1
Roles of Porphyromonas gulae proteases in bacterial and host cell biology
1
Alam Saki Urmi1,Hiroaki Inaba1*, Ryota Nomura,2 Sho Yoshida1, Naoya Ohara3, 4, Fumitoshi 2
Asai4, Kazuhiko Nakano,2 Michiyo Matsumoto-Nakano1 3
4
1Department of Pediatric Dentistry, Okayama University Graduate School of Medicine, 5
Dentistry and Pharmaceutical Sciences, Okayama 700-8556, Japan; 2Department of Pediatric 6
Dentistry, Osaka University Graduate School of Dentistry, Suita-Osaka 565-0871, Japan;
7
3Department of Oral Microbiology, Graduate School of Medicine, Dentistry and Pharmaceutical 8
Sciences and the Advanced Research Center for Oral and Craniofacial Sciences, Dental 9
School, Okayama University, Okayama 700-8558; 4Department of Pharmacology, School of 10
Veterinary Medicine, Azabu University, Sagamihara, Kanagawa 252-5201, Japan;
11
Running title: P. gulae possesses proteolytic activity 12
Key words: P. gulae, protease, hemagglutination, coaggregation, protein degradation 13
Funding: This research was supported by grants-in-aid for Scientific Research (20K09918 to 14
H.I., and 20H03897 to M. M.K.) from the Ministry of Education, Culture, Sports, Science and 15
Technology of Japan.
16 17
Address correspondence to:
18
Hiroaki Inaba, DDS, PhD 19
Department of Pediatric Dentistry 20
Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences 21
2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558, Japan 22
Email: [email protected] 23
24
2 Take away
1
P gulae possess trypsin protease activity as well as that of P. gingivalis.
2
P. gulae proteases are required for bacterial growth.
3
P. gulae proteases have an impact on hemagglutination coaggregation with A. viscosus.
4
Cell contact and adhesion-related human proteins were degraded by P. gulae proteases.
5
P. gulae proteases were shown to cleave γ-globulin, fibrinogen, and fibronectin.
6
3 Abstract
1
Porphyromonas gulae, an animal-derived periodontal pathogen, expresses several virulence 2
factors, including fimbria, lipopolysaccharide (LPS), and proteases. We previously reported 3
that its invasive efficiency was dependent on fimbriae types. In addition, P. gulae LPS 4
increased inflammatory responses via toll-like receptors. The present study was conducted 5
to investigate the involvement of P. gulae proteases in bacterial and host cell biology. P. gulae 6
strains showed an ability to agglutinate mouse erythrocytes and also demonstrated 7
coaggregation with Actinomyces viscosus, while the protease inhibitors antipain, PMSF, TLCK, 8
and leupeptin diminished P. gulae proteolytic activity, resulting in inhibition of hemagglutination 9
and coaggregation with A. viscosus. In addition, specific proteinase inhibitors were found to 10
reduce bacterial cell growth. P. gulae inhibited Ca9-22 cell proliferation in a multiplicity of 11
infection- and time-dependent manner. Additionally, P. gulae-induced decreases in cell 12
contact and adhesion-related proteins were accompanied by a marked change in cell 13
morphology from well spread to rounded. In contrast, inhibition of protease activity prevented 14
degradation of proteins, such as E-cadherin, β-catenin, and focal adhesion kinase, and also 15
blocked inhibition of cell proliferation. Together, these results indicate suppression of the 16
amount of human proteins, such as γ-globulin, fibrinogen and fibronectin, by P. gulae proteases, 17
suggesting that a novel protease complex contributes to bacterial virulence.
18 19
4 1. Introduction
1
The animal biotype of the human periodontal pathogen Porphyromonas gingivalis, referred to 2
as P. gulae, has been recovered from gingival sulcus samples of a variety of species, such as 3
bear, canine, cat, coyote, marsupial, monkey, ovine, and wolf (Fournier et al., 2001; Mikkelsen 4
et al., 2008; Borsanelli et al, 2017). P. gulae is a black-pigmented, Gram-negative 5
coccobacillus, asaccharolytic, anaerobic, non-motile, non-spore-forming organism (Fournier et 6
al., 2001; Senhorinho et al., 2011), with its presence noted in significantly greater numbers in 7
canines and cats with periodontitis as compared to those with healthy gingiva (Senhorinho et 8
al., 2011; Iwashita et al., 2019). Other studies also detected P. gulae in human gingival 9
tissues, both with and without periodontitis (Yamasaki et al., 2012; Gaetti-Jardim et al., 2015).
