Brucella induced upregulation of NO promote macrophages glycolysis through the NF-κB/G6PD pathway.

Increased glycolytic metabolism recently emerged as an essential process driving host defense against Brucella, but little is known about how this process is regulated during infection. We have identified a critical role for nuclear factor kappa B (NF-κB) transcription factor regulation in glycolytic switching during Brucella infection for the first time. Chromatin immunoprecipitation with next-generation sequencing for NF-κB and DNA Pull-Down revealed two novel NF-κB-binding sites in the enhancer region of the Nitric oxide (NO)production-response regulator gene glucose-6-phosphate dehydrogenase (G6PD), which is important for the switch to glycolysis during a Brucella infection. These findings demonstrate that Brucella drives metabolic reprogramming by inhibiting host oxidative phosphorylation (OXPHOS) and enhancing its glycolysis via the NF-κB-G6PD-NO-pathway. These studies provide a theoretical basis for investigating drugs or vaccines to control Brucella colonization and induction of undulant by manipulating host metabolic patterns.

The effect of Brucella abortus on glial activation and cell death in adult male rat's hippocampus.

A zoonotic disease called brucellosis can cause flu-like symptoms and heart inflammation. The bacteria responsible for this disease can also enter the brain, causing a condition called neurobrucellosis that can result in long-term neurological problems. In this study, researchers aimed to determine the changes in the hippocampal cells of rats infected with Brucella. For the study, 24 adult male albino rats were inoculated with 1 × 106 CFU Brucella abortus 544. The rats were then deeply anesthetized, and their hippocampus samples were taken for stereological, histological, and molecular studies. The results showed that the infected rats had increased microgliosis and astrogliosis. Furthermore, a high level of caspase-3 in their hippocampal tissue indicated their susceptibility to apoptosis. Additionally, there was a decrease in expression of Ki67, which further supported this. Sholl's analysis confirmed a significant failure in glial morphology. The study demonstrated that the pathogen has the ability to destroy the hippocampus and potentially affect its normal physiology. However, more research is needed to clarify various aspects of neurobrucellosis.

Protein disulfide isomerase A4 binds to Brucella BtpB and mediates intracellular NAD+/NADH metabolism in RAW264.7 cells.

The Toll/interleukin-1 receptor (TIR) signaling domain is distributed widely in mammalian Toll-like receptors and adaptors, plant nucleotide-binding leucine-rich repeat receptors, and specific bacterial virulence proteins. Proteins that possess TIR domain exhibit NADase activity which is distinct from the canonical signaling function of these domains. However, the effects of bacterial TIR domain proteins on host metabolic switches and the underlying mechanism of NADase activity in these proteins remain unclear. Here, we utilized Brucella TIR domain-containing type IV secretion system effector protein, BtpB, to explore the mechanism of NADase activity in host cells. We showed that using ectopic expression BtpB not only generates depletion of NAD+ but also loss of NADH and ATP in RAW264.7 macrophage cells. Moreover, immunoprecipitation-mass spectrometry, co-immunoprecipitation, and confocal microscope assays revealed that BtpB interacted with host protein disulfide isomerase A4 (PDIA4). The Brucella mutant strain deleted the gene for BtpB, significantly decreased PDIA4 expression. Furthermore, our data revealed that PDIA4 played an important role in regulating intracellular NAD+/NADH levels in macrophages, and PDIA4 overexpression restored the decline of intracellular NAD+ and NADH levels induced by Brucella BtpB. The results provide new insights into the metabolic regulatory activity of TIR domain proteins in the critical human and animal pathogen Brucella.

RNA-seq reveals the important role of transcriptional regulator DeoR1 in regulating Brucella abortus various pathways.

