A Lactobacillus mutant capable of accumulating long-chain polyphosphates that enhance intestinal barrier function

Abbreviations: polyP, polyphosphate; Pi , phosphate; HSP, heat-shock protein; MRS, de Man–Rogosa–Sharpe; SP, soy peptide HK; SCDM, simplified chemically defined medium; GITC, guanidine isothiocyanate; SDS, sodium dodecyl sulfate; ATP, adenosine triphosphate; PPK, polyphosphate kinase; ADP, adenosine diphosphate; NTG, nitrosoguanidine; X-Pi, 5-bromo-4-chloro-3-indolyl-phosphate.

Figures & data

Table 1. PolyP production by lactic acid bacteria and bifidobacterium.

Strain	Poly P (nmol/mg protein)	Strain	Poly P (nmol/mg protein)
Lactobacillus paracasei subsp. paracasei JCM 1163	13.4	Bifidobacterium breve SBC8312	0.3
Bifidobacterium pseudolongum subsp. pseudolongum SBC8868	7.1	Lactobacillus brevis SBC8035	0.2
Lactobacillus delbureckii subsp. delbrueckii SBC8973	6.8	Lactobacillus fermentum SBC8951	0.2
Bifidobacterium animalis subsp. lactis SBC8999	6.1	Lactobacillus reuteri SBC8966	0.2
Lactobacillus brevis SBC8007	5.2	Lactobacillus brevis SBC8206	0.2
Lactobacillus brevis SBC8009	4.2	Bifidobacterium bifidum SBC8311	0.2
Lactobacillus brevis SBC8015	2.4	Lactobacillus brevis SBC8010	0.2
Lactobacillus brevis SBC8005	2.1	Weissella minor SBC8879	0.2
Lactobacillus brevis SBC8012	2.0	Weissella halotolerans SBC8880	0.2
Lactobacillus brevis SBC8025	1.9	Lactobacillus brevis SBC8039	0.2
Lactobacillus brevis SBC8030	1.8	Lactobacillus acidophilus SBC8836	0.2
Lactobacillus brevis SBC8044	1.7	Lactobacillus reuteri SBC8841	0.1
Lactobacillus brevis SBC8001	1.5	Lactobacillus brevis SBC8036	0.1
Lactobacillus reuteri SBC8841	1.4	Lactobacillus fermentum SBC8953	0.1
Lactobacillus brevis SBC8011	1.3	Weissella viridescens SBC8883	0.1
Bifidobacterium animalis subsp. animalis SBC8871	1.2	Lactobacillus brevis SBC8043	<0.01
Lactobacillus brevis SBC8008	1.2	Lactobacillus buchneri SBC8946	<0.01
Weissella confusa SBC8881	1.2	Lactobacillus buchneri SBC8959	<0.01
Lactobacillus brevis SBC8003	1.1	Lactobacillus rhamnosus SBC8886	<0.01
Lactobacillus sp. SBC8873	1.0	Lactobacillus vaginalis SBC8971	<0.01
Lactobacillus brevis SBC8803	0.8	Lactobacillus brevis SBC8998	<0.01
Bifidobacterium longum subsp. infantis SBC8867	0.8	Lactobacillus gasseri group SBC8887	<0.01
Lactobacillus brevis SBC8006	0.7	Lactobacillus brevis SBC8947	<0.01
Lactobacillus brevis SBC8002	0.7	Lactobacillus brevis SBC8954	<0.01
Lactobacillus plantarum SBC8259	0.7	Lactobacillus brevis SBC8993	<0.01
Lactobacillus brevis SBC8014	0.7	Lactobacillus brevis SBC8994	<0.01
Lactobacillus brevis SBC8037	0.7	Lactobacillus brevis SBC8995	<0.01
Lactobacillus brevis SBC8033	0.6	Lactobacillus brevis SBC8996	<0.01
Lactobacillus vaginalis SBC8965	0.6	Lactobacillus brevis SBC8997	<0.01
Weissella cibaria SBC8878	0.5	Lactobacillus brevis SBC8032	<0.01
Bifidobacterium longum subsp. infantis SBC8866	0.5	Weissella paramesenteroides SBC8882	<0.01
Lactobacillus brevis SBC8013	0.5	Weissella hellenica SBC8875	<0.01
Lactobacillus reuteri SBC8970	0.4	Enterococcus mundtii SBC8860	<0.01
Streptococcus faecalis SBC8254	0.4	Enterococcus casseliflavus SBC8852	<0.01
Lactobacillus brevis SBC8017	0.3	Enterococcus faecium SBC8853	<0.01
Lactobacillus brevis SBC8004	0.3	Enterococcus avium SBC8854	<0.01
Lactobacillus brevis SBC8027	0.3	Enterococcus cecorum SBC8855	<0.01
Lactobacillus brevis SBC8029	0.3	Enterococcus durans SBC8856	<0.01
Bifidobacterium longum subsp. longum SBC8314	0.3	Enterococcus gallinarum SBC8857	<0.01
Enterococcus faecalis SBC8256	0.3	Enterococcus hirae SBC8858	<0.01
Lactobacillus brevis SBC8016	0.3	Enterococcus malodoratus SBC8859	<0.01
Bifidobacterium breve SBC8313	0.3

