Brucella abortus virB12 is expressed during infection but is not an essential component of the type IV secretion system

The Brucella abortus virB operon, consisting of 11 genes, virB1 to virB11, and two putative genes, orf12 (virB12) and orf13, encodes a type IV secretion system (T4SS) that is required for intracellular replication and persistent infection in the mouse model. This study was undertaken to determine whether orf12 (virB12) encodes an essential part of the T4SS apparatus. The virB12 gene was found to encode a 17-kDa protein, which was detected in vitro in B. abortus grown to stationary phase. Mice infected with B. abortus 2308 produced an antibody response to the protein encoded by virB12, showing that this gene is expressed during infection. Expression of virB12 was not required for survival in J774 macrophages. VirB12 was also dispensable for the persistence of B. abortus, B. melitensis, and B. suis in mice up to 4 weeks after infection, since deletion mutants lacking virB12 were recovered from splenic tissue at wild-type levels. These results show that VirB12 is not essential for the persistence of the human-pathogenic Brucella spp. in the mouse and macrophage models of infection.     

Identification and characterization of a Brucella abortus ATP-binding cassette transporter homolog to Rhizobium meliloti ExsA and its role in virulence and protection in mice

Brucella abortus is a facultative intracellular bacterial pathogen that causes abortion in domestic animals and undulant fever in humans. The mechanism of virulence of Brucella spp. is not fully understood yet. Furthermore, genes that allow Brucella to reach the intracellular niche and to interact with host cells need to be identified. Using the genomic survey sequence (GSS) approach, we identified the gene encoding an ATP-binding cassette (ABC) transporter of B. abortus strain S2308. The deduced amino acid sequence encoded by this gene exhibited 69 and 67% identity with the sequences of the ABC transporters encoded by the exsA genes of Rhizobium meliloti and Mesorhizobium loti, respectively. Additionally, B. abortus ExsA, like R. meliloti and M. loti ExsA, possesses ATP-binding motifs and the ABC signature domain features of a typical ABC transporter. Furthermore, ortholog group analysis placed B. abortus ExsA in ortholog group 6 of ABC transporters more likely to be involved in bacterial pathogenesis. In R. meliloti, ExsA is an exopolysaccharide transporter essential for alfalfa root nodule invasion and establishment of infection. To test the role of ExsA in Brucella pathogenesis, an exsA deletion mutant was constructed. Replacement of the wild-type exsA by recombination was demonstrated by Southern blot analysis of Brucella genomic DNA. Decreased survival in mice of the Brucella DeltaexsA mutant compared to the survival of parental strain S2308 demonstrated that ExsA is critical for full bacterial virulence. Additionally, the B. abortus exsA deletion mutant was used as a live vaccine. Challenge experiments revealed that the exsA mutant strain induced superior protective immunity in BALB/c mice compared to the protective immunity induced by strain S19 or RB51.     

The role of integrase/recombinase xerD and monofunctional biosynthesis peptidoglycan transglycosylase genes in the pathogenicity of Brucella abortus infection in vitro and in vivo

Brucella abortus clones identified previously using a green fluorescence protein reporter system after 4h macrophage infection provided insight regarding possible genes involved in early host-pathogen interaction. Among identified genes were an integrase/recombinase (xerD) gene involved in cell division, and a monofunctional biosynthesis peptidoglycan transglycosylase (mtgA) gene that catalyzes the final stages of the peptidoglycan membrane synthesis. Here, we evaluate the in vitro and in vivo survival of B. abortus xerD and mtgA insertional mutants. B. abortus xerD::kan and B. abortus mtgA::kan demonstrated no significant growth defects in broth culture when compared to the parental strain, S2308. Also, neither gene was required for B. abortus S2308 replication in RAW 264.7 macrophages. However, experimental evidence using interferon regulatory factor 1 knockout mice, a mouse strain highly susceptible to virulent Brucella, revealed that mice infected with B. abortus xerD::kan or B. abortus mtgA::kan survived longer than mice infected with S2308. Additionally, in immunocompetent BALB/c mice, B. abortus xerD::kan had a significantly lower level of bacterial survival when compared to S2308. Together, these results suggest that B. abortus xerD and mtgA genes play a role during the initial phase of infection in mice.     

