Inhibitors of benzamidine type influence the virulence properties of Porphyromonas gingivalis strains.

Abstract

Synthetic inhibitors of benzamidine type have been found to have inhibiting effects on arginine specific cysteine proteinases of P. gingivalis. The purpose of our study was to assess the effects of these inhibitors on the virulence properties of two P. gingivalis strains, the reference strain ATCC 33277 and JH16-1, a clinical isolate obtained from a patient with severe periodontitis. The inhibitors tested were pentamidine, benzamidine, three bis-benzamidine derivatives with a pentamidine-related structure, one bis-benzamidine derivative with another structure, and one arginine derivative as a negative control, each in the concentrations of 2 microM and 20 microM. As virulence criteria the following parameters were determined: arginine-specific amidolytic activity, growth inhibition, hemagglutination of sheep erythrocytes, adherence to KB cells and immuno-phagocytosis including intracellular killing. Pentamidine and the bis-benzamidine derivatives with pentamidine-related structure showed the most remarkable effects on reduction of amidolytic activity by 35%, growth inhibition and reduced hemagglutination. Except for the arginine derivative all other inhibitors tested enhanced the phagocytosis capacities of granulocytes. No clear influence of the inhibitors on adherence of P. gingivalis to KB cells was seen. Although in vitro effects of the synthetic inhibitors of cysteine proteinases on virulence of P. gingivalis were observed further in vitro tests concerning immunomodulatory effects should be done before these substances are used for therapy in clinically controlled studies.

Full text

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Inhibitors of benzamidine type influence the virulence proper-
ties of Porphyromonas gingivalis strains?
Sigrun Eick?, Wolfgang Pfister, Uta Stürzebecher, Sigrid Jarema and
Jörg Stürzebecher
Institute of Medical Microbiology, Department of Oral Microbiology and Institute of Vascular
Biology and Medicine, University Hospital of Jena, Germany
Received: 30 May, 2003, revised: 04 September, 2003; accepted: 08 September, 2003
Key words: Porphyromonas gingivalis strains, inhibitors of benzamidine type, growth inhibition,
adhesion to KB cells, phagocytosis and intracellular killing
Synthetic inhibitors of benzamidine type have been found to have inhibiting effects
on arginine specific cysteine proteinases of P. gingivalis. The purpose of our study
was to assess the effects of these inhibitors on the virulence properties of two P.
gingivalis strains, the reference strain ATCC 33277 and JH16-1, a clinical isolate ob-
tained from a patient with severe periodontitis. The inhibitors tested were pentami-
dine, benzamidine, three bis-benzamidine derivatives with a pentamidine-related
structure, one bis-benzamidine derivative with another structure, and one arginine
derivative as a negative control, each in the concentrations of 2 ?M and 20 ?M. As vir-
ulence criteria the following parameters were determined: arginine-specific
amidolyticactivity,growthinhibition,hemagglutinationofsheeperythrocytes,adher-
ence to KB cells and immuno-phagocytosis including intracellular killing. Pentami-
dine and the bis-benzamidine derivatives with pentamidine-related structure showed
themostremarkableeffectsonreductionofamidolyticactivityby35%,growthinhibi-
tionandreducedhemagglutination.Exceptfortheargininederivativeallotherinhibi-
tors tested enhanced the phagocytosis capacities of granulocytes. No clear influence
of the inhibitors on adherence of P. gingivalis to KB cells was seen. Although in vitro
effects of the synthetic inhibitors of cysteine proteinases on virulence of P. gingivalis
were observed further in vitro tests concerning immunomodulatory effects should be
done before these substances are used for therapy in clinically controlled studies.
Vol. 50 No. 3/2003
725–734
QUARTERLY
?Presented at the XXX Winter School of Faculty of Biotechnology, Jagiellonian University, Koœcielisko,
Poland, 28th February–4th March, 2003.
?DepartmentofOralMicrobiologyandInstituteofVascularBiologyandMedicine,UniversityHospitalof
Jena,Semmelweistr.4,D-07740Jena,Germany;phone:(493641)933587;fax:(493641)933474;e-mail:
Sigrun.Eick@med.uni-jena.de
Abbreviations: BApNA, N-?-benzoyl-DL-arginine-p-nitroanilide; FBS, foetal bovine serum; MTT, Thia-
zolyl Blue; PBS, phosphate-buffered saline; PMN, polymorphonuclear leukocytes.

