The outer membrane of Brucella ovis shows increased permeability to hydrophobic probes and is more susceptible to cationic peptides than are the outer membranes of mutant rough Brucella abortus strains.

The permeability of the outer membrane (OM) to hydrophobic probes and its susceptibility to bactericidal cationic peptides were investigated for natural rough Brucella ovis and for mutant rough Brucella abortus strains. The OM of B. ovis displayed an abrupt and faster kinetic profile than rough B. abortus during the uptake of the hydrophobic probe N-phenyl-naphthylamine. B. ovis was more sensitive than rough B. abortus to the action of cationic peptides. Bactenecins 5 and 7 induced morphological alterations on the OMs of both rough Brucella strains. B. ovis lipopolysaccharide (LPS) captured considerably more polymyxin B than LPSs from both rough and smooth B. abortus strains. Polymyxin B, poly-L-lysine, and poly-L-ornithine produced a thick coating on the surfaces of both strains, which was more evident in B. ovis than in rough B. abortus. The distinct functional properties of the OMs of these two rough strains correlate with some structural differences of their OMs and with their different biological behaviors in animals and culture cells.     

Antibody and delayed-type hypersensitivity responses to Ochrobactrum anthropi cytosolic and outer membrane antigens in infections by smooth and rough Brucella spp.

Immunological cross-reactions between Brucella spp. and Ochrobactrum anthropi were investigated in animals and humans naturally infected by Brucella spp. and in experimentally infected rams (Brucella ovis infected), rabbits (Brucella melitensis infected), and mice (B. melitensis and Brucella abortus infected). In the animals tested, O. anthropi cytosolic proteins evoked a delayed-type hypersensitivity reaction of a frequency and intensity similar to that observed with B. melitensis brucellin. O. anthropi cytosolic proteins also reacted in gel precipitation tests with antibodies in sera from Brucella natural hosts with a frequency similar to that observed with B. melitensis proteins, and absorption experiments and immunoblotting showed antibodies to both Brucella-specific proteins and proteins common to Brucella and O. anthropi. No antibodies to O. anthropi cytosolic proteins were detected in the sera of Brucella-free hosts. Immunoblotting with sera of Brucella-infected sheep and goats showed immunoglobulin G (IgG) to Brucella group 3 outer membrane proteins and to O. anthropi proteins of similar molecular weight. No IgG to the O-specific polysaccharide of O. anthropi lipopolysaccharide was detected in the sera of Brucella-infected hosts. The sera of sheep, goats, and rabbits infected with B. melitensis contained IgG to O. anthropi rough lipopolysaccharide and lipid A, and B. ovis and O. anthropi rough lipopolysaccharides showed equal reactivities with IgG in the sera of B. ovis-infected rams. The findings show that the immunoresponse of Brucella-infected hosts to protein antigens is not necessarily specific for brucellae and suggest that the presence of O. anthropi or some related bacteria explains the previously described reactivities to Brucella rough lipopolysaccharide and outer membrane proteins in healthy animals.     

Real-time multiplex PCR assay for detection of Brucella spp., B. abortus, and B. melitensis

The identification of Brucella can be a time-consuming and labor-intensive process that places personnel at risk for laboratory-acquired infection. Here, we describe a real-time PCR assay for confirmation of presumptive Brucella isolates. The assay was designed in a multiplex format that will allow the rapid identification of Brucella spp., B. abortus, and B. melitensis in a single test.     

Demonstration of a peptidoglycan-linked lipoprotein and characterization of its trypsin fragment in the outer membrane of Brucella spp.

The sodium dodecyl sulfate (SDS) extraction-trypsin digestion protocol used by Braun and Sieglin (V. Braun and U. Sieglin, Eur. J. Biochem. 13:336-346, 1970) to show the peptidoglycan-linked lipoprotein of Escherichia coli was applied to both Brucella abortus and E. coli. Whereas a single polypeptide of 8,000 molecular weight was obtained from E. coli, several proteins of apparent molecular weight lower than 35,000 were demonstrated by SDS-polyacrylamide gel electrophoresis in B. abortus. These results did not change when the trypsin digestion conditions were modified. On the other hand, when the SDS extractions were performed under conditions more stringent than those used for other gram-negative bacteria, only a polypeptide fragment of apparent molecular weight of 8,000 was obtained from B. abortus. This polypeptide was similar to the trypsin fragment of the E. coli lipoprotein with respect to its behavior in SDS-polyacrylamide gels, isoelectric point in urea, molecular weight, and presence of both ester- and amide-linked fatty acids. Moreover, the amino acid analysis showed an overall similarity with respect to the amino acid composition of E. coli lipoprotein. A polypeptide of the same molecular weight, isoelectric point, and amino acid composition was also obtained from Brucella ovis by the same method. These results demonstrated that B. abortus and B. ovis cell envelopes contain a lipoprotein and strongly support the hypothesis that it is the only major protein covalently linked to the peptidoglycan.     

