The aspartate-semialdehyde dehydrogenase of Edwardsiella ictaluri and its use as balanced-lethal system in fish vaccinology

Abstract

asdA mutants of gram-negative bacteria have an obligate requirement for diaminopimelic acid (DAP), which is an essential constituent of the peptidoglycan layer of the cell wall of these organisms. In environments deprived of DAP, i.e., animal tissues, they will undergo lysis. Deletion of the asdA gene has previously been exploited to develop antibiotic-sensitive strains of live attenuated recombinant bacterial vaccines. Introduction of an Asd(+) plasmid into a ΔasdA mutant makes the bacterial strain plasmid-dependent. This dependence on the Asd(+) plasmid vector creates a balanced-lethal complementation between the bacterial strain and the recombinant plasmid. E. ictaluri is an enteric gram-negative fish pathogen that causes enteric septicemia in catfish. Because E. ictaluri is a nasal/oral invasive intracellular pathogen, this bacterium is a candidate to develop a bath/oral live recombinant attenuated Edwardsiella vaccine (RAEV) for the catfish aquaculture industry. As a first step to develop an antibiotic-sensitive RAEV strain, we characterized and deleted the E. ictaluri asdA gene. E. ictaluri ΔasdA01 mutants exhibit an absolute requirement for DAP to grow. The asdA gene of E. ictaluri was complemented by the asdA gene from Salmonella. Several Asd(+) expression vectors with different origins of replication were transformed into E. ictaluri ΔasdA01. Asd(+) vectors were compatible with the pEI1 and pEI2 E. ictaluri native plasmids. The balanced-lethal system was satisfactorily evaluated in vivo. Recombinant GFP, PspA, and LcrV proteins were synthesized by E. ictaluri ΔasdA01 harboring Asd(+) plasmids. Here we constructed a balanced-lethal system, which is the first step to develop an antibiotic-sensitive RAEV for the aquaculture industry.

Figure 1. Sequence alignment among representative members of the AsdA family.

The secondary structure at the top of the alignment corresponds to the E. ictaluri AsdA enzyme (spirals represent α-helix; arrows represent β-sheet). Conserved amino acids residues are indicated in grey. The stars indicated the key catalytic active site residues (Cys-135, Gln-162, Glu-241, Arg-267, and His-274). The AsdA sequences were obtained from NCBI's Entrez Protein database for Edwardsiella ictaluri YP_002935083.1; Edwardsiella tarda YP_003297386.1; Escherichia coli AP_004358.1; Salmonella Typhi NP_807591.1; Salmonella Paratyphi A YP_152515.1; Salmonella Typhimurium AAB69392.1; Shigella flexnieri YP_690789.1; Shigella sonnei YP_312455.1; Citrobacter koseri YP_001456333.1; Enterobacter cancerogenus ZP_05969786.1; Enterobacter sp. YP_001178547.1; Yersinia pestis NP_671174.1; Yersinia ruckeri ZP_04615435.1; Proteus mirabilis YP_002152826.1; Aeromonas hydrophila ABK39477.1; Aeromonas salmonicida YP_001142146.1; Sodalis glossinidius YP_456010.1; Vibrio cholerae YP_001217562.1; Pseudomonas aeruginosa NP_251807.1; Erwinia carovora atrosepticum YP_052242.1.

Figure 2. Sequence alignment among representative members of the AsdB family.

The secondary structure at the top of the alignment corresponds to the S. mutans AsdB enzyme (spirals represent α-helix; arrows represent β-sheet). Conserved amino acids residues are indicated in grey. The stars indicated the key catalytic active site residues not present in AsdB from Edwardsiella. The AsdB sequences were obtained from NCBI's Entrez Protein database for Streptococcus mutans NP_721384.1; Edwardsiella ictaluri YP_002934124; Edwardsiella tarda YP_003296462; Vibrio cholerae YP_001217630.1; Bacillus cereus YP_085142.1; Legionella longbeachae CBJ10915; Legionella pneumophila YP_096311.1; Xanthomonas axonopodis NP_643032.1; Xanthomonas campestris NP_637897.1; Mycobacterium tuberculosis NP_218225.1; Mycobacterium marinum YP_001853481.1.

