Bacterial effectors are considered potent weapons deployed into plant cells to suppress host immunity. Pseudomonas syringae pv. actinidiae (Psa), the causal agent of kiwifruit bacterial canker, is a devastating pathogen posing a global threat to kiwifruit production. Although Psa delivers multiple effectors into plant cells, the molecular mechanisms governing effector-mediated susceptibility remain poorly understood. Here, we report that HopBB1-1 and HopBB1-2, the sole pair of homologous proteins in PsaM228, function redundantly in virulence. Whereas deletion of either effector individually did not affect bacterial pathogenicity, simultaneous deletion of both significantly reduced bacterial growth in planta. Both effectors can induce cell death and activate a series of immune response individually in Nicotiana benthamiana, and we further found that HopBB1-1 and HopBB1-2 both interacted with a plant protein, DNA damage repair and tolerance, DRT100, in both nonhost and host plants. Overexpression of AcDRT100 enhanced the kiwifruit resistance to Psa, underscoring its positive role in plant immunity and potential as a candidate gene for breeding resistant cultivars. Crucially, the virulence function of HopBB1-1/HopBB1-2 depends on AcDRT100, and NbDRT100 is required for HopBB1-1/HopBB1-2-induced cell death in N. benthamiana, indicating that DRT100 acts as a common target in both plants. Our findings uncover a sophisticated and precise effector-target module in Psa pathogenesis, revealing how redundant virulence functions are directed at a key regulator of host immunity, DRT100, which we identify as a promising candidate for developing disease-resistant varieties.

IntroductionBrucella is a Gram-negative facultative intracellular bacterium that can cause fever, abortion, and other symptoms in humans and various mammals. btpB, a type IV secretion system (T4SS) effector of Brucella, plays a critical role in regulating Brucella infection and inhibiting the host's innate immune response.MethodsIn this study, a btpB mutant strain of Brucella A19 (ΔbtpB) was constructed using homologous recombination, and its biological characteristics, virulence, and immunogenicity were systematically investigated.ResultsThe results showed that ΔbtpB exhibited weakened resistance to in vitro stress, while its growth characteristics did not differ significantly from the wild-type strain A19. In the mouse immunization model, ΔbtpB induced weaker splenic pathological damage, and the splenic bacterial load was significantly lower than that of A19, indicating its reduced virulence. Additionally, ΔbtpB infection elicited stronger humoral and cellular immune responses in mice, including higher antibody levels, increased levels of Th1 cytokines (such as IFN-γ and IL-2), and enhanced proliferation and activity of CD8+ cells. Detection of Th1 and Th2 cells revealed that ΔbtpB induced stronger Th1 and Th2 responses in the spleen in the early stage, but the Th2 response weakened in the middle and late stages of infection. Notably, ΔbtpB infection did not suppress natural killer (NK) cell activity and even significantly enhanced its cytotoxic activity compared to the A19 strain.ConclusionOur research demonstrates that ΔbtpB leads to a reduced survival capacity of Brucella, while enhancing its immunogenicity. This suggests btpB is an important target for the prevention of Brucella.