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Porphyromonas gingivalis has been impli-
cated as an important bacterial species in
pathogenesis of severe forms of periodontitis.
In the last few years a major focus of research
on P. gingivalis has been on the cysteine pro-
teinases with arginine and lysine cleavage
specificity (Travis et al., 1997; Imamura,
2003). The so-called gingipains as a whole
make up 85% of the proteolytic activity of this
bacterium, and the molar concentration of
Arg-gingipain is consistently 2–3-fold higher
than that of Lys-gingipain (Potempa et al.,
1997). While the cysteine proteinase activity
with arginine specificity originates from two
different genes, rgpA and rgpB, the lysine spe-
cific cysteine proteinase activity is derived
from a single gene, kgp (Mikolajczyk-Paw-
linska et al., 1998). rgpB mutants possess
about 50% of the arginine specific cysteine ac-
tivity (Aduse-Opoku et al., 1998; Tokuda et al.,
1996), a rgpA mutant has 40% activity (To-
kuda et al., 1998). The gingipains are associ-
ated with many of the virulence factors of this
species, such as degradation of immunoglo-
bulins G and A (Abe et al., 1998), degradation
of complement factors (Grenier, 1992), en-
hancement of vascular permeability (Rubin-
stein et al., 2001), and formation of fimbriae
(Tokuda et al., 1998).
Antibiotics are widely used as additive ther-
apy in severe cases of P. gingivalis associated
periodontitis. But this usage has limitations,
e.g. because of the development of antibiotic
resistance. Most P. gingivalis strains are carri-
ers of the tet(Q) resistance gene (Chung et al.,
2002). Therapy with tetracycline fibers is un-
able to eradicate P. gingivalis completely
(Mombellietal.,2002).Newideastouseinhib-
itors of major virulence properties have been
discussed (Potempa et al., 2000). The purpose
of this study was to evaluate the effect of in-
hibitors of benzamidine type on the virulence
properties associated with arginine specific
cysteine proteinase activity of P. gingivalis.
Beside the influence on N-?-benzoyl-DL-argi-
nine-p-nitroanilide(BApNA)activitytheeffect
on growth, hemagglutination, adherence to
epithelial cells, and immunophagocytosis
were determined.
MATERIALS AND METHODS
Bacterialstrains.P.gingivalisATCC33277
was obtained from the German strain collec-
tion DSMZ (Braunschweig, Germany). The
JH16-1 strain was a clinical isolate obtained
from a female patient with severe chronic
periodontitis. Analysis for strain diversity
confirmed that the clinical isolate had been
persistentfortwoyearsinthispatient(Eicket
al., 2002). The strains were maintained on
Schaedleragarenrichedwith10%sheepblood
and vitamin K. Bacterial cells used in assays
weregrowntolatelogphase(36h),harvested,
washed twice and resuspended in Medium
199 (Gibco) to an absorbance at 660 nm corre-
sponding to 108or 109bacteria/ml.
Proteinase inhibitors. Seven inhibitors
tested were chosen from a spectrum of sub-
stances with a benzamidine structure, they
were named 1, 2, 3, 4, 6, 7 and 9 (pentami-
dine). The inhibitors 1, 4, and 6 were bis-ben-
zamidines with a pentamidine related struc-
ture. Inhibitor 2 was a bis-benzamidine with
another structure. Inhibitor 7 represented
benzamidine. “Inhibitor” 3 was an arginine
derivative. The tests were carried out as a
blind study. At the time of the study the inves-
tigators did not know either the group of
benzamidines the inhibitors belonged to or
that a negative control was included.
The inhibitors were synthesized by the
group of J. Stürzebecher (Institute of Vascu-
lar Biology and Medicine, University Hospital
of Jena, Germany). The inhibitors had been
tested against purified Arg-gingipain RgpB
(the enzyme was kindly provided by Jan
Potempa, Jagiellonian University, (Kraków,
Poland) before. The inhibition constant (Ki)
was up to 0.45 ?M (inhibitor 1).
Determination of the arginine specific
cysteine amidolytic activity. Proteinase in-
hibitors were added to bacterial suspensions
726S. Eick and others2003

