Hoai Thu Dao; Quang Lam Truong; Van Tan Do; Tae-Wook Hahn

doi:10.4142/jvs.2020.21.e20

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Dao, Truong, Do, and Hahn: Construction and immunization with double mutant Δ apxIBD Δ pnp forms of Actinobacillus pleuropneumoniae serotypes 1 and 5

Original Article

J Vet Sci 2020;21(2):e20.

Published online: 4 March 2020

DOI: https://doi.org/10.4142/jvs.2020.21.e20

Construction and immunization with double mutant Δ apxIBD Δ pnp forms of Actinobacillus pleuropneumoniae serotypes 1 and 5

Hoai Thu Dao^1,^†, Quang Lam Truong^1,^2,^†, Van Tan Do¹, Tae-Wook Hahn^1,^*

¹College of Veterinary Medicine and Institute of Veterinary Science, Kangwon National University, Chuncheon 24341, Korea

²Key Laboratory of Veterinary Medicine, Faculty of Veterinary Medicine, Vietnam National University of Agriculture, Hanoi 100000, Vietnam

^*Corresponding author: Tae-Wook Hahn Department of Veterinary Medicine, College of Veterinary Medicine, Kangwon National University, 1 Kangwondaehak-gil, Chuncheon 24341, Korea. E-mail: twhahn@kangwon.ac.kr

^† These authors distributed equally in this study.

Conflict of Interest The authors declare no conflicts of interest.

Received 16 October 2019 Revised 6 December 2019 Accepted 6 December 2019

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Actinobacillus pleuropneumoniae (APP) causes a form of porcine pleuropneumonia that leads to significant economic losses in the swine industry worldwide. The apxIBD gene is responsible for the secretion of the ApxI and ApxII toxins and the pnp gene is responsible for the adaptation of bacteria to cold temperature and a virulence factor. The apxIBD and pnp genes were deleted successfully from APP serotype 1 and 5 by transconjugation and sucrose counter-selection. The APP1Δ apxIBDΔ pnp and APP5Δ apxIBDΔ pnp mutants lost hemolytic activity and could not secrete ApxI and ApxII toxins outside the bacteria because both mutants lost the ApxI- and ApxII-secreting proteins by deletion of the apxIBD gene. Besides, the growth of these mutants was defective at low temperatures resulting from the deletion of pnp. The APP1Δ apxIBDΔ pnp and APP5Δ apxIBDΔ pnp mutants were significantly attenuated compared with wild-type ones. However, mice vaccinated intraperitoneally with APP5Δ apxIBDΔ pnp did not provide any protection when challenged with a 10-times 50% lethal dose of virulent homologous (APP5) and heterologous (APP1) bacterial strains, while mice vaccinated with APP1Δ apxIBDΔ pnp offered 75% protection against a homologous challenge. The Δ apxIBDΔ pnp mutants were significantly attenuated and gave different protection rate against homologous virulent wild-type APP challenging.

Keywords: Actinobacillus pleuropneumoniae, deletion mutation, apxIBD gene, pnp gene, virulence

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Fig. 1.

PCR analysis of the wild-type and apxIBD, pnp-deleted mutant of APP1 (A) and APP5 (B) using primer pairs P5–P6 and P11–P12. Lane M, DNA molecular weight ladder 1 kb; lane 1, negative control; lane 2, apxIBD gene amplified from genomic DNA of wild-type APP; lane 3, apxIBD gene amplified from genomic DNA of the Δ apxIBDΔ pnp mutant; lane 4, pnp gene amplified from genomic DNA of the wild-type APP; lane 5, pnp gene amplified from genomic DNA of Δ apxIBDΔ pnp mutant. PCR, polymerase chain reaction; APP, Actinobacillus pleuropneumoniae.

Fig. 2.

Phenotypes of the APP mutants. Hemolytic activity of wild-type and mutants of APP1 (A) and APP5 (B). The black arrow indicates the clear zone caused by the hemolytic activity surrounding bacteria on blood agar. Cold shock adaptive ability of wild-type and mutant APP1 (C) and APP5 (D). Open arrow indicates colony growth on BHI agar containing a final amount of 10 µg/mL NAD. APP, Actinobacillus pleuropneumoniae; BHI, brain heart infusion; NAD, nicotinamide adenine dinucleotide.

Fig. 3.

Growth curves of wild-type and mutants of APP1 (A) and APP5 (B). Symbols: ο, wild-type APP; ▲, mutant APP Δ apxIBDΔ pnp. APP, Actinobacillus pleuropneumoniae; OD₆₀₀, optical density at 600 nm.

