Increased Brucella abortus asRNA_0067 expression under intraphagocytic stressors is associated with enhanced virB2 transcription.

Intracellular pathogens like Brucella face challenges during the intraphagocytic adaptation phase, where the modulation of gene expression plays an essential role in taking advantage of stressors to persist inside the host cell. This study aims to explore the expression of antisense virB2 RNA strand and related genes under intracellular simulation media. Sense and antisense virB2 RNA strands increased expression when nutrient deprivation and acidification were higher, being starvation more determinative. Meanwhile, bspB, one of the T4SS effector genes, exhibited the highest expression during the exposition to pH 4.5 and nutrient abundance. Based on RNA-seq analysis and RACE data, we constructed a regional map depicting the 5' and 3' ends of virB2 and the cis-encoded asRNA_0067. Without affecting the CDS or a possible autonomous RBS, we generate the deletion mutant ΔasRNA_0067, significantly reducing virB2 mRNA expression and survival rate. These results suggest that the antisense asRNA_0067 expression is promoted under exposure to the intraphagocytic adaptation phase stressors, and its deletion is associated with a lower transcription of the virB2 gene. Our findings illuminate the significance of these RNA strands in modulating the survival strategy of Brucella within the host and emphasize the role of nutrient deprivation in gene expression.

Post-transcriptional control of the essential enzyme MurF by a small regulatory RNA in Brucella abortus.

Brucella abortus is a facultative, intracellular, zoonotic pathogen that resides inside macrophages during infection. This is a specialized niche where B. abortus encounters various stresses as it navigates through the macrophage. In order to survive this harsh environment, B. abortus utilizes post-transcriptional regulation of gene expression through the use of small regulatory RNAs (sRNAs). Here, we characterize a Brucella sRNAs called MavR (for MurF- and virulence-regulating sRNA), and we demonstrate that MavR is required for the full virulence of B. abortus in macrophages and in a mouse model of chronic infection. Transcriptomic and proteomic studies revealed that a major regulatory target of MavR is MurF. MurF is an essential protein that catalyzes the final cytoplasmic step in peptidoglycan (PG) synthesis; however, we did not detect any differences in the amount or chemical composition of PG in the ΔmavR mutant. A 6-nucleotide regulatory seed region within MavR was identified, and mutation of this seed region resulted in dysregulation of MurF production, as well as significant attenuation of infection in a mouse model. Overall, the present study underscores the importance of sRNA regulation in the physiology and virulence of Brucella.

When mitophagy dictates the outcome of cellular infection: the case of Brucella abortus.

Mitochondria are at the basis of various cellular functions ranging from metabolism and redox homeostasis to inflammation and cell death regulation. Mitochondria therefore constitute an attractive target for invading pathogens to fulfil their infectious cycle. This involves the modulation to their advantage of mitochondrial metabolism and dynamics, including the controlled degradation of mitochondria through mitophagy. Mitophagy might for instance be beneficial for bacterial survival as it can clear bactericidal mitochondrial ROS produced by damaged organelle fragments from the intracellular niche. In the case of the bacterial pathogen Brucella abortus, mitophagy induction has another role in the intracellular lifecycle of the bacteria. Indeed, in our study, we showed that B. abortus triggers an iron-dependent BNIP3L-mediated mitophagy response required for proper bacterial egress and infection of neighboring cells. These results highlight the diversity of mitophagy processes that might be crucial for several stages of cellular infection.

Brucella effectors NyxA and NyxB target SENP3 to modulate the subcellular localisation of nucleolar proteins.

The cell nucleus is a primary target for intracellular bacterial pathogens to counteract immune responses and hijack host signalling pathways to cause disease. Here we identify two Brucella abortus effectors, NyxA and NyxB, that interfere with host protease SENP3, and this facilitates intracellular replication of the pathogen. The translocated Nyx effectors directly interact with SENP3 via a defined acidic patch (identified from the crystal structure of NyxB), preventing nucleolar localisation of SENP3 at late stages of infection. By sequestering SENP3, the effectors promote cytoplasmic accumulation of nucleolar AAA-ATPase NVL and ribosomal protein L5 (RPL5) in effector-enriched structures in the vicinity of replicating bacteria. The shuttling of ribosomal biogenesis-associated nucleolar proteins is inhibited by SENP3 and requires the autophagy-initiation protein Beclin1 and the SUMO-E3 ligase PIAS3. Our results highlight a nucleomodulatory function of two Brucella effectors and reveal that SENP3 is a crucial regulator of the subcellular localisation of nucleolar proteins during Brucella infection, promoting intracellular replication of the pathogen.

