Bacterial Pathogen Brucella Research Articles

Small Molecule Inhibitors against the Bacterial Pathogen Brucella.

Brucellosis remains one of the major zoonotic diseases worldwide. As a causative agent of brucellosis, it has many ways to evade recognition by the immune system, allowing it to replicate and multiply in the host, causing significant harm to both humans and animals. The pathogenic mechanism of Brucella has not been elucidated, making the identification of drug targets from the pathogenic mechanism a challenge. Metalloenzymatic targets and some protein targets unique to Brucella are exploitable in the development of inhibitors against this disease. The development of specific small molecule inhibitors is urgently needed for brucellosis treatment due to the antibiotic resistance of Brucella. This review summarizes the research on small molecule inhibitors of Brucella, which could be instructive for subsequent studies.

Brucella-driven host N-glycome remodeling controls infection

Many powerful methods have been employed to elucidate the global transcriptomic, proteomic, or metabolic responses to pathogen-infected host cells. However, the host glycome responses to bacterial infection remain largely unexplored, and hence, our understanding of the molecular mechanisms by which bacterial pathogens manipulate the host glycome to favor infection remains incomplete. Here, we address this gap by performing a systematic analysis of the host glycome during infection by the bacterial pathogen Brucella spp. that cause brucellosis. We discover, surprisingly, that a Brucella effector protein (EP) Rhg1 induces global reprogramming of the host cell N-glycome by interacting with components of the oligosaccharide transferase complex that controls N-linked protein glycosylation, and Rhg1 regulates Brucella replication and tissue colonization in a mouse model of brucellosis, demonstrating that Brucella exploits the EP Rhg1 to reprogram the host N-glycome and promote bacterial intracellular parasitism, thereby providing a paradigm for bacterial control of host cell infection.

The IRE1α-XBP1 Signaling Axis Promotes Glycolytic Reprogramming in Response to Inflammatory Stimuli.

Immune cells must be able to adjust their metabolic programs to effectively carry out their effector functions. Here, we show that the endoplasmic reticulum (ER) stress sensor Inositol-requiring enzyme 1 alpha (IRE1α) and its downstream transcription factor X box binding protein 1 (XBP1) enhance the upregulation of glycolysis in classically activated macrophages (CAMs). The IRE1α-XBP1 signaling axis supports this glycolytic switch in macrophages when activated by lipopolysaccharide (LPS) stimulation or infection with the intracellular bacterial pathogen Brucella abortus. Importantly, these different inflammatory stimuli have distinct mechanisms of IRE1α activation; while Toll-like receptor 4 (TLR4) supports glycolysis under both conditions, TLR4 is required for activation of IRE1α in response to LPS treatment but not B. abortus infection. Though IRE1α and XBP1 are necessary for maximal induction of glycolysis in CAMs, activation of this pathway is not sufficient to increase the glycolytic rate of macrophages, indicating that the cellular context in which this pathway is activated ultimately dictates the cell's metabolic response and that IRE1α activation may be a way to fine-tune metabolic reprogramming. IMPORTANCE The immune system must be able to tailor its response to different types of pathogens in order to eliminate them and protect the host. When confronted with bacterial pathogens, macrophages, frontline defenders in the immune system, switch to a glycolysis-driven metabolism to carry out their antibacterial functions. Here, we show that IRE1α, a sensor of ER stress, and its downstream transcription factor XBP1 support glycolysis in macrophages during infection with Brucella abortus or challenge with Salmonella LPS. Interestingly, these stimuli activate IRE1α by independent mechanisms. While the IRE1α-XBP1 signaling axis promotes the glycolytic switch, activation of this pathway is not sufficient to increase glycolysis in macrophages. This study furthers our understanding of the pathways that drive macrophage immunometabolism and highlights a new role for IRE1α and XBP1 in innate immunity.

Characterizing the transport and utilization of the neurotransmitter GABA in the bacterial pathogen Brucella abortus.