Brucella spp. is an intracellular bacterium that uses its transcriptional regulator DeoR1 to promote intracellular transport and survival, but the molecular mechanism remains unknown. To analyze the role of DeoR1 in the virulence of B. abortus and the genes regulated by DeoR1, we created a A19ΔdeoR1 mutant of B. abortus A19 (A19). Virulence assay was performed using a murine macrophage cell line (RAW264.7) and mice. We observed that A19ΔdeoR1 mutant is attenuated in RAW264.7 cells and mice. We performed RNA-seq whole transcriptome analysis of A19ΔdeoR1 and A19 from infected RAW264.7 cells. A total of 135 differentially expressed genes were identified, including 100 up-regulated and 35 down-regulated genes. These differentially expressed genes were involved in amino acid synthesis and metabolism, energy production and conversion, stress proteins, chaperonin, hypothetical proteins and protein of unknown function, cell wall/membrane/envelope, intracellular transporting and secretion, and transcriptional regulator. Interestingly, genes involved in the intracellular trafficking and secretion were significantly down-regulated in A19ΔdeoR1. Furthermore, selected RNA-seq results were experimentally confirmed by qRT-PCR. Overall, these results deciphered differential phenomena associated with virulence in A19ΔdeoR1 and A19 from infected RAW264.7 cells, which provided important information for understanding the detailed role of DeoR1 in Brucella pathogenesis. SIGNIFICANCE: Transcriptional regulators are predominant bacterial signal transduction factors. The pathogenicity of Brucella is due to its ability to regulate the expression of virulence related genes. Transcriptional regulators are designed to regulate gene expression and enact an appropriate adaptive physiological response. Here, a total of 135 differentially expressed genes were identified in transcriptional regulator deoR1 mutant.

Beyond its preferential niche: Brucella abortus RNA down-modulates the IFN-γ-induced MHC-I expression in epithelial and endothelial cells.

Brucella abortus (Ba) is a pathogen that survives inside macrophages. Despite being its preferential niche, Ba infects other cells, as shown by the multiple signs and symptoms humans present. This pathogen can evade our immune system. Ba displays a mechanism of down-modulating MHC-I on monocytes/macrophages in the presence of IFN-γ (when Th1 response is triggered) without altering the total expression of MHC-I. The retained MHC-I proteins are located within the Golgi Apparatus (GA). The RNA of Ba is one of the PAMPs that trigger this phenomenon. However, we acknowledged whether this event could be triggered in other cells relevant during Ba infection. Here, we demonstrate that Ba RNA reduced the surface expression of MHC-I induced by IFN-γ in the human bronchial epithelium (Calu-6), the human alveolar epithelium (A-549) and the endothelial microvasculature (HMEC) cell lines. In Calu-6 and HMEC cells, Ba RNA induces the retention of MHC-I in the GA. This phenomenon was not observed in A-549 cells. We then evaluated the effect of Ba RNA on the secretion of IL-8, IL-6 and MCP-1, key cytokines in Ba infection. Contrary to our expectations, HMEC, Calu-6 and A-549 cells treated with Ba RNA had higher IL-8 and IL-6 levels compared to untreated cells. In addition, we showed that Ba RNA down-modulates the MHC-I surface expression induced by IFN-γ on human monocytes/macrophages via the pathway of the Epidermal Growth Factor Receptor (EGFR). So, cells were stimulated with an EGFR ligand-blocking antibody (Cetuximab) and Ba RNA. Neutralization of the EGFR to some extent reversed the down-modulation of MHC-I mediated by Ba RNA in HMEC and A-549 cells. In conclusion, this is the first study exploring a central immune evasion strategy, such as the downregulation of MHC-I surface expression, beyond monocytes and could shed light on how it persists effectively within the host, enduring unseen and escaping CD8+ T cell surveillance.

Revealing metabolic alterations in brucellosis patients by targeted metabolomics.

Brucellosis, a zoonotic disease caused by brucella infection, presents metabolic profile changes in patients that have not been extensively explored. This study utilized an ultra-high performance liquid chromatography tandem mass spectrometry based targeted metabolomic approach to comprehensively investigated metabolic changes in Brucella patients. Serum samples of brucellosis 50 patients and 50 well-matched healthy controls were analyzed for 228 metabolites, revealing significant alterations in 83 metabolites in brucellosis patients. Notably, disruptions were observed in key metabolite pathways, such as amino acid metabolism, urea cycle, tricarboxylic acid cycle (TCA), and fatty acid metabolism. Patients diagnosed with Brucellosis exhibited distinct differences in the levels of aspartate, glutamate, ß-alanine, and asparagine when compared to controls. Within the urea cycle, a significant downregulation of arginine was observed, whereas ornithine levels were considerably upregulated. In the TCA cycle, concentrations of 2-oxoglutarate, succinate, and malate were significantly elevated, while citrate levels demonstrated a notable decrease. Due to the interruption of the TCA cycle, glycolysis was accelerated to compensate for the resultant energy deficit in Brucella patients. Concurrently, there was a significant increase in the levels of short and medium-chain fatty acids, while long-chain fatty acids showed a marked decrease. The study systematically revealed significant metabolic alterations in Brucellosis patients and further explored the potential correlation between these changes and clinic symptoms, including fatigue, muscle soreness and prolonged fever. The results enhanced our understanding of Brucellosis, offering valuable insights potentially beneficial in formulating more effective treatment strategies and improving prognostic approaches.

