HtrA-mediated E-cadherin cleavage is limited to DegP and DegQ homologs expressed by gram-negative pathogens
Abfalter et al. Cell Communication and Signaling
HtrA-mediated E-cadherin cleavage is limited to DegP and DegQ homologs expressed by gram-negative pathogens
Carmen M. Abfalter 0
Maria Schubert 0
Thomas P. Schmidt
0 Equal contributors Division of Microbiology, Department of Molecular Biology, Paris-Lodron University of Salzburg , Billroth Str. 11, A-5020 Salzburg , Austria
Background: The serine proteases HtrA/DegP secreted by the human gastrointestinal pathogens Helicobacter pylori (H. pylori) and Campylobacter jejuni (C. jejuni) cleave the mammalian cell adhesion protein E-cadherin to open intercellular adhesions. A wide range of bacteria also expresses the HtrA/DegP homologs DegQ and/or DegS, which significantly differ in structure and function. Methods: E-cadherin shedding was investigated in infection experiments with the Gram-negative pathogens H. pylori, enteropathogenic Escherichia coli (EPEC), Salmonella enterica subsp. Enterica (S. Typhimurium), Yersinia enterocolitica (Y. enterocolitica), and Proteus mirabilis (P. mirabilis), which express different combinations of HtrAs. Annotated wild-type htrA/degP, degQ and degS genes were cloned and proteolytically inactive mutants were generated by a serine-to-alanine exchange in the active center. All HtrA variants were overexpressed and purified to compare their proteolytic activities in casein zymography and in vitro E-cadherin cleavage experiments. Results: Infection of epithelial cells resulted in a strong E-cadherin ectodomain shedding as reflected by the loss of full length E-cadherin in whole cell lysates and formation of the soluble 90 kDa extracellular domain of E-cadherin (NTF) in the supernatants of infected cells. Importantly, comparing the caseinolytic and E-cadherin cleavage activities of HtrA/DegP, DegQ and DegS proteins revealed that DegP and DegQ homologs from H. pylori, S. Typhimurium, Y. enterocolitica, EPEC and P. mirabilis, but not activated DegS, cleaved E-cadherin as a substrate in vitro. Conclusions: These data indicate that E-cadherin cleavage is confined to HtrA/DegP and DegQ proteins representing an important prevalent step in bacterial pathogenesis.
HtrA; DegP; DegQ; E-cadherin
Human pathogens developed sophisticated strategies to
survive and colonize under extreme conditions or to
conquer host defense mechanisms. The serine proteases
HtrA/DegP are important key players in protein quality
control and stress response through refolding and
degrading misfolded proteins in the periplasm of bacteria
[1, 2]. In E. coli, DegP was identified as an
ATPindependent heat shock protease that maintains protein
homeostasis in the periplasm by combining chaperone
and protease activities. DegP consists of an N-terminal
signal peptide, which is responsible for its periplasmic
localization followed by a conserved chymotrypsin—like
protease domain harboring the catalytic triad composed
of a histidine, a serine and an aspartate residue. In the
C-terminal region, DegP contains two flexible PDZ
(postsynaptic density protein [PSD95], Drosophila disc
large tumor suppressor [Dlg1], and zonula occludens-1
protein [ZO-1]) domains mediating protein-protein
interactions, substrate recognition and substrate binding
[1, 3, 4]. The monomeric E. coli DegP can form trimers,
hexamers, dodecamers, and finally active 24-mers [5, 6].
It was demonstrated that binding of hexameric DegP to
© The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
misfolded proteins leads to the formation of active
12mers and 24-mers . Several substrates for E. coli DegP
were described, including maltose binding protein,
alkaline phosphatase, α-amylase, outer membrane protein
OmpF and OmpC, the pilin subunit PapA or the
acylated precursor of colicin A lysis protein . E. coli also
expresses the HtrA/DegP homologs DegQ (HhoA, HtrA
homolog A) and DegS (HhoB, HtrA homolog B). The
main difference between DegP and DegQ is the length
of the N-terminally positioned LA loop, which lacks 20
amino acids in DegQ [7, 8]. The LA loop is implicated
in the stabilization of the inactive hexameric
conformation of DegP . Although DegQ and DegP exhibit
~60% sequence identity, it is not fully understood
whether they share overlapping function. It was shown
that DegQ is capable of rescuing temperature sensitive
degP-negative strains , while others have postulated
that the substrate specificity of DegQ might be different
since DegQ re-expression could not fully restore the
phenotype of a degP knock-out mutant [8, 11]. DegS is
considered as a regulatory protease targeting the
antisigma factor RseA in the periplasm, which is implicated
in sensing protein folding stress. After detecting
misfolded outer membrane proteins, DegS processes the
anti-sigma factor RseA, which is followed by RseP
cleavage. As a regulated intramembrane proteolysis cascade,
this leads to the sigma-E-mediated expression of factors
involved in protein folding stress in the periplasm and
assembly of outer membrane proteins [3, 12].
In many pathogenic bacteria, HtrA promotes virulence
as reflected by the observation that htrA knock-out
mutants show either an apathogenic phenotype or a
significantly reduced virulence [2, 13]. A widespread
explanation for the HtrA-dependent pathogenesis arose
from the observation that HtrA increases bacterial
survival under stress conditions during infection. Further, it
was suggested that HtrA is involved in the processing of
outer membrane (virulence) factors . For instance,
DegP was identified as a critical factor for IcsA (VirG)
surface presentation in Shigella flexneri (S. flexneri) .
Furthermore, reduced adherence of a C. jejuni htrA
knock-out mutant was observed in vitro [15–18]
suggesting that the expression of adhesins might be
downregulated. However, in a mouse model for C. jejuni
infections, isogenic htrA-negative bacteria colonized
equally well, while host cell apoptosis and the
proinflammatory immune responses were significantly
attenuated [19, 20]. Similar observations were made for a
number of other htrA-negative pathogens in vivo (e.g.
Yersinia pestis, Streptococcus pneumoniae,
Mycobacterium tuberculosis, Listeria monocytogenes, Klebsiella
pneumoniae, etc.) [13, 21]. In Chlamydia trachomatis
(C. trachomatis) HtrA functions as an active chaperone
and serine protease . HtrA is secreted from
chlamydial inclusions into the host cytoplasm
independently of the type-III secretion system  and exhibits a
critical role in the replicative phase of the chlamydial
developmental cycle . These data underline the
crucially important role of HtrA in bacterial pathogenesis.