Inactivation of the type IV secretion system reduces the Th1 polarization of the immune response to Brucella abortus infection

The Brucella abortus type IV secretion system (T4SS), encoded by the virB operon, is essential for establishing persistent infection in the murine reticuloendothelial system. To gain insight into the in vivo interactions mediated by the T4SS, we compared host responses elicited by B. abortus with those of an isogenic mutant in the virB operon. Mice infected with the B. abortus virB mutant elicited smaller increases in serum levels of immunoglobulin G2a, gamma interferon (IFN-gamma), and interleukin-12p40 than did mice infected with wild-type B. abortus. Despite equal bacterial loads in the spleen, at 3 to 4 days postinfection, levels of IFN-gamma were higher in mice infected with wild-type B. abortus than in mice infected with the virB mutant, as shown by real-time PCR, intracellular cytokine staining, and cytokine levels. IFN-gamma-producing CD4(+) T cells were more abundant in spleens of mice infected with wild-type B. abortus than in virB mutant-infected mice. Similar numbers of IFN-gamma-secreting CD8(+) T cells were observed in the spleens of mice infected with B. abortus 2308 or a virB mutant. These results suggest that early differences in cytokine responses contribute to a stronger Th1 polarization of the immune response in mice infected with wild-type B. abortus than in mice infected with the virB mutant.     

Validation of the abbreviated Brucella AMOS PCR as a rapid screening method for differentiation of Brucella abortus field strain isolates and the vaccine strains, 19 and RB51

The Brucella AMOS PCR assay was previously developed to identify and differentiate specific Brucella species. In this study, an abbreviated Brucella AMOS PCR test was evaluated to determine its accuracy in differentiating Brucella abortus into three categories: field strains, vaccine strain 19 (S19), and vaccine strain RB51/parent strain 2308 (S2308). Two hundred thirty-one isolates were identified and tested by the conventional biochemical tests and Brucella AMOS PCR. This included 120 isolates identified as B. abortus S19, 9 identified as B. abortus strain RB51, 57 identified as B. abortus biovar 1, 15 identified as B. abortus bv. 2, 1 identified as B. abortus bv. 2 (M antigen dominant), 7 identified as B. abortus bv. 4, and 22 identified as B. abortus S2308 and isolated from experimentally infected cattle. The Brucella AMOS PCR correctly identified each isolate as RB51/S2308, S19, or a field strain of Brucella.     

Brucella abortus requires the heme transporter BhuA for maintenance of chronic infection in BALB/c mice

The gene annotated BAB2_1150 in the Brucella abortus 2308 genome sequence is predicted to encode a homolog of the well-characterized heme transporter ShuA of Shigella dysenteriae and accordingly has been given the designation bhuA (Brucella heme utilization). Phenotypic analysis of an isogenic bhuA mutant derived from B. abortus 2308 verified that there is a link between BhuA and the ability of the parent strain to use heme as an iron source in in vitro assays. Maximum expression of bhuA in B. abortus 2308 is observed during stationary phase when this strain in cultivated in low-iron minimal medium, and a comparison of the growth characteristics of the B. abortus bhuA mutant and 2308 in this medium suggested that heme serves as an important iron source for the parent strain during stationary phase. The B. abortus bhuA mutant HR1703 exhibits significant attenuation in cultured murine macrophages compared to strain 2308, and unlike its parent strain, the B. abortus bhuA mutant is unable to maintain a chronic spleen infection in experimentally infected BALB/c mice. These experimental findings suggest that heme and/or heme-containing proteins represent important iron sources for B. abortus 2308 during its residence in the mammalian host and that BhuA is required for efficient utilization of these iron sources.     

