Antibacterial activity of antileukoprotease.

Abstract

Antileukoprotease (ALP), or secretory leukocyte proteinase inhibitor, is an endogenous inhibitor of serine proteinases that is present in various external secretions. ALP, one of the major inhibitors of serine proteinases present in the human lung, is a potent reversible inhibitor of elastase and, to a lesser extent, of cathepsin G. In equine neutrophils, an antimicrobial polypeptide that has some of the characteristics of ALP has been identified (M. A. Couto, S. S. L. Harwig, J. S. Cullor, J. P. Hughes, and R. I. Lehrer, Infect. Immun. 60:5042-5047, 1992). This report, together with the cationic nature of ALP, led us to investigate the antimicrobial activity of ALP. ALP was shown to display marked in vitro antibacterial activity against Escherichia coli and Staphylococcus aureus. On a molar basis, the activity of ALP was lower than that of two other cationic antimicrobial polypeptides, lysozyme and defensin. ALP comprises two homologous domains: its proteinase-inhibitory activities are known to be located in the second COOH-terminal domain, and the function of its first NH2-terminal domain is largely unknown. Incubation of intact ALP or its isolated first domain with E. coli or S. aureus resulted in killing of these bacteria, whereas its second domain displayed very little antibacterial activity. Together these data suggest a putative antimicrobial role for the first domain of ALP and indicate that its antimicrobial activity may equip ALP to contribute to host defense against infection.

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INFECTION AND IMMUNITY, Nov. 1996, p. 4520–4524
0019-9567/96/$04.00?0
Copyright ? 1996, American Society for Microbiology
Vol. 64, No. 11
Antibacterial Activity of Antileukoprotease
P. S. HIEMSTRA,1* R. J. MAASSEN,1† J. STOLK,1R. HEINZEL-WIELAND,2
G. J. STEFFENS,2AND J. H. DIJKMAN1
Department of Pulmonology, Leiden University Hospital, Leiden, The Netherlands,1and
Department of Molecular Biology, Gru ¨nenthal GmbH, Aachen, Germany2
Received 3 October 1995/Returned for modification 10 November 1995/Accepted 15 August 1996
Antileukoprotease (ALP), or secretory leukocyte proteinase inhibitor, is an endogenous inhibitor of serine
proteinases that is present in various external secretions. ALP, one of the major inhibitors of serine proteinases
present in the human lung, is a potent reversible inhibitor of elastase and, to a lesser extent, of cathepsin G.
In equine neutrophils, an antimicrobial polypeptide that has some of the characteristics of ALP has been iden-
tified (M. A. Couto, S. S. L. Harwig, J. S. Cullor, J. P. Hughes, and R. I. Lehrer, Infect. Immun. 60:5042–5047,
1992). This report, together with the cationic nature of ALP, led us to investigate the antimicrobial activity of
ALP. ALP was shown to display marked in vitro antibacterial activity against Escherichia coli and Staphylo-
coccus aureus. On a molar basis, the activity of ALP was lower than that of two other cationic antimicrobial poly-
peptides, lysozyme and defensin. ALP comprises two homologous domains: its proteinase-inhibitory activities
are known to be located in the second COOH-terminal domain, and the function of its first NH2-terminal
domain is largely unknown. Incubation of intact ALP or its isolated first domain with E. coli or S. aureus
resulted in killing of these bacteria, whereas its second domain displayed very little antibacterial activity.
Together these data suggest a putative antimicrobial role for the first domain of ALP and indicate that its
antimicrobial activity may equip ALP to contribute to host defense against infection.
