Gene expression changes in Porphyromonas gingivalis W83 after inoculation in rat oral cavity

The development of chronic periodontitis was due to not only periodontal pathogens, but also the interaction between periodontal pathogens and host. The aim of this study is to investigate the alterations in gene expression in Porphyromonas gingivalis (P.gingivalis) W83 after inoculation in rat oral cavity. P.gingivalis W83 inoculation in rat oral cavity caused inflammatory responses in gingival tissues and destroyed host alveolar bone. Microarray analysis revealed that 42 genes were upregulated, and 22 genes were downregulated in the detected 1786 genes in the inoculated P.gingivalis W83. Real-time quantitative PCR detection confirmed the expression alterations in some selected genes. Products of these upregulated and downregulated genes are mainly related to transposon functions, cell transmembrane transportation, protein and nucleic acid metabolism, energy metabolism, cell division and bacterial pathogenicity. P.gingivalis W83 has a pathogenic effect on host oral cavity. Meanwhile, inflammatory oral environment alters P.gingivalis W83 gene expression profile. These changes in gene expression may limit the proliferation and weaken the pathogenicity of P.gingivalis W83, and favor themselves to adapt local environment for survival.


Background
Periodontitis is a chronic inflammatory disorder mediated by host and bacteria interactions and manifested by damage to the periodontal tissues that may progress to tooth loss. The host inflammatory responses stimulated by periodontal pathogens intend to eliminate the invaded bacteria and attribute to the destruction of tooth supporting tissues and tooth loss [1]. Moreover, the local periodontal environment may change the gene expression profile of periodontal pathogens [2][3][4]. To a certain extent, the variation of bacterial gene expression may alter the pathogenic ability of bacteria.
Porphyromonas gingivalis (P.gingivalis) is an opportunistic pathogen of the oral mucosa and a prominent member of the oral biofilms. It is well known that P.gingivalis is implicated in the onset and progression of chronic periodontitis. P.gingivalis can induce immune cells to secrete cytokines when they invade into hosts. These cytokines are present in inflamed gingiva and aggravate the destruction of oral gingival tissues and alveolar bone [5]. In the meantime, the expression of P.gingivalis genes varies under different conditions, such as iron or hemin [6,7], polyphosphate [8], rhein [9]. P.gingivalis may up-regulate or downregulate gene expression to adapt environment and survive [10].
The development of chronic periodontitis was not only due to periodontal pathogens, but also the interaction between periodontal pathogens and host. Most researches focus on periodontal pathogens acting on hosts, but ignore the action of host on P.gingivalis. Actually, the changes in P.gingivalis gene expression may affect the progression of chronic periodontitis. In the present study, the differential gene expression in P.gingivalis W83 inoculated in rat oral cavity and wild strain was analyzed.

Ethical statement
All rats were manipulated in accordance with Animal Research Reporting In Vivo Experiments (ARRIVE) guidelines. The experimental protocols were approved by the ethical committee of China Medical University.

Bacteria and animals
This study was carried out with 6-week-old SPF rats (180 − 220 g) provided by Department of Experimental Animals, China Medical University, and maintained in a temperature-controlled room (23 ± 1°C). P.gingivalis W83 was obtained from the American Type Culture Collection (ATCC) and grown anaerobically (10 % CO 2 , 10 % H 2 , 80 % N 2 ) in enriched brain-heart infusion (BHI) broth containing 5 % fiber-free sheep blood, 1 % vitamin K and hemin, at 37°C.

P.gingivalis W83 inoculation
12 Rats were given azithromycin (10 mg/500 ml) ad libitum for 4 days to reduce the original oral flora. This was followed by a 7-day antibiotic-free period. 6 Rats were then orally challenged with P. gingivalis W83 (1 × 10 9 CFU) by gavage into the esophagus and oral cavity five times every other day [11]. The other 6 rats (control group) were only challenged with BHI broth. All 12 rats received steel wire ligature in cervical part in two sides of first molars and an 8-week high sugar feeding.

Alveolar bone loss analysis
Horizontal bone loss was assessed morphometrically by measuring the distance between the cement − enamel junction and the alveolar bone crest of the first, the second and the third molar. The alveolar bone destruction was detected by morphological and macroscopic observation, radiographic (PLANMECA, Finland) and stereomicroscope (SZX12, Olympus, Japan) fitted with a DIGIMED Viewer imaging measurement system evaluation at 6 sites per molars. Alveolar bone loss of every molar was presented in the figures as mean ± SD. Independent samples t-test was used to calculate the significance among the groups (SPSS Inc., Chicago, IL, USA). P-value < 0.05 was considered statistically significant.

