Whole Genome Comparison between Exopolysaccharide-Producing Prevotella intermedia Strain 17 and Its Non-Producing Variant Strain 17-2

Yamanaka T, Maruyama H, Mashimo C, Ozawa J, Takagi N, Matsumoto K, Sanada I and Nambu T

Published on: 2024-05-14

Abstract

Background

Exopolysaccharides (EPS) as biofilm matrices in many bacteria are known to be crucial for their resistance against host innate immune responses and for establishing persistent infection in the host. Prevotella intermedia, a gram-negative, black-pigmented anaerobic rod, has been implicated in the development of chronic oral infection, and we have previously reported that some clinical isolates of P. intermedia have a capacity to produce EPS, form dense biofilm structures in vitro, and can induce abscess formation in mice experimentally. However, the gene expression events behind the EPS production in this organism still remain to be elucidated. To explore genes involved in the biofilm formation of P. intermedia, we performed a genome comparison between biofilm-forming P. intermedia strain 17 and strain 17-2, a variant of strain 17 that lacked the biofilm-forming capacity.

Methods

Genome sequences of both strains in the NCBI database (strain 17 chromosome I: NC_017860; chromosome II: NC_017861; strain 17-2 chromosome I: AP014926; chromosome II: AP014926) were used in this study. A genome comparison between P. intermedia strains 17 and 17-2 was undertaken with MUMmer.

Results

The comparative analysis showed 213 single nucleotide variants on the whole genome sequence and a deletion of 3,186 base pairs on chromosome II of strain 17-2. The latter larger deletion affected four genes annotated as sugar transferase (locus tag: PIN17_RS10270), capsule biosynthesis protein (RS10275), chain-length determining protein (RS10280), and polysaccharide biosynthesis protein (RS10285), respectively.

Conclusions

These results suggest that the gene cluster highlighted in this study might be involved in the biofilm formation of this organism.

Keywords

Prevotella intermedia; Genome comparison; Biofilm

Introduction

Prevotella intermedia, a gram-negative, black-pigmented anaerobic rod, is frequently isolated from healthy oral cavities as well as chronic periodontitis lesions [1,2] acute necrotizing ulcerative gingivitis [3], pregnancy gingivitis [4], and endodontic lesions [5-7]. This organism possesses a number of virulent factors that contribute to its pathogenic potential [8-12].

P.intermedia strain 17 was initially isolated from a chronic periodontitis lesion in our laboratory as an exopolysaccharide (EPS)-producing strain [13], and some of its phenotypic characteristics were determined [14,15]. The whole genome sequence of strain 17 was determined by the Institute for Genomic Research (TIGR; Rockville, MD, USA) [16].

EPS production as biofilm matrices in many bacteria has been associated with pathogenicity [17-19]. Gene expression cascades behind EPS production have been under intense investigation in many biofilm-forming bacteria for possible therapeutic approaches against persistent biofilm infections. For example, Pseudomonas aeruginosa, an opportunistic pathogen that causes a multitude of infections, exploits the AlgZR two-component system to regulate its alginate production and twitching motility [20]. The curli/cellulose regulator CsgD is known to be behind the biofilm formation of Escherichia coli upright Salmonella [21,22]. In our previous studies, when we compared the microarray expression data obtained from strain 17 as bacterial cells were producing EPS to those of strain 17-2 as EPS non-producing variant, stress-inducible heat shock proteins were up-regulated in strain 17 at a gene transcriptional level [23]. However, we could not particularize the functions of these genes in the biofilm formation of P. intermedia since a genetic transfer system for having gene-targeted mutants of this organism is yet limited [24,25].

Recently, we have determined the complete genome sequence of strain 17-2 [26]. In this study, we performed a whole genome comparison between these two strains in order to obtain a cue to elucidate a cascade of gene expression events associated with the biofilm formation of this organism.

