
6(7), e1001020 (2010). https://doi.org/10.1371/journal.ppat.
1001020
68. Cordillot, M., Dubee, V., Triboulet, S., Dubost, L., Marie, A.,
Hugonnet, J.E., Arthur, M., Mainardi, J.L.: In vitro cross-linking
of Mycobacterium tuberculosis peptidoglycan by L,D-
transpeptidases and inactivation of these enzymes by carbapen-
ems. Antimicrob. Agents Chemother. 57(12), 5940–5945
(2013). https://doi.org/10.1128/AAC.01663-13
69. Harrison, J., Lloyd, G., Joe, M., Lowary, T.L., Reynolds, E.,
Walters-Morgan, H., Bhatt, A., Lovering, A., Besra, G.S.,
Alderwick, L.J.: Lcp1 is a phosphotransferase responsible for li-
gating arabinogalactan to peptidoglycan in Mycobacterium tuber-
culosis. MBio. 7(4), e00972-16 (2016). https://doi.org/10.1128/
mBio.00972-16
70. Bera, A., Herbert, S., Jakob, A., Vollmer, W., Gotz, F.: Why are
pathogenic staphylococci so lysozyme resistant? The
Glycoconj J (2018) 35:421–432
peptidoglycan O-acetyltransferase OatA is the major determinant
for lysozyme resistance of Staphylococcus aureus. Mol.
Microbiol. 55(3), 778–787 (2005). https://doi.org/10.1111/j.
1365-2958.2004.04446.x
71. Kieser, K.J., Baranowski, C., Chao, M.C., Long, J.E., Sassetti,
C.M., Waldor, M.K., Sacchettini, J.C., Ioerger, T.R., Rubin, E.J.:
Peptidoglycan synthesis in Mycobacterium tuberculosis is orga-
nized into networks with varying drug susceptibility. Proc. Natl.
Acad. Sci. U. S. A. 112(42), 13087–13092 (2015). https://doi.org/
10.1073/pnas.1514135112
72. Hayashi, J.M., Luo, C.Y., Mayfield, J.A., Hsu, T., Fukuda, T.,
Walfield, A.L., Giffen, S.R., Leszyk, J.D., Baer, C.E., Bennion,
O.T., Madduri, A., Shaffer, S.A., Aldridge, B.B., Sassetti, C.M.,
Sandler, S.J., Kinoshita, T., Moody, D.B., Morita, Y.S.: Spatially
distinct and metabolically active membrane domain in
mycobacteria. Proc. Natl. Acad. Sci. U. S. A. 113(19), 5400–
5405 (2016). https://doi.org/10.1073/pnas.1525165113
73. Typas, A., Banzhaf, M., Gross, C.A., Vollmer, W.: From the reg-
ulation of peptidoglycan synthesis to bacterial growth and mor-
phology. Nat. Rev. Microbiol. 10(2), 123–136 (2011). https://doi.
org/10.1038/nrmicro2677
74. Prigozhin, D.M., Mavrici, D., Huizar, J.P., Vansell, H.J., Alber, T.:
Structural and biochemical analyses of Mycobacterium tuberculo-
sis N-acetylmuramyl-L-alanine amidase Rv3717 point to a role in
peptidoglycan fragment recycling. J. Biol. Chem. 288(44),
31549–31555 (2013). https://doi.org/10.1074/jbc.M113.510792
75. Kieser, K.J., Rubin, E.J.: How sisters grow apart: mycobacterial
growth and division. Nat. Rev. Microbiol. 12(8), 550–562 (2014).
https://doi.org/10.1038/nrmicro3299
76. Szwedziak, P., Lowe, J.: Do the divisome and elongasome share a
common evolutionary past? Curr. Opin. Microbiol. 16(6), 745–
751 (2013). https://doi.org/10.1016/j.mib.2013.09.003
77. Hett, E.C., Chao, M.C., Steyn, A.J., Fortune, S.M., Deng, L.L.,
Rubin, E.J.: A partner for the resuscitation-promoting factors of
Mycobacterium tuberculosis. Mol. Microbiol. 66(3), 658–668
(2007). https://doi.org/10.1111/j.1365-2958.2007.05945.x
78. Dasgupta, A., Datta, P., Kundu, M., Basu, J.: The serine/threonine
kinase PknB of Mycobacterium tuberculosis phosphorylates PBPA,
a penicillin-binding protein required for cell division. Microbiology.
