Neutrophils as Trojan Horse Vehicles for Brucella abortus Macrophage Infection.

Brucella abortus is a stealthy intracellular bacterial pathogen of animals and humans. This bacterium promotes the premature cell death of neutrophils (PMN) and resists the killing action of these leukocytes. B. abortus-infected PMNs presented phosphatidylserine (PS) as "eat me" signal on the cell surface. This signal promoted direct contacts between PMNs and macrophages (Mϕs) and favored the phagocytosis of the infected dying PMNs. Once inside Mϕs, B. abortus replicated within Mϕs at significantly higher numbers than when Mϕs were infected with bacteria alone. The high levels of the regulatory IL-10 and the lower levels of proinflammatory TNF-α released by the B. abortus-PMN infected Mϕs, at the initial stages of the infection, suggested a non-phlogistic phagocytosis mechanism. Thereafter, the levels of proinflammatory cytokines increased in the B. abortus-PMN-infected Mϕs. Still, the efficient bacterial replication proceeded, regardless of the cytokine levels and Mϕ type. Blockage of PS with Annexin V on the surface of B. abortus-infected PMNs hindered their contact with Mϕs and hampered the association, internalization, and replication of B. abortus within these cells. We propose that B. abortus infected PMNs serve as "Trojan horse" vehicles for the efficient dispersion and replication of the bacterium within the host.

Characterization and Immunogenicity of Outer Membrane Vesicles from Brucella abortus.

Bovine brucellosis is a worldwide spread zoonotic disease. The objectives of this study were characterization of outer membrane vesicles from B. abortus and to evaluate their immunogenicity in mice. For this purpose, OMVs were derived from B. abortus strain 99 using ultracentrifugation method. Isolated OMVs were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transmission electron microscopy which revealed spherical 20-300 nm structures rich in proteins. OMVs also showed immuno-reactivity with mice antisera in Western blot. Further, indirect ELISA showed specific and high-titer immune responses against the antigens present in OMVs suggesting their potential for a safe acellular vaccine candidate.

Structural asymmetry in a conserved signaling system that regulates division, replication, and virulence of an intracellular pathogen.

We have functionally and structurally defined an essential protein phosphorelay that regulates expression of genes required for growth, division, and intracellular survival of the global zoonotic pathogen Brucella abortus. Our study delineates phosphoryl transfer through this molecular pathway, which initiates from the sensor kinase CckA and proceeds through the ChpT phosphotransferase to two regulatory substrates: CtrA and CpdR. Genetic perturbation of this system results in defects in cell growth and division site selection, and a specific viability deficit inside human phagocytic cells. Thus, proper control of B. abortus division site polarity is necessary for survival in the intracellular niche. We further define the structural foundations of signaling from the central phosphotransferase, ChpT, to its response regulator substrate, CtrA, and provide evidence that there are at least two modes of interaction between ChpT and CtrA, only one of which is competent to catalyze phosphoryltransfer. The structure and dynamics of the active site on each side of the ChpT homodimer are distinct, supporting a model in which quaternary structure of the 2:2 ChpT-CtrA complex enforces an asymmetric mechanism of phosphoryl transfer between ChpT and CtrA. Our study provides mechanistic understanding, from the cellular to the atomic scale, of a conserved transcriptional regulatory system that controls the cellular and infection biology of B. abortus. More generally, our results provide insight into the structural basis of two-component signal transduction, which is broadly conserved in bacteria, plants, and fungi.

Intermediate rough Brucella abortus S19Δper mutant is DIVA enable, safe to pregnant guinea pigs and confers protection to mice.

Brucella abortus S19 is a smooth strain used as live vaccine against bovine brucellosis. Smooth lipopolysaccharide (LPS) is responsible for its residual virulence and serological interference. Rough mutants defective of LPS are more attenuated but confers lower level of protection. We describe a modified B. abortus S19 strain, named as S19Δper, which exhibits intermediate rough phenotype with residual O-polysaccharide (OPS). Deletion of perosamine synthetase gene resulted in substantial attenuation of S19Δper mutant without affecting immunogenic properties. It mounted strong immune response in Swiss albino mice and conferred protection similar to S19 vaccine. Immunized mice produced higher levels of IFN-γ, IgG2a and thus has immune response inclined towards Th1 cell mediated immunity. Sera from immunized animals did not show agglutination reaction with RBPT antigen and thus could serve as DIVA (Differentiating Infected from Vaccinated Animals) vaccine. S19Δper mutant displayed more susceptibility to serum complement mediated killing and sensitivity to polymyxin B. Pregnant guinea pigs injected with S19Δper mutant completed full term of pregnancy and did not cause abortion, still birth or birth of weak offspring. S19Δper mutant with intermediate rough phenotype displayed remarkable resemblance to S19 vaccine strain with improved properties of safety, immunogenicity and DIVA capability for control of bovine brucellosis.