Fig. 4.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Apx toxins in wild-type and the mutants of APP1 (A) and APP5 (B). Lane M, Xpert Prestained Protein marker; lane 1, culture supernatant of the wild-type APP before precipitating; lane 2, culture supernatant of the mutants before precipitating; lane 3, RTX toxins of wild-type after precipitating with ammonium sulfate; lane 4, RTX toxins of the mutants after precipitating with ammonium sulfate. APP, Actinobacillus pleuropneumoniae; RTX, repeats in toxin.

Fig. 5.

Evaluation of the stability deletion gene in the genome of APP1Δ apxIBDΔ pnp and APP5Δ apxIBDΔ pnp over 10 passages by PCR using primer pairs P5–P6 and P11–P12 for amplifying apxIBD and pnp genes, respectively. Amplified apxIBD gene (A) and pnp gene (B) from APP1Δ apxIBDΔ pnp. Amplified apxIBD gene (C) and pnp gene (D) from APP5Δ apxIBDΔ pnp. Lane M, DNA Ladder 1kb; lane 1, negative control; lane 2, target gene amplified from genomic DNA of wild-type APP, lanes 3–12, deletion gene amplified from genome DNA of mutant through 10 continuous passages. APP, Actinobacillus pleuropneumoniae; PCR, polymerase chain reaction.

Table 1.

Bacterial strains used in this study

Strain	Relevant characteristics	Source
Escherichia coli
DH5α	Cloning vehicle: supE44 Δ lacU169 (ϕ80 lacZΔ M15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1	Invitrogen
WM3064	Donor strain for conjugation; thrB1004 pro thi rpsL hsdS lacZΔ M15	[37]
	RP4–360_(araBAD)567_ dapA1341::_ erm pir(wt)_
Actinobacillus
pleuropneumoniae
APP1	Serovar 1, an isolate from infected pig in Korea	This study
APP5	Serovar 5, an isolate from infected pig in Korea	This study
APP1Δ apxIBDΔ pnp	Mutation in apxIBD and pnp gene of APP1	This study
APP5Δ apxIBDΔ pnp	Mutation in apxIBD and pnp gene of APP5	This study
APP1Δ apxIBD	Mutation in apxIBD of APP1	This study
APP5Δ apxIBD	Mutation in apxIBD of APP5	This study
APP1Δ pnp	Mutation in pnp gene of APP1	This study
APP5Δ pnp	Mutation in pnp gene of APP5	This study

APP, Actinobacillus pleuropneumoniae.

Table 2.