Materials and Methods

Bacterial Strains and Genome Sequences

Bacterial strains used in this study are listed in (Table 1) complete genome sequences of P. intermedia strains 17 and 17-2 were used in this study. The strain 17-2 is a naturally occurring variant obtained from strain 17 in our laboratory through repetitive passages [23]. The strain 17-2 can not produce either EPS or biofilm-like structures [Fig. 1]. The genome sequences of both strains in the NCBI database (strain 17 chromosome I: NC_017860; chromosome II: NC_017861; strain 17-2 chromosome I: AP014926; chromosome II: AP014925) were compared with MUMmer [27]. The whole genome sequence of P. intermedia OMA14 was used as a reference sequence (Chromosome ?: NC_017860; Chromosome ?: NC_017861) [28].

Table 1:  Stock Strains of P. intermedia in Our Laboratory and a Plasmid Used in This Study.

 

Strain

Relevant description

Reference

 

 

 

P. intermedia

  strain 17

Biofilm-forming clinical isolate

13-16, 23

  strain 17-2

Biofilm-non-forming variant from strain 17

23

  ATCC25611

Type strain

27

  OD1-16

Biofilm-forming clinical isolate in our laboratory

28

pCR2.1-TOPO 

Cloning vector

Invitrogen

Fig 1:  Scanning Electron Micrographs of Prevotella intermedia Strains 17 and 17-2. Stock Cultures of Strain 17 in Our Laboratory Spontaneously Form Meshwork Structures Which Is a Typical Phenotype for Biofilm-Forming Bacteria. Strain 17-2 Is A Naturally Occuring Variant of the Strain 17. This Variant Can Not Produce either EPS or Biofilm-Like Structures.

PCR Amplification and Alignment Comparison

Compared to the strain 17 genome, 3,186-bp region was deleted in the strain 17-2 genome. Therefore, we performed PCR amplification of this region with our stock strains of P. intermedia and compared the alignment to P. intermedia strains 17 and OMA14 to examine whether this DNA fragment is conserved in this organism or not. Stock cultures of P. intermedia ATCC25611 [29] and OD1-16, a clinical isolate from a chronic periodontitis lesion [30], were grown on trypticase soy broth (TSB; BBL Microbiology Systems, Cockeysville, ND) supplemented with 0.5% yeast extract (Difco Laboratories, Detroit, MI), hemin (5 mg/l), L-cystine (400 mg/l), and vitamin K1 (10 mg/l). Bacterial cultures were grown anaerobically in an anaerobic chamber (ANX-3, Hirasawa, Tokyo, Japan) at 37°C in a 5% CO2, 10% H2, and 85% N2 atmosphere. The genomic DNA from our stock strains was purified using the GNOME Kit (Qbiogene Inc., Morgan Irvine, CA). PCR amplification was performed with a primer pair (5’-GTTTTACACTTTCCAGCACA-3’ and 5’-CAATACTGAGCGAAGTAGGA-3’). Conditions for PCR were as follows: 94°C for 5 min, and then 35 cycles of 94°C for 40 s, 60°C for 40 s, and 72°C for 1 min, followed by 72°C for 2 min. The PCR products were electrophoresed, isolated, and cloned using the pCR2.1-TOPO cloning vector (Invitrogen, Carlsbad, CA). Plasmids containing the PCR products were purified using the QIAprep Spin MiniPrep Kit (Qiagen Science, MD). The PCR products were then sequenced using the Applied Biosystems 3730 with a pair of M13 primers. Homology searches of the obtained DNA sequences were performed using the BLAST algorithm in the DDBJ (Mishima, Japan).

Results

When we compared the genome sequences of the strain 17-2 with the strain 17, 213 single nucleotide mutations (Table 2) and one relatively large deletion on chromosome II were found (Fig. 2). The 3,186 bp on chromosome II of strain 17 (NC_01761.1: 1841078–1844263) were deleted on chromosome ? of strain 17-2. This deletion affected four genes annotated as sugar transferase (locus tag: PIN17_RS10270), capsule biosynthesis protein (RS10275), chain-length determining protein (RS10280), and polysaccharide biosynthesis protein (RS10285) (Fig. 2).

