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Original Article

Involvement of PorK, a component of the type IX secretion system, in Prevotella

melaninogenica pathogenicity

Yoshio Kondo1_{, Keiko Sato}2_{, Keiji Nagano}3_{, Miyuki Nishiguchi}1_{, Tomonori Hoshino}1_{*, Taku}

Fujiwara1_{, Koji Nakayama}2

1_{Department of Pediatric Dentistry and} 2_{Department of Microbiology and Oral Infection,}

Graduate School of Biomedical Sciences, Nagasaki University, 1-7-1 Sakamoto, Nagasaki,

852-8588, Japan and 3_{Department of Microbiology, School of Dentistry, Aichi Gakuin}

University 1–100 Kusumoto-cho, Chikusa-ku, Nagoya, Aichi, 464-8650, Japan

This is the author manuscript accepted for publication and has undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1348-0421.12638

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*Present address: Division of Pediatric Dentistry, Department of Human Development and

Fostering, Meikai University School of Dentistry, 1-1 Keyakidai, Sakado, Saitama, 350-0283

Japan

Short Running Title: PorK in P. melaninogenica

Correspondence address:

Yoshio Kondo, Department of Pediatric Dentistry, Nagasaki University Graduate

School of Biomedical Sciences, 1-7-1, Sakamoto, Nagasaki, 852-8588, Japan Tel:

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List of Abbreviations: 2D-PAGE, 2-dimensional polyacrylamide gel electrophoresis;

CB, cacodylate buffer; CBB, coomassie brilliant blue; CDS, coding sequence; CFU,

colony forming unit; CTD, C-terminal domain; Em, erythromycin; Em

r

,

erythromycin-resistant; MS, mass spectrometry; OD, optical density; PBS, phosphate

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ABSTRACT

Prevotella melaninogenica is a Gram-negative anaerobic commensal bacterium that resides in

the human oral cavity and is isolated as a pathogen from suppurative diseases both inside and

outside the mouth. However, little is known about the pathogenic factors of P. melaninogenica.

The periodontal pathogens, Porphyromonas gingivalis and Tanerella forsythia, secrete virulence

factors such as protease and bacterial cell surface proteins via a type IX secretion system (T9SS)

that are involved in pathogenicity. P. melaninogenica also possesses all known orthologs of the

T9SS. In this study, a P. melaninogenica GAI 07411 mutant deficient in the orthologue of the

T9SS-encoding gene, porK, was constructed. In the porK mutant, hemagglutination and biofilm

formation were decreased. Furthermore, following growth on skim milk-containing medium,

the diameter of the halo surrounding the porK mutant was smaller than that of the wild-type

strain suggesting a decrease in the secretion of proteases outside the bacterium. To investigate

this in detail, culture supernatants of wild-type strains and porK mutant strains were purified

and compared by two-dimensional electrophoresis. In the mutant strain, fewer spots were

detected indicating that the number of secreted proteins was decreased. In infection experiments,

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reduced compared with the wild-type strain. These results suggested that P. melaninogenica

secretes potent virulence factors via the T9SS that contribute to the pathogenesis of this

bacterium.

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INTRODUCTION

In many cases, dental infections are complex, involving oral streptococci and anaerobic

bacteria (1, 2). The genus Prevotella is a major dental pathogen, and an increase in

β-lactamase-producing Prevotella spp. has been reported (3-5). Consistent with this, a decrease

in susceptibility to penicillin antibacterial drugs and cephem antibiotics, the first-line drugs used

in the dental field, has been reported as an emerging problem (6). The reduction in susceptibility

to antibacterial drugs of these β-lactams has become a serious problem in the treatment of dental

infections, and there is concern about the progression to serious infectious diseases such as

cellulitis and cervical abscesses.

Prevotella melaninogenica is a black-pigmented anaerobic bacterium that is classified into the

genus Prevotella belonging to the phylum Bacteroidetes. This bacterium is frequently found

among the normal flora of the upper respiratory tract and plays a major role as an

important human pathogen in various anaerobic infections, often along with other aerobic

and anaerobic bacteria. Furthermore, P. melaninogenica has often been associated with

aspiration pneumonia; however, little has been reported regarding its virulence factors.

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reported in the phylum Bacteroidetes. The Porphyromonas gingivalis T9SS includes the PorK,

PorL, PorM, PorN, PorP, PorQ, PorT, PorU, PorV (PG27, LptO), PorW and Sov proteins.

