Joel V. Walker

Genetic Organization and Iron-Responsive Regulation of the Brucella abortus 2,3-Dihydroxybenzoic Acid Biosynthesis Operon, a Cluster of Genes Required for Wild-Type Virulence in Pregnant Cattle

ABSTRACT Brucella abortus reportedly produces the monocatechol siderophore 2,3-dihydroxybenzoic acid (2,3-DHBA) in response to iron limitation. Nucleotide sequence analysis of the cloned DHBA biosynthesis locus from virulent B. abortus 2308 and genetic complementation of defined Escherichia coli mutants were used to identify the B. abortus genes (designated dhbC, -B, and -A) responsible for synthesis of this siderophore. Reverse transcriptase PCR analysis of total RNA with dhb-specific primers demonstrated that dhbC, -B, and -A are transcribed as components of an operon, together with dhbE, a functional homolog of the Escherichia coli entE gene. Homologs of the E. coli entD and Vibrio cholerae vibH genes were also detected in the flanking regions immediately adjacent to the B. abortus dhbCEBA operon, suggesting that B. abortus has the genetic capacity to produce a more complex 2,3-DHBA-based siderophore. Slot blot hybridization experiments and primer extension analysis showed that transcription of the B. abortus dhbCEBA operon originates from two iron-regulated promoters located upstream of dhbC. Consistent with their iron-dependent regulation, both of the dhbCEBA promoter sequences contain typical consensus Fur-binding motifs. Although previously published studies have shown that 2,3-DHBA production is not required for the establishment and maintenance of chronic spleen infection by B. abortus in mice, experimental infection of pregnant cattle with the B. abortus dhbC mutant BHB1 clearly showed that production of this siderophore is essential for wild-type virulence in the natural ruminant host.

A Brucella melitensis high-temperature-requirement A (htrA) deletion mutant is attenuated in goats and protects against abortion.

It has been previously demonstrated that a Brucella melitensis high-temperature-requirement A (htrA) deletion mutant is more susceptible to oxidative killing in vitro than the parental strain and is attenuated in mice. To evaluate the contribution of the B melitensis HtrA protease to virulence in ruminants, the capacity of the B melitensis htrA mutant RWP5 to produce abortion in goats was compared to that of the virulent parental strain 16M. Experimental infection with strain 16M caused abortion in eight of 12 pregnant nannies, while none of the 12 nannies inoculated with RWP5 aborted. Furthermore, intramuscular injection of fetuses in utero with RWP5 led to colonisation of the fetus with subsequent colonisation of the nanny, but no abortion was observed. Nannies vaccinated with RWP5 showed complete protection against abortion when challenged with 16M during the third trimester of pregnancy. However, these animals were not protected from colonisation by 16M. The results presented here clearly indicate that the B melitensis htrA gene product contributes to pathogenesis in goats, but the utility of B melitensis htrA mutants as vaccines in this host appears to be limited.

Safety of Brucella abortus Strain RB51 in Bison

To determine the safety of Brucella abortus strain RB51 (SRB51) vaccine in American bison (Bison bison), 31 animals from a herd with brucellosis were used. In October 1996, 10 adult bison males and seven calves were vaccinated with the standard calfhood cattle dose of 1.8 × 1010 colony forming units (CFU) of SRB51 subcutaneously while the adult females received the standard adult cattle dose of 1 × 109 CFU. Western immunoblot indicated the presence of SRB51 antibodies following vaccination. To evaluate prolonged bacterial colonization of tissues, the adult males, calves, and three adult females were divided into two groups which were slaughtered at either 13 or 16 wk post-vaccination. At necropsy, tissue samples were obtained for B. abortus culture from the liver, spleen, lymph nodes, and reproductive tract of each animal. While B. abortus field strain was cultured from one adult bull, no SRB51 was isolated from any of the animals. Seven pregnant females were monitored until parturition for signs of abortions and fetal lesions. Six cows delivered healthy calves and one delivered a dead full-term calf that was brucellae negative. Based on these results, administration of SRB51 to bison did not cause prolonged bacterial colonization of tissues in calves, adult males, or adult females. Furthermore, SRB51 did not induce abortions following vaccination in the second month of gestation.