Proteinase inhibitors are thought to play an important role
in the regulation of the extracellular action of proteinases, such
as the serine proteinase human leukocyte elastase, that are
released from stimulated neutrophils. Elastase can cause ex-
tensive tissue degradation and has been shown to be involved
in several diseases, including pulmonary emphysema. Protein-
ase inhibitors involved in protecting the lung against the action
of elastase include ?1-proteinase inhibitor, which is produced
mainly in the liver and reaches the lung by passive diffusion
(35), and two locally produced inhibitors, elafin (also known as
skin-derived antileukoproteinase) (13, 31, 38) and antileuko-
protease (ALP) (14). ALP, also known as secretory leukocyte
proteinase inhibitor, is an effective inhibitor of human leuko-
cyte elastase and to a lesser extent of cathepsin G. It is a low-
molecular-weight (Mr, 11,700) cationic nonglycosylated mole-
cule that is composed of two highly homologous domains (33);
the second COOH-terminal domain contains its proteinase
inhibitory site, whereas the function of the first NH2-terminal
domain is largely unknown (7, 16, 26, 37). A recent report
demonstrated that the NH2-terminal domain may aid in stabi-
lizing the elastase-ALP complex and may mediate the en-
hancement of the antiproteinase activity of ALP by heparin
(39). ALP is produced locally in bronchi by serous cells in the
submucosal glands and by Clara and goblet cells of the bron-
chiolar and bronchial lining epithelium (17). Because it is
present in different parts of the airways, ALP is thought to play
an important role in maintaining the proteinase-antiproteinase
balance in the central and possibly also the lower airways (11).
Both domains of ALP contain a characteristic pattern of
cysteine residues; this pattern, known as the four-disulfide core
(FDC), is also found in a variety of other molecules with
miscellaneous functions (6, 33). Collectively this group of pro-
teins is often referred to as the FDC family of proteins. Re-
cently a novel antimicrobial polypeptide from equine neutro-
phils, designated eNAP-2, was identified and shown to be
structurally homologous to members of the FDC protein fam-
ily (3). In addition, eNAP-2 displayed antiproteinase activity
against microbial serine proteinases but not against mamma-
lian proteinases (4). Given this homology of ALP with eNAP-2
and the cationic nature of ALP, we investigated the antibac-
terial activity of ALP.
MATERIALS AND METHODS
Proteins. Recombinant ALP was produced in Escherichia coli and purified as
previously described (29). The separated NH2- and COOH-terminal domains
were obtained by partial acidic hydrolysis. Briefly, desalted ALP (ca. 15 to 20
mg/ml) was incubated in 50% (vol/vol) acetic acid in a water bath at 90?C for 1 h.
The mixture was cooled on ice and then slowly titrated to pH 4.5 with 10 N
NaOH. This solution was then passed over a column of octyl-Sepharose (Phar-
macia LKB Biotechnology, Uppsala, Sweden) equilibrated in 3 M NaCl. The
NH2-terminal domain (domain 1) did not bind to the column and was recovered
from the fall-through. The COOH-terminal domain (domain 2) bound to the
matrix and was eluted with 50 mM sodium acetate (pH 4.5). Both fractions were
desalted by diafiltration using an ultrafiltration cell filled with a membrane with
a nominal cutoff of 5 kDa. They were further purified by cation-exchange chro-
matography on carboxymethyl-Sepharose (Pharmacia LKB Biotechnology), us-
ing 100 mM sodium acetate (pH 4.5) as the starting buffer, and eluted with a
linear gradient of NaCl in the same buffer.
Defensins were purified from an acetic acid extract of purulent sputum.
Briefly, 40 ml of purulent sputum was sonicated, 30% (vol/vol) acetic acid was
added to a final concentration of 20%, and the material was extracted by over-
night incubation at 4?C. Insoluble material was removed by centrifugation for 20
min at 27,000 ? g; the cleared supernatant was dialyzed overnight against 5%
acetic acid in Spectrapor 3 tubing (Spectrum Medical Instruments Inc., Los
Angeles, Calif.) and concentrated to 10 ml by vacuum centrifugation (Speed-
Vac; Savant Instruments Inc., Hicksville, N.Y.). The concentrated acetic acid
sputum extract was fractionated by gel filtration on a Sephacryl S-200 column
(2.5 by 90 cm; Pharmacia) that had been equilibrated in 5% acetic acid. The
fractions were analyzed for protein content by the bicinchonic acid method
(Pierce, Rockford, Ill.) and for lysozyme activity (32) and antibacterial activity
against E. coli ML-35p (kindly provided by R. I. Lehrer, UCLA School of
Medicine, Los Angeles, Calif.) by using a radial diffusion assay (20). The low-
molecular-weight fractions that eluted after lysozyme and displayed antibacterial
activity against E. coli ML-35p were analyzed by Tricine sodium dodecyl sulfate
(SDS)-polyacrylamide gel electrophoresis (PAGE) (reducing conditions [30])
* Corresponding author. Mailing address: Department of Pulmonol-
ogy, Building 1, C3-P, Leiden University Hospital, P.O. Box 9600, 2300
RC Leiden, The Netherlands. Phone: 31-71-5262950. Fax: 31-71-
5248118.