Brucella-driven host N-glycome remodeling controls infection.

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

Intracellular growth of Brucella is mediated by Dps-dependent activation of ferritinophagy.

Bacteria of the genus Brucella cause brucellosis, one of the world's most common zoonotic diseases. A major contributor to Brucella's virulence is the ability to circumvent host immune defense mechanisms. Here, we find that the DNA-binding protein Dps from Brucella is secreted within the macrophage cytosol, modulating host iron homeostasis and mediating intracellular growth of Brucella. In addition to dampening iron-dependent production of reactive oxygen species (ROS), a key immune effector required for immediate bacterial clearance, cytosolic Dps mediates ferritinophagy activation to elevate intracellular free-iron levels, thereby promoting Brucella growth and inducing host cell necrosis. Inactivation of the ferritinophagy pathway by Ncoa4 gene knockout significantly inhibits intracellular growth of Brucella and host cell death. Our study uncovers an unconventional role of bacterial Dps, identifying a crucial virulence mechanism used by Brucella to adapt to the harsh environment inside macrophages.

Bacillus Calmette-Guérin-Trained Macrophages Elicit a Protective Inflammatory Response against the Pathogenic Bacteria Brucella abortus.

The bacillus Calmette-Guérin (BCG) can elicit enhanced innate immune responses against a wide range of infections, known as trained immunity. Brucella abortus is the causative agent of brucellosis, a debilitating disease that affects humans and animals. In this study, we demonstrate that C57BL/6 mouse bone marrow-derived macrophages under BCG training enhance inflammatory responses against B. abortus. BCG-trained macrophages showed increased MHC class II and CD40 expression on the cell surface and higher IL-6, IL-12, and IL-1ß production. The increase in IL-1ß secretion was accompanied by enhanced activation of canonical and noncanonical inflammasome platforms. We observed elevated caspase-11 expression and caspase-1 processing in BCG-trained macrophages in response to B. abortus compared with untrained cells. In addition, these BCG-trained cells showed higher NLRP3 expression after B. abortus infection. From a metabolic point of view, signaling through the Akt/mammalian target of rapamycin/S6 kinase pathway was also enhanced. In addition, BCG training resulted in higher inducible NO synthase expression and nitrite production, culminating in an improved macrophage-killing capacity against intracellular B. abortus. In vivo, we monitored a significant reduction in the bacterial burden in organs from BCG-trained C57BL/6 mice when compared with the untrained group. In addition, previous BCG immunization of RAG-1-deficient mice partially protects against Brucella infection, suggesting the important role of the innate immune compartment in this scenario. Furthermore, naive recipient mice that received BM transfer from BCG-trained donors showed greater resistance to B. abortus when compared with their untrained counterparts. These results demonstrate that BCG-induced trained immunity in mice results in better control of intracellular B. abortus in vivo and in vitro.

Exosomes released by Brucella-infected macrophages inhibit the intracellular survival of Brucella by promoting the polarization of M1 macrophages.

Exosomes, membrane vesicles released extracellularly from cells, contain nucleic acids, proteins, lipids and other components, allowing the transfer of material information between cells. Recent studies reported the role of exosomes in pathogenic microbial infection and host immune mechanisms. Brucella-invasive bodies can survive in host cells for a long time and cause chronic infection, which causes tissue damage. Whether exosomes are involved in host anti-Brucella congenital immune responses has not been reported. Here, we extracted and identified exosomes secreted by Brucella melitensis M5 (Exo-M5)-infected macrophages, and performed in vivo and in vitro studies to examine the effects of exosomes carrying antigen on the polarization of macrophages and immune activation. Exo-M5 promoted the polarization of M1 macrophages, which induced the significant secretion of M1 cytokines (tumour necrosis factor-α and interferon-γ) through NF-κB signalling pathways and inhibited the secretion of M2 cytokines (IL-10), thereby inhibiting the intracellular survival of Brucella. Exo-M5 activated innate immunity and promoted the release of IgG2a antibodies that protected mice from Brucella infection and reduced the parasitaemia of Brucella in the spleen. Furthermore, Exo-M5 contained Brucella antigen components, including Omp31 and OmpA. These results demonstrated that exosomes have an important role in immune responses against Brucella, which might help elucidate the mechanisms of host immunity against Brucella infection and aid the search for Brucella biomarkers and the development of new vaccine candidates.