However, the molecular mechanism remained largely
An additional function of HtrA in several
Gramnegative pathogens of the gastrointestinal tract was
recently described. During infection with H. pylori and C.
jejuni, HtrA is secreted into the microenvironment
[25, 26] and was detected in outer membrane vesicles
(OMVs) [27, 28]. H. pylori and C. jejuni HtrAs
cleave-off the extracellular domain of the cell
adhesion protein E-cadherin on epithelial cells [15, 27, 29,
30]. E-cadherin is an important key molecule in the
establishment and maintenance of an intact epithelial
barrier. Consequently, E-cadherin cleavage disrupts
the barrier function and allows bacterial entry into
the intercellular space and transmigration [31, 32]. In
H. pylori or C. trachomatis, genomic htrA deletions
mutants could not be generated so far. However,
functional small molecule inhibitors and substrate-derived
peptide inhibitors were designed which efficiently blocked
HtrA functions [30, 33, 34]. HtrA-mediated E-cadherin
cleavage was also shown for EPEC and S. flexneri
supporting our hypothesis that E-cadherin ectodomain shedding
might be a prevalent mechanism for pathogenic bacteria
to promote virulence through the interference with
(baso-) lateral domains of epithelial cells .
However, these studies were restricted to HtrA/DegP and
the role of DegQ and DegS in E-cadherin cleavage
was not considered so far. In this report, we
investigated E-cadherin shedding in response to infection
with the Gram-negative gastrointestinal pathogens H.
pylori, EPEC, Y. enterocolitica, S. enterica subsp.
Enterica (S. Typhimurium) and the uropathogenic
bacterium P. mirabilis, which express different
combinations of HtrA proteins.
MKN-28 and NCI-N87 cells were grown in RPMI 1640
medium (Sigma Aldrich) containing 10% FBS (Sigma
Aldrich) in 6-well plates to a confluency of 70 to 80% for 2
days. 16 h prior to the infection, medium was replaced
by serum-free RPMI 1640. H. pylori (Hp26695) was
cultivated on GC-Agar plates containing 10% horse serum
under microaerophilic conditions (CampiGen, Thermo
Scientific) at 37 °C for 2 days. P. mirabilis (ATCC
29906) was grown on nutrient agar, and EPEC (E2348),
Salmonella enterica subsp. Enterica (S. Typhiumurium,
NCTC 12023) and Y. enterocolitica (ATCC 27729) were
cultivated on LB agar plates for 24 h at 37 °C.
Serumstarved cells were infected at a multiplicity of infection
(MOI) of 100 with H. pylori, at a MOI 5 with EPEC or S.
Typhimurium, at a MOI 50 with Y. enterocolitica and at
a MOI 2 with P. mirabilis. Cells were harvested after
indicated time periods in lysis buffer (20 mM Tris pH 7.5,
1 mM EDTA, 100 mM NaCl, 1% Triton X-100, 0.5%
DOC, 0.1% SDS, 0.5% NP-40). Samples were centrifuged
for 10 min at 16000 × g at 4 °C. Pellets were discarded
and lysates were analyzed for full length E-cadherin by
Western blotting. For the detection of the soluble
extracellular E-cadherin fragment, supernatants of infected
cells were collected. Bacteria were harvested in sterile
PBS supplemented and sonicated to prepare bacterial
lysates. Protein amounts were measured using Bradford
(RotiQuant, Carl Roth).
SDS PAGE and western blotting
10 μg of the bacterial lysates or 0.5 μg recombinant
proteins were separated by SDS-PAGE and stained using 1%
Coomassie Brilliant Blue G250 (BioRad). To investigate
E-cadherin cleavage, 50 μg of cell lysates or 100 μl of
supernatants were separated by SDS-PAGE and transferred
onto nitrocellulose membranes. Monoclonal antibodies
recognizing the extracellular domain (ab40772, Abcam)
or intracellular domain (24E10, Cell Signaling) of
Ecadherin were used to detect the NTF in supernatants
or the loss of full length E-cadherin in whole cell lysates,
respectively. ß-actin was detected using a monoclonal
antibody (Sigma Aldrich).
10 μg of the bacterial lysates or 1 μg recombinant
proteins were separated by casein-containing SDS gels
under non-reducing conditions. Subsequently, gels were
renatured in 2.5% Triton X-100 and equilibrated in
developing buffer as previously described . Caseinolytic
activity was visualized after staining with 0.5%
Coomassie Blue R250 (BioRad).
Protein sequences from H. pylori HtrA (G2J5T2), EPEC
DegP (B7UIK8), EPEC DegQ (B7UJW6), EPEC DegS
(B7UJW7), S. Typhimurium HtrA (P26982), Y.
enterocolitica DegP (P74978), P. mirabilis DegQ (B4EXL6), P.
mirabilis DegS (B4EXL5) were retrieved from UniProt
(Table 1). Sequence alignments were performed using
Clustal Omega . Protein domain prediction was
performed using SignalP4.1 and SMART (simple modular
architecture research tool) [36–38]. (*) indicates identical
amino acids in all sequences, conserved amino acid
substitutions are labeled with (:) and semi-conservative
substitutions are marked with (.).