152(Pt 2), 493–504 (2006). https://doi.org/10.1099/mic.0.28630-0
79. Datta, P., Dasgupta, A., Singh, A.K., Mukherjee, P., Kundu, M.,
Basu, J.: Interaction between FtsW and penicillin-binding protein
3 (PBP3) directs PBP3 to mid-cell, controls cell septation and
mediates the formation of a trimeric complex involving FtsZ,
FtsW and PBP3 in mycobacteria. Mol Microbiol 62(6), 1655–
1673 (2006). https://doi.org/10.1111/j.1365-2958.2006.05491.x
80. Meniche, X., Otten, R., Siegrist, M.S., Baer, C.E., Murphy, K.C.,
Bertozzi, C.R., Sassetti, C.M.: Subpolar addition of new cell wall
is directed by DivIVA in mycobacteria. Proc. Natl. Acad. Sci. U.
S. A. 111(31), E3243–E3251 (2014). https://doi.org/10.1073/
pnas.1402158111
81. Santi, I., Dhar, N., Bousbaine, D., Wakamoto, Y., McKinney, J.D.:
Single-cell dynamics of the chromosome replication and cell divi-
sion cycles in mycobacteria. Nat. Commun. 4, 2470 (2013).
https://doi.org/10.1038/ncomms3470
82. Aldridge, B.B., Fernandez-Suarez, M., Heller, D.,
Ambravaneswaran, V., Irimia, D., Toner, M., Fortune, S.M.:
Asymmetry and aging of mycobacterial cells lead to variable
growth and antibiotic susceptibility. Science. 335(6064), 100–
104 (2012). https://doi.org/10.1126/science.1216166
83. Wayne, L.G.: Dormancy of Mycobacterium tuberculosis and latency
of disease. Eur. J. Clin. Microbiol. Infect. Dis. 13(11), 908–914 (1994)
84. Cunningham, A.F., Spreadbury, C.L.: Mycobacterial stationary
phase induced by low oxygen tension: cell wall thickening and
localization of the 16-kilodalton alpha-crystallin homolog. J.
Bacteriol. 180(4), 801–808 (1998)
431
85. Fang, X., Wallqvist, A., Reifman, J.: Modeling phenotypic meta-
bolic adaptations of Mycobacterium tuberculosis H37Rv under
hypoxia. PLoS Comput. Biol. 8(9), e1002688 (2012). https://doi.
org/10.1371/journal.pcbi.1002688
86. Doyle, R.J., Chaloupka, J., Vinter, V.: Turnover of cell walls in
microorganisms. Microbiol. Rev. 52(4), 554–567 (1988)
87. Shah, I.M., Laaberki, M.H., Popham, D.L., Dworkin, J.: A
eukaryotic-like Ser/Thr kinase signals bacteria to exit dormancy
in response to peptidoglycan fragments. Cell. 135(3), 486–496
(2008). https://doi.org/10.1016/j.cell.2008.08.039
88. Nikitushkin, V.D., Demina, G.R., Shleeva, M.O., Kaprelyants,
A.S.: Peptidoglycan fragments stimulate resuscitation of "non-
culturable" mycobacteria. Antonie Van Leeuwenhoek. 103(1),
37–46 (2013). https://doi.org/10.1007/s10482-012-9784-1
89. Dworkin, J., Shah, I.M.: Exit from dormancy in microbial organ-
isms. Nat. Rev. Microbiol. 8(12), 890–896 (2010). https://doi.org/
10.1038/nrmicro2453
90. Yeats, C., Finn, R.D., Bateman, A.: The PASTA domain: a beta-
lactam-binding domain. Trends Biochem. Sci. 27(9), 438 (2002)
91. Mir, M., Asong, J., Li, X., Cardot, J., Boons, G.J., Husson, R.N.:
The extracytoplasmic domain of the Mycobacterium tuberculosis
Ser/Thr kinase PknB binds specific muropeptides and is required
for PknB localization. PLoS Pathog. 7(7), e1002182 (2011).
