Microbiology (2013), 159, 833–847
Review
DOI 10.1099/mic.0.051839-0
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. Palcic2 and Jørgen J. Leisner1
Correspondence
Jørgen J. Leisner
[email protected]
1
Department of Veterinary Disease Biology, Faculty of Health Sciences, University of Copenhagen,
Grønnegaardsvej 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-acetylglucosaminidases, GH20 b-hexosaminidases and b-N-acetylglucosaminidases, 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; Sjögren 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 comprises 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).
051839 G 2013 SGM
The biological roles of bacterial chitinases and CBPs are easily
understood in an environmental context, typically exemplified 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.
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833
ND,
Not determined; CF, cystic fibrosis.
Species
GH18 enzyme
Protein name/ID
Accessory domain*
Endogenous
upregulationD
Reference
Exogenous
upregulationD
Cell culture/in vivo
phenotype
Substrate
Enterococcus faecalis
efChi18A/EF0361
–
ND
Escherichia coli
ChiA/b3338
CBM5/12
ND
F. tularensis
ChiA/FTT_0715
CBM5/12, FnIII
ND
In vivo mice spleen§
Legionella pneumophila
ChiA/lpg1116
–
Virulence regulator
ND
Listeria monocytogenes
ChiA/lmo1883
–
Virulence regulators||
Stationary phase, murine
macrophages, in vivo
mice intestine,
heat-shock, GlcNAc||,
chitin||
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
ChiC/PA2300
CBM5/12, FnIII
Quorum sensing
Artificial CF sputum;
infection of lung
epithelial cells
ND
Chitin
P. aeruginosa
In vitro human urine
and horse blood
Microbiology 159
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ND
Chitin
Mutant with
reduced adhesion
to epithelial cellsd
Mutant not
attenuated in
mice
No effect in cell
cultures; reduced
recovery of
mutant from mice
lungs
No effect in cell
cultures; reduced
mutant recovery
from mice spleen
and liver
Chitin
Chitin
Chitin
Chitin,
LacdiNAc
Leisner et al., 2009; Vebø
et al., 2009; Vebø et al.,
2010
Francetic et al., 2000a;
Tran et al., 2011
Kadzhaev et al., 2009;
Mortensen et al., 2010;
Twine et al., 2006
DebRoy et al., 2006;
Galka et al., 2008
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
Chugani & Greenberg,
2007; Folders et al.,
2001; Lesic et al., 2007;
Salunkhe et al., 2005;
Fung et al., 2010
R. F. Frederiksen and others
834
Table 1. Examples of bacterial GH family 18 chitinases as putative virulence factors
http://mic.sgmjournals.org
*Chitinases may contain other accessory domains (e.g. LysM) than those listed here, but these have not been found in chitinases associated with virulence.
DData from microarray studies if not noted otherwise.
dUnpublished results; Tran et al. (2011).
§Protein analysis.
||Northern blot.
Chitin, LacNAc, Eriksson et al., 2003;
LacdiNAc
Harvey et al., 2011;
Hautefort et al., 2008;
Larsen et al., 2011; B. G.
Storgaard, M. M. Palcic,
J. J. Leisner and others,
unpublished results;
Wright et al., 2009; R. F.
Frederiksen and others,
unpublished results.
Reduced mutant
infection of
epithelial cell
cultures
Epithelial cells,
macrophages, H2O2, in
vivo caecal lumen of
chicken
CBM5/12
ChiA/STM0018
S. Typhimurium
Species
Table 1. cont.
Protein name/ID
Accessory domain*
ND
Endogenous
upregulationD
GH18 enzyme
Exogenous
upregulationD
Cell culture/in vivo
phenotype
Substrate
Reference
Chitinases as virulence factors
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-Nacetylglucosaminidases; EC 3.2.1.96), promoting endohydrolysis 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; Bøhle et al., 2011) and Streptococcus pyogenes
(Collin & Olsén, 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-acetylglucosaminidases/hexosaminidases and chitosanases, respectively, 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 triosephosphate 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
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ND,
Not determined.