Express immunochromatographic detection of antibodies against Brucella abortus in cattle sera based on quantitative photometric registration and modulated cut-off level.

An immunochromatographic test system was developed for rapid detection of the levels of specific IgG antibodies to Brucella abortus lipopolysaccharide, as a tool for diagnosis of brucellosis in cattle. The pilot test strips were examined using blood sera from sick (78 samples) and healthy (35 samples) cows. The results obtained by immunochromatographic assay, using a portable optical densitometer for digital video detection, correlate well with the results obtained by immunoenzyme assay and are in agreement with the results of the disease diagnosis. The new test system allows detection of antibodies within 10 min and can be proposed as an alternative to the methods available for serodiagnosis of brucellosis.

G1-arrested newborn cells are the predominant infectious form of the pathogen Brucella abortus.

Several intracellular pathogens, such as Brucella abortus, display a biphasic infection process starting with a non-proliferative stage of unclear nature. Here, we study the cell cycle of B. abortus at the single-cell level, in culture and during infection of HeLa cells and macrophages. The localization of segregation and replication loci of the two bacterial chromosomes indicates that, immediately after being engulfed by host-cell endocytic vacuoles, most bacterial cells are newborn. These bacterial cells do not initiate DNA replication for the next 4 to 6 h, indicating a G1 arrest. Moreover, growth is completely stopped during that time, reflecting a global cell cycle block. Growth and DNA replication resume later, although bacteria still reside within endosomal-like compartments. We hypothesize that the predominance of G1-arrested bacteria in the infectious population, and the bacterial cell cycle arrest following internalization, may constitute a widespread strategy among intracellular pathogens to colonize new proliferation niches.

A lysozyme-like protein in Brucella abortus is involved in the early stages of intracellular replication.

Secretion of proteins in Gram-negative bacteria is a high-energy-consuming process that requires translocation across two membranes and a periplasmic space composed of a mesh-like layer, the peptidoglycan. To achieve this, bacteria have evolved complex secretion systems that cross these barriers, and in many cases there are specific peptidoglycanases that degrade the peptidoglycan to allow the proper assembly of the secretion machinery. We describe here the identification and characterization of a muramidase in Brucella abortus that participates in the intracellular multiplication in professional and nonprofessional phagocytes. We demonstrated that this protein has peptidoglycanase activity, that a strain with a clean deletion of the gene displayed a defect in the early stages of the intracellular multiplication curve, and that this is dependent on the lytic activity. While neither the attachment nor the invasion of the strain was affected, we demonstrated that it had a defect in excluding the lysosomal marker LAMP-1 but not in acquiring the reticulum endoplasmic marker calnexin, indicating that the gene participates in the early stages of the intracellular trafficking but not in the establishment of the replicative niche. Analysis of the assembly status and functionality of the VirB secretion apparatus indicated that the mutant has affected the proper function of this central virulence factor.

Caspase-2-dependent dendritic cell death, maturation, and priming of T cells in response to Brucella abortus infection.

Smooth virulent Brucella abortus strain 2308 (S2308) causes zoonotic brucellosis in cattle and humans. Rough B. abortus strain RB51, derived from S2308, is a live attenuated cattle vaccine strain licensed in the USA and many other countries. Our previous report indicated that RB51, but not S2308, induces a caspase-2-dependent apoptotic and necrotic macrophage cell death. Dendritic cells (DCs) are professional antigen presenting cells critical for bridging innate and adaptive immune responses. In contrast to Brucella-infected macrophages, here we report that S2308 induced higher levels of apoptotic and necrotic cell death in wild type bone marrow-derived DCs (WT BMDCs) than RB51. The RB51 and S2308-induced BMDC cell death was regulated by caspase-2, indicated by the minimal cell death in RB51 and S2308-infected BMDCs isolated from caspase-2 knockout mice (Casp2KO BMDCs). More S2308 bacteria were taken up by Casp2KO BMDCs than wild type BMDCs. Higher levels of S2308 and RB51 cells were found in infected Casp2KO BMDCs compared to infected WT BMDCs at different time points. RB51-infected wild type BMDCs were mature and activated as shown by significantly up-regulated expression of CD40, CD80, CD86, MHC-I, and MHC-II. RB51 induced the production of cytokines TNF-α, IL-6, IFN-γ and IL12/IL23p40 in infected BMDCs. RB51-infected WT BMDCs also stimulated the proliferation of CD4(+) and CD8(+) T cells compared to uninfected WT BMDCs. However, the maturation, activation, and cytokine secretion are significantly impaired in Casp2KO BMDCs infected with RB51 or Salmonella (control). S2308-infected WT and Casp2KO BMDCs were not activated and could not induce cytokine production. These results demonstrated that virulent smooth strain S2308 induced more apoptotic and necrotic dendritic cell death than live attenuated rough vaccine strain RB51; however, RB51, but not its parent strain S2308, induced caspase-2-mediated DC maturation, cytokine production, antigen presentation, and T cell priming.

