Review

Sweet Talk: Protein

Glycosylation in Bacterial
Interaction With the Host
Qiuhe Lu,1 Shan Li,1,2 and Feng Shao1,*
Pathogenic bacteria encode virulent glycosyltransferases that conjugate various glycans onto substrate proteins via the N- or O-linkage. The HMW system in
nontypeable Haemophilus inuenzae and the Pgl system in Campylobacter
jejuni glycosylate bacterial surface or periplasmic proteins at the eukaryoticlike Asn-X-Ser/Thr motif. The NleB effector from enterobacteria mediates arginine GlcNAcylation of host death-domain proteins to block inammation, representing an atypical N-glycosylation. The large clostridial cytotoxins and
related glucosyltransferase toxins from Legionella and Photorhabdus monoglycosylate a serine/threonine or tyrosine in host Rho GTPase or elongation
factor 1A (eEF1A). The emerging bacterial autotransporter heptosyltransferase
(BAHT) family of heptosyltransferases also catalyses O-glycosylation and modies autotransporters for adhesion to the host. These glycosylations, diverse in
linkages and glycan structures, determine appropriate functioning of bacterial
virulence factors or hijack host cellular processes in pathogenesis.

Trends
Bacteria have evolved chemically
diverse glycosylation systems for
pathogenesis.
Bacterial glycosylation not only allows
adhesion to the host cell but also functions to modulate crucial host cellular
processes.
An enteropathogenic Escherichia coli
(EPEC) type III effector NleB catalyses
arginine GlcNAcylation of host deathdomain proteins to block cell death and
suppress inammation.
The Photorhabdus asymbiotica protein
toxin (PaTox) modies Rho GTPase by
GlcNAcylation of a tyrosine residue.

Introduction to Protein Glycosylation in Pathogenic Bacteria

A large bacterial autotransporter heptosyltransferase (BAHT) family mediates O-linked hyperheptosylation of
bacterial autotransporters through an
unprecedented structural mechanism.

Protein glycosylation, that is, enzyme-catalysed covalent attachment of glycans onto amino acid
side chains in proteins, is one of the most common post-translational modications (PTMs) in all
kingdoms of life. Protein glycosylation occurs via two major chemical forms, either the N-linked
or O-linked glycosylation [1,2]. In the canonical N-linked glycosylation, glycans are conjugated to
the side chain amide nitrogen of an asparagine in an Asn-X-Ser/Thr consensus sequence (X is
any amino acid except for a proline). The O-linked glycosylation generally occurs on the hydroxyl
group of a serine or threonine residue. Historically, protein glycosylation was long believed to be
restricted to eukaryotes, but it is now well established that bacteria and even archaea also bear
multitudinous glycoproteins generated through N- and O-linked glycosylation [36].
Protein glycosylation in pathogenic bacteria has become a subject of heightened attention in the
past 10 years; accumulating evidence indicates a tight association of this type of PTM with
bacterial virulence [4,7]. A large number of glycoproteins have been identied in pathogenic
bacteria, including Pseudomonas aeruginosa [8,9], Neisseria meningitides [10], Haemophilus
inuenzae [11], Campylobacter jejuni [12], Mycobacterium tuberculosis [13], Streptococcus
parasanguis [14], diffusely adhering Escherichia coli (DAEC) [15], and enterotoxigenic E. coli
(ETEC) [16]. Studies in the past several years have also recorded various O-linked glycosylation
modications in Burkholderia [17,18], Francisella [19,20], and Acinetobacter spp. [21,22] with
potential virulence functions (reviewed in [23]). Bacterial pathogens are often endowed with
secreted toxins or effector proteins that function to facilitate infection or modulate host innate
immune defenses. A concept is emerging that the bacterial toxin/effector often harbors a potent
enzymatic activity and induces a novel PTM on key host signaling molecules [24]. It is equally
important to note that some bacterial toxins or virulence factors bear glycosyltransferase

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2015 Elsevier Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.tim.2015.07.003

The serine-rich repeat protein (SRRP)

Fap1 from an oral streptococcal bacterium is sequentially modied by multiple glycosyltransferases, of which
dGT1 adopts a glycosyltransferase fold
different from all known ones.

National Institute of Biological

Sciences, #7 Science Park Rd,
Zhongguancun Life Science Park,
Beijing, 102206, China
2
Taihe Hospital, Hubei University of
Medicine, Shiyan, Hubei, 442000,
China

*Correspondence:
shaofeng@nibs.ac.cn (F. Shao).

activities that are designed to modify their host targets during infection. Here we summarize
bacterial glycosyltransferase and glycosylation involved in pathogenesis and discuss recent
research progress in this area. Owing to the focus of the review and space limitation, we
elaborate on the relatively well-characterized glycosylation systems that have a dened function
in bacterial interaction with the host cell.