Development of multiple reaction monitoring (MRM) assays to identify Brucella abortus proteins in the serum of humans and livestock.

In the present study, a targeted multiple reaction monitoring-mass spectrometry (MRM-MS) approach was developed to screen and identify protein biomarkers for brucellosis in humans and livestock. The selection of proteotypic peptides was carried out by generating in silico tryptic peptides of the Brucella proteome. Using bioinformatics analysis, 30 synthetic peptides corresponding to 10 immunodominant Brucella abortus proteins were generated. MRM-MS assays for the accurate detection of these peptides were optimized using 117 serum samples of human and livestock stratified as clinically confirmed (45), suspected (62), and control (10). Using high throughput MRM assays, transitions for four peptides were identified in several clinically confirmed and suspected human and livestock serum samples. Of these, peptide NAIYDVVTR corresponding to B. abortus proteins: BruAb2_0537 was consistently detected in the clinically confirmed serum samples of both humans and livestock with 100% specificity. To conclude, a high throughput MRM-MS-based protocol for detecting endogenous B. abortus peptides in serum samples of humans and livestock was developed. The developed protocol will help design sensitive assays to accurately diagnose brucellosis in humans and livestock. The data associated with this study are deposited in Panorama Public (https://panoramaweb.org/rNOZCy.url with ProteomeXchange ID: PXD034407).

Mechanism of cyclic ß-glucan export by ABC transporter Cgt of Brucella.

Polysaccharides play critical roles in bacteria, including the formation of protective capsules and biofilms and establishing specific host cell interactions. Their transport across membranes is often mediated by ATP-binding cassette (ABC) transporters, which utilize ATP to translocate diverse molecules. Cyclic ß-glucans (CßGs) are critical for host interaction of the Rhizobiales, including the zoonotic pathogen Brucella. CßGs are exported into the periplasmic space by the cyclic glucan transporter (Cgt). The interaction of an ABC transporter with a polysaccharide substrate has not been visualized so far. Here we use single-particle cryoelectron microscopy to elucidate the structures of Cgt from Brucella abortus in four conformational states. The substrate-bound structure reveals an unusual binding pocket at the height of the cytoplasmic leaflet, whereas ADP-vanadate models hint at an alternative mechanism of substrate release. Our work provides insights into the translocation of large, heterogeneous substrates and sheds light on protein-polysaccharide interactions in general.

Platelet depletion does not alter the course of Brucella abortus infection in vivo.

Brucellosis is a bacterial disease of animals and a zoonotic infection. Thrombocytopenia is a common outcome in long-lasting brucellosis in humans. Likewise, ex vivo experiments have shown that platelets may play a role in Brucella abortus infections. Following these reports, we explored the course of brucellosis in thrombocytopenic mice, using the non-toxic low-molecular-weight aspercetin protein that depletes platelets in vivo. Aspercetin does not induce systemic hemorrhage or inflammation, and when injected into mice, it generates a rapid dose-dependent drop in platelet counts without affecting central organs, disrupting hematological parameters, or the proinflammatory cytokine profile. Compared to the B. abortus infected control group, the infected thrombocytopenic mice did not show significant differences in the hematological profiles, pathological score, spleen, liver histopathology, or bacterial loads. Except for IL-6, which was higher in the infected thrombocytopenic mice, the TNF-α, IFN-γ and IL-10 did not significantly differ with the PBS-infected group. The results indicate that platelets do not play a significant role in modulating Brucella infection in vivo at the early stages of infection, which is commensurate with the stealthy strategy followed by Brucella organisms at the onset of the disease.

