Proinflammatory caspase-2-mediated macrophage cell death induced by a rough attenuated Brucella suis strain.

Brucella spp. are intracellular bacteria that cause an infectious disease called brucellosis in humans and many domestic and wildlife animals. B. suis primarily infects pigs and is pathogenic to humans. The macrophage-Brucella interaction is critical for the establishment of a chronic Brucella infection. Our studies showed that smooth virulent B. suis strain 1330 (S1330) prevented programmed cell death of infected macrophages and rough attenuated B. suis strain VTRS1 (a vaccine candidate) induced strong macrophage cell death. To further investigate the mechanism of VTRS1-induced macrophage cell death, microarrays were used to analyze temporal transcriptional responses of murine macrophage-like J774.A1 cells infected with S1330 or VTRS1. In total 17,685 probe sets were significantly regulated based on the effects of strain, time and their interactions. A miniTUBA dynamic Bayesian network analysis predicted that VTRS1-induced macrophage cell death was mediated by a proinflammatory gene (the tumor necrosis factor alpha [TNF-α] gene), an NF-κB pathway gene (the IκB-α gene), the caspase-2 gene, and several other genes. VTRS1 induced significantly higher levels of transcription of 40 proinflammatory genes than S1330. A Mann-Whitney U test confirmed the proinflammatory response in VTRS1-infected macrophages. Increased production of TNF-α and interleukin 1ß (IL-1ß) were also detected in the supernatants in VTRS1-infected macrophage cell culture. Hyperphosphorylation of IκB-α was observed in macrophages infected with VTRS1 but not S1330. The important roles of TNF-α and IκB-α in VTRS1-induced macrophage cell death were further confirmed by individual inhibition studies. VTRS1-induced macrophage cell death was significantly inhibited by a caspase-2 inhibitor but not a caspase-1 inhibitor. The role of caspase-2 in regulating the programmed cell death of VTRS1-infected macrophages was confirmed in another study using caspase-2-knockout mice. In summary, VTRS1 induces a proinflammatory, caspase-2- and NF-κB-mediated macrophage cell death. This unique cell death differs from apoptosis, which is not proinflammatory. It is also different from classical pyroptosis, which is caspase-1 mediated.

Over-expression of homologous antigens in a leucine auxotroph of Brucella abortus strain RB51 protects mice against a virulent B. suis challenge.

Infection by members of the Gram-negative bacterial genus Brucella causes brucellosis in a variety of mammals. Brucellosis in swine remains a challenge, as there is no vaccine in the USA approved for use in swine against brucellosis. Here, we developed an improved recombinant Brucella abortus vaccine strain RB51 that could afford protection against Brucella suis infection by over-expressing genes encoding homologous proteins: L7/L12 ribosomal protein, Cu/Zn superoxide dismutase [SOD] and glycosyl-transferase [WboA]. Using strain RB51leuB as a platform and an antibiotic-resistance marker free plasmid, strains RB51leuB/SOD, RB51leuB/SOD/L7/L12 and RB51leuB/SOD/WboA were constructed to over-express the antigens: SOD alone, SOD and ribosomal protein L7/L12 or SOD and glycosyl-transferase, respectively. The ability of these vaccine candidates to protect against a virulent B. suis challenge were evaluated in a mouse model. All vaccine groups protected mice significantly (P<0.05) when compared to the control group. Within the vaccine groups, the mice vaccinated with strain RB51leuB/SOD/WboA were significantly better protected than those that were vaccinated with either strain RB51leuB/SOD or RB51leuB/SOD/L7/L12. These results suggest that Brucella antigens can be over-expressed in strain RB51leuB and elicit protective immune responses against brucellosis. Since the plasmid over-expressing homologous antigens does not carry an antibiotic resistance gene, it complies with federal regulations and therefore could be used to develop safer multi-species vaccines for prevention of brucellosis caused by other species of Brucella.

Characterization of recombinant B. abortus strain RB51SOD toward understanding the uncorrelated innate and adaptive immune responses induced by RB51SOD compared to its parent vaccine strain RB51.

