Four recent publications in Molecular Microbiology have begun to address the importance
of a putative protease, BB0104, in the physiology and pathogenesis of Borrelia burgdorferi.
BB0104 is a member of a large family of serine proteases designated as the high-temperature
requirement (HtrA) protease family (reviewed in Singh et al., 2011; Hansen and Hilgenfeld,
2013). Members of this group have some homology at the amino acid level and similar
architecture. All form oligomers (6–24 subunits) and have a central chymotrypsin-like
protease domain followed by one or two PDZ (substrate binding and oligomerization)
domains. Some HtrA homologues have a N-terminal signal peptide and a trans-membrane
domain. In general, these proteases in prokaryotes are responsible for maintaining
an operational bacterial proteome by turning over damaged or incorrectly folded proteins
generated during abortive cellular localization or under conditions of cell stress
(e.g. heat stock, oxidative stress etc.). Additionally, these critical proteases act
as chaperones to correctly promote the folding of proteins as they are being translocated
from the cytosol to the membranes or to the extracellular milieu. Data also suggest
that this chaperone function is required to maintain the correct levels of target
proteins in bacterial membranes, thus promoting a bacterial membrane environment that
is required for optimal membrane function. This is accomplished by the HtrA binding
to specific, target proteins and, if the protein is not able to be inserted in the
membrane in a time-dependent fashion, HtrA will transition from chaperone function
to protease function and turnover the bound protein. By biochemically ‘sensing’ the
integrity of membrane proteins, HtrA proteases provide a monitoring system that allows
the cells to adapt readily to changing environmental conditions. As a result of these
functions, HtrA proteases play a significant role in the overall physiology of the
bacteria.
Recently, it has been shown in different bacterial pathogens that HtrA can play a
significant role in the pathology of infections. For example, in Bacillus anthracis,
a htrA deletion mutant (ΔhtrA) is dramatically reduced in virulence in guinea pigs
(6 orders of magnitude) compared with the wild-type and complemented mutant strains
(Chitlaru et al., 2011). This mutant synthesizes normal levels of capsule, lethal
toxin and oedema toxin but is more sensitive to increased temperature, reactive oxygen
species and osmotic stress (Chitlaru et al., 2011). The decrease in virulence observed
in this strain is attributed to the altered export of key proteins involved in protecting
B. anthracis from stress-related challenges. Another important pathogen, Mycobacterium
tuberculosis, harbours genes encoding three HtrA-like proteases (MtHtrA1, MtHtrA2
and MtHtrA3), which exhibit a variety of functions (Roberts et al., 2013). MtHtrA1,
is a membrane bound protease that is apparently essential for bacterial cell survival
since numerous attempts to disrupt htrA1 have been unsuccessful (Sassetti et al.,
2003; Mohamedmohaideen et al., 2008). Therefore, it has been suggested that MtHtrA1
has protease or chaperone activity for proteins that are essential for the general
cell physiology. On the other hand, htrA2 mutants have been isolated that show no
obvious defects during in vitro growth. However, these mutants are attenuated in mice
suggesting that MtHtrA2 is involved in the processing and localization of key virulence
factors important for the pathology of tuberculosis. htrA3 mutants have no detectable
phenotype when grown in vitro or when tested for infectivity in mice. Interestingly,
while MtHtrA1 localizes exclusively to the membrane fraction of M. tuberculosis, MtHtrA2
and MtHtrA3 localize to the membrane fraction and appear to be secreted (Braunstein
et al., 2000). Other bacterial pathogens like Chlamydia trachomatis and Helicobacter
pylori have also been shown to export HtrA proteases into the host cell cytosol (C. trachomatis)
or extracellularly (H. pylori) (Hoy et al., 2010; Wu et al., 2011). In the case of
H. pylori, HpHtrA is proteolytically active against host E-cadherin which is important
for epithelial adherence junctions and barrier integrity. It has been proposed that
the proteolytic cleavage of E-cadherin by HpHtrA could disrupt the gastric epithelial
lining promoting colonization by H. pylori (Hoy et al., 2012). As data accumulate
on the HtrA family of proteases, it is becoming very clear that these enzymes are
pivotal to the overall physiology and virulence of a variety of bacterial pathogens.