Protein N-Glycosylation in Bacterial Pathogenesis

Glycosylated Adhesins in Nontypeable Haemophilus inuenzae
Adhesion to the host cell surface is important for many bacteria to initiate infection. This is also
true for nontypeable (nonencapsulated) H. inuenzae (NTHi), a common invasive bacterium that
frequently causes otitis media, chronic bronchitis, and community-acquired pneumonia [25].
Development of NTHi disease begins with bacterial colonization of respiratory epithelial cells.
Most clinical isolates of NTHi express two related high molecular weight adhesion proteins
HMW1 and HMW2. HMW1/2 are targeted to the cell surface to mediate bacterial adhesion to
the host cell [26]. The function of HMW1 and HMW2 requires HMW1B/HMW1C and HMW2B/
HMW2C proteins, respectively. HMW1B/HMW2B are inserted into the bacterial outer membrane and mediate surface display of HMW1/HMW2 [27], a system known as the two-partner
secretion (TPS) or the type Vb secretion pathway. Notably, HMW1/2 are glycosylated in the
bacterial cytoplasm in an HMW1C/2C-dependent manner, with glucose and galactose being
the major sugar conjugates [11]. Mass spectrometry analyses have identied 31 glycosylation
sites in HMW1 [28]. The glycans are either a single hexose or di-hexoses that are attached to the
asparagines in the Asn-X-Ser/Thr consensus sequence. HMW1C has been demonstrated to
harbor glycosyltransferase activity and can catalyse N-linked glucosylation or galactosylation of

Table 1. Bacterial Glycosylation Systems Discussed in This Reviewa

Organism

Glycosyltransferase
(GT)

GT
Family

Linkage

Substrates

Residues
Modied

Glycan

Haemophilus
inuenzae

HMW1C and
HMW2C

GT41

HMW1/2 adhesins

Asparagine

Hex and di-Hex

(Hex: Glc or Gal)

Campylobacter
jejuni

PglB

GT66

>60 substrates

Asparagine

Heptasaccharide

EPEC

NleB

NC

FADD/RIPK1/
TRADD/TNFR1

Arginine

GlcNAc

Clostridium spp.

TcdA/B and the

large Clostridial
cytotoxin family

GT44

Rho GTPases

Threonine

Glc or GlcNAc

Photorhabdus
asymbiotica

PaTox

NC

Rho GTPases

Tyrosine

GlcNAc

Legionella
pneumophila

Lgts

GT88

eEF1A

Serine

Glc

DAEC/ETEC/
Citrobacter
rodentium

AAH/TibC/BAHTcr
(the BAHT family)

NC

AIDA-I/TibA/CARC
(autotransporters)

Serine

Hep

Streptococcus
parasanguinis

Gtf1/Gtf2 complex

GT4

Fap1

Serine

GlcNAc

Gtf3

GT8

GlcNAc-Fap1

GlcNAc

Glc

dGT1

NC

Glc-GlcNAc-Fap1

Glc

Glc

Abbreviations: NC, not classied; Hex, hexose; Glc, glucose; Gal, galactose; Hep, heptose; GlcNAc, N-acetylglucosamine; the
heptasaccharide refers to GalNAc-/1,4-GalNAc-/1,4-(Glc-b1,3-)GalNAc-/1,4-GalNAc-/1,4-GalNAc-/1,3-bacillosamine,
in which the GalNAc stands for N-acetylgalactosamine. EPEC, enteropathogenic Escherichia coli; DAEC, diffusely adhering
E. coli; ETEC, enterotoxigenic E.coli; FADD, FAS-associated death domain protein; RIPK1, receptor-interacting protein kinase
1; TNFR1, tumor necrosis factor receptor 1; DD, death domain; BAHT, bacterial autotransporter heptosyltransferase.