10
Additionally, P. gulae as well as P. gingivalis have abilities to adhere to and invade human 11
gingival epithelial cells, though those activities are dependent on fimbriae (Inaba et al., 2019).
12
Bacterial pathogens possess several virulence factors, such as proteases, capsules, pili, 13
lipopolysaccharide, β-lactamase, alkaline phosphatase, phospholipases, and toxins 14
(Casadevall and Pirofski 2001; Webb and Kahler 2008; Bomberger et al., 2009). Bacterial 15
virulence is frequently involved with various factors related to bacterial or host cell biology, 16
such as adhesion and invasion, intracellular and extracellular survival mechanisms, nutrient 17
acquisition, biofilm formation, host cell damage, and virulence regulation (Webb and Kahler 18
2008). The functions of virulence factors are classified into two types, requisite and 19
contributory (Casadevall and Pirofski 2001). Requisite virulence factors, such as toxins and 20
polysaccharide capsules, serve to discriminate pathogens from nonpathogenic organisms and 21
confer pathogenicity to the microbe, leading to development of disease. On the other hand, 22
contributory virulence factors, such as proteases and phospholipases, function to worsen 23
disease conditions (Casadevall and Pirofski 2001).
24
The roles of proteases in bacterial pathogenicity have been reported to be degradation 25
of host tissue components for growth and proliferation, and avoidance of host immune 26
defenses (Sabotič and Kos 2012). Secreted proteases have also been implicated in the 27
bacterial pathogenesis and shown to have several immunomodulating activities, including 28
5 release of pro-inflammatory cytokines, extracellular matrix degradation, immunoglobulin G 1
(IgG) cleavage, and degradation of other immunoglobulins (Sabotič and Kos 2012). Cysteine 2
and serine proteases are known to be important pathogenic factors for periodontal pathogens, 3
including P. gingivalis, Treponema denticola, Tannerella forsythia, and Fusobacterium 4
nucleatum (Holt and Ebersole 2005; da Silva 2017). Moreover, cysteine and serine proteases 5
produced by periodontal pathogens have been reported to be capable of degrading periodontal 6
tissues and inactivation of host defense effectors, which promote pathological alterations 7
associated with development and progression of periodontal disease (Guo et al., 2010; Doron 8
et al., 2014). Additionally, bacterial proteases may be involved in the initial attachment of P.
9
gulae to human oral keratinocytes and gingival epithelial cells (Lenzo et al., 2016; Inaba et al., 10
2019).
11
Here we report that P. gulae proteases were found to play an important role in 12
hemagglutination activity, coaggregation with Actinomyces viscosus, and bacterial growth, as 13
well as degradation of focal contact and adherence junction-related proteins in human gingival 14
epithelial cells, resulting in promotion of morphological changes and inhibition of cell 15
proliferation. Moreover, proteolytic enzymes were shown to cleave γ-globulin, fibrinogen, and 16
fibronectin in vitro. Together, these results suggest P. gulae proteases as a candidate 17
virulence factor.
18 19
6 2. Results
1
2.1. Hemagglutination activity 2
P. gulae exhibits several virulence characteristics similar to those of the human periodontal 3
pathogen P. gingivalis (Yamasaki et al., 2012; Lenzo et al., 2016; Inaba et al., 2019).
4
Furthermore, a number of Porphyromonas species isolated from companion animals have 5
been shown to possess bacterial proteases (Summanen et al., 2005; Summanen et al., 2009;
6
Lenzo et al., 2016). However, P. gulae proteases have not been clearly shown to have effects 7
on either bacterial or host cell biology. Hemagglutination of erythrocytes is considered to be 8
a characteristic feature of some bacterial species (Rajkumar et al., 2016). P. gulae and P.
9
gingivalis reportedly possess protease-related and hemagglutinin genes, while those were not 10
found in other Porphyromonas species (O'Flynn et al., 2015). We first examined P. gulae 11
proteases and the results showed distinct effects on hemagglutination activity in all examined 12
strains of P. gulae (Figure S1), including P. gulae ATCC 51700 as well as P. gingivalis ATCC 13
33377 (Figure 1A). Such activity has been reported to be related to the hemagglutinin- 14
adhesin domains of protease-related genes (Guo et al., 2010). In fact, incubation with P.
