Brucellosis, a globally significant zoonotic disease caused by Brucella infection, relies on the pathogen’s ability to invade and replicate within host cells. This intracellular replication is tightly regulated by transcriptional networks, including the LysR-family regulator VtlR, which is critical for B. abortus virulence but whose role in B. melitensis remains unclear. Here, we constructed vtlR mutant and complemented strains in B. melitensis M5 and demonstrated that VtlR is essential for virulence. Phenotypic assays revealed that vtlR deletion impaired bacterial growth on L-fucose, D-glucose, and meso-erythritol, increased sensitivity to hydrogen peroxide and sodium nitroprusside, and reduced intracellular survival in RAW264.7 macrophages while triggering reactive oxygen species (ROS) production. RNA-seq and RT-qPCR analysis indicated that VtlR positively regulates small RNA AbcR2 and three DUF1127-domain proteins (RS13565, RS04310, RS13280), mirroring its regulatory role in B. abortus. However, overexpression of these targets failed to restore virulence in the vtlR mutant. Notably, the mutant strain elicited protective immunity in mice, suggesting its potential as a live-attenuated vaccine candidate. Collectively, this study elucidates the VtlR regulon in B. melitensis, advancing our understanding of Brucella pathogenesis and vaccine development.

Brucella relies on the type IV secretion system (T4SS) to establish replication niches within host cells. However, the Brucella T4SS effectors and their functions have not been fully identified. In this study, we investigated the function of Brucella RS15060, a novel T4SS effector discovered in our previous study, on the bacterial biological characteristics and pathogenesis by construction of the gene deletion and complementation strains. We found that deletion of the rs15060 gene weakened abilities of Brucella to replicate within host cells and establish chronic infection in mice but enhanced abilities to adhere/invade HeLa cells and evade lysosomal degradation in the early stage of infection. In addition, the rs15060 deletion Brucella strain showed significant changes in bacterial shape, cell wall thickness, and sensitivity to bactericidal factors. Furthermore, the rs15060 deletion strain showed an increased synthesis of bacterial lipopolysaccharide core and induced a stronger host’s inflammatory response. The Brucella rs15060 complementation strain restored the altered biological characteristics. Moreover, BLASTP prediction and 3D structure simulation revealed that the Brucella RS15060 contains NAD(P)-binding and active motifs in structure, which are important for proteins to exert NAD dependent epimerase/dehydratase activity. The complementation strain with mutation on NAD(P)-binding and/or active motifs of RS15060 did not restore the altered characteristics, suggesting that the Brucella RS15060 is a potential NAD dependent epimerase/dehydratase, and the predicted NAD(P)-binding and/or active motifs play an important role on bacterial cell wall and LPS core synthesis, which is crucial for maintaining bacterial morphology and exerting virulence.

S100A4 is a low-molecular-weight protein belonging to the S100 protein family, and is widely expressed in a variety of cells. It plays important regulatory roles in cell physiological processes, including proliferation, adhesion, motility, and the inflammatory and immune responses. In this study, we investigated the roles of S100A4 in host cell proliferation and Brucella infection. We found that Brucella inhibited S100A4 expression in RAW264.7 macrophages in a late stage of infection. Knockout of S100A4 expression significantly enhanced the proliferation of RAW264.7 macrophages, whereas the overexpression of S100A4 inhibited their proliferation. A proteomic analysis indicated that the overexpression of S100A4 in RAW264.7 macrophages affected pathogen infection, signaling pathway regulation, cell mobility and adhesion, and the ferroptosis pathway. We also found that the role of S100A4 in the proliferation of RAW264.7 macrophages is ferroptosis dependent. Fer-1 treatment increased the proliferation of S100A4(OE) cells, which was mediated by the ATF4-SLC7A11-GSH axis. The overexpression of S100A4 promoted the intracellular infection of Brucella, with enhanced bacterial intracellular survival and adhesion, in RAW264.7 macrophages. In summary, we have demonstrated that S100A4 plays vital roles in the growth of RAW264.7 macrophages and an important role in Brucella intracellular infection by enhancing the pathogen's intracellular viability and adhesion ability.

