Development of non-antibiotic-resistant, chromosomally based, constitutive and inducible expression systems for aroA-attenuated Salmonella enterica Serovar Typhimurium

Abstract

Live-vaccine delivery systems expressing two model antigens from Mycoplasma hyopneumoniae, F2(P97) (Adh) and NrdF, were constructed using Salmonella enterica serovar Typhimurium aroA (STM-1), and immunogenicity in mice was evaluated. Recombinant plasmid-based expression (PBE) and chromosomally based expression (CBE) systems were constructed. The PBE system was formed by cloning both antigen genes into pJLA507 to create an operon downstream of temperature-inducible promoters. Constitutive CBE was achieved using a promoter-trapping technique whereby the promoterless operon was stably integrated into the chromosome of STM-1, and the expression of antigens was assessed. The chromosomal position of the operon was mapped in four clones. Inducible CBE was obtained by using the in vivo-induced sspA promoter and recombining the expression construct into aroD. Dual expression of the antigens was detected in all systems, with PBE producing much larger quantities of both antigens. The stability of antigen expression after in vivo passage was 100% for all CBE strains recovered. PBE and CBE strains were selected for comparison in a vaccination trial. The vaccine strains were delivered orally into mice, and significant systemic immunoglobulin M (IgM) and IgG responses against both antigens were detected among all CBE groups. No significant immune response was detected using PBE strains. Expression of recombinant antigens in S. enterica serovar Typhimurium aroA from chromosomally located strong promoters without the use of antibiotic resistance markers is a reliable and effective method of inducing a significant immune response.

FIG. 1.

Cloning schematic for the production of PBE and CBE constructs. E, EcoRI; H, HindIII; N, NdeI; Nc, NcoI; Nt, NotI; S, SalI; X, XhoI; atpE, atpE ribosome binding site; rrnBto, E. coli rRNA terminator; tnp, transposase. The diagram is not to scale.

FIG. 2.

Chromosomal locations of antigen gene constructs. (A) Southern hybridization analysis of HindIII-digested chromosomal DNA from STM-1 vaccine strains using a Dig-labeled nrdF probe. Lanes: 1, STM-1; 2, STM-AN1; 3, STM-AN2; 4, STM-AN3; 5, STM-AN4. Molecular size markers in kb are indicated on the left. (B) PCR of the nrdF-adh operon from the chromosomes of STM-1 vaccine strains. Lanes: 1, STM-1; 2, STM-AN1; 3, STM-AN2; 4, STM-AN3; 5, STM-AN4; 6, STM-sspA. Molecular size markers in kb are indicated on the left. (C) Chromosomal locations of the nrdF-adh operon within STM-1 vaccine strains as determined using inverse PCR. Gene names are indicated. adh, M. hyopneumoniae F2_P97; aroD, 3-dehydroquinase; arsA, arsenite-translocating ATPase; arsB, arsenite efflux membrane protein; dps, stress response DNA binding protein; finO, FinP binding protein; glgA, glycogen synthase; glgC, glucose-1-phosphate adenylyltransferase; glgP, glycogen phosphorylase; glnH, high-affinity glutamine transport protein; glnP, glutamine transport permease protein; nrdF, M. hyopneumoniae ribonucleotide reductase; ompX, outer membrane protein X; orf, hypothetical open reading frame; psspA, promoter for stringent starvation protein A; rrnB, E. coli rRNA terminator; STM3533, putative transcriptional regulator; traI, OriT nickase/helicase; traX, pilin subunit acetylation; trbH, conjugative-transfer protein; ybiF, putative permease; ydiB, quinate/shikimate dehydrogenase; ydiF, putative acetyl-coenzyme A (CoA)/acetoacetyl-CoA transferase beta subunit. The scale in kb is indicated.

FIG. 3.

Growth curve and Western blot analyses of expression from STM-1(pJLA507-AN) and CBE strains. (A and B) Western blot of whole-cell lysates of STM-1(pJLA507-AN) using rabbit polyclonal Adh antisera (A) and rabbit polyclonal NrdF antisera (B). Lane 1, STM-1(pJLA507-AN) preinduction; lane 2, STM-1(pJLA507-AN) 1 h postinduction; lane 3, STM-1(pJLA507-AN) 2 h postinduction; lane 4, STM-1(pJLA507-AN) 4 h postinduction; lane 5, purified His-tagged Adh (200 ng) (A) or purified His-tagged NrdF (5 to 10 μg) (B). (C) Western blot analysis of whole-cell lysates of CBE strains using rabbit polyclonal Adh antisera. Lane 1, STM-1; lane 2, STM-AN1; lane 3, STM-AN2; lane 4, STM-AN3; lane 5, STM-AN4. Molecular mass markers in kDa are shown on the left. (D) Growth curve analysis of PBE strains performed in LB. The strains were grown at 37°C for 1 h prior to induction at 42°C, indicated by the arrow. (E) Growth curves of CBE strains performed in minimal medium supplemented with aromix.

FIG. 4.

Murine serum immunoglobulin responses against purified Adh, NrdF, and whole-cell STM-1 as determined by ELISA and Western blotting. Day 42 responses (shaded bars) and day 70 responses (white bars) are shown. The y axis represents reciprocal titer values. Standard errors are indicated. Statistical significance (P < 0.05) determined by comparison to the PBS group is indicated by asterisks. (A) Serum IgM responses against purified Adh. (B) Serum IgM responses against purified NrdF. (C) Serum IgG responses against purified Adh. (D) Serum IgG responses against purified NrdF. (E) Serum IgG responses against whole-cell STM-1. (F) Western blot analysis of pooled CBE orally immunized-mouse sera (day 42; diluted 1:50) and intraperitoneally immunized-mouse sera (day 42) against M. hyopneumoniae (strain J) whole-cell lysate and purified antigen. Lane 1, CBE orally immunized-mouse sera against M. hyopneumoniae whole-cell lysate; lane 2, NrdF intraperitoneally immunized-mouse sera against M. hyopneumoniae whole-cell lysate; lane 3, Adh intraperitoneally immunized-mouse sera against M. hyopneumoniae whole-cell lysate; lane 4, CBE orally immunized-mouse sera against purified NrdF; lane 5, CBE orally immunized-mouse sera against purified Adh. Molecular mass markers in kDa are shown on the right. The filled arrowhead indicates the molecular mass of intact adhesin protein, and the unfilled arrowhead indicates the molecular mass of intact NrdF protein.