Review

Bacterial chitinases and chitin-binding proteins as

virulence factors
Rikki F. Frederiksen,
1
Dafni K. Paspaliari,
1
Tanja Larsen,
1
Birgit G. Storgaard,
1,2
Marianne H. Larsen,
1
Hanne Ingmer,
1
Monica M. Palcic
2
and Jrgen J. Leisner
1
Correspondence
Jrgen J. Leisner
jjl@sund.ku.dk
1
Department of Veterinary Disease Biology, Faculty of Health Sciences, University of Copenhagen,
Grnnegaardsvej 15, 1870 Frederiksberg C., Denmark
2
Carlsberg Laboratory, Gamle Carlsbergvej 10, 1799 Copenhagen V., Denmark
Bacterial chitinases (EC 3.2.1.14) and chitin-binding proteins (CBPs) play a fundamental role in
the degradation of the ubiquitous biopolymer chitin, and the degradation products serve as an
important nutrient source for marine- and soil-dwelling bacteria. However, it has recently become
clear that representatives of both Gram-positive and Gram-negative bacterial pathogens encode
chitinases and CBPs that support infection of non-chitinous mammalian hosts. This review
addresses this biological role of bacterial chitinases and CBPs in terms of substrate specificities,
regulation, secretion and involvement in cellular and animal infection.
Introduction
Glycoside hydrolases (GHs) are enzymes that cleave the
glycosidic bonds of glycans. They are increasingly being
recognized as virulence factors of bacterial pathogens and
examples include enzymes belonging to GH2 galactosidases,
GH13 pullulanases, GH18 endo-b-N-acetylglucosamini-
dases, GH20 b-hexosaminidases and b-N-acetylglucosami-
nidases, GH30 glycosylceramidases, GH33 sialidases, GH73
muramidases/lysozymes, GH84 hyaluronidases and GH101
N-acetylgalactosidases (Canard et al., 1994; Garbe & Collin,
2012; Kim et al., 2009; Lenz et al., 2003; van Bueren et al.,
2007; Renzi et al., 2011; Sjogren et al., 2011).
Chitinases belong to GH families 18 and 19, and are
ubiquitous enzymes produced by bacteria and other life
forms (Gooday, 1990). They hydrolyse the second most
abundant polysaccharide in nature, chitin, which com-
prises linear b 1,4-N-acetylglucosamine (GlcNAc) residues.
This polymer is a structural component of crustacean
shells, the fungal cell wall, the exoskeletons of arthropods
and the cysts of various protozoans. Chitinases often
work synergistically with chitin-binding proteins (CBPs).
Bacterial CBPs contain carbohydrate-binding modules of
family 33 (CBM33) and sometimes family 5/12 (CBM5/
12). CBM33s are thought to facilitate chitinase accessibility
to crystalline chitinous matrices by introducing breaks in
the chitin chains. Recently, it has become apparent that
some CBM33 domains of bacterial CBPs are able to
enzymically cleave chitin. Therefore, a more descriptive
term, lytic polysaccharide monooxygenases, has been
proposed for these proteins (Aachmann et al., 2012) and
they are now reclassified as auxiliary activity family 10
(AA10) (CAZy, 2013).
The biological roles of bacterial chitinases and CBPs are easily
understood in an environmental context, typically exempli-
fied by chitin cycling in the marine environment by numerous
taxa, including Vibrio spp. (Keyhani & Roseman, 1999).
There is an increasing amount of direct or indirect evidence
suggesting that some chitinases and CBPs additionally serve
as virulence factors for bacterial pathogens during infection.
Examples include chitinases from Legionella pneumophila
(DebRoy et al., 2006), Listeria monocytogenes (Chaudhuri
et al., 2010; Larsen et al., 2010) and Salmonella enterica
serovar Typhimurium (S. Typhimurium) (Larsen et al., 2011)
as well as a CBP from Vibrio cholerae (Kirn et al., 2005). A
summary of cell culture/in vivo indications of this role is
presented in Tables 1 and 2. Potential targets of the chitinases
and CBPs during infection include glycoproteins and
glycolipids of host organisms that contain GlcNAc (Fig. 1),
the monomer that also composes chitin.
The assessment of bacterial chitinases as virulence factors is
complicated by the fact that the majority of bacterial
pathogens that encode GH family 18 and 19 enzymes
harbour up to several different variants of these proteins.
Furthermore, enzymic activity targeting chitin per se may in
some cases be perceived as being involved in virulence. This
concerns bacterial pathogens infecting invertebrate hosts that
contain chitin as a constituent either of their exoskeleton
(arthropods) or their intestinal peritrophic membrane
(annelids and some arthropods). This virulence aspect will
not be discussed further here, as it falls outside the scope of
this review, but it has been dealt with elsewhere (Gooday,
1999). Instead, we will focus on what is known regarding
bacterial chitinases and CBPs as virulence factors through
their interaction with target substrates other than chitin.
Microbiology (2013), 159, 833847 DOI 10.1099/mic.0.051839-0
051839
G
2013 SGM Printed in Great Britain 833
Table 1. Examples of bacterial GH family 18 chitinases as putative virulence factors
ND, Not determined; CF, cystic fibrosis.
