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Published ahead of print on December 20, 2007, doi:10.1165/rcmb.2007-0289OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 38, pp. 609-617, 2008
© 2008 American Thoracic Society
DOI: 10.1165/rcmb.2007-0289OC

Moraxella-Dependent {alpha}1-Antichymotrypsin Neutralization

A Unique Virulence Mechanism

Taras Manolov1, Thuan Tong Tan1,2, Arne Forsgren1 and Kristian Riesbeck1

1 Medical Microbiology, Department of Laboratory Medicine, Malmö University Hospital, Lund University, Malmö, Sweden; and 2 Department of Internal Medicine, Singapore General Hospital, Singapore

Correspondence and reprint requests should be addressed to Kristian Riesbeck, MD, PhD, Medical Microbiology, Department of Laboratory Medicine, Malmö University Hospital, Lund University, SE-205 02 Malmö, Sweden. E-mail: kristian.riesbeck{at}med.lu.se


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
The acute phase reactant and protease inhibitor {alpha}1-antichymotrypsin is considered to play a protective role in the airways, but whether it interacts with respiratory bacteria is not known. We analyzed whether the common respiratory pathogens Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and other bacterial species interact with antichymotrypsin. M. catarrhalis was the only species that bound antichymotrypsin among 25 bacterial species tested by flow cytometry and direct binding assay. We compared a series of clinical isolates in addition to wild-type and ubiquitous surface protein–deficient Moraxella to study the nature of antichymotrypsin binding by the bacteria. Experiments with Moraxella mutants revealed that ubiquitous surface proteins A1 and A2 were responsible for the interaction, and using recombinant fragments, a consensus sequence within ubiquitous surface proteins A1 and A2 was defined. Binding of iodine-labeled antichymotrypsin was dose dependent and strong (dissociation constant [Kd] 24.9–44.8 nM). Moreover, a chymotrypsin activity assay showed that antichymotrypsin, when bound to the bacterial surface, was neutralized. Moraxella antichymotrypsin neutralization is a novel microbial virulence mechanism that may induce excessive inflammation resulting in more exposed extracellular matrix that is beneficial for bacterial colonization.

Key Words: bacteria • inflammation • protease inhibitor



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Moraxella antichymotrypsin neutralization is a novel microbial virulence mechanism that may induce excessive inflammation, resulting in more exposed extracellular matrix that is beneficial for bacterial colonization.

 
{alpha}1-Antichymotrypsin is a serine protease inhibitor (serpin), and the primary target is considered to be neutrophil cathepsin G (1). After tissue damage, the plasma concentration of antichymotrypsin may double within 8 hours, and the protease inhibitor is therefore regarded as an acute phase reactant. Hepatocytes are the main source of antichymotrypsin, but bronchial epithelial cells have also been shown to produce high concentrations of antichymotrypsin upon stimulation (2). Cleavage of antichymotrypsin by proteases within the reactive-site loop results in appearance of chemoattractant activity for neutrophils (3). In contrast, native (free) antichymotrypsin has a suppressive effect on neutrophil chemotaxis (4). Both native and antichymotrypsin in complex with a particular protease are capable of inhibiting superoxide production by neutrophils (5). Moreover, antichymotrypsin is known to inhibit TNF-{alpha}–induced platelet-activating factor synthesis in neutrophils, macrophages, and endothelial cells (6). Antichymotrypsin can be considered as a key player in several inflammatory processes, protecting host tissues from proteolytic and oxidative damage. It is a well-known fact that inherited deficiency of {alpha}1-antitrypsin can cause emphysema (7). Interestingly, there are also indications that antichymotrypsin deficiencies may likewise be associated with a higher risk of developing chronic obstructive pulmonary disease (COPD), at least in certain populations (8).

In addition to nontypeable Haemophilus influenzae and Streptococcus pneumoniae, Moraxella catarrhalis is one of the most important bacterial causes of exacerbations in patients with COPD (9). Much research efforts have been directed at delineating the factors involved in the pathogenesis of M. catarrhalis infections. In particular, the interactions of major outer membrane proteins have been under intense scrutiny (10). Ubiquitous surface proteins (Usp) A1 and A2 are closely related and share significant homology in their central regions (11). These proteins are highly multifunctional and interact with epithelial cells through fibronectin and carcinoembryonic antigen-related cell adhesion molecule-1 (12). UspA1/A2 have also affinity for the basement membrane (13) and contribute to serum resistance (14).

