Salmonella enterica Serovar Typhimurium Expressing a Chromosomally Integrated Copy of the Bacillus anthracis Protective Antigen Gene Protects Mice against an Anthrax Spore Challenge

Helen S. Garmory; Richard W. Titball; Kate F. Griffin; Ulrike Hahn; Reinhard Böhm; Wolfgang Beyer

doi:10.1128/IAI.71.7.3831-3836.2003

DOI: 10.1128/IAI.71.7.3831-3836.2003

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FIG. 1.
Construction of ZpVDL9.3PA83ec and ZpUTKmPA83ec.
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FIG. 2.
Western blot of auxotrophic S. enterica serovar Typhimurium cell lysates and culture supernatants separated by SDS-PAGE and probed with anti-PA monoclonal antibody. ZpVDL9.3PA83ec (plasmid-borne PA gene), ZpUTKmPA83ec (chromosomal PA gene), and Zoosaloral H (parent) were cultured overnight in Luria broth with appropriate antibiotic selection. Harvested cells and supernatants were normalized to similar CFU per milliliter and lysed in SDS-PAGE sample buffer. Lane M, Bio-Rad biotinylated SDS-PAGE standards, low range; lane 1, 1 mg of recombinant PA/ml; lane 2, Zoosaloral H cell lysate; lane 3, ZpVDL9.3PA83ec cell lysate; lane 4, ZpUTKmPA83ec cell lysate; lane 5, Zoosaloral H supernatant; lane 6, ZpVDL9.3PA83ec supernatant; lane 7, ZpUTKmPA83ec supernatant.
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FIG. 3.
IgG1 and IgG2a responses to PA determined by ELISA of serum samples following oral or i.v. immunization of mice. Groups of A/J mice (n = 10) were inoculated i.v. (10⁵ CFU) (A) or orally (10⁹ CFU, with a gavage needle) (B) with S. enterica serovar Typhimurium expressing chromosomally encoded PA83ec-′hlyA. Circles show individual values; mean values are indicated by horizontal lines. Results for mice with responses to PA below the limit of detection are not shown.
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FIG. 4.
Numbers of mice surviving challenge with 100 (A) or 1,000 (B) LD₅₀s of B. anthracis STI spores over 14 days after oral (open circles) or i.v. (open squares) inoculation with S. enterica serovar Typhimurium expressing chromosomally encoded PA83ec-′hlyA. The survival of control mice inoculated with S. enterica serovar Typhimurium Zoosaloral H alone is also shown (closed triangles).

Tables

Figures

TABLE 1.

Codon usage in native PA83, E. coli codon-optimized PA83ec, and S. enterica serovar Typhimurium^a

Amino acid	Codon	Frequency/1,000 codons in:
Amino acid	Codon	PA83	PA83ec	Salmonella
R	CGA	2.7	0.0	3.5
	CGC	0.0	10.8	22.5
	CGG	5.4	0.0	6.2
	CGU	4.1	27.1	19.9
	AGA	21.7	0.0	3.1
	AGG	4.1	0.0	2.0
L	CUA	4.1	0.0	4.6
	CUC	2.7	1.3	9.9
	CUG	1.4	58.4	49.4
	CUU	8.2	14.9	10.5
	UUA	51.6	10.8	13.4
	UUG	9.5	2.7	11.7
S	UCA	19.0	0.0	7.4
	UCC	4.1	25.8	10.9
	UCG	9.5	0.0	8.3
	UCU	28.5	47.5	8.7
	AGC	1.4	13.5	16.4
	AGU	31.2	6.7	7.6
T	ACA	28.5	1.3	6.8
	ACC	8.2	44.8	24.1
	ACG	13.6	8.1	15.5
	ACU	25.8	21.7	8.3
P	CCA	13.6	8.1	6.5
	CCC	4.1	1.3	6.0
	CCG	8.2	24.4	23.7
	CCU	12.2	2.7	7.7
A	GCA	23.1	13.5	13.5
	GCC	2.7	8.1	26.7
	GCG	9.5	10.8	37.2
	GCU	17.7	21.7	14.7
G	GGA	25.8	0.0	7.4
	GGC	2.7	21.7	34.3
	GGG	14.9	0.0	11.3
	GGU	4.1	24.4	19.6
V	GUA	23.1	14.9	12.2
	GUC	2.7	6.7	17.7
	GUG	13.6	10.8	24.8
	GUU	14.9	21.7	15.8
K	AAA	59.8	47.5	36.7
	AAG	17.1	29.8	12.0
N	AAC	21.7	74.7	21.3
	AAU	70.7	17.6	20.2
Q	CAA	35.3	12.2	12.8
	CAG	5.4	28.5	31.0
H	CAC	1.4	8.1	9.4
	CAU	12.2	6.7	11.9
E	GAA	51.6	44.8	38.4
	GAC	8.2	36.6	21.7
D	GAG	16.3	23.0	21.5
	GAU	55.7	27.1	34.1
Y	UAC	8.2	27.1	12.6
	UAU	29.9	10.8	17.6
C	UGC	0.0	0.0	6.3
	UGU	0.0	0.0	5.0
F	UUC	6.8	20.3	15.3
	UUU	25.8	12.2	22.5
I	AUA	21.7	0.0	6.7
	AUC	13.6	48.9	24.8
	AUU	38.0	23.0	28.2
M	AUG	10.9	10.9	25.5
W	UGG	9.5	9.5	11.8

↵ a Data for PA83 codon usage were derived from (Williamson et al., (8 July 2001, British Patent Office). Data for S. enterica serovar Typhimurium codon usage were derived from Tzschaschel et al. (28).