15
gingivalis significantly enhances hemagglutination of erythrocytes, while that is prevented by 16
P. gingivalis gingipain-specific inhibitors (Nakayama et al., 1995; Kadowaki et al., 2004).
17
Therefore, we examined the involvement of proteases in P. gulae hemagglutination activity 18
using protease inhibitors, including antipain, PMSF, TLCK, and leupeptin, which revealed 19
suppressed agglutination of mouse erythrocytes (Figure 1B). These results suggest that 20
bacterial proteases may contribute to the hemagglutination activity of P. gulae.
21
2.2. P. gulae growth in chemically defined medium 22
Previous studies have noted that several bacterial proteases, such as P. gingivalis gingipains, 23
Burkholderia cenocepacia HtrA, Enteroaggregative Escherichia coli Pic, and F. nucleatum 24
fusolisin, are essential for bacterial growth (Flannagan et al., 2007; Harrington et al., 2009;
25
Doron et al, 2014). Among those, P. gingivalis growth mediated by gingipains was reported 26
to increase when the bacterial cells were grown in chemically defined medium (CDM) (Grenier 27
et al. 2001). After inoculation in CDM for the present assays, P. gulae was found to be in the 28
7 exponential phase between 24 and 144 h, and then clearly in the stationary phase from 144 h 1
(Figure 2A). Furthermore, antipain, PMSF, TLCK, and leupeptin inhibited the growth of P.
2
gulae ATCC 51700 (Figure 2B), suggesting that P. gulae proteases may be essential for 3
bacterial growth.
4
2.3. Coaggregation reactions between P. gulae and A. viscosus 5
Coaggregation is known to play a role in formation of macroscopic clumps between different 6
bacterial species (Mutha et al., 2019) and the oral pathogens, such as P. gingivalis, T. denticola, 7
and Streptococcus gordonii, have been reported to produce proteases, leading to 8
coaggregation among oral microorganisms (Kadowaki et al., 2004; Cogoni et al., 2012; Mutha 9
et al., 2019). In addition, coaggregation of A. viscosus with Streptococci, Eikenella corrodens, 10
and P. gingivalis has been noted (Cisar et al., 1979; Ebisu et al., 1988; Kadowaki et al., 2004).
11
Thus, we examined the effects of proteases on coaggregation between P. gulae and A.
12
viscosus. P. gulae ATCC 51700 was found to coaggregate with A. viscosus ATCC 15987 13
(Figure 3A), while inhibition of P. gulae proteases significantly abrogated that activity of the 14
bacterium (Figure 3B), indicating that coaggregation reactions between P. gulae and A.
15
viscosus are regulated via the activity of P. gulae proteases.
16
2.4. P. gulae induces morphological changes and slows proliferation of Ca9-22 cells 17
To investigate the response of gingival epithelial cells (referred to here as Ca9-22 cells) when 18
co-cultured with P. gulae, the morphology of infected cells was examined by light microscopy.
19
P. gulae caused rounding and detachment of some cells in a multiplicity of infection (MOI)- 20
dependent manner (Figure 4A). A previous study reported that the nature of morphological 21
changes is linked to the proliferation rate of host cells infected with microorganisms (Inaba et 22
al., 2009). In a dehydrogenase activity assay, P. gulae inhibited cell proliferation in an 23
increasing MOI- and time-dependent manner (Figure 4B). P. gingivalis protease knockout 24
mutants were previously not found to induce cell rounding and detachment (Inaba et al., 2012), 25
thus we investigated whether P. gulae proteases have a role in the cell proliferation responses 26
of Ca9-22 cells to P. gulae. Pretreatments with the protease inhibitors, such as antipain, 27
PMSF, TLCK, and leupeptin prevented inhibition of Ca9-22 proliferation by P. gulae (Figure 28
8 4C). These results suggest that P. gulae proteases cause morphological changes in Ca9-22 1
cells, leading to inhibition of their proliferation.