Table 2: Number of Single Nucleotide Changes in Strain 17-2 by Type.

Type

Total

SNP

126

MNP

0

INS

36

DEL

51

Mixed

0

Interval

0

Total

213

Fig 2: Schematic Depiction of Deleted Region of P. intermedia Strain 17-2. Gene Annotations Of The Strain 17-2 Are Different From Those Of The Strain 17 Due To The Deletion.

This region was highly conserved in the P. intermedia genome available on the data base (strains OMA14 and ATCC25611). The DNA sequences obtained from our stock strains (Strains OD1-16 and ATCC25611) were highly homologous to those of strain 17 (data not shown). The gene annotation of strain 17-2 was quite different from that of strain 17 because of the deletion (Fig. 2). The sequences of both sides of the deleted region were 5’-GGAAAATG-3’ and 5’-GGAATATG-3’ (Fig. 3), suggesting homologous recombination.

Fig 3: Schematic Depiction of Nucleotide Alignment of the Deleted Region.

Discussion

Bacterial genomes have extraordinary plasticity; therefore, it might be possible that some laboratory strains subcultured for decades since their first isolation might have lost important pathophysiological characteristics [31]. P. intermedia strain 17 was isolated from a chronic periodontitis lesion as an EPS-producing strain in our laboratory. Since then, strain 17 has been producing a large amount of EPS and forming meshwork structures around the cells, which is characteristic of biofilm-forming bacteria. We have previously reported that strain 17 induced highly noticeable abscess lesions in mice at 107 CFU, but non-EPS-producing periodontopathic bacteria, such as P. intermedia ATCC 25611 and Porphyromonas gingivalis strains ATCC 33277, 381, and W83, required 100-fold more bacteria (109 CFU) [32]. Thus, the capacity to produce EPS in P. intermedia might contribute to the pathogenicity of this organism, like other bacteria causing biofilm infection [17-22].

Through repetitive subcultures of strain 17 in our laboratory, we obtained a variant named strain 17-2, which completely lost the biofilm-forming capacity. Environmental stresses such as animal passages, ethanol, or salt stresses did not restore this variant to its original condition [30]. In order to find a reason why the phenotype change in strain 17-2 was irreversible, we determined the whole genome sequence of strain 17-2 [26]. The genome comparison between strain 17 and strain 17-2 highlighted a deletion of 3,186 bp on the strain 17-2 chromosome ?. Judging from the gene annotation, the gene cluster found in this study might be involved in the exopolysaccharide biosynthesis in P. intermedia. We now presume that the deletion was probably caused by simple homologous recombination because of the similar alignment of both ends of the deleted region.

In our laboratory, ATCC25611 does not produce EPS in its cultures, although this region is conserved in the genome. It is well known that point mutations convert non-mucoid P. aeruginosa into an alginate-producing mucoid phenotype [33]. In our previous report with a clinical isolate of Escherichia hermannii obtained from an infected root canal [34], a transposon insertion into wzt, a gene that encodes an ATP-binding cassette transporter, took away the expression of the cell surface-associated meshwork structures and the ability to produce extracellular materials from the cells. Complementation of the disrupted wzt in the mutant with an intact wzt resulted in the restoration of these phenotypes. Therefore, it is highly plausible that mutations other than this deleted region regulate the EPS productivity of P. intermedia.

Conclusion

These results suggest that the gene cluster highlighted in this study might be involved in the biofilm formation of this organism. As for future studies, we must establish a genetic transfer system for having gene-targeted mutants of these strains and perform complementation of the deleted region in strain 17-2 to understand the real function of the gene cluster highlighted in this study. It is also important to note that the data presented were derived from strain 17 and its variant strain 17-2. Therefore, the results might not be representative of the overall mechanism of biofilm formation in this organism. More thorough investigations with other P. intermedia strains are also needed to determine the role of this gene cluster in biofilm formation by this organism.

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