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the asparagines in HMW1 (Table 1) [29]. HMW1C can also generate O-glycosidic bonds
between the two hexoses linked to the asparagine [29].
Nonglycosylated HMW1 is partially degraded and sheds from the bacterial surface, suggesting
that glycosylation is required for HMW1 stability and tethering to the bacterial surface [11].
Deciency in HMW1 glycosylation reduces bacterial adhesion to host epithelial cells. An early
report suggests that HMW1 is recognized by a sialylated glycoprotein receptor on human
epithelial cells [30]. Thus, HMW1 in NTHi represents a novel mechanism in pathogenesis, in
which glycosylation of both the adhesin and its host cell receptor mediates bacterial interaction
with the host.
By sequence homology, HMW1C and HMW1C-like proteins belong to the GT41 family of
glycosyltransferases, but mainly catalyse N-linked glycosylation rather than the O-GlcNAcylation
seen with other GT41-family members. HMW1C-like glycosyltransferases are prevalent among
bacteria and can be grouped into two categories [31]. In the rst category, the glycosyltransferase, such as HMW1C in NTHi, is part of the TPS system, which has been found in several
bacterial species, including ETEC, Yersinia, and Burkholderia spp. Among this category, the
ETEC protein EtpC glycosylates EtpA, a coproduced adhesin from the TPS system, which also
confers bacterial adhesion to intestinal epithelial cells [32]. For the second category of HMW1Clike proteins, the genomic organization is not suggestive of the glycosylation target. One protein
in this category from Actinobacillus pleuropneumoniae (ApHMW1C) glycosylates bacterial
autotransporters and outer membrane proteins [33], indicating that this type of HMW1C-like
protein may also modify bacterial surface proteins. The crystal structure of ApHMW1C conrms
that HMW1C-like enzymes are type B glycosyltransferases and provides mechanistic insights
into the sugar transfer reaction [34]. The HMWC-like proteins are also widespread in Pasteurellaceae and Neisseriaceae families; it will be interesting for future studies to examine whether
and how their glycosyltransferase activities mediate the interaction of the bacteria with the host
(see Outstanding Questions).
A General Glycosylation Pathway in Campylobacter jejuni Pathogenesis
C. jejuni is a leading cause of bacterial gastroenteritis worldwide with a high incidence that
sometimes surpasses that of Salmonella and Shigella infections [35]. One virulence factor shared
by different C. jejuni strains is a unique N-linked protein glycosylation (pgl) system [3,12].
Modication catalysed by the pgl system features a heptasaccharide, composed of GalNAc/1,4-GalNAc-/1,4-(Glc-b1,3-)GalNAc-/1,4-GalNAc-/1,4-GalNAc-/1,3-bacillosamine (GalNAc, N-acetylgalactosamine), which is also conjugated onto the asparagine within the eukaryotic-like Asn-X-Ser/Thr glycosylation consensus motif in target C. jejuni proteins [36]. The pgl
locus contains 12 open reading frames (ORFs) and is functionally sufcient to produce N-linked
glycoproteins in E. coli [37]. The 12 ORFs encode several glycosyltransferases and sugar
biosynthetic enzymes that work together to generate a lipid-linked heptasaccharide precursor.
The heptasaccharide, once it crosses the bacterial inner membrane into the periplasm, is
transferred onto the target asparagine by PglB (Table 1), a structural homologue of the
STT3 catalytic subunit of Saccharomyces cerevisiae oligosaccharyltransferase complex [38].
Genetic mutation in the pgl locus, particularly pglB, disrupts protein glycosylation in C. jejuni,
which results in diminished C. jejuni adhesion and invasion of human epithelial cells and
consequently much reduced bacterial colonization of mice and chicken intestinal tracts [39
41]. Since its discovery, more than 60 periplasmic and membrane-bound proteins in C. jejuni
have been found to be N-glycosylated by the pgl system [42]. However, which glycoprotein(s)
plays a critical role in C. jejuni interaction with the host is still a question (see Outstanding
Questions). Given the large number of glycosylation targets, it is possible that the virulence
function of the pgl system is a result of pleiotropic effects of multiglycosylated proteins. A recent
study has proposed an attracting hypothesis that Pgl-mediated N-glycosylation of C. jejuni

