Comparative Analysis of the Complete Genome of Lactobacillus plantarum GB-LP2 and Potential Candidate Genes for Host Immune System Enhancement

Abstract

Acute respiratory virus infectious diseases are a growing health problem, particularly among children and the elderly. Much effort has been made to develop probiotics that prevent influenza virus infections by enhancing innate immunity in the respiratory tract until vaccines are available. Lactobacillus plantarum GB-LP2, isolated from a traditional Korean fermented vegetable, has exhibited preventive effects on influenza virus infection in mice. To identify the molecular basis of this strain, we conducted a whole-genome assembly study. The single circular DNA chromosome of 3,284,304 bp was completely assembled and 3,250 protein-encoding genes were predicted. Evolutionarily accelerated genes related to the phenotypic trait of anti-infective activities for influenza virus were identified. These genes encode three integral membrane proteins, a teichoic acid export ATP-binding protein and a glucosamine - fructose-6-phosphate aminotransferase involved in host innate immunity, the nonspecific DNA-binding protein Dps, which protects bacteria from oxidative damage, and the response regulator of the three-component quorum-sensing regulatory system, which is related to the capacity of adhesion to the surface of the respiratory tract and competition with pathogens. This is the first study to identify the genetic backgrounds of the antiviral activity in L. plantarum strains. These findings provide insight into the anti-infective activities of L. plantarum and the development of preventive probiotics.

Introduction

Respiratory infectious diseases caused by influenza viruses are increasingly becoming a global health concern [10]. They have considerable negative effects on lifestyles and economies [26]. Since the pandemics of avian-origin H5N1 and swine-origin H1N1 influenzas [11,27], researchers have begun to focus more on developing preventive agents for use until vaccines are developed. Many researchers have explored the potential use of probiotics, including Lactobacillus strains that exhibit antiviral activities, using different delivery methods in animals and humans [3,14,18,23,25]. Increasing evidence suggests that intranasal administration of Lactobacillus species could protect against influenza virus infection by modulating respiratory immune responses in animal models [9,12,34,35]. A recent study found that both live and dead forms of Lactobacillus rhamnosus increase innate immune responses and attenuate pulmonary damage in respiratory tissues [35].

Previous research using genome sequencing and functional analyses has revealed the underlying mechanisms of how specific strains exhibit better performance in preventing and treating infectious intestinal diseases via physical, biochemical, and immunomodulatory interactions [17,19]. However, genomic evidence is not yet available to support their anti-infective activities related to the influenza virus.

In this study, we sequenced the complete genome of Lactobacillus plantarum GB-LP2 isolated from a traditional Korean fermented vegetable. Previous research has demonstrated that it exhibits antiviral effects on influenza virus infection in mice following intranasal administration [9]. Whole-genome assembly of the complete genome sequence of L. plantarum GB-LP2 was conducted and genomic contents were identified. A comparative phylogenetic tree revealed the evolutionary relationship between L. plantarum GB-LP2 and other previously reported L. plantarum strains. Additionally, a comparative genomic analysis was performed with seven other complete genome sequences of L. plantarum strains. We identified evolutionarily accelerated genes that might affect the phenotypic trait related to the antiinfective activities of influenza virus. This is the first report of a complete sequence and comparative analysis related to the anti-infective activities of influenza virus in L. plantarum species. The results will help clarify the anti-infective activities of probiotics and related genes.

 

Materials and Methods

Strain Isolation and Whole Genome Sequencing

Genomic DNA of the GB-LP2 strain was isolated and purified using an UltraClean Microbial DNA Isolation Kit (MoBio, Carlsbad, CA, USA) according to the manufacturer’s protocol. The concentration and purity of the extracted DNA were determined using a NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE, USA). Approximately 5 μg of the extracted genomic DNA was sheared mechanically into 8–12 kb fragments using a Hydroshear system (Digilab, Marlborough, MA, USA). SMRTbell libraries were prepared for SMRT sequencing with C4 chemistry on a PacBio RS II system (Pacific Biosciences, Menlo Park, CA, USA). Libraries were purified using 0.45× AMPure XP beads to remove s hort i nserts o f <1.5 k b . The s ize distribution o f the sheared DNA template was characterized using an Agilent 12000 DNA Kit (Applied Biosystems, Santa Clara, CA, USA), and the concentration was determined using Invitrogen Qubit (Carlsbad, CA, USA). The sequencing primers were annealed to the templates at a final concentration of 5 nM template DNA, and DNA polymerase enzyme C4 was added according to the manufacturer’s recommendations for small-scale libraries. A DNA/Polymerase Binding Kit P6 (Pacific Biosciences) was used to load the enzyme template-complexes and libraries onto 75,000 zero-mode waveguides. A DNA Sequencing Reagent 2.0 Kit (Pacific Biosciences) was used to sequence SMRT cells using a 120-min sequence capture protocol along with a stage start to maximize the subread length with PacBio RS II.