Notes:

PolyP was extracted from lactic acid bacteria with glass milk. The extracted polyP was converted to ATP by PPK under the condition of high ADP concentration (0.5 mM). ATP concentration was determined by the luminescence induced by luciferase reaction. SBC: Sapporo Breweries Culture Collection.

Fig. 1. Chain length determination of polyP from L. paracasei JCM 1163 using polyacrylamide gel electrophoresis.

Notes: The polyacrylamide gel was electrophoresed at 10 mA. After electrophoresis, the gel was stained with staining solution [0.05% Toluidine Blue O dissolved in 25% methanol] for at least 15 min, and destained with destaining solution (25% methanol) for at least 10 min several times until a clear background was obtained. PolyP with average chain length of 700, 65, and 25 Pi residues was used as standards.

Fig. 2. Time course of polyP production by L. paracasei JCM 1163.

Notes: L. paracasei JCM 1163 was cultured in MRS medium at 37 °C. After centrifugation, the polyP content in the bacterial cells (A) and culture supernatant (B) were measured.

Fig. 3. PolyP production in Pi-starved L. paracasei JCM 1163 upon Pi supplementation.

Notes: L. paracasei JCM 1163 pre-cultured in MRS medium was transferred to SP medium and cultured until the Pi concentration in the SP medium reached less than 100 μM (4 h at 37 °C). Next, Pi was added into the culture medium and the polyP content in cells (A) and OD₆₀₀ (B) was periodically measured.

Fig. 4. PolyP production by NTG-treated L. paracasei JCM 1163.

Notes: (A) L. paracasei JCM 1163 at the early logarithmic growth phase was treated with 0.5 mg/ml nitrosoguanidine (NTG) for 1 h, and then seeded in MRS medium containing 0.005% 5-bromo-4-chloro-3-indolyl-phosphate (X-Pi). Mutants that showed blue-colored colonies on the MRS medium were selected and their polyP contents were measured. (B) High polyP-accumulating mutant No. 14 was further treated with NTG for 1 h, and then seeded in MRS medium containing 0.005% X-Pi.

Fig. 5. Rescue of intestinal barrier function with polyP accumulated by lactic acid bacteria.

Notes: An oxidant (0.3 mM NH₂Cl) exposure to mouse intestinal loops increases their mannitol flux. The effect of polyP (0.1 or 1 μM) extracted from L. paracasei JCM 1163 on [³H]-mannitol flux was investigated (n = 5, each group). All values are expressed as mean ± standard errors of the mean (SEM). **Significantly different from corresponding control group (NH₂Cl without polyP) at p < 0.01.