Re-examination of the role of the Brucella melitensis HtrA stress response protease in virulence in pregnant goats

Based on previously reported studies describing the experimental infection of pregnant goats with B. melitensis strain RWP5, we proposed that the HtrA protease plays an important role in the virulence of B. melitensis in its natural ruminant host. Subsequent studies, however, have shown that RWP5 is actually an htrA cycL double mutant. In order to definitively evaluate the role of the B. melitensis htrA in virulence, we constructed an authentic htrA mutant and examined this strain in pregnant goats. The findings of these studies indicate that the contribution of the htrA gene product to the virulence of B. melitensis in its natural host is not as great as was previously proposed.

Attenuation and immunogenicity of a Brucella abortus htrA cycL double mutant in cattle.

PHE1 is a htrA cycL double gene deletion mutant of virulent Brucella abortus strain 2308 (S2308) which has previously been evaluated in the murine and caprine models of bovine brucellosis. This report describes the results of studies conducted with this mutant in the natural bovine host. Six sexually mature, non-gravid heifers were inoculated via the conjunctival sac with 1 x 10(10) colony forming units (CFU) of either the parental S2308 or the htrA cycL gene deletion mutant, PHE1. At 4, 7 and 11 days post-inoculation, PHE1 was found to colonize the bovine host at lower levels than S2308. In a second experiment, eight heifers in mid-gestation were infected with 1 x 10(7) CFU of either strain via the conjunctival sac. The virulent S2308 caused abortions or weak calves in 4/4 cows, while all four cows infected with PHE1 had healthy calves. Furthermore, PHE1 exhibited decreased resistance to killing by cultured bovine neutrophils and macrophages compared to the parental strain. These studies demonstrate that the B. abortus htrA cycL gene deletion mutant PHE1 is highly attenuated in the bovine host when compared to the virulent parental S2308.

Behaviour of a high-temperature-requirement A (HtrA) deletion mutant of Brucella abortus in goats.

Previous studies have shown that high-temperature-requirement A (HtrA) mutants of Brucella abortus are more sensitive to oxidative killing in vitro, are less able to survive in cultured murine macrophages and are attenuated in BALB/c mice. To measure the effect of an HtrA mutation on the virulence of B abortus in ruminants, pregnant goats in late gestation were exposed to infection by the conjunctival route with B abortus 2308 or an isogenic htrA mutant, PHE1. Infection with either 2308 or PHE1 resulted in abortion, but the serological responses to infection were consistent with 2308 but variable with PHE1. Strain 2308 was recovered post mortem both from aborted fetuses and infected dams, whereas PHE1 was recovered from neither. Nevertheless, short term studies revealed that PHE1 could be recovered from infected goats for up to two weeks after infection, suggesting that although the HtrA mutation may change the colonising ability of B abortus, the virulence of the mutant in pregnant goats is not reduced.

Evaluating the virulence of a Brucella melitensis hemagglutinin gene in the caprine model

With the completion of the genomic sequence of Brucella melitensis 16M, a putative hemagglutinin gene was identified which is present in 16M and absent in Brucella abortus. The possibility of this hemagglutinin being a potential virulence factor was evaluated via gene replacement in B. melitensis yielding 16MΔE and expression in trans in B. abortus 2308-QAE. Utilizing the caprine brucellosis model, colonization and pathogenesis studies were performed to evaluate these strains. B. melitensis 16M hemagglutinin gene expression in trans in 2308-QAE revealed a significant (p≤0.05) increase in colonization and abortion rates when compared to B. abortus 2308, mimicking B. melitensis 16M virulence in pregnant goats. The B. melitensis disruption mutants colonization and abortion rates demonstrated no attenuation in colonization but displayed a 28% reduction in abortions when compared to parental B. melitensis 16M.

Characterization of a Putative Hemagglutinin Gene in the Caprine Model for Brucellosis

With the completion of the genomic sequences of Brucella melitensis 16M and B. abortus 2308 and the vaccine strain RB51, a putative hemagglutinin gene was identified that is present in 16M and absent in B. abortus. The possibility of this hemagglutinin being a potential host specificity factor was evaluated via expression in trans in B. abortus 2308-QAE and RB51-QAE. Using the caprine brucellosis model, colonization and pathogenesis studies were performed to evaluate the strains.