Coding sequences (CDSs) encoding proteins homologous to the P. gingivalis T9SS proteins are

present in the genomes of several bacteria in the phylum Bacteroidetes (7). P. gingivalis

secretes a variety of virulence factors, such as the extracellular and cell-surface cysteine

proteinases Arg-gingipain and Lys-gingipain via the T9SS (8, 9). Tanerella forsythia also

secretes some virulence-related proteins such as surface layer (S-layer) glycoproteins (TfsA and

TfsB) and the leucine-rich-repeat protein BspA (10). In these secreted proteins, ~70 amino acid

residues are conserved and this is known as the C-terminal domain (CTD). Recently, the CTD

was reported to not only relate to cell surface binding protein, but was also considered to

function as a secretion signal as it passes through the outer membrane (11).

In this study, genomic analysis showed that P. melaninogenica possess the genes encoding all

known components of the T9SS. Furthermore, a mutant strain of porK, a constituent protein of

T9SS, was constructed to investigate the involvement of T9SS in P. melaninogenica

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MATERIALS AND METHODS

Bacterial strains and culture conditions

All bacterial strains and plasmids used in this study are listed in Table 1. P. melaninogenica was

maintained on Brucella HK agar (Kyokuto Pharmaceutical Industrial Co., Tokyo, Japan)

supplemented with 5% (v/v) heat-inactivated rabbit blood at 37C under anaerobic conditions (10% CO2, 10% H2 and 80% N2). When P. melaninogenica cells were used in these experiments,

they were cultivated in TS broth (Becton, Dickinson, and Co., Sparks, MD) supplemented with

hemin (5 µg ml-1_{) or modified GAM broth ((Nissui Pharmaceutical Co., Tokyo, Japan). For the}

selection and maintenance of the erythromycin (Em)-resistant P. melaninogenica strain, Em was

added to the medium at a concentration of 5 µg ml˗1.

Construction of plasmids and bacterial strains

The P. melaninogenica porK deletion mutant was constructed as follows. A 1.0 kb upstream

region of porK was amplified by PCR from the chromosomal DNA of P. melaninogenica with

the primer pair 5-porK_up-XhoI and 3-porK_up-BamHI using a PCR kit (Advantage-HF 2 PCR

kit; TAKARA Bio Inc., Shiga, Japan). The amplified DNA was then cloned into a T-vector

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fragment was then inserted into the XhoI and BamHI sites of pBluesript II SK(-) to generate

pML001. A 1.0 kb downstream region of porK was amplified from the chromosomal DNA of P.

melaninogenica with the primer pair 5-porK_dn-BamHI and 3-porK_dn-NotI. The amplified

DNA was cloned into T-vector and digested with BamHI and NotI. The resulting fragment was

then inserted into the BamHI and NotI sites of pML001 to generate pML002. The 1.1 kb BamHI

ermF DNA cassette was inserted into the BamHI site of pML002, resulting in pML003. Then,

pML003 was digested with XhoI and NotI and inserted into the XhoI–NotI site of pTCB plasmid

(12), resulting in pML004. After mating of Escherichia coli S17-1 (13) pML004 with P.

melaninogenica, an Emr_{transconjugant was obtained and double recombination resulted in}

deletion of porK (KMD001).

Reverse transcription (RT)-PCR analysis

RT-PCR analysis was performed as described previously (14). The primers for RT-PCR analysis

(Table S1) were designed using Primer3 software (http://primer3.sourceforge.net/).

Antiserum preparation. Preparation of anti-PorK antisera has been described previously (15).

Antiserum was made against the amino acid sequence F387_{GLYDMAGNVAEWT}400_{from PorK}

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PorK-specific antibody in this study for use with P. melaninogenica.

Genome sequencing of P. melaninogenica GAI 07411

Bacterial cells of P. melaninogenica GAI 07411 were inoculated into TS broth and incubated

under anaerobic conditions. Genomic DNA was extracted using a MasterPure Gram-positive

DNA purification kit (Lucigen, WI, USA) according to the manufacturer’s instructions.

Single-molecule real-time sequencing reads were generated using a PacBio RSII sequencer

(Pacific Biosciences, CA, USA). Whole genome sequencing produced a total of 130,972 reads

with a mean length of 8,169 bp. Subsequent de novo assembly utilizing the HGAP3 protocol

yielded three scaffolds with 250-, 246- and 222-fold average reference coverage, respectively.

