Bacterial serine protease HtrA as a promising new target for antimicrobial therapy?
Wessler et al. Cell Communication and Signaling
Bacterial serine protease HtrA as a promising new target for antimicrobial therapy?
Silja Wessler 0
Gisbert Schneider 2
Steffen Backert 1
0 Department of Molecular Biology, Division of Microbiology, Paris-Lodron University of Salzburg , Billroth Str. 11, A-5020 Salzburg , Austria
1 Division of Microbiology, University of Erlangen-Nuremberg , Staudtstr. 5, D-91058 Erlangen , Germany
2 Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH) , Vladimir-Prelog-Weg 4, CH-8093 Zürich , Switzerland
Recent studies have demonstrated that the bacterial chaperone and serine protease high temperature requirement A (HtrA) is closely associated with the establishment and progression of several infectious diseases. HtrA activity enhances bacterial survival under stress conditions, but also has direct effects on functions of the cell adhesion protein E-cadherin and extracellular matrix proteins, including fibronectin and proteoglycans. Although HtrA cannot be considered as a pathogenic factor per se, it exhibits favorable characteristics making HtrA a potentially attractive drug target to combat various bacterial infections.
HtrA proteins and their orthologues represent an
important class of heat-shock-induced serine proteases
and chaperones protecting protein structures. They are
expressed in both prokaryotic and eukaryotic species,
including plants and humans [1–3]. Whereas HtrA
orthologues commonly display proteolytic activities
against multiple target proteins, their structural
architecture and physiological functions are rather miscellaneous
and differ between species. In many bacteria, HtrA
proteases are composed of an N-terminal signal peptide,
followed by a trypsin-like serine protease domain and
one or two C-terminal PDZ (postsynaptic density
protein [PSD95], Drosophila disc large tumor suppressor
[Dlg1], and zonula occludens-1 protein [ZO-1]) modules
which permit intermolecular protein-protein interactions
[4, 5] (Fig. 1). In Gram-negative bacteria, HtrA proteases
are generally transported into the periplasm, where they
form proteolytic active multimers with known functions
in protein quality control. The best characterized HtrA
proteins are the Escherichia coli DegP, DegQ, and DegS
orthologues [6, 7]. All these different HtrAs display a
high degree of sequence identity in their protease
domain, but exhibit numerous specific features and
activities . DegP and DegQ harbor two PDZ domains,
while DegS often contains a transmembrane domain and
only one PDZ domain [1, 8] (Fig. 1). DegP is well
characterized as a protease with ATP-independent chaperone
functions. Its active oligomers assemble upon target
binding and hydrolyze unfolded or misfolded proteins
into small peptides [9, 10]. DegS represents a regulatory
protease which cleaves the anti-sigma factor RseA, while
the physiological functions of DegQ are not fully
understood . Inactivation of the htrA gene by mutation
causes an increased sensitivity to stress, e.g., elevated
temperature, of all bacteria investigated to date [12–18].
(Patho)-physiological function of bacterial HtrA
Until recently, it has been commonly accepted that HtrA
family members of bacteria are strictly acting inside the
periplasm. However, we have recently unraveled a hitherto
unknown function of HtrA during bacterial infection.
Campylobacter jejuni and its close relative Helicobacter
pylori actively secrete HtrA proteins in the extracellular
environment, where they target host cell factors [19–21].
HtrA was also identified in outer membrane vesicles
released by C. jejuni, H. pylori, Vibrio cholera, Chlamydia
muridarum or Borrelia burgdorferi [22–26]. Infection
experiments with polarized cell monolayers in vitro
suggested that H. pylori and C. jejuni HtrA can disrupt
the epithelial barrier by opening cell-to-cell junctions.
This remarkable effect is achieved by cleaving-off the
extracellular domain of the surface adhesion protein and
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Fig. 1 Domain structure of HtrA proteins in Gram-negative bacteria.
Monomeric DegP and DegQ proteins harbor an N-terminal signal
peptide (SP), an ATP-independent trypsin-like protease domain
followed by two PDZ domains. Many DegS proteins are composed
of a transmembrane domain (TMD), protease domain and one
tumor suppressor E-cadherin, and probably other
junctional proteins by HtrA, followed by paracellular bacterial
transmigration [20, 21]. The deletion of the htrA gene in
C. jejuni led to a defect in E-cadherin shedding and causes
impaired transmigration of the bacteria across monolayers
of polarized epithelial cells in vitro [19, 21].