Cytokine expression profile of B. melitensis-infected goat monocyte-derived macrophages.

Brucella parasitize the macrophage where is able to replicate and modulate the immune response in order to establish a chronic infection. The most adequate response to control and eliminate Brucella infection is a type 1 (Th1) cell-mediated effector immunity. Research in immune response of B. melitensis-infected goats is relatively scarce. In this study, we first evaluated changes in the gene expression of cytokines, a chemokine (CCL2) and the inducible nitric oxide synthase (iNOS) of goat macrophage cultures derived from monocytes (MDMs) infected for 4 and 24 h with Brucella melitensis strain 16 M. TNFα, IL-1ß and iNOS, and IL-12p40, IFNγ and also iNOS were significantly expressed (p < 0.05) at 4 and 24 h respectively, in infected compared to non-infected MDMs. Therefore, the in vitro challenge of goat MDMs with B. melitensis promoted a transcriptional profile consistent with a type 1 response. However, when the immune response to B. melitensis infection was contrasted between MDM cultures phenotypically restrictive or permissive to intracellular multiplication of B. melitensis 16 M, it was observed that the relative IL-4 mRNA expression was significantly higher in permissive macrophage cultures with respect to restrictive cultures (p < 0.05), independently of the time p.i. A similar trend, although non-statistical, was recorded for IL-10, but not for pro-inflammatory cytokines. Thus, the up-expression profile of inhibitory instead of pro-inflammatory cytokines could explain, in part, the difference observed in the ability to restrict intracellular replication of Brucella. In this sense, the present results make a significant contribution to the knowledge of the immune response induced by B. melitensis in macrophages of its preferential host species.

The Brucella Effector BspI Suppresses Inflammation via Inhibition of IRE1 Kinase Activity during Brucella Infection.

Mammalian GTPase-activating proteins (GAPs) can inhibit innate immunity signaling in a spatiotemporal fashion; however, the role of bacterial GAPs in mediating innate immunity remains unknown. In this study, we show that BspI, a Brucella type IV secretion system (T4SS) effector protein, containing a GAP domain at the C terminus, negatively regulates proinflammatory responses and host protection to Brucella abotus infection in a mouse model. In macrophages, BspI inhibits the activation of inositol-requiring enzyme 1 (IRE1) kinase, but it does not inhibit activation of ATF6 and PERK. BspI suppresses induction of proinflammatory cytokines via inhibiting the activity of IRE1 kinase caused by VceC, a type IV secretion system effector protein that localizes to the endoplasmic reticulum. Ectopically expressed BspI interacts with IRE1 in HeLa cells. The inhibitory function of BspI depends on its GAP domain but not on interaction with small GTPase Ras-associated binding protein 1B (RAB1B). Collectively, these data support a model where BspI, in a GAP domain-dependent manner, inhibits activation of IRE1 to prevent proinflammatory cytokine responses.

Brucella activates the host RIDD pathway to subvert BLOS1-directed immune defense.

The phagocytosis and destruction of pathogens in lysosomes constitute central elements of innate immune defense. Here, we show that Brucella, the causative agent of brucellosis, the most prevalent bacterial zoonosis globally, subverts this immune defense pathway by activating regulated IRE1α-dependent decay (RIDD) of Bloc1s1 mRNA encoding BLOS1, a protein that promotes endosome-lysosome fusion. RIDD-deficient cells and mice harboring a RIDD-incompetent variant of IRE1α were resistant to infection. Inactivation of the Bloc1s1 gene impaired the ability to assemble BLOC-1-related complex (BORC), resulting in differential recruitment of BORC-related lysosome trafficking components, perinuclear trafficking of Brucella-containing vacuoles (BCVs), and enhanced susceptibility to infection. The RIDD-resistant Bloc1s1 variant maintains the integrity of BORC and a higher-level association of BORC-related components that promote centrifugal lysosome trafficking, resulting in enhanced BCV peripheral trafficking and lysosomal destruction, and resistance to infection. These findings demonstrate that host RIDD activity on BLOS1 regulates Brucella intracellular parasitism by disrupting BORC-directed lysosomal trafficking. Notably, coronavirus murine hepatitis virus also subverted the RIDD-BLOS1 axis to promote intracellular replication. Our work establishes BLOS1 as a novel immune defense factor whose activity is hijacked by diverse pathogens.

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.

MicroRNA-155 expression with Brucella infection in vitro and in vivo and decreased serum levels of MicroRNA-155 in patients with brucellosis.

