Intracellular Survival Of Brucella Research Articles

Brucella Manipulates Host Cell Ferroptosis to Facilitate Its Intracellular Replication and Egress in RAW264.7 Macrophages.

Brucella virulence relies on its successful intracellular life cycle. Modulating host cell death is a strategy for Brucella to survive and replicate intracellularly. Ferroptosis is a novel regulated cell death characterized by iron-triggered excessive lipid peroxidation, which has been proven to be associated with pathogenic bacteria infection. Thus, we attempted to explore if smooth-type Brucella infection triggers host cell ferroptosis and what role it plays in Brucella infection. We assessed the effects of Brucella infection on the lactate dehydrogenase release and lipid peroxidation levels of RAW264.7 macrophages; subsequently, we determined the effect of Brucella infection on the expressions of ferroptosis defense pathways. Furthermore, we determined the role of host cell ferroptosis in the intracellular replication and egress of Brucella. The results demonstrated that Brucella M5 could induce ferroptosis of macrophages by inhibiting the GPX4-GSH axis at the late stage of infection but mitigated ferroptosis by up-regulating the GCH1-BH4 axis at the early infection stage. Moreover, elevating host cell ferroptosis decreased Brucella intracellular survival and suppressing host cell ferroptosis increased Brucella intracellular replication and egress. Collectively, Brucella may manipulate host cell ferroptosis to facilitate its intracellular replication and egress, extending our knowledge about the underlying mechanism of how Brucella completes its intracellular life cycle.

A genome-wide CRISPR screen identified host genes essential for intracellular Brucella survival.

Brucella is a zoonotic intracellular bacterium that poses threats to human health and economic security. Intracellular infection is a hallmark of the agent Brucella and a primary cause of distress, through which the bacterium regulates the host intracellular environment to promote its own colonization and replication, evading host immunity and pharmaceutical killing. Current studies of Brucella intracellular processes are typically premised on bacterial phenotype such as intracellular bacterial survival, followed by biochemical or molecular biological approaches to reveal detailed mechanisms. While such processes can deepen the understanding of Brucella-host interaction, the insights into host alterations in infection would be easily restricted to known pathways. In the current study, we applied CRISPR Cas9 screen to identify host genes that are most affected by Brucella infection on cell viability at the genomic level. As a result of CRISPR screening, we firstly identified that knockout of the negatively selected genes GOLGA6L6, DEFB103B, OR4F29, and ERCC6 attenuate the viability of both the host cells and intracellular Brucella, suggesting these genes to be potential therapeutic targets for Brucella control. In particular, knockout of DEFB103B diminished Brucella intracellular survival by altering host cell autophagy. Conversely, knockout of positive screening genes promoted intracellular proliferation of Brucella. In summary, we screened host genes at the genomic level throughout Brucella infection, identified host genes that are previously not recognized to be involved in Brucella infection, and provided targets for intracellular infection control.IMPORTANCEBrucella is a Gram-negative bacterium that infects common mammals causing arthritis, myalgia, neuritis, orchitis, or miscarriage and is difficult to cure with antibiotics due to its intracellular parasitism. Therefore, unraveling the mechanism of Brucella-host interactions will help controlling Brucella infections. CRISPR-Cas9 is a gene editing technology that directs knockout of individual target genes by guided RNA, from which genome-wide gene-knockout cell libraries can be constructed. Upon infection with Brucella, the cell library would show differences in viability as a result of the knockout and specific genes could be revealed by genomic DNA sequencing. As a result, genes affecting cell viability during Brucella infection were identified. Further testing of gene function may reveal the mechanisms of Brucella-host interactions, thereby contributing to clinical therapy.

Erythronate utilization activates VdtR regulating its metabolism to promote Brucella proliferation, inducing abortion in mice.