Background: Brucellosis is a zoonotic bacterial disease primarily controlled through quarantine, culling, and vaccination. Live attenuated vaccines remain the most effective countermeasure, yet their application is limited by residual virulence and diagnostic interference. This study developed three rough-type attenuated Brucella melitensis mutants (G7, G8, G16) and evaluated their potential as DIVA (Differentiating Infected from Vaccinated Animals) vaccine candidates. Methods: Rough phenotypes were characterized through heat agglutination, acridine orange staining, and immunoblotting. Macrophage cytotoxicity was assessed via LDH release assays, while RT-qPCR analyzed macrophage activation capacity. Mouse infection and immunization-challenge experiments, complemented by histopathology, evaluated residual virulence and protective immunity. Antibody profiles were determined by ELISA, and DIVA capability was verified using LPS-coated ELISA. Results: G7 and G8 exhibited complete rough phenotypes, whereas G16 retained partial O-antigen (semi-rough). All rough mutants induced macrophage cytotoxicity and activation. The strains showed attenuated virulence with no viable bacteria recovered from spleens at 4 weeks post-inoculation. Histopathology revealed no liver lesions at 6 weeks post-inoculation. Immunized mice predominantly produced IgG2a-dominated Th1-type responses. The immune protection levels of G7 and G16 matched the reference vaccine M5–90Δ26, while G8 showed slightly lower efficacy. LPS-ELISA effectively differentiated vaccinated from infected animals via concurrent IgM/IgG detection. Conclusions: This study demonstrates that the rough-type B. melitensis mutants G7 and G16 serve as promising DIVA vaccine candidates, offering strong protection with low residual virulence while enabling serological differentiation between vaccinated and infected animals, highlighting their potential as effective vaccines for brucellosis control.

Brucella is an intracellular parasitic pathogen that causes the worldwide zoonotic disease brucellosis. The type IV secretion system (T4SS) is utilized to secrete various effectors to help Brucella form Brucella-containing vacuoles within the cell and accomplish intracellular trafficking and replication. Brucella has fewer recognized effector proteins than other intracellular parasites in the Proteobacteria, indicating that Brucella may contain a large number of unidentified effector proteins. In this study, the optimal conditions for inducing protein secretion from Brucella were screened, and the secreted proteins of 2308 and the T4SS-deficient mutant SV123 under optimal conditions were collected for comparative proteomics analysis. By label-free quantitative proteomics, we identified 15 differential proteins. Through the β-lactamase TEM1 assay and indirect immunofluorescence assay, we identified RS15060 and RS10635 as novel T4SS effectors. Furthermore, by constructing mutation strains and performing cell/mouse infection experiments, we found that deletion of the rs15060 gene reduced the capacity of Brucella to replicate in cells and cause chronic infection in mice. In conclusion, a novel Brucella T4SS effector protein, RS15060, was identified to be associated with virulence in this study, and the discovery of effector proteins is conducive to a more comprehensive elucidation of T4SS function as well as to uncovering the cryptic strategies of Brucella survival in cells.

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.

B. abortus is a facultative intracellular bacterium that replicates within macrophages. Intracellular survival is one of the important indexes to evaluate the virulence of Brucella. Ferroptosis is a type of programmed cell death induced by the accumulation of free iron, reactive oxygen species (ROS), and toxic lipid peroxides, play roles on cancers, cardiovascular diseases, and inflammatory diseases. In this study, we found that Brucella rough strain RB51 induced ferroptosis on macrophages with reduced levels of host glutathione and glutathione peroxidase 4 (Gpx4), together with increased ferrous iron, lipid peroxidation, and ROS. The inhibitor ferrostatin-1 significantly reduced the ferroptosis of RB51-infected macrophages, confirming that ferroptosis occurred during infection with Brucella RB51. Furthermore, we found that RB51 infection induced ferroptosis is regulated by P53-Slc7a11-Gpx4/GSH signal pathway. Inhibiting P53 decreased the levels of ROS and lipid peroxidation, while the levels of Slc7a11, Gpx4 and GSH were rescued. More importantly, inhibiting ferroptosis by different ferroptosis inhibitors increased the intracellular survival of Brucella RB51, indicating ferroptosis functions on the attenuation of Brucella intracellular survival. Collectively, our observations demonstrate that Brucella RB51 infection induces ferroptosis on macrophages, which is regulated by P53-Slc7a11-Gpx4/GSH signal pathway and functions on the attenuation of intracellular survival of Brucella.