Brucella is a Gram-negative facultative intracellular pathogen that causes widespread zoonotic infections. Macrophages are crucial immune and antigen-presenting cells that differentiate into proinflammatory M1-type or anti-inflammatory M2-type cells in different microenvironments. The BtpB protein of Brucella is a type IV secretion system effector protein that modulates host inflammatory responses by inhibiting Toll-like receptor signaling and controlling dendritic cell activation. A high expression of intracellular proinflammatory factors is induced in B. suis deficient in BtpB (B. suis mutant strain ΔbtpB). However, the role of BtpB in macrophage polarization triggered by Brucella infection is uncertain. In this study, RAW264.7 macrophages were utilized as a model to investigate the impact of BtpB on macrophage polarization. The cells were infected with wild-type B. suis strain S2, B. suis mutant strain ΔbtpB, or the B. suis complemented strain C-ΔbtpB followed by flow cytometry, reverse transcription quantitative PCR (RT-qPCR), western blotting, immunohistochemistry, and metabolic detection analyses. The expression of BtpB suppressed M1 polarization and promoted M2 macrophage polarization. Moreover, BtpB upregulated the expression of signal transducer and activator of transcription 3 (STAT3) in host cells. Inhibition of STAT3 promoted the expression of nitric oxide synthase (NOS2) in cells infected with either B. suis or C-ΔbtpB, with no observable differences compared to cells infected with ΔbtpB. Conversely, STAT3 overexpression resulted in a downregulation of NOS2 levels in both wild-type and mutant cells. These findings were corroborated by messenger RNA (mRNA) assay and enzyme-linked immunosorbent assay (ELISA) that confirmed the regulatory role of STAT3 in modulating NOS2 expression. Additionally, intracellular proliferation assays under STAT3-modulated conditions indicated that STAT3 suppresses replication of Brucella. In conclusion, the results demonstrate that BtpB inhibits M1 polarization in macrophages by regulating STAT3 expression which provides a strong foundation for improved understanding of Brucella infection mechanisms.

Brucellosis, a severe zoonotic infectious disease, poses substantial economic and health threats globally. The intracellular survival strategy of Brucella complicates disease control, highlighting the need for novel immunotherapeutic strategies such as antibody-based therapies and multi-epitope vaccines. This study generated two IgM monoclonal antibodies (D3 and F5) against the conserved outer membrane protein OMP16 of Brucella using hybridoma technology. Peptide scanning and Western blot identified their linear epitopes (D3: 77TLSKQAQW84; F5: 120RDFLASRG127), which are highly conserved among major Brucella species. Integrated approaches—including molecular docking, alanine-scanning mutagenesis, and dot-blot assays—revealed key residues at the epitope interface that form stable bonds with antibody complementarity-determining regions (CDRs). Functionally, both antibodies activated the complement system, with F5 exhibiting significant complement-dependent bacteriolytic activity in vitro. Furthermore, in the presence of complement, D3 and F5 enhanced macrophage-mediated opsonophagocytosis and intracellular killing of Brucella abortus A19. In a mouse infection model, passive immunization with either antibody significantly alleviated infection-induced weight loss and splenomegaly and reduced bacterial load in the spleen. Our study underscores the role of IgM antibodies in combating Brucella infection, offers insights for antibody-based immunotherapy, and provides a theoretical foundation for developing multi-epitope vaccines based on the conserved epitopes and critical residues.

Brucella spp., the causative agents of brucellosis, are facultative intracellular pathogens lacking classical virulence factors, and the disease represents one of the most prevalent zoonoses worldwide. Nevertheless, Brucella efficiently modulates the expression of genes associated with host cellular metabolism and immune evasion. The SIRT1 protein is a member of the NAD(+)-dependent class III histone deacetylase family that regulates apoptosis, autophagy, and inflammatory responses during cellular stress. In this study, we demonstrate that B. abortus infection of macrophage cells downregulates the expression of SIRT1. SIRT1 negatively regulated B. abortus intracellular proliferation. Infection reduced NAD(+) levels in macrophages, but stimulation of SIRT1 expression with resveratrol attenuated this inhibitory effect. SIRT1 negatively regulated B. abortus-induced acetylation modification of the FOXO1 transcription factor. Furthermore, B. abortus infection promoted the increase of the LC3-II and p62 autophagy markers in macrophages while inhibiting expression of the Atg7 marker. Conversely, resveratrol treatment decreased B. abortus-induced LC3-II and p62 expression and promoted Atg7 expression, whereas knockdown of SIRT1 exerted the opposing effects. In addition, B. abortus infection impaired lysosomal function by inhibiting key proteins m-CTSB and m-CTSD, reducing phagosome-lysosome fusion, and promoting phagosome accumulation. Resveratrol-mediated SIRT1 activation alleviated B. abortus-induced inhibition of m-CTSB and m-CTSD expression. In conclusion, our findings reveal that B. abortus modulates intracellular proliferation and macrophage lysosomal function through SIRT1, which leads to autophagic modulation, thereby providing a novel therapeutic target for treatment of brucellosis.