Brucella is a facultative intracellular pathogen that preferentially colonizes reproductive organs and utilizes erythritol as a preferred carbon source for its survival and proliferation. In this study, we identified a virulence-related DeoR-family transcriptional regulator (VdtR) and an erythronate metabolic pathway responsible for four-carbon acid sugar metabolism of D-erythronate and L-threonate in Brucella. We found that VdtR plays an important role in Brucella intracellular survival and trafficking to the endoplasmic reticulum in RAW 264.7 macrophages and in virulence in a mouse model. More importantly, we found that VdtR negatively regulates the erythronate metabolic pathway to promote extracellular proliferation of Brucella, depending on utilization of D-erythronate, an oxidative product of erythritol in the host. In a pregnant mouse model, the erythronate metabolic pathway was shown to cooperate with erythritol metabolism and play a crucial role in Brucella proliferation in the placenta, inducing placentitis and finally resulting in abortion or stillbirth. Our results demonstrate that, in addition to erythritol, erythronate is a preferred carbon source for Brucella utilization to promote its extracellular proliferation. This discovery updates the information on the preferential colonization of reproductive organs by Brucella and provides a novel insight into the Brucella-associated induction of abortion in pregnant animals. IMPORTANCE Brucella is an intracellular parasitic bacterium causing zoonosis, which is distributed worldwide and mainly characterized by reproductive disorders. Erythritol is found in allantoic fluid, chorion, and placenta of aborted animals, preferentially utilized by Brucella to cause infertility and abortion. However, the erythritol metabolism-defected mutant was unable to function as a vaccine strain due to its residual virulence. Here, we found that erythronate, an oxidative product of erythritol in the host, was also preferentially utilized by Brucella relying on the function of a deoxyribonucleoside regulator-family transcriptional regulator VdtR. Erythronate utilization activates VdtR regulation of the erythronate metabolic pathway to promote Brucella extracellular proliferation, inducing placentitis/abortion in mice. Double mutations on Brucella erythritol and D-erythronate metabolisms significantly reduced bacterial virulence. This study revealed a novel mechanism of Brucella infection-induced abortion, thus providing a new clue for the study of safer Brucella attenuated vaccines.

Brucella induces heme oxygenase-1 expression to promote its infection.

Brucellosis is a zoonotic and contagious infectious disease caused by Brucella spp, which causes substantial economic losses to animal husbandry and leads to severe public health problems. Brucella have evolved multiple strategies to escape host immunity and survive within host cells. Elucidating the immune evasion strategies during Brucella infection will facilitate the control of brucellosis. The host enzyme, heme oxygenase-1 (HO-1), is a multifunctional protein that functions during inflammatory diseases and microbial infections. However, how HO-1 functions during Brucella infection is rarely studied. In this study, we evaluated the role of HO-1 during Brucella infection. We found that Brucella infection induced HO-1 expression in macrophages. We further showed that HO-1 was regulated by PI3K, AMPK kinase, and nuclear erythroid-related factor 2 (Nrf2) in macrophages. Interestingly, knocking out HO-1 or inhibiting the activity of HO-1 significantly decreased Brucella intracellular growth. Inducing the expression of HO-1 by treatment with CoPP promoted Brucella intracellular growth. Mechanistic analyses indicated that the effect of HO-1 was not meditated by HO-1 metabolites, but by decreasing the production of reactive oxygen species (ROS), TNF-α, and IL-1β. Moreover, Brucella induced HO-1 expression in bone marrow-derived macrophages (BMDMs) and mice. When the expression of HO-1 was knocked down in BMDMs, the intracellular survival of Brucella was reduced. Furthermore, the induction of HO-1 by CoPP significantly increased bacterial loads in vivo. Thus, we demonstrated that Brucella induced HO-1 expression to promote its survival and growth in vitro and in vivo. This study also identified HO-1 as a novel innate immune evasion factor during Brucella infection.

Nucleomodulin BspJ as an effector promotes the colonization of Brucella abortus in the host.

Background Brucella infection induces brucellosis, a zoonotic disease. The intracellular circulation process and virulence of Brucella mainly depend on its type IV secretion system (T4SS) expressing secretory effectors. Secreted protein BspJ is a nucleomodulin of Brucella that invades the host cell nucleus. BspJ mediates host energy synthesis and apoptosis through interaction with proteins. However, the mechanism of BspJ as it affects the intracellular survival of Brucella remains to be clarified.ObjectivesTo verify the functions of nucleomodulin BspJ in Brucella's intracellular infection cycles.MethodsConstructed Brucella abortus BspJ gene deletion strain (B. abortus ΔBspJ) and complement strain (B. abortus pBspJ) and studied their roles in the proliferation of Brucella both in vivo and in vitro.ResultsBspJ gene deletion reduced the survival and intracellular proliferation of Brucella at the replicating Brucella-containing vacuoles (rBCV) stage. Compared with the parent strain, the colonization ability of the bacteria in mice was significantly reduced, causing less inflammatory infiltration and pathological damage. We also found that the knockout of BspJ altered the secretion of cytokines (interleukin [IL]-6, IL-1β, IL-10, tumor necrosis factor-α, interferon-γ) in host cells and in mice to affect the intracellular survival of Brucella.ConclusionsBspJ is extremely important for the circulatory proliferation of Brucella in the host, and it may be involved in a previously unknown mechanism of Brucella's intracellular survival.