Infection with Brucella is characterized by the inhibition of host immune responses. MicroRNA-155 (miR-155) has been implicated in the immune response to many diseases. In this study, its expression during Brucella 16M infection of macrophages and mice was analyzed. Expression of miR-155 was significantly induced in macrophages at 24 h post infection. Further, an analysis of infected mice showed that miR-155 was inhibited at 7 and 14 days but induced at 28 days. Interestingly, this trend in induction or inhibition was reversed at 7 and 14 days in 16M△virB-infected mice. This suggested that decreased expression of miR-155 at an early stage of infection was dependent on intracellular replication. In humans with brucellosis, serum levels of miR-155 were significantly decreased compared to those in individuals without brucellosis and healthy volunteers. Significant correlations were observed between serum level of miR-155 and serum anti-Brucella antibody titers and the sweating symptom. This effect suggests that Brucella interferes with miR-155-regulated immune responses via a unique mechanism. Taken together, data from this study indicate that Brucella infection affects miR-155 expression and that human brucellosis patients show decreased serum levels of miR-155.

Clinical Effect of Doxycycline Combined with Compound Sulfamethoxazole and Rifampicin in the Treatment of Brucellosis Spondylitis.

OBJECTIVE: The purpose of this study was to determine the clinical value of triple antibiotic therapy consisting of doxycycline, compound sulfamethoxazole and rifampicin in the treatment of brucellosis spondylitis. METHODS: A retrospective analysis was performed on 100 patients with brucellosis spondylitis admitted to the First Affiliated Hospital of Hebei North University from March 2016 to June 2019. Patients were divided into the following two groups: the control group (n = 50) treated with dual antibiotic therapy (rifampicin + compound sulfamethoxazole), and the observation group (n = 50) treated with triple antibiotic therapy (rifampicin + doxycycline + compound sulfamethoxazole). The treatment effect, low back pain relief, levels of erythrocyte sedimentation rate (ESR), procalcitonin (PCT) and C-reactive protein (CRP), as well as the adverse reactions were compared between the two groups. RESULTS: The response rate of the observation group was significantly higher than that of the control group (P < 0.05). Before treatment, there was no significant difference in the low back pain assessed by the visual analogue scale (VAS), or levels of ESR, PCT and CRP between the two groups (P > 0.05). But after treatment, the VAS score and the levels of ESR, PCT and CRP in observation group were lower than those in the control group (P < 0.05). No significant difference was found in the incidence of adverse reactions (P > 0.05). CONCLUSION: The triple antibiotic therapy of doxycycline, compound sulfamethoxazole and rifampicin is effective in the treatment of brucellosis spondylitis. It can significantly alleviate patients' back pain and inflammation with a high safety profile, which is worthy of clinical application.

Omp31 of Brucella Inhibits NF-κB p65 Signaling Pathway by Inducing Autophagy in BV-2 Microglia.

Neurobrucellosis is a serious central nervous system (CNS) inflammatory disorder caused by Brucella, and outer membrane protein-31 (Omp31) plays an important role in Brucella infection. This study aims to determine whether Omp31 can induce autophagy in BV-2 microglia. Another goal of the study is to further examine the effect of autophagy on the nuclear transcription factor κB (NF-κB) p65 signaling pathway. We observed that Omp31 stimulated autophagy by increasing microtubule-associated protein 1 light chain 3B (LC3B-II) levels and inducing autophagosome formation at 6 h and 12 h. Concomitantly, Omp31 induced tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) expression in a time-dependent manner but reduced the expression of TNF-α at 6 h. We utilized Omp31 with or without rapamycin or 3-methyladenine (3-MA) to treat BV-2 microglia, and it demonstrated further that Omp31 induced autophagy by promoting LC3B-II, Beclin-1 proteins expression and inhibiting the p62 protein levels. Furthermore, we explored the effects of autophagy on the NF-κB p65 pathway through western blot analysis, RT-qPCR assay, enzyme-linked immunosorbent assay (ELISA) and immunofluorescence. The data suggest that Omp31 as well as rapamycin, the autophagy inducer, can decrease TNF-α levels through the inhibition of the NF-κB p65 signaling pathway. Taken together, Omp31 can function as a catalyst in both autophagy induction and NF-κB p65 signal inhibition. Furthermore, Omp31-induced autophagy may inhibit the expression of TNF-α by negatively regulating NF-κB p65 signaling pathway.

