Porphyromonas gingivalis laboratory strains and clinical isolates exhibit different distribution of cell surface and secreted gingipains

Figures & data

Table 1. Fold-change increase of the Arg- and Lys-gingipain activity of P. gingivalis whole cells following cysteine and glycylglycine addition to the assay

Additives (Original assay concentration: Test assay concentration)^a
Cys (mM)		0:20	0:200	0:0	0:20	0:200	0:20	0:200	0:0	0:20	0:200
GG (mM)		0:0	0:0	0:300	0:300	300:300	0:0	0:0	0:180	0:180	180:180
P. gingivalis	Country of origin [33]	Whole-cell Arg-gingipain activity					Whole-cell Lys-gingipain activity
84–3	Sudan	5.3	12	11.1	37.9	32.1	2.4	4.6	4.3	6.3	7.1
15–9	Romania	3.2	6.1	7.3	23.3	21.5	1.8	3.1	3.5	6.5	6.5
W50	Germany	6.8	11.4	18	43.6	42.6	2.2	3.8	3.9	7.9	6.8
7B TORR	USA	4.1	8.6	11.4	34.4	23.4	2.5	4.7	3.9	6.7	7.7
ATCC 33277	USA	7.4	14.7	14	53	48	2.3	4.6	4.8	9.4	7.4
A7AI-28	USA	3.5	10.6	12	45.3	42.3	2.8	3.9	4.2	5	4.9
11A	Romania	4.5	10.8	10.3	37.9	35.7	2	3.4	3.2	6.9	5.8
3–3	USA	3.2	6.1	8.2	20.5	17.9	2.2	3.8	4	6.6	6.6
381	USA	3.7	6.8	17.4	21	19.8	1.5	2.8	2.7	4.1	3.7
ATCC 49417	Canada	6	15.6	13.7	59.7	51.5	4.1	5.6	6.8	12.1	11.3
YH522	Japan	5.5	12.6	11.7	40.8	32.1	1.8	3.6	3.3	5.8	5.5
13–1	Sudan	3.9	7.2	8.8	23.3	16.7	1.5	1.9	2.1	5.1	2.8
Afr-5B1	Sudan	2.4	9	7.5	39.7	35.9	1.8	3.6	3.3	5.8	5.5
3A1	Norway	3	5.7	9	21.6	19.9	2.3	4.6	4.1	7.2	6.1
Mean		4.5	9.8	11.5	35.9	31.4	2.2	3.9	3.9	6.8	6.3
Std Dev		1.5	3.2	3.4	12.5	11.7	0.66	0.93	1.1	2.0	2.0
Parameter a		20 Cys	200 Cys	300 GG	20 Cys, 300 GG	300 GG	20 Cys	200 Cys	180 GG	20 Cys 180 GG	180 GG
Parameter b		200 Cys	300 GG	20 Cys, 300 GG	200 Cys, 300 GG	200 Cys, 300 GG	200 Cys	180 GG	20 Cys, 180 GG	200 Cys, 180 GG	200 Cys, 180 GG
P value		< 0.001	> 0.05	< 0.001	< 0.001	< 0.001	< 0.001	> 0.05	< 0.001	< 0.05	< 0.001

a.

1. Cys, cysteine; GG, glycylglycine

Figure 1. Colony phenotype of P. gingivalis isolates. Cells were spread on HBA plates and incubated anaerobically before imaging at weekly intervals. (a) week 1, (b) week 2, and (c) week 3

Table 2. Comparison of whole-cell Arg-gingipain V_max in the presence of 200 mM of cysteine (Cys) or glycylglycine (GlyGly) with 1 mM BApNA substrate

P. gingivalis	Vmax (nM/s/5 x 10^6 cells)		Vmax Ratio
	GlyGly 200 mM	Cys 200 mM	(GlyGly/Cys)
84–3	18.8 ± 0.2	13.9 ± 1.2	1.4^a
15–9	40.1 ± 0.3	29.2 ± 1.0	1.4^a
W50	38.9 ± 0.4	22.1 ± 1.3	1.8^a
7B TORR	45.6 ± 0.3	33.5 ± 2.0	1.4^a
ATCC 33277	33.5 ± 0.2	26.6 ± 0.6	1.3^a
A7A1-28	20.5 ± 0.2	19.9 ± 0.7	1.0
11A	42.6 ± 0.4	27.9 ± 0.4	1.5^a
3–3	13.6 ± 0.1	13.4 ± 0.2	1.0
381	24.2 ± 0.3	18.0 ± 0.5	1.3^a
ATCC 49417	22.2 ± 0.2	17.0 ± 0.8	1.3^a
YH522	27.5 ± 0.1	25.2 ± 0.7	1.1^a
13–1	6.8 ± 0.1	7.1 ± 0.2	1.0
Afr-5B1	11.5 ± 0.1	11.0 ± 0.3	1.0
3A1	21.4 ± 0.2	16.4 ± 0.8	1.3^a

a. P < 0.05, Student’s t-test (unpaired parametric)