Species GH18 enzyme Reference
Protein name/ID Accessory domain* Endogenous
upregulationD
Exogenous
upregulationD
Cell culture/in vivo
phenotype
Substrate
Enterococcus faecalis efChi18A/EF0361 ND In vitro human urine
and horse blood
ND Chitin Leisner et al., 2009; Veb
et al., 2009; Veb et al.,
2010
Escherichia coli ChiA/b3338 CBM5/12 ND Mutant with
reduced adhesion
to epithelial cellsd
Chitin Francetic et al., 2000a;
Tran et al., 2011
F. tularensis ChiA/FTT_0715 CBM5/12, FnIII ND In vivo mice spleen Mutant not
attenuated in
mice
Chitin Kadzhaev et al., 2009;
Mortensen et al., 2010;
Twine et al., 2006
Legionella pneumophila ChiA/lpg1116 Virulence regulator ND No effect in cell
cultures; reduced
recovery of
mutant from mice
lungs
Chitin DebRoy et al., 2006;
Galka et al., 2008
Listeria monocytogenes ChiA/lmo1883 Virulence regulators|| Stationary phase, murine
macrophages, in vivo
mice intestine,
heat-shock, GlcNAc||,
chitin||
No effect in cell
cultures; reduced
mutant recovery
from mice spleen
and liver
Chitin,
LacdiNAc
Chatterjee et al., 2006;
Chaudhuri et al., 2010;
Kazmierczak et al.,
2003; Larsen et al., 2010;
Mraheil et al., 2011;
B. G. Storgaard, M. M.
Palcic, J. J. Leisner and
others, unpublished
results; Toledo-Arana
et al., 2009; van der
Veen et al., 2007
ChiB/lmo0105 CBM5/12, FnIII Virulence regulators|| Stationary phase, in vivo
mice intestine, in vitro
human blood, chitin||
No effect in cell
cultures; reduced
mutant recovery
from mice spleen
and liver
Chitin,
LacdiNAc
P. aeruginosa ChiC/PA2300 CBM5/12, FnIII Quorum sensing Artificial CF sputum;
infection of lung
epithelial cells
ND Chitin Chugani & Greenberg,
2007; Folders et al.,
2001; Lesic et al., 2007;
Salunkhe et al., 2005;
Fung et al., 2010
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An additional issue that complicates the picture is that
most bacterial chitinases are classified in the GH family 18
(CAZy, 2013; Cantarel et al., 2009), which also harbours
some bacterial endoglycosidases (Fig. 2) (endo-b-N-
acetylglucosaminidases; EC 3.2.1.96), promoting endohy-
drolysis of the chitobiose core of various N-linked
glycoproteins. The endoglycosidases resemble chitinases
in amino acid sequences but no, or only little, chitinolytic
activity has been demonstrated for these enzymes.
Examples of such endoglycosidases include enzymes
produced by Enterococcus faecalis (Collin & Fischetti,
2004; Bhle et al., 2011) and Streptococcus pyogenes
(Collin & Olsen, 2001). We will not describe the role of
these enzymes as virulence factors in any detail, since this
has been the topic of another recent review (Garbe &
Collin, 2012).
Reports in the literature also describe some of the enzymes
in the GH families 20 and 46 as chitinases. They are not
included in this review as these families in our opinion
should more appropriately be regarded as b-N-acetylglu-
cosaminidases/hexosaminidases and chitosanases, respect-
ively, as outlined by Henrissat (1999).
Classification of hydrolytic domains of chitinases
As mentioned above, based on their primary structures
the hydrolytic domains of chitinases can be allocated to the
GH families 18 and 19. These families attain the triose-
phosphate isomerase (TIM) barrel and bilobal secondary
structures, respectively, and differ in their reaction
mechanisms, with family 18 enzymes operating with
overall retention of the anomeric configuration at the
cleavage point, and family 19 operating by inversion of the
anomeric configuration (Henrissat, 1999). The two families
most likely evolved from different ancestors (Bussink et al.,
2007). Family 18 chitinases occur in viruses, bacteria,
archaea and eukaryotes (Fig. 2), while family 19 chitinases
have been associated mainly with plants, although it is now
clear that many bacteria encode these enzymes as well.
Based on sequence analyses the GH family 18 enzymes have
been separated into different groups (Karlsson & Stenlid,
2009; Svitil & Kirchman, 1998; Watanabe et al., 1993). This
classification illuminates the phylogenetic relationships
between catalytic domains, but has so far not offered
insights regarding distinct roles of biological significance.
The TIM barrel of GH family 18 enzymes contains in the
active site a catalytic motif of DXDXE residues (Fig. 2) that
includes a glutamic acid which protonates the oxygen in
scissile glycosidic bonds (Vaaje-Kolstad et al., 2004). Based
on conservation of the active site structure and essential
catalytic residues, the GH 18 family enzymes are suggested
to be part of an enzyme superfamily also including GH 20
b-hexosaminidases and N-acetylglucosaminidases, GH 25
lysozymes, GH 56 hyaluronidases, GH 84 O-GlcNAcases
and GH 85 endo-b-N-glucosaminidases (Martinez-Fleites
et al., 2009). GHs can be classified as either exo or endo
depending on whether the enzyme cleaves the substrate T
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Chitinases as virulence factors
http://mic.sgmjournals.org 835
Table 2. Examples of bacterial CBPs/lytic polysaccharide monooxygenases as putative virulence factors
ND, Not determined.
Species Chitin-binding protein Reference
Protein name/
ID
Domain* Secretion
mechanism
Endogenous regulationD Exogenous
upregulationD
Cell culture/in vivo
phenotypes
Substrate (oxidation
and/or binding)
Up Down
V. cholerae GbpA/VCA0811 CBM33,
CBM5/12
Type II ND Quorum
sensingd
MucinD Reduced mutant
adherence to
epithelial cells;
reduced
colonization of
mice intestine
Chitin and chito
oligosaccharides
GlcNAc of mucin
Kirn et al., 2005;
Bhowmick et al.,
2008; Jude et al.,
2009; Wong et al.,
2012
Listeria
monocytogenes
Lmo2467 CBM33,
FnIII,
CBM5/12
ND ND ND Macrophage cytosol,
stationary phase
Reduced recovery
of mutant from
mice spleen and
liver
ND Chaudhuri et al.,
2010; Chatterjee
et al., 2006;
Toledo-Arana
et al., 2009
P. aeruginosa CbpD/PAO852 CBM33 Type II Quorum
sensingd
Biofilm
formation
Upregulated in CF
isolates but not in
wound isolatesd
ND Chitin Folders et al.,
2000; Sriramulu
et al., 2005; Kay
et al., 2006
Serratia marcescens CBP21 CBM33 ND ND ND Chitin, GlcNAc
uptake
Reduced mutant
adherence to
epithelial cells
Chitin Kawada et al.,
2008
Enterococcus faecalis efCBM33A/
EF0362
CBM33 ND ND ND In vitro human urine
and horse blood
ND Chitin Veb et al., 2009;
Veb et al., 2010
*CBM33 is now classified as AA10.