Recent papers published in Molecular Microbiology present data that begin to shed
light on the role of an HtrA homolgue, BB0104 (BbHtrA), in the physiology and pathogenesis
of the Lyme disease agent, Borrelia burgdorferi (Coleman et al., 2013; Kariu et al.,
2013; Russell et al., 2013; Russell and Johnson, 2013). Lyme disease is characterized
by initial pathogen colonization of the dermis BbHtrA at the tick bite site, followed
by dissemination to secondary tissues and, in some cases, by clinical manifestations
such as neuroborreliosis and Lyme arthritis (LA). These reports outline a possible
role for BbHtrA in these processes. The first paper, by Kariu et al., suggests a potential
role for BbHtrA in the proteolytic processing of BB0323 (Kariu et al., 2013). BB0323
has been shown to be required for normal cell growth and division, and BB0323 mutants
are avirulent in mice (Zhang et al., 2009). Ostberg et al. have previously shown that
BB0323 is potentially processed by the carboxyl-terminal peptidase CtpA (Ostberg et
al., 2004). In that study, analyses of a CtpA mutant indicated that BB0323 is still
processed (from ∼46 to ∼32 kDa) but not to the 29 kDa protein observed in wild-type
cells (Ostberg et al., 2004). In the more recent publication by Kariu et al., data
presented suggest that BB0323 is also processed by purified, recombinant BbHtrA, into
fragments of 30 and 15 kDa but not to ∼27 kDa protein detected in wild-type cells.
Additionally, they provide data that indicate that the N-terminal domain of BB0323
is required for normal protein function while the C-terminal, LysM domain is required
for infectivity in mice (Kariu et al., 2013). Kariu et al. propose a model suggesting
that initial processing of BB0323 requires BbHtrA activity while the final processing
into enzymatically active polypeptides requires CtpA. However, it was not possible
to isolate a BbHtrA mutant to confirm their model.
In a very recent study, Coleman et al. took a biochemical approach to understanding
the function of BbHtrA in B. burgdorferi (Coleman et al., 2013). First, they showed
that BbHtrA forms a trimer in solution when no substrate is present consistent with
structural analyses originally done on the BbHtrA homologue, DegP from E. coli (Hansen
and Hilgenfeld, 2013). Second, they were able to demonstrate caseinolytic activity
that was both dependent on a conserved catalytic serine (S198) in the protease domain
and increased temperature. Third, they present data which show that BbHtrA localizes
to the soluble and membrane fractions, and, more importantly, that significant levels
of BbHtrA was secreted into the growth media. Previously, several research groups
have shown that in Gram-negative bacteria HtrA proteases localize primarily to the
periplasmic space where they perform their normal protease and chaperone functions.
Yet, in other bacteria, HtrA proteases localize not only to the periplasmic space
but also to cell membrane fractions, the cell surface and, in specific cases, also
to the extracellular milieu (Roberts et al., 2013). The mechanism by which these proteins
are secreted has not been determined and, as pointed out by the authors, B. burgdorferi
also has no obvious mechanism or secretory machinery to secrete BbHtrA from the cell.
Finally, using BbHtrA antibody, the authors used an immunoprecipitation enrichment
to identify proteins from B. burgdorferi that bound to BbHtrA. Several proteins including
outer surface protein A (OspA), outer surface protein B (OspB), basic membrane protein
D (BmpD), chemotaxis protein X (CheX), flagellar basal body protein (FilL), BB0365
(lpA7) and BB0690 (NapA) were enriched using this technique. Of these, BmpD and CheX
were proteolytically degraded by purified BbHtrA. Taken together, these data suggest
that BbHtrA functions as a chaperone and protease similar to other members of the
HtrA family and that it interacts with a subset of cellular proteins that are essential
for chemotaxis and motility. Like Kariu et al. these authors could not isolate an
BbHtrA mutant. Considering the putative substrates reported for BbHtrA in these two
reports, it seems likely that isolating an BbHtrA mutant is not possible.
Two new research articles published in this issue of Molecular Microbiology by Russell
and Johnson, and Russell et al. provide data that indicate that BbHtrA degrades an
interesting variety of host proteoglycans (Russell et al., 2013; Russell and Johnson,
2013). In their first paper, Russell and Johnson describe the binding and cleavage
of aggrecan, the most abundant proteoglycan in the extracellular matrix (ECM) of joint
and connective tissues. They first were able to demonstrate binding of aggrecan to
intact B. burgdorferi cells and, subsequently targeting two specific proteins that
were significantly enriched using aggrecan-affinity chromatography and mass spectrometry.
One protein, identified was BB0588 (a glycosaminoglycan binding protein designated
Bgp), which has been previously described (Parveen and Leong, 2000; Parveen et al.,
2003). The second protein was identified as BbHtrA (BB0104). Russell and Johnson then
demonstrated caseinolytic activity for BbHtrA similar to that described by Coleman
et al. Recombinant BbHtrA and BbHtrA S226A [the same serine residue describe above
(S198) but numbered based upon the full length protein] were purified and used in
protease assays against purified aggrecan. Like the human proteases (HtrA1, AT5 and
MMP2) that have been shown to degrade aggrecan, BbHtrA was able to proteolytically
cleave recombinant human and natural bovine aggrecan into three fragments with molecular
masses similar to those generated by the other proteases tested (Russell and Johnson,
2013). This suggested that BbHtrA yielded products from specific cleavage sites within
aggrecan near those previously described for the human proteases. To determine the
cleavage sites, Russell and Johnson isolated proteolytic fragments generated from
the incubation of purified BbHtrA with aggrecan and the putative sites were mapped
using monoclonal antibodies targeting the newly generated, exposed aggrecan neoepitopes
and identified by protein sequencing. These data indicate that BbHtrA cleaved aggrecan
within a small region of the interglobular domain at or near arginine-374. Importantly,
Hu et al. had previously detected fragments of aggrecan, which had been cleaved at
this same site in the synovial fluid of patients with LA (Hu et al., 2001). Hu et al.
