Do acute hepatopancreatic necrosis disease-causing PirAB^VP toxins aggravate vibriosis?

Figures & data

Table 1. Bacteria species used in this study.

Bacteria species
Aeromonas hydrophila LVS3
Vibrio harveyi
BB120 (wild type)
BB152 (Mutation in LuxM: HAI-1 synthase)
JMH603 (Mutation in CqsA: CAI-1 synthase)
MM30 (Mutation in LuxS: AI-2 synthase)
JMH634 (Mutation in LuxM, LuxS and CqsA synthase)
Vibrio alginolyticus AQ13-91 (Wild type)
Vibrio campbellii LMG21363 (Wild type)
Vibrio parahaemolyticus CAIM170 (Wild type)
Vibrio proteolyticus LMG10942 (Wild type)
Vibrio anguillarum NB10 (Wild type)

Table 2. Survival (%) of Artemia franciscana at different time points after being challenged with V. harveyi wild type and its quorum sensing mutants (mean ± SD, n = 12).

Treatments	12 h	24 h	36 h	48 h	60 h
Control	99.2 ± 2.9^a	99.2 ± 2.9^a	99.2 ± 2.9^b	90.8 ± 6.7^e	62.5 ± 12.2^e
V. harveyi strains
BB120 (Wild type)	95.8 ± 5.2^a	95.0 ± 6.8^a	85.8 ± 9.9^a	43.3 ± 16.1^c	7.5 ± 8.7^b
BB152 (HAI-1^-)	97.5 ± 6.2^a	95.0 ± 6.7^a	88.3 ± 10.3^a	44.2 ± 10.8^c	13.3 ± 12.3^bc
JMH603 (CAI-1^-)	99.2 ± 2.9^a	99.2 ± 2.9^a	96.7 ± 4.9^b	57.5 ± 8.7^c	7.5 ± 7.5^b
MM30 (AI-2^-)	98.3 ± 3.9^a	98.3 ± 3.9^a	97.5 ± 4.5^b	75.0 ± 7.9^d	21.7 ± 15.4^c
JMH634 (triple QS mutant)	97.5 ± 4.5^a	97.5 ± 4.5^a	97.5 ± 4.5^b	91.7 ± 8.4^e	69.2 ± 9.0^e
V. alginolyticus AQ13-91	99.2 ± 2.9^a	99.2 ± 2.9^a	95.8 ± 7.9^b	60.0 ± 11.3^b	14.2 ± 9.0^bc
V. campbellii LMG21363	100^a	99.2 ± 2.9^a	93.3 ± 9.8^ab	31.8 ± 15.9^b	4.2 ± 5.2^b
V. parahaemolyticus CAIM170	100^a	99.2 ± 2.9^a	89.2 ± 13.1^a	38.3 ± 17.5^b	5.8 ± 5.2^b
V. proteolyticus LMG10942	99.2 ± 2.9^a	99.2 ± 2.9^a	92.5 ± 9.7^ab	11.7 ± 15.3^a	0.8 ± 2.9^a
V. anguillarum NB10	99.2 ± 2.9^a	99.2 ± 2.9^a	97.5 ± 4.5^b	80.0 ± 6.0^d	44.2 ± 7.9^d

Note: The non-challenged group served as control. Significant differences within the column between control and treatment bacterial groups at each sampling point are indicated with different superscript (p < 0.05).

Table 3. Average (±SD) cell counts and dead cells percentage of V. harveyi wild type BB120 and its derivatives with or without PirAB^VP (PirAB⁺ or PirAB^-), obtained from flow cytometer (n = 3).

	Total count (×10^3 cells/ml)		Dead cells (%)
	PirAB^-	PirAB^+	PirAB^-	PirAB^+
BB120 (wild type)	559.3 ± 38.5^a	500.9 ± 10.3^a	2.7 ± 0.4^A	2.4 ± 0.3^A
BB152 (HAI-1^-)	792.1 ± 10.0^b	734.8 ± 5.7^a	1.5 ± 0.3^A	1.7 ± 0.2^A
JMH603 (CAI-1^-)	650.8 ± 18.5^b	580.8 ± 12.8^a	1.5 ± 0.2^A	1.9 ± 0.1^A
MM30 (AI-2^-)	943.2 ± 27.6^a	1051.0 ± 15.7^b	1.3 ± 0.1^A	1.8 ± 0.2^B
JMH634 (triple QS mutant)	377.9 ± 6.0^b	144.4 ± 3.7^a	5.2 ± 0.2^A	5.0 ± 0.1^A

Note: Different superscripts indicate significant difference (p < 0.05).

Table 4. The effect of PirAB^VP on virulence factors of V. harveyi wild type BB120 and its derivatives (n = 3).