Cloning, mutagenesis and protein purification
Cloning, mutagenesis and protein purification was
performed as described before . Briefly, genes encoding
H. pylori HtrA (HpHtrA aa 18–475), EPEC DegP
(EpDegP aa 27–474), EPEC DegQ (EpDegQ aa 29–455),
EPEC DegS (EpDegS aa 28–355), S. Typhimurium HtrA
(StHtrA aa 27–475), Y. enterocolitica DegP (YeDegP aa
21–478), P. mirabilis DegQ (PmDegQ aa 31–463), P.
mirabilis DegS (PmDegS aa 1–356) lacking predicted
signal peptides were amplified. Primer sequences are
shown in Table 1. PCR fragments flanked by restriction
sites for BamHI/EcoRI (HpHtrA, EpDegS, YeDegP and
PmDegQ), BamHI/XmaI (HpHtrA) or EcoRI/XhoI
(PmDegS) were ligated into pGEX-6P-1 (GE Healthcare)
for the expression of N-terminally tagged GST fusion
proteins. Generation of inactive HtrA proteases
(HpHtrAS221A, EpDegPS236A, EpDegQS214A, EpDegSS201A,
StHtrAS237A, YeHtrAS238A, PmDegQS219A, PmDegSS199A)
was performed by S → A mutations in the active center
using a site directed mutagenesis kit (Agilent) (Table 1). E.
coli BL21 has been transformed with generated constructs
and purification of the proteins was performed as
previously described . In brief, transformed E. coli was
grown in 300 ml LB medium to an OD600 of 0.6 and the
expression was induced by the addition of 0.1 mM
isopropylthiogalactosid (IPTG). The bacterial culture was
pelleted at 6000 × g for 30 min at 4 °C and lysed in 10 ml
PBS by sonication. The lysate was cleared by
centrifugation and the supernatants were incubated with glutathione
sepharose (GE Healthcare Life Sciences) at 4 °C overnight.
GST-tagged proteins were cleaved with 180 U Prescission
protease (GE Healthcare Life Sciences) for 16 h at 4 °C to
remove the GST tag. RseA (residues 121–216) fused to an
N-terminal His6-tag was kindly provided by Tim Clausen
(IMP, Vienna) and has been described previously .
RseA was expressed in E. coli BL21 and purified via
ProBond NiNTA sepharose (Invitrogen). RseA was washed
and eluted with 250 mM imidazole. All purified proteins
were rebuffered in the respective cleavage buffer
compatible with following cleavage experiments.
A polyclonal antibody recognizing HpHtrA was
generated by the immunization of rabbits with recombinant
HpHtrA (Paul-Ehrlich Institute, Langen, Germany).
Polyclonal antibodies for the detection of StHtrA,
YeDegP, EpDegP or PmDegQ were produced by
immunization of rabbits with recombinant StHtrAS237A,
YeDegPS238A, EpDegPS236A and PmDegQ proteins (David’s
Biotechnology GmbH, Regensburg, Germany).
In vitro cleavage assays
For in vitro cleavage studies, 50 ng of recombinant
human E-cadherin (R&D) was incubated with 500 ng of
Table 1 Proteins analyzed in this study
Pathogen Helicobacter pylori EPEC
Strain Hp26695 E2348/69
NCTC National Collection of Type Cultures, ATCC American Type Culture Collection
asequences of HtrA proteins; brestriction recognition sites are underlined; csubstituted nucleotides are underlined
Name HtrA DegP DegQ
Reference   This work
recombinant proteases in 50 mM Hepes (pH 7.4)
containing 1 mM EDTA at 37 °C for 24 h. As indicated,
proteolytic inactive proteins were included as controls.
Cleavage of E-cadherin was detected by Western blot
analyses. To demonstrate EpDegS activity, 7 μg EpDegS
was incubated with 9 μg recombinant RseA protein in
the presence of 100 μM YFF (DNRLGLVYFF) activator
peptide  for 16 h at 37 °C in 100 mM NaPO4
(pH 7.5), 200 mM NaCl, 5 mM MgCl2, 1 mM DTT und
10% glycerol. Where indicated, 300 ng E-cadherin was
added. Aliquots of the samples were analyzed by
Western blotting for E-cadherin cleavage, while RseA
degradation was detected by coomassie-stained SDS PAGEs.
H. pylori only harbors a DegP homolog, whereas EPEC,
S. Typhimurium and Y. enterocolitica express DegP,
DegS and DegQ, and the genome of P. mirabilis
contains DegQ and DegS. To analyze their capacity to
induce E-cadherin ectodomain shedding during infection,
epithelial cells were colonized with selected pathogens
and E-cadherin cleavage was investigated through
detection of the loss of full length E-cadherin (E-cadFL) in
whole cell lysates and the formation of the soluble
Nterminal fragment (E-cadNTF) in the supernatants of
infected cells. To demonstrate equal protein amounts in
whole cell lysates, β-actin was shown. As reported
previously , H. pylori (Fig. 1a) and EPEC (Fig. 1b) induced
efficient E-cadherin shedding as monitored by increase
of E-cadNTF and, partially, by the corresponding decrease
of E-cadFL after indicated time periods of infection. Cells
infected with S. Typhimurium showed an increased
amount of the cleaved soluble E-cadNTF in supernatants
after 6 h and after 8 h. The amount of E-cadFL
Fig. 1 E-cadherin cleavage during infection with Gram-negative pathogens. Human epithelial cells were infected with (a) H. pylori (Hp) at a MOI
100, (b) EPEC (Ep) at a MOI 5, (c) S. Typhimurium (St) at a MOI 5, (d) Y. enterocolitica (Ye) at a MOI 50 and (e) P. mirabilis (Pm) at a MOI 2. Different
MOIs were chosen after careful titration of infection doses to minimize bacterial overgrowth during infection. After indicated time periods, cells
were lysed and full length E-cadherin (EcadFL) was detected by Western blot analyses using an antibody against the intracellular domain. Aliquots
of supernatants were analyzed for the soluble extracellular E-cadherin fragment (EcadNTF) using an antibody against the extracellular domain.
β-actin served as a loading control
detectable in cell lysates decreased correspondingly
(Fig. 1c) indicating that infections with S. Typhimurium
induces E-cadherin ectodomain shedding during
infection as well. Similar observations were made for cells
infected with Y. enterocolitica (Fig. 1d). Compared to
non-infected cells, E-cadFL slightly decreased, while
EcadNTF in the supernatants of infected cells appeared
(Fig. 1d). As a Gram-negative uropathogen, P. mirabilis
was included in this study. P. mirabilis induced a very
strong decline of E-cadFL in whole cell lysates and
correspondingly, the amount of E-cadNTF drastically
increased indicating an efficient cleavage of E-cadherin
during colonization (Fig. 1e). These data imply that
Ecadherin shedding occurs frequently during bacterial
E-cadherin shedding can be induced by host proteases
[30, 41] or by bacterial proteases, such as HtrA proteins
[15, 27, 30]. To evaluate if HtrAs of S. Typhimurium, Y.
enterocolitica, or P. mirabilis are expressed and capable
of E-cadherin cleavage, we analyzed the expression of
proteolytic active proteases by casein zymography in a
first step. H. pylori expressed caseinolytically active
monomeric and oligomeric HtrA at 50 kDa and
>170 kDa, which have been previously identified by
mass-spectrometry . In lysates of S. Typhimurium,
three different activities at 85 kDa, 45 kDa and 28 kDa
were observed. Four proteolytic activities (90 kDa,
55 kDa, 30 kDa and 20 kDa) were found in Y.
enterocolitica, while P. mirabilis exhibited caseinolytically active
proteases of approximately 75 kDa, 55 kDa and 25 kDa.