Abstract Brucellosis, a major global zoonosis causing over 2.1 million human infections annually, poses a significant threat to public health and livestock economies. Conventional vaccines face substantial limitations, including residual virulence and diagnostic interference in live attenuated vaccines, and poor immunogenicity in subunit vaccines. To address these issues, we developed BP26-OMP16Es, a self-assembling nanoparticle vaccine constructed by fusing immunodominant B- and T-cell epitopes from OMP16 to the self-assembling antigen BP26 via flexible linkers, yielding single-copy (1×) and double-copy (2×) epitope tandem nanoparticles. These particles formed homogeneous, barrel-shaped structures (14–23 nm) with strong immunoreactivity against Brucella-specific antibodies. In murine models, the vaccine elicited potent humoral and cellular immune responses, characterized by high antibody titers, enhanced IFN-γ+ CD8+ T cell proliferation, a balanced Th1/Th2 profile, and the induction of immunological memory for durable protection. Immune sera also mediated effective clearance of infected macrophages via antibody-dependent cellular cytotoxicity (ADCC). Challenge experiments confirmed that the vaccine significantly reduced splenic Brucella burden and alleviated pathological damage, with the 2× construct offering superior protection. In summary, this study integrates self-assembling nanotechnology with a multiepitope strategy to create a safe and potent vaccine platform. The 2× design enhances epitope density, boosting immunogenicity and conferring solid protection against Brucella infection. This approach provides valuable insights for developing vaccines against brucellosis and other intracellular pathogens.

Persister cells constitute a subpopulation of dormant cells that are transiently tolerant to antibiotics and associated with chronic infection. We have known (p)ppGpp synthetase Rsh promoted persister cells formation during rifampicin exposure in Brucella, but the metabolic regulation mechanism by which Rsh promotes persister cells formation is unknown. In this study, we firstly characterized the proteome and metabolome of persister cells formation during rifampicin exposure between B. abortus A19 (WT) and Δrsh strains. We found that sulfur metabolism-related protein (NAD(P)/FAD-dependent sulfite reductase, cysI) was significantly down-regulated in Δrsh compared with WT strain during persister cells formation (P < 0.01). The Rsh significantly affected hydrogen sulfide consumption by hydrogen sulfide measurement (P < 0.01). And then we found Rsh positively regulated CysI that its promoter region was 122 bp of upstream of cysI gene. In addition, the cysI knockout strain (ΔcysI) was constructed which significantly reduced persister cells formation (P < 0.01) and also reduced consumption of hydrogen sulfide compared with WT strain. Taken together, we firstly reported that (p)ppGpp synthetase Rsh promotes persister cells formation through regulating sulfur metabolism mediated by sulfite reductase CysI in B. abortus. Our results provide a potential target and new strategy for clearing persister cells as well as prevention of Brucella infection.

Brucella spp. are intracellular pathogens utilizing a unique, asymmetric cell cycle to survive within host macrophages. While the biogenesis of the replicative niche is well-studied, the intrinsic regulatory circuits that control the Brucella cell cycle remain incompletely understood. Phosphodiesterase (PDEs) modulates c-di-GMP levels and thereby contribute to the bacterial cell cycle. However, little is known about the role of PDEs in the cell cycle and mechanisms of B. abortus. In this study, we firstly demonstrate that BpdE is a high conserved protein across major Brucella species. Deletion of bpdE (ΔbpdE) significantly accelerated bacterial proliferation in vitro and enhanced intracellular survival within host macrophages. We found that ΔbpdE mutant exhibited an accumulation of DNA content compared to B. abortus A19 (WT) and complemented strain (CΔbpdE). BpdE significantly affected the transcription of cell cycle regulators, including the min system. Additionally, we confirmed that the minCDE genes constitute a single operon in B. abortus. Furthermore, β-galactosidase reporter assays demonstrated that BpdE positively regulates the activity of the minCDE promoter. Taken together, we reported that the c-di-GMP phosphodiesterase BpdE represses bacterial proliferation by regulating minCDE operon in B. abortus. Our results provide a novel regulatory axis for bacterial proliferation and offer new insights into the intricate mechanisms of Brucella pathogenesis.