Differential requirements for VirB1 and VirB2 during Brucella abortus infection

The Brucella abortus virB operon, encoding a type IV secretion system (T4SS), is required for intracellular replication and persistent infection in the mouse model. The products of the first two genes of the virB operon, virB1 and virB2, are predicted to be localized at the bacterial surface, where they could potentially interact with host cells. Studies to date have focused on characterization of transposon mutations in these genes, which are expected to exert polar effects on downstream genes in the operon. In order to determine whether VirB1 and VirB2 are required for the function of the T4SS apparatus, we constructed and characterized nonpolar deletion mutations of virB1 and virB2. Both mutants were shown to be nonpolar, as demonstrated by their ability to express the downstream gene virB5 during stationary phase of growth in vitro. Both VirB1 and VirB2 were essential for intracellular replication in J774 macrophages. The nonpolar virB2 mutant was unable to cause persistent infection in the mouse model, demonstrating the essential role of VirB2 in the function of the T4SS apparatus during infection. In contrast, the nonpolar virB1 mutant persisted at wild-type levels, showing that the function of VirB1 is dispensable in the mouse model of persistent infection.     

Construction of Cu-Zn superoxide dismutase deletion mutants of Brucella abortus: analysis of survival in vitro in epithelial and phagocytic cells and in vivo in mice.

Cu-Zn superoxide dismutase (SOD) deletion mutants of Brucella abortus S2308, a virulent strain, and S19, a vaccine strain, were generated by gene replacement. A deletion plasmid, pBA delta sodknr, was constructed by excising the Cu-Zn SOD gene (Cu-Zn sod) from a 2.3-kb B. abortus DNA fragment of plasmid pBA20-1527 and inserting a 1.4-kb DNA fragment encoding kanamycin resistance into the Cu-Zn sod excision site. The deletion plasmid was introduced into B. abortus by electroporation, and Southern blot analysis confirmed that the antibiotic resistance fragment had replaced Cu-Zn sod in kanamycin-resistant colonies. The survival and growth of Cu-Zn SOD mutant strains were compared with that of the parental strains in HeLa cells and in the mouse macrophagelike cell line J774. The survival and growth of the Cu-Zn SOD mutant strains were similar to those of their respective parental strains in HeLa and J774 cell lines. The kinetics of infection with these strains were examined in BALB/c mice. The splenic levels of the S19 Cu-Zn SOD mutant recovered from intraperitoneally infected BALB/c mice were approximately 10-fold lower than those of the parental strain through 26 days postinfection. Thereafter, infection sharply declined in both groups, and by 105 days postinfection, no organisms were detected. The splenic levels of the S2308 Cu-Zn SOD mutant were lower than those of wild-type S2308-infected mice. The spleen weights of mice infected with the S2308 Cu-Zn SOD mutant were consistently lower than those of wild-type S2308-infected mice. These results suggest that the antioxidant enzyme Cu-Zn SOD plays a role in the survival and pathogenicity of B. abortus in vivo.     

Lymphocyte proliferation in response to immunodominant antigens of Brucella abortus 2308 and RB51 in strain 2308-infected cattle.

Lymphocyte proliferation in response to proteins from the Brucella abortus strain 2308 (S2308) and the lipopolysaccharide (LPS) O-antigen-deficient mutant of S2308, strain RB51 (SRB51), was measured in S2308-infected cattle following abortion. Supramammary and superficial cervical lymph node lymphocytes from infected cattle proliferated most when incubated with 27- to 18-kDa proteins of S2308 or SRB51. Proteins of SRB51, which contained no LPS O antigens, induced lymphocyte proliferation similar to that induced by S2308 proteins, which contained LPS O antigens. These results indicate that 27- to 18-kDa proteins, but not LPS O antigens, of S2308 and SRB51 are immunodominant in S2308-infected cattle as assessed by lymphocyte proliferation assays.     

Intracellular rescuing of a B. melitensis 16M virB mutant by co-infection with a wild type strain

Brucella is a broad-range, facultative intracellular pathogen that can survive and replicate in an endoplasmic reticulum (ER)-derived replication niche by preventing fusion of its membrane-bound compartment with late endosomes and lysosomes. This vacuolar hijacking was demonstrated to be dependent on the type IV secretion system VirB but no secreted effectors have been identified yet. A virB mutant is unable to reach its ER-derived replicative niche and does not multiply intracellularly. In this paper, we showed that, by co-infecting bovine macrophages or HeLa cells with the wild type (WT) strain of Brucella melitensis 16M and a deletion mutant of the complete virB operon, the replication of DeltavirB is rescued in almost 20% of the co-infected cells. Furthermore, we demonstrated that co-infections with the WT strains of Brucella abortus or Brucella suis were equally able to rescue the replication of the B. melitensis DeltavirB mutant. By contrast, no rescue was observed when the WT strain was given 1h before or after the infection with the DeltavirB mutant. Finally, vacuoles containing the rescued DeltavirB mutant were shown to exclude the LAMP-1 marker in a way similar to the WT containing vacuoles.     