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surface proteins may function to protect bacterial proteins from digestion by gut proteases,
thereby promoting tness of the bacterium in the host environment [43]. Moreover, the free
oligosaccharides generated from the Pgl pathway have been suggested to have an independent
function in osmotic protection [44]. The Pgl system in Campylobacter species has been a
prototype for appreciating the important role of N-glycosylation in bacterial interaction with their
respective hosts.
Bacteria-Catalysed Arginine GlcNAcylation in Blocking Host Inammation
The type III secretion system (T3SS) is a major virulence secretion system in many Gramnegative bacterial pathogens for injecting effector proteins into host cells; these effectors
function to manipulate host signaling pathways and evade immune defenses. In studying
the role of T3SS in enteropathogenic Escherichia coli (EPEC) pathogenesis, two independent
groups discovered a critical function of the NleB effector in blocking host death receptor
signaling and suppressing the inammatory responses [45,46]. Most interestingly, NleB acts
as an N-acetylglucosamine (GlcNAc) transferase to modify the death domains (DDs) in TNFR1
(tumor necrosis factor receptor 1) and more efciently its downstream adaptors, including
TNFR1-associated death domain protein (TRADD), FAS-associated death domain protein
(FADD), and receptor-interacting protein kinase 1 (RIPK1) (Table 1). This nding not only explains
the previous observation that NleB could selectively inhibit tumor necrosis factor-/ (TNF/) but
not interleukin (IL)-1b activation of the nuclear factor kB (NF-kB) proinammatory signaling
[47,48], but also suggests a biologic function of NleB in blocking the death receptor-instructed
host cell death. TNF/ is well known for inducing cell death through TNFR1 by forming a
caspase-8-activating death-inducing signaling complex (DISC) containing TRADD/RIPK1/
FADD. Formation of the DISC is mediated by homotypic/heterotypic DDDD interactions among
TRADD, RIPK1, and FADD. GlcNAcylated DDs by NleB lose the ability to interact with one
another, as a result of which, DISC formation in EPEC-infected cells, upon stimulation by TNF/
or other death receptor ligands, is disrupted by NleB-catalysed GlcNAcylation of the DDcontaining proteins (Figure 1, Key Figure).
Bacterial infection is often accompanied by host cell death. Under certain circumstances, death
of infected host cells helps to restrict the infection, thereby serving as a defense mechanism [49].
Thus, pathogens have evolved sophisticated strategies to modulate the death signaling in host
cells. Blocking host death receptor pathways by the GlcNAc transferase effector NleB suggests
a critical role of GlcNAc modication of DDs in facilitating EPEC infection. Citrobacter rodentium
is an EPEC-like mucosal pathogen of mice and also harbors the NleB effector. In mouse
infection, wild-type C. rodentium, compared to the isogenic DnleB mutant, reduces caspase-8 cleavage and activation; Fas-decient mice develop more severe diarrhea and tissue
damage when the infection is performed with the NleB-positive C. rodentium strain [46].
Consistently, C. rodentium colonization of the mouse intestine is severely reduced by disrupting
the GlcNAc transferase activity in NleB [45].
Protein GlcNAcylation is universal from E. coli to mammals, and the underlying biochemical
mechanism has been well established. Different from known GlcNAc modications that exclusively occur on a serine/threonine hydroxyl group (termed O-GlcNAcylation) [50], NleB transfers
the GlcNAc onto a specic arginine conserved in the DDs of TNFR1, TRADD, FADD, and RIPK1
[45,46] (Figure 1, Key Figure). This unusual form of GlcNAcylation was demonstrated by
extensive mass spectrometry analyses of NleB-modied TRADD and FADD both in vitro and
during EPEC infection. Notably, NleB is unable to catalyse O-GlcNAcylation; NleB-modied
TRADD resists detection by the O-GlcNAc-specic antibody [45]. Studies of protein O-GlcNAcylation have established that enzyme-catalysed GlcNAc transfer proceeds with the nucleophilic
substitution reaction. Given that the arginine guanidine group is an extremely poor nucleophile,
future structural investigations of NleB modication of the DD shall be revealing in understanding

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Key Figure

Two Distinct Glycosylation Modifications Are Required for Citrobacter

rodentium Colonization in Mice

Key:
Serine

BAHT

Heptose
ADP-heptose

Autotransporter
NleB

TRADD

RIPK1

TNFR1

Adhesion and
colonizaon

DD

NleB

R -GlcNAc

NleB
+ UDP-GlcNAc

DD

TRAF2

RIPK1

NleB

TRADD

RIPK1

TRADD

RIP3
NF-B

FADD

FADD

Caspase-8

Caspase-8

Apoptosis

RIPK1
RIP3
MLKL

Necroptosis

Figure 1. Citrobacter rodentium is a mouse model of human attachingeffacing enteropathogens, including enteropathogenic Escherichia coli (EPEC). The initial step in C. rodentium infection is adherence to intestinal epithelial cells, mediated by
an autotransporter (named CARC) on the bacterial surface. The autotransporter, prior to crossing the bacterial inner
membrane, is heptosylated by a BAHT (bacterial autotransporter heptosyltransferase)-family member on numerous serine
residues in the passenger domain, and the modication is required for the adhesion. C. rodentium also harbors a type III
secretion system (T3SS) effector NleB that monoglycosylates the death domain (DD) of TNFR1-associated death domain
protein (TRADD), FAS-associated death domain protein (FADD), and receptor-interacting protein kinase 1 (RIPK1) downstream of tumor necrosis factor receptor 1 (TNFR1) or other death receptors. NleB is a unique GlcNAc transferase that
modies a conserved arginine in the DDs (inset). GlcNAcylated DDs are decient in homotypic/heterotypic oligomerization,
thereby blocking the assembly of signaling complexes downstream of the death receptor. As a result, NleB disrupts multiple
tumor necrosis factor-/ (TNF/)-activated signaling events, including activation of nuclear factor kB (NF-kB), apoptosis and
necroptosis to counteract host immunity. The two glycosylation modications mediated by the BAHT and NleB are both
critically required for C. rodentium colonization of the gastrointestinal tract in infected mice.

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this unique protein glycosylation (see Outstanding Questions). The two studies on NleB [45,46]
together illustrate a new bacterial virulence mechanism mediated by glycosylation of host
proteins, and also uncover a novel PTM, i.e., arginine GlcNAcylation. NleB-homologous effectors are also present in certain Salmonella and Shigella species, suggesting that the modication
is not unique to EPEC [45]. Along this idea, a stimulating question arises whether arginine
GlcNAcylation is generally involved in regulating cellular processes in prokaryotes and/or
eukaryotes (see Outstanding Questions). The recent success in generating the antibody that
can efciently and specically recognize arginine GlcNAcylation independently of the surrounding sequences [51] will be helpful for answering the question.