Background: Brucella abortus is the main causative agent for bovine brucellosis. B. abortus A19 is a widely used vaccine strain to protect cows from Brucella infection in China. However, A19 has a similar lipopolysaccharide (LPS) antigen to that of the field virulent Brucella strain, whose immunization interferes with the serodiagnosis of vaccinated and infected animals. [Aim] To develop a novel Brucella DIVA vaccine candidate. Study design and methods: The B. abortus mutant A19mut2 with the formyltransferase gene wbkC is replaced by an acetyltransferase gene wbdR from E. coli O157 using the bacterial homologous recombination technique, generating a modified O-polysaccharide that cannot induce antibodies in mice against wild-type Brucella LPS. The biological phenotypes of the A19mut2 were assessed using a growth curve analysis, agglutination tests, Western blotting, and stress resistance assays. Histopathological changes and bacterial colonization in the spleens of vaccinated mice were investigated to assess the residual virulence and protection of the A19mut2. Humoral and cellular immunity was evaluated by measuring the levels of IgG, IgG subtypes, and the release of cytokines IFN-γ and IL10 in the splenocytes of the vaccinated mice. ELISA coated with wild-type LPS can distinguish mouse antibodies induced by A19 and A19mut2 immunization. Results: The A19mut2 showed a decreased residual virulence in mice, compared to the A19 strain, but induced significant humoral and cellular immune responses, as the A19 immunization did. The protection efficacy of A19mut2 immunization against B. abortus S2308 NalR infection was similar to that of A19 immunization. Conclusion: The A19mut2 has potential as a novel DIVA vaccine candidate in the future.

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 is an intracellular parasitic bacterium that uses multiple strategies to evade the host’s defense mechanisms. However, how Brucella manipulates the host-induced oxidative stress and relevant biological processes are still poorly understood. In this study, a comparative transcriptome assay of macrophages infected with Brucella abortus S2308 and its rough mutant RB14 was performed to investigate the differentially expressed genes which might be associated with the pathogenic mechanism of Brucella. Our results showed that numerous host pro-oxidative and antioxidative stress genes were differentially expressed in macrophages infected with B. abortus S2308 and mutant RB14 at 4, 8, 24, and 48 h post-infection. Interestingly, we found that several ferroptosis-associated genes were differentially expressed during B. abortus RB14 infection. Moreover, we found that the rough mutant RB14-induced macrophage death was associated with reduced levels of host glutathione and glutathione peroxidase 4, together with increased free iron, lipid peroxidation, and ROS, all of which are important hallmarks of ferroptosis. The ferroptosis occurring during infection with RB14 was reduced by treatment with the inhibitor ferrostatin-1. However, B. abortus S2308 infection did not induce these hallmarks of ferroptosis. Taken together, our results demonstrate that ferroptosis is involved in rough B. abortus infection. Investigating how Brucella manipulates oxidative stress and ferroptosis in its host will be helpful to clarify the pathogenicity of B. abortus.

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.

