The Interaction between Guavaextract and Aggregatibacter
actinomycetemcomitans Leukotoxin
Authors: Cardo Toma, Genet Kidane.
Primary tutor: Dr. Anders Johansson, Department of Odontology/Molecular
Periodontology, Faculty of Medicine, Umea University, Sweden.
External tutor: Dr. Rolf Claesson, Department of Odontology/Oral Microbiology,
Umea University, Sweden.
ABSTRACT
Previous studies have shown that the highly leukotoxic JP2 clone of Aggregatibacter
actinomycetemcomitans (Aa) was strongly associated with the initiating of aggressive
periodontitis amongst adolescents in Ghana. In this population, conventional
periodontal treatment cannot be performed because of limited resources. The focus of
the present study has been on developing a preventive tool for eliminating Aa
leukotoxin (LtxA). Recent studies indicate that extract from Guava inhibits the activity
of the Aa leukotoxicity. However, it requires more knowledge about Guava LtxA
neutralizing properties in order to be used in future prevention strategies of aggressive
periodontitis. Our aim has been to determine the persistence of the inhibitory effect of
guava on leukotoxicity of Aa.
We examined the bindings of LtxA-neutralizing Guava components to different isolates
of the Aa bacterium including the high leukotoxic JP2 clone. The experiments began
by culturing the Aa bacteria as a biofilm on a plastic surface which then became
exposed to guava extract for 30 min. The guava extract was then replaced with a plane
culture medium for different times before we added cultured macrophages (THP-1
cells). The cytolysis effect of LtxA was quantified as the releases of cytosolic lactate
dehydrogenase (LDH) or decrease in the neutral red uptake of Aa-exposed (2 hr)
cultures of human macrophages (THP-1 cells).
All experiments showed that leukotoxicity caused by the tested isolates of Aa became
significantly reduced in the presence of guava-extract. The results also showed that the
persistence of guava on leukotoxicity from Aa remained for 24 hr after removal of the
unbound guava components. The persistence of the LtxA neutralizing effect for at least
24 h indicate a possibility in using guava components as a preventive tool for aggressive
periodontitis associated with the presence of Aa.
INTRODUCTION
The Aggregatibacter actinomycetemcomitans and its leukotoxin
Aggregatibacter actinomycetemcomitans (Aa) is a gram-negative bacterium, which is
present in the oral cavity of humans (Henderson, et al., 2010). Initially it colonizes oral
mucosa as a facultative intracellular pathogen (Fine et al., 2007). Among the different
species found in oral cavity, Aa is a bacterium associated with aggressive forms of
periodontitis, and it produces a leukotoxin (LtxA) that specifically affects human
leukocytes (Johansson 2011). When Aa becomes located in the subgingival biofilm, it
releases components that induce processes in the host response, which can result in
attachment loss of the tooth (Nishihara and Koseki 2004).
LtxA activates a pro-inflammatory death of human monocytes/macrophages
(Johansson 2011). Different types of Aa have quantitatively different abilities in
expressing its LtxA (Höglund Åberg et al., 2014a). A particular clone of this bacterium
(JP2) has a significantly enhanced expression of LtxA. Thus, it has a strong association
with aggressive periodontitis among adolescents (Haubek et al., 2008; Höglund Åberg
et al., 2014b).
Periodontitis among adolescents in Ghana
The two major categories of periodontal disease caused by Aa are gingivitis and
periodontitis (Lindhe et al., 2008). Epidemiological studies have shown that
periodontitis occurs predominantly in a slowly progressing form (Lindhe et al., 2008).
The more severe and rapidly progressive form of periodontitis is denoted as aggressive
periodontitis (Armitage 1999). A recent study by Höglund Åberg et al., (2014b) showed
that aggressive periodontitis was common among adolescents in Ghana. The presence
of Aa was significantly associated with an enhanced risk for initiation and progression
of aggressive periodontitis. The individuals who were carriers of the JP2 genotype of
Aa showed the highest risk for the initiation of aggressive periodontitis and progression
of attachment loss (Höglund Åberg et al., 2014b).
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Guava as LtxA neutralizing element
Guava (Psidium guajava) is a native plant of tropical America (Gutiérrez et al., 2008).