Deletion of the BCSP31 gene of Brucella abortus by replacement.

The 31-kDa salt-extractable immunogenic protein, BCSP31, was deleted from several Brucella abortus strains by replacement with a marker gene encoding resistance to the antibiotics kanamycin and neomycin. The BCSP31 gene replacement plasmids, constructed with ColE1-derived vectors, were introduced by electroporation into B. abortus strain 19 (S19), into a rough variant of B. abortus S19, and into B. abortus S2308, and antibiotic-resistant transformants were isolated. B. abortus S19 is an attenuated strain used as a vaccine for prevention of bovine brucellosis in the United States, and B. abortus S2308 is a commonly used challenge strain. The antibiotic-resistant isolates were all obtained by recombination; none were spontaneous mutants. Loss of the gene encoding BCSP31 and presence of the marker gene were confirmed by Southern analysis. Vector sequences were either absent or linked to the genome, indicating that ColE1-derived plasmids are not maintained in B. abortus. Survival of B. abortus mutant strains in the macrophagelike cell line J774 and in HeLa cells was examined and shown to be indistinguishable from that of the parental strain.     

Brucella abortus tandem repeated ATP-binding proteins, BapA and BapB, homologs of haemophilus influenzae LktB, are not necessary for intracellular survival

Brucella abortus actively secretes materials and uptakes nutrients to maintain the survival and multiplication of the bacteria in host cells. ATP-binding cassette (ABC) transporters can uptake or secrete diverse materials across the bacterial membrane, and thus, ABC transporters may be important for survival of the pathogen in the host. In the present study, the B. abortus genes encoding tandem repeated Brucella ATP-binding proteins, BapA and BapB, were identified. The deduced amino acid sequences of these two genes place BapA and BapB into group 6 containing RTX toxin transporters and cyclic beta-1,2-glucan transporters, one of 25 ABC transporter ortholog groups. One of the ortholog group 6 proteins, Haemophilus influenzae LktB, shows the highest similarity and identity with these two Brucella proteins. To test the role of these putative tandem repeated ABC transporters in Brucella pathogenesis, a bap deletion mutant was constructed and used to infect murine RAW 264.7 macrophages and mice. The number of cfu from RAW 264.7 cells and spleens of BALB/c mice infected with wild type or the bap deletion mutant was similar during the course of infection, suggesting the bap genes are not necessary to maintain the pathogenesis of B. abortus, or alternative compensatory mechanisms may exist to permit the intracellular survival of B. abortus in vitro and in vivo. This is the first molecular approach to investigate the role of putative ABC transporters classified into ortholog group 6 in Brucella pathogenesis.     

Gene discovery through genomic sequencing of Brucella abortus

Brucella abortus is the etiological agent of brucellosis, a disease that affects bovines and human. We generated DNA random sequences from the genome of B. abortus strain 2308 in order to characterize molecular targets that might be useful for developing immunological or chemotherapeutic strategies against this pathogen. The partial sequencing of 1,899 clones allowed the identification of 1,199 genomic sequence surveys (GSSs) with high homology (BLAST expect value < 10(-5)) to sequences deposited in the GenBank databases. Among them, 925 represent putative novel genes for the Brucella genus. Out of 925 nonredundant GSSs, 470 were classified in 15 categories based on cellular function. Seven hundred GSSs showed no significant database matches and remain available for further studies in order to identify their function. A high number of GSSs with homology to Agrobacterium tumefaciens and Rhizobium meliloti proteins were observed, thus confirming their close phylogenetic relationship. Among them, several GSSs showed high similarity with genes related to nodule nitrogen fixation, synthesis of nod factors, nodulation protein symbiotic plasmid, and nodule bacteroid differentiation. We have also identified several B. abortus homologs of virulence and pathogenesis genes from other pathogens, including a homolog to both the Shda gene from Salmonella enterica serovar Typhimurium and the AidA-1 gene from Escherichia coli. Other GSSs displayed significant homologies to genes encoding components of the type III and type IV secretion machineries, suggesting that Brucella might also have an active type III secretion machinery.     