Brucella abortus is a Gram-negative, facultative intracellular pathogen for several mammals, including humans. Live attenuated B. abortus strain RB51 is currently the official vaccine used against bovine brucellosis in the United States and several other countries. Overexpression of protective B. abortus antigen Cu/Zn superoxide dismutase (SOD) in a recombinant strain of RB51 (strain RB51SOD) significantly increases its vaccine efficacy against virulent B. abortus challenge in a mouse model. An attempt has been made to better understand the mechanism of the enhanced protective immunity of RB51SOD compared to its parent strain RB51. We previously reported that RB51SOD stimulated enhanced Th1 immune response. In this study, we further found that T effector cells derived from RB51SOD-immunized mice exhibited significantly higher cytotoxic T lymphocyte activity than T effector cells derived from RB51-immunized mice against virulent B. abortus-infected target cells. Meanwhile, the macrophage responses to these two strains were also studied. Compared to RB51, RB51SOD cells had a lower survival rate in macrophages and induced lower levels of macrophage apoptosis and necrosis. The decreased survival of RB51SOD cells correlates with the higher sensitivity of RB51SOD, compared to RB51, to the bactericidal action of either Polymyxin B or sodium dodecyl sulfate (SDS). Furthermore, a physical damage to the outer membrane of RB51SOD was observed by electron microscopy. Possibly due to the physical damage, overexpressed Cu/Zn SOD in RB51SOD was found to be released into the bacterial cell culture medium. Therefore, the stronger adaptive immunity induced by RB51SOD did not correlate with the low level of innate immunity induced by RB51SOD compared to RB51. This unique and apparently contradictory profile is likely associated with the differences in outer membrane integrity and Cu/Zn SOD release.

Simultaneous expression of homologous and heterologous antigens in rough, attenuated Brucella melitensis.

The possibility of expressing a homologous antigen and a heterologous antigen simultaneously in an attenuated Brucella melitensis strain was investigated. The Brucella wboA gene encoding a mannosyltransferase involved in biosynthesis of lipopolysaccharide O-antigen, and the Bacillus anthracis pag gene encoding the protective antigen (PA) were cloned into plasmid pBBR4MCS. The resulting plasmid was introduced into O-antigen deficient B. melitensis strain WRRP1 to produce strain WRSPA. Strain WRSPA produced O-antigen and a series of PA products, induced protection in BALB/c mice against challenge with B. melitensis strain 16M, but failed to protect A/J mice against challenge with B. anthracis Sterne strain.

The putative penicillin-binding proteins 1 and 2 are important for viability, growth and cell morphology of Brucella melitensis.

The penicillin-binding proteins (PBPs) are enzymes that regulate the assembly of the peptidoglycan layer of the bacterial cell wall. The genome of Brucella melitensis strain 16M possesses seven pbp genes: three in pbp-1 family (designated as 1A, 1B, and 1C); one in pbp-2 family; and three in pbp-6 family (designated as 6A, 6B, and 6C). We investigated the importance of pbp-1 and pbp-2 genes to viability, cell morphology and infectivity of B. melitensis. A recombinant B. melitensis strain (designated 16MDeltapbp1C) was generated by disrupting the pbp-1C of strain 16M by allelic exchange. This strain produced nearly 20% smaller colonies on trypticase soy agar plates, and grew slower in trypticase soy broth compared to the strain 16M. Electron microscopy revealed that strain 16M exhibited native cocco-bacillus morphology, while 16MDeltapbp1C possessed a spherical morphology. Strain 16MDeltapbp1C did not differ from strain 16M in terms of recovery from infected mouse macrophage cell line J774.1, or recovery from spleens of infected BALB/c mice, suggesting that pbp-1C is dispensable for intracellular persistence of B. melitensis. Expression of mRNA of fixR, the gene downstream of pbp-1C was similar between the strains 16M and 16MDeltapbp1C suggesting that disruption of pbp-1C did not induce any polar effects. Multiple attempts to mutate pbp-1A, pbp-1B, or pbp-2 genes failed, most probably because these genes are indispensable for viability of B. melitensis. Our findings suggest that pbp-1C regulates in vitro growth and cell morphology, whereas pbp-1A, pbp-1B, and pbp-2 are essential for viability of B. melitensis.

Brucella abortus strain RB51 leucine auxotroph as an environmentally safe vaccine for plasmid maintenance and antigen overexpression.