Species
Chitin-binding protein
Protein name/
ID
Domain*
Secretion
mechanism
Endogenous regulationD
Up
V. cholerae
Exogenous
upregulationD
CBM33,
CBM5/12
Type II
ND
Quorum
sensingd
MucinD
Lmo2467
CBM33,
FnIII,
CBM5/12
ND
ND
ND
Macrophage cytosol,
stationary phase
CbpD/PAO852
CBM33
Type II
Quorum
sensingd
Biofilm
formation
Serratia marcescens
CBP21
CBM33
ND
ND
Enterococcus faecalis
efCBM33A/
EF0362
CBM33
ND
ND
P. aeruginosa
Cell culture/in vivo Substrate (oxidation
phenotypes
and/or binding)
Down
GbpA/VCA0811
Listeria
monocytogenes
Reference
Reduced mutant
adherence to
epithelial cells;
reduced
colonization of
mice intestine
Reduced recovery
of mutant from
mice spleen and
liver
Chitin and chito
oligosaccharides
GlcNAc of mucin
Kirn et al., 2005;
Bhowmick et al.,
2008; Jude et al.,
2009; Wong et al.,
2012
ND
Upregulated in CF
isolates but not in
wound isolatesd
ND
Chitin
ND
Chitin, GlcNAc
uptake
Reduced mutant
adherence to
epithelial cells
Chitin
Chaudhuri et al.,
2010; Chatterjee
et al., 2006;
Toledo-Arana
et al., 2009
Folders et al.,
2000; Sriramulu
et al., 2005; Kay
et al., 2006
Kawada et al.,
2008
ND
In vitro human urine
and horse blood
ND
Chitin
*CBM33 is now classified as AA10.
DData from microarray studies if not noted otherwise.
dProtein analysis.
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Vebø et al., 2009;
Vebø et al., 2010
R. F. Frederiksen and others
836
Table 2. Examples of bacterial CBPs/lytic polysaccharide monooxygenases as putative virulence factors
Chitinases as virulence factors
Lipid-anchored
glycans
Key
B
N-glycans
A
Endocytosis
C
Phagosome
O-glycans
N-acetylglucosamine
Glycosaminoglycans
N-acetylgalactosamine
Glycosaminoglycans
β4
NS
Exocytosis
NS
Nucleus
D
Bacteria
β4
β3
β4
β4
α4
β4
α4
2S
β4
NS
α4
β3
β3
6S
β3
β4
6S β3
β4
β4
β3
β4
β4
β3
β3
β4
β4
β3
β3
β4
β4
6S
GH family 18 targets
6S
6S
N-glycans
LacdiNAc (GlcNAc)2 LacNAc
6S
Lipid-anchored
glycans
α3
β4
α2
α3
β4
β4
α6
α3
α6
α3 β4
β4 β4 β3
β4
β4
β2 β2
β6
α3
β4 α6
α6
β4
α6
β4
α2 α2 α2
α3
α3
Asn
Asn
High mannose-type Complex-type
α3 β4 α3
β4 β3
α3 α6 β4
β4
α6
α6β6
β4
β4 α6
α3
Asn
Hybrid-type
5
α3
β4
α2 α2
β3
β4
α3
β3
β3
α3
β3 α3
β4
α3
β6
Ser/Thr Ser/Thr
Core 1 Core 2
α3
β4
6S
β3
β4
β3
β4
β4
β3
Ser/Thr
Core 3
β3
β3
β6
Ser/Thr
Core 4
α2
α6
α4
β4
Glycolipids
2
α3
β4
β4
β4
6S
Hyaluronic Ser
Ser
Heparan
acid
Keratan
1
Glucose
Xylose
Glucuronic acid
Iduronic acid
Ceramide
Fucose
Mannose
Sialic acid
Galactose
3
Et
P
NH2
PI
GPI-anchor
4
O-glycans
α3
α3
β4
β2
β4
β3
Ser/Thr
Ser/Thr
O-mannose O-fucose 6
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].
from a terminus or randomly at internal sites along the
polymer, respectively. The endochitinases tend to be nonprocessive and have shallow substrate-binding clefts, while
exochitinases are often processive with deep substratebinding 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 superfamily 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; Wohlkönig et al., 2010).
The catalytic domains of these enzyme families share no
overall sequence similarity, but all attain a common fold
http://mic.sgmjournals.org
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
(Wohlkönig 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 carbohydrates are, based on primary sequence similarity, classified
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Microbiology 159
Fig. 2. Phylogenetic tree of catalytic domains of GH family 18 chitinases and endoglycosidases from selected organisms. In
orange, endoglycosidases; in blue, slime moulds; in green, viruses; in purple, trypanosomatids. Proteins lacking predicted signal
peptides are indicated by an asterisk. Amino acid positions of catalytic domains are also indicated after the accession numbers.