† Deceased.
4520

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and acid urea-PAGE (20); in addition, their reactivity with a rabbit antiserum
raised against human defensins (a generous gift from T. Ganz, UCLA School of
Medicine) was determined. This analysis demonstrated that these fractions con-
sisted of a mixture of the human defensins HNP-1, HNP-2, and HNP-3.
Human leukocyte elastase was isolated from purulent sputum as previously
described (8, 15), and lysozyme from human colostrum was purchased from
Sigma (St. Louis, Mo.).
Inhibitory activity of intact ALP and its domains against human leukocyte
elastase. Increasing amounts of ALP and its fragments were incubated with 8 nM
elastase in 0.1 M Tris-HCl–0.2 M NaCl–0.05% (vol/vol) Triton X-100 (pH 7.8)
for 15 min at 25?C. Residual elastase activity was determined by using 0.33 mM
methoxysuccinyl-alanyl-alanyl-prolyl-valine p-nitroanilide, and hydrolysis of this
substrate was monitored at 405 nm. Kinetic constants were calculated as de-
scribed previously (1).
Antibacterial assays. The antibacterial activity of the proteins was investigated
by using logarithmic-phase E. coli ML-35p (18) and Staphylococcus aureus 42D
(kindly provided by R. van Furth, Leiden University Hospital, Leiden, The
Netherlands) (21). To obtain bacteria in the mid-logarithmic phase, 50 to 200 ?l
of an overnight culture made in tryptic soy broth (TSB; Difco Laboratories,
Detroit, Mich.) was added to 50 ml of TSB and incubated for 2.5 h at 37?C with
shaking. The bacteria were then washed in 10 mM sodium phosphate buffer
(NAPB; pH 7.4), and their concentration was estimated by spectrophotometry at
A620on the basis of the relationship A6200.2 ? 5 ? 107/ml. The proteins that had
been dialyzed against 0.01% (vol/vol) acetic acid were dried by vacuum centrif-
ugation (Speed-Vac) in the tube that was used for the assay, and 100 ?l of
log-phase bacteria at 5 ? 104/ml in 10 mM NAPB (pH 7.4) containing 1%
(vol/vol) TSB was added. At the start of the incubation and after 2 h of incuba-
tion at 37?C, the number of CFU was determined by plating serial dilutions. All
negative cultures were assigned the value 100 CFU/ml, which was the lowest
value that could be detected by this procedure. The percentage control CFU was
calculated by the formula Nexp/Ncontrol? 100, in which Nexpor Ncontrolis the
number of bacteria obtained after a 2-h incubation in the presence or absence,
respectively, of the polypeptides. All incubations were performed in duplicate.
Other methods. Protein sequence alignments and charge analysis of the iso-
lated ALP domains were obtained by using the PC/GENE and GeneWorks
programs (Intelligenetics, Mountain View, Calif.).
Analysis. Data from the antibacterial assays are expressed in percent control
CFU as mean ? standard error of the mean (SEM). Statistical analysis of the
data was performed with the GraphPad Instat program (GraphPad Software,
San Diego, Calif.). Results from individual antibacterial assays were used to
construct point-to-point curves from which the concentration of peptide causing
50% decrease in CFU (50% inhibitory concentration) for each of the individual
experiments was calculated. This 50% inhibitory concentration was expressed as
mean (95% confidence interval). Differences among treatment groups were
examined by using a one-way analysis of variance followed by a Student-New-
man-Keuls multiple-comparisons test. Differences were considered to be statis-
tically significant at P values less than 0.05.