EPEC lysates contained proteolytic activities at 50 kDa,
30 kDa and 20 kDa (Fig. 2, upper panel), of which the
50 kDa protease was identified as active DegP previously
. Equal protein amounts were demonstrated by a
coomassie-stained SDS PAGE (Fig. 2, lower panel).
The serine protease HtrA or DegP is a highly
conserved protease. Sequence alignments of HtrAs and
DegPs of H. pylori (HpHtrA), Y. enterocolitica (YeDegP),
EPEC (EpDegP) and S. Typhimurium (StHtrA)
demonstrated high similarities (Fig. 3). All HtrA/DegP
proteases harbor a predicted N-terminal signal peptide
(orange), a proteolytic domain (green) with the catalytic
triad containing a histidine, an aspartate and a serine
(red). The protease domain was followed by two
Cterminal PDZ domains (purple). In comparison to DegQ
proteases (Additional file 1: Figure S1A), the LA loop
(blue) in DegP proteases contained additional 20 amino
acids . Interestingly, the LA loop of H. pylori HtrA
lacked 22 amino acids suggesting that H. pylori HtrA
might be a DegQ protein rather than a DegP protein.
However, HpHtrA shows a higher identity with EpDegP
(43% identity, E = 2e-87) compared to the alignment of
HpHtrA with EpDegQ (37% identity, E = 8e-81), while a
comparison of EpDegQ and PmDegQ uncovered an
Fig. 2 Active proteases expressed by pathogens. H. pylori (Hp), EPEC
(Ep), S. Typhimurium (St), Y. enterocolitica (Ye) and P. mirabilis (Pm)
were sonicated and protein lysates were analyzed by casein
zymography (upper panel). As a control, proteins were separated by
SDS PAGE followed by coomassie staining to show equal protein
loading (lower panel)
identity of 66% (Additional file 1: Figure S1A). DegS has
a different domain architecture [1, 3]. DegS proteins
often contain a transmembrane domain instead of a
signal peptide and only one PDZ domain (Fig. 4a).
Comparison of the amino acid sequence of EpDegS and
PmDegS (Additional file 1: Figure S1B) showed an
identity of 59% (E = 5e-144). However, a signal peptide has
been predicted for EpDegS and a putative
transmembrane domain for PmDegS (Additional file 1: Figure
S1B), which might indicate that they also have different
Fig. 3 Sequence alignment of the different HtrA/DegP proteins. Signal peptides (orange), proteolytic domains (green) containing the catalytic
triad (red) and two PDZ domains (purple) of H. pylori HtrA (HpHtrA), Y. enterocolitica DegP (YeDegP), EPEC DegP (EpDegP) and S. Typhimurium
HtrA (StHtrA) are indicated. The LA loop region is highlighted in blue
functions. To investigate the different bacterial HtrA/
DegP proteins, we cloned, overexpressed and purified
DegP proteins from H. pylori (Hp), S. Typhimurium (St),
Y. enterocolitica (Ye), and EPEC (Ep) and analyzed the
caseinolytic activity in casein zymography experiments
(Fig. 4b). In P. mirabilis (Pm), DegP was not annotated;
hence, the degQ gene was cloned. Additionally,
proteolytic inactive proteases (HpHtrAS221A, EpDegPS236A,
StHtrAS237A, YeHtrAS238A, PmDegQS219A) were
generated by the exchange of the serine by an alanine in the
active center. Recombinant wildtype proteases (rHtrAwt)
and their corresponding inactive mutants (rHtrASA)
were examined by casein zymography (Fig. 4b, upper
panel) and coomassie-stained SDS PAGE (Fig. 4b, lower
panel). In fact, all rHtrAwt proteins were caseinolytically
active to different extents. A strong activity was
observed for HpHtrAwt, StHtrAwt and PmDegQwt, while
YeHtrAwt and EpDegPwt were less active. As expected,
the proteolytic inactive rHtrASA mutants did not show
any activities (Fig. 4b, upper panel). In our previous
studies, we already identified an auto-processed H. pylori
HtrA (sHtrA, short HtrA) by mass-spectrometry 
Fig. 4 Recombinant HtrA’s/DegP’s are proteolytically active and cleave E-cadherin in vitro. a Domain architecture of HtrA/DegP, DegQ and DegS
proteins. SP, signal peptide (orange); protease domain (green); PDZ domains (purple); TMD, transmembrane domain (red). b The proteolytic activity
of recombinant HtrA/DegP (rHtrA) wildtype proteins (wt) of H. pylori (Hp), S. Typhimurium (St), Y. enterocolitica (Ye), EPEC (Ep) and DegQ of P. mirabilis
(Pm) was analyzed by casein zymography and compared to their corresponding inactive mutants (SA) (upper panel). Coomassie-stained SDS PAGEs
demonstrated equal protein loading (lower panel). Self-processed proteins (black asterisks) exhibiting proteolytic activity (white asterisks) are indicated.
c Recombinant HtrAs/DegPs (wt) were investigated in in vitro cleavage assays using E-cadherin (E-cadFL) as a substrate and compared with
the corresponding inactive variants (SA) as a control. Fragments of E-cadherin were detected using an antibody recognizing the extracellular
domain domain. HtrA/DegP proteins were detected using corresponding polyclonal antibodies
(Fig. 4b, lower panel, black asterisk), which was
proteolytically active (Fig. 4b, upper panel, white asterisk).
Auto-cleavage of DegP as part of a physiological process
was also described for E. coli  and was also detected
for EpDegP in this study (Fig. 4b, lower panel). In
contrast to HpHtrA, auto-cleavage of EpDegP was almost
complete, but led to an inactivation of DegP. A similar
picture was observed for StHtrA and YeHtrA. Only the
full length versions of StHtrA and YeHtrA were
proteolytically active, while the truncated proteins exhibited no
activities. This is in a slight contrast to PmDegQ.