The histidine kinase PdhS controls cell cycle progression of the pathogenic alphaproteobacterium Brucella abortus.

Bacterial differentiation is often associated with the asymmetric localization of regulatory proteins, such as histidine kinases. PdhS is an essential and polarly localized histidine kinase in the pathogenic alphaproteobacterium Brucella abortus. After cell division, PdhS is asymmetrically segregated between the two sibling cells, highlighting a differentiation event. However, the function(s) of PdhS in the B. abortus cell cycle remains unknown. We used an original approach, the pentapeptide scanning mutagenesis method, to generate a thermosensitive allele of pdhS. We report that a B. abortus strain carrying this pdhS allele displays growth arrest and an altered DivK-yellow fluorescent protein (YFP) polar localization at the restrictive temperature. Moreover, the production of a nonphosphorylatable PdhS protein or truncated PdhS proteins leads to dominant-negative effects by generating morphological defects consistent with the inhibition of cell division. In addition, we have used a domain mapping approach combined with yeast two-hybrid and fluorescence microscopy methods to better characterize the unusual PdhS sensory domain. We have identified a fragment of the PdhS sensory domain required for protein-protein interaction (amino acids [aa] 210 to 434), a fragment sufficient for polar localization (aa 1 to 434), and a fragment (aa 527 to 661) whose production in B. abortus correlates with the generation of cell shape alterations. The data support a model in which PdhS acts as an essential regulator of cell cycle progression in B. abortus and contribute to a better understanding of the differentiation program inherited by the two sibling cells.

Identification of genes contributing to the intracellular replication of Brucella abortus within HeLa and RAW 264.7 cells.

Brucella abortus, the causative agent of brucellosis, can survive and replicate within host cells. Understanding bacterial virulence factors and bacteria-host cell interactions is critical for controlling brucellosis, yet very little is known about the virulence strategies and signaling pathways activated in phagocytes during infection to ensure their growth and survival. B. abortus was mutagenized by mini-Tn5Km2 transposon mutagenesis to identify virulence genes related to the internalization and intracellular replication of the bacteria. Of the total 2300 mutants used to infect HeLa cells, 23 mutants defective for intercellular growth and the mutated genes were identified. Sequence analysis of DNA flanking the transposon showed various insertion sites in bacterial genes that might be associated with virulence, including genes associated with lipoproteins, amino acid metabolism, translation, transcription, carbohydrate transport, coenzyme transport, inorganic ion transport, energy metabolism, membrane transport, and cell wall/membrane biogenesis. Moreover, mutants were classified into class I, class II and class III as higher, similar, and lower internalization, respectively, into HeLa cells. Furthermore, defective mutants for intracellular growth in HeLa cells were found to be defective in RAW 264.7 cells. Taken together, we suggest that the identified virulence associated genes might contribute to the intracellular growth and survival of B. abortus in phagocytes.

The alkylation response protein AidB is localized at the new poles and constriction sites in Brucella abortus.

BACKGROUND: Brucella abortus is the etiological agent of a worldwide zoonosis called brucellosis. This alpha-proteobacterium is dividing asymmetrically, and PdhS, an essential histidine kinase, was reported to be an old pole marker. RESULTS: We were interested to identify functions that could be recruited to bacterial poles. The Brucella ORFeome, a collection of cloned predicted coding sequences, was placed in fusion with yellow fluorescent protein (YFP) coding sequence and screened for polar localizations in B. abortus. We report that AidB-YFP was systematically localized to the new poles and at constrictions sites in B. abortus, either in culture or inside infected HeLa cells or RAW264.7 macrophages. AidB is an acyl-CoA dehydrogenase (ACAD) homolog, similar to E. coli AidB, an enzyme putatively involved in destroying alkylating agents. Accordingly, a B. abortus aidB mutant is more sensitive than the wild-type strain to the lethality induced by methanesulphonic acid ethyl ester (EMS). The exposure to EMS led to a very low frequency of constriction events, suggesting that cell cycle is blocked during alkylation damage. The localization of AidB-YFP at the new poles and at constriction sites seems to be specific for this ACAD homolog since two other ACAD homologs fused to YFP did not show specific localization. The overexpression of aidB, but not the two other ACAD coding sequences, leads to multiple morphological defects. CONCLUSIONS: Data reported here suggest that AidB is a marker of new poles and constriction sites, that could be considered as sites of preparation of new poles in the sibling cells originating from cell division. The possible role of AidB in the generation or the function of new poles needs further investigation.