The genome sequence was annotated by DFAST (16) and deposited as P. melaninogenica GAI

07411 chromosome 1, chromosome 2 and the plasmid pMEL0001 in the

DDBJ/EMBL/GenBank database under accession numbers AP018049, AP018050 and

AP018051, respectively.

Hemagglutination assay

The hemagglutination assay was performed using rabbit blood as described previously (17).

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The biofilm formation assay was performed according to methods described previously (18).

Briefly, an overnight culture was diluted 1:100 into fresh TS broth, and incubated at 37°C for

24 hr. Following incubation, adherent cells were washed three times with PBS, stained with 100

µl of 0.1% crystal violet in water for 10 min and washed three times with PBS. The dye was

dissolved by the addition of 30% acetic acid (100 µl) and the optical density for each strain at

570 nm was determined by a Multi-Detection Microplate Reader. Data are expressed as the

mean and standard deviation of three independent experiments with three wells per strain. The

data of biofilm formation assays were analyzed using Student’s t-test.

Subcellular fractionation

Subcellular fractionation of P. melaninogenica cells was performed as previously described

(19).

Transmission electron microscopy (TEM)

Whole cell and ultrathin section samples were prepared for TEM. For the preparation of whole

cell samples, bacterial cells were washed in PBS, placed on a carbon support film grid, and

negatively stained with ammonium molybdate (containing 5 mM molybdenum), pH 7.0. Then,

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with 4% paraformaldehyde, 2.5% glutaraldehyde and 0.5% tannic acid in 100 mM sodium

cacodylate buffer pH 7.4 (CB) overnight at 4°C. After washing in CB, they were post-fixed with

1% osmium tetroxide in CB for 6 hr. After washing in CB, the fixed cells were dehydrated in a

series of 25%–100% ethanol and embedded in an epoxy resin ((Epok 812; Okenshoji Co., Ltd.,

Tokyo, Japan). The ultrathin sections were stained with 1% uranyl acetate and 1% lead citrate.

The stained samples (whole cells and ultrathin sections) were observed using a JEM1400 Plus

Electron Microscope (JEOL, Tokyo, Japan).

Two-dimensional gel electrophoresis (2D-PAGE) and MS spectrometry

2D-PAGE was performed as described previously (11). Briefly, a P. melaninogenica cell culture

was centrifuged at 6,000g for 30 min at 4C and the supernatant was separated from the pelleted cells. The proteins in the supernatant were concentrated by ammonium sulfate

precipitation (80% saturation) and harvested by centrifugation. The precipitated proteins were

resuspended in a cell lysis solution (7 M urea, 2 M thiourea, 4% CHAPS, 1 mM EDTA and 5

mM tributylphosphine). Proteins were identified by peptide-mass fingerprinting after in-gel

tryptic digestion as previously described (15).

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The virulence of P. melaninogenica wild-type strain and the porK mutant strain was measured

by mouse subcutaneous infection experiments (20-23). Bacterial cells were grown at 37°C until

an OD550_{of 1.0 was reached. The cells were then harvested, resuspended and adjusted to a}

concentration of approximately 1  1012_{CFU/ml in enriched TS broth. Female BALB/c mice (8} to 10 weeks of age) were challenged with subcutaneous injection of 0.2 ml of bacterial

suspension onto the depilated dorsal surface. Injected mice were examined daily for survival.

These experiments were repeated independently three times. For data analysis, Kaplan–Meier

plots were constructed and the log-rank test was used to evaluate the differences in mean

survival rates in the three experiments between mice infected with the wild-type and those

infected with the porK mutant.

RESULTS

Construction of a P. melaninogenica mutant deficient in T9SS protein

To investigate the role of PorK in P. melaninogenica, we attempted to construct a porK mutant

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25845, GAI 96524, GAI 00278, GAI 00319, GAI 07400, GAI 07402, GAI 07404, GAI 07406,

GAI 07408, GAI 07410 and GAI 07411 (Fig. 1A). We only obtained Em-resistant (Emr₎

transconjugants from P. melaninogenica strain GAI 07411. The replacement of porK was

confirmed in the Emr_{transconjugant by PCR of the extracted genomic DNA (Fig. 1B). The}

whole cell lysates of wild-type cells or Emr_{transconjugant cells were subjected to SDS-PAGE}

and immunoblot analysis with anti-PorK antiserum. An anti-PorK-immunoreactive protein was

detected at a molecular mass of 54 kDa in the wild-type strain that was not detected in the porK

mutant (Fig. 1C). These results confirmed that the Emr_{transconjugant was a porK mutant.}