The neurotransmitter gamma-aminobutyric acid (GABA) is the most abundant inhibitory neurotransmitter in the human brain; however, it is becoming more evident that this non-proteinogenic amino acid plays multiple physiological roles in biology. In the present study, the transport and function of GABA is studied in the highly infectious intracellular bacterium Brucella abortus. The data show that 3H-GABA is imported by B. abortus under nutrient limiting conditions and that the small RNAs AbcR1 and AbcR2 negatively regulate this transport. A specific transport system, gts, is responsible for the transport of GABA as determined by measuring 3H-GABA transport in isogenic deletion strains of known AbcR1/2 regulatory targets; however, this locus is unnecessary for Brucella infection in BALB/c mice. Similar assays revealed that 3H-GABA transport is uninhibited by the 20 standard proteinogenic amino acids, representing preference for the transport of 3H-GABA. Metabolic studies did not show any potential metabolic utilization of GABA by B. abortus as a carbon or nitrogen source, and RNA sequencing analysis revealed limited transcriptional differences between B. abortus 2308 with or without exposure to GABA. While this study provides evidence for GABA transport by B. abortus, questions remain as to why and when this transport is utilized during Brucella pathogenesis.

IFN-γ-dependent nitric oxide suppresses Brucella-induced arthritis by inhibition of inflammasome activation.

Brucellosis, caused by the intracellular bacterial pathogen Brucella, is a globally important zoonotic disease for which arthritis is the most common focal complication in humans. Wild-type mice infected systemically with Brucella typically do not exhibit arthritis, but mice lacking IFN-γ develop arthritis regardless of the route of Brucella infection. Here, we investigated mechanisms by which IFN-γ suppresses Brucella-induced arthritis. Several cell types, including innate lymphoid cells, contributed to IFN-γ production and suppression of joint swelling. IFN-γ deficiency resulted in elevated joint IL-1β levels, and severe joint inflammation that was entirely inflammasome dependent, and in particular, reliant on the NLRP3 inflammasome. IFN-γ was vital for induction of the nitric oxide producing enzyme, iNOS, in infected joints, and nitric oxide directly inhibited IL-1β production and inflammasome activation in Brucella-infected macrophages in vitro. During in vivo infection, iNOS deficiency resulted in an increase in IL-1β and inflammation in Brucella-infected joints. Collectively, this data indicate that IFN-γ prevents arthritis both by limiting Brucella infection, and by inhibiting excessive inflammasome activation through the induction of nitric oxide.

Caspase-1 and Caspase-11 Mediate Pyroptosis, Inflammation, and Control of Brucella Joint Infection.

Brucellosis, caused by the intracellular bacterial pathogen Brucella, is a zoonotic disease for which arthritis is the most common focal complication in humans. Here we investigated the role of inflammasomes and their effectors, including interleukin-1 (IL-1), IL-18, and pyroptosis, on inflammation and control of infection during Brucella-induced arthritis. Early in infection, both caspase-1 and caspase-11 were found to initiate joint inflammation and proinflammatory cytokine production. However, by 1 week postinfection, caspase-1 and caspase-11 also contributed to control of Brucella joint infection. Inflammasome-dependent restriction of Brucella joint burdens did not require AIM2 (absent in melanoma 2) or NLRP3 (NLR family, pyrin domain containing 3). IL-1R had a modest effect on Brucella-induced joint swelling, but mice lacking IL-1R were not impaired in their ability to control infection of the joint by Brucella In contrast, IL-18 contributed to the initiation of joint swelling and control of joint Brucella infection. Caspase1/11-dependent cell death was observed in vivo, and in vitro studies demonstrated that both caspase-1 and caspase-11 induce pyroptosis, which limited Brucella infection in macrophages. Brucella lipopolysaccharide alone was also able to induce caspase-11-dependent pyroptosis. Collectively, these data demonstrate that inflammasomes induce inflammation in an IL-18-dependent manner and that inflammasome-dependent IL-18 and pyroptosis restrict Brucella infection.

Inflammasomes confer protection via IL-18 and pyroptosis, and are negatively regulated by IFN-γ-dependent nitric oxide during Brucella infection