Molecular characterization, occurrence, and immunogenicity in infected sheep and cattle of two minor outer membrane proteins of Brucella abortus.

Screening of a Brucella abortus genomic library with two sets of monoclonal antibodies allowed the isolation of the genes corresponding to two minor outer membrane proteins (OMP10 and OMP19) found in this bacterial species. Sequence analysis of the omp10 gene revealed an open reading frame capable of encoding a protein of 126 amino acids. The nucleotide sequence of the insert producing the OMP19 protein contains two overlapping open reading frames, the largest of which (177 codons) was shown to encode the protein of interest. Analysis of the N-terminal sequences of both putative proteins revealed features of a bacterial signal peptide, and homology to the bacterial lipoprotein processing sequence was also observed. Immunoblotting with monoclonal antibodies specific for OMP10 or OMP19 showed that both proteins are present in the 34 Brucella strains tested, representing all six Brucella species and all their biovars. The OMP19 detected in the five Brucella ovis strains examined migrated at an apparent molecular weight that is slightly higher than those of the other Brucella species, confirming the divergence of B. ovis from these species. OMP10 and OMP19 were produced in recombinant Escherichia coli and purified to homogeneity for serological analysis. A large fraction of sera from sheep naturally infected with Brucella melitensis were reactive with these proteins in an enzyme-linked immunosorbent assay, whereas sera from B. abortus-infected cattle were almost completely unreactive in this assay.     

Effect of omp10 or omp19 deletion on Brucella abortus outer membrane properties and virulence in mice

The distinctive properties of Brucella outer membrane have been considered to be critical for Brucella sp. virulence. Among the outer membrane molecules possibly related to these properties, Omp10 and Omp19 are immunoreactive outer membrane lipoproteins. Moreover, these proteins of Brucella could constitute a new family of outer membrane proteins specifically encountered in the family RHIZOBIACEAE: We evaluated the impact of omp10 or omp19 deletion on Brucella abortus outer membrane properties and virulence in mice. The omp10 mutant was dramatically attenuated for survival in mice and was defective for growth in minimal medium but was not impaired in intracellular growth in vitro, nor does it display clear modification of the outer membrane properties. Significantly fewer brucellae were recovered from the spleens of mice infected with the omp19 mutant than from those of mice infected with the parent strain at 4 and 8 weeks postinfection. The omp19 mutant exhibited an increase in sensitivity to the polycation polymyxin B and to sodium deoxycholate. These results indicate that inactivation of the omp19 gene alters the outer membrane properties of B. abortus.     

Immune response to porin in cattle immunized with whole cell, outer membrane, and outer membrane protein antigens of Brucella abortus combined with trehalose dimycolate and muramyl dipeptide adjuvants.

The immune response of cattle to nonliving vaccines derived from Brucella abortus rough strain 45/20 was studied. Vaccines contained trehalose dimycolate and a derivative of muramyl dipeptide. N-acetylmuramyl-L-alpha-aminobutyryl-D-isoglutamine. A factorial experiment was designed to test the effects of type of antigen, quantity of antigen, and quantity of mineral oil on the immune response to porin. Muramyl dipeptide was kept constant at 5 mg per dose, and 1 part of trehalose dimycolate was incorporated for two parts of dry matter. Over a 10-week period, blastogenesis responses to porin were largest in cattle immunized with outer membranes; the highest antibody titers to the porin-lipopolysaccharide complex were achieved by immunization with detergent-extracted outer membrane proteins. There was no advantage in the use of 25, rather than 5, mg of any of the antigens, but antibody responses were improved by increasing the quantity of oil from 0.6 to 1.8 ml per dose. In other animals, blastogenesis and antibody responses were sustained at high levels longer than 3 months after two vaccinations with outer membrane proteins. Intradermal injection of porin evoked inflammatory reactions histologically consistent with delayed-type hypersensitivity. Cross-reactions in cases of delayed-type hypersensitivity occurred with porin derived from a smooth strain of B. abortus but were less extensive than in the blastogenesis test. The magnitude of the delayed-type hypersensitivity and blastogenesis responses induced by vaccination exceeded those observed after natural or experimental infections. No ill effects were observed after vaccination. These findings provide a basis for the use of trehalose dimycolate and muramyl dipeptide adjuvants in evaluating nonviable vaccines for bovine brucellosis.     