Comparable to HpHtrA, we detected large amounts of active
full length and a smaller fraction of active
autoprocessed PmDegQ (Fig. 4b). These data imply that
auto-proteolytic processing leads to an inactivation of
StHtrA, YeHtrA and EpDegP, but not of HpHtrA or
PmDegQ. Recombinant HtrA/DegP proteins were further
used for the production of polyclonal antisera recognizing
the individual proteins (Additional file 1: Figure S2). In
order to evaluate their E-cadherin cleavage capability,
purified DegP homologs (rHtrAwt) and the corresponding
inactive mutants (rHtrASA) were then examined in in vitro
cleavage experiments using recombinant E-cadherin
(rEcad) as a substrate. Incubation of rEcad with rHtrA/
DegP from H. pylori, S. Typhimurium, Y. enterocolitica
and EPEC induced the typical fragmentation pattern of
rEcad indicating that the DegP homologs of the tested
Gram-negative pathogens can directly target E-cadherin
as a substrate. As expected, the inactive HtrA/DegPSA
proteins did not cleave rEcad (Fig. 4c). Polyclonal
antibodies detecting the individual HtrA/DegP proteins
(Additional file 1: Figure S2) showed equal loading of
HtrA/DegPwt and HtrA/DegPSA proteins (Fig. 4c).
Since it is unclear whether HtrA homologs have
overlapping functions in bacteria, we compared the
Ecadherin cleavage activity of the HtrA homologs DegP,
DegQ and DegS from EPEC and P. mirabilis. Both, DegP
and DegQ proteins, but not DegS or the corresponding
inactive mutants from EPEC and P. mirabilis were
caseinolytically active (Additional file 1: Figure S3A and
Additional file 1: Figure S3B). Comparing the E-cadherin
cleavage activity of the EPEC HtrA proteins DegP, DegQ
and DegS revealed that EpDegP cleaved E-cadherin more
efficiently than EpDegQ. Compared to EpDegP, EpDegQ
induced weak fragmentation of E-cadherin in vitro.
EpDegS did not mediate E-cadherin cleavage. HpHtrA
was used as a positive control. The polyclonal
antiEpDegP antibody detected EpDegPwt and EpDegPSA and
showed weak cross-reactivity to EpDegQ and EpDegS
(Fig. 5a). To underline the finding that DegQ proteases
also cleave E-cadherin, we compared the E-cadherin
cleavage activity of EpDegP, EpDegQ, EpDegS with
PmDegQ and PmDegS. In fact, PmDegQ directly cleaved
rEcad, which was comparable to EpDegP and EpDegQ
(Fig. 5b). Corresponding to EpDegS, PmDegS did not
fragment rEcad (Fig. 5b). It has been demonstrated that
DegS activity requires stimulation by activator peptides
. The fact that recombinant DegS from EPEC is an
active protease was demonstrated in an in vitro cleavage
experiment using the DegS substrate RseA (Fig. 5c).
Upon stimulation with the YFF activator peptide ,
EpDegS efficiently degraded RseA (Fig. 5c, middle
panel). In parallel, rEcad was added as indicated.
However, rEcad was not targeted by active DegS (Fig. 5c,
upper panel). These data underline that E-cadherin
shedding is mainly mediated by bacterial DegP and
DegQ homologs, while activated DegS failed to target
Ecadherin as a substrate.
HtrA proteases are crucially important for bacterial
pathogenesis. Their periplasmic chaperone functions
facilitate bacterial viability and survival by refolding and
degradation of misfolded proteins under stress
conditions [1, 3]. Furthermore, HtrA proteins are also
implicated in the modulation of pathogen-host interaction by
processing of surface-presented virulence factors or
adhesins [14, 16, 17]. Another important function was
Fig. 5 DegP and DegQ, but not DegS cleave E-cadherin in vitro. a DegP, DegQ and DegS wildtype (wt) of EPEC (Ep) and the corresponding
inactive mutants (SA) were tested in in vitro cleavage assays using E-cadherin (rEcad) as a substrate (upper panel). EpDegPwt and EpDegPSA were
detected using anti-EpDegP antibody (lower panel). b The E-cadherin-cleavage activity of EPEC (Ep) DegP, DegQ and DegS was compared with
the activity of P. mirabilis (Pm) DegQ and DegS. EpDegP and PmDegQ were detected using polyclonal antibodies. c The selective activity of
EpDegS was shown in in vitro cleavage experiments using 7 μg EpDegS and 9 μg recombinant RseA (rRseA) as a substrate. To stimulate the activity of
EpDegS, 100 μM YFF activator peptide or equal amounts of diluent (−) were added as indicated. 300 ng rEcad was included in the reactions where
indicated. Aliquots of samples were analyzed by Western blotting to detect E-cadherin (upper panel) and the remaining sample was separated by SDS
PAGE following coomassie staining to detect the degradation of RseA (middle panel) and EpDegS proteins (lower panel). The asterisk (*) indicates GST
protein co-purified with the EpDegS protein
observed for secreted or outer-membrane
vesicleassociated HtrA from H. pylori and C. jejuni, which
directly cleaves the extracellular domain of E-cadherin on
host cells [15, 27, 29, 30]. HtrA-mediated E-cadherin
cleavage opens intercellular adherens junctions allowing
bacterial transmigration across the epithelial barrier [15,
29, 30, 33]. Cleavage of E-cadherin has been additionally
observed for HtrA expressed by EPEC and S. flexneri
during infection of cultured epithelial cells and in vitro
 indicating that HtrA-induced E-cadherin shedding
represents a prevalent mechanism in bacterial
pathogenesis. In contrast to H. pylori or C. jejuni, many
pathogens express more than one HtrA homolog, namely
DegP, DegQ and DegS and it is completely unclear,
which of these homologs target E-cadherin. Therefore,
we investigated the cleavage activity of the three
different bacterial HtrA homologs and found that (i.)
additional Gram-negative pathogens S. Typhimurium, Y.
enterocolitica and P. mirabilis express
E-cadherinfragmenting HtrA proteases and (ii.) that DegP and
DegQ homologs, but not DegS, cleave E-cadherin.