Plasmid and primers used in this study

Plasmids, primers	Characteristics	Source
Plasmids
pBluescriptII SK(+)	E. coli cloning vector carrying an ampicillin resistance determinant	Stratagene
pEMOC2	Transconjugation vector: ColE1 ori mob RP4 sacB, Amp ^r Cam ^r	[38]
pEMOC2Δ apxIBD	pEMOC2 including the truncated apxIBD	This study
pEMOC2Δ pnp	pEMOC2 including the truncated pnp	This study
Primers for constructing APP1Δ apxIBDΔ pnp
P1-F	5′-ACGCGTCGACCGTTCTCTTAAACTCTCCG-3′ (Sal I site underlined)	Amplified an upstream sequence of apxIB (892 bp) in APP1
P2-R	5′-CGGGATCCCGCTATTTGATCACCGAGC-3′ (Bam HI underlined)
P3-F	5′-CGGGATCCCGAGGATTATGCGTTTCCAA-3′ (Bam HI site underlined)	Amplified a downstream sequence of apxID (1,003 bp) in APP1
P4-R	5′-ATGCGCGGCCGCGACGGTGGTGTAGGCAATG-3′ (Not I underlined)
P5-F	5′-GCCAACTCATTTACCGCTT-3′	Amplified 3,759 bp in parent and 1,389 bp in mutant. These
P6-R	5′-GGTAACGGAAACGACCGAT-3′	primers were used to confirm the deletion of apxIBD in APP1
P7-F	5′-ATACGTCGACGCCATAACGCTCGGTACG-3′ (Sal I site underlined)	Amplified an upstream sequence of pnp (1,052 bp) in APP1
P8-R	5′-CATTAATCGGGATCCCGTAAAGGTGGTGCGGTAAT-3′ (Bam HI site underlined)
P9-F	5′-CGGGATCCCGACCTTCACGTTTGAAGA-3′ (Bam HI site underlined)	Amplified a downstream sequence of pnp (1,142 bp) in APP1
P10-R	5′-ATGCGCGGCCGCACATCACGCTTGCTTCGG-3′ (Not I site underlined)
P11-F	5′-CATTTGTGCCCCAGAACTCA-3′	Amplified 3,885 bp in parent and 2,423 bp in mutant. These
P12-R	5′-ATTGAGCCTTTGCCTTGCTC-3′	primers were used to confirm the deletion of pnp in APP1
Primers for constructing APP5Δ apxIBDΔ pnp
P1-F	5′-ACGCGTCGACACTTTGGTTGAAAGGCTAT-3′ (Sal I site underlined)	Amplified an upstream sequence of apxIB in APP5 (922 bp)
P2-R	5′-CGTTACCGGGATCCCGGTACGGTTCAGCAACTT-3′ (Bam HI underlined	d)
P3-F	5′-ACCGTACCGGGATCCCGGTAACGAGCCTAAAAT-3′ (Bam HI site underlined)	Amplified a downstream sequence of apxID in APP5 (922 bp)
P4-R	5′-ATGCGCGGCCGCTCGGGAAGAAGACTACGG-3′ (Not I site underlined	)
P5-F	5′-GCCTGCCATCACAGGTAA-3′	Amplified 4,308 bp in parent and 2,080 bp in mutant. These
P6-R	5′-CGGTCCATTAGCTTACAGC-3′	primers were used to confirm the deletion of apxIBD in APP5
P7-F	5′-ACGCGTCGACGCCATAACGCTCGGTACG-3′ (Sal I site underlined)	Amplified an upstream sequence of pnp in APP5 (1,077 bp)
P8-R	5′-CATTAATCGGGATCCCGTAAAGGTGGTGCGGTAAT-3′ (Bam HI underlined)
P9-F	5′-CACCTTTACGGGATCCCGGATTAATGTTTCGCCTTC-3′ (Bam HI site underlined)	Amplified a downstream sequence of pnp in APP5 (1,171 bp)
P10-R	5′-ATGCGCGGCCGCCGTTGATATTGTGCGGCG-3′ (Not I site underlined)
P11-F	5′-CGTTTCGGCACGCTTAAT-3′	Amplified 3,828 bp in parent and 2,393 bp in mutant. These
P12-R	5′-GGCAATATCGGCTTTAGGG-3′	primers were used to confirm the deletion of pnp in APP5

Table 3.

Virulence of the wild-type and mutant of APP in mice^*

Strain tested	LD₅₀† (CFU)	Fold attenuation^‡
APP1 wild-type	1.6 × 10⁶	1
APP1Δ apxIBD	6.3 × 10⁷	39
APP1Δ pnp	4.4 × 10⁶	3
APP1Δ apxIBDΔ pnp	1.5 × 10⁸	93
APP5 wild-type	6.5 × 10⁶	1
APP5Δ apxIBD	1.4 × 10⁸	21
APP5Δ pnp	1.6 × 10⁷	2
APP5Δ apxIBDΔ pnp	3.1 × 10⁸	48

APP, Actinobacillus pleuropneumoniae; LD₅₀, 50% lethal dose; CFU, colony-forming unit.

^* Groups of five mice were injected intraperitoneally with 200 mL of bacterial suspension containing various doses of APP. The numbers of surviving mice were recorded 10 days after inoculation.

^† LD₅₀ was calculated by the Reed-Muench method;

^‡ Fold attenuation was normalized to the wild-type APP.

Table 4.

Protection of mice immunized with APP1 and APP5 mutants challenged with homologous and heterologous serotypes of APP^*

Group	Injection dose (CFU)	Challenge (10 × LD₅₀)	Number of survival/number of tested (%)
APP1Δ apxIBDΔ pnp	1 × 10⁷	APP1	6/8 (75%)
		APP5	0/8 (0%)
APP5Δ apxIBDΔ pnp	1 × 10⁷	APP1	0/8 (0%)
		APP5	0/8 (0%)
Control	TSB	APP1	0/8 (0%)
		APP5	0/8 (0%)

APP, Actinobacillus pleuropneumoniae; CFU, colony-forming unit; LD₅₀, 50% lethal dose.

^* Mice were immunized twice intraperitoneally with the mutant or culture media (controls) and challenged at 2 weeks after vaccination with homologous or heterologous virulent strains. Surviving mice were recorded at 10 days after challenge.

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