Category of in vitro tests	In the presence of PirAB^VP
	BB120 (Wild type)	BB152 (HAI-1^-)	JMH603 (CAI-1^-)	MM30 (AI-2^-)	JMH634 (Triple mutant)
Biofilm formation	NSE	NSE	↑*	NSE	ND
Swimming motility	↑**	↑**	↑***	↑**	ND
Haemolysis activity	↓*	NSE	NSE	NSE	ND
Caseinase activity	↑***	↑**	↓*	↓**	ND
Lipase activity	↑***	NSE	↓*	NSE	↑***

Note: ND = not detected, NSE = no significant effect (p > 0.05), ↑: increase, ↓: decrease; *(p < 0.05), **(p < 0.01), ***(p < 0.001).

Figure 1. Survival (%) of Artemia franciscana at different time points after being challenged with PirAB^VP toxin @ 5.2 µg/100 µl and Vibrio harveyi strains (A) PirAB^VP toxin and V. harveyi wild type BB120 strain, (B) PirAB^VP toxin and HAI-1 deficient mutant BB152 strain, (C) PirAB^VP toxin and CAI-1 deficient mutant JMH603 strain, (D) PirAB^VP toxin and AI-2 deficient mutant MM30 strain and (E) PirAB^VP toxin and QS triple mutant JMH634 strain. The error bars represent the standard deviation values of 12 replicates. Asterisks on the top of error bars indicate the significant in vivo interaction, either synergistic or antagonistic interaction between toxin and bacteria, and based on output interaction plot obtained from two-way ANOVA analysis *(p < 0.05), **(p < 0.01), ***(p < 0.001). The effect of the toxin or the Vibrio is not statistically analysed and hence not presented here, only the interactive compound of the 2-way ANOVA is presented. For an interactive statistical effect to be considered of biological relevance, only data at timepoints for which the survival in all treatments was above 10% are considered.

Figure 2. Survival (%) of A. franciscana at different time points after being challenged with PirAB^VP toxin @ 5.2 µg/100 µl and (A) V. alginolyticus AQ13-91, (B) V. campbellii LMG21363 (C) V. parahaemolyticus CAIM170, (D) V. proteolyticus LMG10942 and (E) V. anguillarum NB10. The error bars represent the standard deviation values of 12 replicates. Asterisks on the top of error bars indicate the significant in vivo interaction, either synergistic or antagonistic interaction between toxin and bacteria, and based on output interaction plot obtained from two-way ANOVA analysis *(p < 0.05), **(p < 0.01), ***(p < 0.001). The effect of the toxin or the Vibrio is not statistically analysed and hence not presented here, only the interactive compound of the 2-way ANOVA is presented. For an interactive statistical effect to be considered of biological relevance, only data at timepoints for which the survival in all treatments was above 10% are considered.

Table 5. Average (±SD) cell counts and dead cells percentage of V. alginolyticus AQ1391, V. campbellii LMG21363, V. proteolyticus LMG10942, V. parahaemolyticus CAIM170 and V. anguillarum NB10 strains with or without PirAB^VP (PirAB⁺ or PirAB^-), obtained from flow cytometer (n = 3).

Bacterial species	Average cell count (×10^3 cells/ml)		Dead cells (%)
	PirAB^-	PirAB^+	PirAB^-	PirAB^+
V. alginolyticus AQ13-91	4267.0 ± 67.8 ^a	5942.7 ± 72.8^b	2.1 ± 1.2 ^A	0.5 ± 0.1^A
V. campbellii LMG21363	4339.1 ± 36.7^a	5639.7 ± 62.4^b	0.5 ± 0.0 ^B	0.3 ± 0.0^A
V. parahaemolyticus CAIM170	3383.4 ± 143.2 ^b	2090.8 ± 68.8 ^a	2.4 ± 0.5^A	3.0 ± 0.2^A
V. proteolyticus LMG10942	4027.0 ± 82.7^a	4894.0 ± 69.1^b	7.1 ± 0.2 ^B	4.7 ± 0.2^A
V. anguillarum NB10	1197.4 ± 22.7^a	999.5 ± 46.8 ^a	1.4 ± 0.2^A	1.1 ± 0.0^A

Note: Different superscripts indicate significant difference (p < 0.05).

Table 6. The effect of PirAB^VP on virulence factors of the studied Vibrio spp (n = 3).

Category of in vitro tests	In the presence of PirAB^VP
	V. alginolyticus AQ1391	V. campbellii LMG21363	V. parahaemolyticus CAIM170	V. proteolyticus LMG10942	V. anguillarum NB10
Biofilm formation	ND	ND	ND	ND	ND
Swimming motility	NSE	NSE	↑*	Overgrew	↓*
Haemolysis activity	ND	↑*	↓*	ND	NSE
Caseinase activity	ND	NSE	NSE	ND	NSE
Lipase activity	ND	ND	ND	ND	ND

Note: ND = not detected, NSE = no significant effect (p > 0.05), ↑: increase, ↓: decrease; *(p < 0.05), **(p < 0.01), ***(p < 0.001).