In Ghana, the guava plant grows in the middle and southern parts of the country. The
plant is used as chewing sticks for mechanical cleaning of the teeth (Akpona et al.,
2009). A previous study showed that guava extract had a neutralizing effect on the LtxA
(Kwamin et al., 2012). This study showed that ethanol and water extracts of leafs or
twigs from the Psidium guajava tree had a neutralizing effect on the LtxA in vitro. It
has not been determined yet by any studies which guava components exactly that are
responsible for the neutralizing effects on the LtxA. It requires further future research
to identify the active guava components.
The objective of this study:

The general purpose of the study was to evaluate the interaction of guava extract
with LtxA by studying the properties of the LtxA neutralizing guava
components.

The specific objective was to determine how persistent the LtxA neutralizing
effect is.
MATERIAL AND METHOD
Literature search
We searched articles on PubMed using the following MeSH terms; aggressive
periodontitis, Aggregatibacter actinomycetemcomitans, JP2 clone, Psidium
guajava. Manual searches were done on references found in relevant articles. We
selected 37 articles in the beginning then we included 14 of them by excluding
the rest because of their limited relevance to our study. Different exclusion
criteria like looking to the objectives and purpose of the studies were used. Thus,
we found 14 articles that fitted our interest of study. Our tutors also gave articles
to us.
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Bacterial cultures
Clinical isolates of Aa from previous studies of African adolescents were used in the
study. We intended to use the Aa as dead and later live bacteria to see if that could
affect the neutralization of LtxA. The dead Aa was killed by UV-light and was from
strain HK1519 (JP2-genotype) and the live Aa culture was of rough phenotype (clinical
isolate). In the cell culture assay it was determined which concentration of UV-Aa
(HK1519) that was needed to kill phorbol myristate acetate (PMA, Sigma-Aldrich, St
Louis, MI, USA) differentiated THP-1 cells (macrophages). For the rough phenotype
of Aa, we cultivated the bacteria on a blood agar plate for 48-72 hr. We harvested then
the bacteria with a sterile cotton swab, and suspended it in PYG-broth in a density of
OD 600nm ≈ 0.5, which corresponds to about 109 bacteria/ml. We added one hundred
µl of this suspension to each well of a 96-welled microtiter plate and allowed it to form
biofilms during 24 h incubation at 37°C.
Preparation of guava extracts
Guava extract from leaves and twigs taken from local guava trees in Ghana was
produced previously and stored in the freezer in the department of molecular
periodontology at Umea University. Briefly, the minced guava samples (1 g) were
extracted with four mL of 70% ethanol. Then they were agitated over-night at room
temperature before filtered through a 0.45μm sprout filter (Sarstedt AG and Co,
Nümbrecht, Germany). This procedure has previously been described in detail by
Gribing and Jansson (2013).
Cell culture
Materials and reagents where collected and placed in a bio-safety hood. We counted
THP-1 cells (human monocyte carcinoma cells) in a Bürker chamber and then
centrifuged them. The cell pellet was diluted to a concentration of 106 cells/ml in a
RPMI (Sigma-Aldrich, St Louis, MO, USA) culture media with 10% Foetal Bovine
Serum (FBS). Then we added PMA (Sigma-Aldrich) to a final concentration of 50 nM
5
PMA (Sigma-Aldrich), mixed and placed 100 μl/well in a 96 well plate. Lastly, we
incubated the plate for 24 hr at 37°C, to let cells differentiate towards macrophages.
Cytotoxicity tests
Neutral red up-take
THP-1 cells were quantified, transferred to a microtiter plate and allowed to
differentiate toward macrophages in presence of PMA, as described above. Afterwards,
we changed the culture medium and added bacteria (UV-Aa) in various concentrations
by performing a serial dilution in RPMI1640. The ratio of bacteria/cell was calculated.
Then we incubated the plate for 60 min at 37°C.