To avoid potentiating the spread of an antibiotic resistance marker, a plasmid expressing a leuB gene and a heterologous antigen, green fluorescent protein (GFP), was shown to complement a leucine auxotroph of cattle vaccine strain Brucella abortus RB51, which protected CD1 mice from virulent B. abortus 2308 and elicited GFP antibodies.

A disruption of ctpA encoding carboxy-terminal protease attenuates Burkholderia mallei and induces partial protection in CD1 mice.

Burkholderia mallei is the etiologic agent of glanders in solipeds (horses, mules and donkeys), and incidentally in carnivores and humans. Little is known about the molecular mechanisms of B. mallei pathogenesis. The putative carboxy-terminal processing protease (CtpA) of B. mallei is a member of a novel family of endoproteases involved in the maturation of proteins destined for the cell envelope. All species and isolates of Burkholderia carry a highly conserved copy of ctpA. We studied the involvement of CtpA on growth, cell morphology, persistence, and pathogenicity of B. mallei. A sucrose-resistant strain of B. mallei was constructed by deleting a major portion of the sacB gene of the wild type strain ATCC 23344 by gene replacement, and designated as strain 23344DeltasacB. A portion of the ctpA gene (encoding CtpA) of strain 23344DeltasacB was deleted by gene replacement to generate strain 23344DeltasacBDeltactpA. In contrast to the wild type ATCC 23344 or the sacB mutant 23344DeltasacB, the ctpA mutant 23344DeltasacBDeltactpA displayed altered cell morphologies with partially or fully disintegrated cell envelopes. Furthermore, relative to the wild type, the ctpA mutant displayed slower growth in vitro and less ability to survive in J774.2 murine macrophages. The expression of mRNA of adtA, the gene downstream of ctpA was similar among the three strains suggesting that disruption of ctpA did not induce any polar effects. As with the wild type or the sacB mutant, the ctpA mutant exhibited a dose-dependent lethality when inoculated intraperitoneally into CD1 mice. The CD1 mice inoculated with a non-lethal dose of the ctpA mutant produced specific serum immunoglobulins IgG1 and IgG2a and were partially protected against challenge with wild type B. mallei ATCC 23344. These findings suggest that CtpA regulates in vitro growth, cell morphology and intracellular survival of B. mallei, and a ctpA mutant protects CD1 mice against glanders.

Outcome and immune responses after Brucella abortus infection in young adult and aged mice.

Aging results in a general waning of immunity and enhanced susceptibility to many intracellular pathogens. However, in some instances, aging is accompanied by alternative immune responses that can be equal to, or even more effective, than those of young adults. Brucella spp. are intracellular bacteria and important human and animal pathogens, but there are no data regarding the effect of age on host defense in brucellosis. Young or old adult mice (DBA/2 or BALB/c) were infected with either an attenuated B. abortus strain that over-expressed the Brucella superoxide dismutase (strain RB51-SOD) or a fully virulent strain (strain 2308). Survival, organism burden in the spleen, and immune responses were assessed. All young adult and aged mice survived infection with RB51-SOD (up to 6 x 10(8) cfu) or strain 2308 (up to 8 x 10(8) cfu). Old mice had a lower organism burden in the spleen than young adult mice five or more weeks after infection. Antibody and cytokine responses were Th1-focused in young adult mice, but Th-mixed in older mice, including evidence of the newly defined Th17 subtype immune response. Immunization with the RB51-SOD strain provided protection vs. strain 2308 challenge in young and aged BALB/c, but only young adult DBA/2 mice. Thus, clinical outcomes of Brucella infection in aged mice are equal or superior to those of young adult mice; immune responses in older mice are less-Th1 specific suggesting alternate pathways may contribute to host defense vs. Brucella in aged mice.

Reduced cerebral infection of Neospora caninum in BALB/c mice vaccinated with recombinant Brucella abortus RB51 strains expressing N. caninum SRS2 and GRA7 proteins.