In this article we wanted to examine whether respiratory pathogens can interact with antichymotrypsin. We demonstrate that M. catarrhalis, but not pneumococci or H. influenzae, binds antichymotrypsin. The interaction is mediated via the outer membrane proteins UspA1 and A2, and it results in the suppression of the enzyme inhibitory function of antichymotrypsin. Our findings suggest a novel and unique virulence mechanism of a human respiratory pathogen that may influence inflammatory events at a site of infection.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Strains and Reagents
See the online supplement for details.

Gel Electrophoresis and Detection of Proteins on Membranes
See the online supplement for details.

Flow Cytometry
See the online supplement for details.

Absorption of Plasma to M. catarrhalis and Elution of Bound Proteins
See the online supplement for details.

Protein Labeling, and Bacterial Binding of [125I]-Labeled ACT
See the online supplement for details.

Chymotrypsin Activity and Chymotrypsin Inhibition Assay
For analysis of the ACT interaction with recombinant UspA1/A2, 12.5 nM UspA1360–480 or UspA2200–480 was incubated with equimolar concentration of ACT in 0.1 M Tris-HCl, 0.96 M NaCl, 10 mM CaCl2, pH 8.3 buffer for 10 minutes at 25°C. Thereafter, equimolar amounts of chymotrypsin were added. After 10 minutes of incubation, the reaction mixture was pre-heated for 1 minute at 37°C, and 0.1 µM S-2586 chromogenic chymotrypsin substrate (Chromogenix, Orangeburg, NY) was added. The mixture was incubated for 10 minutes at 37°C and terminated by 20% acetic acid, followed by A405 determination.

To analyze the M. catarrhalis interaction with ACT, bacteria were washed with 50 mM Tris-HCl-buffered saline (pH 8.3), containing 10 mM of CaCl2. Bacteria (2 x 109) were resuspended in buffer containing 12.5 nM ACT and incubated for 10 minutes at 25°C. Thereafter, the bacteria were spun down. Supernatants were incubated with the chymotrypsin solution, followed by incubation with chromogenic substrate and determination of A405 as described above. The bacterial pellet was washed once and dispersed in the same buffer and equivalent amount of chymotrypsin was added. After incubation for 10 minutes at 25°C, bacteria were spun down and chymotrypsin activity was determined in the supernatant.

Enzyme-Linked Immunosorbent Assay
See the online supplement for details.


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Antichymotrypsin Binding Is a Unique Property of M. catarrhalis
Since antichymotrypsin has a role in protection against excessive airway inflammation (15), we wanted to determine whether three common respiratory pathogens interact with antichymotrypsin. Binding of antichymotrypsin by S. pneumoniae, H. influenzae, and M. catarrhalis was analyzed by flow cytometry. Interestingly, M. catarrhalis was able to efficiently bind antichymotrypsin, whereas the other two respiratory pathogens were not (Figure 1). To determine whether other bacteria also bound antichymotrypsin, we tested related Moraxella subspecies as well as several common human pathogens (including, e.g., Staphylococcus aureus, Streptococcus pyogenes, and Pseudomonas aeruginosa) for antichymotrypsin binding by flow cytometry and in an [125I]-labeled antichymotrypsin binding assay. Interestingly, M. catarrhalis was the only species that significantly bound antichymotrypsin among all the different bacterial species analyzed (Table 1).


Figure 1
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  		Figure 1. Moraxella catarrhalis binds {alpha}1-antichymotrypsin, whereas Streptococcus pneumoniae and Haemophilus influenzae do not. Clinical isolates were analyzed for antichymotrypsin binding by flow cytometry. (A) Antichymotrypsin binding by M. catarrhalis RH4 was set to 100%. Background fluorescence without antichymotrypsin was subtracted. Bacteria (5 x 107) were incubated with 10% human plasma followed by addition of a specific sheep anti-antichymotrypsin antiserum and a fluorescein isothiocyanate–conjugated donkey anti-sheep secondary pAb. Similar results were also obtained with purified antichymotrypsin (20 µg/ml) instead of plasma. One representative experiment out of two is shown. All strains were tested on two separate occasions with similar results. Error bars correspond to SEM. Representative flow cytometry profiles for M. catarrhalis (strain BBH18) (B), S. pneumoniae (strain S6-4950/95) (C), and finally nontypable H. influenzae (D) (strain 3655) are shown. Bacteria were incubated with 20 µg/ml antichymotrypsin as mentioned above. Background profiles are filled with black. Mean fluorescent intensities are indicated in the different panels and show raw data without the background subtracted.