Table 3. The V_max of P. gingivalis whole-cell and VFSN Arg- and Lys-gingipains

P. gingivalis	Arg-gingipain		Lys-gingipain
	Whole cell (nM/s/5 x 10^6 cells)	VFSN (µM/s)	Whole cell (nM/s/5 x 10^6 cells)	VFSN (µM/s)
84–3	110 ± 1.2	13.9 ± 0.153	13 ± 0.3	6.69 ± 0.131
15–9	68 ± 0.7	10.6 ± 0.331	11 ± 0.2	3.88 ± 0.063
W50	117 ± 1.3	10.6 ± 0.182	16 ± 0.4	0.78 ± 0.014
7B TORR	57 ± 0.9	3.87 ± 0.042	10 ± 0.3	1.06 ± 0.014
ATCC 33277	106 ± 0.8	2.44 ± 0.013	15 ± 0.2	0.17 ± 0.005
A7AI-28	70 ± 0.5	2.38 ± 0.028	13 ± 0.5	0.66 ± 0.007
11A	66 ± 0.8	1.30 ± 0.033	13 ± 0.1	0.03 ± 0.002
3–3	55 ± 0.6	2.67 ± 0.029	10 ± 0.3	0.19 ±.0002
381	51 ± 0.5	1.02 ± 0.020	10 ± 0.4	0.34 ± 0.007
ATCC 49417	110 ± 1.5	0.64 ± 0.032	17 ± 0.2	0.07 ± 0.002
YH522	74 ± 1	0.32 ± 0.006	11 ± 0.1	0.02 ± 0.001
13–1	20 ± 0.2	0.18 ± 0.004	8 ± 0.2	0.08 ± 0.002
Afr-5B1	54 ± 1.9	0.17 ± 0.007	9 ± 0.4	0.64 ± 0.013
3A1	49 ± 0.5	0.13 ± 0.004	11 ± 0.2	0.018 ± 0.004

Table 4. The comparison of Arg- and Lys- gingipain V_max for whole cells (WC) and VFSN

P. gingivalis	WC (Arg/Lys)	VFSN (Arg/Lys)	Arg (VFSN/WC)	Lys (VFSN/WC)
84–3	8.5	2.0	126	515
15–9	6.0	2.7	155	353
7B TORR	5.7	3.7	68	106
W50	7.3	14	91	49
A7A1-28	5.4	3.6	34	51
Afr-5B1	6.0	0.26	3.0	71
381	5.0	3.0	20	34
3–3	5.5	14	49	19
ATCC 33277	7.0	14	23	12
13–1	2.5	2.0	9.0	10
ATCC 49417	6.5	9.0	6.0	4.0
11A	5.0	45	20	2.0
YH522	7.0	14	4.0	2.0
3A1	4.5	7.0	3.0	2.0
Mean	5.9	9.6	44	88
Std dev	1.4	11	49	153

Figure 2. (a) The kinetics curves for P. gingivalis whole-cell Arg-gingipain activities using BApNA as substrate. (b) The kinetics curves for P. gingivalis whole-cell Lys-gingipain activities using GPKNA as substrate

Figure 3. Correlation of the log₁₀ transformed Arg-and Lys-gingipain activities of P. gingivalis (a) whole cells and (b) VFSN. Dashed lines indicate 95% PI with the model

Table 5. The K_m for substrates of whole-cell (WC) and VFSN Arg- and Lys-gingipains of P. gingivalis.

P. gingivalis	Arg-specific gingipain			Lys-specific gingipain
	Whole cell	VFSN	P-value	Whole cell	VFSN	P-value
	Km (µM)			Km (µM)
84–3	65 ± 4	70 ± 7	> 0.05	185 ± 16	193 ± 13	> 0.05
15–9	53 ± 4	69 ± 13		203 ± 14	192 ± 11
W50	59 ± 5	67 ± 8		189 ± 9	186 ± 12
7B TORR	57 ± 5	67 ± 11		232 ± 16	228 ± 13
ATCC 33277	60 ± 2.5	66 ± 7		210 ± 12	212 ± 12
11A	54 ± 4	55 ± 13		234 ± 9	223 ± 12
381	45 ± 3	57 ± 11		181 ± 10	166 ± 12
ATCC 49417	64 ± 2	68 ± 6		198 ± 12	215 ± 14
YH522	53 ± 4	50 ± 8		161 ± 10	171 ± 14
13–1	51 ± 4	49 ± 8		201 ± 12	187 ± 13
3A1	43 ± 3	56 ± 9		224 ± 12	220 ± 14
A7A1-28	40 ± 2	57 ± 6	< 0.05	142 ± 12	139 ± 8
3–3	53 ± 4	70 ± 11		161 ± 12	166 ± 12
Afr-5B1	41 ± 3	57 ± 11		201 ± 13	189 ± 13