Neospora caninum, an obligate intracellular protozoan parasite, is the causative agent of bovine neosporosis, an important disease affecting the reproductive performance of cattle worldwide. Currently there is no effective vaccine available to prevent N. caninum infection in cattle. In this study, we examined the feasibility of developing a live, recombinant N. caninum vaccine using Brucella abortus vaccine strain RB51 as the expression and delivery vector. We generated two recombinant RB51 strains each expressing SRS2 (RB51/SRS2) or GRA7 (RB51/GRA7) antigens of N. caninum. BALB/c mice immunized by single intraperitoneal inoculation of the recombinant RB51 strains developed IgG antibodies specific to the respective N. caninum antigen. In vitro stimulation of splenocytes from the vaccinated mice with specific antigen resulted in the production of interferon-gamma, but not IL-5 or IL-10, suggesting the development of a Th1 type immune response. Upon challenge with N. caninum tachyzoites, mice vaccinated with strain RB51/SRS2, but not RB51/GRA7, showed significant resistance to cerebral infection when compared to the RB51 vaccinated mice, as determined by the tissue parasite load using a real-time quantitative TaqMan assay. Interestingly, mice vaccinated with either strain RB51 or RB51/GRA7 also contained significantly lower parasite burden in their brains compared to those inoculated with saline. Mice vaccinated with strain RB51/SRS2 or RB51/GRA7 were protected to the same extent as the strain RB51 vaccinated mice against challenge with B. abortus virulent strain 2308. These results suggest that a recombinant RB51 strain expressing an appropriate protective antigen(s), such as SRS2 of N. caninum, can confer protection against both neosporosis and brucellosis.

Brucella suis urease encoded by ure1 but not ure2 is necessary for intestinal infection of BALB/c mice.

BACKGROUND: In prokaryotes, the ureases are multi-subunit, nickel-containing enzymes that catalyze the hydrolysis of urea to carbon dioxide and ammonia. The Brucella genomes contain two urease operons designated as ure1 and ure2. We investigated the role of the two Brucella suis urease operons on the infection, intracellular persistence, growth, and resistance to low-pH killing. RESULTS: The deduced amino acid sequence of urease-alpha subunits of operons-1 and -2 exhibited substantial identity with the structural ureases of alpha- and beta-proteobacteria, Gram-positive and Gram-negative bacteria, fungi, and higher plants. Four ure deficient strains were generated by deleting one or more of the genes encoding urease subunits of B. suis strain 1330 by allelic exchange: strain 1330Deltaure1K (generated by deleting ureD and ureA in ure1 operon), strain 1330Deltaure2K (ureB and ureC in ure2 operon), strain 1330Deltaure2C (ureA, ureB, and ureC in ure2 operon), and strain 1330Deltaure1KDeltaure2C (ureD and ureA in ure1 operon and ureA, ureB, and ureC in ure2 operon). When grown in urease test broth, strains 1330, 1330Deltaure2K and 1330Deltaure2C displayed maximal urease enzyme activity within 24 hours, whereas, strains 1330Deltaure1K and 1330Deltaure1KDeltaure2C exhibited zero urease activity even 96 h after inoculation. Strains 1330Deltaure1K and 1330Deltaure1KDeltaure2C exhibited slower growth rates in tryptic soy broth relative to the wild type strain 1330. When the BALB/c mice were infected intraperitoneally with the strains, six weeks after inoculation, the splenic recovery of the ure deficient strains did not differ from the wild type. In contrast, when the mice were inoculated by gavage, one week after inoculation, strain 1330Deltaure1KDeltaure2C was cleared from livers and spleens while the wild type strain 1330 was still present. All B. suis strains were killed when they were incubated in-vitro at pH 2.0. When the strains were incubated at pH 2.0 supplemented with 10 mM urea, strain 1330Deltaure1K was completely killed, strain 1330Deltaure2C was partially killed, but strains 1330 and 1330Deltaure2K were not killed. CONCLUSION: These findings suggest that the ure1 operon is necessary for optimal growth in culture, urease activity, resistance against low-pH killing, and in vivo persistence of B. suis when inoculated by gavage. The ure2 operon apparently enhances the resistance to low-pH killing in-vitro.

Cloning, expression and characterization of immunogenic aminopeptidase N from Brucella melitensis.