Genome analysis of the T9SS-related gene group in P. melaninogenica GAI 07411

The whole genome sequence of P. melaninogenica GAI 07411 was obtained and deposited in

the DDBJ / EMBL / GenBank database (Fig. S1 and Table 2). The orthologs of T9SS-related

genes were identified by reciprocal BLASTP analysis (maximum E-value at 1e˗5) using the P.

gingivalis protein sequences (Table 3). Proteins secreted by T9SS can often be identified by

their conserved CTD, which targets them for secretion (24, 25). The majority of these CTDs

belong to the TIGRFAM protein domain family TIGR04183 (referred to as Por_secretion_tail).

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which were predicted proteases.

We performed RT-PCR using gene-specific primers for porL, porM and porN to investigate

whether these genes were transcribed in the porK mutant. We could amplify fragments using

each of the primer sets Fw_porL-RT and Rv_porL-RT, Fw_porM-RT and Rv_porM-RT, and

Fw_porN-RT and Rv_porN-RT, suggesting that the porL, porM and porN genes were

transcribed in the porK mutant (Fig. S2). These results indicate that porK was correctly deleted

and that the expression of the porL, porM, porN genes was unaffected at the transcriptional

level in the porK mutant.

Properties of the porK mutant

The porK mutant showed no pigmentation on blood agar plates (Fig. 2A) and no

hemagglutination (Fig. 2B). Biofilm formation, which can occur after infection with pathogenic

bacteria, is thought to be an important factor in the pathogenesis. Hence, in this study, a biofilm

formation assay was performed and the porK mutant was found to exhibit lower levels of

biofilm formation than the wild-type strain (Fig. 2C).

Membrane structure

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between the wild-type strain and the porK mutant (Fig. S3A). Furthermore, TEM observations

of ultrathin sections revealed that there was no clear difference in membrane structure between

the wild-type and porK mutant (Fig. S3B).

Reduction of protease activity in the porK mutant

After growth on Brucella agar plates containing skim milk, the halo around the porK mutant

was smaller than that around the wild-type strain (Fig. 2D). This suggested that the amount of

secreted protein, such as peptidase, produced by the porK mutant was decreased compared with

the wild-type strain. Next, the secreted proteins were compared by 2D-PAGE and the number of

spots stained by Coomassie Brilliant Blue (CBB) were decreased in the porK mutant compared

with the wild-type strain (Fig. 3). These results indicated that in the porK mutant the protein

secreted via the T9SS was not released outside of the bacterial cells. MS spectrometry was

performed to identify the wild-type strain-specific proteins (Table 5). Although some peptidases

were included in it, these did not contain the CTD proteins predicted by genome analysis.

Cell morphology of the porK mutant

The bacterial cells were negatively stained with ammonium molybdate and analyzed by

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surface (Fig. 4A). The porK mutant also possessed a pilus-like structure, but it was much longer

than that of the wild-type strain (Fig. 4B).

Contribution of PorK to virulence in P. melaninogenica

BALB/c mice were challenged with subcutaneous injections of P. melaninogenica bacterial

suspension (2  1011 _{CFU per animal), and their survival was monitored for 7 days. In total,} 80% of the mice challenged with the wild-type strain died within 2 days (Fig. 5). By contrast,

only 20% of the mice inoculated with the porK mutant died within 2 days. These results

suggested that PorK is involved in P. melaninogenica virulence and that this involvement might

be an indirect effect through the activities of the proteins secreted via the T9SS.

DISCUSSION

P. melaninogenica is a member of the normal human oral flora and can be grown from the

tongue, gingival crevices, saliva and plaques of healthy individuals (26-29). P. melaninogenica

is described as a "potential pathogen" (30-33) because it is commonly cultured as the sole

infectious agent in extraoral abscesses such as spondyl osteomyelitis, nematitis, peritoneal

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melaninogenica is also frequently cultured in the context of polymicrobial diseases including

brain abscesses, pleuropulmonary infections, endocarditis, illicit drug injection sites,

intra-abdominal infections, wound infections, necrotizing fasciitis, pyogenic infections,

decubitus and diabetic ulcers (35, 38-42). P. melaninogenica is also one of the most common

and abundant anaerobic species found in respiratory specimens from individuals with cystic

fibrosis (31, 43-45).