Isolating culture and acquiring plaque
After P.gingivalis W83 inoculation in rat oral cavity for 8 weeks, plaques were acquired from periodontal pockets of first molar using toothpicks and put into 0.5 ml transfer tube. The plaques were dispersed by oscillator. 100 μl ten-fold serial dilutions were inoculated on BHI culture medium anaerobically at 37°C for 5-7 days. The morphology of colonies was observed in primary cultures. P.gingivalisW83 colonies were identified by their black pigmentation, gram staining and PCR. The single clone was purified in BHI medium for subcultures in order to detect the differences in the gene of P.gingivalisW83.

Microarray hybridization
3 samples were picked up from wild strain P.gingivalis W83 and inoculated P.gingivalis W83, respectively. The total RNA was extracted and labeled with Klenow, and then hybridism with P.gingivalis W83 chip. The commercial GeneChip P.gingivalis W83 Genome Array used here was provided by CapitalBio Corporation (http:// www.capitalbio.com/, Beijing, China), a service provider authorized by Roche NimbleGen (Wisconsin, USA). Array hybridization, washing, scanning and data analysis were performed at the CapitalBio Corporation, Beijing, China and carried out according to the Nimble-Gen's Expression user's guide.

Real-time quantitative PCR
To independently confirm the expression data generated by the microarray experiments, we performed real-time quantitative PCR analyses for 14 genes differentially regulated. Total RNA was extracted. Quality and concentration of the RNA were determined by measuring its absorbance at 260 and 280 nm using a microplate reader (M-200, Tecan, Switzerland). Total bacterial RNA was subsequently reverse-transcribed using the M-MLV RTase cDNA Synthesis Kit (Takara, China) following the manufacturer's protocol. Real-time quantitative PCR analysis was conducted in an ABI Prism 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) in combination with the SYBR® Premix Ex TaqTM II PCR Master Mix Reagents Kit (Takara), as recommended by the manufacturer of the Wall Clear PCR Strip Tubes (Axygen, USA). The primers for the real-time quantitative PCR analysis were designed using Primer3 (http://bioinfo.ut.ee/primer3/) ( Table 1). P.gingivalis W83 16 s DNA was used as the internal reference. Real-time quantitative PCR was performed three times for each sample. The data were analyzed according to relative gene expression by the 2-ΔΔ Ct method.

Statistics
Significantly differentially expressed genes between the inoculated periodontitis and wild strains were identified using two class unpaired method in the Significant Analysis of Microarray software (SAM, version 3.02). Genes were determined to be significantly differentially expressed with a selection threshold of false discovery rate, FDR < 5 % and fold change > 2.0 in the SAM output result.
Identification of the inoculated P.gingivalis W83 in rat with periodontitis Suspicious gram-negative bacilli were taken from plaque culture. After pure subculture, gram staining and polymerase chain reaction (PCR) proved that the bacteria were P.gingivalis W83. PCR fragment length of the product was 857 bp, as shown in Fig. 2.
Genes upregulated in the inoculated P.gingivalis W83 We determined the expression of 1786 genes by microarray analysis in P.gingivalis wild strain and P.gingivalis inoculated in oral cavity. The complete list of gene expression values has been deposited in NCBI's Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/ query/acc.cgi?acc=GSE67608). The detection showed that 42 genes were up-regulated in the inoculated P.gingivalis W83 compared with wild strain (Table 2) (Fig. 3). In these upregulated genes, 30 expressed hypothetical proteins. Among other 12 genes, PG0874, PG0009, PG0427, PG0942 and PG0590 function as transposons. PG0009, PG0427, PG0942 and PG0590 encode ISPg5 transposases; and PG0874 encodes an int protein as a mobilizable transposon.
Genes downregulated in the inoculated P.gingivalis W83 Compared with wild strain, 22 genes were downregulated in the inoculated strain (Table 3) (Fig. 3). Among these genes, PG0682, PG0683, PG0684, PG0282 and PG0946 encode ABC transporters. Products of PG0682, PG0683 and PG0684 are putative permease proteins; and products of PG0282 and PG0946 are ATPbinding proteins. In addition, PG2008 and PG0283 also encode transport and binding proteins. All these proteins are involved in cell transmembrane transportation. Some downregulated genes encode proteins related to protein and nucleic acid metabolism, including PG1129, PG1993, PG0001 and PG0522. Products of other genes are related to energy metabolism (PG1042), cell division (PG0141),bacterial pathogenicity (PG1975), and so on.