A 97-kDa purified aminopeptidase N (PepN) of Brucella melitensis was previously identified to be immunogenic in humans. The B. melitensis pepN gene was cloned, expressed in Escherichia coli and purified by affinity chromatography. The recombinant PepN (rPepN) exhibited the same biochemical properties, specificity and susceptibility to inhibitors as the native PepN. rPepN was evaluated as a diagnostic antigen in an indirect enzyme-linked immunosorbent assay (ELISA) using sera from patients with acute and chronic brucellosis. The specificity of the ELISA was determined with sera from healthy donors. The ELISA had a cutoff value of 0.156 with 100% specificity and 100% sensitivity. Higher sensitivity was obtained using rPepN compared with crude extract from B. melitensis. Anti-PepN sera did not exhibit serological cross-reaction to crude extracts from Rhizobium tropici, Ochrobactrum anthropi, Yersinia enterocolitica 09 or E. coli O157H7.

Establishment of a gene expression system in Ochrobactrum anthropi.

Genetic studies of Ochrobactrum anthropi are hindered by the lack of a suitable gene expression system. We constructed a set of vectors containing several promoters and a His tag fusion in the N terminus to facilitate protein detection and purification. The new vectors should significantly enhance the genetic manipulation and characterization of O. anthropi.

Brucella melitensis triggers time-dependent modulation of apoptosis and down-regulation of mitochondrion-associated gene expression in mouse macrophages.

Brucella spp. are facultative intracellular bacteria that cause brucellosis in humans and other animals. Brucella spp. are taken up by macrophages, and the outcome of the macrophage-Brucella interaction is a basis for establishment of a chronic Brucella infection. Microarrays were used to analyze the transcriptional response of the murine macrophage-like J774.A1 cell line to infection with virulent Brucella melitensis strain 16M. It was found that most significant changes in macrophage gene transcription happened early following infection, and global macrophage gene expression profiles returned to normal between 24 and 48 h postinfection. These findings support the observation that macrophages kill the majority of Brucella cells at the early infection stage, but the surviving Brucella cells are able to avoid macrophage brucellacidal activity inside replicative phagosomes at the later infection stage. At 4 h postinfection, macrophage genes involved in cell growth, metabolism, and responses to endogenous stimuli were down-regulated, while the inflammatory response (e.g., tumor necrosis factor alpha and Toll-like receptor 2), the complement system, the responses to external stimuli, and other immune responses were up-regulated. It is likely that the most active brucellacidal activity happened between 0 and 4 h postinfection. Mitochondrion-associated gene expression, which is involved in protein synthesis and transport, electron transfer, and small-molecule transfer, and many other mitochondrial functions were significantly down-regulated at 4 h postinfection. Although there were both pro- and antiapoptosis effects, B. melitensis 16M appears to inhibit apoptosis of macrophages by blocking release of cytochrome c and production of reactive oxygen species in the mitochondria, thus preventing activation of caspase cascades.

Efficacy of strain RB51 vaccine in heifers against experimental brucellosis.

With the goal of providing an additional tool for controlling bovine brucellosis in Brazil and evaluating the full calf dose in adult cattle, the efficacy of the rough Brucella abortus strain RB51 vaccine was tested in heifers. Thirty-three females of approximately 24 months of age were divided in two groups: one group (n=20) received the RB51 vaccine and the other group (n=13) were used as non-vaccinated control. Animals in the vaccinated group were split in two sub-groups. One sub-group (n=12) was vaccinated subcutaneously with 1.5x10(10) colony forming units (CFU) of RB51 at Day 0 of the experiment and the other sub-group (n=8) was vaccinated subcutaneously with 1.6x10(10) CFU of RB51 at 60 days of gestation (Day 260 of the experiment). All cattle were challenged between 6 and 7 months of pregnancy with 3x10(8) CFU of the virulent strain 2308 of B. abortus by the conjunctival route. Vaccination with RB51 vaccine did not result in the production of any antibodies against the O-side chain of lipopolysaccharide (LPS), as measured by conventional serological tests (rose bengal plate agglutination test (RBPAT), standard tube agglutination test (STAT), and 2-mercaptoethanol test (2ME)). A total of 25% cumulative incidence of abortions was found in the vaccinated group, whereas in the control group the cumulative incidence was 62%. B. abortus RB51 was not isolated from any sample, and no abortions were produced by RB51 vaccination of females at 60 days of pregnancy. The results indicate that vaccination with RB51 prevented 59.4% of abortions, 58.6% of cow infections, and 61.0% of fetal infections. The relative risk (RR) revealed that non-vaccinated animals have 2.462 (95% CI 1.029-5.889) times higher risk of aborting than RB51-vaccinated animals.