The finding that DegP and DegQ, but not DegS, are
active E-cadherin proteases is interesting since it
indicates a specific and economical mechanism through
which bacteria can interfere directly with host cells
functions. Generally, the amino acid sequences of DegP and
DegQ proteases show high similarities indicating
conserved roles in bacteria. Sequence alignment revealed
that HpHtrA lacks 22 amino acids in the LA loop
leading to the assumption it could be a DegQ homolog
rather than a DegP protein. However, HpHtrA exhibits a
higher similarity with DegP proteins. Therefore, it
remains vague whether HpHtrA represents a DegP or
DegQ protein. From the literature, it is apparently not
clear whether HtrA homologs have redundant functions.
Consistently described, deletion of degP led to a higher
sensitivity of the bacteria toward elevated temperatures
[43–45]. It has been previously suggested that DegQ can
compensate for lacking DegP functions . In other
studies, degP, degQ and degS mutants did not show the
same phenotype  suggesting that the HtrA homologs
have different roles. Further, DegQ or DegS
reexpression did not fully replace DegP functions in a
knock-out mutant [8, 11] implying that DegP and DegQ
have different roles in the bacterial periplasm. In our
report, we found that only DegP from H. pylori, EPEC, S.
Typhimurium, Y. enterocolitica, and DegQ proteases
expressed by EPEC and P. mirabilis target E-cadherin as
a substrate. Since these pathogens interfere with host
cell functions via different mechanisms, it needs to be
investigated in future, how HtrA-mediated E-cadherin
cleavage contributes to the infections with the individual
pathogens. Importantly, the opening of the intercellular
space can facilitate the contact between pathogens and
basolaterally expressed host factors or cells of the
immune system. Interestingly, P. mirabilis does not express
a DegP protein, but an extremely active DegQ protein.
Furthermore, active DegP and DegQ proteases also
induced a similar fragmentation pattern of E-cadherin
indicating that they target identical calcium binding and
substrate recognition sites, which have been recently
identified for HpHtrA [33, 47]. DegS proteases from
EPEC and P. mirabilis failed to cleave E-cadherin in
vitro. The domain architecture of the DegS proteins
differs considerably. A transmembrane domain was
predicted in PmDegS, while EpDegS contains a putative
signal peptide. Following the highly conserved protease
domain, DegS proteins harbor only one PDZ domain
[1, 3]. The fact that DegS did not cleave E-cadherin
leads to the hypothesis that either the variation on
the N-terminus or the second PDZ domain is
implicated in the recognition and/or binding of E-cadherin.
Based on these observations, we conclude that DegP
and DegQ proteins, but not DegS exhibit an
Ecadherin-cleaving activity. Our findings were mainly
obtained from in vitro experiments as bacterial
pathogens harboring genomic deletions of the individual
degP, degQ and degS genes are not available to
investigate the individual impact of HtrA proteins on bacterial
pathogenesis. Still, in infection experiments using
Gramnegative pathogens, which express different combinations
of DegP, DegQ, and/or DegS, it became apparent that (i.)
pathogens do not need DegS and (ii.) pathogens require at
least DegP or DegQ for efficient E-cadherin cleavage.
E-cadherin cleavage during infection has been described
for H. pylori, C. jejuni, EPEC and S. flexneri [15, 27, 29].
In this study, we added S. Typhimurium, Y.
enterocolitica, and P. mirabilis to the collection of
E-cadherintargeting pathogens. Those gastrointestinal bacteria
colonize the epithelium of the intestine as the first
barrier. E-cadherin shedding could promote bacterial
virulence of these pathogens through providing entry
through the polarized epithelium where specific
virulence and pathogenic factors then interfere with host cell
functions [31, 32]. Hence, it would be highly interesting
to investigate the influence of the different HtrA
homologs in their respective experimental animal models in
vivo as HtrA proteins represent attractive therapeutic
target molecules. The finding that the uropathogen P.
mirabilis also induces E-cadherin shedding through its
highly active DegQ protein also suggests a possible role
for HtrA proteins in pathogens, which colonize
nonintestinal epithelia. Therefore, future studies are
necessary to study the function of HtrAs during the
colonization of pathogens targeting the epithelium of
Additional file 1: Show an additional alignment of DegQ and DegS
proteins (Figure S1), validation of anti-HtrA antibodies (Figure S2), and
the activity of recombinant proteases (Figure S3). (PDF 1620 kb)
DegP/Q/S: Periplasmic serine endoproteases; Dlg1: Drosophila disc large
tumor suppressor; EPEC: Enteropathogenic escherichia coli; HtrA: High
temperature requirement A; NTF: N-terminal fragment; PDZ: Postsynaptic
density protein (PSD95); ZO-1: Zonula occludens-1 protein
We thank Markus Hell (Landeskliniken Salzburg) for providing Yersinia
enterocolitica. We are also very grateful to Tim Clausen (IMP, Vienna) for the
RseA plasmid and the DegS activator peptide.
Conceived and designed the experiments: CMA, GP, SW. Performed the
experiments: MS, CMA, CG, GP, TPS. Wrote the paper: CMA, SW. All authors
read and approved the final manuscript.
1. Clausen T , Kaiser M , Huber R , Ehrmann M. HTRA proteases: regulated proteolysis in protein quality control . Nat Rev Mol Cell Biol . 2011 ; 12 ( 3 ): 152 - 62 .
2. Skorko-Glonek J , Zurawa-Janicka D , Koper T , Jarzab M , Figaj D , Glaza P , Lipinska B. HtrA protease family as therapeutic targets . Curr Pharm Des . 2013 ; 19 ( 6 ): 977 - 1009 .
3. Hansen G , Hilgenfeld R. Architecture and regulation of HtrA-family proteins involved in protein quality control and stress response . Cell Mol Life Sci . 2013 ; 70 ( 5 ): 761 - 75 .
4. Sawa J , Heuck A , Ehrmann M , Clausen T. Molecular transformers in the cell: lessons learned from the DegP protease-chaperone . Curr Opin Struct Biol . 2010 ; 20 ( 2 ): 253 - 8 .
5. Krojer T , Sawa J , Schafer E , Saibil HR , Ehrmann M , Clausen T. Structural basis for the regulated protease and chaperone function of DegP . Nature . 2008 ; 453 ( 7197 ): 885 - 90 .