GH 18 active site consensus sequences are shown to the right. Bar, 0.1 substitutions per nucleotide position.
AAK74656.1:238-573
AEQ25108.1:112-445
897
AAO79989.1:62-323
AAP10651.1:34-338
1000
AAO80224.1:42-325
1000
AAO80224.1:42-325
998
CAC99961.1:45-327
505
NP_250990.1:27-311
1000
1000
BAA76623.1:25-305
710
429
ACA66994.1:20-318
CAL09784.1:296-581
CAL08082.1:402-687
1000
1000
AAL18982.1:53-465
CAD01171.1:53-465
993
EGL73180.1:52-463
855
YP_455154.1:51-479
XP_003284291.1:50-453
586
EFA83839.1:50-458
963
997
AAY61262.1:29-398
1000
CBJ90617.1:74-495
835
CBJ90620.1:154-603
NP_048613.3:16-374
ABK39408.1:158-549
961
53
960
389
BAA31567.1:158-544
ABV13340.1:157-547
895
NP_047523.1:149-534
1000
1000
619
CAD79454.1:148-533
ACP07011.1:159-575
833
AAA26720.1:241-595
AAP07469.1:39-450
901
649
BAA31568.1:16-408
859
CAC98320.1:38-436
536
CAJ68298.1:381-682
999
236
1000
ABK38659.1:297-747
ACP05347.1:385-832
ACP07620.1:319-776
593
CAL08731.1:162-553
320
ABS77295.1:101-288
NP_979924.1:104-412
Q5SEJ6:60-396
827
ABA49451.1:34-336
936
ACR63998.1:587-892
1000
ABS77692.2:337-650
326
AAU28282.1:27-323
999
AAO82555.1:47-301
104
1000
AAA26738.1:51-305
AAU27202.1:482-771
Streptococcus pneumoniae TIGR4, Endo H (GH_85ENGASE)
Streptococcus pyogenes Alab49, EndoS
Enterococcus faecalis V583, EF0114
Bacillus cereus ATCC14579, chitinase A
Vibrio cholerae M66-2, chiA
Enterococcus faecalis V583, chitinase EF0361
Listeria monocytogenes EGD-e, ChiA
Pseudomonas aeruginosa PAO1, ChiC*
Serraa marcescens , chitinase C*
Yersinia pseudotuberculosis YPIII, glycoside hydrolase family 18
Francisella tularensis subsp. tularensis FSC198, chitinase*
Francisella tularensis subsp. tularensis FSC198, FTF0066
Salmonella enterica subsp. enterica serovar Typhimurium str. LT2, ChiA*
Salmonella enterica subsp. enterica serovar Typhi, putative chitinase*
Cronobacter sakazaki i E899, CSE899_07525*
Sodalis glossinidius str. ‘morsitans’, exochitinase*
Dictyostelium purpureum , DICPUDRAFT_96643*
Polysphondylium pallidum PN500,glycosylhydrolase*
Rickesia felis URRWXCal2 chitinase*
Xenorhabdus nematophila ATCC 19061, putative chitinase*
Xenorhabdus nematophila ATCC 19061, Chi*
Paramecium bursaria Chlorella virus 1
Aeromonas hydrophila subsp. hydrophila ATCC 7966, chitodextrinase
Serraa marcescens, chitinase A
Citrobacter koseri ATCC BAA-895, CKO_02217
Bombyx mori nucleopolyhedrovirus, chitinase
Autographa californica nucleopolyhedrovirus, ChiA
Vibrio cholerae M66-2, chitinase
Streptomyces plicatus, chitinase 63
Bacillus cereus ATCC 14579, chitinase C
Serraa marcescens, chitinase B*
Listeria monocytogenes EGD-e,ChiB
Clostridium difficile , peroxiredoxin/chitinase*
Aeromonas hydrophila subsp. hydrophila ATCC 7966, chiA*
Vibrio cholerae M66-2, chitinase
Vibrio cholerae M66-2, chitodextrinase
Francisella tularensis subsp.tularensis FSC198, chitinase family 18 protein
Coxiella burnei, Dugway 5J108-111, chitinase
Bacillus cereus ATCC 10987, glycosylhydrolase*
Leishmania mexicana, chitinase
Burkholderia pseudomallei 1710b, glycosyl hydrolase
Escherichia coli BW2952, periplasmic endochitinase
Coxiella burnei, Dugway 5J108-111, chitinase*
Legionella pneumophila subsp. pneumophila str. Philadelphia 1
Enterococcus faecalis V583, EF2863
Streptomyces plicatus, EndoH
Legionella pneumophila subsp. pneumophila str. Philadelphia 1, ChiA
0.1
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D
D
D
D
D
D
D
D
D
D
N
S
S
S
S
G
T
K
S
T
N
D
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N
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R. F. Frederiksen and others
838
GH_18 consensus sequence
Chitinases as virulence factors
Lysozyme-like domain
12gb|AEF50770.1| 24-162
1000
8gi|16502196 23-165
10gb|AAL19843.1| 24-165
14gb|ACA66512.1| 36-168
988
3gb|ABU76288.1| 40-167
5gb|AEQ13640.1| 40-167
467
1000
6gi|150963892|gb|ABR85917.1|
7gb|ABR81692.1| 42-191
611
1000 9gi|16501515 83-340
790
11gb|AAL19197.1| 83-340
996
969
4gb|ABU78377.1| 80-337
1000
17gb|ABV13338.1| 100-357
1000
1gb|ABK38513.1| 210-467
2gb|ABK38655.1| 82-342
1000
1000
13gb|ACP05048.1| 90-350
15gi|178465709 93-294
1000
16gi|178465710 72-273
Serratia plymuthica AS12, lytic enzyme
Salmonella enterica subsp. enterica serovar 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 secreted chitinase
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 ATCC 7966, carbohydrate-binding domain