 

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  		TABLE 1. M. catarrhalis IS A UNIQUE ANTICHYMOTRYPSIN-BINDING BACTERIUM

 
M. catarrhalis–Dependent Binding of Antichymotrypsin Is Related to UspA1/A2
Since UspA1 and A2 are multifunctional proteins and have been shown to interact with several host cell components (12, 14, 16–21), we chose to analyze antichymotrypsin binding to the clinical isolate M. catarrhalis RH4 (22) in addition to a corresponding mutant devoid of UspA1 and A2. Flow cytometry analyses revealed that M. catarrhalis RH4 bound antichymotrypsin (Figure 2A), whereas a UspA1/A2-deficient ({Delta}uspA1/A2) double mutant did not bind antichymotrypsin (Figure 2B). Similar results were obtained with another M. catarrhalis strain (BBH18) and its derived UspA1/A2 mutant (results not shown).


Figure 2
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  		Figure 2. Binding of antichymotrypsin to M. catarrhalis depends on ubiquitous surface proteins A1 and A2. (A) M. catarrhalis RH4 wild type and (B) the corresponding double mutant {Delta}uspA1/A2 were analyzed for antichymotrypsin binding by flow cytometry. Bacteria (5 x 107) were incubated with 10% plasma as described in Figure 1. Shaded profiles represent background fluorescence without plasma, but with all antibodies supplemented. Profiles from one representative experiment out of three are shown. Similar results were obtained with plasma and purified antichymotrypsin (20 µg/ml). Experiments with M. catarrhalis BBH18 and the corresponding {Delta}uspA1/A2 double mutant showed results to those seen with M. catarrhalis RH4. (C) Bound antichymotrypsin can also be eluted from UspA1/A2-expressing M. catarrhalis surface. Bacteria (109) were incubated with plasma and washed. Thereafter, bound plasma proteins were eluted under acidic conditions. Resulting eluates were run in SDS-PAGE (5-µl aliquots each) and analyzed by Western blot using an anti-antichymotrypsin pAb. For comparison, E. coli BL21 was included as a negative control. Purified antichymotrypsin (0.5 µg per lane) and human plasma (5 µl of plasma diluted 1:3 in PBS) were included as positive controls. (D) Clinical M. catarrhalis isolates (n = 14) were analyzed for antichymotrypsin binding and UspA1/A2 expression. Solid and open circles represent wild type and UspA1/A2 mutant bacteria, respectively. Bacteria were incubated with antichymotrypsin, followed by detection with a donkey anti-antichymotrypsin antiserum and flow cytometry analysis. In parallel, M. catarrhalis UspA1/A2 expression was analyzed using a rabbit anti-UspA1/A2 antiserum. Control experiments without antichymotrypsin or without anti-UspA1/A2 amtiserum were run for each strain or mutant, and background fluorescences were subtracted accordingly.

 
To confirm that UspA1/A2 were the responsible outer membrane proteins binding antichymotrypsin, human plasma was incubated with the M. catarrhalis RH4 and the corresponding UspA1/A2 mutant, followed by acidic elution and Western blot analysis. The wild-type strain bound antichymotrypsin from human plasma, whereas the {Delta}uspA1/A2 double mutant did not (Figure 2C). To exclude the possibility that the UspA1/A2-dependent antichymotrypsin binding was not merely a function of a single isolate, we selected a random series of clinical and collection M. catarrhalis strains and tested them for binding of purified antichymotrypsin. In parallel, the same strains were analyzed for relative UspA1/A2 expression, and it was found to correlate well with the antichymotrypsin binding (Figure 2D). Taken together, these data suggest that UspA1/A2 are solely responsible for antichymotrypsin binding of M. catarrhalis, and the amount of bound antichymotrypsin correlated well with the UspA1/A2 expression.

To further investigate the antichymotrypsin binding property of M. catarrhalis, we incubated bacteria with increasing concentrations of [125I]-antichymotrypsin. M. catarrhalis–bound radiolabeled antichymotrypsin in a dose-dependent and saturable manner (Figure 3). To obtain information about the binding strength and capacity of the M. catarrhalis/antichymotrypsin interaction, we chose to use a hyperbolic curve, representing a one site ligand binding model. The quality of the fit was good for the BBH18 strain (R2 = 0.99) and less so for RH4 (R2 = 0.86). The M. catarrhalis BBH18 strain bound slightly more than strain RH4, namely 1.07 x 1012 ± 2.19 x 1010 (best fit ± SEM) against 6.46 x 1011± 3.75 x 1010 (best fit ± SEM) antichymotrypsin molecules. Dissociation constants were estimated to 44.8 ± 4.5 nM and 24.9 ± 4.5 nM, respectively (values presented as best fit ± SEM).


