ABSTRACT
The aroC gene of the facultative intracellular pathogenBrucella suis was cloned and sequenced. The clonedaroC gene complements Escherichia coli andSalmonella enterica serovar Typhimurium aroCmutants. A B. suis aroC mutant was found to be unable to grow in a defined medium without aromatic compounds. The mutant was highly attenuated in tissue culture (THP1 macrophages and HeLa cells) and murine virulence models.
The attenuation of bacterial pathogens by auxotrophic mutations was first demonstrated by Bacon et al. with “Bacterium typhosa” 50 years ago (1). Over 2 decades after these original observations, Hoiseth and Stocker (13) showed that a Salmonella enterica serovar Tyhpimirium aroA(5-enolpyruvylshikimate 3-phosphate [EPSP] synthase) mutant was both attenuated and an excellent live vaccine in the mouse typhoid model. This enzyme is part of the aro pathway, which leads, through shikimic acid, to chorismic acid. Chorismate is a branching point from which separate pathways lead to the aromatic amino acids, topara-aminobenzoic acid and hence folic acid, to vitamin K, to ubiquinone and hence the electron transport systems, and to dihydroxybenzoic acid, which is the first step in the biosynthesis of the siderophore enterochelin (18, 25). The shikimate pathway occurs in prokaryotes, yeasts and filamentous fungi, apicomplexan parasites (16), and the plastids of plants and algae. The aro pathway is not, however, present in vertebrates, meaning that these animals must obtain the essential products derived from chorismic acid from their diet and that the intermediates of this pathway are not available to complement the requirements of an auxotroph. Mutations in the aroC gene (7), encoding chorismate synthase, the final enzyme in this pathway, which catalyzes the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) to chorismic acid, and also in aroD andaroB are equally attenuating, confirming the key role of thearo pathway for the virulence of Salmonella(11).
Brucella spp. are gram-negative bacteria responsible of animal brucellosis in a variety of mammalian hosts. A major characteristic of this intracellular pathogen is its ability to survive and replicate in the macrophages of the host, where it remains enclosed in phagocytic compartments. Little is known about the genes implicated in the virulence of Brucella. In a previous study, we used signature-tagged mutagenesis (STM) to identify genes required for the intracellular growth and survival of Brucella in a macrophage infection model (10). Several mutants which multiplied poorly or not at all in THP1 (human macrophage-like cells) and HeLa cells were isolated. In one of them, the transposon interrupted a gene that was highly similar to known aroCgenes. In the present work, we characterized this gene and the consequence of its mutation on the virulence of Brucella suis.
Cloning and sequencing of the B. suis aroC gene.The genomic DNA from anaroC::Tn5 mutant identified by STM was extracted, and a 3.5-kb EcoRV genomic fragment containing the mini-Tn5 Km2 transposon was cloned in pUC18-SmaI-BAP (Amersham Pharmacia) to transformEscherichia coli DH5α. Sequences flanking the transposon were determined using transposon primers P6 and P7 (12) and the direct and reverse primers of pUC18, showing that the transposon was inserted between bp 201 and 215 of the coding sequence and that it had created a deletion of the 14 bases. A digoxigenin-labeled probe was generated by PCR using primers P7 and M13 direct as described previously (10). This allowed us to identify a clone from a B. suis cosmid library in pSuperCos containing genomic inserts of approximately 45 kb (15). The complete sequence of aroC was obtained directly by sequencing the cosmid DNA by primer walking. The aroC gene sequence is 1,038 bp long with a G+C content of 61%. Approximately 500 bp of upstream sequence and 600 bp of downstream sequence were determined. No consensus sequence for Brucella promoters has been described; however a putative ribosome-binding site is located 6 bp upstream from the initiation ATG codon in the aroC gene. There are no detectable open reading frames (ORFs) in the 500 bp upstream. A possible stem-loop is found immediately downstream of the stop codon and an ORF encoding a putative riboflavin biosynthesis protein was detected 150 bp downstream, suggesting that in Brucella aroCis not part of an operon (data not shown). The sequence encodes a single protein of 345 amino acids with an estimated molecular mass of 36.6 kDa. The deduced amino acid sequence has considerable homology with chorismate synthase sequences from other bacteria (55% identity and 79% homology with AroC of both E. coli andS. enterica serovar Typhimurium) (5).