RESULTS
Incubation of ALP with E. coli resulted in a marked de-
crease in the number of CFU (Fig. 1). Comparison of the 50%
inhibitory concentration for each of the polypeptides revealed
that the mean value of ALP was higher than that of defensins
and lysozyme (P ? 0.05) (50% inhibitory concentrations [95%
confidence intervals]: ALP, 4.7 ?M [1.0 to 8.4]; lysozyme 1.8
?M [0.9 to 2.8]; and defensins, 1.4 ?M [?0.7 to 3.5]). Micro-
scopic evaluation indicated that the decrease in CFU in mix-
tures containing ALP was not likely caused by clumping of
bacteria. The antibacterial activity of ALP was blocked by the
presence of 0.15 M NaCl in the assay buffer (data not shown).
To investigate in which part of the ALP molecule this anti-
bacterial activity is located, ALP was cleaved by mild acid
treatment, resulting in the formation of two major fragments
representing the first NH2-terminal (residues Ser-1 to Asp-49)
and second COOH-terminal (residues Pro-50 to Ala-107) do-
mains of ALP (37). Inhibition experiments using elastase dem-
onstrated that all elastase inhibitory activity was present in the
second domain (data not shown).
Next, intact ALP and the isolated domains were incubated in
various concentrations with E. coli and S. aureus to compare
their activities on a molar basis. Intact ALP and its first domain
displayed antibacterial activity against E. coli, whereas the sec-
ond domain was much less active (Fig. 2A). Comparison of the
50% inhibitory concentrations for each of the ALP prepara-
tions demonstrated that both intact ALP and the first domain
were significantly more active against E. coli than the second
domain (P ? 0.01 and ? 0.05, respectively) (50% inhibitory
concentrations [95% confidence intervals]: ALP, 4.2 ?M [1.3
to 7.0]; first domain, 12.1 ?M [0.5 to 23.7]; and second domain,
23.7 ?M [9.4 to 38.0]). It must be noted that in one of the four
FIG. 1. Antibacterial activity of ALP, lysozyme, and defensins against E. coli
ML-35p. Log-phase bacteria were incubated for 2 h at 37?C with the indicated
concentrations of ALP (open circles), lysozyme (closed circles), or defensins
(closed triangles), and the number of CFU was determined and used to calculate
the percent control. The mean numbers of bacteria at the start and the end of the
incubation were (5.4 ? 1.7) ? 104and (81.7 ? 46.4) ? 104CFU/ml, respectively.
Data are presented as mean ? SEM of four separate experiments.
FIG. 2. Antibacterial activity of ALP and its isolated first and second do-
mains against E. coli (A) and S. aureus (B). Log-phase bacteria were incubated
for 2 h at 37?C with the indicated concentrations of intact ALP (Mr, 11,700; open
circles) and its first (Mr, 5,400; closed circles) and second (Mr, 6,300; closed
triangles) domains, and the number of CFU was determined and used to calcu-
late the percent control. The mean numbers of bacteria at the start and the end
of the incubation were (5.9 ? 2.2) ? 104and (71.7 ? 21.0) ? 104CFU/ml,
respectively for E. coli; for S. aureus, (16 ? 3.1) ? 104and (40.7 ? 7.9) ? 104
CFU/ml were present at the start and the end of the incubation. Data are
presented as mean ? SEM of four (A) or three (B) separate experiments.
VOL. 64, 1996ANTIBACTERIAL ACTIVITY OF ANTILEUKOPROTEASE 4521

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experiments it was not possible to calculate a 50% inhibitory
concentration for the second domain because of lack of activ-
ity. Therefore, the value for this experiment was arbitrarily set
at the highest concentration tested (31.9 ?M). The apparent
difference in 50% inhibitory activity between ALP and its first
domain did not reach statistical significance. Using S. aureus as
the target microorganism, the second domain was even less
active (Fig. 2B). Therefore, it was not possible to calculate a
50% inhibitory concentration. The difference between these
values for intact ALP and its first domain did not reach statis-
tical significance (50% inhibitory concentrations [95% confi-
dence intervals]: intact ALP, 7.6 ?M (0.9 to 14.4]; and first
domain, 31.2 ?M [7.6 to 54.8]).