Protein O-Glycosylation in Bacterial Pathogenesis

Large Clostridial Glucosylating Toxins
Clostridium difcile, a Gram-positive, anaerobic and spore-forming bacillus, is the leading cause
of hospital-acquired infectious diarrhea and pseudomembranous colitis. The predominant
virulence factors of C. difcile are two homologous and functionally similar cytotoxins: toxin
A (TcdA) and toxin B (TcdB) [52,53]. The functional region in TcdA/B is the N-terminal glucosyltransferase domain that has been biochemically and structurally characterized in detail [54]. Both
TcdA and TcdB confer a toxic and cell-rounding effect in intoxicated host cells owing to the
action of the glucosyltransferase domain. The toxins enter into host cells through clathrinmediated endocytosis [55], following which the glucosyltransferase domain is released into the
cytosol by autoproteolytic cleavage [54]. TcdA/B selectively modify Rho GTPases, including Rho
(RhoA/B/C), Rac (Rac13), and Cdc42, by covalent addition of a glucose moiety (Table 1). The
glucosylation occurs on the side chain hydroxyl group of Thr-37 in RhoA and the equivalent Thr35 in Rac/Cdc42 [56,57]. TcdA/B-catalysed glucosylation causes multifaceted inhibitory effects
on Rho GTPase activation, including preventing their activation by guanine nucleotide exchange
factors (GEFs), uncoupling from the downstream effectors, and blocking their cytosol-membrane cycling [58,59] (Figure 2). These biochemical consequences claim the cell rounding,
membrane blebbing, and ultimately death of TcdA/B-stimulated host cells [52].
TcdA (308.0 kDa) and TcdB (269.6 kDa) are the prototypes of the large clostridial glucosylating
cytotoxin family, which additionally includes Clostridium sordellii lethal toxin (TcsL) and hemorrhagic toxin (TcsH), Clostridium novyi alpha toxin (Tcn/) and Clostridium perfringens large
cytotoxin TpeL [52,54,60,61]. These toxins also inactivate small GTPases by glucosylation or
GlcNAcylation on the threonine residue equivalent to Thr-37 in RhoA. A recent study identied a
new toxin from entomopathogenic Photorhabdus asymbiotica, which bears a glycosyltransferase domain structurally similar to that of TcdA/B [62]. The P. asymbiotica protein toxin (PaTox)
also modies Rho GTPase by GlcNAcylation, but uniquely modies a tyrosine residue (Tyr-32 in
RhoA) near the threonine targeted by TcdA/B (Table 1). The modication results in inactivation of
Rho GTPase signaling similar to that by TcdA/B. This nding highlights the commonality and
diversity of targeting small GTPases for glycosylation in bacterial pathogenesis.
While TcdA/B-catalysed O-glycosylation of Rho GTPases represents an important virulence
mechanism for C. difcile, this virulence activity of TcdA/B is also subjected to innate immune
detection by the inammasome pathway in host macrophages [63] (Figure 2). Inammasome
sensing of TcdA/B leads to the activation of an inammatory caspase (caspase-1); activated
caspase-1 then processes IL-1/18 for maturation and triggers a lytic form of inammatory cell
death called pyroptosis, mounting a strong immune response. Pyrin, encoded by the familial
Mediterranean fever (FMF) disease gene MEFV, serves as the critical immune sensor responsible
for detecting TcdA/B glucosylation and inactivation of Rho [63] (Figure 2). Thus, the pyrin
inammasome suggests a novel link between bacterial glycosylation of host proteins and
activation of the host innate immune system.

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Clostri
dium

Clostridial TcdA/B

Glucose

Pi

Glucose
Endocytosis

Clostri
dium

um
di
tri
s
o
Cl

GAP

Rho
GDP

Rho
GTP

Eectors

Acn cytoskeleton
and other funcons

GEF

Rho
GDP
GDP

GDI
Clostridial TcdA/B

Pyrin
ASC

GTP

PYRIN
PYRIN

Pro-caspase-1

Pseudo-TRIM
CARD
CARD

Caspase domain

B30.2

Pyrin
inammasome

p10
p20
p20
IL-1

Acvated
caspase-1
p10

IL-18

Anbacterial
defense

Pro-IL-1
Pro-IL-18

Pyroptosis

Figure 2. Glucosylation of Rho GTPases by Clostridium difcile Toxin A/B and Its Detection by the Pyrin
Inammasome. Toxin A/B (TcdA/B), the major virulence factors of C. difcile, enters into host cells through the endocytic
route. TcdA/B features a glucosyltransferase domain that monoglucosylates Rho GTPases on a threonine residue (Thr-37 in
RhoA). The modication inhibits the interaction of Rho GTPases with the guanine nucleotide dissociation inhibitor (GDI) as
well as their nucleotide cycling catalysed by the GTPase-activating protein (GAP) and the guanine nucleotide exchange
factors (GEFs). Glucosylated Rho GTPases are also impaired in activating their downstream effectors and are therefore
functionally decient in regulating actin cytoskeleton dynamics and other cellular processes. TcdA/B-mediated glucosylation and inactivation of Rho GTPases are detected by a cytosolic immune sensor, pyrin. Pyrin forms an inammasome
complex with the ASC (Apoptosis-associated Speck-like Protein Containing a Caspase Recruitment Domain) adaptor to
activate caspase-1, an inammatory caspase. Activated capase-1 processes pro-IL-1b/18 for maturation and triggers
macrophage inammatory death (pyroptosis), mounting antibacterial immune responses.