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(109/ml) in a final concentration of 2 ?M and
20 ?M. After mixing these suspensions were
incubated at 37°C for 1 h. After that each 100
?l of suspension was placed into a well of a 96
well-microtitreplate.Eachwell contained125
?l of reaction mixture for determination of
theargininespecificcysteineamidolyticactiv-
ity(0.5mMBApNA,10mM L-cysteine,10mM
CaCl2and 100 mM Tris/HCl, 1% agar, pH 7.6,
according to Grenier & Turgeon (1994)).
The plate was incubated at 37°C for 1 h.
Then the bacterial suspension was removed
and the plates were washed three times with
phosphate-buffered saline (PBS). The argi-
nine specific amidolytic activity was mea-
suredbymeansofaspectrophotometerat405
nm.
Growth inhibition by proteinase inhibi-
tors. Bacteria (100 ?l of a 108/ml suspension)
were added to tubes containing 10 ml of
Schaedler broth with 10% sheep blood and vi-
tamin K and a proteinase inhibitor (final con-
centration 0.05–20 ?M), including a negative
controlwithoutinhibitor.Afterincubationfor
24 h in an anaerobic atmosphere at 37°C col-
ony forming units (cfu) were determined. The
MIC50and MIC90of the inhibitor were calcu-
lated.
Hemagglutination. For hemagglutination
assays to bacterial suspensions (109/ml)
proteinase inhibitors were added (final con-
centration2?Mand20?M)andincubatedfor
1 h. After that the mixtures were diluted in a
twofold series with PBS. Aliquots (100 ?l) of
each dilution were mixed with an equal vol-
ume of a sheep erythrocyte suspension (2% in
PBS) and incubated in a round-bottomed
microtitre plate at room temperature for 3 h.
Hemagglutination was evaluated visually.
Adherence assay. KB cells were seeded
into two 24-well tissue culture plates at a den-
sity of about 104cells per well, the cells were
grown to confluent monolayer in EMEM
(Gibco) enriched with 10% foetal bovine se-
rum (FBS). The cells were washed twice with
PBS. Bacterial suspension (108/ml) in Me-
dium 199 was mixed with EMEM 1:4.
Proteinase inhibitors were added to a final
concentration of 2 ?M and 20 ?M and each
well was inoculated with 1 ml of this suspen-
sion. Bacteria were co-cultured with KB cells
at 37°C for 1 h. After that the infected mono-
layers were washed five times with PBS. The
number of adherent P. gingivalis was counted
as cfu after cell lysis.
Additionally the MTT tetrazolium salt colori-
metric assay described by Mosmann (1983)
wasusedtomeasurecytotoxicity.Themethod
is based on the capacity of mitochondrial en-
zymes of viable cells to reduce the yellow solu-
ble salt MTT to a blue insoluble formazan pre-
cipitate, which is quantified after dissolution
in an organic solvent. The MTT-formazan pro-
duction correlates directly with the count of
viable cells. Proteinase inhibitors were mixed
with EMEM + 10% FCS to a final concentra-
tion 2 and 20 ?M and incubated with KB cells
for 24 h in a 96-well microtiter plate. Then 10
?l of a MTT solution (5 mg/ml) was added to
100?lofculturemedia.Afterincubationfor2
h at 37°C the suspension was removed and 20
?l of 3% SDS solution and 100 ?l of isopro-
panole/0.04 M HCl were added and incubated
for 1 h at 37°C. Finally the MTT reaction was
examined photometrically at 570 nm.
Immunophagocytosis. The method used
was based on the descriptions by Smith &
Rommel(1977)andPantazis&Kniker(1979).
Freshlydrawnbloodobtainedfrom10healthy
subjectswaslayedonacoverslip.Thecellsad-
heredat37°Cwith5%CO2and100%humidity
for 25 min. Thereafter the soft blood clot was
removed.
Bacterial suspensions were adjusted photo-
metrically to 109bacteria per ml with PBS.
Bacterial suspension (0.2 ml) was mixed with
0.2mlhumanAnti-AB-serumforopsonization
for 30 min. These mixtures were centrifuged
at 2000 ? g for 10 min and then the pellets
were washed and resuspended in 1 ml PBS.
Finally, 0.2 ml of foetal bovine serum and the
proteinase inhibitor to be tested at a final con-
centrationof20?Mwereadded.Thebacterial
suspensions were transferred to the cover
Vol. 50
Porphyromonas gingivalis strains and benzamidine727