DData from microarray studies if not noted otherwise.
dProtein analysis.
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from a terminus or randomly at internal sites along the
polymer, respectively. The endochitinases tend to be non-
processive and have shallow substrate-binding clefts, while
exochitinases are often processive with deep substrate-
binding clefts. The closed architecture of the exochitinases
seems to be correlated with the presence of an insertion
domain in the TIM barrel of these enzymes (Horn et al.,
2006; Suzuki et al., 1999).
Based on tertiary structure, the catalytic domain of family
19 chitinases has been allocated to the lysozyme super-
family which also includes GH 22, GH 23 and GH 24
lysozymes, and the GH 46 chitosanases. Enzymes of this
superfamily are believed to have diverged from a common
ancestor (Holm & Sander, 1994; Wohlkonig et al., 2010).
The catalytic domains of these enzyme families share no
overall sequence similarity, but all attain a common fold
constituted by an all a-helix domain containing a catalytic
glutamate residue and a b-hairpin. Considering the
chemical similarity between chitin and peptidoglycan, it
is not so surprising that lysozymes can often hydrolyse
chitin, and that chitinases sometimes cleave peptidoglycan,
though less efficiently than their natural substrates
(Wohlkonig et al., 2010). In agreement with that, the
phylogenetic tree of the bacterial family 19 chitinases (Fig.
3) illustrates that some enzymes belonging to this family
may more appropriately be annotated as lysozymes or as
bifunctional chitinases/lysozymes.
Classification of chitin-binding modules of chitinases
and CBPs
Protein domains responsible for the binding of carbohy-
drates are, based on primary sequence similarity, classified
A
C
Phagosome
Endocytosis
Nucleus
Exocytosis
Bacteria
1
D
B
Key
Lipid-anchored
glycans
Fucose
Mannose
Sialic acid
Galactose
N-acetylglucosamine
N-acetylgalactosamine
Glucose
Xylose
Glucuronic acid
Iduronic acid
Ceramide
GH family 18 targets
Lipid-anchored
glycans
Glycolipids GPI-anchor
PI
2 4
NH
2
LacdiNAc LacNAc
3
(GlcNAc)
2
N-glycans
O-glycans
Glycosaminoglycans
Glycosaminoglycans
Ser/Thr Ser/Thr Ser/Thr
Core 3
Core 2 Core 1 Core 4 O-mannose O-fucose 6
5
Asn
Hybrid-type
Asn
Complex-type
Asn
High mannose-type
N-glycans
Ser Ser
NS
NS
NS
2S
6S
6S
6S
6S
4
3
3
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3 3
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Hyaluronic
acid Keratan
Ser/Thr
Ser/Thr Ser/Thr
O-glycans
Fig. 1. Overview of potential glycan substrates for GH family 18 and GH family 19 enzymes and a schematic outline of their
location in relation to mammalian host cells. The three potential GH family 18 disaccharide targets in glycoproteins, glycolipids
and glycosaminoglycans are also shown. A, secreted glycoproteins; B, membrane-associated glycosylated proteins; C,
membrane-associated glycoproteins and glycolipids in phagosomes; D, extracellular glycosaminoglycans [these might be
synthesized by membrane synthases (hyaluronic acid) or by the Golgi].
Chitinases as virulence factors
http://mic.sgmjournals.org 837

GH_18 consensus sequence

D G X D X D X E
Streptococcus pneumoniae TIGR4, EndoH(GH_85ENGASE) - - - - - - - -
Streptococcus pyogenes Alab49, EndoS D G L D V D V E
Enterococcus faecalis V583, EF0114 D G L D I D M E
Bacillus cereus ATCC14579, chitinase A D G I D I D L E
Vibrio cholerae M66-2, chiA D G L D V D L E
Enterococcus faecalis V583, chitinase EF0361 D G L D I D L E
Listeria monocytogenes EGD-e, ChiA D G L D I D L E
Pseudomonas aeruginosa PAO1, ChiC* D G L D I D L E
Serraa marcescens , chitinase C* D G L D I D L E
Yersinia pseudotuberculosis YPIII, glycosidehydrolase family 18 D G L D I D L E
Francisella tularensis subsp. tularensis FSC198, chitinase* D G F D F D L E
Francisella tularensis subsp. tularensis FSC198, FTF0066 N G I N F D L S
Salmonella enterica subsp. enterica serovar Typhimurium str. LT2, ChiA* S E V D I D W E
Salmonella enterica subsp. enterica serovar Typhi, putative chitinase* S E V D I D W E
Cronobacter sakazaki i E899, CSE899_07525* S E V D I D W E
Sodalis glossinidius str. morsitans, exochitinase* S E I D I D W E
Dictyostelium purpureum, DICPUDRAFT_96643* G E I D L D W E
Polysphondylium pallidum PN500,glycosylhydrolase* T E I D L D W E
Rickesia felis URRWXCal2 chitinase* K Q I D L D W E
Xenorhabdus nematophila ATCC19061, putativechitinase* S H L D I D W E
Xenorhabdus nematophila ATCC19061, Chi* T A V D I D W E
Paramecium bursaria Chlorella virus 1 N S I S L D W E
Aeromonas hydrophila subsp. hydrophila ATCC7966, chitodextrinase D G V D I D W E
Serraa marcescens, chitinase A D G V D I D W E
Citrobacter koseri ATCCBAA-895, CKO_02217 D G V D I D W E
Bombyx mori nucleopolyhedrovirus, chitinase D G V D V D W E
Autographa californica nucleopolyhedrovirus, ChiA D G V D V D W E
Vibrio cholerae M66-2, chitinase D G V D I D W E
Streptomyces plicatus, chitinase 63 D G I D L D W E
Bacillus cereus ATCC14579, chitinase C D G V D L D W E
Serraa marcescens, chitinase B* D G V D I D W E