Figure 3
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  		Figure 3. Antichymotrypsin is bound to UspA1/A2-expressing M. catarrhalis in a dose-dependent and saturable manner. M. catarrhalis BBH18 wild type and double UspA1/A2 mutant (5 x 107) were incubated with increasing concentrations of [125I]- antichymotrypsin, washed, and followed by analysis of bound [125I]-antichymotrypsin. Each concentration was tested in duplicate. Mean binding by the UspA1/A2 mutant was regarded as background and was subtracted from mean antichymotrypsin binding by M. catarrhalis for each concentration. Data shown are from two independent experiments; the one-site binding model curve is fit with the help of GraphPad software. Insert: binding of antichymotrypsin to M. catarrhalis RH4. Data from one representative experiment out of two are shown. Each concentration was tested in triplicate, and background binding to empty tubes was subtracted for all concentrations.

 
To analyze whether the interaction between UspA1/A2 and antichymotrypsin is specific to ACT or serpins in general, three typical serpins (human {alpha}1-antitrypsin, human antithrombin, and chicken ovalbumin) were also tested for UspA1/A2-dependent binding to the bacteria. M. catarrhalis BBH18 and the corresponding UspA1/A2 mutant was incubated with 5% plasma or 0.25 mg/ml chicken ovalbumin and tested for serpin binding by flow cytometry. The fluorescence of the M. catarrhalis {Delta}uspA1/A2 mutant was subtracted from the data obtained with the M. catarrhalis wild type. None of the three other serpins bound more than 11% as compared with the binding of antichymotrypsin, which was set as 100% (results not shown). Thus, we cannot fully exclude the possibility that M. catarrhalis UspA1/A2 may bind other serpins than antichymotrypsin, although these interactions are at a considerably lower level.

Bacterial Interaction with Antichymotrypsin from Bronchoalveolar Lavage
M. catarrhalis is commonly isolated from the lower respiratory tract of patients with COPD. To analyze whether M. catarrhalis UspA1/A2 can bind antichymotrypsin obtained from the lower respiratory tract, bronchoalveolar lavage (BAL) from patients with clinically diagnosed pneumonia was tested for antichymotrypsin content by enzyme-linked immunosorbent assay (ELISA) (Figure 4A). Four representative samples were selected corresponding to different concentrations of antichymotrypsin. M. catarrhalis wild type and the corresponding double UspA1/A2 mutant were incubated with BAL at physiological conditions for 1 hour at 37°C and analyzed by flow cytometry. In parallel with the results obtained with human plasma and purified antichymotrypsin (Figures 1 and 2), M. catarrhalis was able to bind antichymotrypsin also from BAL, whereas the M. catarrhalis {Delta}uspA1/A2 mutant did not bind. The level of antichymotrypsin binding (expressed as mean fluorescence intensity) in flow cytometry was related to the antichymotrypsin concentration in BAL (Figure 4). Thus, M. catarrhalis UspA1/A2 binds both antichymotrypsin originating from plasma and the lower respiratory tract.


Figure 4
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  		Figure 4. M. catarrhalis binds antichymotrypsin present in bronchoalveolar lavage (BAL). (A) Four BAL samples with high and low concentrations of antichymotrypsin as determined by enzyme-linked immunosorbent assay (ELISA) were randomly selected for incubation with M. catarrhalis BBH18 wild type and the corresponding UspA1/A2 mutant. Solid bars represent antichymotrypsin concentrations, shaded bars show antichymotrypsin binding by the M. catarrhalis wild type, and open bars correspond to the binding of antichymotrypsin by the UspA1/A2 mutant. In B and C, representative flow cytometry profiles are shown for the M. catarrhalis BBH18 wild type and the corresponding UspA1/A2 mutant incubated with BAL1; D and E correspond to BAL2. In ELISA, monoclonal anti-antichymotrypsin Ab were coated on microtiter plates (10 µg/ml), blocked with BSA-PBS, and incubated with BAL or an antichymotrypsin calibration solution. Bound antichymotrypsin was detected with unconjugated polyclonal sheep anti-antichymotrypsin antibodies and donkey horseradish peroxidase (HRP)-conjugated anti-sheep antibodies. For flow cytometry experiments, bacteria were incubated with 10% BAL for 1 hour at 37°C with subsequent addition of anti-antichymotrypsin pAb and secondary fluorescent antibodies followed by flow cytometry analysis.