Abstract Brucellosis, caused by the intracellular bacterial pathogen Brucella, is a zoonotic disease for which arthritis is the most common focal complication in humans. Here we show caspase-1 (casp1) and caspase-11 (casp11) initiate Brucella-induced joint inflammation, but later in infection also restrict Brucella replication. In contrast, AIM2 and NLRP3 were dispensable for control of joint infection by Brucella. Both IL-1 and IL-18 promoted joint inflammation, however only IL-18 contributed to protection against Brucella infection of the joint. In vitro studies demonstrated that casp1 and casp11 both induce pyroptosis which limited Brucella infection in macrophages. While IFN-γ production in part required casp1/11 and IL-18, casp1/11-dependent clearance of Brucella was only partially IFN-γ dependent. However, IFN-γ deficiency resulted in severe inflammation that was entirely inflammasome dependent, and in particular reliant on NLRP3. IFN-γ was vital for induction of the nitric oxide (NO) producing enzyme, iNOS, in infected joints, and NO inhibited casp1 activation in Brucella-infected macrophages in vitro. During Brucella infection in vivo, iNOS inhibition exacerbated arthritis, and administration of a NO donor reduced joint inflammation in IFN-γ-deficient mice. Collectively these data demonstrate inflammasomes induce early inflammation in an IL-18 and IL-1 dependent manner, and inflammasome-dependent IL-18 and pyroptosis restrict Brucella burdens in the joint. Moreover, IFN-γ reduces inflammation by inhibiting excessive casp1 activation through production of NO. Thus NO donors, in combination with antibiotics, may be an effective therapy for treating Brucella-induced inflammation.

Inflammasomes promote the initiation of Brucella induced arthritis in an IL-18 dependent manner and mediate Brucella clearance independently of IFN-γ

Abstract Brucellosis, caused by the intracellular bacterial pathogen Brucella, is a zoonotic disease for which infection of the joints is the most common focal complication in humans. The purpose of this study was to identify if Brucella activation of inflammasomes and in turn caspase1/11 activation is responsible for inducing arthritis. Here we report caspase-1/11−/− (casp1/11−/−) mice had reduced swelling, inflammation and inflammatory cytokine levels early in infection relative to wild-type “WT” mice. Later in infection, joint inflammation was similar between WT and casp1/11−/− mice, but bacterial loads and swelling were greater in casp1/11−/− joints. While IL-1Rdeficiency reduced joint swelling, this effect occurred later and was not of the same magnitude as casp1/11 deficiency, indicating an IL-1 independent role of casp1/11. Casp1/11 induced inflammation was partially dependent on IL-18, and IFN-γ levels were reduced in both casp1/11−/− and IL-18-deficient mice relative to WT mice early in infection. However, while IFN-γ and casp1/11 both played a role in controlling joint Brucella burdens, an additive effect of these deficiencies was observed indicating that the protective effects of IFN-γ and casp1/11 on bacterial clearance are not interdependent. Enhanced arthritis and musculoskeletal inflammation was observed in IFN-γ-deficient, relative to WT mice. However, neutralization of IFN-γ in casp1/11−/− mice did not result in enhanced inflammation. Collectively these data demonstrate casp1/11 induces early inflammation in an IL-18 dependent manner. Moreover, there is an IFN-γ independent role of casp1/11 in joint Brucella clearance, and casp1/11 is responsible for enhanced inflammation observed in IFN-γ-deficient mice.

Postreplication Roles of the Brucella VirB Type IV Secretion System Uncovered via Conditional Expression of the VirB11 ATPase.

ABSTRACTBrucella abortus, the bacterial agent of the worldwide zoonosis brucellosis, primarily infects host phagocytes, where it undergoes an intracellular cycle within a dedicated membrane-bound vacuole, the Brucella-containing vacuole (BCV). Initially of endosomal origin (eBCV), BCVs are remodeled into replication-permissive organelles (rBCV) derived from the host endoplasmic reticulum, a process that requires modulation of host secretory functions via delivery of effector proteins by the Brucella VirB type IV secretion system (T4SS). Following replication, rBCVs are converted into autophagic vacuoles (aBCVs) that facilitate bacterial egress and subsequent infections, arguing that the bacterium sequentially manipulates multiple cellular pathways to complete its cycle. The VirB T4SS is essential for rBCV biogenesis, as VirB-deficient mutants are stalled in eBCVs and cannot mediate rBCV biogenesis. This has precluded analysis of whether the VirB apparatus also drives subsequent stages of the Brucella intracellular cycle. To address this issue, we have generated a B. abortus strain in which VirB T4SS function is conditionally controlled via anhydrotetracycline (ATc)-dependent complementation of a deletion of the virB11 gene encoding the VirB11 ATPase. We show in murine bone marrow-derived macrophages (BMMs) that early VirB production is essential for optimal rBCV biogenesis and bacterial replication. Transient expression of virB11 prior to infection was sufficient to mediate normal rBCV biogenesis and bacterial replication but led to T4SS inactivation and decreased aBCV formation and bacterial release, indicating that these postreplication stages are also T4SS dependent. Hence, our findings support the hypothesis of additional, postreplication roles of type IV secretion in the Brucella intracellular cycle.