Listeria monocytogenes EGD-e,ChiB D F V D L D W E
Clostridium dicile, peroxiredoxin/chitinase* D G I D I D W E
Aeromonas hydrophila subsp. hydrophila ATCC7966, chiA* D G V D V D Y E
Vibrio cholerae M66-2, chitinase D G V D I D Y E
Vibrio cholerae M66-2, chitodextrinase D G V D I D Y E
Francisella tularensis subsp. tularensis FSC198, chitinase family 18protein N G I D I D W E
Coxiella burnei, Dugway 5J108-111, chitinase G G V D I D W E
Bacillus cereus ATCC 10987, glycosylhydrolase* R D I H F D F E
Leishmania mexicana, chitinase D G I D F N W E
Burkholderia pseudomallei 1710b, glycosyl hydrolase D G F D F D L E
Escherichia coli BW2952, periplasmic endochitinase K V L D F D I E
Coxiella burnei, Dugway 5J108-111, chitinase* D A L D F D V E
Legionella pneumophila subsp. pneumophila str. Philadelphia 1 N V L D F D I E
Enterococcus faecalis V583, EF2863 D G I D F D D E
Streptomyces plicatus, EndoH D G V D F D D E
Legionella pneumophila subsp. pneumophila str. Philadelphia 1, ChiA D G F D I D I E
AAK74656.1:238-573
AEQ25108.1:112-445
AAO79989.1:62-323
AAP10651.1:34-338
AAO80224.1:42-325
AAO80224.1:42-325
CAC99961.1:45-327
NP_250990.1:27-311
BAA76623.1:25-305
ACA66994.1:20-318
CAL09784.1:296-581
CAL08082.1:402-687
AAL18982.1:53-465
CAD01171.1:53-465
EGL73180.1:52-463
YP_455154.1:51-479
XP_003284291.1:50-453
EFA83839.1:50-458
AAY61262.1:29-398
CBJ90617.1:74-495
CBJ90620.1:154-603
NP_048613.3:16-374
ABK39408.1:158-549
BAA31567.1:158-544
ABV13340.1:157-547
NP_047523.1:149-534
CAD79454.1:148-533
ACP07011.1:159-575
AAA26720.1:241-595
AAP07469.1:39-450
BAA31568.1:16-408
CAC98320.1:38-436
CAJ68298.1:381-682
ABK38659.1:297-747
ACP05347.1:385-832
ACP07620.1:319-776
CAL08731.1:162-553
ABS77295.1:101-288
NP_979924.1:104-412
Q5SEJ6:60-396
ABA49451.1:34-336
ACR63998.1:587-892
ABS77692.2:337-650
AAU28282.1:27-323
AAO82555.1:47-301
AAA26738.1:51-305
AAU27202.1:482-771
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into distinct CBMs. Chitin-binding properties have been
reported in CBMs belonging to families 1, 2, 3, 5, 12, 14,
18, 19, 33, 37 and 50 (CAZy, 2013). CBM5 is related to 12,
and will hence be referred to as CBM5/12. In bacterial
genomes, mainly chitin-binding modules belonging to
CBM33 and CBM5/12 are found. As described below, these
occur both as accessory domains of chitinases and as CBPs.
Members of the CBM33 family are mostly found in
bacteria and viruses as domains of secreted CBPs. Although
originally conceived as non-catalytic, recent data show that
the CBPs from Serratia marcescens (Vaaje-Kolstad et al.,
2010) and Enterococcus faecalis (Vaaje-Kolstad et al., 2012)
can act as enzymes by oxidative cleavage of chitin. Based on
the mechanism of action, it has recently been suggested
that these CBM33 domains together with the GH61
domains of fungi compose a family of lytic polysaccharide
monooxygenases, sharing certain features such as copper
dependency (Aachmann et al., 2012). They are now
reclassified as AA10 (CAZy, 2013).
In contrast, chitin-binding domains of families CBM5/12
and CBM2 are generally considered as non-catalytic acces-
sory modules that may accompany the catalytic domain of
chitinases, as well as the CBM33 of CBPs. CBM2 domains are
found in bacterial family 18 chitinases, while CBM5/12s are
frequently found in both family 18 and 19 chitinases, as well
as in some CBPs (CAZy, 2013; Punta et al., 2012; Karlsson &
Stenlid, 2009). Functional studies on chitin-binding prop-
erties of CBM2 and CBM5/12 have mainly focused on the
role of the latter, but both domains have been shown to
enhance substrate affinity and increase catalytic efficiency of
chitinases (Watanabe et al., 1994; Hashimoto et al., 2000;
Svitil & Kirchman, 1998; Nakamura et al., 2008), in a manner
likely dependent on certain conserved surface-exposed
aromatic residues (Morimoto et al., 1997).
Besides the chitin-binding domains, both chitinases and
CBPs can contain additional domains, as discussed below.
Expression and secretion of chitinases and CBPs
during infection
From DNA microarray studies as well as deletion mutant
studies, it is clear that a number of endogenous factors may
promote expression of chitinase-encoding genes during
infection in Listeria monocytogenes, Legionella pneumophila,
Pseudomonas aeruginosa and Chromobacterium violaceum
(Table 1) (Stauff & Bassler, 2011; Winson et al., 1995; Kay
et al., 2006). External parameters are also important in this
regard. Transcription of the virulence-related chitinase
ChiB of Listeria monocytogenes and a chitinase from
Enterococcus faecalis (efChi18A) has been shown to be
upregulated in vitro in body fluids, such as blood and urine
(Toledo-Arana et al., 2009; Veb et al., 2009; Veb et al.,
2010). Similarly, chitinase upregulation has also been seen
during growth of P. aeruginosa in artificial cystic fibrosis
sputum (Fung et al., 2010), which is in agreement with the
fact that the expression of a P. aeruginosa chitinase is
enhanced in clinical isolates as compared with that in a
laboratory strain (Salunkhe et al., 2005).