Brucella abortus Depends on l-Serine Biosynthesis for Intracellular Proliferation.

l-Serine is a nonessential amino acid and a key intermediate in several relevant metabolic pathways. In bacteria, the major source of l-serine is the phosphorylated pathway, which comprises three enzymes: d-3-phosphoglycerate dehydrogenase (PGDH; SerA), phosphoserine amino transferase (PSAT; SerC), and l-phosphoserine phosphatase (PSP; SerB). The Brucella abortus genome encodes two PGDHs (SerA-1 and SerA-2), involved in the first step in l-serine biosynthesis, and one PSAT and one PSP, responsible for the second and third steps, respectively. In this study, we demonstrate that the serA1 serA2 double mutant and the serC and serB single mutants are auxotrophic for l-serine. These auxotrophic mutants can be internalized but are unable to replicate in HeLa cells and in J774A.1 macrophage-like cells. Replication defects of auxotrophic mutants can be reverted by cell medium supplementation with l-serine at early times postinfection. In addition, the serB mutant is attenuated in the murine intraperitoneal infection model and has an altered lipid composition, since the lack of l-serine abrogates phosphatidylethanolamine synthesis in this strain. Taken together, these results reveal that limited availability of l-serine within the host cell impairs proliferation of the auxotrophic strains, highlighting the relevance of this biosynthetic pathway in Brucella pathogenicity.

Occurrence and repair of alkylating stress in the intracellular pathogen Brucella abortus.

It is assumed that intracellular pathogenic bacteria have to cope with DNA alkylating stress within host cells. Here we use single-cell reporter systems to show that the pathogen Brucella abortus does encounter alkylating stress during the first hours of macrophage infection. Genes encoding direct repair and base-excision repair pathways are required by B. abortus to face this stress in vitro and in a mouse infection model. Among these genes, ogt is found to be under the control of the conserved cell-cycle transcription factor GcrA. Our results highlight that the control of DNA repair in B. abortus displays distinct features that are not present in model organisms such as Escherichia coli.

Brucella infection regulates peroxiredoxin-5 protein expression to facilitate intracellular survival by reducing the production of nitric oxide and reactive oxygen species.

Peroxiredoxin-5 (Prdx5) is a multifunctional protein involved in oxidative stress, apoptosis and inflammatory responses. However, how Prdx5 functions during microbial infections is rarely reported. In this study, we demonstrate that Brucella infection increased Prdx5 expression to promote its intracellular growth in macrophages. Further study show that B. abortus infection promoted its intracellular growth by decreasing the production of nitric oxide and reactive oxygen species. In addition, the expression of Prdx5 was independent on live Brucella and the type IV secretion system of Brucella. Instead, its expression was regulated by the lipopolysaccharide of Brucella. Moreover, Brucella infection increased Prdx5 expression in primary macrophage and mice. Collectively, these findings demonstrate for the first time that Prdx5 promotes Brucella intracellular growth by decreasing the production of NO and ROS. This finding provides new insights into the evasive strategies of Brucella and will be useful for the development of novel effective therapeutic approaches to treat Brucella infections.

Brucella abortus nitric oxide metabolite regulates inflammasome activation and IL-1ß secretion in murine macrophages.

NLRP3 inflammasome is a protein complex crucial to caspase-1 activation and IL-1ß and IL-18 maturation. This receptor participates in innate immune responses to different pathogens, including the bacteria of genus Brucella. Our group recently demonstrated that Brucella abortus-induced IL-1ß secretion involves NLRP3 inflammasome and it is partially dependent on mitochondrial ROS production. However, other factors could be involved, such as P2X7-dependent potassium efflux, membrane destabilization, and cathepsin release. Moreover, there is increasing evidence that nitric oxide acts as a modulator of NLRP3 inflammasome. The aim of this study was to unravel the mechanism of NLRP3 inflammasome activation induced by B. abortus, as well as the involvement of bacterial nitric oxide (NO) as a modulator of this inflammasome pathway. We demonstrated that NO produced by B. abortus can be used by the bacteria to modulate IL-1ß secretion in infected murine macrophages. Additionally, our results suggest that B. abortus-induced IL-1ß secretion depends on a P2X7-independent potassium efflux, lysosomal acidification, cathepsin release, mechanisms clearly associated to NLRP3 inflammasome. In summary, our results help to elucidate the molecular mechanisms of NLRP3 activation and regulation during an intracellular bacterial infection.