Genome Assembly and Annotation

Raw sequence data from the PacBio RS II system were filtered and assembled using the PacBio SMRT portal system ver. 2.3.0. The “RS_HGAP_assembly.3” algorithm [8] was employed and the genome size parameter was set to 3,300,000 bp using the Compute Minimum Seed Read Length option. Other parameters were set to default. Assembled contigs with a short contig length (<20,000 bp) and low coverage (<50×) were filtered for further analysis. To remove errors in the pre-assembled GB-LP2 genome sequence, an iterative polishing process was conducted until no genomic variants were identified. Genome annotation was completed using the RAST annotation system [1] with default settings. COG annotation was conducted using WebMGA [32] and an annotation map was generated using DNA Plotter [6].

Comparative Analysis

For the comparative analysis, seven complete genomes and 24 draft genome sequences of Lactobacillus plantarum strains were downloaded from the NCBI database (http://www.ncbi.nlm.nih.gov/genome/genomes/154). Average nucleotide identity (ANI) values were calculated for all 32 strains using JSpecies ver. 1.2.1 [24]. An ortholog gene set for the eight complete genomes was built using the MESTORTHO method [15] and PRANK [16] was employed for the multiple sequence alignment of each ortholog gene. After poorly aligned sites were eliminated using GBlocks [30], orthologous sequences were concatenated to one sequence to construct a phylogenetic tree. MEGA6 [31] was used to build the phylogenetic tree with the neighbor-joining method, and bootstrap analysis was performed on the combined dataset sequences. The maximum likelihood method (codeml of PAML4) [33] was used to estimate the dN (rate of non-synonymous substitution), dS (rate of synonymous substitution), and evolutionarily accelerated genes under the branch and branch-site model.

Strain Deposition and Complete Genome Sequence Accession Number

The L. plantarum GB-LP2 strain was deposited in the Korea Agriculture Culture Collection (KACC, Korea) under number KACC 18511. The NCBI accession number of the complete genome sequence of this strain is PRJNA295633.

 

Results

General Features of Lactobacillus Plantarum GB-LP2

The L. plantarum GB-LP2 genome consists of a single circular DNA chromosome of 3,284,304 bp with a GC content of 44.57%. The GB-LP2 genome contains 3,250 open reading frames (ORFs), 76 tRNAs, and 16 rRNAs (Fig. 1 and Table 1). Among the predicted ORFs, 2,435 genes (77.0%) were predicted as functional genes and 815 (33%) were unknown and hypothetical genes. Fig. 2 presents the categorization of predicted ORFs based on the SEED subsystem categorization and COG functional categorization. Of the 2,435 genes, 1,922 ORFs were assigned to 25 SEED subsystem categories. One hundred twenty-four ORFs were assigned to the Cell Wall and Capsule category related to host antigen reaction; 34 ORFs were responsible for Capsular and Extracellular Polysaccharides; 55 ORFs were responsible for Cell Wall and Capsule; and 35 ORFs were responsible for Gram-Positive Cell Wall Components. One hundred thirty ORFs were categorized into Cofactors, Vitamins, Prosthetic Group, and Pigments, and 11 ORFs were responsible for Thiamin Biosynthesis related to one of the main nutrient factors in the traditional Korean fermented vegetable. Fifty-one ORFs were assigned to Virulence, Disease, and Defense Subsystem, including resistance to antibiotics and toxic compounds such as bile, tetracycline, fluoroquinolones, and beta-lactam antibiotics. Based on the COG functional categorization, 2,535 ORFs (78.0% of all predicted ORFs) were classified into COG functional categories. Among these, 875 ORFs (40.8% of the COG-assigned ORFs) belonged to five major COG functional categories: 220 ORFs in category E (amino acid transport and metabolism), 275 ORFs in category G (carbohydrate transport and metabolism), 179 ORFs in category L (replication, recombination, and repair), 123 ORFs in category P (inorganic ion transport and metabolism), and 238 ORFs in category K (transcription).

