Intact purine biosynthesis pathways are required for wild-type virulence of Brucella abortus 2308 in the BALB/c mouse model

Abstract

Brucella abortus 2308 derivatives with mini-Tn5 insertions in purE, purL, and purD display significant attenuation in the BALB/c mouse model, while isogenic mutants with mini-Tn5 insertions in pheA, trpB, and dagA display little or no attenuation in cultured murine macrophages or mice. These experimental findings confirm the importance of the purine biosynthesis pathways for the survival and replication of the brucellae in host macrophages. In contrast to previous reports, however, these results indicate that exogenous tryptophan and phenylalanine are available for use by the brucellae in the phagosomal compartment.

FIG. 1.

Intracellular survival and replication of B. abortus strains 2308 (•), AR54 (2308 purL::miniTn5) (▪), AR875 (2308 purH::miniTn5) (▴), and AR975 (2308 purE::miniTn5) (×) (A) and of B. abortus 2308, AR975 (2308 purE::miniTn5), and AR975R (AR975 purE::miniTn5→purE⁺) (B) in cultured resident peritoneal macrophages from BALB/c mice. Macrophages were isolated and plated at a density of 5 × 10⁵ cells per well in 96-well microtiter plates and infected with brucellae opsonized with hyperimmune murine serum at a multiplicity of infection of 100:1, using the methods described by Elzer et al. (10). Results are expressed as percent survival, which was calculated by dividing the number of intracellular brucellae present at a particular sampling time by the number of intracellular brucellae present immediately after phagocytosis (t = 0). *, P < 0.05; **, P < 0.01 for comparisons of 2308 versus AR54, AR875, or AR975. †, P < 0.05 for comparisons of AR975R versus AR975 by using the Student t test (26).

FIG. 2.

Spleen colonization profiles of B. abortus strains 2308 (•), AR54 (2308 purL::miniTn5) (□), AR875 (2308 purH::miniTn5) (×), and AR975 (2308 purE::miniTn5) (▴) (A) and B. abortus 2308 (•), AR54 (2308 purL::miniTn5) (▴), AR54R (AR54 purL::miniTn5→purL) (⧫), AR975 (2308 purE::miniTn5) (▪), and AR975R (AR975 purE::miniTn5→purE) (▪) (B) in experimentally infected BALB/c mice. Brucella strains were grown on SBA or SBAk, and infection doses were prepared as described elsewhere (9). Seven- to 9-week-old female BALB/c mice were infected with 5 × 10⁴ (A) or 2 × 10⁴ (B) CFU of brucellae via the intraperitoneal route. At 1, 4, 8, and 12 weeks postinfection, three to five mice from each experimental challenge group were sacrificed by isoflurane overdose, their spleens were removed and homogenized, and the total number of brucellae per spleen was determined by serial dilution and plating onto SBA and/or SBAk. Results are expressed as total CFU per spleen ± the standard deviation. *, P < 0.05; **, P < 0.01 for comparisons of 2308 versus AR54, AR875, or AR975. †, P < 0.05 for comparisons of AR54R versus AR54; ‡‡, P < 0.01 for comparisons of AR975R versus AR975 (Student's t test [26]).

FIG. 3.

Intracellular survival and replication of B. abortus strains 2308 (•), AR93 (2308 trpB::miniTn5) (▪), AR408 (2308 pheA::miniTn5) (⧫), AR536 (2308 dagA::miniTn5) (▴), and AR943 (2308 ilvD::miniTn5) (×) in cultured murine resident peritoneal macrophages. Macrophages were isolated and plated at a density of 5 × 10⁵ cells per well in 96-well microtiter plates and infected with brucellae opsonized with hyperimmune murine serum at a multiplicity of infection of 100:1 using the methods described by Elzer et al. (10). Results are expressed as percent survival, which was calculated by dividing the number of intracellular brucellae present at a particular sampling time by the number of intracellular brucellae present immediately after phagocytosis (t = 0). **, P < 0.01 for comparisons of 2308 versus AR943 by using the Student t test (26).

FIG. 4.

Spleen colonization profiles of B. abortus strains 2308 (•), AR93 (2308 trpB::miniTn5) (▪), AR408 (2308 pheA::miniTn5) (⧫), AR536 (2308 dagA::miniTn5) (▴), and AR943 (2308 ilvD::miniTn5) (×) in experimentally infected BALB/c mice. Brucella strains were grown on SBA or SBAk, and infection doses were prepared as described elsewhere (11). Seven- to 9-week-old female BALB/c mice were infected with 5 × 10⁴ CFU brucellae via the intraperitoneal route. At 1, 4, 8, and 12 weeks postinfection, three to five mice from each experimental challenge group were sacrificed by isoflurane overdose, their spleens were removed and homogenized, and the total number of brucellae per spleen was determined by serial dilution and plating onto SBA and/or SBAk. Results are expressed as total CFU per spleen ± the standard deviation. **, P < 0.01 for comparisons of 2308 versus AR943 or AR536 by using the Student t test (26).