The B. suis aroC gene complementsSalmonella and E. coli aroC mutants.A 1,162-bp fragment containing the complete aroC gene and 60 bp upstream and downstream was amplified by PCR using primers Aro1 (5′ GGC CGG TAA AAG AAA CTG GT 3′) and Aro2 (5′ ATT ATT TTC AGG CGC GGC CA 3′) and cloned in the pGEMT-Easy vector (Promega). AnApaI-SacI fragment containing the gene was subcloned into pBBR1MCS (17), a low-copy-number, broad-host-range vector which replicates in Brucella. These two plasmids containing the B. suis aroC gene were used to transform an S. enterica serovar Typhimurium LT2 ΔaroC strain and an E. coli BRD049 ΔaroC strain by electroporation. Transformants were assayed for growth on M9 minimal medium (27) and M9 supplemented with para-aminobenzoic acid, dihydroxybenzoic acid (100 μg/ml), phenylalanine, tryptophan, and tyrosine (40 μg/ml). The B. suis aroC gene, either on a high- (pGEM) or low-copy-number (pBBR1MCS) vector restored the ability of bothSalmonella and E. coli aroC mutants to grow on minimal medium. This shows that the Brucella gene has the same function as the S. enterica serovar Typhimurium andE. coli genes and that it is aroC. We cannot say whether expression was driven by the natural promoter or by a plasmid promoter.
Brucella is a very fastidious organism, and there is no simple minimum medium. We took advantage of the fact that minimal essential medium (MEM) used for tissue culture does not contain any aromatic compound. MEM base (Life Technologies), supplemented with glucose, was mixed with an equal volume of M9 medium (to limit the alkalinization of the medium in an atmosphere without carbon dioxide). This defined medium was sufficiently rich to support the growth of wild-type B. suis in liquid culture in an orbital shaker but did not support the growth of the aroC mutant. pBBR1MCS containing the B. suis aroC gene was introduced into thearoC mutant by electroporation and was found to restore the ability of the mutant to grow in the defined medium.
B. suis aroC mutant multiplies slowly in infected cells.The B. suis aroC mutant was identified as attenuated in a STM screen. We monitored the growth of the mutant in both human macrophages and HeLa cells. As shown in Fig.1, the aroC mutant enters HeLa cells and THP1 macrophages at levels similar to those of the wild type but over the next 48 h multiplies very slowly in the cells, reaching levels of between 1.5 and 2 log units less than those of the wild type. The presence of pBBR1MCS-aroC restored the capacity of the mutant to multiply in infected cells.
The B. suis aroC mutant is attenuated in cell infection models. (a) HeLa cells. (b) THP1 macrophages. Cells were infected as previously described with multiplicities of infection of 100 for HeLa cells and 20 for THP1 cells (10). Data points are the means of three wells with the standard deviations. These data sets are from one of three independent experiments with similar results.
B. suis aroC mutant is attenuated in BALB/c mice.A mouse model allowed us to confirm the participation ofaroC in the virulence of Brucella. Groups of BALB/c mice were infected intraperitoneally (i.p.) with 5 × 105 wild-type, aroC mutant, or complementedaroC mutant B. suis cells. The bacterial counts in spleens and the weights of spleens were determined at 1, 7, 35, and 56 days postinfection. A multivariate statistical analysis and a profile analysis using PROC GLM (1996 version; Statistical Analysis System Institute Inc.) yielded significant differences (P < 0.006) for pairwise comparisons of individual profiles. The wild-type strain multiplied over 1,000-fold over the first week of infection and then persisted at very high levels with induction of splenomegaly (Table 1) for the duration of the experiment (8 weeks). The aroC mutant colonized spleens and appeared to multiply very slowly over the first week postinfection. After the first week, the spleens of the mice became enlarged and the mice slowly eliminated the bacteria. At 5 weeks postinfection, there was large mouse-to-mouse variation, with some animals still colonized and others clear, but at 8 weeks the spleens of all four mice were clear (limit of detection, 1 CFU) ofBrucella. The complemented mutant multiplied as rapidly as the wild type during the first week but then was unable to maintain the high levels of colonization of the wild type at later points (here again there was large variation between animals). It is not clear why complementation was not complete; the plasmid was stable since all the bacteria recovered from the spleens were chloramphenicol resistant, and the DNA sequence argues against a polar effect on a downstream gene. A possibility is that expression of aroC was not optimal in vivo.