Microarray result confirmation by real-time quantitative PCR
Among the upregulated and downregulated genes, we picked up 14 genes to detect the expression by realtime quantitative PCR. Consistent with microarray hybridization, real-time quantitative PCR detection showed similar expression trends in these genes (Table 4).

Discussion
Chronic periodontitis is initiated by periodontal pathogens, including P.gingivalis. Our study showed that P.gingivalis W83 induced rat gingival tissue inflammation, and alveolar bone loss, which is the key feature of periodontitis. Therefore, our study demonstrates that P.gingivalis W83 has pathogenic effects on rat oral cavity. After inoculation in rat oral cavity for 8 weeks, P.gingivalis W83 were isolated, and analyzed by microarray. In the detected 1786 genes, 42 genes were upregulated, whereas 22 genes were downregulated, indicating that the local periodontal environment can change the gene expression profile of P.gingivalis W83.
In the 42 upregulated genes, 30 expressed hypothetical proteins. Among other 12 genes, PG0874, PG0009, PG0427, PG0942 and PG0590 are in the same class in JCVI cell function classification. They all function as mobile extrachromosomal factor: transposon. Transposon   is a removable genome DNA sequence, which can "jump" in genome from one location to another through the process of cutting and integration. Transposition is generally known to be triggered by cellular stress [12][13][14], therefore upregulation of these transposons suggests that P.gingivalis W83 inoculated in rat oral cavity may adapt local environment for its own survival, which is consistent with some other studies [15,16].
In the 22 downregulated genes, 7 genes encode transport and binding proteins. All these proteins are involved in cell transmembrane transportation. They can transport many substrates, such as metabolites, ion, sugar, amino acids, lipids, cholesterol and drugs [17]. PG2008 encodes a TonB dependent receptor protein, responsible for iron transmembrane transportation [18]. As iron ion is necessary for the breeding and spreading of P.gingivalis W83, downregulation of PG2008 suggests the subdued iron transferring and proliferation of P.gingivalis W83. There are 4 downregulated genes encoding proteins related to nucleic acid and protein metabolism. PG1129 encodes a nucleotide reductase, which is related to purine, pyrimidine, nucleotide and DNA metabolism, and plays a regulating role in cell proliferation. Products of PG1993 and PG0001 are related to the metabolism of DNA, such as copy, restructuring and repair. Therefore, downregulation of these genes means that the proliferation of inoculated P.gingivalis W83 is in certain obstacles.
PG1042 encodes a putative glycogen synthase, involved in biosynthesis and degradation of polysaccharides. Downregulation of PG1042 suggests a disturbed energy metabolism. PG0141 encodes a spoOJ protein related to  Fig. 3 Genes analyzed by microarray in the inoculated P.gingivalis W83. Fluorescence signal strength values in X axes and Y axes represent control groups and experimental groups, respectively. Each data point was behalf of a gene chip hybridization signal. Red marking data points were T/C value ≥2, representing upregulated genes, and green marking data points were T/C value ≤0.5, representing downregulated genes cell division, and PG1975 encodes hemagglutinin HagC related to pathogenicity of P.gingivalis W83. In addition, PG1982 encodes a CRISPR protein related to CAS1 family. CRISPR/CAS system can protect bacteria against the encroachment by phage, and resist other chromosome genetic material and prevent from the expression of their genes [19][20][21]. Downregulation of PG1982 suggests a decrease in the defense capability of P.gingivalis W83.
It should be noted that gene expression observed in this study was in mRNA level. As we have known, alterations in mRNA expression are not always consistent with those in protein expression. Therefore, observations in protein level of gene expression will be more convincing. However, it is impracticable to analyze the protein expression of all 64 genes with RNA expression alteration. Moreover, some products of these genes are still hypothetical proteins. Because the inoculated P.gingivalis was cultured outside the rat oral cavity for some days before RNA extraction, the RNA samples cannot exactly reflect the changes in gene expression after inoculation,

Conclusions
Our study shows that P.gingivalis W83 has pathogenic effects on host, and local inflammatory oral environment alters the gene expression profile of P.gingivalis W83. Products of these upregulated and downregulated genes are mainly related to transposon functions, cell transmembrane transportation, protein and nucleic acid metabolism, energy metabolism, cell division and bacterial pathogenicity. These changes may lead to decreased proliferation and pathogenicity of P.gingivalis W83, and favor themselves to adapt local environment for survival.