In particular, E-cadherin showed to be an important
factor for establishing and maintaining epithelial
integrity in the host. E-cadherin is a single transmembrane
protein, which consists of an intracellular domain (IC), a
transmembrane domain (TD), and five extracellular
domains (EC) . EC domains establish homophilic
interactions in cis and trans that require calcium binding to
the linker region between the EC domains. We have
recently identified the cleavage sites of H. pylori HtrA in
E-cadherin. Mass-spectrometry-based proteomics and
Edman degradation revealed three signature motifs
containing the [VITA]-[VITA]-x-x-D-[DN] sequence pattern
as preferentially cleaved by HtrA . The results of our
studies also suggest that the presence of calcium ions
blocks HtrA-mediated cleavage by interfering with the
accessibility of calcium-binding regions between the
individual EC domains harboring the HtrA cleavage sites
. Investigating C. jejuni ΔhtrA deletion mutants in in
vivo studies, it was demonstrated that HtrA plays a crucial
role during infection by triggering host cell apoptosis and
immunopathology in mice [30, 31]. Similarly, HtrA is
critical for the virulence of many other pathogens including
Brucella abortus , Yersinia enterocolitica ,
Salmonella enterica , Legionella pneumophila ,
Shigella flexneri , Klebsiella pneumoniae , Listeria
monocytogenes , Burkholderia cenocepacia ,
Chlamydia trachomatis , Borrelia burgdorferi ,
Mycobacterium tuberculosis  and Haemophilus
parasuis . In contrast, the deletion of the htrA gene in H.
pylori has not yet been reported, and the generation of
ΔhtrA knockout mutants was found to be lethal [40, 41].
Given the fact that H. pylori htrA is an essential
bifunctional gene with crucial intracellular and extracellular
functions, it may be justified to consider HtrA as a new
target for future anti-bacterial therapy.
Why is HtrA inhibition a step forward in the fight against
With the exception of Mycoplasma genitalium and
Methanococcus janaschii, it seems that all bacterial
pathogens and commensals in the microbiota express HtrA
proteins; a fact that evades the classical and precise
definition of virulence or pathogenic factors .
Consequently, this observation leads to the question if such a
factor might also serve as a potent macromolecular drug
target? In fact, targeting HtrA offers some potential
(i.) it is secreted into the extracellular micro-milieu or
presented on the bacterial cell surface and therefore
accessible to drug compounds [43, 44],
(ii.) it has a defined enzymatic active site and substrate
recognition [19, 20, 45, 46],
(iii.) it cleaves E-cadherin, proteoglycans and fibronectin
as host factors with important functions for bacterial
pathogenesis [19–21, 47], and
(iv.) it is an essential enzyme in H. pylori
physiology [40, 41].
These characteristics make HtrA a potentially
attractive candidate for novel therapeutic approaches to treat
The current model of HtrA function in bacterial
pathogenesis is based on the hypothesis that
HtrAmediated E-cadherin cleavage represents a central step
in bacterial pathogenesis prior to and/or after the
interference of virulence factors (e.g., effector proteins,
cytotoxins, adhesins) with the integrity of the polarized
epithelium [48, 49]. These complex pathogen-host
interactions require sophisticated and coordinated
mechanisms to provide access to laterally expressed E-cadherin
and subsequently to basolaterally presented host cell
receptors or circulating cells of the immune system in
deeper regions of the tissues. In principle, the opening of
tight junctions has been shown to be HtrA-independent
in H. pylori  and C. jejuni , indicating that
additional bacterial factors are involved in the disruption of
the epithelial polarity. In H. pylori infections, soluble
factors such as vacuolating cytotoxin A (VacA),
cytotoxinassociated gene A (CagA) and urease were previously
described to open up tight junctions [50–52],
underlining that the interplay of various pathogenic factors and
HtrA is responsible for disrupting the lateral junctions
between epithelial cells. The mechanism by which C.
jejuni opens tight junctions is yet unknown. For both
pathogens, an HtrA-mediated transmigration process
was observed [20, 21, 28], enabling bacterial contact with
basolaterally expressed receptors, such as α5β1 integrins
or fibronectin [53, 54], but also allowing the bacteria to
directly interact with cells of the immune system. It is
currently being investigated whether C. jejuni prefers the
transcellular migration or paracellular route, or whether
this pathogen combines two pathways to overcome the
epithelial barrier . However, HtrA-mediated
Ecadherin cleavage in concert with activated host
proteases has been shown to promote pathogenesis in vitro
for H. pylori [20, 55, 56] and in C. jejuni animal models
[30, 31], which has been summarized in several review
articles [49, 57]. Beta1-integrins and fibronectin have
already been identified as important binding partners for
a number of additional pathogens including Yersinia
pseudotuberculosis, Staphylococcus aureus, Klebsiella
pneumoniae, and others , indicating the importance
of opening intercellular adhesion complexes. The
observation that additional gastrointestinal pathogens
(Shigella flexneri, enteropathogenic Escherichia coli
[EPEC], Yersinia enterocolitica, Salmonella enterica sub.