Stimulation of total cell lysates from P. melaninogenica showed low levels of cytokine

responses (IL-1α, IL-6 and TNFα in human monocytes and human gingival fibroblasts) via the

TLR2 signaling pathway but not the TLR4 pathway (46, 47). Therefore, P. melaninogenica may

have TLR2 agonist properties. Another study reported that P. melaninogenica supernatants

impaired phagocytosis by polymorphonuclear leukocytes (48). These studies show the effects of

complex mixtures on cellular responses, but no investigations have been made into the

individual pathogenic factors.

Despite this association with a wide variety of infectious diseases and cellular responses, little is

known about the contribution of P. melaninogenica to disease progression and molecular

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study, we succeeded in constructing a mutant strain of P. melaninogenica GAI 07411, which

will contribute greatly to the study of P. melaninogenica pathogenicity.

Exogenous DNA elimination mechanism in the recipient strain may influence mutant

construction. In this study, the porK mutant was successfully constructed using strain GAI

07411, whereas no mutant has been obtained from the other strains used including strain ATCC

25845. Since genome sequence of strain ATCC 25845 was available, genomic comparison in

exogenous DNA elimination mechanism between strains GAI 07411 and ATCC 25845 was

carried out. Two CRISPR-Cas regions were detected from strain GAI 07411, but not from strain

ATCC 25845. However, since the spacer sequence in CRISPR of strain GAI 07411 has no DNA

region highly homologous to pML004, it is suggested that pML004 is not excluded by the

CRISPR-Cas systems. On the other hand, regarding the restriction-modification system, two

and four regions were detected in strains GAI 07411 and ATCC 25845, respectively. This

suggests that the exclusion of external DNA is weaker in GAI 07411 than in ATCC 25845.

Furthermore, the plasmid pMEL0001 in strain GAI 07411 is the first plasmid found in P.

melaninogenica strains, which also suggests that strain GAI 07411 is tolerant to exogenous

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Twenty % of isolates of Prevotella spp. have been reported to be resistant to tetracycline (49).

Strain GAI 07411 was sensitive to tetracycline (0.7 μg ml˗1_{) and the genome of GAI 07411 has}

no tetracycline-resistant genes including tetQ.

We attempted to construct a porK complementary strain using three different approaches. 1) A

DNA fragment comprising an upstream region (2.0 kb) of porK, the whole porK gene, the tetQ

gene and a downstream region (2.0 kb) of porK was introduced into the porK mutant by

electroporation. 2) A shuttle plasmid (pTCB plasmid containing the promoter region of the

catalase-encoding gene in Porphyromonas glue, the whole porK gene, and a downstream region

(0.2 kb) of porK) was constructed and transconjugated into the porK mutant. 3) The plasmid

originally possessed by P. melaninogenica GAI 07411, pMEL0001, was purified and combined

with pKD375 (containing the tetQ DNA cassette in pUC19) (50) to construct a potent shuttle

plasmid. The DNA fragment (containing the promoter region of the catalase-encoding gene in P.

glue, the whole porK gene, and a downstream region (0.2 kb) of porK) was inserted into this

plasmid and introduced into the porK mutant by transformation. So far, a porK complementary

strain has not been obtained from any of the three methods. However, the expression of the porL,

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phenotype of the porK mutant is affected only by deficiency in porK.

The T9SS is commonly found among members of the phylum Bacteroidetes (7, 51), which

possess a cluster of T9SS genes in the following order: porK, porL, porM and porN (7, 52).

Most of the other T9SS genes are not clustered together and are not located near the porKLMN

operon. In this study, a component of the T9SS of P. melaninogenica GAI 07411 was identified

by genome analysis.

Some members in the genera Prevotella and Porphyromonas form black pigmented colonies

on the blood agar and heme derivatives, μ-oxobisheme and hematin are related to the black

pigments of P. gingivalis (53) and Prevotella intermedia (54), respectively. Many

microorganisms can produce heme via complex in vivo heme biosynthetic pathways. However,

all bacteria of the genera Prevotella and Porphyromonas lack enzymes that synthesize their own

heme. Prevotella spp. such as P. intermedia cannot survive without protoporphyrin IX-based

iron such as heme, hemoglobin and myoglobin, cytochrome c and catalase (55, 56). P.

melaninogenica, like other Prevotella spp. including P. intermedia, require heme for growth

(57). In this study, growth of the porK mutant appeared as white colonies. Proteins secreted via

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investigate the iron demand of P. melaninogenica and its ability to utilize iron sources from the

host.