These experiments also revealed that the effect of ALP on
E. coli is bactericidal, since after incubation with the highest
concentration of intact ALP (17.1 ?M) for 2 h at 37?C, no cul-
turable bacteria were recovered (?100 CFU/ml, on the basis of
the detection limit of the assay), whereas at the start of the
incubation, (5.9 ? 2.2) ? 104CFU/ml was present. The effect
ALP on S. aureus is also bactericidal, since after 2 h in presence
of 17.1 ?M intact ALP, significantly fewer bacteria were
present than at the start of the incubation ([0.4 ? 0.4] ? 104
and [16 ? 3.1] ? 104CFU/ml, respectively; P ? 0.05, paired
Student’s t test).
In a subsequent set of experiments, E. coli and S. aureus
were incubated with a fixed concentration of intact ALP, the
isolated first and second domains, and the combination of the
first and second domains (Fig. 3). Both isolated domains, alone
or in combination, were significantly less active than intact
ALP against either E. coli or S. aureus. The first domain was
significantly more active than the second domain; the second
domain did not display significant antibacterial activity at this
concentration. Although the combined domains appeared to
be a little more active than the isolated first domain, this
difference did not reach statistical significance.
The two isolated domains were subjected to SDS-PAGE and
acid urea-PAGE analysis (Fig. 4). Whereas both domains mi-
grated similarly on SDS-PAGE, on acid urea-PAGE, the elec-
trophoretic mobility of the first domain was higher than that of
the second domain. This result, indicating that the first domain
has a higher cationic charge than the second domain, was
confirmed by estimation of the charge of domain 1 and 2 at pH
7, showing that the net charge of domain 1 (?7) was higher
than that of domain 2 (?5). In addition, the charge profile
showed a clustering of positive charge in the NH2-terminal half
of domain 1 (data not shown).
DISCUSSION
The main function of ALP is most likely to provide tissues
protection against degradation by serine proteinases that are
released from neutrophils during inflammation. The results
from this study demonstrate that ALP also displays antibacte-
rial activity: at concentrations that are found in various exter-
nal secretions (10), ALP caused marked killing of both gram-
negative (E. coli) and gram-positive (S. aureus) bacteria. Using
isolated domains that were obtained after acid treatment of
ALP, we found that the antibacterial activity of ALP is located
in the first NH2-terminal domain of ALP. Previous studies (7,
16, 26, 37) and this study demonstrated that the proteinase
inhibitory activity is located in the second, COOH-terminal
domain. This finding indicates that the antibacterial and pro-
teinase inhibitory activities of ALP are represented by different
domains. We observed that on a molar basis, the antibacterial
activity of the first ALP domain is lower than that of the intact
molecule. Combination of the two isolated domains did not
reconstitute the full activity of the intact ALP molecule. This
may in part be explained by a possible (conformational) change
in the first domain induced by the cleavage procedure of native
ALP; alternatively, it may indicate that the second domain
plays a role in the antibacterial activity in the intact molecule.
Although the two ALP domains are homologous to each other,
they apparently exert different functions. Analysis of the iso-
lated domains by acid urea-PAGE demonstrated that the first
domain had a higher electrophoretic mobility than the second
domain, indicating a more cationic character as previously
suggested (37). This finding, which was supported by the re-
sults of computer-assisted charge analysis of the protein se-
quence, may in part explain why the first domain displays
antibacterial activity whereas the second domain is largely de-
void of such activity. The possible involvement of the cationic
properties in the activity of ALP is also supported by our
observation that higher salt concentrations inhibit the antibac-
terial activity of ALP. Inhibition of antimicrobial activity at
higher ionic strength has been reported for a variety of other
antimicrobial proteins (19, 22). Although ionic interactions
may be involved, the mechanism whereby ALP causes killing of
bacteria is not yet clear. It has previously been reported that
recombinant ALP is toxic to E. coli when expressed in this
bacterium in high amounts (27). This toxicity was found to be
due to its ability to bind to mRNA and DNA, resulting mainly
FIG. 3. Effect of intact ALP and its isolated domains on E. coli and S. aureus.
Log-phase bacteria were incubated with 10 ?M intact ALP, its isolated first (1D)
and second (2D) domains, and the combination of the two domains (1D ? 2D),
each at 10 ?M. After 2 h of incubation at 37?C, the number of CFU was
determined and used to calculate the percent control. The mean numbers of
bacteria at the start and the end of the incubation were (4.5 ? 1.2) ? 104and
(97.2 ? 32.5) ? 104CFU/ml, respectively, for E. coli; for S. aureus, (3.6 ? 0.2)
? 104and (16.4 ? 1.3) ? 104CFU/ml were present at the start and the end of
the incubation. Data are presented as mean ? SEM of four (E. coli) or three
(S. aureus) separate experiments. *, P ? 0.05 versus intact ALP; **, P ? 0.01
versus intact ALP; ***, P ? 0.001 versus intact ALP.