Differential secretion of interleukin-12 (IL-12) subunits and heterodimeric IL-12p70 protein by CD-1 mice and murine macrophages in response to intracellular infection by Brucella abortus

The secretion of interleukin-12 (IL-12) following intracellular infection with virulent Brucella abortus strain 2308 was investigated in CD-1 mice and in CD-1 cultured peritoneal macrophages. Bioactive IL-12p70 and free non-immunoactive p40 subunits (IL-12p40) were determined by enzyme-linked immunosorbent assays. In CD-1 mice, B. abortus 2308 was a potent inducer of IL-12p40 (maximum levels were 5.9 and 3.4 ng/ml in sera and spleen homogenates, respectively). Secretion of IL-12p70 was also demonstrated in vivo, although at much lower levels (216.6 and 198.9 pg/ml in sera and spleen homogenates, respectively). Production of IL-12 over the first 7 days after infection was accompanied by active multiplication of B. abortus in the spleens of infected mice. CD-1 cultured peritoneal macrophages secreted only IL-12p40 (878.4 pg/10(7) macrophages) in response to B. abortus infection and no production of IL-12p70 was observed. In contrast, CD-1 peritoneal macrophages secreted detectable amounts of IL-12p70 (16.2 pg/10(7) macrophages) in response to purified lipopolysaccharide (S-LPS) from B. abortus 2308. The macrophages also secreted significant amounts of interferon-gamma (IFN-gamma) (520.1 pg/10(7) macrophages) in response to intracellular B. abortus. These results indicate that B. abortus 2308 is not a potent inducer of IL-12p70 production, whereas purified S-LPS from B. abortus 2308 induces the secretion of this bioactive form of IL-12 in cultured peritoneal macrophages. CD-1 peritoneal macrophages were able to secrete IFN-gamma, as well as high amounts of IL-12p40, in response to intracellular infection by B. abortus.     

Brucella abortus cyclic beta-1,2-glucan mutants have reduced virulence in mice and are defective in intracellular replication in HeLa cells

Null cyclic beta-1,2-glucan synthetase mutants (cgs mutants) were obtained from Brucella abortus virulent strain 2308 and from B. abortus attenuated vaccinal strain S19. Both mutants show greater sensitivity to surfactants like deoxycholic acid, sodium dodecyl sulfate, and Zwittergent than the parental strains, suggesting cell surface alterations. Although not to the same extent, both mutants display reduced virulence in mice and defective intracellular multiplication in HeLa cells. The B. abortus S19 cgs mutant was completely cleared from the spleens of mice after 4 weeks, while the 2308 mutant showed a 1.5-log reduction of the number of brucellae isolated from the spleens after 12 weeks. These results suggest that cyclic beta-1,2-glucan plays an important role in the residual virulence of the attenuated B. abortus S19 strain. Although the cgs mutant was cleared from the spleens earlier than the wild-type parental strain (B. abortus S19) and produced less inflammatory response, its ability to confer protection against the virulent strain B. abortus 2308 was fully retained. Equivalent levels of induction of spleen gamma interferon mRNA and anti-lipopolysaccharide (LPS) of immunoglobulin G2a (IgG2a) subtype antibodies were observed in mice injected with B. abortus S19 or the cgs mutant. However, the titer of anti-LPS antibodies of the IgG1 subtype induced by the cgs mutant was lower than that observed with the parental S19 strain, thus suggesting that the cgs mutant induces a relatively exclusive Th1 response.     