Generation and characterization of β1,2-gluco-oligosaccharide probes from Brucella abortus cyclic β-glucan and their recognition by C-type lectins of the immune system.

The β1,2-glucans produced by bacteria are important in invasion, survival and immunomodulation in infected hosts be they mammals or plants. However, there has been a lack of information on proteins which recognize these molecules. This is partly due to the extremely limited availability of the sequence-defined oligosaccharides and derived probes for use in the study of their interactions. Here we have used the cyclic β1,2-glucan (CβG) of the bacterial pathogen Brucella abortus, after removal of succinyl side chains, to prepare linearized oligosaccharides which were used to generate microarrays. We describe optimized conditions for partial depolymerization of the cyclic glucan by acid hydrolysis and conversion of the β1,2-gluco-oligosaccharides, with degrees of polymerization 2–13, to neoglycolipids for the purpose of generating microarrays. By microarray analyses, we show that the C-type lectin receptor DC-SIGNR, like the closely related DC-SIGN we investigated earlier, binds to the β1,2-gluco-oligosaccharides, as does the soluble immune effector serum mannose-binding protein. Exploratory studies with DC-SIGN are suggestive of the recognition also of the intact CβG by this receptor. These findings open the way to unravelling mechanisms of immunomodulation mediated by β1,2-glucans in mammalian systems.

Inhibition studies of bacterial, fungal and protozoan β-class carbonic anhydrases with Schiff bases incorporating sulfonamide moieties

A series of new Schiff bases derived from sulfanilamide, 3-fluorosulfanilamide or 4-(2-aminoethyl)-benzenesulfonamide containing either a hydrophobic or a hydrophilic tail, have been investigated as inhibitors of three β-carbonic anhydrases (CA, EC 4.2.1.1) from three different microorganisms. Their antifungal, antibacterial and antiprotozoan activities have been determined against the pathogenic fungus Cryptococcus neoformans, the bacterial pathogen Brucella suis and the protozoan parasite Leishmania donovani chagasi, responsible for Leishmaniasis.The results of these inhibition studies show that all three enzymes were efficiently inhibited by the Schiff base sulfonamides with KI values in the nanomolar or submicromolar range, depending on the nature of the tail, coming from the aryl/heteroaryl moiety present in the starting aldehyde employed in the synthesis. Furthermore, the compounds hereby investigated revealed high β-CAs selectivity over the ubiquitous, physiologically relevant and off-target human isoforms (CA I and II) and to be more potent as antifungal and antibacterial than as antiprotozoan potential drugs.

The Brucella TIR domain containing proteins BtpA and BtpB have a structural WxxxE motif important for protection against microtubule depolymerisation

BackgroundThe TIR domain-containing proteins BtpA/Btp1/TcpB and BtpB are translocated into host cells by the facultative intracellular bacterial pathogen Brucella. Here, they interfere with Toll like receptor signalling to temper the host inflammatory response. BtpA has also been found to modulate microtubule dynamics. In both proteins we identified a WxxxE motif, previously shown to be an essential structural component in a family of bacterial type III secretion system effectors that modulate host actin dynamics by functioning as guanine nucleotide exchange factors of host GTPases. We analysed a role for the WxxxE motif in association of BtpA and BtpB with the cytoskeleton.ResultsUnlike BtpA, ectopically expressed BtpB did not show a tubular localisation, but was found ubiquitously in the cytoplasm and the nucleus, and often appeared in discrete punctae in HeLa cells. BtpB was able to protect microtubules from drug-induced destabilisation similar to BtpA. The WxxxE motif was important for the ability of BtpA and BtpB to protect microtubules against destabilising drugs. Surprisingly, ectopic expression of BtpA, although not BtpB, in HeLa cells induced the formation of filopodia. This process was invariably dependent of the WxxxE motif. Our recent resolution of the crystal structure of the BtpA TIR domain reveals that the motif positions a glycine residue that has previously been shown to be essential for interaction of BtpA with microtubules.ConclusionsOur results suggest a structural role for the WxxxE motif in the association of BtpA and BtpB with microtubules, as with the WxxxE GEF family proteins where the motif positions an adjacent catalytic loop important for interaction with specific Rho GTPases. In addition, the ability of ectopically expressed BtpA to induce filopodia in a WxxxE-dependent manner suggests a novel property for BtpA. A conserved WxxxE motif is found in most bacterial and several eukaryotic TIR domain proteins. Despite the similarity between ectopically expressed BtpA and WxxxE GEFs to modulate host actin dynamics, our results suggest that BtpA is not part of this WxxxE GEF family. The WxxxE motif may therefore be a more common structural motif than thus far described. BtpA may provide clues to cross-talk between the TLR and GTPase signalling pathways.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-014-0053-y) contains supplementary material, which is available to authorized users.