The glyceraldehyde-3-phosphate dehydrogenase and the small GTPase Rab 2 are crucial for Brucella replication.

The intracellular pathogen Brucella abortus survives and replicates inside host cells within an endoplasmic reticulum (ER)-derived replicative organelle named the "Brucella-containing vacuole" (BCV). Here, we developed a subcellular fractionation method to isolate BCVs and characterize for the first time the protein composition of its replicative niche. After identification of BCV membrane proteins by 2 dimensional (2D) gel electrophoresis and mass spectrometry, we focused on two eukaryotic proteins: the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the small GTPase Rab 2 recruited to the vacuolar membrane of Brucella. These proteins were previously described to localize on vesicular and tubular clusters (VTC) and to regulate the VTC membrane traffic between the endoplasmic reticulum (ER) and the Golgi. Inhibition of either GAPDH or Rab 2 expression by small interfering RNA strongly inhibited B. abortus replication. Consistent with this result, inhibition of other partners of GAPDH and Rab 2, such as COPI and PKC iota, reduced B. abortus replication. Furthermore, blockage of Rab 2 GTPase in a GDP-locked form also inhibited B. abortus replication. Bacteria did not fuse with the ER and instead remained in lysosomal-associated membrane vacuoles. These results reveal an essential role for GAPDH and the small GTPase Rab 2 in B. abortus virulence within host cells.

Brucella coopts the small GTPase Sar1 for intracellular replication.

The pathogen Brucella abortus resides inside macrophages within a unique, replication-permissive organelle that is derived from the endoplasmic reticulum (ER). Although dependent on the Brucella type IV secretion system VirB, the mechanisms governing the biogenesis of this compartment remain elusive. Here, we investigated a putative role of the early secretory pathway in ER membrane accretion by the Brucella-containing vacuoles (BCVs). We show that BCVs interact with ER exit sites (ERES), and blockade of Sar1 activity, which disrupts ERES, prevents intracellular replication of Brucella. In cells expressing the dominant interfering form Sar1[T39N], BCVs do not acquire ER membranes, suggesting that they are unable to mature into replicative organelles. By contrast, treatments that block subsequent secretory events do not affect bacterial replication. We propose that Sar1-dependent ERES functions, but not subsequent secretory events, are essential for the biogenesis of the Brucella replicative compartment and, thus, bacterial replication. These results assign an essential role for Sar1 in pathogenesis of an intracellular bacterium.

Laboratory maintenance of Brucella abortus.

This unit provides protocols for growth of Brucella abortus on solid or in liquid media and for long-term storage of laboratory stocks. Two issues affecting the culture and storage of isolates of this slow-growing bacterium are emphasized: contamination of cultures and outgrowth of attenuated variants lacking a complete lipopolysaccharide. Laboratories planning to work with B. abortus should be aware that Biosafety Level 3 facilities are required. Furthermore, this organism is classified in the U.S. as a Select Agent, which restricts its use to laboratories registered with the U.S. government's Select Agent programs.

Survival of Brucella abortus strain RB51 lyophilized and as liquid vaccine under different storage conditions.