Host intracellular environments can also promote chitinase
transcription, as demonstrated for Listeria monocytogenes
during infection of macrophage cell cultures (Mraheil et al.,
2011), and for epithelial and macrophage cell cultures
infected by S. Typhimurium (Eriksson et al., 2003;
Hautefort et al., 2008). Other microarray data show,
however, that the distinct Salmonella chitinase-encoding
gene, chiA, is not always expressed during intracellular
infection, as no upregulation was observed for Salmonella
enterica serovar Typhi infecting macrophage cells (Faucher
et al., 2006).
Serratia plymuthica AS12, lytic enzyme
Salmonella enterica subsp. entericaserovar Typhi, putative bacteriophage
Salmonella Typhimurium str. LT2, putative Fels-1 prophage chitinase
Yersinia pseudotuberculosis YPIII, putative endolysin
Cronobacter sakazakii ATCC BAA-894 ESA_01019
Escherichia coli O7:K1 str. CE10, putative phage endolysin
Pseudomonas aeruginosa PA7, lytic enzyme
Pseudomonas aeruginosa PA7, conserved hypothetical protein
Salmonella enterica subsp. enterica serovar Typhi, putative secretedchitinase
Salmonella Typhimurium str. LT2, putative endochitinase
Cronobacter sakazakii ATCC BAA-894 ESA_03154
Citrobacter koseri ATCC BAA-895, CKO_02215
Aeromonas hydrophila subsp. hydrophila ATCC 7966, endochitinase
Aeromonas hydrophila subsp. hydrophila ATCC7966, carbohydrate-binding domain
Vibrio cholerae M66-2, putative chitinase
Streptomyces griseus subsp. griseus NBRC13350, chitinase C
Streptomyces griseus subsp. griseus NBRC 13350, putative chitinase
12gb|AEF50770.1| 24-162
8gi|16502196 23-165
10gb|AAL19843.1| 24-165
14gb|ACA66512.1| 36-168
3gb|ABU76288.1| 40-167
5gb|AEQ13640.1| 40-167
6gi|150963892|gb|ABR85917.1|
7gb|ABR81692.1| 42-191
9gi|16501515 83-340
11gb|AAL19197.1| 83-340
4gb|ABU78377.1| 80-337
17gb|ABV13338.1| 100-357
1gb|ABK38513.1| 210-467
2gb|ABK38655.1| 82-342
13gb|ACP05048.1| 90-350
15gi|178465709 93-294
16gi|178465710 72-273
1000
467
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1000
1000
1000
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Fig. 3. Phylogenetic tree of catalytic domains of GHfamily 19 chitinases frompathogenic bacteria. The chitinases can be allocated
into two distinct groups: 1) small proteins around 200220 residues having lysozyme-like catalytic domains that may originate from
bacteriophages and are characterized by lack of signal peptides and 2) larger proteins with or without signal peptides. Residue
positions of catalytic domains are indicated after the accession numbers. Bar, 0.05 substitutions per nucleotide position.
Chitinases as virulence factors
http://mic.sgmjournals.org 839
A Northern blot study demonstrated that the central
Listeria monocytogenes virulence regulators PrfA and sB
significantly increase transcription of the two chitinase
genes chiA and chiB in this organism (Larsen et al., 2010).
However, the overall evaluation of the role of Listeria
monocytogenes chitinases as virulence factors is complicated
by the fact that the expression of both enzymes is also
induced by chitin, while only expression of chiA and not
chiB is induced by GlcNAc (Larsen et al., 2010).
The above-described in vitro data are supported by in vivo
findings for several pathogens, such as chitinase gene
upregulation in Listeria monocytogenes infecting mice
intestine (Toledo-Arana et al., 2009) and in S. Typhi-
murium infecting chicken intestines (Harvey et al., 2011).
Similarly, the chitinase from Francisella tularensis, the
agent causing tularaemia, has been shown by proteomics to
be highly expressed during in vivo infection of mice spleens
(Twine et al., 2006).
Secretion to the outside of the bacterial cell is needed
during the infection process for the chitinases to interact
with host cell glycans. Information on secretion mechan-
isms for bacterial chitinases so far comes mostly from
bioinformatical prediction of the presence of signal
sequences. Such analyses have predicted that the efChi18A
chitinase (Table 1) from the Gram-positive Enterococcus
faecalis is secreted by the Sec pathway. The Gram-negative
Escherichia coli, Legionella pneumophila and V. cholerae
chitinases also carry signal sequences and are secreted by
means of the type II secretion system (Table 1) (DebRoy
et al., 2006; Francetic et al., 2000b; Sikora et al., 2011),
whereas this does not seem to be the case for P.
aeruginosa (Folders et al., 2001). A number of chitinases
(Fig. 2) lack the typical signal peptide sequence but most
of these are predicted by bioinformatics tools
(SecretomeP 2.0 Server) to be secretory proteins, which
suggests that their substrates are outside the bacterial cell.
It remains to be determined whether these enzymes are
indeed secreted or instead are located on the outer
bacterial surface (Larsen et al., 2011; B. G. Storgaard, M.