To determine the viability of the cells at the end of the exposure period we used the
neutral red uptake method as describe previously (Repetto et al., 2008). Briefly, we
replaced the medium with fresh media containing neutral red and then we incubated the
plate again for 90 min at 37°C. When that was done we aspirated the medium and
washed the wells with 150 µl PBS/well. Now the viable cells should be remained in the
bottom of the wells. 150 μl of lysis solution was added to each well and the intensity of
the accumulated red color was documented according to a spectrophotometric analysis
at 550 nm. The percentage of viable cells was calculated in relation to the red color
accumulated in the control cells (100%).
L-(+)-LDH-assay
We chose this method because cytosolic lactate dehydrogenase (LDH) is a large
cytosolic molecule (140 kDa) and a marker for increased membrane leakage
(Wroblewski and LaDue, 1955). First, a substrate buffer was made of 30 ml 100 mM
phosphate-buffer with 83 μg/ml Na-pyruvate and 166 μg/ml NADH. Then in a 96welled microtiter plates it was added 25 µl sample + 180 µl substrate buffer. LDH
catalyze the oxidation of NADH, and cause a decrease in the absorbance at 340 nm.
(NADH has high absorbance while NAD has low) (Wroblewski and LaDue, 1955).
Therefore, a reading of the absorbance at 340 nm kinetic (2 min interval time) during 6
min was made.
6
Analyses of extracellular release of LDH determined the cell injury from the activity of
lactate dehydrogenase (LDH), which was released extracellularly in the reaction
mixtures (Wroblewski and LaDue, 1955). The mixtures were centrifuged and the
supernatants were analyzed for LDH activity. Incubating cells in the absence of bacteria
in the medium made a negative control. The maximum LDH activity was also
considered as it was obtained in supernatants of THP-1 cells treated with 0.1 % Triton
X-100 for 1 hr. Lastly, a calculation was made of the percent LDH release in relation
to triton-exposed control (100 %) and negative control (0 %) (Wroblewski and LaDue,
1955).
Examinations of leukotoxin neutralization by guava extracts
Guava in a concentration needed to neutralize the leucotoxic effect of 100 UV-Aa was
added to cultures of PMA-differentiated THP-1 cells. The viability of the cells was
examined with the neutral red uptake method at the end of the incubation period. For
determination of the persistence of binding of the guava compound to the LtxA, guava
extracts were added to a biofilm of rough Aa for 30 min. Then the guava extract was
removed, and the biofilm was rinsed with PBS and incubated in PBS or PYG-broth for
up to 24 h before the THP-1 cells were added. The release of LDH from the THP-1
cells after 120 min exposure to the Aa biofilm was quantified as described above in
order to determine the persistence of the LtxA neutralization.
Ethical considerations
Previous studies have identified high frequency of Aa and attachment loss in African
adolescents. The ethical committee in both Umeå (Dnr 2010-188-31M) and Ghana
(IRBnr 000 1276) approved these studies. As these studies show large treatment needs
without enough resources, new strategies are needed to prevent the development of
periodontitis. If LtxA have proven to be a key molecule for this disease development,
we consider it essential to develop findings of LtxA neutralizing components, which
can be used in clinical prevention.
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Statistical analyses
Significant LtxA neutralizing effects of guava extract were calculated with student´s ttest using the Microsoft Excel 2010 software. P-values ≤ 0.05 was considered as
significant differences.
RESULTS
Dosedepent effect of leukotoxicity and neutralization by guava extract
First, we determined the concentration of UV-killed Aa (strain HK1519) that was
needed to kill a sufficient amount of PMA-differentiated macrophages. That was in
order to find a suitable concentration of Aa to examine neutralizing effects of guava
(Figure 1a). Based on the previous determination results we selected a concentration of
100 Aa/macrophage to examine the LtxA neutralizing effect of guava. In these
experiments, we used different concentrations of guava in presence of Aa and found an
optimal LtxA neutralizing effect at a concentration of 0.5% extract (Figure 1b).
Persistence of guava extract on inhibition of leukotoxicity
Different Aa strains differ in their activity of producing LtxA. The highly leukotoxic
JP2 strain produces most LtxA, and it is also the most pathogenic Aa. However, the
leukotoxicity of all tested isolates of Aa was neutralized in the presence of guavaextract components (Figure 2). In these experiments, Aa (rough phenotype) were added
to form a biofilm in PYG-growth medium before we added guava extract. After
removal of the guava extract, the Aa-biofilm was incubated for additional 24 h before
we added the target cells (THP-1). The result from these experiments showed that guava
neutralizes the leukotoxicity of all tested isolates and that the effect persists for at least
24 h (Figure 2).