Molecular cloning, nucleotide sequence, and occurrence of a 16.5-kilodalton outer membrane protein of Brucella abortus with similarity to pal lipoproteins.

Recombinant lambda gt11 phages were selected by screening a genomic library of Brucella abortus DNA with monoclonal antibodies specific for a 16.5-kDa Brucella outer membrane protein (Omp16). The corresponding gene, named pal, was subcloned on a 0.7-kb AluI fragment. Immunoblotting confirmed the expression of a recombinant Omp16 in the transformants. DNA sequence analysis revealed an open reading frame of 168 codons. The deduced amino acid sequence agrees with an internal peptide sequence of native Omp16 and contains a potential lipoprotein signal peptide cleavage site, giving rise to a predicted mature protein of 144 amino acids. The predicted sequence of Omp16 also shows a remarkable degree of similarity to the sequences of three peptidoglycan-associated bacterial lipoproteins. In immunoblotting with a monoclonal antibody specific for Omp16, we demonstrated that Omp16 was expressed in the 34 Brucella strains tested, representing all six species and known biovars.     

BvrR/BvrS-controlled outer membrane proteins Omp3a and Omp3b are not essential for Brucella abortus virulence

The Brucella abortus two-component regulatory system BvrR/BvrS controls the expression of outer membrane proteins (Omp) Omp3a (Omp25) and Omp3b (Omp22). Disruption of bvrS or bvrR generates avirulent mutants with altered cell permeability, higher sensitivity to microbicidal peptides, and complement. Consequently, the role of Omp3a and Omp3b in virulence was examined. Similar to bvrS or bvrR mutants, omp3a and omp3b mutants displayed increased attachment to cells, indicating surface alterations. However, they showed unaltered permeability; normal expression of Omp10, Omp16, Omp19, Omp2b, and Omp1; native hapten polysaccharide; and lipopolysaccharide and were resistant to complement and polymyxin B at ranges similar to those of the wild-type (WT) counterpart. Likewise, omp3a and omp3b mutants were able to replicate in murine macrophages and in HeLa cells, were resistant to the killing action of human neutrophils, and persisted in mice, like the WT strain. Murine macrophages infected with the omp3a mutant generated slightly higher levels of tumor necrosis factor alpha than the WT, whereas the bvrS mutant induced lower levels of this cytokine. Since the absence of Omp3a or Omp3b does not result in attenuation, it can be concluded that BvrR/BvrS influences additional Brucella properties involved in virulence. Our results are discussed in the light of previous works suggesting that disruption of omp3a generates attenuated Brucella strains, and we speculate on the role of group 3 Omps.     

Comparison of sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles and antigenic relatedness among outer membrane proteins of 49 Brucella abortus strains.

Immunoglobulin A (IgA) antibodies in human sera with binding specificity for the C-terminal R-D-Ala-D-Ala sequence of the precursor peptide from bacterial cell wall peptidoglycan were detected by an enzyme-linked immunosorbent assay (ELISA). Specificity of the test system was proved by comparing the high binding of specific IgA to albumin-(D-Ala3) as an antigen with the failure to bind to albumin-(L-Ala3), by binding inhibition studies with L-Ala3, D-Ala3, or peptides with structural analogy to peptidoglycan peptide subunit peptides as inhibitors, and by excluding binding of peroxidase-labeled anti-human IgA to immunoglobulin classes others than IgA. Interference of rheumatoid factors of IgA class was excluded by an ELISA for assaying IgA-rheumatoid factor and by the fact that an IgA fraction essentially free of IgG and IgM was isolated from a serum reacting strongly positive in the ELISA for measuring specific IgA to the peptide subunit of peptidoglycan. This isolated IgA again exhibited binding specificity in the ELISA, thus corroborating the existence of specific IgA in human serum to the C-terminal R-D-Ala-D-Ala sequence of peptidoglycan precursor peptide. The existence of IgA antibodies with specificity for bacterial peptidoglycan was further proved by preadsorption of serum to peptidoglycans and subsequent measurement of specific IgA in the ELISA. Screening of human sera for IgA antibodies with specificity for R-D-Ala-D-Ala peptides revealed that specific antibodies directed against this sequence of bacterial cell wall peptidoglycan may be detected in several human sera.     