Characterization of the urease operon of Brucella abortus and assessment of its role in virulence of the bacterium

Most members of the genus Brucella show strong urease activity. However, the role of this enzyme in the pathogenesis of Brucella infections is poorly understood. We isolated several Tn5 insertion mutants deficient in urease activity from Brucella abortus strain 2308. The mutations of most of these mutants mapped to a 5.7-kbp DNA region essential for urease activity. Sequencing of this region, designated ure1, revealed the presence of seven open reading frames corresponding to the urease structural proteins (UreA, UreB, and UreC) and the accessory proteins (UreD, UreE, UreF, and UreG). In addition to the urease genes, another gene (cobT) was identified, and inactivation of this gene affected urease activity in Brucella. Subsequent analysis of the previously described sequences of the genomes of Brucella spp. revealed the presence of a second urease cluster, ure2, in all them. The ure2 locus was apparently inactive in B. abortus 2308. Urease-deficient mutants were used to evaluate the role of urease in Brucella pathogenesis. The urease-producing strains were found to be resistant in vitro to strong acid conditions in the presence of urea, while urease-negative mutants were susceptible to acid treatment. Similarly, the urease-negative mutants were killed more efficiently than the urease-producing strains during transit through the stomach. These results suggested that urease protects brucellae during their passage through the stomach when the bacteria are acquired by the oral route, which is the major route of infection in human brucellosis.     

Brucella abortus HtrA functions as an authentic stress response protease but is not required for wild-type virulence in BALB/c mice

A second mutation has recently been identified in the previously described Brucella abortus htrA mutant PHE1. As a result of this finding, a new B. abortus htrA mutant, designated RWP11, was constructed to evaluate the biological function of the Brucella HtrA protease. RWP11 is more sensitive to oxidative killing in vitro and less resistant to killing by cultured murine neutrophils and macrophages than the virulent parental strain 2308 but is not attenuated in BALB/c mice through 4 weeks postinfection. The in vitro phenotype of B. abortus RWP11 is consistent with the proposed function of bacterial HtrA proteases as components of a secondary line of defense against oxidative damage. The in vivo phenotype of this mutant, however, indicates that, unlike the corresponding Salmonella and Yersinia proteins, Brucella HtrA does not play a critical role in virulence in the mouse model.     

Deletion of znuA virulence factor attenuates Brucella abortus and confers protection against wild-type challenge

znuA is known to be an important factor for survival and normal growth under low Zn(2+) concentrations for Escherichia coli, Haemophilus spp., Neisseria gonorrhoeae, and Pasteurella multocida. We hypothesized that the znuA gene present in Brucella melitensis 16 M would be similar to znuA in B. abortus and questioned whether it may also be an important factor for growth and virulence of Brucella abortus. Using the B. melitensis 16 M genome sequence, primers were designed to construct a B. abortus deletion mutant. A znuA knockout mutation in B. abortus 2308 (DeltaznuA) was constructed and found to be lethal in low-Zn(2+) medium. When used to infect macrophages, DeltaznuA B. abortus showed minimal growth. Further study with DeltaznuA B. abortus showed that its virulence in BALB/c mice was attenuated, and most of the bacteria were cleared from the spleen within 8 weeks. Protection studies confirmed the DeltaznuA mutant as a potential live vaccine, since protection against wild-type B. abortus 2308 challenge was as effective as that obtained with the RB51 or S19 vaccine strain.     

Identification of genes required for chronic persistence of Brucella abortus in mice

The genetic basis for chronic persistence of Brucella abortus in lymphoid organs of mice, cows, and humans is currently unknown. We identified B. abortus genes involved in chronic infection, by assessing the ability of 178 signature-tagged mutants to establish and maintain persistent infection in mice. Each mutant was screened for its ability to colonize the spleens of mice at 2 and 8 weeks after inoculation. Comparison of the results from both time points identified two groups of mutants attenuated for chronic infection in mice. The first group was not recovered at either 2 or 8 weeks postinfection and was therefore defective in establishing infection. Mutants in this group carried transposon insertions in genes involved in lipopolysaccharide biosynthesis (wbkA), in aromatic amino acid biosynthesis, and in type IV secretion (virB1 and virB10). The second group, which was recovered at wild-type levels 2 weeks postinfection but not 8 weeks postinfection was able to establish infection but was unable to maintain chronic infection. One mutant in this group carried a transposon insertion in a gene with homology to gcvB of Mycobacterium tuberculosis, encoding glycine dehydrogenase, an enzyme whose activity is increased during the state of nonreplicating persistence. These results suggest that some mechanisms for long-term persistence may be shared among chronic intracellular pathogens. Furthermore, identification of two groups of genes, those required for initiating infection and those required only for long-term persistence, suggests that B. abortus uses distinct sets of virulence determinants to establish and maintain chronic infection in mice.