Identification of plasmalogens in Bifidobacterium longum, but not in Bifidobacterium animalis

Plasmalogens are glycerophospholipids that contain a vinyl ether bond at the sn-1 position of glycerol backbone instead of an ester bond. Plasmalogens are indicated to have many important functions in mammalian cells. On the other hand, it is suggested that some gut microbiota plays many probiotic functions to human health. Presence of plasmalogens in Clostridium strains in gut microbiota is well-known, but presence of plasmalogens in Bifidobacterium longum (B. longum) strain, one of the most important probiotic gut microbiota, has not been reported. We identified plasmalogens in lipid extract from some B. longum species, but not from Bifidobacterium animalis (B. animalis) species which are another important strain of probiotic bifidobacteria. Major phospholipid classes of plasmalogens in B. longum species were cardiolipin, phosphatidylglycerol and phosphatidic acid. Almost all of the phospholipids from B. longum examined were indicated to be plasmalogens. Although major phospholipid classes of plasmalogens in human brain and major phospholipid classes of plasmalogens in B. longum are different, it is interesting to note that many reported functions of microbiota-gut-brain axis on human neurodegenerative diseases and those functions of plasmalogens on neurodegenerative diseases are overlapped. The presence of plasmalogens in B. longum species may play important roles for many probiotic effects of B. longum to human health.


Results
Differences of phospholipid composition among gut microbiota. The HPLC-ELS D method used in the present study can separate almost all major phospholipids present in animal tissues by a single run of HPLC (Fig. 1a), but phospholipids from bacteria did not separate clearly each other ( Fig. 1b-d). Causes of the incomplete separation of bacterial phospholipids by the HPLC-ELSD method were not clear, but may be due to presence of some other unidentified phospholip id classes 22 . However, phospholipid compositions were clearly different among B. longum, B. animalis and Clostridium (Fig. 1b-d). Furthermore, we experienced that the patterns of phospholipids composition and/or phospholipid classes on the chromatograms of the same species of B. longum often changed after re-cultured by the same culture medium after storage at a refrigerator or a deep freezer (−30 °C).
Presence of plasmalogens in some of B. longum species. Chromatograms of B. longum BB536 were showed in Fig. 2. Phosphatidylglycerol (PG) and cardiolipin (CL) were the most abundant phospholipids, but phospholipid classes of the other peaks were not identified. Phospholipid chromatograms of B. longum subsp. infantis and B. longum subsp. suis were similar to those of B. longum BB536 (Fig. 2). However, phospholipid chromatograms of B. longum subsp. longumwere different from those of the other B. longum species (Fig. 2). The main phospholipid of B. longum subsp. longum was not PG nor CL, but it was phosphatidic acid (PA). The PA peak remained after hydrolysis with PLA1, indicating the PA peak was an ether phospholipid. Hydrolysis of total phospholipids with phospholipase A1 (PLA1) resulted in most of the phospholipids of these B. longum species including B. longum subsp. longum that remained on chromatograms (Fig. 2), which indicated that most of these main phospholipids of B. longum species were ether phospholipids. Treatment of the PLA1 hydrolyzed phospholipids with 2,4-dinitrophenylhydrazine-hydrochloride (DNPH-HCl) caused disappearance of main peaks on the chromatograms (Fig. 2), indicating that most of phospholipids of these B. longum species were plasmalogens. Appearance of lysophospholipid peaks (lyzo-p-lipids) after DNPH-HCl treatment (Fig. 2) also indicates presence of plasmalogens in these B. longum species.
Absence of plasmalogens in B. animalis. Figure 3 shows chromatograms of phospholipids of B. animalis subsp. lactis JCM 10602 and B. animalis subsp. animalis JCM 1190. Chromatograms of these two species of B. animalis were identical (Fig. 3) and peaks of PG and CL were relatively small as compared to unknown peaks of the later retention times. Hydrolysis of these phospholipids with PLA1 caused disappearance of all peaks (Fig. 3), which indicated that all phospholipids peaks of these B. animalis species were diacyl phospholipids.
