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.Brucella abortus is a facultative, gram-negative pathogen of humans and animals that inhabits macrophages (2). The capacity to withstand nutritional deprivation would be expected to be particularly important for the survival of B. abortus, since this organism does not escape from the phagosome into the nutrient-rich environment of the host cell cytoplasm (2). Indeed, previous studies suggest that the brucellae encounter a considerable degree of nutritional deprivation during their long-term residence in host macrophages. Brucella melitensis purE (5) and Brucella suis aroC (12) mutants, for example, are unable to maintain chronic infection in experimentally infected mice, and the attenuation of the B. melitensis purE mutant purE201 extends to the natural ruminant host (4) and nonhuman primates (M. Nikolich, personal communication). These observations are also consistent with those seen with auxotrophic mutants of Salmonella and Mycobacterium and indicate that the availability of certain nutrients is severely restricted in the phagosomal compartment of host macrophages (3,11,22).In an effort to gain a better understanding of the metabolic versatility required for sustained intracellular residence by the brucellae in host macrophages, transposon mutagenesis and an in vitro screen were employed to identify B. abortus 2308 derivatives with mini-Tn5 insertions in genes required for resistance to nutrient deprivation. Transposon mutagenesis of this strain was performed by conjugative transfer of the mini-Tn5 derivative Km1 by employing pUT as the delivery vector and Escherichia coli S17-1pir as the conjugal donor strain (6,14,28). Approximately 1,000 B. abortus mini-Tn5 mutants were patched with sterile toothpicks onto Schaedler agar supplemented with 5% defibrinated bovine blood (SBA), SBA supplemented with 45 g of kanamycin/ml (SBAk), and Gerhardt's minimal medium (13) supplemented with 1.5% agar (GMMA). Plates were incubated at 37°C with 5% CO 2 and examined for growth after 4, 7, and 10 days of incubation. Schaedler agar, the basal medium for SBA, is a complex culture medium containing enzymatic digests of casein, animal and plant tissues, yeast extract, glucose, cystine, and hemin (Difco manual, 10th ed., Difco Laboratories, Detroit, Mich.). In contrast, GMM, the base for GMMA, is a defined medium formulated during a study aimed at defining the minimal in vitro growth requirements of Brucella strains (1...

Booster vaccination with safe, modified, live-attenuated mutants of Brucella abortus strain RB51 vaccine confers protective immunity against virulent strains of B. abortus and Brucella canis in BALB/c mice

“…2b). The results presented here are consistent with our previous findings (Truong et al, 2014(Truong et al, , 2015 and the work of other investigators (Alcantara et al, 2004;Yang et al, 2010) demonstrating the important roles of purine biosynthesis (purD, purE) and ABC-type WCAs of strain RB51 or B. canis S26 in sera from vaccinated mice. Results are shown as mean¡SD.…”

Systematic Targeted Mutagenesis ofBrucella melitensis16M Reveals a Major Role for GntR Regulators in the Control ofVirulence

“…It may be suggested that LysR18 is involved in the control of purine catabolism genes. Until now, only the synthesis of purines was shown to be required for the virulence of Brucella in both cellular and murine models (1,11,30,31).…”

Deletion of znuA Virulence Factor Attenuates Brucella abortus and Confers Protection against Wild-Type Challenge

“…Attempts to develop live brucellae vaccines have met with varied success. For instance, inactivation of the amino acid biosynthesis pathway genes pheA, trpB, and dagA displayed little or no attenuation in cultured murine macrophages or in mice (1). The mutants of purine biosynthesis pathway genes purL, purD, and purE (1) displayed significant attenuation in BALB/c mice, but live brucellae remained viable after 12 weeks, suggesting that their virulence was not sufficiently attenuated for adoption as a livestock vaccine.…”