Spleen weights of mice infected i.p. with wild typeB. suis S1, the B. suis ΔaroC strain, and the B. suis ΔaroC complemented strain
The present work confirms our preliminary data that aB. suis aroC mutant is attenuated for replication within cultured cells (10); moreover, it causes limited infection when introduced into BALB/c mice. The in vivo virulence ofaro mutants varies with bacterial species, concentration of nutrients available in infected tissues or intracellular compartments, and the location of the mutation in the aromatic amino acid biosynthetic pathway (19, 21). Salmonella enterica serovar Typhi, Shigella flexneri, andYersinia enterocolitica aromatic acid auxotrophs are less virulent than the wild types (3, 4, 20), butListeria monocytogenes aromatic auxotrophic mutants are only slightly attenuated (21). Microbial acquisition of nutrients is a central feature of the host-parasite relationship, and bacterial pathogenicity is in part dependent on the availability of the nutrient. These differences in attenuation may be due to different levels of availability of aromatic compounds at the site of infection in the animal host or to different abilities of the bacteria to take up the low levels of aromatic compounds from the environment. The attenuation of aro mutants and especially thearoC mutant may have consequences in terms of antimicrobial therapy. An effect on virulence similar to those described for thearoC mutant might be obtained with a specific inhibitor of chorismate synthase or a competitive substrate such as (6S)-6-fluoro-5-enolpyruvylshikimate-3-phosphate (2). Specific inhibitors of EPSP synthase (encoded by aroA) such as carbaphosphonate and its derivatives already exist (26).
The in vivo behavior of the aroC mutant is similar to those of Salmonella and Yersinia aroA andaroC mutants (3, 20). These bacteria multiply very slowly in spleens over the first week after infection and then are slowly eliminated from spleens in the following weeks (Fig.2). This elimination is accompanied by a marked splenomegaly, although one that is less marked in the B. suis aroC mutant than that induced by aro Salmonellastrains (24). The presently available liveBrucella vaccines used in veterinary medicine are all attenuated empirically (22). An ideal live vaccine is a compromise between attenuated virulence and ability to induce an immune response sufficient to protect against challenge with virulent organisms; if the vaccine strain is too attenuated it will not protect (23, 24). The in vivo behavior of the B. suis aroC mutant (low-level residual virulence and splenomegaly, indicating the induction of a cellular immune response, followed by total clearance) suggests that Brucella aro mutants may be good candidates for the development of live vaccines. The efficiency of the aroC mutant as a live vaccine is under investigation. No suitable live vaccine is available for human use; here, for safety, a mutant with two different attenuating lesions is required (7, 14, 20, 28). The construction of a double mutation in B. suis affecting both aroC and, for example,purE (6, 8), htrA (9), or any auxotrophic genes that we previously identified as being involved in amino acid biosynthesis (carAB,leuA) or glutamine metabolism (glnD) will bring new insight to the development of a live vaccine candidate, since all these genes were previously said to be required for the intracellular survival of B. suis (10).
Kinetic survival in BALB/c mice of wild-type B. suis versus that of the ΔaroC mutant and ΔaroC complemented strain. Mice were infected i.p. with a dose of 5 × 105 CFU. At different times postinfection the bacterial load was determined by plating appropriate dilutions of homogenized spleens on agar. Each point represents the geometric mean with standard deviations for four mice.
Nucleotide sequence accession number.The aroCsequence obtained in this study has been assigned GenBank accession no.AF276655 .
ACKNOWLEDGMENTS
We thank Patrick Michel and Hilde Cassiman for providing technical support and Frank Boelaert, Jean-Yves Paquet, and Niko Speybrouck for helpful discussion and statistical analysis.
This work was supported by INSERM and by the EEC (BIO4 CT960144).
Notes
Editor: V. J. DiRita
FOOTNOTES
- Received 5 July 2000.
- Returned for modification 29 August 2000.
- Accepted 12 October 2000.
- Copyright © 2001 American Society for Microbiology