Inhibition of beta-carbonic anhydrases from the bacterial pathogen Brucella suis with inorganic anions

The bacterial pathogen Brucella suis encodes two carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the β-class, bsCA1 and bsCA2, which are crucial for its life cycle. Sulfonamides, strong inhibitors of these enzymes, were shown to block the growth of the pathogen in vitro. Here we report the inhibition of these two CAs by inorganic and complex anions and other molecules interacting with zinc proteins, such as sulfamide, sulfamic acid, and phenylboronic/arsonic acids. The enzyme bsCA1 was inhibited in the low micromolar range by sulfamide, sulfamic acid, phenylboronic/arsonic acid, and in the submillimolar range by diethyldithiocarbamate. Isoform bsCA2 generally showed a stronger inhibition with most of these anions, with several low micromolar and many submillimolar inhibitors detected. Micromolar inhibition against bsCA2 was observed for sulfamide and sulfamic acid, whereas diethyldithiocarbamate, perruthenate, pyrovanadate, tellurate and phenylarsonic acid showed inhibition constants in the range of 0.29–1.52mM. These inhibitors may be used as leads for developing anti-Brucella agents with a diverse mechanism of action compared to clinically used antibiotics.

Brucella “Hitches a Ride” with Autophagy

Autophagy involves lysosomal-mediated degradation of cellular components and contributes to host immunity. Some pathogens avoid autophagy-mediated killing, while others exploit it to acquire host cell nutrients. Starr etal. reveal that the intracellular bacterial pathogen Brucella abortus can "hitch a ride" with autophagy, subverting autophagy machinery to spread from cell to cell (Starr etal., 2012).

A new β-carbonic anhydrase from Brucella suis, its cloning, characterization, and inhibition with sulfonamides and sulfamates, leading to impaired pathogen growth

A β-carbonic anhydrase (CA, EC 4.2.1.1) from the bacterial pathogen Brucella suis, bsCA II, has been cloned, purified, and characterized kinetically. bsCA II showed high catalytic activity for the hydration of CO 2 to bicarbonate, with a k cat of 1.1 × 10 6, and k cat/ K m of 8.9 × 10 7 M −1 s −1. A panel of sulfonamides and sulfamates have been investigated for inhibition of this enzyme. All types of activities, from the low nanomolar to the micromolar, have been detected for these derivatives, which showed inhibition constants in the range of 7.3 nM–8.56 μM. The best bsCA II inhibitors were some glycosylated sulfanilamides, aliphatic sulfamates, and halogenated sulfanilamides, with inhibition constants of 7.3–87 nM. Some of these dual inhibitors of bsCA I and II, also inhibited bacterial growth in vitro, in liquid cultures. These promising data on live bacteria allow us to propose bacterial β-CA inhibition as an approach for obtaining anti-infective agents with a new mechanism of action compared to classical antibiotics.

PdhS, an Old-Pole-Localized Histidine Kinase, Recruits the Fumarase FumC in Brucella abortus

The bacterial pathogen Brucella abortus was recently demonstrated to recruit the essential cytoplasmic histidine kinase PdhS to its old pole. Here, we report identification of the fumarase FumC as a specific partner for the N-terminal "sensing" domain of PdhS, using an ORFeome-based yeast two-hybrid screen. We observed that FumC and PdhS colocalize at the old pole of B. abortus, while the other fumarase FumA is not polarly localized. FumC is not required for PdhS localization, and polar FumC localization is not FumA dependent. FumC homologs are not polarly localized in Sinorhizobium meliloti and Caulobacter crescentus, suggesting that polar recruitment of FumC by PdhS is evolutionarily recent.