The Legionella pneumophila Glucosyltransferase Family

Gram-negative L. pneumophila is a facultative intracellular pathogen causing an atypical form of
pneumonia known as Legionnaires disease. L. pneumophila delivers more than 300 effector
proteins into the host cell through the Dot/Icm type IV secretion system (T4SS) [64]. These
effectors manipulate host immune defense and vesicular trafcking pathways to create a niche
supportive of intracellular growth of the bacterium [65]. In a biochemical search for new
Legionella virulence factors with UDP-glucosyltransferase activity, a 60-kDa Lgt1 (Legionella
glucosyltransferase 1) protein was identied from the bacterial culture supernatant by its
glucosylation of a 50-kDa protein in eukaryotic cytoplasmic fractions [66]. Lgt1 shares catalytic
core residues of the clostridial large glucosylating toxins, including the DxD motif conserved in
type A glycosyltransferases [67]; the crystal structure of Lgt1 conrms its high structural
homology to the TcdB glycosyltransferase domain [68]. A total of 13 Lgt1-like ORFs are present
in six sequenced L. pneumophila strains, and can be grouped into three families: Lgt1 to Lgt3.
The most studied Philadelphia-1 strain harbors lgt1, lgt2, and lgt3, whereas other strains
possess only lgt1 and lgt3 [69]. The 13 Lgts are all substrates of the Dot/Icm T4SS, suggesting
their function is to target host proteins for glycosylation.

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The 50-kDa in vitro substrate of Lgt1 was identied as the eukaryotic elongation factor 1A
(eEF1A), in which Ser-53 is mono-O-glucosylated [67]. Lgt1 can also glucosylate eEF1A in L.
pneumophila-infected macrophages [70]. Other Lgts, including those in the Lgt2 and Lgt3
families, all modify eEF1A by glucosylation of the same serine residue (Table 1) [71]. eEF1A is a
large GTPase and a component of the elongation complex in protein synthesis, playing a key role
in delivering aminoacyl-tRNAs to the A-site of the ribosome [72]. Ser-53 is located within the
GTPase domain of eEF1A; Lgt-catalysed glucosylation may prevent the conformational change
of the GTPase domain, thereby causing eEF1A inactivation. Recombinant Lgt1, 2, and 3 all can
inhibit eukaryotic protein synthesis in a cell-free translation system [71]. Delivery of the Lgt protein
into mammalian cells induces eEF1A glucosylation, and at the same time causes inhibition of
host protein synthesis as well as host cell death [67,71]. Lgt1 efciently glucosylates a decapeptide of eEF1A [73], but a ternary complex of aminoacyl-tRNA, GTP, and eEF1A is a more
preferred substrate [70]. Although the exact function of translational inhibition for L. pneumophila
infection is unknown, identication of the Lgts suggests that O-linked glucosylation of protein
synthesis machinery is exploited by bacterial pathogens such as L. pneumophila to hijack host
functions.
A Large Bacterial Autotransporter Heptosyltransferase (BAHT) Family
Gram-negative bacteria display various autotransporter virulence factors onto the cell surface via
the type Va secretion system [74]. All classical autotransporters are characterized by an Nterminal passenger domain and a C-terminal b-domain mediating translocation of the passenger
domain across the outer membrane. The passenger domain determines the functionality of the
autotransporter. It has long been known that some autotransporters are glycosylated in their
passenger domains, among which AIDA-I from DAEC 2787 and TibA from ETEC H10407 are
the two earliest identied ones [15,16,75]. Very recently, a large BAHT family was identied in
several Gram-negative pathogens, including DAEC 2787 and ETEC H10407 [76]. The BAHT
family uses ADP-heptose generated in the lipopolysaccharide (LPS) biosynthesis pathway as the
sugar donor to modify the autotransporter. The gene encoding the BAHT always precedes that
of its substrate autotransporter in the same operon in the genome. BAHT modies numerous
serine residues within repeated units of a 19-residue consensus sequence in the autotransporter
passenger domain (Figure 1, Key Figure), which has been clearly demonstrated with AIDA-I and
TibA (Table 1) [76]. Heptosylation of AIDA-I is catalysed by AAH and its paralog AAH2, both of
which are BAHT-family members. Heptosylated AIDA-I confers a tight adhesion of the bacterium
to the host cell surface, in which the activity of AAH plays a dominant role. An AIDA-I-like
autotransporter in C. rodentium (named CARC) mediates bacterial colonization of the mouse
gastrointestinal tract [76]. Similar to AIDA-I, CARC is hyperheptosylated by its cognate BAHT,
and this modication is critically required for C. rodentium colonization in mice (Figure 1, Key
Figure).
Similar to AIDA-I modication by AAH, TibA in ETEC is heptosylated by the BAHT-family member
TibC (Table 1). The crystal structure of TibC-modied TibA passenger domain identied 35
heptose moieties, which are exclusively on serine residues and form patterned and solenoid-like
arrays on the surface of a b-helix structure [76]. Recombinant TibC and AAH, as well as other
BAHTs, can readily catalyse heptosylation of a synthetic peptide derived from the passenger
domain of AIDA-I or TibA, conrming that BAHTs are bona de heptosyltransferases. Interestingly, recombinant BAHT proteins, including AAH and TibC, all show a brownish color due to the
presence of one ferric ion in each BAHT molecule [76]. Several recombinant BAHT proteins have
been shown to adopt a dodecamer assembly. The crystal structure and cryoelectron microscopy (cryo-EM) structure of TibC have further revealed a characteristic ring shape of the
dodecamer [77]. Each TibC protomer bears an N-terminal b-barrel domain, a catalytic domain,
a b-hairpin thumb, and a unique iron-nger motif. Two adjacent protomers contact each other in
a back-to-back manner through the iron-nger motif-mediated dimerization; six symmetric