Brucella Infection Regulates Thioredoxin-Interacting Protein Expression to Facilitate Intracellular Survival by Reducing the Production of Nitric Oxide and Reactive Oxygen Species.

Thioredoxin-interacting protein (TXNIP) is a multifunctional protein that functions in tumor suppression, oxidative stress, and inflammatory responses. However, how TXNIP functions during microbial infections is rarely reported. In this study, we demonstrate that Brucella infection decreased TXNIP expression to promote its intracellular growth in macrophages by decreasing the production of NO and reactive oxygen species (ROS). Following Brucella abortus infection, TXNIP knockout RAW264.7 cells produced significantly lower levels of NO and ROS, compared with wild-type RAW264.7 cells. Inducible NO synthase (iNOS) inhibitor treatment reduced NO levels, which resulted in a dose-dependent restoration of TXNIP expression, demonstrating that the expression of TXNIP is regulated by NO. In addition, the expression of iNOS and the production of NO were dependent on the type IV secretion system of Brucella Moreover, Brucella infection reduced TXNIP expression in bone marrow-derived macrophages and mouse lung and spleen. Knocked down of the TXNIP expression in bone marrow-derived macrophages increased intracellular survival of Brucella These findings revealed the following: 1) TXNIP is a novel molecule to promote Brucella intracellular survival by reducing the production of NO and ROS; 2) a negative feedback-regulation system of NO confers protection against iNOS-mediated antibacterial effects. The elucidation of this mechanism may reveal a novel host surveillance pathway for bacterial intracellular survival.

Adenosine receptor Adora2b antagonism attenuates Brucella abortus 544 infection in professional phagocyte RAW 264.7 cells and BALB/c mice

Brucella as a stealthy intracellular pathogen avoids activation of innate immune response. Here we investigated the contribution of an adenosine receptor, Adora2b, during Brucella infection in professional phagocyte RAW 264.7 cells and in a murine model. Adora2b-deficient cells showed attenuated Brucella internalization and intracellular survival with enhanced release of IL-6, TNF-α, IL-12 and MCP-1. In addition, blockade of Adora2b using MRS 1754 treatment in mice resulted in increased total weight of the spleens but suppressed bacterial burden in these organs accompanied by elevated levels of IL-6, IFN-γ, TNF-α, IL-12 and MCP-1, while reduced IL-10. Overall, we proposed that the Adora2b participates in the successful phagocytic pathway and intracellular survival of Brucella in RAW 264.7 cells, and could be a potential therapeutic target for the treatment of acute brucellosis in animals.

A small non-coding RNA facilitates Brucella melitensis intracellular survival by regulating the expression of virulence factor

Brucella species are the causative agents of brucellosis, a worldwide zoonotic disease that affects a broad range of mammals and causes great economic losses. Small regulatory RNAs (sRNAs) are post-transcriptional regulatory molecules that participate in the stress adaptation and pathogenesis of Brucella. In this study, we characterized the role of a novel sRNA, BSR1141, in the intracellular survival and virulence of Brucella melitensis. The results show that BSR1141 was highly induced during host infections and under in vitro stress situations that simulated the conditions encountered within host phagocytes. In addition, a BSR1141 mutant showed reduced survival both under in vitro stress conditions and in mice, confirming the role of BSR1141 in Brucella intracellular survival. Bioinformatic and experimental approaches revealed that BSR1141 affects the expression of many target genes, including the Brucella virulence component virB2. These data indicate that BSR1141 could influence the expression of virB2, which is important for B. melitensis pathogenesis and intracellular survival. This work provides new insight into the mechanism of adaptation to environmental stress and into the pathogenesis of intracellular pathogens.

Interaction between Brucella melitensis 16M and small ubiquitin-related modifier 1 and E2 conjugating enzyme 9 in mouse RAW264.7 macrophages.