Hemagglutination is caused by the attachment of bacterial molecules to two or more

erythrocytes and hemagglutination assays are widely used as a model to test for bacterial

adhesion to the host cells. In the porK mutant strain, hemagglutination was decreased compared

with the wild-type strain. Although lactose, galactose and raffinose-containing carbohydrates on

erythrocytes have been reported to be involved in the hemagglutination of P. melaninogenica

(58), the associated bacterial proteins remain to be identified. The results of this study showed

that proteins secreted via the T9SS are involved in the hemagglutination of P. melaninogenica.

Once biofilm is formed, P. melaninogenica can further damage the host tissue by the

production of collagenase (59) and lipase (both contributing to abscess formation). In the porK

mutant strain, biofilm formation was decreased compared with the wild-type strain, along with a

concomitant reduction in pathogenicity. In addition, it has also been reported that T9SS is

involved in biofilm formation in the periodontal pathogen Capnocytophaga ochracea (60).

Following growth on skim milk-containing agar plates, the halo formed around the wild-type

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peptidases secreted from the bacteria are decreased in the mutant. Furthermore, with 2D-PAGE

analysis, fewer spots were detected for the porK mutant indicating a decrease in secreted

proteins. Proteins secreted by the T9SS have a conserved CTD and pass through the outer

membrane (11, 25, 52, 61, 62). The CTD is necessary and sufficient for secretion by the T9SS. P.

gingivalis HBP35 and F. johnsoniae ChiA which lack their CTDs are not secreted and

heterologous fusion proteins with HBP35 and ChiA CTDs are efficiently secreted (61, 63).

Many T9SS CTDs of F. johnsoniae and P. gingivalis belong to the TIGRFAM protein domain

family TIGR04183 (7, 11, 52). However, the T9SS CTDs seem diversified, not everything falls

within the boundary of TIGR04183. For example, F. johnsoniae SprB is secreted by the T9SS

(64), but its C-terminal region shows no similarity with the TIGR04183 family members, but

rather belongs to an unrelated domain family TIGR04131 (24). Also, T. forsythia PorU is

secreted outside the bacterium via the T9SS, but its C-terminal region shows no similarity to

TIGR04183 (10). In T. forsythia, it seems that non-CTD family proteins are also secreted via

T9SS (10). Therefore, it is plausible that P. melaninogenica possesses T9SS cargo proteins that

do not belong to the TIGRFAM protein domain family TIGR04183. However, analysis of the

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the proteins T9SS-dependently secreted. This may be due to the fact that the culture

supernatants used in this study contained vesicles.

The results presented here show that PorK, a component of the T9SS, is involved in the

pathogenicity of P. melaninogenica. Secreted proteins appear to be important in the disease

process. In addition, a pilus-like structure was observed on the bacterial surface of P.

melaninogenica cells by electron microscopy, and the porK mutant strain showed a pilus-like

structure longer than that in the wild-type strain. Proteins secreted by the T9SS may affect the

construction of the pilus-like structure. We also found that biofilm formation is decreased in the

porK mutant, suggesting that the long pilus-like structure may disturb the biofilm formation in

the porK mutant; however, there is no evidence for a causal relationship between the elongated

pilus-like structure and poor biofilm formation in the porK mutant.

What proteinaceous factor(s) secreted by the T9SS in P. melaninogenica contribute to

pathogenesis of the bacterium remains to be determined, and further research is needed although

the animal experiment in this study strongly suggests the involvement of the T9SS in the

pathogenesis. Our findings in this study may provide novel insights into the development of

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ACKNOWLEDGEMENTS

We thank Prof. Kaori Tanaka (Division of Anaerobe Research, Life Science Support Center,

Gifu University, Japan) for the generous gift of P. melaninogenica strains GAI96524, GAI00278,

GAI00319, GAI07400, GAI07402, GAI07404, GAI07406, GAI07408, GAI07410 and

GAI07411. This work was supported by the Japan Society for the Promotion of Science

Kakenhi Grants (Grant IDs 16K11808 to TH and 15K20598 to YK).We thank Kate Fox, DPhil,

from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.