Enterica) utilize the HtrA homologs DegP and DegQ for
E-cadherin cleavage during infection of cultured
epithelial cells and in vitro underlines a function of HtrA
proteins as “virulence- or pathogenicity-promoting” factors
[19, 59]. Based on this hypothesis, it is enticing to
surmise that pharmacological inhibitors blocking
extracellular HtrA activity could stop bacterial transmigration
and tissue invasion in vivo, while leaving the microbiota
unaffected. Consequently, selective pharmacological
inhibition of HtrA might facilitate antibiotic treatment by
preventing bacterial access to deeper regions of
gastrointestinal tissues. Possibly, bacterial HtrAs could also
target additional substrates. For Chlamydia trachomatis,
it was demonstrated that HtrA is secreted into the
chlamydia-containing vesicles and into the host
cytoplasm. Although substrates for HtrA were not identified,
inhibition of HtrA efficiently affected the bacterial life
cycle and survival [60, 61]. With the availability of
highresolution structural models of the various HtrAs from
relevant pathogens, structure-based inhibitor design
should become feasible (Fig. 2).
In contrast to other investigated bacterial species, H.
pylori HtrA synthesis appears to be crucially important
for bacterial physiology and survival since any
intervention via mutagenesis or deletion of the htrA gene in the
genome of H. pylori has not been successful up to date
[20, 40, 41]. Correspondingly, a naturally occurring
htrAnegative H. pylori isolate was not found in a
comprehensive screening of more than 990 samples . These
observations point to the question whether
pharmacological inhibition of HtrA could tackle H. pylori physiology
specifically? Helicobacter HtrA inhibitor (HHI) was the
first described small molecule compound inhibiting H.
pylori HtrA , which blocked HtrA-mediated
EFig. 2 Structural model of the H.pylori HtrA monomer. The model is
based on a preliminary X-ray crystal structure of the apo-enzyme
containing one of the two PDZ domains . The cartoon structure
(a) shows the protease domain with the catalytic residue Ser221
highlighted. The interface between the protease domain and PDZ1 is
mediated by helix-helix interactions. The surface representation (b) has
the same orientation as in (a). Temperature coloring is according to
the computed “ligandability” [45, 64]. A potential ligand interaction
“hot spot” is predicted inside the active site (approximated by the
dashed circle). This model and related computational analyses support
the design of H. pylori HtrA inhibitors. The graphics were prepared with
MacPyMol (v1.7, Schrödinger LLC, New York, NY, USA)
cadherin cleavage and subsequent bacterial transmigration
across a polarized epithelial monolayer. However, HHI did
not affect the bacterial survival  and it is unknown,
whether HHI is actually taken up by the bacteria. A first
step in the direction of a future targeted H. pylori therapy
has recently been made by demonstrating that compound
1 drastically affected H. pylori survival and/or growth
[41, 62]. The data obtained suggest that compound 1
penetrates the bacterial cell wall to block periplasmic
HtrA activity and subsequently H. pylori survival. Further
research will be necessary to identify and optimize small
molecule HtrA inhibitors as anti-H. pylori
pharmacological lead compounds.
New strategies are urgently needed to combat bacterial
infections. At the first glance, targeting a widespread
bacterial enzyme does not appear to be straightforward.
However, considering the HtrA-mediated host cell factor
processing as a central step in the pathogenesis of many
different infectious bacteria opens up a new perspective.
Inhibiting extracellular HtrA by compounds that do not
penetrate the bacterial membrane will likely not affect
the colonization and survival of commensals; thus solely
interference of pathogens with their individual
virulence/pathogenic factors with the epithelium will be
limited. Potent HtrA inhibitors penetrating the periplasm of
H. pylori might pave the way towards a targeted anti-H.
pylori treatment owed to the fact that H. pylori
physiology essentially requires functional HtrA activity. While
many of the current antibiotics affect all bacteria
independently of assets and drawbacks for the colonized
host, pathogen-selective HtrA inhibitors might present a
drug discovery opportunity.
DegP/Q: Periplasmic serine endoproteases; HtrA: High temperature
requirement A; PDZ: Postsynaptic density protein (PSD95), Drosophila disc
large tumor suppressor (Dlg1), and zonula occludens-1 protein (ZO-1).
The work of S.W. was supported by the grants P_24074 and W_1213 from
the Austrian Science Fund (FWF). The work of G.S. was supported by the
OPO-Foundation Zurich. The work of S.B. was supported by the Deutsche
Forschungsgemeinschaft (B10 in CRC-796 and A04 in CRC-1181).
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