Role in virulence of a Brucella abortus protein exhibiting lectin-like activity.

Brucella abortus is a facultative, intracellular zoonotic pathogen which can cause undulant fever in humans and abortions in cattle. A 14-kDa protein of B. abortus was previously identified to be immunogenic in animals infected with Brucella spp. In this study, we discovered that the 14-kDa protein possessed immunoglobulin binding and hemagglutination properties that appeared to be based on the protein's lectin-like properties. Hemagglutination inhibition experiments suggested that the 14-kDa protein has affinity towards mannose. Disruption of the gene encoding the 14-kDa protein in virulent B. abortus strain 2308 induced a rough-like phenotype with an altered smooth lipopolysaccharide (LPS) immunoblot profile and a significant reduction in the bacterium's ability to replicate in mouse spleens. However, the mutant strain was stably maintained in mouse spleens at 2.0 to 2.6 log(10) CFU/spleen from day 1 to week 6 after intraperitoneal inoculation with 4.65 log(10) CFU. In contrast to the case for the smooth virulent strain 2308, in the rough attenuated strain RB51 disruption of the 14-kDa protein's gene had no effect on the mouse clearance pattern. These findings indicate that the 14-kDa protein of B. abortus possesses lectin-like properties and is essential for the virulence of the species, probably because of its direct or indirect role in the synthesis of smooth LPS.

Induction of antigen-specific Th1-type immune responses by gamma-irradiated recombinant Brucella abortus RB51.

Brucella abortus strain RB51 is an attenuated rough mutant used as the live vaccine against bovine brucellosis in the United States and other countries. We previously reported the development of strain RB51 as a bacterial vaccine vector for inducing Th1-type immune responses against heterologous proteins. Because safety concerns may preclude the use of strain RB51-based recombinant live vaccines, we explored the ability of a gamma-irradiated recombinant RB51 strain to induce heterologous antigen-specific immune responses in BALB/c mice. Exposure of strain RB51G/LacZ expressing Escherichia coli beta-galactosidase to a minimum of 300 kilorads of gamma radiation resulted in complete loss of replicative ability. These bacteria, however, remained metabolically active and continued to synthesize beta-galactosidase. A single intraperitoneal inoculation of mice with 10(9) CFU equivalents of gamma-irradiated, but not heat-killed, RB51G/LacZ induced a beta-galactosidase-specific Th1-type immune response. Though no obvious differences were detected in immune responses to B. abortus-specific antigens, mice vaccinated with gamma-irradiated, but not heat-killed, RB51G/LacZ developed significant protection against challenge with virulent B. abortus. In vitro experiments indicated that gamma-irradiated and heat-killed RB51G/LacZ induced maturation of dendritic cells; however, stimulation with gamma-irradiated bacteria resulted in more interleukin-12 secretion. These results suggest that recombinant RB51 strains exposed to an appropriate minimum dose of gamma radiation are unable to replicate but retain their ability to stimulate Th1 immune responses against the heterologous antigens and confer protection against B. abortus challenge in mice.

Carboxyl-terminal protease regulates Brucella suis morphology in culture and persistence in macrophages and mice.