Brucella is an intracellular pathogen that invades a host and settles in its immune cells; however, the mechanism of its intracellular survival is unclear. Modification of small ubiquitin-related modifier (SUMO) occurs in many cellular activities. E2 conjugating enzyme 9 (Ubc9) is the only reported ubiquitin-conjugating enzyme that links the SUMO molecule with a target protein. Brucella's intracellular survival mechanism has not been studied with respect to SUMO-related proteins and Ubc9. Therefore, to investigate the relationship between Brucella melitensis 16M and SUMO, we constructed plasmids and cells lines suitable for overexpression and knockdown of SUMO1 and Ubc9 genes. Brucella 16M activated SUMO1/Ubc9 expression in a time-dependent manner, and Brucella 16M intracellular survival was inhibited by SUMO1/Ubc9 overexpression and promoted by SUMO1/Ubc9 depletion. In macrophages, Brucella 16M-dependent apoptosis and immune factors were induced by SUMO1/Ubc9 overexpression and restricted by SUMO1/Ubc9 depletion. We noted no effect on the expressions of SUMO1 and Ubc9 in B. melitensis 16M lipopolysaccharide-prestimulated mouse RAW264.7 macrophages. Additionally, intracellular survival of the 16M△VirB2 mutant was lower than that of Brucella 16M (p < 0.05). VirB2 can affect expression levels of Ubc9, thereby increasing intracellular survival of Brucella in macrophages at the late stage of infection. Collectively, our results demonstrate that B. melitensis 16M may use the VirB IV secretion system of Brucella to interact with SUMO-related proteins during infection of host cells, which interferes with SUMO function and promotes pathogen survival in host cells.

Brucella induces unfolded protein response and inflammatory response via GntR in alveolar macrophages.

Brucella is an intracellular bacterium that causes the zoonosis brucellosis worldwide. Alveolar macrophages (AM) constitute the main cell target of inhaled Brucella. Brucella thwarts immune surveillance and evokes endoplasmic reticulum (ER) stress to replicate in macrophages via virulence factors. The GntR regulators family was concentrated as an important virulence factor in controlling virulence and intracellular survival of Brucella. However, the detailed underlying mechanism for the host-pathogen interaction is poorly understood. In this study the BSS2_II0438 mutant (ΔGntR) was constructed. The type IV secretion system (T4SS) virulence factor genes (VirB2, VirB6, and VirB8) were down-expression in ΔGntR. ΔGntR could infect and proliferate to high titers in GAMs without a significant difference compared with the parental strain. ΔGntR infection increased the expression of ER stress marker genes GRP78, ATF6, and PERK in the early stages of its intracellular cycle but decreased the expression of these genes in the late stages. ΔGntR increased greatly the number of Brucella CFUs in the inactive ER stress state in GAMs. Meanwhile, ΔGntR infection increased the levels of IFN-γ, IL-1β, and TNF-α, indicating ΔGntR could induce the secretion of inflammatory but not anti-inflammatory cytokines IL-10. Taken together, our results clarified the role of the GntR in B. suis. S2 virulence expression and elucidated that GntR is potentially involved in the signaling pathway of the Brucella-induced UPR and inflammatory response in GAMs.

Transcriptional regulator GntR of Brucella abortus regulates cytotoxicity, induces the secretion of inflammatory cytokines and affects expression of the type IV secretion system and quorum sensing system in macrophages

The pathogenic mechanisms of Brucella are still poorly understood. GntR is a transcriptional regulator and plays an important role in the intracellular survival of Brucella. To investigate whether GntR is involved in the cytotoxicity of Brucella abortus (B. abortus), we created a 2308ΔgntR mutant of B. abortus 2308 (S2308). Lactate dehydrogenase (LDH) cytotoxicity assays using a murine macrophage cell line (RAW 264.7) show that high-dose infection with the parental strain produces a high level of cytotoxicity to macrophages, but the 2308ΔgntR mutant exhibits a very low level of cytotoxicity, indicating that mutation of GntR impairs the cytotoxicity of B. abortus to macrophages. After the macrophages are infected with 2308ΔgntR, the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-8 (IL-8) increase and are slightly higher than that for the S2308 infected group, indicating that the 2308ΔgntR mutant could induce the secretion of inflammatory cytokines. The virulence factor detection experiments indicate that genes involved in the type IV secretion system (T4SS) and quorum sensing system (QSS) are down-regulated in 2308ΔgntR. The lower levels of survival of 2308ΔgntR under various stress conditions and the increased sensitivity of 2308ΔgntR to polymyxin B suggest that GntR is a virulence factor and that deletion of gntR reduces of B. abortus to stress conditions. Taken together, our results demonstrate that GntR is involved in the cytotoxicity, virulence and intracellular survival of B. abortus during its infection.