 
Antichymotrypsin Is Inactivated when Bound to the M. catarrhalis Surface
To characterize in detail the bacterial binding of antichymotrypsin, the M. catarrhalis wild type and the UspA1/A2 deficient mutant were preincubated with antichymotrypsin and spun down. The supernatants were collected and tested for antichymotrypsin activity by incubation with chymotrypsin, followed by a chymotrypsin activity assay as readout. As shown in Figure 5A, M. catarrhalis wild type efficiently removed most of the antichymotrypsin (i.e., chymotrypsin was still active). In contrast, the mutant M. catarrhalis {Delta}uspA1/A2 did not absorb any antichymotrypsin from the supernatant, resulting in a preserved antichymotrypsin-dependent inhibition of chymotrypsin.


Figure 5
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  		Figure 5. Antichymotrypsin is efficiently absorbed by M. catarrhalis and does not inhibit chymotrypsin when bound to the M. catarrhalis cell surface. In A, M. catarrhalis BBH18 wild type and the corresponding UspA1/A2 mutant were incubated with antichymotrypsin and spun down. Supernatants were incubated with chymotrypsin, and chymotrypsin activity was determined by a chromogenic substrate. Chymotrypsin and antichymotrypsin-inhibited chymotrypsin were included as positive and negative controls, respectively. The uninhibited chymotrypsin activity was set to 100%. Means of two separate experiments is shown, each run in triplicates. Error bars indicate SD. In B, bacteria were treated with antichymotrypsin, washed, and incubated with chymotrypsin, followed by centrifugation. Chymotrypsin activity was determined in the supernatant. The mean values of two experiments are shown, each run in triplicate. Error bars indicate SD.

 
Since antichymotrypsin was bound to M. catarrhalis, the next question was whether it could inhibit chymotrypsin at the bacterial surface. Bacteria were pretreated with antichymotrypsin, followed by incubation with equivalent amounts of chymotrypsin. Thereafter, the bacteria were spun down and chymotrypsin activity was analyzed in the supernatants (Figure 5B). No significant difference in chymotrypsin activity was found between antichymotrypsin -pretreated M. catarrhalis wild type or the UspA1/A2 mutant strain. These experiments suggested that antichymotrypsin did not inhibit chymotrypsin when bound to the bacterial surface.

Host proteins can potentially affect growth of bacterial pathogens. To exclude the possibility that antichymotrypsin would interfere with M. catarrhalis, bacterial survival was studied after incubation with 1 µM antichymotrypsin for up to 4 hours. When compared with control bacteria without added antichymotrypsin, neither survival of the M. catarrhalis wild type nor of the UspA1/A2 mutant was affected after incubation with antichymotrypsin (results not shown).

Both UspA1 and A2 Have Two Separate Domains, which Are Responsible for M. catarrhalis–Dependent Antichymotrypsin Binding
To pinpoint the antichymotrypsin-binding site within the UspA1 and UspA2 molecules, antichymotrypsin binding was studied in ELISA using truncated recombinant proteins spanning UspA150–770 and UspA230–539 (Figures 6A and 6B). Interestingly, two regions in both UspA1 and UspA2 appeared to be important for the interaction with antichymotrypsin. These sequences were localized within UspA1299–452 and UspA1557–704, and within UspA2101–318 and UspA2302–458. When the protein sequences of the antichymotrypsin binding regions of the UspA1 and A2 were aligned, we found that they shared a single highly homologous 32–amino acid sequence (Figure 6C). The two conserved repetitive amino acid sequences found in UspA1/A2 of several different M. catarrhalis strains are included in this sequence (23).


