Stable transfection of the bovine NRAMP1 gene into murine RAW264.7 cells: effect on Brucella abortus survival

Abstract

Genetically based natural resistance to brucellosis in cattle provides for novel strategies to control zoonotic diseases. Bovine NRAMP1, the homologue of a murine gene (Bcg), has been identified as a major candidate for controlling the in vivo resistant phenotype. We developed an in vitro model for expression of resistance- and susceptibility-associated alleles of bovine NRAMP1 as stable transgenes under the regulatory control of the bovine NRAMP1 promoter in the murine RAW264.7 macrophage cell line (Bcg(s)) to analyze the regulation of the NRAMP1 gene and its role in macrophage function. We demonstrated that the 5'-flanking region of bovine NRAMP1, despite the lack of TATA and CAAT boxes, has a functional promoter capable of driving the expression of a transgene in murine macrophages. A polymorphism within a microsatellite in the 3' untranslated region critically affects the expression of bovine NRAMP1 and the control of in vitro replication of Brucella abortus but not Salmonella enterica serovar Dublin. We did not observe any differences in the production of NO by resting or gamma interferon (IFN-gamma)- and IFN-gamma-lipopolysaccharide (LPS)-treated transfected cell lines, yet the resistant transfected cell lines produced significantly less NO than other cell lines, following stimulation with LPS at 24 and 48 h.

FIG. 1

Sequence of the 5′-flanking region, exon 1, intron 1, exon 2, intron 2, and part of exon 3 of bovine NRAMP1. The transcription start site in exon 1 is shown by the first capital boldface G with +1. The putative binding sequence motifs for transcription factors are underlined with the name of the corresponding factor shown. The exon sequences are indicated by boldface uppercase letters. The complete sequence is available in GenBank under the temporary accession number bankit321467.

FIG. 2

RPA analysis for expression of GFP under the control of the bovine NRAMP1 promoter in pΔ3Bo-GFP transfectants of RAW264.7 cells. The message for GFP is easily detectable in all four clones with visual GFP activity (lanes 3 through 6); no signal for GFP is obtained with the parental cells (lanes 1 and 2). The lower dominant band is β-actin. The respective sizes of the protected fragments are 308 (GFP) and 250 (β-actin; positive control) bases. Exposure is 3 h.

FIG. 3

RPA analysis for expression of bovine NRAMP1-R in stable RAW264.7 transfectants. Lane 3, parental RAW264.7 cells; lanes 4 through 11, stable bovine NRAMP1-R transfected clones. A protected fragment of the expected size is observed for the clones in lanes 4, 5, 7, 8, and 9; no signal is obtained with the parental cells (lane 3). The presence of a larger protected fragment can be attributed to the presence of secondary structure in the antisense probe. The lower dominant band is β-actin. The respective sizes of the protected fragments are 289 (NRAMP1-R) and 250 (β-actin; positive control) bases. Full-length antisense probes for β-actin and NRAMP1-R are shown in lanes 1 and 2. Exposure is 3 h.

FIG. 4

RPA analysis for expression of bovine NRAMP1-S16 in stable RAW264.7 transfectants (lanes 2 through 5). The protected fragments for actin (250 bases) and bovine NRAMP1-S16 (295 bases) are indicated. Lane 1, parental cell line. Exposure is 12 h.

FIG. 5

Survival of Salmonella serovar Dublin in three NRAMP1-R- or NRAMP1-S16-transfected clones of the murine RAW264.7 macrophage cell line. Bacterial survival is expressed as a percentage of survival in the parental RAW264.7 cells. The box plot shows the median and quartiles from two independent experiments; in each experiment, the cell lines were assayed in triplicate, and the survival rates were pooled for the three clones from each genotype. The cells were challenged at a MOI of 20. There is no significant difference in survival rates between the three R and the three S16 clones.

FIG. 6

Survival of B. abortus in three NRAMP1-R or NRAMP1-S16 transfected clones of the murine RAW264.7 macrophage cell line. Bacterial survival is expressed as the percentage of survival in the parental RAW264.7 cells. The box plot shows the median, quartiles, and outside values (*) from three independent experiments; in each experiment, the cell lines were assayed in triplicate, and the survival rates were pooled for the three clones from each genotype. The difference in survival rates between the three R and the three S16 clones is significant at P < 0.05.

FIG. 7

Colony morphology of B. abortus recovered from NRAMP1-R transgenic (A) and RAW264.7 (B) cells. B. abortus was grown on tryptic soy agar plates for 4 days. Note the normal appearance of B. abortus colonies (B) and the wide variation in colony size (A).

FIG. 8

Nitrite levels in supernatants from bovine NRAMP1-R (R), NRAMP1-S16 (S16), vector-transfected RAW264.7 cells (V), and parental RAW264.7 cells (P) left untreated (No) or stimulated for 48 h with LPS or IFN-γ–LPS. In each experiment, the cells were assayed in triplicate, and the results are represented as the mean ± standard deviation of three independent experiments.