The Rab1 in host cells modulates Brucella intracellular survival and binds to Brucella DnaK protein.

The intracellular pathogen Brucella abortus (B. abortus) survives and replicates inside host cells within the Brucella-containing vacuole, in which membrane contains a small GTPase Rab1. Here, we reported that Rab1 mediates B. abortus intracellular growth. Furthermore, B. abortus DnaK was identified to interact with Rab1 using GST pull-down and mass spectrometry analysis. This interaction was confirmed by co-immunoprecipitation and immunofluorescence. Through DnaK-CyaA fusion protein translocation assay and immunofluorescence confocal microscopy, the B. abortus DnaK was proved to be a virB-dependent translocated substrate.

Macrophage activation induced by Brucella DNA suppresses bacterial intracellular replication via enhancing NO production

Brucella DNA can be sensed by TLR9 on endosomal membrane and by cytosolic AIM2-inflammasome to induce proinflammatory cytokine production that contributes to partially activate innate immunity. Additionally, Brucella DNA has been identified to be able to act as a major bacterial component to induce type I IFN. However, the role of Brucella DNA in Brucella intracellular growth remains unknown. Here, we showed that stimulation with Brucella DNA promote macrophage activation in TLR9-dependent manner. Activated macrophages can suppresses wild type Brucella intracellular replication at early stage of infection via enhancing NO production. We also reported that activated macrophage promotes bactericidal function of macrophages infected with VirB-deficient Brucella at the early or late stage of infection. This study uncovers a novel function of Brucella DNA, which can help us further elucidate the mechanism of Brucella intracellular survival.

Ontology-based representation and analysis of host-Brucella interactions.

BackgroundBiomedical ontologies are representations of classes of entities in the biomedical domain and how these classes are related in computer- and human-interpretable formats. Ontologies support data standardization and exchange and provide a basis for computer-assisted automated reasoning. IDOBRU is an ontology in the domain of Brucella and brucellosis. Brucella is a Gram-negative intracellular bacterium that causes brucellosis, the most common zoonotic disease in the world. In this study, IDOBRU is used as a platform to model and analyze how the hosts, especially host macrophages, interact with virulent Brucella strains or live attenuated Brucella vaccine strains. Such a study allows us to better integrate and understand intricate Brucella pathogenesis and host immunity mechanisms.ResultsDifferent levels of host-Brucella interactions based on different host cell types and Brucella strains were first defined ontologically. Three important processes of virulent Brucella interacting with host macrophages were represented: Brucella entry into macrophage, intracellular trafficking, and intracellular replication. Two Brucella pathogenesis mechanisms were ontologically represented: Brucella Type IV secretion system that supports intracellular trafficking and replication, and Brucella erythritol metabolism that participates in Brucella intracellular survival and pathogenesis. The host cell death pathway is critical to the outcome of host-Brucella interactions. For better survival and replication, virulent Brucella prevents macrophage cell death. However, live attenuated B. abortus vaccine strain RB51 induces caspase-2-mediated proinflammatory cell death. Brucella-associated cell death processes are represented in IDOBRU. The gene and protein information of 432 manually annotated Brucella virulence factors were represented using the Ontology of Genes and Genomes (OGG) and Protein Ontology (PRO), respectively. Seven inference rules were defined to capture the knowledge of host-Brucella interactions and implemented in IDOBRU. Current IDOBRU includes 3611 ontology terms. SPARQL queries identified many results that are critical to the host-Brucella interactions. For example, out of 269 protein virulence factors related to macrophage-Brucella interactions, 81 are critical to Brucella intracellular replication inside macrophages. A SPARQL query also identified 11 biological processes important for Brucella virulence.ConclusionsTo systematically represent and analyze fundamental host-pathogen interaction mechanisms, we provided for the first time comprehensive ontological modeling of host-pathogen interactions using Brucella as the pathogen model. The methods and ontology representations used in our study are generic and can be broadened to study the interactions between hosts and other pathogens.Electronic supplementary materialThe online version of this article (doi:10.1186/s13326-015-0036-y) contains supplementary material, which is available to authorized users.