Elicitation of Th1/Th2 related responses in mice by chitosan nanoparticles loaded with Brucella abortus malate dehydrogenase,outer membrane proteins 10 and 19

Brucella spp. is the causative agent of brucellosis, one of the worldwide diseases. The pathogen infects humans and animals mainly through the digestive or respiratory tract. Therefore, induction of mucosal immunity is required as the first line of defense. In this study, three Brucella abortus recombinant proteins, malate dehydrogenase (rMdh), outer membrane proteins (rOmp) 10 and 19 were loaded in mucoadhesive chitosan nanoparticles (CNs) and induction of mucosal and systemic immunity were investigated after intranasal immunization of BALB/c mice. These antigens were also coimmunized as cocktail (rCocktail) to evaluate multiple antigen specific vaccine candidates. At 6-weeks post-immunization (wpi), antigen specific total IgG was increased in all of the immunized groups, predominantly IgG1. In addition, spleenocyte from rMdh-, rOmp19-, and rCocktail-immunized groups significantly produced IFN-γ and IL-4 suggesting the induction of a mixed Th1-Th2 response. For mucosal immunity, anti-Mdh IgA from nasal washes and fecal excretions, and anti-Omps IgA from sera, nasal washes, genital secretions and fecal excretions were significantly increased in single antigen immunized groups. In the rCocktail-immunized group, anti-Mdh IgA were significantly increased while anti-Omps IgA was not. Collectively, this study indicates that comprise of B. abortus antigen-loaded CNs elicited the antigen-specific IgA with a Th2-polarized immune responses and combination of the highly immunogenic antigens elicited IgG specific to each type of antigen.     

Immune responses and resistance to brucellosis in mice vaccinated orally with Brucella abortus RB51.

Immune responses and resistance to infection with Brucella abortus 2308 (S2308) were measured in mice following oral or intraperitoneal (i.p.) vaccination with strain RB51 (SRB51). Bacteria persisted in the parotid lymph node for 4 weeks following oral vaccination of mice with 5 x 10(8) or 5 x 10(6) CFU of SRB51. Bacteria did not appear in the spleen during 12 weeks after oral vaccination, whereas they did appear in the spleen for 8 weeks following i.p. vaccination of mice with SRB51 (5 x 10(8) or 5 x 10(6) CFU). Increased resistance to S2308 infection occurred at 12 to 20 weeks in mice vaccinated i.p. with SRB51 (5 x 10(8) or 5 x 10(6) CFU) but occurred at 12 weeks only in mice vaccinated orally with SRB51 (5 x 10(8) CFU). Oral SRB51 vaccination induced lower levels of antibodies to the surface antigens of intact SRB51 bacteria than did i.p. vaccination. However, neither route of vaccination induced anamnestic antibody responses to the surface antigens of intact S2308 bacteria after challenge infection of the vaccinated mice with S2308. Mice vaccinated orally with SRB51 and challenged with S2308 at 12 to 20 weeks had lower and less persistent spleen cell proliferation and production of gamma interferon in response to S2308 and certain immunodominant S2308 proteins (32 to < or = 18 kDa) than did mice vaccinated i.p. with SRB51. However, mice vaccinated orally or i.p. with SRB51 and challenged with S2308 had similar spleen cell tumor necrosis factor alpha production. These results indicate that oral vaccination of mice with SRB51 was effective in inducing protective immunity to S2308 infection, although the immunity was lower and less persistent than that induced by i.p. vaccination. The lower protective immunity induced by oral vaccination may have resulted from lower and less persistent cell-mediated immunity and gamma interferon production in response to S2308 and S2308 proteins.     