2
2.5. Degradation of human focal contact and adherence proteins by P. gulae 3
Focal contact and adherence junction components, including E-cadherin, β-catenin, focal 4
adhesion kinase (FAK) and paxillin, have been reported to be associated with epithelial 5
morphology (Deakin and Turner 2011; Howard et al., 2011; Golubovskaya et al., 2012), and 6
also shown to be cleaved following bacterial protease stimulation in assays of 7
enteroaggregative E. coli Pet, group A Streptococci SpeB, P. gingivalis gingipains, and 8
Campylobacter jejuni HtrA (Cappello et al., 2011; Sumitomo et al., 2013; Zhou et al., 2015;
9
Elmi et al., 2015). Therefore we investigated whether human gingival epitheliolysis are 10
proteolytically processed upon bacterial challenge. Following P. gulae challenge at an MOI 11
of 500 for the indicated times, Ca9-22 cell lysates were analyzed by immunoblotting with equal 12
amounts of total protein. As shown in Figure 5A, β-catenin and FAK were markedly degraded 13
in a time-dependent manner, while E-cadherin and paxillin underwent discrete proteolysis by 14
P. gulae from 4 to 6 h after stimulation. In contrast, no proteolysis was detectable in the 15
control cells (bacteria free). Furthermore, P. gulae-induced proteolytic capacity varied in an 16
MOI-dependent manner (Figure 5B). To test whether the observation of proteolysis in Ca9- 17
22 cells was triggered by bacterial proteinases, we next examined the effects of protease 18
inhibitors against proteolytic attack. Pretreatment of Ca9-22 cells with the protease inhibitors 19
(100 μM), such as antipain, PMSF, TLCK, and leupeptin, prevented cleavage of β-catenin and 20
paxillin induced by P. gulae stimulation (Figure 5C), while the combination of TLCK and 21
leupeptin enhanced those effects. On the other hand, combined antipain and PMSF 22
prevented degradation of E-cadherin and FAK, whereas TLCK and leupeptin together showed 23
only weak effects (Figure 5C). These findings suggest that P. gulae may be associated with 24
protein degradation, resulting in cellular contraction and rounding.
25
2.6. Degradation of human glycoprotein proteins by P. gulae 26
Fibronectin and fibrinogen are essential extracellular matrix glycoproteins involved in various 27
platelet functions, including adhesion, migration, and aggregation, resulting in the involvement 28
9 of immune responses (To & Midwood 2011; Induruwa et al., 2018). As for mucosal immunity, 1
IgG has been strongly implicated as an important factor in the immune system (Sistig et al., 2
2002), while λ-globulin has been assessed as an indicator of IgG (Chorfi et al., 2004). Other 3
previous reports have noted that several different human glycoproteins were degraded 4
following bacterial protease stimulation, such as Neisseria meningitides IgA1, Pseudomonas 5
aeruginosa elastase, P. gingivalis gingipain, and T. denticola PrtP (Lin et al., 1997;
6
Schmidtchen et al., 2003; Kadowaki et al., 2004; Miao et al., 2011). Therefore, we examined 7
whether the human glycoproteins λ-globulin, fibrinogen, and fibronectin are proteolytically 8
processed upon P. gulae challenge (Figure 6A and B). λ-globulin was degraded in a bacterial 9
number- and time-dependent manner, while proteolysis of both fibrinogen and fibronectin was 10
apparent as early as 1 h following P. gulae stimulation, thus protease inhibitors were used to 11
examine involvement in glycoprotein degradation (Figure 6C, D, E and Figure S2).
12
Furthermore, degradation of γ-globulin was completely inhibited by antipain, TLCK, and 13
leupeptin, though PMSF did not have such an effect, while degradation of fibrinogen and 14
fibronectin was diminished by those inhibitors. Together, these findings suggest that P. gulae 15
proteases process human proteins in a proteolytic manner.
16 17
10 3. Discussion
1
Periodontal diseases, including gingivitis and chronic periodontitis, are highly prevalent 2
infectious diseases that occur in humans and animals (Pihlstrom et al. 2005; Yamasaki et al., 3
2012). P. gulae and P. gingivalis have been found to be associated with gingival bleeding and 4
bone loss (Gaetti-Jardim et al., 2015), while periodontal destruction, including gingival 5
connective tissue and bone, as well as loss of supporting gingival connective tissue and bone 6
in association with periodontitis are important consequences of the presence of subgingival 7
bacterial flora (Holt and Ebersole 2005). Additionally, bacterial proteolytic enzymes have 8
been clearly shown to be major players in that destruction (Holt and Ebersole 2005).
9
Although the function of P. gulae proteases have yet to be investigated, based on biochemical 10
and functional similarities with P. gingivalis observed in this study, including enzyme 11
characteristics, hemagglutination, and degradation of host proteins, some conclusions can be 12
drawn.