Trends in Microbiology, October 2015, Vol. 23, No. 10

637

dimers are further linked together via b-hairpin thumb-mediated hand-in-hand contacts to
assume the ring-shape assembly. The catalytic domain structurally resembles type B glycosyltransferase except for insertions of the b-hairpin thumb and the iron-nger motif. The structure
of TibC bound with the sugar donor identied a conserved aspartate that acts as the catalytic
base to activate the substrate acceptor to initiate the sugar transfer reaction. In the cryo-EM
structure of the TibC/TibA enzymesubstrate complex, the two TibC molecules of a back-toback dimer bind to one TibA located inside of the ring, resulting in a TibC12TibA6 assembly.
Molecular dynamics simulation of the TibC12TibA6 complex indicates a highly efcient processive hyperglycosylation reaction of six autotransporter molecules simultaneously by the
dodecamer enzyme. Thus, bacteria-mediated O-glycosylation can also target their own virulence factors, particularly the autotransporters, which involves distinct biochemical and structural mechanisms.
O-Glycosylated Serine-Rich Repeat Proteins (SRRPs) in Gram-Positive Bacteria
SRRPs are a family of large and surface-expressed glycoproteins exclusively found in Grampositive pathogenic and commensal bacteria, such as the streptococcal, staphylococcal, and
lactobacillus species [78]. SRRPs are mainly involved in bacterial attachment to eukaryotic cells,
inter/intra-species aggregation, and biolm formation [78,79]. For instance, the serine-rich
glycoprotein SrpA mediates the binding of Streptococcus sanguinis to human platelets, serving
as a key virulence determinant for infective endocarditis caused by the bacteria [80]. SRRPs of
Group B Streptococcus contribute to bacterial adherence and invasion of human brain microvascular endothelial cells as well as penetration of the bloodbrain barrier in the development of
streptococcal meningitis [81]. Biogenesis and appropriate functioning of the SRRPs requires the
glycosylation modication.
SRRPs mediate adhesion by forming large stalk structures projecting outwards from the
bacterial surface, resembling the pili in Group B Streptococcus [82]. The SRRP family typically
includes an extended signal sequence at the N-terminus, one or two species-specic nonrepeat regions (the NR domain) responsible for adhesion, two serine-repeat regions (one of
them makes the large size of the SRRP), and a C-terminal cell-wall anchoring domain [79]. Fap1
(mbriae-associated protein 1) is the rst SRRP identied from an oral streptococcal bacterium,
S. parasanguinis FW213 [14,83]. S. parasanguinis colonizes the tooth surface in the human oral
cavity and is important for dental plaque development [84]. Adhesion of S. parasanguinis is
mediated by Fap1 whose two serine-rich repeat regions are extensively modied by O-linked
glycans on numerous serine residues. The glycan structure has been recently determined as
Rha1-3Glc1-(Glc1-3GlcNAc1-)2,6Glc1-6GlcNAc (Rha, rhamnose) [85]. The fap1 locus
includes a cluster of 11 genes encoding several glycosyltransferases and proteins involved
in the secretion process [86,87]. gtf1 and gtf2 located downstream of fap1 encode two
glycosyltransferases that form a complex to transfer the rst GlcNAc to native Fap1 (Table 1)
[88]. Gtf3 then adds glucose to the GlcNAcylated Fap1 [89]; subsequently, dGT1, adopting a
novel glycosyltransferase fold, catalyses an additional glucosyl conjugation onto the GlcGlcNAc-modied Fap1 at the branching point (Table 1) [85]. S. parasanguinis mutants devoid
of the glycosyltransferases are decient in producing mature glycosylated Fap1 [88,89]. Similar
loci are present in a variety of other streptococcal and staphylococcal species, presumably
playing a similar role in biogenesis of the corresponding glycosylated SRRP [78]. It is the NR
domain in SRRPs that mediates adhesion and bacterial aggregation; the exact function of the
glycosylation, which occurs on the serine-repeat regions, is not clearly dened yet (see
Outstanding Questions). Notwithstanding, SRRPs are a growing family of O-linked glycoproteins that likely have important functions in bacterial physiology and pathophysiology. The
biosynthesis pathway of glycosylated SRRPs also provides a new angle for developing
therapeutics for SRRP-mediated infectious diseases such as bacterial endocarditis, meningitis, and pneumonia.