DISCLOSURE

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FIGURE LEGENDS

Fig. 1. Authenticity of the P. melaninogenica porK mutant. (A) Chromosomal structure at the

porK locus of the porK deletion mutant. (B) PCR analysis to confirm that the porK gene was

mutated by insertion of the erythromycin (ermF) cassette. The regions amplified from the

genomic DNA are shown in panel (A). (C) Whole cell lysates of the wild-type (lane 1) and porK

mutant (lane 2) were subjected to SDS-PAGE and immunoblot analysis with an anti-PorK

antibody. Left panel, CBB staining. Right panel, immunoblot membrane reacted with the

anti-PorK antiserum.

Fig. 2. Properties of the porK mutant. (A). Colony pigmentation. P. melaninogenica cells

were anaerobically grown on rabbit blood agar plates at 37C for 4 days. (B) Hemagglutination. P. melaninogenica cells were grown in TS broth, washed with PBS, and resuspended in PBS at

an optical density at 0.4. The suspension and its dilutions in a 2-fold series were applied to the

wells of a microtiter plate from left to right and mixed with a rabbit erythrocyte suspension.

Rows: wild-type and porK mutant. (C) Biofilm formation of the wild-type and porK mutant

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were stained with crystal violet (left). The quantitative determination of biofilm formation

(right). *: P<0.001. (D) Skim milk plate assay to evaluate the protease activity by P.

melaninogenica strains.

Fig. 3. Comparative analysis of proteins in the supernatant of Prevotella melaninogenica.

2D-PAGE analysis with IPG strips covering isoelectric points 4–7 was performed using the

membrane fraction from wild-type and porK mutant cells. Each sample was prepared from the

culture supernatant incubated at the same concentration and with the same sample volume.

Proteins were stained with CBB R250. No spots corresponding to protein spots with red circles

in the wild-type sample were observed in the porK mutant sample.

Fig. 4. Transmission electron micrographs. The whole cells of the wild-type (A) and porK

mutant strains (B) were negatively stained, then observed by TEM. Scale bars indicate 200 nm.

Fig. 5. Survival rates of mice challenged with the wild-type and porK mutant strain of P.

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cells into their back (approximately 2 × 1011_{CFU), and then their survival was monitored daily}

for up to 7 days. Five mice were used for each bacterial strain, and the experiments were

performed three times. *, P = 0.02 versus the corresponding values for the wild-type littermates,

as determined by the log-rank test.

Supporting Information

Fig. S1. Circular maps of the chromosomes and plasmid of Prevotella melaninogenica GAI 07411. (A) Circular maps of the chromosomes. The first and second circles (counted from the

outside in) indicate CDSs on the plus and minus strands, respectively. Common and

non-common CDSs are indicated in blue and red, respectively, compared with ATCC 25845.

The third and fourth circles show rRNA operons (red) and tRNA genes (green), respectively.

The GC content and GC skew [(G − C)/(G + C)] are shown in the sixth and seventh circles,

respectively. Note that dnaA is placed at the top of the first chromosome circle. (B)

Plasmid map. Arrows indicate CDSs on the plus and minus strands, respectively.

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RT-PCR. (B) RNA was purified from P. melaninogenica strains and reverse transcribed. PCR

was performed with each primer set using cDNA, genomic DNA and RNA as templates. Lane 1,

wild-type; lane 2, porK mutant.

Fig. S3. Membrane structure of P. melaninogenica. Membrane fractions of the wild-type and

porK mutant were subjected to SDS-PAGE and stained with CBB R250. (B) Ultrathin sections

of the samples were prepared and observed by TEM. The scale bars are shown in the lower left

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strain or plasmid description source or reference

P. melaninogenica

ATCC 25845 Wild-type American Type Culture Collection

GAI 96524 Wild-type Dr. Tanaka K

KMD001 GAI 07411∆porK::ermF; Emr _{this study}

E. coli

XL1-Blue Host strain for cloning Stratagene

S17-1 RP4-2-Tc::Mu aph::Tn7 recA, Smr ₍₁₃₎

Plasmids

pBluescript II SK(-)