Brucella abortus strain RB51 (SRB51) is a new cattle vaccine that is approved for use in the U.S. for prevention of brucellosis. At the present time, other countries are implementing or considering the use of SRB51 vaccine in their brucellosis control programs. In the current study, the effect of three stabilizing media, two fill volumes (1 and 3 ml), and three storage temperatures (-25, 4 and 25 degrees C) on the viability of lyophilized SRB51 over a 52 week period was determined. The effects of three concentrations of bacteria (5 x 10(8), 1 x 10(9), or 5 x 10(9) cfu/ml) and two storage temperatures (4 or 25 degrees C) on viability of liquid SRB51 vaccine were also determined. For lyophilized strain RB51 vaccine, fill volume did not influence viability (P> 0.05) during lyophilization. Although fill volume did not influence viability during storage in World Health Organization (WHO) media or media containing both WHO and Lactose Salt (LS) media, 1 ml fill volumes of SRB51 in LS media had greater (P< 0.05) viability when compared to 3 ml fill volumes. Lyophilized SRB51 vaccine stored at 25 degrees C had a more rapid decline in viability (P< 0.05) when compared to vaccine stored at -25 or 4 degrees C. With the exception of the 3-ml fill volumes of LS media, all three stabilizing media were similar in maintaining viability of SRB51 at -25 degrees C storage temperatures. However, when compared to WHO or WHO/LS media, stabilization in LS media was associated with a more rapid decline in viability during storage at 4 or 25 degrees C (P< 0.05). Initial SRB51 concentration in liquid vaccine did not influence (P> 0.05) viability during storage at 4 or 25 degrees C. When compared to liquid SRB51 vaccine stored at 25 degrees C, storage at 4 degrees C was associated with a slower decline in viability (P< 0.05) during 12 weeks of storage. Biochemical and morphological characteristics of SRB51 were stable under the storage conditions utilized in the present study. This study suggests that viability of SRB51 can be readily maintained during storage as a lyophilized or liquid brucellosis vaccine.

Isolation and characterization of the UDP-glucose 4'-epimerase-encoding gene, galE, from Brucella abortus 2308.

The UDP-glucose 4'-epimerase-encoding gene, galE or exoB, was isolated from Brucella abortus 2308 by complementation of an exoB mutant of Sinorhizobium meliloti. Confirmation of the identity was done by constructing an in-frame deletion of 660 bp with galE of the B. abortus genome by marker exchange. The resulting galE mutant lacked UDP-glucose 4'-epimerase activity. This activity was restored by in trans complementation with the intact gene. The B. abortus gal E mutant is not altered in colony morphology compared to wt 2308. The lack of UDP-glucose 4'-epimerase activity in the mutant and PCR analysis strongly suggest that only one copy of galE exists in B. abortus 2308. The galE sequence of B. abortus 2308 is more similar to galE from other animal-inhabiting bacteria than it is to exoB from the Sinorhizobium legume symbionts. We propose that galE in B. abortus evolved by lateral transfer from other animal-inhabiting bacteria rather than from a common ancestor of Brucella and Sinorhizobium.

T lymphocyte mediated protection against facultative intracellular bacteria.

Acquired immunity against intracellular bacteria is T cell dependent. T cells play a major role in protection against intracellular bacteria, but bacterial antigens recognized by T cells have been studied less extensively than bacterial antigens recognized by B cells. Using T lymphocytes from animals immunized against Brucella abortus, we have screened a bacterial genomic library for genes encoding antigens recognized by T cells. Lymphocytes that proliferated to B. abortus proteins were characterized for phenotype and cytokine activity. Bovine and murine lymphocytes recognized common bacterial antigens and possessed similar cytokine profiles, suggesting an analogous immune response in these two animal species. In vivo protection afforded by a particular cell type is dependent on the bacterial antigens presented and mechanisms of antigen presentation. MHC class I and class II gene knockout animals infected with B. abortus have demonstrated that protection to B. abortus is especially dependent on CD8+ T cells. Knowing the cells required for protection, vaccines can be designed to elicit the protective subset of lymphocytes. Currently, we are testing several recombinant B. abortus proteins using different immunization strategies. Finally, bacterial genes activated following intracellular phagocytosis are being examined using a novel, reporter system adapted to B. abortus.

The role of the differential complement fixation test, using rough and smooth brucella antigens, in the anamnestic test.

To assess the ability of the differential complement fixation test to distinguish vaccinal reactors from infected cattle, approximately 1,000 heifers were tested by the complement fixation test (CFT) using rough and smooth brucella antigens, before the injection of 45/20 vaccine and at 3 and 6 or 10 weeks after vaccination. Before vaccination 91.5% of heifers were negative to the rough antigen but 0.6% were positive with high titre (greater than or equal to 128). By 10 weeks after injection of 45/20 vaccine 97.6% of heifers were positive to the rough CF antigen, at greater than or equal to 8, a majority reaching greater than or equal to 128. Nineteen pre-vaccinal reactors to the standard CFT were killed and Brucella abortus was isolated from the tissues of 14. Twenty-six post-vaccinal reactors were killed and B. abortus was isolated from the tissues of 8. In the 22 B. abortus infected animals the differential CFT classified 9 correctly as infected, 5 incorrectly as vaccination reactions and 8 as inconclusive. The differential CF was ineffective in distinguishing titres resulting from vaccination with 45/20 vaccine from those due to infection.