13
P. gingivalis gingipains are thought to play critical roles in the pathogenesis and 14
development of periodontitis (Greiner et al., 2003, Imamura, 2003). It has also been shown 15
that proteolytic enzymes of P. gingivalis are capable of producing large amounts of thrombin 16
and can induce multiple pro-inflammatory responses in periodontitis pathogenesis (Holt and 17
Ebersole 2005). P. gingivalis gingipains are reportedly involved in degradation of focal 18
contact and adherence junction components, such as E-cadherin, paxillin, FAK, and β-catenin 19
(Hintermann et al., 2002; Inaba et al., 2004; Katz et al., 2002; Nakagawa et al., 2006; Elmi et 20
al., 2015; Zhou et al., 2015). Additionally, these phenomena might play important roles in the 21
pathogenesis of periodontal disease (Katz et al., 2002). In the present study, P. gulae 22
proteases were found to be associated with protein degradation, including E-cadherin, paxillin, 23
FAK, and β-catenin, resulting in reduced cell-cell contact and thus gingival epithelial cell 24
proliferation defects (Figure 5 and 6). The host proteins, such as γ-globulin, fibrinogen, and 25
fibronectin, have been were previously shown to be digested by P. gingivalis proteases 26
(Kadowaki et al., 2004). Since γ-globulin contributes to host defense mechanisms, these 27
findings suggest that their degradation is related to facilitation and prolongation in periodontal 28
11 disease (Kadowaki and Yamamoto 2003). During the course of inflammatory periodontal 1
disease, fibronectin is cleaved following protease stimulation (Stanley et al., 2008), while 2
periodontal tissues, including a large amount of fibrinogen, are damaged and spontaneous 3
bleeding is frequent (Bamford et al., 2007). Our results showed that P. gulae proteases 4
caused loss of γ-globulin, fibrinogen, and fibronectin (Figure 6). Together, these finding may 5
implicate involvement of P. gulae proteases in periodontal pathogenesis.
6
Hemagglutination is the process of aggregation of erythrocytes caused by bacterial 7
virulence factors adhering to two or more erythrocytes, and considered to contribute to 8
bacterial pathogenicity (Haraldsson et al., 2005). Those activities of the bacterial strains, 9
such as P. gingivalis, F. nucleatum and Prevotella melaninogenica, have been found to differ 10
(Chandad et al., 1996; Roques et al., 2000; Haraldsson et al., 2005), and those findings 11
indicated that hemagglutination of P. gingivalis and F. nucleatum may be linked to bacterial 12
pathogenicity, while the relationship of P. melaninogenica hemagglutination with its 13
pathogenicity is at a low level. In our study, the hemagglutination titers of the P. gulae strains, 14
except for D066, were higher than that of P. gingivalis ATCC 33277 (Figure 1 and 1S), 15
suggesting that P. gulae pathogenicity may be equivalent to or greater than that of P. gingivalis.
16
Bacterial proteases produced by microorganisms, such as P. gingivalis, E. coli, Shigella flexneri, 17
and Vibrio species, have been reported to show hemagglutinin activity with erythrocytes from 18
a variety of species (Nakayama et al., 1995; Miyoshi, 2013; Ruiz-Perez and Nataro, 2013).
19
Here, we found that cysteine and serine protease inhibitors had a greater decrease of 20
hemagglutination (Figure 1B). Our results suggest that hemagglutinin activity may be 21
induced in vitro by P. gulae proteases as well as P. gingivalis gingipains.
22
Hemin has been shown to be an important nutrient for P. gingivalis growth (Genco et al., 23
1994), while hemin acquisition mechanisms are known to be involved in hemagglutination, 24
hemolysis, binding, and degradation of the hemoglobin molecule (Lewis et al., 1999). All P.
25
gulae strains reportedly possess hemolytic activity, such as β-hemolysis, which has been 26
defined as a clear zone around the area of bacterial growth. (Senhorinhoet al., 2012).
27
Previous studies have also noted that P. gingivalis gingipains facilitate hemolysis and growth 28
12 in nutrient poor conditions (Lewis et al., 1999; Shi et al., 1999; Greiner et al., 2003; Imamura, 1
2003). The present results suggest a role for P. gulae proteases based on findings showing 2
that several protease inhibitors inhibited bacterial growth (Figure 2B). When these findings 3
are considered, P. gulae proteases may be required for growth of the bacterium.
4
Previous reports have noted the possibility that bacterial coaggregation promotes 5
interactions between oral microorganisms, and speculated that to be a mechanism involved in 6
formation of dental plaque and other types of biofilm (Bradshaw et al., 1998; Kramer et al., 7
2000; Mutha et al., 2019). Several bacterial proteases, such as P. gingivalis gingipains, T.
8
denticola chymotrypsin-like proteases, and S. gordonii Challisin, have been reported to play 9
important roles in coaggregation with different oral species, leading to synergy in microbial 10
community development and host tissue pathogenesis (Kadowaki et al., 2004; Cogoni et al., 11
2012; Mutha et al., 2019). Actinomyces species are known to be initial colonizers and play a 12
special role as scaffold builders during initial biofilm formation on an enamel surface (Tronstad 13
and Sunde, 2004; Khemaleelakul et al., 2006). Among them, A. viscosus has also been 14
implicated to be involved in development of root canal caries and periodontal disease (Dung 15
and Liu, 1999; Noiri Y and Ebisu, 2000). It is also possible that oral pathogens, such as P.