https://doi.org/10.1371/journal.ppat.1002182
92. Nikitushkin, V.D., Demina, G.R., Shleeva, M.O., Guryanova,
S.V., Ruggiero, A., Berisio, R., Kaprelyants, A.S.: A product of
RpfB and RipA joint enzymatic action promotes the resuscitation
of dormant mycobacteria. FEBS J. 282(13), 2500–2511 (2015).
https://doi.org/10.1111/febs.13292
93. Kana, B.D., Mizrahi, V.: Resuscitation-promoting factors as lytic
enzymes for bacterial growth and signaling. FEMS Immunol.
Med. Microbiol. 58(1), 39–50 (2010). https://doi.org/10.1111/j.
1574-695X.2009.00606.x
94. Eoh, H., Wang, Z., Layre, E., Rath, P., Morris, R., Branch Moody,
D., Rhee, K.Y.: Metabolic anticipation in Mycobacterium tuber-
culosis. Nat. Microbiol. 2, 17084 (2017). https://doi.org/10.1038/
nmicrobiol.2017.84
95. Rani, C., Khan, I.A.: UDP-GlcNAc pathway: potential target for
inhibitor discovery against M. tuberculosis. Eur. J. Pharm. Sci. 83,
62–70 (2016). https://doi.org/10.1016/j.ejps.2015.12.013
96. Soni, V., Suryadevara, P., Sriram, D., Consortium, O., Kumar, S.,
Nandicoori, V.K., Yogeeswari, P.: Structure-based design of di-
vers e inhibito rs of Mycobacterium tuberculosis N-
acetylglucosamine-1-phosphate uridyltransferase: combined mo-
lecular docking, dynamic simulation, and biological activity. J.
Mol. Model. 21(7), 174 (2015). https://doi.org/10.1007/s00894-
015-2704-3
97. Schumacher, C.E., Harris, P.W.R., Ding, X.B., Krause, B., Wright,
T.H., Cook, G.M., Furkert, D.P., Brimble, M.A.: Synthesis and
biological evaluation of novel teixobactin analogues. Org.
Biomol. Chem. 15(41), 8755–8760 (2017). https://doi.org/10.
1039/c7ob02169k
98. Eldholm, V., Pettersson, J.H., Brynildsrud, O.B., Kitchen, A.,
Rasmussen, E.M., Lillebaek, T., Ronning, J.O., Crudu, V.,
Mengshoel, A.T., Debech, N., Alfsnes, K., Bohlin, J., Pepperell,
C.S., Balloux, F.: Armed conflict and population displacement as
drivers of the evolution and dispersal of Mycobacterium tubercu-
losis. Proc. Natl. Acad. Sci. U. S. A. 113(48), 13881–13886
(2016). https://doi.org/10.1073/pnas.1611283113
99. Abedon, S.T.: Lysis from without. Bacteriophage. 1(1), 46–49
(2011). https://doi.org/10.4161/bact.1.1.13980
100. Lai, M.J., Liu, C.C., Jiang, S.J., Soo, P.C., Tu, M.H., Lee, J.J.,
Chen, Y.H., Chang, K.C.: Antimycobacterial activities of
Endolysins derived from a Mycobacteriophage, BTCU-1.
Molecules. 20(10), 19277–19290 (2015). https://doi.org/10.
3390/molecules201019277
432
101. Dubee, V., Triboulet, S., Mainardi, J.L., Etheve-Quelquejeu, M.,
Gutmann, L., Marie, A., Dubost, L., Hugonnet, J.E., Arthur, M.:
Inactivation of Mycobacterium tuberculosis l,d-transpeptidase
LdtMt(1) by carbapenems and cephalosporins. Antimicrob.