Rapid field detection assays for Bacillus anthracis,Brucella spp., Francisella tularensis and Yersinia pestis

Rapid detection is essential for timely initiation of medical post-exposure prophylactic measures in the event of intentional release of biological threat agents. We compared real-time PCR assay performance between the Applied Biosystems 7300/7500 and the RAZOR instruments for specific detection of the causative agents of anthrax, brucellosis, tularemia and plague. Furthermore, an assay detecting Bacillus thuringiensis, a Bacillus anthracis surrogate, was developed for field-training purposes. Assay sensitivities for B. anthracis, Brucella spp., Francisella tularensis and Yersinia pestis were 10-100 fg of target DNA per reaction, and no significant difference in assay performance was observed between the instrument platforms. Specificity testing of the diagnostic panels with both instrument platforms did not reveal any cross-reactivity with other closely related bacteria. The duration of thermocycling with the RAZOR instrument was shorter, i.e. 40 min as compared with 100 min for the Applied Biosystems 7300/7500 instruments. These assays provide rapid tools for the specific detection of four biological threat agents. The detection assays, as well as the training assay for B. thuringiensis powder preparation analysis, may be utilized under field conditions and for field training, respectively.     

Role of disulfide bonding in outer membrane structure and permeability in Chlamydia trachomatis

The outer membrane of Chlamydia trachomatis can be efficiently solubilized by a variety of mild detergents in the presence of the reducing agent dithiothreitol. This allows purification of the chlamydial major outer membrane protein at high yield in very gentle conditions by using its differential solubility in Sarkosyl and octylglucoside in the presence of dithiothreitol. The major outer membrane protein of the L2 serovar is an acidic protein with a pI of ca. 5. It contains three cysteine residues that allow it to form a disulfide-linked proteinaceous network responsible for the characteristic rigid outer membrane of the elementary body. By the use of an in vitro reconstitution assay developed by Nikaido and his co-workers, it was shown that the outer membrane contains pores with an "exclusion limit" between molecular weights 850 and 2,250. In addition, the "opening-closing" of the pores was shown to be controlled through a simple reduction-oxidation mechanism. A model that outlines the role of disulfide bonding in the physiology of chlamydial development is presented.     

Role of Toll-like receptor 4 in induction of cell-mediated immunity and resistance to Brucella abortus infection in mice

Initial host defense to bacterial infection is executed by innate immunity, and therefore the main goal of this study was to examine the contribution of Toll-like receptors (TLRs) during Brucella abortus infection. CHO reporter cell lines transfected with CD14 and TLRs showed that B. abortus triggers both TLR2 and TLR4. In contrast, lipopolysaccharide (LPS) and lipid A derived from Brucella rough (R) and smooth (S) strains activate CHO cells only through TLR4. Consistently, macrophages from C3H/HePas mice exposed to R and S strains and their LPS produced higher levels of tumor necrosis factor alpha (TNF-alpha) and interleukin-12 compared to C3H/HeJ, a TLR4 mutant mouse. The essential role of TLR4 for induction of proinflammatory cytokines was confirmed with diphosphoryl lipid A from Rhodobacter sphaeroides. Furthermore, to determine the contribution of TLR2 and TLR4 in bacterial clearance, numbers of Brucella were monitored in the spleen of C3H/HeJ, C3H/HePas, TLR2 knockout, and wild-type mice at 1, 3, and 6 weeks following B. abortus infection. Interestingly, murine brucellosis was markedly exacerbated at weeks 3 and 6 after infection in animals that lacked functional TLR4 (C3H/HeJ) compared to C3H/HePas that paralleled the reduced gamma interferon production by this mouse strain. Finally, by mass spectrometry analysis we found dramatic differences on the lipid A profiles of R and S strains. In fact, S lipid A was shown to be more active to trigger TLR4 than R lipid A in CHO cells and more effective in inducing dendritic cell maturation. In conclusion, these results indicate that TLR4 plays a role in resistance to B. abortus infection and that S lipid A has potent adjuvant activity.     

Immunization with recombinant Brucella species outer membrane protein Omp16 or Omp19 in adjuvant induces specific CD4+ and CD8+ T cells as well as systemic and oral protection against Brucella abortus infection