16
gingivalis, T. denticola, and A. viscosus, are related to biofilm formation in the plaque-free zone 17
located at the bottom of human periodontal pockets as well as progression of periodontitis 18
(Noiri Y and Ebisu, 2000). Furthermore, P. gingivalis fimbriae and gingipains have been 19
reported to mediate coaggregation with A. viscosus (Goulbourne and Ellen, 1991; Kadowaki 20
et al., 2004). In the present study, the coaggregation of P. gulae with A. viscosus was 21
dependent on the presence of P. gulae proteases (Figure 3). Collectively, these findings 22
suggest that oral bacterial proteases mediate adherence of the bacterium.
23
In conclusion, P. gulae proteases are possible crucial virulence factors related to 24
colonization and survival of invading bacteria, as well as host defense and tissue destruction, 25
and may be an important therapeutic target. Additionally, previously findings that bacterial 26
invasion and cellular inflammatory responses are mediated by P. gulae virulence factors, such 27
as fimbriae and LPS. The present findings suggest that P. gulae may be involved in both the 28
13 pathogenesis and continuing presence of periodontal disease. Additional studies are needed 1
to directly validate the associations of protease functions and periodontal pathogenesis.
2 3
14 4. Experimental procedures
1
4.1. Bacterial and cell cultures 2
The bacterial strains used in the present study were P. gingivalis ATCC 33277, P. gulae ATCC 3
51700, D040, D044, D049, D066, and D077, and A. viscosus ATCC 15987. Bacterial cells 4
were grown in Trypticase soy broth supplemented with yeast extract (1 mg/ml), menadione (1 5
g/ml), and hemin (5 g/ml), as described previously (Inaba et al., 2009). Ca9-22 cells, 6
originated from human gingival epithelia, were obtained from the Japanese Collection of 7
Research Bioresources (Tokyo, Japan) and cultured in DMEM (Wako, Osaka, Japan) 8
supplemented with 10% fetal bovine serum (FBS) at 37C in 5% CO2. 9
4.2. Chemicals 10
Antipain, a cysteine and serine protease inhibitor, N-p-tosyl-L-Lysine chloromethyl ketone 11
(TLCK), a serine protease inhibitor, and phenylmethylsulfonyl fluoride (PMSF), a serine 12
protease inhibitor, were purchased from Sigma-Aldrich (St. Louis, MO). Leupeptin, a cysteine 13
and serine protease inhibitor, was purchased from Peptide Institute (Osaka, Japan). Samples 14
were preincubated with various concentrations (100 μM) of the inhibitors at 37°C for 2 h prior 15
to addition of substrates. All inhibitors were dissolved in 0.1% dimethyl sulfoxide (DMSO).
16
4.3. Hemagglutination assay 17
Hemagglutination assays were performed as previously described (Nakayama et al., 1995), 18
with some modifications. Briefly, cultured P. gulae cells were centrifuged, washed, and 19
resuspended in phosphate-buffered saline (PBS) at an optical density of 0.25 at 600 nm.
20
Serial two-fold dilutions of the bacterial suspension in PBS were prepared; 100 µl aliquot of 21
each dilution mixed with an equal volume of 2.5% mouse erythrocytes. The suspension was 22
then incubated in a round-bottom microtiter plate for 3 h.
23
4.4. Growth curve assays for determination of general proteinase activity 24
Growth curve assays were carried out in CDM as previously described (Grenier et al., 2001), 25
with some modifications. CDM contains NaH2PO4 (10 mM), KCl (10 mM), citric acid (2 mM), 26
boric acid (5 μM), CaCl2 (20 μM), MgCl2 (1.25 mM), 3% pancreatic hydrolysate of casein 27
15 (tryptone), ɑ-ketoglutarate (50 mM), hemin (7.5 μM), and menadione (3 μM). P. gulae cells 1
were adjusted at OD600 of 1.0, then the bacterial suspension was diluted 1:5 with fresh CDM 2
and incubated anaerobically at 37ºC. Bacterial growth was monitored every 12 h at 600 nm 3
using a Novaspec III spectrophotometer (Biochrom, Holliston, MA, USA) for up to192 h.