Figure 3. Phylogenetic tree constructed by the unweighted pair group method with arithmetic mean.

Bootstrap values indicate the number of times that a given node was detected out of 100. The Asd sequences were obtained from NCBI's Entrez Protein database for Edwardsiella ictaluri YP_002935083.1; Edwardsiella tarda YP_003297386.1; Escherichia coli AP_004358.1; Salmonella Typhi NP_807591.1; Salmonella Paratyphi A YP_152515.1; Salmonella Typhimurium AAB69392.1; Shigella flexnieri YP_690789.1; Shigella sonnei YP_312455.1; Citrobacter koseri YP_001456333.1; Enterobacter cancerogenus ZP_05969786.1; Enterobacter sp. YP_001178547.1; Yersinia pestis NP_671174.1; Yersinia ruckeri ZP_04615435.1; Proteus mirabilis YP_002152826.1; Aeromonas hydrophila ABK39477.1; Aeromonas salmonicida YP_001142146.1; Sodalis glossinidius YP_456010.1; Vibrio cholerae YP_002810714.1; Pseudomonas aeruginosa NP_251807.1; Erwinia carovora atrosepticum YP_052242.1; Streptococcus mutans NP_721384.1; Edwardsiella ictaluri YP_002934124; Edwardsiella tarda YP_003296462; Vibrio cholerae YP_001217630.1; Bacillus cereus YP_085142.1; Legionella longbeachae CBJ10915; Legionella pneumophila YP_096311.1; Xanthomonas axonopodis NP_643032.1; Xanthomonas campestris NP_637897.1; Mycobacterium tuberculosis NP_218225.1; Mycobacterium marinum YP_001853481.1; Chlamydia trachomatis YP_002887982.1.

Figure 4. Deletion of asdA gene in E. ictaluri.

A. Deletion map of ΔasdA01; B. Genotype verification of J112 ΔasdA01 by PCR; C. Phenotype of E. ictaluri J111 ΔasdA01 and J112 ΔasdA01 mutants. The strains were grown in BHI at 28°C with agitation (180 r.p.m.).

Figure 5. Complementation of representative ΔasdA mutant strains with E. ictaluri asdA gene.

(A–D) Growth of representative ΔasdA mutant strains complemented with asdA from E. ictaluri. pEZ140 (SD-asdA); pEZ142 (P_asdA-asdA); The strains were grown in BHI at 28°C with agitation (180 r.p.m.); (E) Promoter region of asdA gene from E. ictaluri and representative strains.

Figure 6. Growth of E. ictaluri ΔasdA01 complemented with asdB from Streptococcus mutans.

The strains were gown in BHI at 28°C with agitation (180 r.p.m.).

Figure 7. Complementation of asdA gene with Asd⁺ vectors.

(A) Plasmid profile of E. ictaluri ΔasdA01 complemented with AsdA⁺ vectors of different copy number. pEI1 (5.7 kb), pEI2 (4.9 kb), pYA3620 (3169 bp), pYA3493 (3113 bp), pYA3341 (2595 bp); Supercoiling ladder, from the top to the bottom: 16210 bp, 14174 bp, 12138 bp, 10102 bp, 8066 bp, 7045 bp, 6030 bp, 5012 bp, 3990 bp, 2972, 2067 bp; (B) Growth of E. ictaluri ΔasdA01 complemented with different AsdA⁺ vectors; The strains were grown in BHI at 28°C with agitation (180 r.p.m.).

Figure 8. Synthesis of heterologous antigens in E. ictaluri J112 ΔasdA01 by using AsdA⁺ expression vectors.

A. Plasmid profile of J112 (pYA3994); B. Expression of GFP J112 (pYA3994); C. Expression and secretion of Y. pestis LcrV antigen by J112 (pYA3840); D. Expression and secretion of S. pneumoniae PspA-Rx1 antigen by J112 (pYA4088).