TceSR two-component regulatory system of Brucella melitensis 16M is involved in invasion, intracellular survival and regulated cytotoxicity for macrophages.

The mechanisms of invasion and intracellular survival of Brucella are still poorly understood. Previous studies showed that the two-component regulatory systems (TCSs) play an important role in the intracellular survival of Brucella. To investigate if TCSs involve in the virulence and cytotoxicity of Brucella melitensis, we introduced a mutation into one of the TCSs in chromosome II in Br. melitensis 16M strain, and generated 16MΔTceSR, a mutant of Br. melitensis 16M strain. In vitro infection experiments using murine macrophage cell line (RAW 264.7) showed that the survival of 16MΔTceSR mutant in macrophages decreased 0·91-log compared with that of wild type Br. melitensis 16M strain at 2 h postinfection, replication of 16MΔTceSR mutant in macrophages was 5·65-log, which was much lower than that wild type strain. Results of lactate dehydrogenase cytotoxicity assays in macrophages demonstrated high dose infection with wide type strain produced high level cytotoxicity to macrophages, but 16MΔTceSR mutant had very low level cytotoxicity, indicating mutation of TCSs impaired the cytotoxicity of Br. melitensis to macrophages. Animal experiments showed that the spleen colonization of 16MΔTceSR was significantly reduced compared with its wild type strains. The lower levels of survival of 16MΔTceSR in various stress conditions suggested that the mutation of the TCSs of Br. melitensis was the causative factor of its reduced resistance to stress conditions. Taken together, our results demonstrated TCS TceSR involves in the intracellular survival, virulence and cytotoxicity of Br. melitensis during its infection. Significance and impact of the study: Two-component systems (TCSs) are predominant bacterial signal transduction mechanisms. The pathogenicity of Brucella is due to its ability to adapt to the intracellular environment including low levels of acidic pH, high-salt and heat shock. TCSs are designed to sense diverse stimuli, transfer signals and enact an appropriate adaptive physiological response. Here, we show that Br. meilitensis TCS TceSR is not only involved in regulation of Br. meilitensis virulence and adaptation of environmental stresses, but also can regulate cytotoxicity in macrophages.

The role of TREM-2 in internalization and intracellular survival of Brucella abortus in murine macrophages

Triggering receptor expressed on myeloid cells-2 (TREM-2) is a cell surface receptor primarily expressed on macrophages and dendritic cells. TREM-2 functions as a phagocytic receptor for bacteria as well as an inhibitor of Toll like receptors (TLR) induced inflammatory cytokines. However, the role of TREM-2 in Brucella intracellular growth remains unknown. To investigate whether TREM-2 is involved in Brucella intracellular survival, we chose bone marrow derived macrophages (BMDMs), in which TREM-2 is stably expressed, as cell model. Colony formation Units (CFUs) assay suggests that TREM-2 is involved in the internalization of Brucella abortus (B. abortus) by macrophages, while silencing of TREM-2 decreases intracellular survival of B. abortus. To further study the underlying mechanisms of TREM-2-mediated bacterial intracellular survival, we examined the activation of B. abortus-infected macrophages through determining the kinetics of activation of the three MAPKs, including ERK, JNK and p38, and measuring TNFα production in response to lipopolysaccharide (LPS) of Brucella (BrLPS) or B. abortus stimulation. Our data show that TREM-2 deficiency promotes activation of Brucella-infected macrophages. Moreover, our data also demonstrate that macrophage activation promotes killing of Brucella by enhancing nitric oxygen (NO), but not reactive oxygen species (ROS) production, macrophage apoptosis or cellular death. Taken together, these findings provide a novel interpretation of Brucella intracellular growth through inhibition of NO production produced by TREM-2-mediated activated macrophages.

The key roles of toll-like receptor (TLR) for intracellular survival of Brucella

Brucella spp. are facultative intracellular pathogens that have the ability to survive and multiply in professional and nonprofessional phagocytes and cause abortion in domestic animals and undulant fever in humans. Brucella species can survive in a variety of cells, including macrophages and their virulence and chronic infections are thought to be due to their ability to avoid the killing mechanisms within macrophages. Inhibition of phagosome-lysosome fusion has been proposed as a mechanism for intracellular survival of Brucella in professional and nonprofessional phagocytes. Toll-like receptors (TLRs) are part of a skillful system for detection of invasion by microbial pathogens. Recognition of microbial components by TLRs triggers signaling pathways that promote expression of genes and regulate innate immune responses. Recent studies for the interac- tion between TLRs- Brucella have indicated the importance of control of Brucella infection. Here, we review selected aspects of TLRs- Brucella interaction, which may be helpful to understanding the mechanism of Brucella pathogenesis.