6. Spiess C , Beil A , Ehrmann M. A temperature-dependent switch from chaperone to protease in a widely conserved heat shock protein . Cell . 1999 ; 97 ( 3 ): 339 - 47 .
7. Wrase R , Scott H , Hilgenfeld R , Hansen G. The Legionella HtrA homologue DegQ is a self-compartmentizing protease that forms large 12-meric assemblies . Proc Natl Acad Sci U S A . 2011 ; 108 ( 26 ): 10490 - 5 .
8. Waller PR , Sauer RT . Characterization of degQ and degS, Escherichia coli genes encoding homologs of the DegP protease . J Bacteriol . 1996 ; 178 ( 4 ): 1146 - 53 .
9. Figaj D , Gieldon A , Polit A , Sobiecka-Szkatula A , Koper T , Denkiewicz M , Banecki B , Lesner A , Ciarkowski J , Lipinska B , et al. The LA loop as an important regulatory element of the HtrA (DegP) protease from Escherichia coli: structural and functional studies . J Biol Chem . 2014 ; 289 ( 22 ): 15880 - 93 .
10. Kolmar H , Waller PR , Sauer RT . The DegP and DegQ periplasmic endoproteases of Escherichia coli: specificity for cleavage sites and substrate conformation . J Bacteriol . 1996 ; 178 ( 20 ): 5925 - 9 .
11. Bass S , Gu Q , Christen A. Multicopy suppressors of prc mutant Escherichia coli include two HtrA (DegP) protease homologs (HhoAB), DksA, and a truncated R1pA . J Bacteriol . 1996 ; 178 ( 4 ): 1154 - 61 .
12. Alba BM , Leeds JA , Onufryk C , Lu CZ , Gross CA. DegS and YaeL participate sequentially in the cleavage of RseA to activate the sigma (E)-dependent extracytoplasmic stress response . Genes Dev . 2002 ; 16 ( 16 ): 2156 - 68 .
13. Frees D , Brondsted L , Ingmer H. Bacterial proteases and virulence . Subcell Biochem . 2013 ; 66 : 161 - 92 .
14. Purdy GE , Fisher CR , Payne SM . IcsA surface presentation in shigella flexneri requires the periplasmic chaperones DegP , Skp, and SurA . J Bacteriol . 2007 ; 189 ( 15 ): 5566 - 73 .
15. Hoy B , Geppert T , Boehm M , Reisen F , Plattner P , Gadermaier G , Sewald N , Ferreira F , Briza P , Schneider G , et al. Distinct roles of secreted HtrA proteases from gram-negative pathogens in cleaving the junctional protein and tumor suppressor E-cadherin . J Biol Chem . 2012 ; 287 ( 13 ): 10115 - 20 .
16. Baek KT , Vegge CS , Brondsted L. HtrA chaperone activity contributes to host cell binding in campylobacter jejuni . Gut pathogens . 2011 ; 3 : 13 .
17. Brondsted L , Andersen MT , Parker M , Jorgensen K , Ingmer H. The HtrA protease of campylobacter jejuni is required for heat and oxygen tolerance and for optimal interaction with human epithelial cells . Appl Environ Microbiol . 2005 ; 71 ( 6 ): 3205 - 12 .
18. Boehm M , Lind J , Backert S , Tegtmeyer N. Campylobacter jejuni serine protease HtrA plays an important role in heat tolerance, oxygen resistance, host cell adhesion , invasion, and transmigration. Eur J Microbiol Immunol . 2015 ; 5 ( 1 ): 68 - 80 .
19. Heimesaat MM , Alutis M , Grundmann U , Fischer A , Tegtmeyer N , Bohm M , Kuhl AA , Gobel UB , Backert S , Bereswill S. The role of serine protease HtrA in acute ulcerative enterocolitis and extra-intestinal immune responses during campylobacter jejuni infection of gnotobiotic IL-10 deficient mice . Front Cell and Infect Microbiol . 2014 ; 4 : 77 .
20. Heimesaat MM , Fischer A , Alutis M , Grundmann U , Boehm M , Tegtmeyer N , Gobel UB , Kuhl AA , Bereswill S , Backert S. The impact of serine protease HtrA in apoptosis, intestinal immune responses and extra-intestinal histopathology during campylobacter jejuni infection of infant mice . Gut pathogens . 2014 ; 6 : 16 .
21. Ingmer H , Brondsted L. Proteases in bacterial pathogenesis . Res Microbiol . 2009 ; 160 ( 9 ): 704 - 10 .
22. Huston WM , Swedberg JE , Harris JM , Walsh TP , Mathews SA , Timms P. The temperature activated HtrA protease from pathogen Chlamydia trachomatis acts as both a chaperone and protease at 37° C. FEBS Lett . 2007 ; 581 ( 18 ): 3382 - 6 .
23. Wu X , Lei L , Gong S , Chen D , Flores R , Zhong G. The chlamydial periplasmic stress response serine protease cHtrA is secreted into host cell cytosol . BMC Microbiol . 2011 ; 11 : 87 .
24. Patel P , De Boer L , Timms P , Huston WM . Evidence of a conserved role for Chlamydia HtrA in the replication phase of the chlamydial developmental cycle . Microbes Infect . 2014 ; 16 ( 8 ): 690 - 4 .
25. Lower M , Weydig C , Metzler D , Reuter A , Starzinski-Powitz A , Wessler S , Schneider G. Prediction of extracellular proteases of the human pathogen helicobacter pylori reveals proteolytic activity of the Hp1018/19 protein HtrA . PLoS One . 2008 ; 3 ( 10 ): 23 .
26. Boehm M , Haenel I , Hoy B , Brondsted L , Smith TG , Hoover T , Wessler S , Tegtmeyer N. Extracellular secretion of protease HtrA from campylobacter jejuni is highly efficient and independent of its protease activity and flagellum . Eur J Microbiol Immunol . 2013 ; 3 ( 3 ): 163 - 73 .
27. Elmi A , Nasher F , Jagatia H , Gundogdu O , Bajaj-Elliott M , Wren B , Dorrell N. Campylobacter jejuni outer membrane vesicle-associated proteolytic activity promotes bacterial invasion by mediating cleavage of intestinal epithelial cell E-cadherin and occludin . Cell Microbiol . 2016 ; 18 ( 4 ): 561 - 72 .