Available vaccines against Brucella spp. are live attenuated Brucella strains. In order to engineer a better vaccine to be used in animals and humans, our laboratory aims to develop an innocuous subunit vaccine. Particularly, we are interested in the outer membrane proteins (OMPs) of B. abortus: Omp16 and Omp19. In this study, we assessed the use of these proteins as vaccines against Brucella in BALB/c mice. Immunization with lipidated Omp16 (L-Omp16) or L-Omp19 in incomplete Freund's adjuvant (IFA) conferred significant protection against B. abortus infection. Vaccination with unlipidated Omp16 (U-Omp16) or U-Omp19 in IFA induced a higher degree of protection than the respective lipidated versions. Moreover, the level of protection induced after U-Omp16 or U-Omp19 immunization in IFA was similar to that elicited by live B. abortus S19 immunization. Flow cytometric analysis showed that immunization with U-Omp16 or U-Omp19 induced antigen-specific CD4(+) as well as CD8(+) T cells producing gamma interferon. In vivo depletion of CD4(+) or CD8(+) T cells in mice immunized with U-Omp16 or U-Omp19 plus IFA resulted in a loss of the elicited protection, indicating that both cell types are mediating immune protection. U-Omp16 or U-Omp19 vaccination induced a T helper 1 response, systemic protection in aluminum hydroxide formulation, and oral protection with cholera toxin adjuvant against B. abortus infection. Both immunization routes exhibited a similar degree of protection to attenuated Brucella vaccines (S19 and RB51, respectively). Overall these results indicate that U-Omp16 or U-Omp19 would be a useful candidate for a subunit vaccine against human and animal brucellosis.     

Molecular cloning and characterization of cgt, the Brucella abortus cyclic beta-1,2-glucan transporter gene, and its role in virulence

The animal pathogen Brucella abortus contains a gene cgt, which complemented Sinorhizobium meliloti nodule development (ndvA) and Agrobacterium tumefaciens chromosomal virulence (chvA) mutants. Complemented strains recovered the presence of anionic cyclic beta-1,2-glucan, motility, tumor induction in A. tumefaciens, and nodule occupancy in S. meliloti, all traits strictly associated with the presence of cyclic beta-1,2-glucan in the periplasm. Nucleotide sequencing revealed that B. abortus cgt contains a 1,797-bp open reading frame coding for a predicted membrane protein of 599 amino acids (65.9 kDa) that is 58.5 and 59.9% identical to S. meliloti NdvA and A. tumefaciens ChvA, respectively. Additionally, B. abortus cgt, like S. meliloti ndvA and A. tumefaciens chvA possesses ATP-binding motifs and the ABC signature domain features of a typical ABC transporter. Characterization of Cgt was carried out by the construction of null mutants in B. abortus 2308 and S19 backgrounds. Both mutants do not transport cyclic beta-1,2-glucan to the periplasm, as shown by the absence of anionic cyclic glucan, and they display reduced virulence in mice and defective intracellular multiplication in HeLa cells. These results suggest that cyclic beta-1,2-glucan must be transported into the periplasmatic space to exert its action as a virulence factor.     

Comparison of immune responses and resistance to brucellosis in mice vaccinated with Brucella abortus 19 or RB51.

Immune responses and resistance to infection with Brucella abortus 2308 (S2308) were measured in mice following vaccination with B. abortus 19 (S19) or the lipopolysaccharide (LPS) O-antigen-deficient mutant, strain RB51 (SRB51). Live bacteria persisted for 8 weeks in spleens of mice vaccinated with 5 x 10(6) or 5 x 10(8) CFU of SRB51, whereas bacteria persisted for 12 weeks in mice vaccinated with 5 x 10(6) CFU of S19. Mice vaccinated with 5 x 10(6) or 5 x 10(8) CFU of SRB51 had increased resistance to infection with S2308 at 12, 16, and 20 weeks after vaccination, but the resistance was lower than that induced by vaccinating mice with 5 x 10(6) CFU of S19. Spleen cells obtained from mice vaccinated with S19 or SRB51 generally exhibited similar proliferative responses to S2308 bacteria or bacterial proteins (106 to 18 kDa) following challenge of mice with S2308 at 12, 16, or 20 weeks after vaccination. Mice vaccinated with S19 had antibody to S2308 bacteria and S2308 smooth LPS at 4, 8, and 12 weeks after vaccination. In contrast, mice vaccinated with either dose of SRB51 did not produce antibody to S2308 smooth LPS. In addition, only mice vaccinated with the highest dose of SRB51 (5 x 10(8) CFU) had antibody responses to S2308 bacteria, although the responses were lower and less persistent than those in mice vaccinated with S19. Collectively, these results indicate that SRB51-vaccinated mice have similar cell-mediated immune responses to S2308 but lower resistance to infection with S2308 compared with S19-vaccinated mice. The lower resistance in SRB51-vaccinated mice probably resulted from a combination of rapid clearance of SRB51 and an absence of antibodies to S2308 LPS.     

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.