Effect of growth medium on guava neutralized leukotoxicity
The LtxA neutralization was more effective after 24 h incubation in PBS than in PYGgrowth medium. Probably, the different neutralization effect was a result of a
proliferation of Aa in PYG were the new bacteria expresses new LtxA that can not be
neutralized due to that the unbound guava extract has been removed. Repeated analyzes
8
performed on the reference strains D7S (low leukotoxic) and JP3 (highly leukotoxic)
showed that the neutralizing effect of guava exposure was significant, except for the
D7S-strain in PBS (Figure 3). D7S in PBS p=0.104, JP3 in PBS p<0.001, D7S in PYG
p=0.010, JP3 in PYG, p=0.001.
DISCUSSION
It has previously been shown that extracts of guava leaves and twigs contains
components that efficiently neutralizes the leukotoxicity of Aa (Kwamin et al., 2012).
In the present study, we showed that this LtxA neutralizing property of guava extracts
is stable and persist for at least 24 h. This finding was determined in an experimental
model of Aa cultured in a biofilm and exposed to guava extract before the target cells
were added. The LtxA neutralizing effect persisted for 24 h after the unbound guava
components were removed and the biofilm incubated in physiological buffers. These
stable properties of the guava extract strengthen the possibility for this tool to be used
in preventive strategies for aggressive periodontitis.
The LtxA has previously been shown to be a powerful tool that causes an imbalance in
the host inflammatory response (Johansson 2011). Young individuals infected with Aa
have been shown to have a significantly enhanced risk to be affected by attachment loss
(Haubek et al., 2008; Höglund Åberg et al., 2014b). The highest odds ratio for the
development of attachment loss was for individuals colonized with highly leukotoxic
Aa (Höglund Åberg et al., 2014a). These results indicate an important role of LtxA in
the pathogenicity of aggressive periodontitis and an interesting target for future
preventive strategies. Taken together, clinical and experimental data indicate a role of
LtxA as a risk factor for periodontal attachment loss.
We hypothesize therefore that neutralization of the LtxA in adolescence in these
populations will be of significant preventive value. Conventional periodontal treatment
in line with that used in the industrial countries will not be possible to carry out in
developing countries. The traditional methods for oral hygiene in many of these
countries include the usages of chewing sticks made from selected plants with
suggested positive health effects (Akpona et al., 2009). One of these plants is guava,
which is widespread in the tropical zones of the world, could be an easily available tool
for self-prevention of periodontal disease in the developing countries. It has recently
9
been shown that there is a great treatment need for periodontal disease in Ghanaian
adolescents (Höglund Åberg 2013).
In conclusion, we hope that our result showing the high persistence of guava on
neutralization of LtxA could contribute to limit the effects of Aa infections in the
tropical countries. In order to determine the potential for this strategy for preventive
purpose, a clinical trial has to be made.
ACKNOWLEDGMENTS
First and foremost, we would like to thank our primary tutor of this project, Dr. Anders
Johansson for the valuable guidance and advice. His willingness to motivate us
contributed tremendously to our project. We also would like to thank him for being
such a kind, humble and patient source of knowledge for us during our journey.
Besides, we would like to thank our secondary tutor, Dr. Rolf Claesson for helping us
with bacterial preparations and sharing his good mood with us. Also, we would like to
take this opportunity to thank the Department of Odontology/ Molecular
Periodontology, Faculty of Medicine, Umea University for offering us this opportunity
and providing us with environment and facilities to complete this scientific work. It
gave us a chance to participate and learned about future preventive strategies against
aggressive periodontitis that could be practical in developing countries, like Ghana,
where conventional periodontal treatment can hardly be performed.
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REFERENCES
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folk classification and pharmacological properties of plant species used as chewing
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Armitage GC (1999). Development of a classification system for periodontal diseases
and conditions. Ann Periodontol 4,1-6.
Gribing N, Jansson K (2013). Guava: A Tool for Prevention of Periodontitis in
Adolescents Infected with Aggregatibacter actinomycetemcomitans? Department of
Odontology, Umea University.