Inhibition studies of a β-carbonic anhydrase from Brucella suis with a series of water soluble glycosyl sulfanilamides

A β-carbonic anhydrase (CA, EC 4.2.1.1) from the bacterial pathogen Brucella suis, bsCA 1, has been cloned, purified characterized kinetically and for inhibition with a series of water soluble glycosylated sulfanilamides. bsCA 1 has appreciable activity as catalyst for the hydration of CO 2 to bicarbonate, with a k cat of 6.4 × 10 5 s −1 and k cat/ K m of 3.9 × 10 7 M −1 s −1. All types of inhibitory activities have been detected, with K Is in the range of 8.9–110 nM. The best bsCA 1 inhibitor were the galactose and ribose sulfanilamides, with inhibition constants of 8.9–9.2 nM. Small structural changes in the sugar moiety led to dramatic differences of enzyme inhibitory activity for this series of compounds. One of the tested glycosylsulfonamides and acetazolamide significantly inhibited the growth of the bacteria in cell cultures.

Cloning, Characterization, and Inhibition Studies of a β-Carbonic Anhydrase from Brucella suis

A beta-carbonic anhydrase (CA, EC 4.2.1.1) from the bacterial pathogen Brucella suis, bsCA 1, has been cloned, purified, and characterized kinetically. bsCA 1 has appreciable activity as catalyst for the hydration of CO(2) to bicarbonate, with a k(cat) of 6.4 x 10(5) s(-1) and k(cat)/K(m) of 3.9 x 10(7) M(-1).s(-1). A panel of 38 sulfonamides and one sulfamate have been investigated for inhibition of this new beta-CA. All types of activities have been detected, with K(I)s in the range of 17 nM to 5.87 microM. The best bsCA 1 inhibitors were ethoxzolamide (17 nM), celecoxib (18 nM), dorzolamide (21 nM), valdecoxib, and sulpiride (19 nM). Whether bsCA 1 inhibitors may have application in the fight against brucellosis, an endemic disease and the major bacterial zoonosis, producing debilitating infection in humans and animals, warrants further studies.

Analysis of TenBrucellaGenomes Reveals Evidence for Horizontal Gene Transfer Despite a Preferred Intracellular Lifestyle

The facultative intracellular bacterial pathogen Brucella infects a wide range of warm-blooded land and marine vertebrates and causes brucellosis. Currently, there are nine recognized Brucella species based on host preferences and phenotypic differences. The availability of 10 different genomes consisting of two chromosomes and representing six of the species allowed for a detailed comparison among themselves and relatives in the order Rhizobiales. Phylogenomic analysis of ortholog families shows limited divergence but distinct radiations, producing four clades as follows: Brucella abortus-Brucella melitensis, Brucella suis-Brucella canis, Brucella ovis, and Brucella ceti. In addition, Brucella phylogeny does not appear to reflect the phylogeny of Brucella species' preferred hosts. About 4.6% of protein-coding genes seem to be pseudogenes, which is a relatively large fraction. Only B. suis 1330 appears to have an intact beta-ketoadipate pathway, responsible for utilization of plant-derived compounds. In contrast, this pathway in the other species is highly pseudogenized and consistent with the "domino theory" of gene death. There are distinct shared anomalous regions (SARs) found in both chromosomes as the result of horizontal gene transfer unique to Brucella and not shared with its closest relative Ochrobactrum, a soil bacterium, suggesting their acquisition occurred in spite of a predominantly intracellular lifestyle. In particular, SAR 2-5 appears to have been acquired by Brucella after it became intracellular. The SARs contain many genes, including those involved in O-polysaccharide synthesis and type IV secretion, which if mutated or absent significantly affect the ability of Brucella to survive intracellularly in the infected host.

Enhanced efficacy of DNA vaccines against an intracellular bacterial pathogen by genetic adjuvants.

After 200 years of practice, vaccinology has proved to be very effective in preventing infectious diseases. However, several human and animal pathogens exist for which vaccines need to be improved or simply have not yet been discovered. The era of molecular genetic has given a new breath for vaccine development with the achievement of the "Third Generation of Vaccines": the DNA vaccine. In this article, we reviewed strategies that have been used to improve and modulate the immune response induced by DNA vaccines, using as a model the intracellular bacterial pathogen Brucella abortus. First, we described different approaches used to isolate and to identify genes that encode potential immunogens. Secondly, we reported the use of cytokine genes and genetic adjuvants that could improve the immunogenicity of target genes. And finally, we discussed the "Expression Library Immunization"-(ELI) strategy and the recent results obtained against Brucella abortus infection.