Brucella is a facultative intracellular bacterium lacking classical virulence factors; its virulence instead depends on its ability to invade and proliferate within host cells. After entering cells, Brucella rapidly modulates the expression of a series of genes involved in metabolism and immune evasion. Here, a novel LysR-family transcriptional regulator, designated Brucellavirulence-related transcriptional regulator (BvtR), was found to be associated with Brucella abortus virulence. We first successfully constructed a BvtR mutant, ΔbvtR, and a complemented strain, ΔbvtR-Com. Subsequently, we performed cell infection experiments, which indicated that the ΔbvtR strain exhibited similar adhesion, invasion and survival within HeLa cells or RAW264.7 macrophages to those of the wild-type strain. In stress resistance tests, the ΔbvtR strain showed enhanced sensitivity to sodium nitroprusside and sodium dodecyl sulfate, but not to hydrogen peroxide, cumene hydroperoxide, polymyxin B and natural serum. Mouse infection experiments indicated that the virulence of the ΔbvtR strain significantly decreased at 4 weeks post-infection. Finally, we analyzed differentially expressed genes regulated by BvtR with RNA-seq, COG classification and KEGG pathway analysis. Nitrogen metabolism, siderophore biosynthesis and oligopeptide transport were found to be the predominantly altered functions, and key metabolic and regulatory networks were delineated in the ΔbvtR mutant. Thus, we identified a novel Brucella virulence-related regulator, BvtR, and demonstrated that BvtR regulation affects Brucella resistance to killing by sodium nitroprusside and sodium dodecyl sulfate. The differentially expressed genes responding to BvtR are involved in diverse functions or pathways in Brucella, thus, suggesting the breadth of BvtR's regulatory functions. This study provides novel clues regarding Brucella pathogenesis.

Brucellosis is a bacterial infectious zoonosis which is spread worldwide, caused by Brucella, with infertility and abortion in domestic animals. Protein-tyrosine phosphatase (PTPs) have been discovered in many kinds of bacterial species, which play crucial roles in many aspects, such as bacterial physiology and virulence. However, no PTPs have been identified in Brucella to date. Here, we identified a novel gene BM28_RS15985 in Brucella melitensis that encodes a homolog of a low weight molecular PTP. Enzyme activity analysis showed that this PTP is a dual specific phosphatase, removing phosphate group from phosphotyrosine and phosphoserine/phosphothreonine peptides, which was designated as Dsp1. The optimal pH of the Dsp1 enzyme activity were 5.5, suggesting that the Dsp1 is an acidic phosphatase, and the optimal reaction temperature of the Dsp1 was 35.0 °C. Besides, the Michaelis constant and maximum reaction velocity of the Dsp1 were 40.17 mM and 24.33 nM/min/mg, respectively. In further study, we investigated the role of Dsp1 in B. melitensis phenotype and virulence. Growth curve and resistance test exhibited that the dsp1 had no role in Brucella growth and resisting bactericidal factors. Cell and animal infection experiment showed that the dsp1 deletion did not affect the intracellular survival and virulence of B. melitensis. In summary, we identified a novel acidic dual specific phosphatase in B. melitensis and evaluated its characteristics of the enzyme activity, this study will expand the understanding of Brucella phosphatase.

本研究通过免疫印迹试验分析流产布鲁菌感染小鼠不同阶段的血清中以及临床牛羊布病阳性血清中抗体生成规律和特点,确定布鲁菌主要免疫原性蛋白的相对区域.结果显示:布鲁菌感染小鼠2周后抗体逐渐产生,在6～8周达到顶峰,之后维持稳定状态;结合临床牛羊血清分析发现布鲁菌免疫原性蛋白主要集中在相对分子量40～70 kDa和7～20 kDa的两个区域;通过SDS-PAGE分离布鲁菌总蛋白,对照免疫印迹试验结果,切取免疫原性蛋白条带进行质谱分析,共鉴定到10个布鲁菌免疫原性蛋白;在大肠杆菌中成功表达和纯化了免疫原性蛋白SSB;免疫印迹试验显示,纯化的SSB蛋白可与临床牛布病阳性血清产生较好的反应,但检测阳性样本时也会产生阴性反应,检测效率与虎红平板凝集试验相比略差,在检测阳性样本时,与虎红平板凝集试验的符合率约为82％.该研究为布病诊断试剂研发奠定基础.