Figure 6
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  		Figure 6. Two antichymotrypsin binding sites exist in both M. catarrhalis UspA1 and A2. Antichymotrypsin binding was studied in ELISA using recombinant truncated proteins spanning UspA150–770 (A) and UspA230–539 (B). A series of truncated UspA1 and UspA2 proteins were coated on microtiter plates and incubated with antichymotrypsin, followed by incubation with sheep anti-human antichymotrypsin and HRP-conjugated donkey anti-sheep/goat pAbs. The mean values of three separate experiments are shown. Error bars correspond to SD. (C) A conserved antichymotrypsin-binding repeat exists in UspA1 and A2. M. catarrhalis UspA1 and A2 peptide sequences with significant antichymotrypsin binding capacity have highly similar amino acid motifs. The homology between peptides is shown in the bottom line; positions where the sequences are identical are shown by the consensus amino acid, and positions where conservative replacements exist are shown by a colon (:). CLUSTAL W software was used for alignment of protein sequences (http://workbench.sdsc.edu/) (60). In (D), synthetic UspA2244–275 and recombinant truncated UspA2101–318 containing the consensus sequence were coated on microtiter plates and analyzed for antichymotrypsin binding by ELISA as described in the legend to Figure 5. An unrelated control peptide—vitronectin341–370 (APRPSLAKKQRFRHRNRKGYRSQRGHSRGR) (61)—was used to asess the unspecific binding to a short peptide. The mean values of three separate experiments are shown. Error bars correspond to SD.

 
To investigate whether the homologous sequences within the antichymotrypsin-binding regions of UspA1/A2 (Figure 6C) were responsible for the antichymotrypsin binding, a synthetic peptide corresponding to amino acids 244–275 of UspA2 was made and tested for antichymotrypsin binding in ELISA. UspA2244–275 bound antichymotrypsin, although the binding efficacy was approximately 3.5-fold lower as compared with recombinant truncated UspA2101–318 (Figure 6D). The lower antichymotrypsin binding by the UspA2244–275 peptide may be explained by the absence of the correct folding and tertiary structure as has been shown with, for example, streptococcal M protein fragments (24).

Recombinant UspA1 and A2 Inactivate Antichymotrypsin In Vitro
Since both UspA1360–680 and UspA2200–480 contain two homologous repeats, which are found in the regions of UspA1/A2 that are responsible for antichymotrypsin binding (Figure 6C), these recombinant proteins were selected for analysis of the interaction in a cell-free system. Recombinant UspA1360–680 and UspA2200–480 were incubated at equimolar concentrations with antichymotrypsin before determination of the antichymotrypsin inhibitory activity. As demonstrated in Figure 7, recombinant UspA1360–680 and UspA2200–480 inactivated antichymotrypsin in vitro (i.e., chymotrypsin was still active after incubation with antichymotrypsin that had been pretreated with UspA1/A2). Thus, these data support that M. catarrhalis binds and inactivates antichymotrypsin via UspA1/A2, as measured by the means of chymotrypsin enzymatic activity.


Figure 7
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  		Figure 7. UspA1 and A2 inactivate antichymotrypsin in vitro. Antichymotrypsin was preincubated with equimolar concentrations of recombinant UspA1360–480 or UspA2200–480 before incubation with an equimolar concentration of chymotrypsin. Thereafter, chymotrypsin activity was determined. Recombinant UspA1/A2 fragments without antichymotrypsin did not have any chymotrypsin activity, nor did they inhibit chymotrypsin (results not shown). Positive and negative chymotrypsin activity controls are defined as in the legend to Figure 6. One representative experiment out of two is shown with the mean values of three independent samples. Error bars indicate SD.

 

   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
In the present article we demonstrate a novel and potentially important interaction between the serine protease inhibitor {alpha}1-antichymotrypsin and the significant human respiratory pathogen Moraxella catarrhalis. The enzyme inhibitor antichymotrypsin is mainly active against host enzymes, and is thus not directly related to antimicrobial defense but part of a regulatory system to protect the host from excessive inflammation and in particular neutrophil activity to avoid damage of the tissue (25). The Moraxella-dependent binding of antichymotrypsin is unique (Figure 1), and to our knowledge no other microbe has been described to bind and neutralize antichymotrypsin.

Antichymotrypsin inhibits the respiratory burst of neutrophils (5) and if antichymotrypsin is degraded and consequently removed from the focal infection site, the suppression of neutrophil superoxide production will be absent. This might lead to an uncontrolled respiratory burst of neutrophils that will damage redox-sensitive components of host tissue cells as well as extracellular proteins (e.g., other serum proteinase inhibitors, complement factors, or extracellular matrix components), hence further increasing the inflammatory process. Native antichymotrypsin is able to partially inhibit chemotaxis of neutrophils to a variety of chemoattractants (4). The protease inhibitor also inhibits cathepsin G–dependent chemotaxis of mononuclear cells (26). In addition to an increased respiratory burst, the absence of native antichymotrypsin may increase the influx of neutrophils as well as monocytes that might lead to an increased probability of tissue damage.