M. Palcic, J. J. Leisner and others, unpublished results).
Five CBPs with suspected roles in virulence (Table 2) all
carry signal peptides associated with Sec- or Tat-dependent
export mechanisms. Lmo2467 from the Gram-positive
Listeria monocytogenes is probably secreted by the Sec
pathway, as secretion by the Tat pathway is a rare event in
this species (Desvaux & Hebraud, 2006). The CbpD and
GbpA proteins from the Gram-negatives P. aeruginosa and
V. cholerae, respectively, are secreted by the type II
secretion system (Folders et al., 2000; Kirn et al., 2005),
while the secretion mechanism for CPB21 from Serratia
marcescens and efCBM33A from Enterococcus faecalis is
unknown. The endogenous factors that regulate expression
of these CBPs are not well described. GbpA is, as most
virulence factors in V. cholerae are, detected at low cell
density only, and is cleaved by quorum sensing-regulated
proteases at high cell density (Jude et al., 2009). CbpD is
likewise expressed in concert with quorum-sensing regu-
lated virulence factors, however, at high cell density (Kay
et al., 2006). Expression of the CBP efCBM33A from
Enterococcus faecalis is upregulated in the presence of blood
and urine (Veb et al., 2009; Veb et al., 2010) and has
been proposed as a potential virulence factor (Paulsen et al.,
2003).
Taken together, these results indicate that for several
bacterial pathogens, the expression/secretion of chitinases
and CBPs is related to the infective ability of the organisms.
What do we know from cellular and animal studies?
A straightforward approach to assess the effect of chitinases
or CBPs on bacterial virulence consists of comparing wild-
type strains with deletion mutants. Data from such studies
are presented in Tables 1 and 2. In some instances
chitinase-encoding genes are important for virulence in
in vivo animal models but not in in vitro cellular assays.
Examples include the intracellular pathogens Legionella
pneumophila and Listeria monocytogenes in which chitinase
deficiency causes defective growth in mice lungs and
spleens, respectively (Chaudhuri et al., 2010; DebRoy et al.,
2006). However, the ability of the Legionella pneumophila
mutants to survive in macrophage cell cultures is similar to
that of the wild-type. This is also the case for Listeria
monocytogenes mutants infecting epithelial and macro-
phage cell cultures (Chaudhuri et al., 2010; DebRoy et al.,
2006; Larsen et al., 2010), despite the notion of enhanced
intracellular chitinase transcription in macrophages
(Mraheil et al., 2011). The in vivo virulence effects of
chitinases might not be limited to bacteria, as the
trypanosomatid protozoan pathogen, Leishmania mexi-
cana, produces a chitinase that contributes to formation of
lesions in a mouse model as well as promoting survival in
host macrophage cells (Joshi et al., 2005).
However, bacterial chitinases may also affect the infection
of cell cultures. The transcription of the S. Typhimurium
LT2 chiA gene is enhanced upon infection of epithelial and
macrophage cells (Eriksson et al., 2003; Hautefort et al.,
2008). We have very recently found that deletion of chiA in
this strain moderately reduces the ability of Salmonella to
invade epithelial cells and macrophages (R. F. Frederiksen
and others, unpublished results). The role of this pheno-
menon in an in vivo model has not been investigated.
Animal studies show that absence of two CBPs, the GbpA
and Lmo2467 from V. cholerae and Listeria monocytogenes,
respectively, attenuates bacterial virulence in mice (Chaudhuri
et al., 2010; Kirn et al., 2005). The possible underlying
mechanisms will be discussed below.
Substrate specificities of catalytic domains relevant to
virulence
In several studies chitinases have been suggested as virulence
factors although the target molecules in the host have not
been identified. Indeed, the absence of endogenous chitin in
R. F. Frederiksen and others
840 Microbiology 159
vertebrate hosts immediately suggests that other GlcNAc-
containing glycans are the target(s). Bacterial pathogens
encounter host GlcNAc-containing glycans in the form of
glycolipids, glycoproteins and/or as part of extracellular
matrices during infection. Extracellular pathogens interact
with such substrates located in tissues or lumens of, for
example, the intestine or blood vessels, while intracellular
pathogens encounter substrates also within the host cells,
e.g. in phagosomes (Fig. 1). The GlcNAc residues are found
in the two major types of protein-linked glycans, the
asparagine-linked glycans (N-glycans) and the serine/
threonine-linked glycans (O-glycans) as well as in glycoli-
pids and extracellular matrix proteins, such as glucos-
aminoglycans (Fig. 1).
N-glycans contain a common pentasaccharide structure com-
posed of an asparagine-linked chitobiose core [(GlcNAc)
2
]
and three terminal mannose residues [(Man)
3
], which act as
the starting point for carbohydrate branches (Fig. 1). N-
glycans carrying branches of only mannose residues are
termed high-mannose N-glycans, while complex-type N-
glycans include other residues, e.g. GlcNAc, galactose (Gal)
and sialic acid. Many N-glycans have features shared by both
high-mannose and complex-type and are classified as hybrid-
type. O-glycans are based on a serine/threonine-linked N-
acetylgalactosamine (GalNAc) residue extended with, for
example, galactose and GlcNAc residues resulting in eight
common core structures. Both the chitobiose core, N,N9-
diacetyllactosamine (LacdiNAc) (GalNAcb1-4GlcNAc) and
N-acetyllactosamine (LacNAc) (Galb1-4GlcNAc) structures
of O-glycans and N-glycans are putative targets for bacterial
GH family 18 enzymes.
The chitobiose core of N-glycans can be cleaved by GH18
family endoglycosidases, which, as mentioned previously,
are phylogenetically related to chitinases (Fig. 2) (Collin &
Olsen, 2001; Garbe & Collin, 2012). It would be of interest
to examine whether true chitinases exhibit endoglycosi-
dase-like activity towards chitobiose cores.
It has recently been demonstrated that a diverse range of
bacterial chitinases hydrolyse GlcNAc-containing model
substrates of LacdiNAc and to a lesser extent also LacNAc,
though at low rates (Larsen et al., 2011; Murata et al., 2005;
Shoda et al., 2006; B. G. Storgaard, M. M. Palcic, J. J.