Fig. 1.Genome map of L. plantarum GB-LP2. Circles, from outer to inner, represent the COG distribution, CDS in the leading strand, CDS in the lagging strand, tRNA, rRNA, and the GC content ratio. Functional genes are labeled around the outer circle as follows: evolutionarily accelerated genes in blue, and genes related to antimicrobial activity in green. The scale is base pair.

Table 1.Comparison of the chromosomal properties of L. plantarum strains.

Fig. 2.Functional categorization of all predicted ORFs in the L. plantarum GB-LP2 genome based on (A) SEED and (B) COG databases.

Comparative Tree Analysis

Two ANI trees and one phylogenetic tree were constructed for a comparative tree analysis of the GB-LP2 strain. Two ANI trees were built with the 31 available genome sequences and seven available complete genome sequences in the NCBI database, respectively (Figs. 3A and 3B). Six strains, namely 16, P8, UCMA3037, TIFN101, IPLA88, and ZJ316, were grouped together with GB-LP2, and ZJ316 was regarded as the closest neighbor of GB-LP2 (99.45% of the ANI value). ZJ316 is a probiotic strain isolated from infant fecal samples; a previous study revealed that it has probiotic effects on pig growth and pork quality [29]. The EDG-AQ4 and AY01 strains had 79.5% and 85.82% of the ANI value, respectively, and the results indicated that the genome sequences may differ even in the same microbial species. A phylogenetic tree using orthologous genes was generated for eight complete genome sequences using the neighbor-joining method and bootstrapping 1,000 times (Fig. 3C). The bootstrap value for each node was 1.000, except for one node (0.992) between STIII and JDM1. The closest neighbor of GB-LP2 was ZJ316, as in the ANI tree, but the tree topology pattern of the phylogenetic tree did not exactly match that of the ANI tree. In the ANI tree, JDM1 was clustered with WCFS1 and B21. In the phylogenetic tree, however, JDM1 was clustered with STIII.

Fig. 3.Comparative tree analyses. (A) ANI tree analysis of 32 available genome sequences in the L. plantarum strain using Jspecies. (B) ANI tree analysis of GB-LP2 with seven available complete genome sequences of L. plantarum. (C) Phylogenetic tree analysis of GB-LP2 with seven available complete genome sequences.

Comparative Genomics Using dN/dS Analysis

To identify the evolutionarily accelerated genes in the GB-LP2 strain, dN/dS analysis, based on two models (branch and branch-site), was conducted for the orthologous gene set. dN/dS analysis based on the branch model revealed 10 evolutionarily accelerated genes (Table 2). Among the 10 evolutionarily accelerated genes, six (PTS system, cellobiose-specific IIC component (E.C. 2.7.1.69), phosphomevalonate kinase (E.C. 2.7.4.2), exodeoxyribonuclease VII large subunit (E.C. 3.1.11.6), teichoic acid export ATP-binding protein TagH (E.C. 3.6.3.40), ribokinase (E.C. 2.7.1.15), and glucosamine--fructose-6-phosphate aminotransferase (isomerizing) (E.C. 2.6.1.16)) were assigned an enzyme commission number. In the branch-site model, three genes were identified as evolutionarily accelerated genes (Table 3): two were integral membrane proteins and the other was the nonspecific DNA-binding protein Dps/iron-binding ferritin-like antioxidant protein/ferroxidase. Table 3 lists the differences in the amino acid sequences of the three genes. In the first integral membrane protein, amico acid 222 of GB-LP2 was changed to aspartic acid from glycine. Glycine is included in the nonpolar amino acid group and aspartic acid is included in the polar group, as it has a negative charge. In the second integral membrane protein, amico acid 245 of GB-LP2 was changed to glutamine from lysine. Lysine is included in the positively charged polar group and glutamine is classified into the uncharged polar group. In genes related to ferroxidase, amico acids 256, 290, and 293 of GB-LP2 were changed to aspartic acid, valine, and threonine from asparagine, threonine, and valine, respectively. Valine is included in the nonpolar amino acid group and threonine is included in the uncharged polar group.

Table 2.Evolutionarily accelerated genes identified in the branch model and related information for L. plantaum GB-LP2.

Table 3.*Indicate posterior probability > 0.95.