Presence of plasmalogens in Clostridium beijerinckii. Presence of plasmalogens in this Clostridium was already reported by the different methodology 15 . By the present method, a small peak of PG and large peaks of CL, and two large peaks of unidentified phospholipids peaks were observed (Fig. 4). Treatment of total phospholipids by PLA1 showed that a large part of CL peak and the other peaks including PG remained after PLA1 hydrolysis, indicating that these peaks were ether phospholipids. The peak of CL was somewhat changed by treatment with PLA1 which may indicate presence of diacyl type CL. Furthermore, the peaks after PLA1 treatments disappeared by DNPH-HCl, indicating that these ether phospholipids were plasmalogens. Appearance of lysophospholipids after HCl treatment also indicates presence of plasmalogens (Fig. 4).
Occurrence of aldehydes from plasmalogens after acid hydrolysis. Occurrence of aldehydes after DPNH-HCl hydrolysis also indicates presence of plasmalogens in B. longum species. Figure 5 shows HPLC chromatograms of aldehydes after DNPH-HCl hydrolysis of PLA1 treated phospholipids of different bacteria. Peaks of short retention time were nonspecific short chain aldehydes. Peaks of the later retention times, which were seen in B. longum BB536, B. longum subsp. infantis and B. longum subsp. longum, and B. longum subsp. suis indicate aldehydes generated from plasmalogens, but B. animalis species showed no occurrence of aldehyde after hydrolysis with DNPH-HCl treatment of total phospholipids. Among the aldehydes generated from plasmalogens of B. longum species, C:14 aldehyde was the highest in all species of B. longum, and a small amount of C:12 and C:17 was found in all species of B. longum examined.
Fatty acid composition of total lipids from B. longum BB536. Fatty acid composition of B. longum BB536 and B. anilalis subsp animalis JCM 1190 was showed in Table 1. By the method used in the present study, an odd numbered fatty acid (C17:0) was observed in both B. longum and B. animalis species. No polyunsaturated acids were detected.

Discussion
The present study showed presence of plasmalogens in some species of B. longum strain. Identification of plasmalogens in a Clostridium species with the same method may verify that the present methodology can identify plasmalogens.
After confirmation of the presence of plasmalogens in some B. longum species, we found a literature that indicated presence of plasmalogens in B. animalis subsp. lactis 23 . The report used gas chromatography-mass spectrometry (GC/MS) for detection of aldehydes after acid hydrolysis of total lipid extract from the bacteria. Aldehydes were observed after acid hydrolysis, but phospholipid class of the plasmalogen was not been identified 23 . However, we did not find any plasmalogens in B. animalis subsp. lactis (Fig. 3) nor generation of aldehydes after HCl hydrolysis of the phospholipids from the bacteria (Fig. 5).  Probably, both B. longum strain and B. animalis strain are the most important probiotic gut-microbiota in human [19][20][21] . Many beneficial effects of Bifidobacterium to human health have been reported [19][20][21][24][25][26] . It is interesting to note that plasmalogens were found only in B. longum species, but not in B. animalis species. It is not clear that there are any differences of probiotic effects to human health between B. longum and B. animalis.
Biosynthesis pathway of plasmalogens in anaerobic bacteria is different from that in animals [11][12][13][14] . In the animal tissues, biosynthesis of plasmalogen begins with acyl-CoA-dependent acylation of dihydroxyacetone-phosphate (DHAP) to form 1-0-acyl DHAP by DHAP acyl transferase which is a peroxisomal enzyme 13 . The acyl chain is then displaced by a long-chain fatty alcohol. In anaerobic bacteria, the dihydroxyacetone phosphate (DHAP) could not be an intermediate in the plasmalogen synthesis. Diacyl-glyceropho spholipids, phosphatidic acid (PA), cytidine5′-diphosphate (CDP)-diacylglycerol (DAD) and diacyl phosphatidyl glycerol (PG) may be precursors of plasmalogens in anaerobic bacteria 11,13,14 . The results of B. longum species in the present s tudy show the presence of PA, PG and Cl plasmalogens in this anaerobic bacteria.