The Brucella effector BspL targets the ER-associated degradation (ERAD) pathway and delays bacterial egress from infected cells.

Perturbation of the endoplasmic reticulum (ER), a central organelle of the cell, can have critical consequences for cellular homeostasis. An elaborate surveillance system known as ER quality control ensures that cells can respond and adapt to stress via the unfolded protein response (UPR) and that only correctly assembled proteins reach their destination. Interestingly, several bacterial pathogens hijack the ER to establish an infection. However, it remains poorly understood how bacterial pathogens exploit ER quality-control functions to complete their intracellular cycle. Brucella spp. replicate extensively within an ER-derived niche, which evolves into specialized vacuoles suited for exit from infected cells. Here we present Brucella-secreted protein L (BspL), a Brucella abortus effector that interacts with Herp, a central component of the ER-associated degradation (ERAD) machinery. We found that BspL enhances ERAD at the late stages of the infection. BspL targeting of Herp and ERAD allows tight control of the kinetics of autophagic Brucella-containing vacuole formation, delaying the last step of its intracellular cycle and cell-to-cell spread. This study highlights a mechanism by which a bacterial pathogen hijacks ERAD components for fine regulation of its intracellular trafficking.

Brucella melitensis UGPase inhibits the activation of NF-κB by modulating the ubiquitination of NEMO.

BACKGROUND: UTP-glucose-1-phosphoryl transferase (UGPase) catalyzes the synthesis of UDP-glucose, which is essential for generating the glycogen needed for the synthesis of bacterial lipopolysaccharide (LPS) and capsular polysaccharide, which play important roles in bacterial virulence. However, the molecular function of UGPase in Brucella is still unknown. RESULTS: In this study, the ubiquitination modification of host immune-related protein in cells infected with UGPase-deleted or wild-type Brucella was analyzed using ubiquitination proteomics technology. The ubiquitination modification level and type of NF-κB Essential Modulator (NEMO or Ikbkg), a molecule necessary for NF-κB signal activation, was evaluated using Coimmunoprecipitation, Western blot, and dual-Luciferase Assay. We found 80 ubiquitin proteins were upregulated and 203 ubiquitin proteins were downregulated in cells infected with B. melitensis 16 M compared with those of B. melitensis UGPase-deleted strain (16 M-UGPase-). Moreover, the ubiquitin-modified proteins were mostly enriched in the categories of regulation of kinase/NF-κB signaling and response to a bacterium, suggesting Brucella UGPase inhibits ubiquitin modification of related proteins in the host NF-κB signaling pathway. Further analysis showed that the ubiquitination levels of NEMO K63 (K63-Ub) and Met1 (Met1-Ub) were significantly increased in the 16 M-UGPase--infected cells compared with that of the 16 M-infected cells, further confirming that the ubiquitination levels of NF-κB signaling-related proteins were regulated by the bacterial UGPase. Besides, the expression level of IκBα was decreased, but the level of p-P65 was significantly increased in the 16 M-UGPase--infected cells compared with that of the 16 M- and mock-infected cells, demonstrating that B. melitensis UGPase can significantly inhibit the degradation of IκBα and the phosphorylation of p65, and thus suppressing the NF-κB pathway. CONCLUSIONS: The results of this study showed that Brucella melitensis UGPase inhibits the activation of NF-κB by modulating the ubiquitination of NEMO, which will provide a new scientific basis for the study of immune mechanisms induced by Brucella.

A Brucella effector modulates the Arf6-Rab8a GTPase cascade to promote intravacuolar replication.

Remodeling of host cellular membrane transport pathways is a common pathogenic trait of many intracellular microbes that is essential to their intravacuolar life cycle and proliferation. The bacterium Brucella abortus generates a host endoplasmic reticulum-derived vacuole (rBCV) that supports its intracellular growth, via VirB Type IV secretion system-mediated delivery of effector proteins, whose functions and mode of action are mostly unknown. Here, we show that the effector BspF specifically promotes Brucella replication within rBCVs by interfering with vesicular transport between the trans-Golgi network (TGN) and recycling endocytic compartment. BspF targeted the recycling endosome, inhibited retrograde traffic to the TGN, and interacted with the Arf6 GTPase-activating Protein (GAP) ACAP1 to dysregulate Arf6-/Rab8a-dependent transport within the recycling endosome, which resulted in accretion of TGN-associated vesicles by rBCVs and enhanced bacterial growth. Altogether, these findings provide mechanistic insight into bacterial modulation of membrane transport used to promote their own proliferation within intracellular vacuoles.