Proteases are well-known virulence factors of bacteria. These enzymes disrupt the host tissue structure and provide bacteria with soluble substrates and extracellular matrix proteins for adhesion. In addition, proteases inactivate the host defense system. The ability of M. catarrhalis to bind and neutralize antichymotrypsin functions similarly to production of an endogenous enzyme, except that in the present case the host cells synthesize the protease (i.e., chymotrypsin), which would be beneficial for the bacterium. An array of pathogens, for example, Legionella pneumophila, Pseudomonas aeruginosa, Bacillus subtilis, and Porphyromonas gingivalis can inactivate antichymotrypsin by endogenously produced proteases (27–30), suggesting that inactivation of antichymotrypsin by microbes is an important virulence mechanism. Previous studies have shown that some of the bacterial proteases specifically degrade antichymotrypsin, resulting mostly in one or two breakdown products. The capability of bacterial pathogens to enzymatically destroy antichymotrypsin, and the specificity of the proteolysis, suggests that this property of microbes has evolved particularly to counteract the antichymotrypsin at a site of infection.

The binding of antichymotrypsin by M. catarrhalis seems to be unique, but other bacteria can interact with other proteinase inhibitors. Secretory leukocyte proteinase inhibitor (SLPI) was shown to be bound and inactivated by Streptococcus pyogenes inhibitor of complement (SIC) (31), a clearly beneficial event for the SLPI-susceptible bacterium. Another proteinase inhibitor, {alpha}2-macroglobulin, is bound by S. pyogenes while retaining its activity (32), which also benefits S. pyogenes, especially in skin infections (33). It is known that Yersinia enterocolitica can bind {alpha}1-antitrypsin through the YopM protein, which does not affect the inhibitory activity of the serpin (34). {alpha}1-Antitrypsin can also interact with Escherichia coli–secreted proteins B and D and inhibit the formation of the translocation pore, and thus interferes with the type III secretion apparatus (35), which in this particular case appears to be beneficial for the host.

The exact mechanism of antichymotrypsin binding to M. catarrhalis is not known. When bound to M. catarrhalis or recombinant UspA1/A2, the complexed antichymotrypsin is not active as a proteinase inhibitor. It can then be assumed that the active site of the serpin is responsible for, or sterically blocked by, the interaction. Antichymotrypsin does not readily dissociate from Sepharose-bound recombinant truncated UspA1 at a high salt concentration (1.5 M NaCl) (Manolov T, Forsgren A, Riesbeck K, unpublished data). This allows us to suggest that ionic forces most likely do not play any important role in the interaction. Analysis of the amino acid content of common motifs known for antichymotrypsin binding support this hypothesis. It is evident that the charged residues are relatively less conserved and are substituted with uncharged polar residues. Therefore, differences in charge between the motifs make it unlikely that ionic forces are responsible for the binding. Uncharged polar residues also vary between the motifs, making hydrogen bonds less likely. Hydrophobic residues, on the other hand, appear to be more conserved, making the hydrophobic interactions more feasible as a responsible mechanism for the antichymotrypsin binding. Further experimental work is, however, necessary to address this issue in detail.

M. catarrhalis binds antichymotrypsin via the outer membrane proteins UspA1 and A2, which also have been shown to interact with several important host proteins (12, 14, 16–21). UspA1/A2 share various repetitive amino acid sequences that are conserved among different M. catarrhalis strains (23). The conservation of these amino acid sequences may be the evidence of importance and ubiquity of this property for M. catarrhalis. Since M. catarrhalis is sometimes a commensal while at other times clearly a pathogen (36–40), it can be envisaged that the UspA1/A2 are opportunistic virulent factors with respect to antichymotrypsin. A prior viral infection might set off the inflammatory cascade that M. catarrhalis will further promote (via inhibition of antichymotrypsin), as it will enhance the milieu in which it can thrive more successfully (i.e., more extracellular matrix ligands would be available). The interaction may be of little significance at an intact epithelial surface without inflammation at times when M. catarrhalis is just a commensal.