Brucella vaccination is one of the most important strategies for controlling brucellosis in livestock. The A19 strain was the effective vaccine used to control brucellosis in China. However, the characteristics of physiological and attenuated virulence of the A19 strain are not investigated in detail. In this study, we compared the phenotypic characteristics of the A19 to the wild-type strain S2308. Virulence test showed that the A19 was significantly attenuated at chronic infection stage in infected mouse model. In growth analysis, the A19 exhibited a quick growth at exponential phase and premature at stationary phase. The inflammatory response of macrophages infected by the A19 was detected using TaqMan qPCR assay, indicating that the inflammatory level of the A19-infected macrophages was higher than that of the S2308 infection. Cell death analysis showed that the A19 was not cytotoxic for macrophages. Cell infection showed that the A19 reduced its ability to invade, survive and traffic within host cells, and the intracellular A19 hardly excludes lysosome-associated marker LAMP-1, suggesting that the A19 can't escape the lysosome degradation within host cells. In further study, the sensitivity test exhibited that the A19 is more sensitive to stress and bactericidal factors than the S2308 strain, Western blot and silver staining analysis exhibited that the A19 has a different expression pattern of OMPs and reduces LPS O-antigen expression relative to the S2308 strain. Those data give us a more detailed understanding about the A19 vaccine strain, which will be beneficial for improvement of current Brucella vaccine and overcoming its defects.

[Background] Brucellosis is a zoonotic infectious disease caused by Brucella spp. and threats the development of animal husbandry and human health. A novel plasmid was constructed for promoter activity determination in Brucella based on NanoLuc luciferase gene (nluc), which is important for research on regulatory mechanism of Brucella virulence genes. [Objective] Preparation of rabbit polyclonal antibody of Nluc, construction of a Nluc reporter plasmid for promoter activation determination in Brucella, and verification of the Nluc reporter plasmid for Brucella bcsp31 gene and virB promoter. [Methods] The nluc gene was ligated into prokaryotic expression vector pET-28a and constructed as recombinant vector pET-Nluc. The New Zealand rabbit was immunized to prepare polyclonal antibody of Nluc protein. The plasmids pNluc, pBcsp31-Luc and pVirB-Luc were constructed based on a broad-host-range vector pBBR1MCS. Brucella recombinant strains S2308(Nluc), S2308(Bcsp31-Nluc) and S2308(VirB-Nluc) were constructed by electrotransformation of plasmids. The promoter activity of bcsp31 and virB were detected in TSB. The activity of virB promoter in Brucella was compared in TSB and within RAW264.7 cells. [Results] The Nluc protein was expressed and purified. The ELISA titer of polyclonal antibody was approximately to 1:100 000. The plasmid pNluc, pBcsp31-Luc, pVirB-Luc and the S2308(Nluc), S2308(Bcsp31-Nluc), S2308(VirB-Nluc) strains were constructed successfully. The results of bcsp31 and virB promoter activity in TSB showed that promoter activity can be detected accurately in pNluc plasmid. The result of virB promoter activity in intracellular Brucella showed that the activity of virB promoter is enhanced significantly. [Conclusion] In this study, a plasmid for promoter activity determination of Brucella genes was constructed successfully. The results of this study showed that the promoter activity of Brucella genes can be detected accurately in pNluc plasmid. This study provides a novel strategy for determining promoter activity of Brucella virulence genes, which may benefit investigation of Brucella pathogenesis.