Long-chain alcohols are utilized for biosynthesis of plasmalogens in animal cells; however, long-chain alcohols were not readily incorporated into plasmalogens in bacteria 11,14 . In contrast, long-chain aldehydes and fatty acids were readily incorporated into both the ether linked chains and acyl chains of plasmalogens in Clostridium beijerinckii 11,14 . Aldehydes generated from B. longum species included C:14, C:12 and C:17 aldehydes (Fig. 5). These aldehydes indicate aliphatic chains of the sn-1 position of plasmalogens in the B. longum species. Fatty acids analysis of total lipids from B. longum BB536 indicated that no polyunsaturated fatty acids and fatty alcohols were present in B. longum species (Table 1). On the other hand, C14:0 and C17:0 alkyl glycerols have been successfully used as alternative plasmalogen precursors 27,28 .
Increased Bacteroides and decreased Bifidobacterium were observed in fecal samples of AD as compared to those of age-and sex-matched controls 39 . Gut microbiot a influences both the production and absorption of neurotransmitters such a s serotonin and GABA by increasing their bioavailability to the brain. Some components of the microbiota synthesize and release amyloid peptides and lipopolysaccharides, which in turn activate inflammatory signaling through the release of cytoki nes, with potential effects on pathophysiological cascade of AD 41 . The increased permeability of the gut and blood brain barrier induced by microbiota dysbiosis may mediate or affect pathogenesis of A D and other neurodegenerative disorders 42 . In addition, the microbiota can secrete large amounts of amyloids and lipopolysaccharides, which might contribute to the pathogenesis of AD 42,43 . Nutrients www.nature.com/scientificreports www.nature.com/scientificreports/ have been shown to affect the composition of gut microbiota as well as the formation and aggregation of cerebral amyloid-β 42 . Oral administration of Bifidobacterium breve strain A1 to AD mice reversed the impairment of attention behavior in Y maze test and the reduced latency time in a passive avoidance test, indicating prevention of cognitive dysfunction 43 .
Colonic bacterial composition is changed in PD 42 . Disturbance of a microbiota-gut-brain axis has been linked to specific microbial products that are related to gut inflammation and neuro-inflammation [44][45][46] . Fecal short chain fatty acid concentrations were significantly reduced in PD and short chain fatty acids from some gut-microbiota significantly reduced bowel symptoms in PD 47 . Gut-microbiota regulates motor deficits and neuro-inflammation in model of PD, suggesting that alterations in human microbiome represent a risk factor for PD 48 . Dysregulation of the brain-gut-microbiota axis in PD may be associated with the pathogenesis of PD itself 48 . Excessive stimulation of the innate immune system resulting from gut dysbiosis and/or small intestinal bacterial overgrowth and increased intestinal permeability may induce systemic inflammation, while activation enteric neurons and enteric glial cells may contribute to the initiation of alpha-synuclein misfolding in PD 49 .
All of the mentioned reports indicated a close relationship of microbiota-gut-brain axis to AD and PD. On the other hand, a close relationship between AD and plasmalogens has been indicated by the observations of decreased ethanolamine plasmalogen (plsPE) in the affected brain regions of AD [50][51][52][53] . Furthermore, decreased levels of plsPE in plasma or serum have been reported in AD 54,55 . Decrease of erythrocyte plasmalogen in AD was also indicated 56 . Decreased levels of plasma plasmalogens in PD was reported 57 . A plasmalogen precursor analog treatment reduced levodopa-induced dyskinesia in PD monkey 58 .
It has been indicated that chronic inflammation is involved in causes of AD and PD 3,5,6 . It is also reported that plasmalogens inhibit amyloid precursor protein procession by inhibiting γ-secretase activity 59,60 . We reported that oral ingestion of plasmalogens showed anti-inflammatory/anti-amyloidogenic effects of plasmalogens in lipopolysaccharide-induced neuroinflammation in adult mice 61 . These results were confirmed recently by the observation that oral ingestion of plasmalogens could attenuate the lipopolysaccharide-induced memory loss and microglial activation 62 . We previously reported that oral administration of purified ether phospholipids extracted from scallop improved cognitive functions of patients with AD 63 and mild cognitive impairment (MCI) 64 .