Cathepsin G, which is the primary target of antichymotrypsin, is not needed for antibacterial resistance against Staphylococcus aureus, Klebsiella pneumoniae, and E. coli in mice (41). Survival of cathepsin G–deficient mice was not different from the wild type after intraperitoneal injection with bacteria, although there was a (not statistically significant) trend toward less deaths of cathepsin G mutant mice that were inoculated with S. aureus or K. pneumoniae. On the other hand, cathepsin G negatively interferes with bacterial clearance in a murine model of P. aeruginosa lung infection—that is, cathepsin G contributes to an increased bacterial burden in wild-type mice (42). The various results could perhaps be explained by different approaches and experimental settings; also, the role of proteinase inhibitors in lung infection may be more important than in peritoneal infections. The significance of neutrophil proteinases as a negative factor in anti-bacterial immunity is supported by reports about neutrophil elastase interfering with bacterial clearance (43, 44). Although cathepsin G was found to be necessary in protection against fungi (45), it may reflect different susceptibility to proteolytic lysis inside phagolysocomes between bacteria and fungal spores (46). Thus, unregulated cathepsin G may thus not only provide M. catarrhalis with damaged extracellular matrix for attachment and colonization, but may also reduce the removal of bacteria from the respiratory tract.

The pathogenesis of chronic obstructive pulmonary disease (COPD) involves an interaction of a range of agents and factors. These include cigarette smoke, viral and bacterial pathogens, inflammatory mediators, and last but not least the balance between anti-proteases and proteases (47–50). In fact, {alpha}l-antitrypsin deficiency is one of the genetic factors that has been linked to COPD (7, 51). Single-nucleotide polymorphisms in a gene, encoding another member of the serine protease inhibitors family, SERPINE2, were recently shown also to be associated with COPD (52). Mutations in the antichymotrypsin-encoding gene were also linked in some studies with an increased risk for developing COPD. The importance of different polymorphisms, however, appears to be dependent on the particular population (8, 53–55).

The importance of the novel interaction of M. catarrhalis described in this article depends on the possibility that the antichymotrypsin concentration is limited in situ. It is widely accepted that {alpha}1-antitrypsin deficiency causes emphysema and in rare cases is linked to the disease progress of patients with COPD. There might be a possibility that this is also valid for antiproteases in general. Apart from decreased {alpha}1-antitrypsin levels due to gene polymorphisms, {alpha}1-antitrypsin is sensitive for oxidation (56). Although speculative, this fact regarding {alpha}1-antitrypsin may suggest that oxidation is sufficient for disturbing the balance between the antiproteinase and the corresponding target enzyme. Therefore, unregulated neutrophil elastase may cleave antichymotrypsin (25). In addition, it has been shown that antichymotrypsin may exist in lungs of patients with COPD in an inactive "latent" form (57), and it has been demostrated that proteinase inhibitors in BAL fluid do not control cathepsin G as efficiently as neutrophil elastase (58). Interestingly, when the release of single neutrophil granules was analyzed, it was found that secreted neutrophil elastase can greatly overwhelm its inhibitor {alpha}1-antitrypsin in the pericellular space before diffusion decreases the concentration of the enzyme (59). A similar mechanism may occur for antichymotrypsin and cathepsin G, suggesting that even slight decrease in antichymotrypsin concentration in the immediate vicinity of M. catarrhalis may increase probability of proteolytic damage.

Among the bacterial agents contributing to exacerbations and progression of COPD, M. catarrhalis has been recognized as an important pathogen. The finding of a neutrophilic inflammatory response in the airways of patients with COPD during exacerbations with M. catarrhalis supports its role in the pathogenesis (48). The novel finding reported in this article, on the interaction with M. catarrhalis UspA1/A2 that inactivates the protease inhibitor antichymotrypsin, might explain how M. catarrhalis contributes to COPD by causing an increased inflammation during exacerbations. While speculative, the tipping of the balance toward an increased protease activity because of the specific antichymotrypsin inactivation may account for the success of M. catarrhalis in the pathogenesis of COPD. Further studies are warranted to in detail monitor the inflammatory response during exacerbations caused by bacteria in patients with COPD.


   Footnotes
 
This work was supported by grants from the Alfred Österlund, the Anna and Edwin Berger, the Claes Groschinsky, the Crafoord, and the Greta and Johan Kock Foundations, the Swedish Medical Research Council, the Swedish Society of Medicine, and the Cancer Foundation at the University Hospital in Malmö.

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1165/rcmb.2007-0289OC on December 20, 2007

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form July 28, 2007

Accepted in final form November 14, 2007


   References
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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