Endocrine modulation of Brucella abortus-infected osteocytes function and osteoclastogenesis via modulation of RANKL/OPG.

Osteoarticular brucellosis is the most frequent complication of active disease. A large amount of cells in bone are osteocytes. Since bone remodeling process is regulated by hormones we sought to study the effect of cortisol and DHEA in Brucella abortus-infected osteocytes. Cortisol treatment inhibited the expression of IL-6, TNF-α, MMP-2 and RANKL in B. abortus-infected osteocytes. DHEA could reverse the inhibitory effect of cortisol on MMP-2 production. B. abortus infection inhibited connexin 43 (Cx43) expression in osteocytes. This expression was increased when cortisol was incorporated during the infection and DHEA treatment partially reversed the effect of cortisol. Osteocytes-infected with B. abortus induced osteoclast's differentiation. Yet, the presence of cortisol, but not DHEA, during osteocyte infection inhibited osteoclastogenesis. Glucocorticoid receptor (GR) is implicated in the signaling of cortisol. Infection with B. abortus was able to increase GRα/ß ratio. Levels of intracellular cortisol are not only dependent on GR expression but also a result of the activity of the isoenzymes 11ß-hydroxysteroid dehydrogenase (11ß-HSD)-1 (cortisone to cortisol conversion), 11ß-HSD2 (cortisol to cortisone conversion). B. abortus infection increased 11ß-HSD 1/2 ratio and cortisone mimicked the effect of cortisol. Our results indicated that cortisol and DHEA could modulate osteocyte responses during B. abortus infection.

Brucella Hijacks Host-Mediated Palmitoylation To Stabilize and Localize PrpA to the Plasma Membrane.

Brucellaceae are a group of pathogenic intracellular bacteria with the ability to modulate the host response, both at the individual cell level and systemically. One of the hallmarks of the virulence process is the capacity of the bacteria to downregulate the adaptive and acquired host immune response through a plethora of virulence factors that directly impact several key signaling cascades. PrpA is one of those virulence factors that alters, via its polyclonal B-cell activity, the humoral and cellular immune responses of the host, ultimately favoring the establishment of a chronic infection. Even though PrpA affects B cells, it directly targets macrophages, triggering a response that ultimately affects B lymphocytes. In the present article we report that PrpA is S-palmitoylated in two N-terminal cysteine residues by the host cell and that this modification is necessary for its biological activity. Our results demonstrate that S-palmitoylation promotes PrpA migration to the host cell plasma membrane and stabilizes the protein during infection. These findings add a new mechanism exploited by this highly evolved pathogen to modulate the host immune response.

Defining the regulatory mechanism of NikR, a nickel-responsive transcriptional regulator, in Brucella abortus.

Metals are essential micronutrients for virtually all forms of life, but metal acquisition is a double-edged sword, because high concentrations of divalent cations can be toxic to the cell. Therefore, the genes involved in metal acquisition, storage and efflux are tightly regulated. The present study characterizes a nickel-responsive transcriptional regulator in the intracellular mammalian pathogen, Brucella abortus. Deletion of bab2_0432 (nikR) in B. abortus led to alterations in the nickel-responsive expression of the genes encoding the putative nickel importer NikABCDE and, moreover, NikR binds directly to a specific DNA sequence within the promoter region of nikA in a metal-dependent manner to control gene expression. While NikR is involved in controlling the expression of nikA, nikR is not required for the infection of macrophages or mice by B. abortus. Overall, this work characterizes the role of NikR in nickel-responsive gene expression, as well as the dispensability of nikR for Brucella virulence.

Characterization of Three Small Proteins in Brucella abortus Linked to Fucose Utilization.