Thus, many aspects of the reported functions of microbiota-gut-brain axis on AD and those functions of plasmalogens on AD are overlapped. Major phospholipid classes of plasmalogens in human tissues including brain are plsPE and plsPC, but major phospholipid classes of plasmalogens in gut microbiota are plsPG and plsCL. It is not known yet whether the presence of plasmalogens in B. longum strain is related to beneficial functions of B. longum strain on gut-brain axis to human health and human neurodegenerative diseases. The presence of plasmalogens in B. longum strain may play roles for many probiotic effects of B. longum to human health.

Materials and Methods
Methodology for identification of plasmalogens. Phospholipase A 1 (PLA1) hydrolyzes acyl bond of the sn-1 position of glycerophospholipids, but it does not act on alkenyl and alkyl bond of phospholipids. Therefore, treatment of total lipids from bacteria with PLA1 leaves only intact ether phospholipids of all classes of glycerophospholipids. On the other hand, only alkenyl bond (vinyl ether bond) is susceptible to acid hydrolysis among the three subclasses of glycerophospholipids. Therefore, it is possible to identify glycerophospholipid subclasses including alkylacyl and alkenylacyl phospholipids by combination of PLA1 hydrolysis and acid hydrolysis of glycerophospholipids. Phospholipid classes were separated by HPLC-ELSD method which was developed previously by us 65,66 . Lysophospholipids generated by acid hydrolysis of plasmalogens could be detected by the HPLC-ELSD method, and aldehydes generated from plasmalogens by acid hydrolysis were labelled with DNPH and were detected by another HPLC method with UV detector.
Cultivations of bacteria. Some of anaerobic bacteria in several different Yogurts and in powdery supplements, which were commercially available in market places, were cultured in TOS Propionate Agar Medium (Yakult Co. Japan) under anaerobic condition using AnaeroPak-Anaero (Mitsubishi Gas Chemical Co, Japan) at 40 °C for 2 days. Several single colonies were isolated and cultured again under the same condition. The Isolate was identified using amplification and subsequently sequencing of 16S rRNA gene using oligonucleotide primers 518F and 800R. One of the Bifidobacterium longum was Bifidobacterium BB536, which was cultured from yogurt made by Morinaga Milk Co (Japan). Bifidobacterium longum subsp. longum JCM1217, Bifidobacterium longum subsp. Infantis JCM1210, Bifidobacterium longum subsp. suis JCM1269. Bifidobacterium animalis subsp. animalis JCM1190 and Bifidobacterium animalis subsp. lactis JCM10602 were provided by RIKEN BRC through the National Bio-Resource Project of the MEXT/AMED, Japan. These bacteria were cultured in TOS Propionate Agar Medium (Yakult Co. Japan) under anaerobic condition using AnaeroPack-Anaero (Mitsubishi Gas Chemical Co. Japan) at 40 °C for 2 days. Clostridium beijerinckii (ATCC8015) was purchased from American Type Culture Identification of PA, PG and CL by LC-MS/MS. LC-MS/MS system was a Waters system consisted of Acquity UPLC H-Class and Xevo TQ-S micro. Column used was KINNETEX HILIC (100 × 4.6 mm, 2.6 µm, Waters Co.). Mobile phase A;10 mM ammonium formate in water containing 0.5% formic acid, and mobile phase B; isopropanol/acetonitrile (5:2) containing 10 mM ammonium formate and 0.5% formic acid. Gradient elution; 5% A was increased to 50% in 20 min, 50% A was kept until 23 min and decreased to the initial condition in 2 min. Flow rate was 0.3 mL/min and column temperature was 50 °C. PA and PG was detected by neutral loss scan at 172 Da in positive mode. CL was detected by precursor ion scan at 153 Da in negative mode.