Apr_{; cloning vector (pBSSK)} _Stratagene

pTCB Apr _Tcr_{, E. coli-P. melaninogenica shuttle plasmid (12)}

pML001

Apr_{; pBSSK containing 1.0-kb porK upstream}

region

this study

pML002

Apr_{; pBSSK containing 1,0-kb porK upstream and}

downstream regions

this study

pML003

Apr_Emr_{; ermF casette was inserted into BamHI}

site of pML002

this study

pML004

Apr_Emr_Tcr_{; pTCB containing 1.0-kb porK}

upstream region, ermF, and 1.0-kb porK downstream region

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Em

r

; erythromycin-resistant

Sm

r

_{; streptomycin resistant}

Ap

r

_{; ampicillin-resistant}

Tc

r

; tetracyclin-resistant

Table 2. General genomic features of Prevotella melaninogenica GAI 07411

chromosome First Second Plasmid Total

size (bp) 17,37,294 13,44,922 8,110 30,90,326

CDSs 1,377 (242*₎ _{1,021 (269*)} ₁₃ _{2411 (511*)}

rRNA operons 3 1 0 4

tRNA genes 38 14 0 52

accession number AP018049.1 AP018050.1 AP018051.1

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F. joh P. gin P. mel

amino acid identity (%) between P. gin

and P. mel gldK(porK) Fjoh_1853 PGN_1676 PMEL1_00291 57 gldL(porL) Fjoh_1854 PGN_1675 PMEL1_00292 32 gldM(porM) Fjoh_1855 PGN_1674 PMEL1_00293 44 gldN(porN) Fjoh_1856 PGN_1673 PMEL1_00294 39 sprA(sov) Fjoh_1653 PGN_0832 PMEL1_01109 45 sprB Fjoh_0979 PGN_1317 PMEL1_00863 38 sprE(porW) Fjoh_1051 PGN_1877 PMEL1_00493 32 porP Fjoh_3477 PGN_1677 PMEL1_00290 35 porQ Fjoh_2755 PGN_0645 PMEL_200249 33 porT(sprT) Fjoh_1466 PGN_0778 PMEL1_00437 35 porU Fjoh_1556 PGN_0022 PMEL1_00129 30 porX Fjoh_2906 PGN_1019 PMEL1_00492 58 porY Fjoh_1592 PGN_2001 PMEL1_00679 47

F. joh; Flavobacterium johnsoniae UW101 P. gin; Porphyromonas gingivalis ATCC 33277 P. mel; Prevotella melannogenica GAI 07411

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locus tag Protein

PMEL1_00129 peptidase C25, porU PMEL1_00246 hypothetical protein PMEL1_00417 hypothetical protein PMEL1_01073 hypothetical protein PMEL1_01214 streptopain PMEL_200096 protease PMEL_200233 internalin PMEL_200234 peptidase S8 PMEL_200422 endo-beta-N-acetylglucosaminidase PMEL_200423 endo-beta-N-acetylglucosaminidase PMEL_200424 endo-beta-N-acetylglucosaminidase PMEL_200426 hypothetical protein

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spot locus tag symbol annotation

1 PMEL1_00538 peptidase M16 inactive domain protein 2 PMEL1_01329 peptidase M16 inactive domain protein 3 PMEL_200173 pflB formate C-acetyltransferase

4 PMEL1_0012307 hypothetical protein 5 PMEL1_0012307 hypothetical protein

6 PMEL1_00816 peptidase, S9A/B/C family, catalytic domain protein

7 PMEL1_00237 peptidase family M13

8 PMEL1_00766 groL chaperonin GroL 9 PMEL1_01023 pepD Xaa-His dipeptidase

10 PMEL1_00944 peptidase, M24 family

11 PMEL1_00534 SusD family protein

12 PMEL1_00537 thioredoxin

13 PMEL1_01328 metK methionine adenosyltransferase 14 PMEL_200933 tetratricopeptide repeat protein 15 PMEL1_00993 peptidase dimerization domain protein

16 PMEL_200748 type I phosphodiesterase / nucleotide pyrophosphatase 17 PMEL_200748 type I phosphodiesterase / nucleotide pyrophosphatase 18 PMEL_200063 F5/8 type C domain protein

19 PMEL1_00822 glyA glycine hydroxymethyltransferase 20 PMEL1_00398 gdh glutamate dehydrogenase, NAD-specific 21 PMEL_200718 putative glucose-1-phosphatase

22 PMEL1_00309

lactate/malate dehydrogenase, NAD binding domain protein

23 PMEL_200928 pdxB 4-phosphoerythronate dehydrogenase 24 PMEL1_00449 tetratricopeptide repeat protein

25 PMEL1_01274 pyridoxal 5'-phosphate synthase, synthase subunit Pdx1 26 PMEL1_00449 tetratricopeptide repeat protein