Leisner and others, unpublished results). LacNAc struc-
tures have been identified in O-glycans and N-glycans of
glycoproteins as well as in glycolipids of eukaryotic cells
(Varki et al., 2009), and are abundant in proteins of the
human immune system (Marth & Grewal, 2008). The
possibility that bacteria might be using LacNac structures
in O-glycans as substrates has been proposed for a
commensal of the human gut, Bacteroides thetaiotaomicron
(Martens et al., 2008). This species encodes GH family 18
enzymes predicted to cleave LacNAc repeats of O-glycan
chains. LacNAc also constitutes part of the carbohydrate
backbone of mucins, which form a protective physical
barrier of the gastrointestinal tract, and chitinase B from
Serratia marcescens has been described to target mucins
(Sanders et al., 2007). The mechanism of hydrolysis has not
been elucidated.
LacdiNAc is present in the N-glycans of some human host
proteins, e.g. lactoferrin, but is in general more commonly
found in the insect glycome (Van den Eijnden et al., 1995).
So far, activity towards LacdiNAc and/or LacNAc struc-
tures in specific glycoproteins or glycolipids has not been
demonstrated.
The non-sulfated glycosaminoglycan hyaluronan, a chief
component of many extracellular matrices, could also be a
target. However, the S. Typhimurium ChiA and Listeria
monocytogenes ChiA and B enzymes do not show hydrolytic
activity towards this substrate (B. G. Storgaard, M. M.
Palcic, J. J. Leisner and others, unpublished results). Other
GlcNAc-containing glycosaminoglycans, such as heparin
and keratan sulfate, have not yet been tested as substrates
for GH family 18 and 19 enzymes.
Unexpected targets may also exist. Interestingly, 28S rRNA
N-glucosidase activity, characteristic for ribosome-inactiv-
ating protein, has been shown for some plant chitinases
(Shih et al., 1997; Xu et al., 2008). These enzymes were,
nevertheless, not fully characterized.
Chitinases from bacterial pathogens may also target the
peptidoglycan-containing cell wall of other members of the
gastrointestinal microbiome or their own cell wall to
accommodate cell growth and division. Only a few studies
on a Bacillus pumilus GH family 18 chitinase and two P.
aeruginosa chitinases of unspecified GH family have,
however, shown such hydrolytic activity (Ghasemi et al.,
2011; Wang & Chang, 1997), whereas Enterococcus faecalis,
S. Typhimurium and Listeria monocytogenes GH family 18
chitinases were inactive in this regard (Leisner et al., 2009;
Larsen et al., 2011). It is of interest that the bacterial GH
family 19 chitinases include a group annotated as having
similarities with the lysozymes belonging to the lysozyme
superfamily (Fig. 3). Unfortunately, data on lysozymic
activity of GH family 19 chitinases are currently only
available for chitinases of plant origin (Beintema &
Terwisscha van Scheltinga, 1996).
Interestingly, for most of the virulence-related chitinases
found in Table 1 (efChi18A, B3338, FTT-0715, Lpg1116,
Lmo1883 and PA2300) homology modelling (Phyre2
server; data not shown) predicted the absence of an
insertion domain in their hydrolytic domain, which
indicates that these enzymes are endochitinases. It is
conceivable that the open architecture of the catalytic
domains of these predicted endochitinases might be prone
to accommodate recognition of non-chitinous host
glycans, but experimental evidence is warranted.
Substrate specificities of chitin-binding domains
relevant to virulence
Although CBM33 (AA10) domains of CBPs are mainly
associated with binding and cleavage of chitin, alternative
Chitinases as virulence factors
http://mic.sgmjournals.org 841
substrates such as cellulose have been observed (Forsberg
et al., 2011). Host-related alternative binding substrates
have also been described, with the virulence-related GbpA
protein (Table 2) from V. cholerae as an example. GbpA
consists of two domains allocated to attachment to the
bacterial cell surface, and two domains capable of binding
to chitin, of which the CBM33 domain also binds GlcNAc
residues of mucin. This tripartite binding capacity indicates
that the protein may be involved in adhesion to chitinous
exoskeletons as well as matrix proteins of host cells (Wong
et al., 2012; Bhowmick et al., 2008). A CBP consisting of a
single CBM33 module from Lactobacillus plantarum is also
able to bind both chitinous and cell-surface-type sub-
strates, such as mucin (Sa nchez et al., 2011). Also, bacterial
CBPs have been proposed to mediate adhesion to host cells
through interactions with mammalian chitinases or
chitolectins (Kawada et al., 2008). It remains to be studied
whether the observed monooxygenase activity towards
chitin (Vaaje-Kolstad et al., 2010; Vaaje-Kolstad et al.,
2012) might be of relevance regarding related substrates in
the host.
CBMs as accessory domains of bacterial enzymes function-
ing as virulence factors and toxins have been reported. As
might be expected, these domains contribute to virulence
through sugar-binding properties that promote adhesion
to extracellular or intracellular targets (Guillen et al., 2010).
Chitin-binding domains, other than CBM33, that accom-
pany chitinases and CBPs might contribute to virulence in
this manner. As described earlier, these domains belong to
CBM2 and CBM5/12 (CAZy, 2013), but so far only CBM
5/12 is found to be associated with bacterial chitinases and
CBPs related to virulence (Tables 1 and 2). It is conceivable
that these domains as part of virulent chitinases could play
a role in adhesion similar to that proposed for the CBM33
chitin-binding domains of CBPs such as GbpA and
Lmo2467 (Bhowmick et al., 2008; Chaudhuri et al.,
2010). However, it should be noted that the CBM5/12
domain (domain 4) of GbpA was found to be dispensable
for virulence, and thus such a role remains to be
demonstrated (Wong et al., 2012).