and Behera et al. were also able to identify similar fragments generated by incubating
B. burgdorferi cells with human cartilage explants and show that this degradation
was not generated by human aggrecanases/proteases which proteolytically attack aggrecan
during inflammatory arthritis (Hu et al., 2001; Behera et al., 2006). As demonstrated
by Coleman et al., Russell and Johnson show that antibodies to BbHtrA are commonly
detected in patients with Lyme disease. These results indicate an important role for
BbHtrA in the pathogenesis of LA.
In their second paper, Russell et al. expand their characterization of BbHtrA by examining
in-depth the interaction of the protease with ECM components. Because of the homology
of BbHtrA with human HtrA1, the authors speculate that BbHtrA might have enzymatic
activity against other important structural components of the ECM in addition to aggrecan.
In vitro enzyme assays using purified BbHtrA provided direct evidence that proteoglycans
biglycan, decorin, neuorcan, brevican and versican were degraded by purified BbHtrA
(Russell et al., 2013). These proteoglycans are widely distributed in ECMs present
in various tissues, joints and the CNS (Wu et al., 2005; Frischknecht and Seidenbecher,
2008). Compromising the structural integrity of the ECM at various sites would facilitate
penetration and colonization by B. burgdorferi. Additionally, BbHtrA was able to degrade
E-cadherin, an important glycoprotein that promotes efficient epithelial cell adhesion.
Hoy et al. demonstrated that some bacterial pathogens are able to degrade E-cadherin
and they suggest that this degradative process promotes dissemination (Hoy et al.,
2012). Another glycoprotein which was susceptible to degradation by BbHtrA was fibronectin.
The degradation of fibronectin by purified BbHtrA yielded peptide fragments which
harboured fibronectin type III repeats 13 and 14 (FnIII13–14) and a N-terminal fragment
designated Fn-f 29. FnIII 13–14 have been shown to cause damage to connective tissue
by inducing human MMP protease and aggrecanases (Sofat et al., 2011) while Fn-f29
is pro-inflammatory (Su et al., 2005). Previous reports have shown that fibronectin
fragments generated by HtrA1 stimulated the production of numerous cytokines and chemokines
(Grau et al., 2005; Pulai et al., 2005; Austin et al., 2009; Tiaden et al., 2012).
Therefore, it seemed plausible to the authors that BbHtrA generated fragments of fibronectin
might be able to stimulate a similar response. Therefore purified BbHtrA was incubated
with ECM producing, cultured chondrocytes and indeed culture supernatant contained
increased levels of chemokines (CXCL1, CCL1, CCL2, CCL5 and IL-8) and cytokines (IL-6
and slCAM-1). Chemokine and cytokine levels have been shown to be elevated in the
epidermis of patients with erythema migrans, in the cerebrospinal fluid patients with
neuroborreliosis and in the synovial fluid of patients with LA (Grygorczuk et al.,
2005; Mullegger et al., 2007; Zhao et al., 2007; Strle et al., 2009). These results
thus suggest that BbHtrA, along with B. burgdorferi lipoproteins, contribute to the
pathology of neuroborreliosis and LA. However, these data should not be over interpreted
before BbHtrA's role in these manifestations of Lyme disease are confirmed in vivo.
The accumulating data published in Molecular Microbiology regarding BbHtrA demonstrate
the importance of this protein in the physiology and pathogenesis of B. burgdorferi
( Fig. 1). The protein is reported to be involved in the localization and processing
of putative virulence factors, such as BB0323 and BmpD (Coleman et al., 2013; Kariu
et al., 2013). Additionally, proteolytic activity of BbHtrA against aggrecan, a key
structural proteoglycan present in connective tissue and joints, suggests a direct
role in LA has been shown (Russell and Johnson, 2013). More importantly, Russell et al.
provide convincing data that HtrA is able to degrade fibronectin and various proteoglycans
found in joints, epidermis and neurological tissues. Most significant, purified recombinant
BbHtrA interacted with chondrocytes triggering the release of chemokines and pro-inflammatory
cytokines (Russell et al., 2013). These data indicate that HtrA is potentially important
in cell physiology (motility, chemotaxis), protein processing (BB0323), bacterial
dissemination (e.g. degradation of E-cadherin and aggrecan) and actively contributes
to release of inflammatory cytokines (IL-6 and slCAM-1) and chemokines (e.g. CXCL1,
CCL1, etc.) from cultured chondrocytes. It seems very clear from these data that BbHtrA
is critical in the physiology and pathogenesis of B. burgdorferi.
Fig 1
A model for the different activities linked to BbHtrA.