Haubek D, Ennibi OK, Poulsen K, Væth M, Poulsen S, Kilian M (2008). Risk of
aggressive periodontitis in adolescent carriers of the JP2 clones of Aggregatibacter
(Actinobacillus) actinomycetemcomitans in Morocco: a prospective longitudinal
cohort study. Lancet 371: 237-42.
Henderson B, Ward JM, Ready D (2010). Aggregatibacter actinomycetemcomitans: a
triple A*periodontopathogen? Periodontal 2000 54; 78–105.
Höglund Åberg C (2013). Exotoxins of Aggregatibacter actinomycetemcomitans and
periodontal attachment loss in adolescents. (Dissertation) Umeå, Sweden: Umea
University.
Höglund Åberg C, Haubek DKwamin F, Johansson A, Claesson R (2014a).
Leukotoxic Activity of Aggregatibacter actinomycetemcomitans and Periodontal
Attachment Loss.
Höglund Åberg C, Kwamin F, Claesson R, Dahlén G, Johansson A, Haubek D
(2014b). Progression of attachment loss is strongly associated with presence of the
JP2 genotype of Aggregatibacter actinomycetemcomitans: a prospective cohort study
of a young adolescent population. J Clin Periodontol 41: 232-241.
Johansson A. Aggregatibacter actinomycetemcomitans Leukotoxin: A Powerful Tool
with Capacity to Cause Imbalance in the Host Inflammatory Response. Toxins 2011;
3: 242-259.
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Kwamin F, Gref R, Haubek D, Johansson A (2012). Interactions of extracts from
selected chewing stick sources with Aggregatibacter actinomycetemcomitans. BMC
Res Notes 5: 203.
Lindhe J, Lang NP, Karring T (2008). Clinical Periodontology and Implant Dentistry
(fifth edition). Vol.1: Basic concepts 133-242. UK, Blackwell Munksgaard, ISBN:
9781405160995.
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FIGURES AND FIGURE LEGENDS
140
Viable cells (%)
120
100
80
60
40
20
0
0.0
1.4
4.1
12.3
37
111
Ratio Aa HK1519/THP-1 cell
333
1000
0.5
1
Figure 1a
120
Viable cells (%)
100
80
60
40
20
0
Control
0
0.03
0.06
0.12
0.25
Guava extract concentration (%)
Figure 1b
Figures 1: Viability test (neutral red uptake) of THP-1 cells differentiated to
macrophages with PMA and exposed to a) different concentration of UV-killed A.
actinomycetemcomitans strain HK1519 or (± SD of 4 replicates) or b) for a constant
concentration of 100 HK1519/THP-1 cell in various concentrations of guava extracts
for 2 hr.
13
LDH-release (%)
40
35
30
25
20
15
10
5
0
Without G
With G
G in Pyg 24 h
G in PBS 24 h
Aa strain
Figure 2
Figure 2. LDH release of THP-1cells cultured for 2 hr on biofilms of different “rough” strains
of A. actinomycetemcomitans in presence or absence of guava extract. The bacteria were
cultured in PYG-broth for 24 hr at 37ºC in a 96-welled microtiter plate. The biofilms of bacteria
were exposed to 0.5% guava extract for 30 min before the different test procedures were
initiated. Blue bars is experiments without guava extracts, red bars is experiments in presence
of 0.5% guava extracts, green bars is experiments where the guava extracts has been replaced
with PYG incubated for 24 hr at 37ºC before the THP-1 cells were added and violette bars is
experiments where the guava extracts have been replaced with PBS and incubated for 24 hr at
37ºC before the THP-1 cells were added.
14
Figure 3
Figure 3. LDH release of THP-1cells cultured for 2 hr on biofilms of two different
“rough” strains of A. actinomycetemcomitans in presence or absence of guava extract.
The bacteria were cultured in PYG-broth for 24 hr at 37ºC in a 96-welled microtiter
plate. The biofilms of bacteria were exposed to 0.5% guava extract for 30 min before
the guava extracts was replaced with PYG or PBS and incubated for 24 hr at 37ºC
before the THP-1 cells were added (± SD of 4 replicates).
15