Brucella is a facultative intracellular bacterium, dividing into smooth- and rough-type Brucella. Smooth-type Brucella can dissociate into rough mutants with cytotoxicity for macrophages during infection, which is critical for Brucella egress and dissemination. However, the mechanism of cytotoxicity infected by rough Brucella is incomplete. In this study, we verified that a rough-type Brucella (RB14 strain) was cytotoxic for macrophages dependent on Type IV secretion system (T4SS). Two specific T4SS VirB4 and VirB11 mutants were constructed, which affect the secretion of T4SS effectors, but not the expression of T4SS components. Cytotoxicity analysis showed that RB14- induced macrophages death depends on T4SS secretion activity. In a further study, 15 reported T4SS effectors were evaluated in inducing macrophage death using over-expression and transfection methods, the results showed that 15 recombinant strains with over-expression of respective effector were not cytotoxicity. In addition, 10 effectors transfected individually, or co-transfected with five effectors barely induced macrophage death, suggesting that all 15 effectors were not associated with macrophage death. Besides, we also evaluated endoplasmic reticulum (ER) stress, Txnip- or Caspase-2 roles in RB14-induced macrophages death. The results showed that inhibition of ER stress, Caspase or Caspase-2 activation was not associated with RB14-infected macrophages death. The casp2 and txnip knockout cells also showed death when infected by the RB14 strain. In all, the RB14-induced macrophage death depends on the secretion activity of T4SS, but not on ER stress, Txnip- or Caspase-2 signal pathway. This study provides a deep insight for rough Brucella-induced macrophage death, which favors for elucidating Brucella infection lifecycle.

Brucellosis is an important zoonotic bacterial disease widespread in the world. The key step of control this disease is accurate diagnosis and elimination of diseased animals. The classic diagnostic methods, such as tube agglutination test, are inaccurate and nonspecific, because of cross-reaction with Yersinia enterocolitica serotype O:9. Previously, several proteins were reported as Brucella main immunogens. In this study, we used animal infection model to evaluate antibody production against OMP16, BP26, BLS, BCSP31, VirB12, SodC and GroEL proteins and investigated their application in diagnosis of brucellosis. The results showed that the BP26 and BLS are two best immunogenic proteins. In further study, we detected 44 clinical bovine sera using western blot, showing that the BP26 and BLS reacted with 30 Brucella-positive sera, but false-positive results were also shown in 14 Brucella-free sera. In an indirect ELISA assay, compared to lipopolysaccharide-based ELISA, the conformance of the BP26-based ELISA was 92.68 % in Brucella-positive sera, but only 52.94 % in Brucella-free sera. The BLS-based ELISA can hardly differentiate positive sera from negative sera. Besides, truncated fragments of the BP26 protein cannot exclude false-positive results in detection of Brucella-free sera. Altogether, although Brucella main immunogenic proteins have good reaction with Brucella-positive sera, false-positive reaction with Brucella-free sera may lead to misdiagnosis of brucellosis, suggesting that it should be more careful to use these immunogenic proteins as antigen targets to diagnosis of brucellosis.

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.

Brucella rough mutants are reported to induce infected macrophage death, which is type IV secretion system (T4SS) dependent. T4SS and its secretory proteins play a major role in host-bacteria interactions, but the crucial secretory proteins to promote macrophage death during Brucella rough mutant infection have not been characterized. In this study, we found that T4SS components played no role for macrophage death induced by Brucella rough mutant infection, but some T4SS effectors did. Proteomics of secretory proteins from Brucella rough mutants Delta rfbE and Delta rfbE Delta virB123 was analyzed by liquid chromatography/tandem mass spectrometry and 861 unique proteins were identified, among which 37 were differential secretory proteins. Gene ontology and pathway analysis showed that differential secretory proteins involved in cellular process and metabolic process, distributed in the cell and membrane, possessed molecular function of catalytic activity and binding, and were associated with ribosome, NOD-like receptor signaling pathway, two-component system and bacterial secretion system. Cell death analysis showed that T4SS effector VceC, and two differential secretory proteins OmpW family protein (BAB1_1579) and protein BAB1_1185 were associated with Brucella cytotoxicity. This study provides new insights into the molecular mechanisms associated with Brucella cytotoxicity and valuable information for screening vaccine candidates for Brucella. Significance: Brucella rough mutants induce infected macrophage death, which is T4SS dependent. In the present report, a comparative proteomics analysis revealed 37 differential secretory proteins between Brucella rough mutants Delta rfbE and Delta rfbE Delta virB123. Further study demonstrated OmpW family protein (BAB1_1579) and uncharacterized protein BAB1_1185, two differential secretory proteins, were associated with Brucella cytotoxicity. This study provides novel information of the secretory proteins from the Brucella rough mutants and their effects on the Brucella cytotoxicity.