FIG. 4. Analysis of the isolated ALP domains by SDS-PAGE and acid urea-
PAGE. Five micrograms of intact ALP and of first (1D) and second (2D)
domains were subjected to Tricine SDS-PAGE (A) and acid urea-PAGE (B) and
stained with Coomassie brilliant blue. Mr’s are indicated on the left.
4522HIEMSTRA ET AL.INFECT. IMMUN.

Page 4

in translation inhibition. Since in our study ALP was added
exogenously to E. coli, such a mechanism alone cannot explain
the observed antibacterial activity.
Antimicrobial properties of proteinase inhibitors have been
previously reported. The cationic proteinase inhibitor aproti-
nin, present in bovine lung and other organs, displays antibac-
terial activity against both gram-positive and gram-negative
bacteria (28). This activity of aprotinin did not require its pro-
teinase inhibitory activity and was shown to be probably related
to its cationic properties. In addition, a synthetic peptide mim-
icking the cysteine proteinase inhibitory site of human cystatin
C was found to kill group A streptococci (2). Another example
of an antimicrobial proteinase inhibitor is the aforementioned
eNAP-2, a cationic polypeptide purified from equine neutro-
phils having limited sequence homology with ALP (3, 4). Both
ALP and eNAP-2 contain a pattern of cysteine residues that is
characteristic for members of the FDC family of proteins (3,
33). Alignment of the protein sequences of ALP and eNAP-
2 demonstrated that most of the homology was found in the
second proteinase inhibitory domain, not in the first antibac-
terial domain, of ALP.
Previous studies have suggested a role for human serine
proteinase inhibitors in host defense against infections; these
studies demonstrated that proteinase inhibitors are able to
inhibit the elastase-mediated degradation of opsonins and re-
ceptors involved in phagocytosis (23, 36). A recent study in
which saliva components exhibiting anti-human immunodefi-
ciency virus type 1 activity were investigated identified ALP as
the major active component (25). This antiviral activity of ALP
is most likely not due to a direct interaction with the virus but
was suggested to be the result of an interaction with the host
cell. The results from the present study show that ALP may act
directly as an antibacterial agent. Thus, ALP may be an addi-
tion to the array of locally produced antimicrobial polypeptides
that have been identified in secretions and that include ly-
sozyme, lactoferrin, and tracheal antimicrobial peptide (5, 34).
Interestingly, the distribution of ALP in human tissues is much
like that of another antimicrobial polypeptide, lysozyme (9,
12). In addition to locally produced antimicrobial polypeptides,
especially purulent secretions may contain various antimicro-
bial polypeptides that are derived from neutrophils, including
defensins.
At present, ALP is being considered as a therapeutic agent
for treatment of inflammatory lung disease. Recombinant ALP
has been administered to cystic fibrosis patients in a short-term
study (24), but its clinical efficacy remains to be established.
Since such diseases are often associated with (recurrent) bac-
terial infections, ALP may not only aid in restoring the balance
between proteinases and proteinase inhibitors but also act as
an antimicrobial agent.
In summary, the results from this study show that intact ALP
and its isolated first NH2-terminal domain efficiently kill bac-
teria in vitro. This finding indicates that ALP may contribute to
host defense against infection through its antibacterial activity.
Additional studies will be required to determine whether ALP
also kills lung pathogens.
ACKNOWLEDGMENTS
We thank M. Couto and R. I. Lehrer (Department of Medicine,
UCLA School of Medicine) for helpful discussions and T. Ganz (De-
partment of Medicine, UCLA School of Medicine) for help in the
charge analysis of the isolated domains. We also thank T. J. N. Hil-
termann and J. K. Sont for helpful discussions relating to the statistical
analysis of the data.
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Editor: V. A. Fischetti
4524HIEMSTRA ET AL.INFECT. IMMUN.