Induction of DnaK and GroEL in Brucella Ovis under Various Stress Conditions

Brucella ovis is a facultative intracellular pathogen, able to survive and replicate within macrophages. The role of molecular chaperones in Brucella intracellular survival has been previously mentioned, but nothing was published on B. ovis in this respect, as far as we know. In the present work, in order to understand any possible contribution of the molecular chaperones DnaK and GroEL for Brucella ovis stress survival and intracellular persistance, the expression on different culture media under different stress conditions were analysed at the transcriptional and translational levels and compared with that of bacteria grown inside murine J774A.1 cells. The transcription analyses showed different levels of dnaK and groEL induction under various stress conditions (e.g., macrophages survival, acidic pH shock, high temperature, oxidative and osmotic shock) and a significant influence of the media composition. These results were consistent with the level of DnaK and GroEL expression analysed by the traditional proteomic methods. Here we demonstrate the molecular chaperones involvement in macrophage-B. ovis interaction, their expression under stress conditions suggesting the chaperones role in the bacterial resistance to such unfavourable environmental conditions.

The 16MΔvjbR as an ideal live attenuated vaccine candidate for differentiation between Brucella vaccination and infection

Brucellosis brings great economic burdens for developing countries. Live attenuated vaccines are the most efficient means for prevention and control of animal Brucellosis. However, the difficulties of differentiating of infection from vaccine immunization, which is essential for eradication programs, limit their applications. Therefore, the development of a vaccine that could differentiate infection from immunization will overcome the limitations and get extensive application. VjbR is a quorum sensing regulator involving in Brucella's intracellular survival. The vjbR∷Tn5 mutants have been proven effective against wild type strain challenge, implying its possibility of use in vaccine candidate development. To further evaluate this candidate gene, in the present study, the antigenicity of purified recombinant VjbR protein was analyzed. Antibodies to Brucella melitensis VjbR could be detected in sera from patients and animals with brucellosis but not in control ones, implying the potential use of this protein as a diagnostic antigen. Then a vjbR mutant of B. melitensis 16M was constructed by replacing the vjbR with kanamycin gene. The mutant showed reduced survival in macrophage and mice. Vaccination of BALB/c mice with 16MΔvjbR conferred significant protective immunity against B. melitensis strain 16M challenges, being equivalent to which induced by the license vaccine Rev.1. The vjbR deletion mutant elicited an anti- Brucella-specific immunoglobulin G response and induced the secretion of gamma interferon and interleukin-10. The most importance is that, the use of vjbR mutants as vaccines in association with diagnostic tests based on the VjbR antigen would allow the serological differentiation between infected and vaccinated animals. These results suggest that 16MΔvjbR is an ideal live attenuated vaccine candidate against B. melitensis and deserves further evaluation for vaccine development.

The type IV secretion system affects the expression of Omp25/Omp31 and the outer membrane properties ofBrucella melitensis

The type IV secretion system (T4SS) contributes to Brucella intracellular survival through its effector proteins. Comparative proteomic analysis showed that intracellular survival proteins are expressed differentially in a virB mutant. Interestingly, several outer membrane proteins (OMPs) are also differentially expressed, implying that T4SS might affect the OM properties of Brucella. To further evaluate the impact of T4SS on OM, in the present study, the OM proteomes were isolated and compared. Many more products of OMPs, particularly different products of the Omp25/Omp31 family, were found to be altered in the virB mutant. The transcription profiles of Omp25/Omp31 were different from those of their protein products, implying their regulation by virB at both transcriptional and post-transcriptional levels. The virB mutant aggregates at a high cell density and produces exopolysaccharide, a phenotype resembling that of the vjbR mutant. The virB mutant showed increased sensitivity to polymyxin B and decreased survival under oxidative, high-salt and high-osmolarity stresses, indicating drastic membrane alterations. These results indicated that in addition to being an effector protein secretion system, T4SS affects OM properties that might be important for the adaptation of Brucella to both in vitro and in vivo hostile environments.