The putative carboxyl-terminal processing protease (CtpA) of Brucella suis 1330 is a member of a novel family of endoproteases involved in the maturation of proteins destined for the cell envelope. The B. suis CtpA protein shared up to 77% homology with CtpA proteins of other bacteria. A CtpA-deficient Brucella strain (1330DeltactpA), generated by allelic exchange, produced smaller colonies on enriched agar plates and exhibited a 50% decrease in growth rate in enriched liquid medium and no growth in salt-free enriched medium compared to the wild-type strain 1330 or the ctpA-complemented strain 1330DeltactpA[pBBctpA]. Electron microscopy revealed that in contrast to the native coccobacillus shape of wild-type strain 1330, strain 1330DeltactpA possessed a spherical shape, an increased cell diameter, and cell membranes partially dissociated from the cell envelope. In the J774 mouse macrophage cell line, 24 h after infection, the CFU of the strain 1330DeltactpA declined by approximately 3 log(10) CFU relative to wild-type strain 1330. Nine weeks after intraperitoneal inoculation of BALB/c mice, strain 1330DeltactpA had cleared from spleens but strain 1330 was still present. These observations suggest that the CtpA activity is necessary for the intracellular survival of B. suis. Relative to the saline-injected mice, strain 1330DeltactpA-vaccinated mice exhibited 4 to 5 log(10) CFU of protection against challenge with virulent B. abortus strain 2308 or B. suis strain 1330 but no protection against B. melitensis strain 16 M. This is the first report correlating a CtpA deficiency with cell morphology and attenuation of B. suis.

Putative outer membrane autotransporter protein influences survival of Brucella suis in BALB/c mice.

In Gram-negative bacteria, autotransporters are secreted proteins able to translocate themselves through the inner- and outer-membranes to the cell surface or to the extracellular environment. The influence of the putative outer membrane autotransporter (OmaA) protein to the persistence of Brucella suis was investigated. Sequence analyses revealed that the OmaA protein of B. suis strain 1330 consists of a signal peptide, a passenger alpha-domain, and a transporter beta-domain, which are the characteristic components of an autotransporter protein. The transporter beta-domain consists of 14 individual amphipathic beta-strands, and a 46-amino acid long alpha-helix lies upstream of the transporter domain, indicating that the B. suis OmaA is a type-I classical autotransporter. BLAST search and phylogenetic analyses revealed that the B. suis OmaA protein shares more similarities with adhesin autotransporter proteins than with protease autotransporter proteins of other bacteria. An OmaA-deficient strain (1330DeltaomaA) was generated by disrupting the DNA region encoding the passenger alpha-domain of the OmaA protein of B. suis wild type strain 1330. The omaA gene encoding the full-length OmaA protein was cloned and used to complement the OmaA-deficient strain. The OmaA-deficient strain did not differ from the wild type strain in terms of persistence in J774 macrophage cell line 24 and 48 h after inoculation, or clearance from the spleens of BALB/c mice at 1 week after intraperitoneal inoculation. These observations suggest that the function of the OmaA protein is dispensable during the acute phase of B. suis infection. However, the OmaA-deficient strain was cleared from the spleens of BALB/c mice faster than the wild type strain between the third and the ninth week after intraperitoneal inoculation, indicating that the OmaA may be important during the chronic phase of B. suis infection. Relative to the BALB/c mice injected with saline, those vaccinated with the OmaA-deficient strain exhibited 3.0-3.9log10 colony forming units protection against a challenge with B. suis strain 1330. This study is the first report correlating an autotransporter protein deficiency with persistence of B. suis in vitro and in vivo.

Immune responses of mice to vaccinia virus recombinants expressing either Listeria monocytogenes partial listeriolysin or Brucella abortus ribosomal L7/L12 protein.

The Brucella abortus L7/L12 gene encoding ribosomal protein L7/L12 and the Listeria monocytogenes partial hly gene encoding the protective region of the hemolysin (partial listeriolysin, pLLO) were cloned into vaccinia virus by homologous recombination to produce recombinants WRL7/L12 and WRpLLO, respectively. The ability of these recombinants to induce humoral, cell mediated and protective immune response in mice was assessed. Although mice inoculated with WRL7/L12 recombinant produced antibodies specific to vaccinia virus and L7/L12 antigens, they were not protected against a virulent challenge with B. abortus 2308 strain. In contrast, mice inoculated with WRpLLO were protected against a challenge with virulent L. monocytogenes. Stimulation with purified fusion listeriolysin protein (MBP-LLO), but not with unrelated control protein (MBP), induced splenocytes from WRpLLO-inoculated mice to secrete significantly higher amounts of IFN-gamma than saline inoculated mice. Mice inoculated with either WRpLLO or WRL7/L12 recombinants produced predominantly IgG2a isotype antibody responses, indicative of a Th1 type of immune response. The protective potential of the WRpLLO recombinant correlated with the level of IFN-gamma produced in these mice.