28. Olofsson A , Vallstrom A , Petzold K , Tegtmeyer N , Schleucher J , Carlsson S , Haas R , Backert S , Wai SN , Grobner G , et al. Biochemical and functional characterization of helicobacter pylori vesicles . Mol Microbiol . 2010 ; 77 ( 6 ): 1539 - 55 .
29. Boehm M , Hoy B , Rohde M , Tegtmeyer N , Baek KT , Oyarzabal OA , Brondsted L , Wessler S , Backert S. Rapid paracellular transmigration of campylobacter jejuni across polarized epithelial cells without affecting TER: role of proteolytic-active HtrA cleaving E-cadherin but not fibronectin . Gut pathogens . 2012 ; 4 ( 1 ): 1757 - 4749 .
30. Hoy B , Lower M , Weydig C , Carra G , Tegtmeyer N , Geppert T , Schroder P , Sewald N , Backert S , Schneider G , et al. Helicobacter pylori HtrA is a new secreted virulence factor that cleaves E-cadherin to disrupt intercellular adhesion . Embo Rep . 2010 ; 11 ( 10 ): 798 - 804 .
31. Backert S , Boehm M , Wessler S , Tegtmeyer N. Transmigration route of Campylobacter jejuni across polarized intestinal epithelial cells: paracellular, transcellular or both? Cell Commun signal: CCS . 2013 ; 11 : 72 .
32. Posselt G , Backert S , Wessler S. The functional interplay of Helicobacter pylori factors with gastric epithelial cells induces a multi-step process in pathogenesis . Cell Commun Signal: CCS . 2013 ; 11 : 77 .
33. Schmidt TP , Perna AM , Fugmann T , Bohm M , Jan H , Haller S , Gotz C , Tegtmeyer N , Hoy B , Rau TT et al. Identification of E-cadherin signature motifs functioning as cleavage sites for Helicobacter pylori HtrA . Sci Rep . 2016 ; 6 : 23264 .
34. Gloeckl S , Ong VA , Patel P , Tyndall JD , Timms P , Beagley KW , Allan JA , Armitage CW , Turnbull L , Whitchurch CB , et al. Identification of a serine protease inhibitor which causes inclusion vacuole reduction and is lethal to Chlamydia trachomatis . Mol Microbiol . 2013 ; 89 ( 4 ): 676 - 89 .
35. Sievers F , Wilm A , Dineen D , Gibson TJ , Karplus K , Li W , Lopez R , McWilliam H , Remmert M , Soding J , et al. Fast , scalable generation of high-quality protein multiple sequence alignments using Clustal Omega . Mol Syst Biol . 2011 ; 7 ( 539 ): 75 .
36. Letunic I , Doerks T , Bork P. SMART: recent updates, new developments and status in 2015 . Nucleic Acids Res . 2015 ; 43 (Database issue):9.
37. Schultz J , Milpetz F , Bork P , Ponting CP . SMART, a simple modular architecture research tool: identification of signaling domains . Proc Natl Acad Sci U S A . 1998 ; 95 ( 11 ): 5857 - 64 .
38. Petersen TN , Brunak S , von Heijne G , Nielsen H. SignalP 4.0: discriminating signal peptides from transmembrane regions . Nat Methods . 2011 ; 8 ( 10 ): 785 - 6 . doi:10.1038/nmeth.1701.
39. Hasselblatt H , Kurzbauer R , Wilken C , Krojer T , Sawa J , Kurt J , Kirk R , Hasenbein S , Ehrmann M , Clausen T. Regulation of the sigmaE stress response by DegS: how the PDZ domain keeps the protease inactive in the resting state and allows integration of different OMP-derived stress signals upon folding stress . Genes Dev . 2007 ; 21 ( 20 ): 2659 - 70 .
40. Wilken C , Kitzing K , Kurzbauer R , Ehrmann M , Clausen T. Crystal structure of the DegS stress sensor: how a PDZ domain recognizes misfolded protein and activates a protease . Cell . 2004 ; 117 ( 4 ): 483 - 94 .
41. Schirrmeister W , Gnad T , Wex T , Higashiyama S , Wolke C , Naumann M , Lendeckel U. Ectodomain shedding of E-cadherin and c-Met is induced by Helicobacter pylori infection . Exp Cell Res . 2009 ; 315 ( 20 ): 3500 - 8 .
42. Skorko-Glonek J , Zurawa D , Tanfani F , Scire A , Wawrzynow A , Narkiewicz J , Bertoli E , Lipinska B. The N-terminal region of HtrA heat shock protease from Escherichia coli is essential for stabilization of HtrA primary structure and maintaining of its oligomeric structure . Biochim Biophys Acta . 2003 ; 1649 ( 2 ): 171 - 82 .
43. Lipinska B , Fayet O , Baird L , Georgopoulos C. Identification , characterization, and mapping of the Escherichia coli htrA gene, whose product is essential for bacterial growth only at elevated temperatures . J Bacteriol . 1989 ; 171 ( 3 ): 1574 - 84 .
44. Li SR , Dorrell N , Everest PH , Dougan G , Wren BW . Construction and characterization of a yersinia enterocolitica O:8 high-temperature requirement (htrA) isogenic mutant . Infect Immun . 1996 ; 64 ( 6 ): 2088 - 94 .
45. Diaz-Torres ML , Russell RR . HtrA protease and processing of extracellular proteins of Streptococcus mutans . FEMS Microbiol Lett . 2001 ; 204 ( 1 ): 23 - 8 .
46. Mo E , Peters SE , Willers C , Maskell DJ , Charles IG . Single, double and triple mutants of salmonella enterica serovar typhimurium degP (htrA), degQ (hhoA) and degS (hhoB) have diverse phenotypes on exposure to elevated temperature and their growth in vivo is attenuated to different extents . Microb Pathog . 2006 ; 41 ( 4-5 ): 174 - 82 .
47. Schmidt TP , Goetz C , Huemer M , Schneider G , Wessler S. Calcium binding protects E-cadherin from cleavage by Helicobacter pylori HtrA . Gut pathogens . 2016 ; 8 : 29 .