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Concluding Remarks and Future Perspectives

Outstanding Questions

Early studies show that glycosyltransferases present in bacteria mainly modify their surface
proteins, such as HMW1/2 in NTHi and SRRPs, in streptococci and staphylococci. The new
BAHT family has extended the list of glycosylation targets to a subfamily of autotransporters,
including the prototypical AIDA-I and TibA in enterobacterial pathogens [76,77]. Despite the
diverse pathophysiologies of the bacteria involved, glycosylated bacterial surface proteins and
autotransporters, regardless of being mono- or polyglycosylated, often act as adherence factors
for bacterial attachment to the host cell. Bacterial glycosyltransferases not only participate in this
initial step of infection, but they also modulate host responses by directly modifying host
proteins. The classic example is the large clostridial glucosylating toxins that modify Rho
GTPases to manipulate host actin cytoskeleton dynamics [54]. Discovering the Lgt family in
Legionella [69] and the NleB effector in enteropathogens [45,46] has broadened the spectrum of
host proteins targeted by bacterial glycosylation. Lgts glucosylate eEF1A of the protein synthesis
machinery; NleB adds a GlcNAc onto the death domain of TRADD, FADD, and RIPK1 to block
inammation. It is likely that future studies will identify other host proteins modied by bacterial
glycosyltransferase effectors in bacterial manipulation of host physiology and defense. It is worth
noting that C. rodentium requires both autotransporter glycosylation and glycosylation of
intracellular host proteins for colonizing the gastrointestinal tract (Figure 1, Key Figure).

What are the functions of HMW1C/2Clike proteins in other bacterial species?

Bacteria-catalysed glycosylation is diverse in both the glycosidic linkage and the glycan structure. The recently identied PaTox toxin shows that the O-linked glycosylation can occur on the
tyrosine hydroxyl group in addition to the canonical serine/threonine residues [62]. The NleB
effector GlcNAcylates the arginine residue [45,46], representing an atypical N-linked glycosylation. Structural enzymology studies are needed to further understand why NleB can target an
arginine guanidine, an extremely poor nucleophile, for glycosylation (see Outstanding Questions). The BAHT family is unique in that it uses ADP-heptose as the sugar donor [76]. The
diverse glycans in bacterial glycosylation are consistent with the presence of many dedicated or
nondedicated sugar biosynthesis systems in different bacteria, but it is not well understood how
the glycan structure is selected for a particular functional need (see Outstanding Questions).
Microbial genome sequencing has predicted a large number of putative bacterial glycosyltransferases. Thus, additional new forms of protein glycosylation involved in bacteriahost
interaction will be identied in future research on bacterial pathogenesis. Furthermore, bacteria-specic glycosylation may be a target of host immune surveillance and also exploited as a
possible target of new anti-infection agents.

How does PglB selectively modify

more than 60 C. jejuni proteins, and
which glycoprotein product plays a
critical role in C. jejuni pathogenesis?
What is the structural basis for NleBcatalysed arginine GlcNAcylation?
Are there other arginine GlcNAcylated
proteins in bacteria or other systems?
What are the host cell receptors for
BAHT-modied adhesins, or more
generally, the host targets for many
glycosylated
bacterial
surface
proteins?
What is the function of SRRPs glycosylation in bacterial infection since the
modication is outside of the domain
responsible for adhesion?
What is the role of the glycan in bacterial glycoprotein-mediated interaction
with the host cell?
Could glycosylated bacterial surface
proteins have virulence functions other
than adhesion to the host cell?
How are different glycan structures
selected to meet different functional
purposes in bacterial pathogens?

Acknowledgments
The authors apologize to colleagues whose work could not be cited owing to space limitation and the focus of the review.
Work in the authors laboratories is funded by the Strategic Priority Research Program of the Chinese Academy of Sciences
(XDB08020202), the China National Science Foundation grants (31225002 and 31461143006), and the National Basic
Research Program of China 973 Programs (2012CB518700 and 2014CB849602). F.S. is also supported in part by an
International Early Career Scientist grant from Howard Hughes Medical Institute.

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