4
4.5. Coaggregation assay 5
P. gulae were subjected to an assay of coaggregation with A. viscosus, as previously described 6
(Kadowaki et al., 2004). Briefly, bacterial cells were resuspended to 5×108 cells per ml in 7
coaggregation buffer (10 mM Tris-HCl, pH 8.0, 0.1 mM CaCl2, 0.1 mM MgCl2, 0.02% NaN3, 8
0.15 M NaCl). Equal volumes (500 µl) of two bacterial suspensions were mixed together, and 9
incubated at room temperature for 48 h. Coaggregation was monitored at 550 nm using a 10
Novaspec Plus spectrophotometer (Amersham Biosciences, Little Chalfont, UK).
11
4.6. Cell proliferation assay 12
Infected or control Ca9-22 cells proliferation was incubated with a Cell Counting Kit (Dojindo, 13
Kumamoto, JAPAN) for 1 h at 37°C and absorbance at 450 nm recorded on SH-1000 Lab 14
microplate reader (Corona Electric) at 450 nm.
15
4.7. Western blotting 16
Ca9-22 cells were solubilized in cell lysis/extraction reagent (Sigma-Aldrich) containing a 17
protease inhibitor cocktail (Thermo Scientific). Immunoblotting was performed as described 18
previously (Inaba et al. 2009). Briefly, blots were probed at 4°C overnight with the following 19
primary antibodies: β-catenin, 1:1000; E-cadherin, 1:1000; FAK, 1:1000; and paxillin, 1:1000 20
(Cell Signaling Technology, Beverly, MA). Blots were stripped and probed with anti-β-actin 21
antibody (Cell Signaling Technology) as a loading control. Proteins were detected using a 22
Pierce ECL Substrate (Thermo Scientific).
23
4.8. Host protein cleavage assay 24
Recombinant human γ-globulin and fibrinogen were purchased from Sigma-Aldrich, and 25
recombinant human fibronectin from Fujifilm Wako. These proteins (5 µg) were incubated 26
with bacterial suspensions at 37°C for 6 h. Following incubation, P. gulae cells were removed 27
by centrifugation and the supernatants were collected. Protein samples were then subjected 28
16 to SDS-PAGE and transferred electrophoretically to polyvinylidene fluoride (PVDF) membrane.
1
Staining of PVDF-bound proteins was performed with Coomassie brilliant blue R250.
2
4.9. Statistical analyses 3
Quantitative data are presented as means ± standard deviations (SDs). Statistical analyses 4
were performed using an unpaired Student t test.
5 6
Acknowledgments 7
This research was supported by grants-in-aid for Scientific Research (20K09918 to H.I., and 8
20H03897 to M.MN.) from the Ministry of Education, Culture, Sports, Science and Technology 9
of Japan. We dedicate this work to the deceased Dr. Kato. We have no conflicts of interest 10
to declare.
11 12
Author contributions 13
HI conceived, designed and performed the experiments, analyzed the data and wrote the 14
manuscript. ASU, RN, SY performed the experiments and analyzed the data. FA and KN 15
conceived of the study and oversaw the project. MMN conceived of the study, oversaw the 16
project and wrote the manuscript. All authors contributed to revisions of the article, and 17
approved the final version.
18 19
Conflict of interest 20
The authors have no conflicts of interest to declare.
21 22
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26 Figure legends
1
Figure 1. Hemagglutinating activity of P. gulae ATCC 51700.
2
(A) P. gulae ATCC 51700 and P. gingivalis ATCC 33277 cells were grown in TSB, then washed 3
with and resuspended in PBS at an optical density at 600 nm of 0.25. Suspensions and its 4
dilutions in a 2-fold series were applied to the wells of a microtiter plate from left to right, then 5
mixed with a mouse erythrocyte suspension and incubated at room temperature for 3 h. (B) 6
P. gulae ATCC 51700 cells were preincubated in PBS with/without protease inhibitors (100 μM), 7
such as antipain, PMSF, TLCK, and leupeptin, at 37°C for 2 h. Next, an equal volume of 1/32 8
diluted erythrocyte suspension was applied as described for panel A. Data are representative 9
of results from three independent experiments.
10 11
Figure 2. Anaerobic growth profile of P. gulae ATCC 51700.