Agents Chemother. 56(8), 4189–4195 (2012). https://doi.org/10.
1128/AAC.00665-12
102. Mattoo, R., Lloyd, E.P., Kaushik, A., Kumar, P., Brunelle, J.L.,
Townsend, C.A., Lamichhane, G.: LdtMav2, a nonclassical
transpeptidase and susceptibility of Mycobacterium avium to car-
bapenems. Future Microbiol. 12, 595–607 (2017). https://doi.org/
10.2217/fmb-2016-0208
103. Bianchet, M.A., Pan, Y.H., Basta, L.A.B., Saavedra, H., Lloyd,
E.P., Kumar, P., Mattoo, R., Townsend, C.A., Lamichhane, G.:
Structural insight into the inactivation of Mycobacterium tubercu-
losis non-classical transpeptidase LdtMt2 by biapenem and
tebipenem. BMC Biochem. 18(1), 8 (2017). https://doi.org/10.
1186/s12858-017-0082-4
104. Knox, J.R., Moews, P.C., Frere, J.M.: Molecular evolution of bac-
terial beta-lactam resistance. Chem. Biol. 3(11), 937–947 (1996)
105. Voladri, R.K., Lakey, D.L., Hennigan, S.H., Menzies, B.E.,
Edwards, K.M., Kernodle, D.S.: Recombinant expression and
characterization of the major beta-lactamase of Mycobacterium
tuberculosis. Antimicrob. Agents Chemother. 42(6), 1375–1381
(1998)
106. Flores, A.R., Parsons, L.M., Pavelka Jr., M.S.: Genetic analysis of
the beta-lactamases of Mycobacterium tuberculosis and
Mycobacterium smegmatis and susceptibility to beta-lactam anti-
biotics. Microbiology. 151(Pt 2), 521–532 (2005). https://doi.org/
10.1099/mic.0.27629-0
107. Kasik, J.E., Weber, M., Winberg, E., Barclay, W.R.: The synergis-
tic effect of dicloxacillin and penicillin G on murine tuberculosis.
Am. Rev. Respir. Dis. 94(2), 260–261 (1966). https://doi.org/10.
1164/arrd.1966.94.2.260
108. Hugonnet, J.E., Tremblay, L.W., Boshoff, H.I., Barry 3rd, C.E.,
Blanchard, J.S.: Meropenem-clavulanate is effective against ex-
tensively drug-resistant Mycobacterium tuberculosis. Science.
323(5918), 1215–1218 (2009). https://doi.org/10.1126/science.
1167498
109. Chambers, H.F., Moreau, D., Yajko, D., Miick, C., Wagner, C.,
Hackbarth, C., Kocagoz, S., Rosenberg, E., Hadley, W.K.,
Nikaido, H.: Can penicillins and other beta-lactam antibiotics be
used to treat tuberculosis? Antimicrob. Agents Chemother. 39(12),
2620–2624 (1995)
110. Zhang, D., Wang, Y., Lu, J., Pang, Y.: In vitro activity of beta-
lactams in combination with beta-lactamase inhibitors against
multidrug-resistant Mycobacterium tuberculosis isolates.
Antimicrob. Agents Chemother. 60(1), 393–399 (2016). https://
doi.org/10.1128/AAC.01035-15
111. Kurz, S.G., Wolff, K.A., Hazra, S., Bethel, C.R., Hujer, A.M.,
Smith, K.M., Xu, Y., Tremblay, L.W., Blanchard, J.S., Nguyen,
L., Bonomo, R.A.: Can inhibitor-resistant substitutions in the
Mycobacterium tuberculosis beta-lactamase BlaC lead to
clavulanate resistance?: a biochemical rationale for the use of
beta-lactam-beta-lactamase inhibitor combinations. Antimicrob.