Vibrio cholerae M66-2, putative chitinase
Streptomyces griseus subsp. griseus NBRC 13350, chitinase C
Streptomyces griseus subsp. griseus NBRC 13350, putative chitinase
0.05
Fig. 3. Phylogenetic tree of catalytic domains of GH family 19 chitinases from pathogenic bacteria. The chitinases can be allocated
into two distinct groups: 1) small proteins around 200–220 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.
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 accessory 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 properties 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).
http://mic.sgmjournals.org
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; Vebø et al., 2009; Vebø 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).
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R. F. Frederiksen and others
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. Typhimurium 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 mechanisms 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 & Hébraud, 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
840
likewise expressed in concert with quorum-sensing regulated 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 (Vebø et al., 2009; Vebø 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 wildtype 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 macrophage 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 mexicana, 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 phenomenon 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
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Chitinases as virulence factors
vertebrate hosts immediately suggests that other GlcNAccontaining 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 glycolipids and extracellular matrix proteins, such as glucosaminoglycans (Fig. 1).
N-glycans contain a common pentasaccharide structure composed 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). Nglycans carrying branches of only mannose residues are
termed high-mannose N-glycans, while complex-type Nglycans 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 hybridtype. O-glycans are based on a serine/threonine-linked Nacetylgalactosamine (GalNAc) residue extended with, for
example, galactose and GlcNAc residues resulting in eight
common core structures. Both the chitobiose core, N,N9diacetyllactosamine (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 &
Olsén, 2001; Garbe & Collin, 2012). It would be of interest
to examine whether ‘true’ chitinases exhibit endoglycosidase-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 structures 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
http://mic.sgmjournals.org
(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 structures 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-inactivating 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
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R. F. Frederiksen and others
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 substrates, such as mucin (Sánchez 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 functioning 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 (Guillén et al., 2010).
Chitin-binding domains, other than CBM33, that accompany 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 functionality 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 protein–protein interactions, but the
842
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 protein–protein 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 pathogens (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 &
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Chitinases as virulence factors
Boraston 2012). In this scenario they may play a particular
role in the host–bacteria intervention by mediating
adhesion of the entire bacterial cell.
References
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.
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We would like to thank Dr Tim Evison at scientificillustration.net
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and suggestions. Work performed in the laboratories of the authors
is supported by grants from The Brødrene Hartmann Foundation,
Aase and Ejnar Danielsen’s 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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