12
(A) Overnight culture of P. gulae ATCC 51700 in TSB was diluted 5-fold with CDM and 13
incubated anaerobically at 37°C. Growth was monitored by measuring optical density at 600 14
nm every 12 h up to 192 h. Data are shown as the mean ± SD of three independent 15
experiments. (B) Bacterial suspensions were incubated with/without protease inhibitors (100 16
μM), such as antipain, PMSF, TLCK, and leupeptin, at 37°C for 144 h. Data are shown as 17
the mean ± SD of three independent experiments and were analyzed with a t-test. * and **, 18
P < 0.05 and P < 0.01 (Student’s t test) compared to P. gulae with DMSO without inhibitors 19
samples.
20 21
Figure 3. Coaggregation reaction between P. gulae ATCC 51700 and A. viscosus ATCC 22
15987.
23
(A) P. gulae ATCC 51700 cells were mixed with an equal volume of A. viscous ATCC 15987 24
cells and incubated for the indicated times. Coaggregation was monitored by measuring 25
optical density at 550 nm and represented as percentage. Data are shown as the mean ± SD 26
of three independent experiments. (B) P. gulae ATCC 51700 cell suspensions were 27
preincubated with/without different protease inhibitors (100 μM), such as antipain, PMSF, 28
27 TLCK, and leupeptin, at 37°C for 2 h. Next, each was mixed with an equal volume of A.
1
viscous ATCC 15987 and incubated for 48 h at room temperature. Coaggregation was 2
monitored and represented as described for panel A. Data are shown as the mean ± SD of 3
three independent experiments. *P <0.05, **P <0.01 (Student’s t test), as compared with 4
untreated P. gulae and A. viscous.
5 6
Figure 4. P. gulae ATCC 51700 induces morphological changes and slows proliferation 7
in Ca9-22 cells.
8
(A) Light microscopy showing morphology of Ca9-22 cells infected with P. gulae ATCC 51700 9
at an MOI of 100, 200, and 500 for 6 h. (B) Proliferation of Ca9-22 cells measured by 10
tetrazolium following infection with P. gulae ATCC 51700 at the times and moi indicated. Data 11
are expressed as relative ratio of infected/uninfected are means ± SD from three independent 12
experiments analyzed with a t-test. *P < 0.01 (Student’s t test) compared with uninfected 13
cells (0 h). (C) The protease inhibitors antipain, PMSF, TLCK, and leupeptin (100 μM) were 14
added to Ca9-22 cells 2 h prior to challenge with P. gulae ATCC 51700. Proliferation of Ca9- 15
22 cells measured by tetrazolium following infection with P. gulae ATCC 51700 treated with 16
each of the inhibitors at an MOI of 500 for 6 h. Data are shown as the mean ± SD of three 17
independent experiments. *P <0.01 compared with uninfected and untreated cells.
18 19
Figure 5. Degradation of focal adhesion and cell-cell adhesion proteins of epithelial cells 20
by P. gulae ATCC 51700.
21
(A) Ca9-22 cells were infected with P. gulae ATCC 51700 at an MOI of 500 for the indicated 22
times, and then lysates of infected and uninfected cells were subjected to immunoblotting.
23
(B) Ca9-22 cells were infected with P. gulae ATCC 51700 at an MOI of 100, 200, or 500 for 6 24
h. The lysates were analyzed by immunoblotting with antibodies. (C) The protease 25
inhibitors antipain PMSF, TLCK, and leupeptin (100 μM) were added to Ca9-22 cells 2 h prior 26
to bacterial challenge with P. gulae ATCC 51700 at an MOI of 500 for 6 h. Expression profiles 27
of focal adhesion and cell-cell adhesion molecules were examined by immunoblotting. β- 28
28 actin was included as a loading control. Data are representative of three independent 1
experiments.
2 3
Figure 6. Degradation of various human proteins by P. gulae ATCC 51700.
4
(A) The recombinant human proteins γ-globulin, fibrinogen, and fibronectin (5 µg) were 5
incubated with P. gulae ATCC 51700 (1x107, 5x107, 1x108 cells) at 37ºC for 6 h. (B) 6
Recombinant human proteins (5 µg), shown in panel A, were incubated with P. gulae ATCC 7
51700 (5x107 cells) at 37ºC for the indicated times. (C-E) The protease inhibitors antipain, 8
PMSF, TLCK and leupeptin (100 μM) were added 2 h prior to addition of the bacterial 9
suspension. Recombinant human proteins (5 µg), shown in panel A, were incubated with P.
10
gulae ATCC 51700 (5x107 cells) after treatment with the inhibitors described for panel C-E, at 11
37ºC for 6 h. Expression of protein profile was analyzed by SDS-PAGE. Following transfer 12
to PVDF membranes, proteins were visualized by Coomassie brilliant blue R250 staining.
13
Data are representative of three independent experiments.
14