Functional roles of other accessory modules
The majority of bacterial chitinases exhibit a complex,
multi-domain architecture, including sometimes catalytic
domains belonging to other GH families, but, more
frequently, additional domains with no obvious catalytic
role. Besides the chitin-binding domains, these include
other domains of essentially unknown function, such as
fibronectin type III (FnIII) or cadherin-like domains and
polycystic kidney disease (PKD) domains. The function-
ality of such domains is mostly deduced by their
annotation and further descriptive studies are clearly
warranted.
PKD domains have been suggested to directly mediate
binding to chitin (Orikoshi et al., 2005). PKD domains
may also promote proteinprotein interactions, but the
importance of this ability with regard to bacterial chitinases
has not been investigated.
FnIII-like domains are widely distributed among family 18
bacterial chitinases and other glycosyl hydrolases (Little
et al., 1994), but have not been reported for any family 19
chitinases (CAZy, 2013; Punta et al., 2012). They appear
to contribute to chitin hydrolysis (Watanabe et al., 1994),
possibly by functioning as a flexible linker between the
catalytic and chitin-binding domain (Jee et al., 2002).
Such FnIII-like domains are probably acquired from
animals (Bork & Doolittle, 1992; Jee et al., 2002), in which
they are involved in cell adhesion and extracellular matrix
assembly, mainly through proteinprotein interactions
between domains of fibronectin, integrin and heparin
(Pankov & Yamada, 2002; Potts & Campbell, 1996). It
remains to be investigated whether bacterial FnIII
domains, despite some structural differences (Jee et al.,
2002), have retained animal-like properties and thereby
serve analogous roles during infection as hypothesized for
the Campylobacter jejuni FlpA protein, which binds
fibronectin and facilitates host epithelial cell adhesion
(Konkel et al., 2010).
Conclusions
The work summarized in this review indicates that GH
family 18 chitinases and CBPs from bacterial pathogens
play roles as virulence factors, although not necessarily
through a single unifying mechanism. We do not yet have
data for GH family 19 chitinases to evaluate their role in
virulence in this regard. However, the frequent occurrence
of family 19 chitinases in plants and their often noticed
anti-fungal activity suggest that these enzymes may
primarily be involved in resistance towards fungal patho-
gens (Gooday, 1999; Kawase et al., 2006). The GH family
18 chitinases of bacteria may exert easily understood roles
during infection of chitin-containing hosts, whereas their
biological function in hosts lacking chitin cannot be
explained simply by chitinolytic activity. In the case of
GH family 18 chitinases, the existence of non-chitinolytic
activity is suggested by a structure/function relationship
that may easily direct the development of new functions
from the scaffold of the parent proteins (Funkhouser &
Aronson, 2007). The substrate specificities of several
bacterial chitinases towards LacNAc and particularly
LacdiNAc model substrates serve as a possible example of
such new functions. However, additional studies are
needed to confirm that alternative substrates of bacterial
chitinases do play a role during infection. Also, it should be
mentioned that the molecular mechanisms may involve
only attaching to the proper substrate, such as is most
likely the case for some of the bacterial CBPs, e.g. the V.
cholerae GbpA protein (Kirn et al., 2005).
It should also be considered that some bacterial CBPs and
chitinases that serve as virulence factors may be attached to
the bacterial cell surface as suggested for other bacterial
glycosyl hydrolases with similar roles (Ficko-Blean &
R. F. Frederiksen and others
842 Microbiology 159
Boraston 2012). In this scenario they may play a particular
role in the hostbacteria intervention by mediating
adhesion of the entire bacterial cell.
Some of the enzymes belonging to the GH family 18 are
described as endoglycosidases (e.g. endo-H) rather than
chitinases. Indeed, significant chitinolytic activity remains
to be demonstrated by most, if not all, such enzymes.
Future studies should clarify to what extent bacterial GH
18 enzymes defined as endoglycosidases or chitinases have
similar or different substrate specificities with regard to
their roles as virulence factors.
It is evident that any firm assessment of the biological role
of bacterial chitinases and CBPs requires a careful
assessment of their importance during infection, by use
of both cellular and mammalian animal models. Here, also
host-specific reactions need to be considered. One example
involves a gene encoding a human CPB, a chitinase 3-like 1
protein, that may interact with bacterial components (e.g.
CBPs) (Kawada et al., 2008; Tran et al., 2011) and appears
to have a functional role during bacterial infections
(Johansen et al., 2005).
Mammalian chitinases and chitolectins, initially thought
of as primarily providing a defence towards chitinous
pathogens, i.e. moulds (Lee et al., 2008), exert numerous
other effects in the mammalian host, including patho-
logical conditions such as autoimmune, neurological or
lysosomal storage diseases in humans (Brinkman et al.,
2005; Boot et al., 1999; Bussink et al., 2006; Di Rosa et al.,
2006; Lautner et al., 2011; Lee et al., 2011, 2012;
Mattsson et al., 2011; Sotgiu et al., 2008; Verbeek et al.,
2010) and crystalline pneumonia in mice (Hoenerhoff
et al., 2006; Liu et al., 2009). It will be of interest to
examine such biologically relevant activities of mam-
malian chitinases and chitolectins in order to improve
the understanding of the roles of similar proteins from
bacterial pathogens.
A thorough examination of the bacterial chitinases, as
discussed above, will hopefully lead to the ultimate goal: a
precise definition of the extent to which they can be
perceived as virulence factors. This will provide an escape
from the situation outlined by Lewis Caroll, aptly used by
Dubos (1945) in his pioneering treatise of bacterial
virulence: When I use a word, Humpty Dumpty said, in
a rather scornful voice, It means just what I choose it to
mean neither more nor less.
Acknowledgements
We would like to thank Dr Tim Evison at scientificillustration.net
for providing Fig. 1, and two anonymous reviewers for comments
and suggestions. Work performed in the laboratories of the authors
is supported by grants from The Brdrene Hartmann Foundation,
Aase and Ejnar Danielsens Foundation, The Axel Muusfeldts
Foundation, The Danish Council for Independent Research, The
Danish Council for Strategic Research and The Danish Pasteur
Society.
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