Elucidating the function of proteins <50 amino acids in length is no small task. Nevertheless, small proteins can play vital roles in the lifestyle of bacteria and influence the virulence of pathogens; thus, the investigation of the small proteome is warranted. Recently, our group identified the Brucella abortus protein VtlR as a transcriptional activator of four genes, one of which is the well-studied small regulatory RNA AbcR2, while the other three genes encode hypothetical small proteins, two of which are highly conserved among the order Rhizobiales This study provides evidence that all three genes encode authentic small proteins and that all three are highly expressed under oxidative stress, low-pH, and stationary-phase growth conditions. Fractionation of the cells revealed that the proteins are localized to the membranes of B. abortus We demonstrate that the small proteins under the transcriptional control of VtlR are not accountable for attenuation observed with the B. abortusvtlR deletion strain. However, there is an association between VtlR-regulated genes and growth inhibition in the presence of the sugar l-fucose. Subsequent transcriptomic analyses revealed that B. abortus initiates the transcription of a locus encoding a putative sugar transport and utilization system when the bacteria are cultured in the presence of l-fucose. Altogether, our observations characterize the role of the VtlR-controlled small proteins BAB1_0914, BAB2_0512, and BAB2_0574 in the biology of B. abortus, particularly in the capacity of the bacteria to utilize l-fucose.IMPORTANCE Despite being one of the most common zoonoses worldwide, there is currently no human vaccine to combat brucellosis. Therefore, a better understanding of the pathogenesis and biology of Brucella spp., the causative agent of brucellosis, is essential for the discovery of novel therapeutics against these highly infectious bacteria. In this study, we further characterize the virulence-associated transcriptional regulator VtlR in Brucella abortus Our findings not only shed light on our current understanding of a virulence related genetic system in Brucella spp. but also increase our knowledge of small proteins in the field of bacteriology.

Lipocalin 2 (Lcn2) interferes with iron uptake by Brucella abortus and dampens immunoregulation during infection of RAW 264.7 macrophages.

Lipocalin 2 (Lcn2) is an important innate immunity component against bacterial pathogens. In this study, we report that Lcn2 is induced by Brucella (B.) abortus infection and significantly contributes to the restriction of intracellular survival of Brucella in macrophages. We found that Lcn2 prevented iron uptake by B. abortus through two distinct mechanisms. First, Lcn2 is secreted to capture bacterial siderophore(s) and abrogate iron import by Brucella. Second, Lcn2 decreases the intracellular iron levels during Brucella infection, which probably deprives the invading Brucella of the iron source needed for growth. Suppression of Lcn2 signalling resulted in a marked induction of anti-inflammatory cytokine, interleukin 10, which was shown to play a major role in Lcn2-induced antibrucella immunity. Similarly, interleukin 6 was also found to be increased when Lcn2 signalling is abrogated; however, this induction was thought to be an alternative pathway that rescues the cell from infection when the effective Lnc2 pathway is repressed. Furthermore, Lcn2 deficiency also caused a marked decrease in brucellacidal effectors, such as reactive oxygen species and nitric oxide but not the phagolysosome fusion. Taken together, our results indicate that Lcn2 is required for the efficient restriction of intracellular B. abortus growth that is through limiting iron acquisition and shifting cells to pro-inflammatory brucellacidal activity in murine macrophages.

Brucella Rough Mutant Induce Macrophage Death via Activating IRE1α Pathway of Endoplasmic Reticulum Stress by Enhanced T4SS Secretion.

Brucella is a Gram-negative facultative intracellular pathogen that causes the worldwide zoonosis, known as brucellosis. Brucella virulence relies mostly on its ability to invade and replicate within phagocytic cells. The type IV secretion system (T4SS) and lipopolysaccharide are two major Brucella virulence factors. Brucella rough mutants reportedly induce the death of infected macrophages, which is T4SS dependent. However, the underlying molecular mechanism remains unclear. In this study, the T4SS secretion capacities of Brucella rough mutant and its smooth wild-type strain were comparatively investigated, by constructing the firefly luciferase fused T4SS effector, BPE123 and VceC. In addition, quantitative real-time PCR and western blotting were used to analyze the T4SS expression. The results showed that T4SS expression and secretion were enhanced significantly in the Brucella rough mutant. We also found that the activity of the T4SS virB operon promoter was notably increased in the Brucella rough mutant, which depends on quorum sensing-related regulators of VjbR upregulation. Cell infection and cell death assays revealed that deletion of vjbR in the Brucella rough mutant absolutely abolished cytotoxicity within macrophages by downregulating T4SS expression. This suggests that up-regulation of T4SS promoted by VjbR in rough mutant ΔrfbE contribute to macrophage death. In addition, we found that the Brucella rough mutant induce macrophage death via activating IRE1α pathway of endoplasmic reticulum stress. Taken together, our study provide evidence that in comparison to the Brucella smooth wild-type strain, VjbR upregulation in the Brucella rough mutant increases transcription of the virB operon, resulting in overexpression of the T4SS gene, accompanied by the over-secretion of effecter proteins, thereby causing the death of infected macrophages via activating IRE1α pathway of endoplasmic reticulum stress, suggesting novel insights into the molecular mechanisms associated with Brucella rough mutant-induced macrophage cytotoxicity.