Glycoconj J (2018) 35:421–432
Agents Chemother. 57(12), 6085–6096 (2013). https://doi.org/
10.1128/AAC.01253-13
112. Diacon, A.H., van der Merwe, L., Barnard, M., von Groote-
Bidlingmaier, F., Lange, C., Garcia-Basteiro, A.L., Sevene, E.,
Ballell, L., Barros-Aguirre, D.: beta-lactams against
tuberculosis–new trick for an old dog? N. Engl. J. Med. 375(4),
393–394 (2016). https://doi.org/10.1056/NEJMc1513236
113. Mishra, S., Shukla, P., Bhaskar, A., Anand, K., Baloni, P., Jha,
R.K., Mohan, A., Rajmani, R.S., Nagaraja, V., Chandra, N.,
Singh, A.: Efficacy of beta-lactam/beta-lactamase inhibitor com-
bination is linked to WhiB4-mediated changes in redox physiolo-
gy of Mycobacterium tuberculosis. Elife. 6, e25624 (2017).
https://doi.org/10.7554/eLife.25624
114. Ramakrishnan, G., Chandra, N.R., Srinivasan, N.: Recognizing
drug targets using evolutionary information: implications for
repurposing FDA-approved drugs against Mycobacterium tuber-
culosis H37Rv. Mol. BioSyst. 11(12), 3316–3331 (2015). https://
doi.org/10.1039/c5mb00476d
115. Garcia-Fernandez, E., Koch, G., Wagner, R.M., Fekete, A.,
Stengel, S.T., Schneider, J., Mielich-Suss, B., Geibel, S.,
Markert, S.M., Stigloher, C., Lopez, D.: Membrane microdo-
main disassembly inhibits MRSA antibiotic resistance. Cell.
171(6), 1354–1367 e1320 (2017). https://doi.org/10.1016/j.
cell.2017.10.012
116. Eniyan, K., Dharavath, S., Vijayan, R., Bajpai, U., Gourinath, S.:
Crystal structure of UDP-N-acetylglucosamine-enolpyruvate re-
ductase (MurB) from Mycobacterium tuberculosis. Biochim.
Biophys. Acta. 1866(3), 397–406 (2018). https://doi.org/10.
1016/j.bbapap.2017.11.013
117. Singh, V., Dhar, N., Pato, J., Kolly, G.S., Kordulakova, J.,
Forbak, M., Evans, J.C., Szekely, R., Rybniker, J., Palcekova,
Z., Zemanova, J., Santi, I., Signorino-Gelo, F., Rodrigues, L.,
Vocat, A., Covarrubias, A.S., Rengifo, M.G., Johnsson, K.,
Mowbray, S., Buechler, J., Delorme, V., Brodin, P., Knott,
G.W., Ainsa, J.A., Warner, D.F., Keri, G., Mikusova, K.,
McKinney, J.D., Cole, S.T., Mizrahi, V., Hartkoorn, R.C.:
Identification of aminopyrimidine-sulfonamides as potent
m o d u l a t o r s o f Wa g 3 1 - m e d i a t e d c e l l e l o n g a t i o n i n
mycobacteria. Mol. Microbiol. (2016). https://doi.org/10.
1111/mmi.13535
118. Linares, J.F., Gustafsson, I., Baquero, F., Martinez, J.L.:
Antibiotics as intermicrobial signaling agents instead of weapons.
Proc. Natl. Acad. Sci. U. S. A. 103(51), 19484–19489 (2006).
https://doi.org/10.1073/pnas.0608949103
119. D'Costa, V.M., King, C.E., Kalan, L., Morar, M., Sung, W.W.,
Schwarz, C., Froese, D., Zazula, G., Calmels, F., Debruyne, R.,
Golding, G.B., Poinar, H.N., Wright, G.D.: Antibiotic resistance is
ancient. Nature. 477(7365), 457–461 (2011). https://doi.org/10.
1038/nature10388
120. Okano, A., Isley, N.A., Boger, D.L.: Peripheral modifications of
[Psi[CH2NH]Tpg4]vancomycin with added synergistic mecha-
nisms of action provide durable and potent antibiotics. Proc.
Natl. Acad. Sci. U. S. A. (2017). https://doi.org/10.1073/pnas.
1704125114