 

Discussion

Modulation of Innate Immunity in the Host

L. plantarum protects human intestinal cells from the invasion of pathogens via competitive adhesion, modulation of dendritic cells and T cells, and tight junction integrity [20]. Additionally, the microdomain of the integral membrane protein (IMP) of L. plantarum plays a major role in the capacity of adhesion in intestinal epithelial cells, particularly in binding to the mannose receptor, which inhibits the Toll-like receptor (TLR) 5 pathway-mediated p38 MAPK signaling pathway after enteropathogenic Escherichia coli infection [21]. We speculated that the evolutionarily accelerated genes of IMP in L. plantarum GB-LP2 that alter the binding capacity to receptors related to innate immunity could protect bronchial cells from the development of influenza virus infection and secondary infection, which may be associated with the preventive effect of GB-LP2 in mice challenged by influenza H1N1 [9]. A teichoic acid export ATP-binding protein (TagH) (E.C. 3.6.3.40) is a subunit of the ABC transporter complex that exports teichoic acid, which is converted to wall teichoic acid (WTA) in the peptidoglycan layer and lipoteichoic acid (LTA) anchored to the cytoplasmic membrane. WTA and LTA bind to TLRs involved in the innate immune response [5], which may be associated with the capacity to transport teichoic acid through the accelerated gene-coded TagH-transporter complex. A glucosamine - fructose-6-phosphate aminotransferase (GlmS) (E.C. 2.6.1.16) has been evolutionarily accelerated in GB-LP2 compared with other L. plantarum strains. GlmS is a rate-limiting enzyme in the biosynthesis of peptidoglycan, which constructs the mesh-like layer in bacterial cell walls and induces the host innate immune response [2,5]. A change in the kinetics or modulation of the accelerated GlmS may be related to the capacity of peptidoglycan biosynthesis in GB-LP2.

Survival of L. plantarum GB-LP2 in the Respiratory Tract Prevents Secondary Infection

The nonspecific DNA-binding protein Dps plays a crucial role in protecting microorganisms from oxidative damage via mechanisms such as ferroxidase activity and DNA binding, increasing survival under harsh conditions such as starvation and exposure to H2O2 [7]. The evolutionarily accelerated gene Dps, if positively selected, may increase the survival of GB-LP2 in the respiratory tract, a nutrient-poor environment unlike the intestine, and prevent the host defense system against H2O2 produced by macrophages and neutrophils. Secondary infection caused by bacteria and influenza viruses increases the risk of morbidity and mortality [13], and disruption of the balance between commensal bacteria and pathogens induces the development of infection on the bronchial epithelial cell barrier [4]. The evolutionary acceleration of Dps suggests that GB-LP2 survival may be related to the niche of overgrowth of pathogens in airways.

Altering a Three-Component Quorum-Sensing Regulatory System Involved in Plantaricin Production

The GB-LP2 chromosome contains all three genes related to a three-component quorum-sensing regulatory system [22]. An S-ribosylhomocysteine lyase (E.C. 4.4.1.31) produces autoinducer-2, a signaling molecule, to communicate among intraspecies and interspecies to modulate population density and gene expression. The location of a histidine protein kinase gene and a response regulator gene in the cluster of bacteriocins of L. plantarum (plantaricins) indicates the quorum-sensing-mediated regulation of plantaricin production [28]. Interestingly, a response regulator gene of GB-LP2 has been evolutionarily accelerated in an environment of vegetablebased fermentation (i.e., solid surface fermentation), which may increase the capacity of adhesion to the surface of the respiratory tract and competition with pathogens in the enhanced quorum-sensing-mediated regulation response compared with other L. plantarum strains [22]

Adaptation to the Plant Environment

A comparative genomic analysis detected GB-LP2-specific genes such as the cellobiose-specific IIC component in the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) (E.C. 2.7.1.69). The PTS is a major carbohydrate active-transport system in bacteria that catalyzes the phosphorylation of sugar substrates to cross the microbial cell membrane. L. plantarum GB-LP2 has a distinguished cellobiose-specific IIC component rather than the lactose-specific form, indicating the evolutionarily accelerated adaptation of GB-LP2 to vegetable-based fermentation.

In conclusion, this comparative analysis yielded potential candidate genes of L. plantarum GB-LP2 that may prevent infection and mortality from the influenza virus among humans via evolutionarily accelerated genes related to the innate immune response, survival, and quorum-sensing regulatory systems in the respiratory tract. Further research focusing on these evolutionarily accelerated genes will help clarify their preventative mechanisms against influenza virus and secondary infection in airways, and may help prevent seasonal flu and epidemic respiratory virus infections.