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slips and the phagocytosis assay was incu-
bated under the conditions mentioned above
for 15 min.
Then the slips were stained with acridine or-
ange (2.5 mg/7.5 ml PBS) for 1 min. After de-
canting the solution they were covered with
0.01% crystal violet in PBS to quench extra-
cellular fluorescence, washed and fixed. A to-
tal of 100 polymorphonuclear leukocytes
(PMN) were immediately examined by fluo-
rescence microscopy. Viable and killed bacte-
ria in granulocytes were distinguished by
their uptake of acridine orange, viable bacte-
ria appeared green and dead bacteria were
red. The numbers of PMNs containing bacte-
ria were counted. These phagocytosing cells
were separated into groups by the following
criteria: (i) cells with less than 10, cells with
10–20, and cells with more than 20 ingested
bacteria and (ii) granulocytes with less than
50%andwithmorethan50%viablebacteria.
Statistical analysis. The statistical analy-
sis was done using SPSS 10.0 software. The
significance of the differences between each
group with addition of an inhibitor (2 ?M and
20 ?M) and the controls (without inhibitor)
was determined by the Mann-Whitney test.
The WILCOXON test was used to assess the
significance of the results between paired
samples in phagocytosis assays.
RESULTS
Amidolytic activity
Inhibitor 1 showed the highest reduction of
theargininecysteineamidolyticactivityofthe
two P. gingivalis strains, the reduction being
35.5%fortheATCCstrainat2?M.Alsoinhib-
itor 4, another pentamidine-related inhibitor,
reduced the amidolytic activity of the two
strains in the lower concentration of 2 ?M. In-
hibitor 6 in both concentrations showed ef-
fectsontheJH16-1strain,andpentamidineat
20 ?M reduced the amidolytic activity of both
strains (Table 1).
Growth curves
Only the inhibitors of the pentamidine-re-
lated group were able to inhibit the growth of
the bacteria by 50% and 90% after 24 h. Pent-
amidinewasmosteffective.Nosignificantdif-
ferencesingrowthinhibitionwasobservedbe-
tween the two strains (Table 2).
728S. Eick and others2003
Table 1. Effect of proteases inhibitors on BApNA-reaction activity produced by P. gingivalis.
Activity of the non-treated suspension of bacterial cells was taken as 100%.

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Hemagglutination
Without the addition of inhibitors hemag-
glutinationwasobserveduptoadilutionof1:32
fortheATCC33277strainandupto1:64forthe
JH16-1 strain. Inhibitors 3 and 7 did not influ-
ence hemagglutination. Both tested concentra-
tions of the pentamidine-related substances (1,
4, 6,and pentamidine)
hemagglutination of the ATCC strain. The ef-
fects on the clinical isolate were not so clear.
Substance 1 at concentrations of 2 ?M and 20
?M of the substance 1 reduced hemag-
inhibited
glutination of this strain, only the higher con-
centration of the other pentamidine-related in-
hibitorshadaslighteffectonhemagglutination.
Adherence to KB cells
Following incubation with KB cells in me-
dium 2.2 ? 105cfu per well of adherent
P. gingivalis ATCC 33277 and 1.5 ? 104cfu of
adherent P. gingivalis JH16-1 were enumer-
ated. Only pentamidine and inhibitor 7 at 20
?M were able to reduce the adherence of the
ATCC 33277 strain, conversely, inhibitor 4 in
bothconcentrationstestedenhancedthenum-
ber of adherent bacteria (P < 0.05). A reduced
adherence of the clinical isolate JH16-1 strain
was observed after addition of inhibitors 1, 2,
3, and 7 in both concentrations and of the
pentamidine-related inhibitors 4 and 6 at 20
?M (P < 0.05, Fig. 1).
The MTT values showed no cytotoxicity for
the inhibitors with a pentamidine-related
structure. Reduced MTT values were mea-
sured for inhibitor 2 in both concentrations
and the inhibitor 3 in the higher concentra-
tion. So it can be concluded that these inhibi-
tors are cytotoxic in the concentrations men-
tioned above.
Immunophagocytosis
No differences of phagocytosing capacities
of peripheral blood PMNs were observed be-
tween the two bacterial strains without inhibi-
tors added. So, in medium 59% of the PMNs
had ingested more than 20 P. gingivalis ATCC
33277 or JH16-1. Inhibitor 3 (arginine deriva-
tive) was the only substance tested with no ef-
fect on phagocytosis. All other inhibitors en-
hanced the percentage of PMNs with more
than 20 ingested P. gingivalis ATCC 33277 (P
< 0.05). In contrast, the inhibitor 6 did not in-
fluence the phagocytotic capacity of granulo-
cytes to the JH16-1 strain. Pentamidine and
inhibitors 1, 2, 4 and 7 promoted the phago-
cytosis of this clinical isolate (P < 0.05). The
results are presented in Fig. 2.
In medium, 46% of the phagocytosing PMNs
had more than 50% killed P. gingivalis ATCC
33277 and 41% of these cells had more than
50% killed P. gingivalis JH16-1. The difference
between the two strains was significant (P <
0.05).Onlyinhibitor4reducedthisnumberto
31% (ATCC 33277) and 24% (JH16-1) (P <
0.05). All other inhibitors showed no effects
on killing of bacteria (not shown).
DISCUSSION
The purpose of the study was to determine
the effects of inhibitors of benzamidine type
Vol. 50
Porphyromonas gingivalis strains and benzamidine729
Table 2. Inhibitory concentration of proteases inhibitors with pentamidine-related structure which
suppress the growth of P. gingivalis strains by 50 and 90%