Multiple t-tests (unpaired nonparametric)

Table 6. The calculated K_m for BApNA and V_max of P. gingivalis whole-cell Arg-gingipains with cysteine addition to the assay

P. gingivalis	Km (µM)^a		Vmax (nM/s/5 x 10^6 cells)^b
	Cys 20 mM	Cys 200 mM	Cys 20 mM	Cys 200 mM
84–3	7 ± 3	11 ± 2	8 ± 0.2	14 ± 0.1
15–9	6 ± 2	24 ± 3	16 ± 0.5	28.5 ± 0.1
W50	5 ± 1	12 ± 1	9 ± 0.2	27 ± 0.1
7B TORR	4 ± 1	14 ± 1	14 ± 0.1	29.5 ± 0.2
ATCC 33277	7 ± 1	24 ± 2	14 ± 0.4	32 ± 0.3
A7A1-28	12 ± 2	17 ± 1	9.5 ± 0.1	19.5 ± 0.1
11A	6 ± 1	21 ± 2	20 ± 0.2	26.4 ± 0.3
3–3	13 ± 1	15 ± 3	8.8 ± 0.1	11.3 ± 0.4
381	13 ± 3	22 ± 2	8.5 ± 0.2	19 ± 0.2
ATCC 49417	10 ± 1	11 ± 2	7 ± 0.5	16 ± 0.4
YH522	4 ± 1	11 ± 1	12 ± 0.2	24 ± 0.2
13–1	14 ± 2	22 ± 3	2.5 ± 0.1	7 ± 0.2
Afr-5B1	13 ± 3	22 ± 4	5.4 ± 0.1	8.5 ± 0.3
3A1	8 ± 1	19 ± 2	7.5 ± 0.1	11.2 ± 0.3

a. P < 0.05

b. P < 0.05

Table 7. The K_m and V_max of P. gingivalis whole-cell Arg-gingipains in the presence of 300 mM glycylglycine and 20 mM DTT or 20 mM L-cysteine

P. gingivalis	Km (µM)^a		Vmax (nM/s/5 x 10^6 cells)^b		Ratios of Vmax
	L-cysteine	DTT	DTT	L-cysteine	Cys/DTT
84–3	65 ± 4	41 ± 3	30 ± 1.0	110 ± 1.2	3.7
15–9	53 ± 4	67 ± 2	59 ± 1.5	68 ± 0.7	1.2
W50	59 ± 5	61 ± 4	65 ± 1.0	117 ± 1.3	1.8
7B TORR	57 ± 5	62 ± 3	54 ± 0.4	57 ± 0.9	1.1
ATCC 33277	60 ± 2.5	64 ± 5	63 ± 1.0	106 ± 0.8	1.7
A7AI-28	40 ± 2	41 ± 2	28 ± 0.5	70 ± 0.5	2.5
11A	54 ± 4	58 ± 4	57 ± 0.3	66 ± 0.8	1.2
3–3	53 ± 4	56 ± 5	37 ± 0.6	55 ± 0.6	1.5
381	45 ± 3	29 ± 3	28 ± 0.8	51 ± 0.5	1.8
ATCC 49417	64 ± 2	51 ± 5	38 ± 0.7	110 ± 1.5	2.9
YH522	53 ± 4	29 ± 5	38 ± 0.5	74 ± 1	1.9
13–1	51 ± 4	33 ± 6	9 ± 0.5	20 ± 0.2	2.2
Afr-5B1	41 ± 3	30 ± 4	25 ± 0.5	54 ± 1.9	2.2
3A1	43 ± 3	31 ± 4	33 ± 0.5	49 ± 0.5	1.5

a. P < 0.05, 84–3, 381, ATCC 49417, YH522, 13–1, Afr-5B1, 3A1.

b. P < 0.05

Figure 4. The kinetics of P. gingivalis whole-cell Arg-gingipains using BApNA substrate in the presence of 20 mM DTT and 300 mM glycylglycine. (a) The aggregate kinetic curves for 14 strains using 20 mM DTT as a reducing agent. (b) Control assays using strain W50 and ATCC 33277 cells in the presence of 300 mM glycylglycine and 20 mM DTT or 20 mM cysteine. The data shows DTT is less effective at stimulating the Arg-gingipain activity of these strains than cysteine. (P < 0.05)

Dashper SG, Mitchell HL, Seers CA, et al. Porphyromonas gingivalis uses specific domain rearrangements and allelic exchange to generate diversity in surface virulence factors. Front Microbiol. 2017;8:48.

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