Protective efficacy by various doses of a new brucellosis vaccine candidate based on Salmonella strains expressing Brucella abortus BSCP31, Omp3b and superoxide dismutase against brucellosis in murine model.

Brucella species are important etiological agents of zoonotic diseases. Attenuated Salmonella strains expressing Brucella abortus BCSP31, Omp3b and superoxide dismutase proteins were tested as vaccine candidates in this study. In order to determine the optimal dose for intraperitoneal (IP) inoculation required to obtain effective protection against brucellosis, mice were immunized with various doses of a mixture of the three vaccine strains. Fifty BALB/c mice were divided into five equal groups (groups A-E). Group A mice were intraperitoneally inoculated with 100 µL of sterile phosphate-buffered saline. Group B, C, D and E mice were intraperitoneally immunized with approximately 1.2 × 105 colony-forming units (CFU) mL-1 of Salmonella containing pMMP65 in 100 µL and with 1.2 × 104 CFU mL-1, 1.2 × 105 CFU mL-1 and 1.2 × 106 CFU mL-1 of the mixture of the three strains in 100 µL, respectively. Serum IgG, tumor necrosis factor alpha and interferon gamma concentrations were significantly higher in group E than in groups A-D. Following challenge with B. abortus 544, the challenge strain was not detected in the spleen of any mouse from group E. Thus, IP immunization with 1.2 × 106 CFU mL-1 of the mixture of the three vaccine strains induced immune responses and provided effective protection against brucellosis in mice.

Brucella abortus Induces a Warburg Shift in Host Metabolism That Is Linked to Enhanced Intracellular Survival of the Pathogen.

Intracellular bacterial pathogens exploit host cell resources to replicate and survive inside the host. Targeting these host systems is one promising approach to developing novel antimicrobials to treat intracellular infections. We show that human macrophage-like cells infected with Brucella abortus undergo a metabolic shift characterized by attenuated tricarboxylic acid cycle metabolism, reduced amino acid consumption, altered mitochondrial localization, and increased lactate production. This shift to an aerobic glycolytic state resembles the Warburg effect, a change in energy production that is well described in cancer cells and also occurs in activated inflammatory cells. B. abortus efficiently uses lactic acid as its sole carbon and energy source and requires the ability to metabolize lactate for normal survival in human macrophage-like cells. We demonstrate that chemical inhibitors of host glycolysis and lactate production do not affect in vitro growth of B. abortus in axenic culture but decrease its survival in the intracellular niche. Our data support a model in which infection shifts host metabolism to a Warburg-like state, and B. abortus uses this change in metabolism to promote intracellular survival. Pharmacological perturbation of these features of host cell metabolism may be a useful strategy to inhibit infection by intracellular pathogens.IMPORTANCEBrucella spp. are intracellular bacterial pathogens that cause disease in a range of mammals, including livestock. Transmission from livestock to humans is common and can lead to chronic human disease. Human macrophage-like cells infected with Brucella abortus undergo a Warburg-like metabolic shift to an aerobic glycolytic state where the host cells produce lactic acid and have reduced amino acid catabolism. We provide evidence that the pathogen can exploit this change in host metabolism to support growth and survival in the intracellular niche. Drugs that inhibit this shift in host cell metabolism inhibit intracellular replication and decrease the survival of B. abortus in an in vitro infection model; these drugs may be broadly useful therapeutics for intracellular infections.

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.