The host defense system produces high levels of oxidation products, including reactive oxygen species (ROS) and reactive nitrogen species (RNS), which disrupt bacterial metabolism and exert lethal effects. Crucially, Brucella counteracts oxidative stress by upregulating antioxidant enzymes-such as superoxide dismutase, catalase, and peroxidase. Central to this defense is OxyR, a peroxide-sensing transcriptional regulator that orchestrates antioxidant enzyme expression to mitigate oxidative damage. To define OxyR's role in Brucella homeostasis and host interactions, we constructed oxyR deletion and complemented strains using homologous recombination. Our study evaluated OxyR's impact on growth, stress responses (particularly oxidative stress), intracellular survival, and modulation of host inflammation. Results revealed that oxyR deletion caused significant growth retardation and reduced tolerance to acid stress, polymyxin B, and oxidative stress, concurrently downregulating both expression and activity of key antioxidant enzymes. Although intracellular survival within murine macrophages remained unaffected, oxyR deletion substantially suppressed critical inflammatory cytokine expression. In summary, OxyR is essential for optimal growth and stress resistance, exhibiting dual functionality in mitigating oxidative damage and modulating host inflammatory responses. These findings provide novel insights into Brucella immune evasion strategy.

Brucellosis, a global zoonosis, faces significant challenges from lacking safe vaccines and therapies. OMP16, a key Brucella virulence factor essential for bacterial survival, also functions as a protective antigen capable of activating host immunity. However, not all OMP16-targeting antibodies confer immunoprotection; antibodies against non-protective epitopes may consume immune resources and even exacerbate infection. To validate the protective potential of two OMP16-targeting monoclonal antibodies (H4/E6) and identify their corresponding epitopes, we established in vitro models showing both mediate macrophage opsonophagocytosis, bactericidal activity, and NK cell-mediated ADCC. Notably, E6 additionally triggered complement-dependent bacteriolysis. Murine challenge studies confirmed that both mAbs reduce bacterial loads and mitigate splenic/hepatic pathology. Based on this protection, we then employed integrated epitope mapping, interaction modeling, and alanine-scanning mutagenesis, revealing two linear B-cell epitopes essential for immunoprotection, alongside critical binding residues. This work not only provides foundational evidence for OMP16-based antibody therapy but also reports the first two validated protective OMP16 epitopes. By deciphering molecular mechanisms of these mAbs, we deliver precision epitopes for next-generation multi-epitope vaccines, offering targeted solutions against brucellosis.

Arbutin is a naturally present antioxidant derived from plants. The objective of this study was to examine the effect of arbutin on boar sperm during storage at 17°C and the underlying mechanisms. Our results showed that the addition of arbutin to extenders markedly enhanced the sperm (progressive) motility and plasma membrane and acrosomal integrity on Days 9 and 13 of preservation (p < 0.05), with the most pronounced effect of arbutin at the concentration of 100 μmol/L. The addition of 100 μmol/L arbutin also reduced the level of ROS and elevated the levels of ATP and MMP in boar sperm on Days 9 and 13 of preservation (p < 0.05). The subsequent sperm oxidative damage experiment showed that the addition of 100 μmol/L arbutin significantly alleviated the decrease in sperm (progressive) motility and in plasma membrane and acrosomal integrity caused by H2O2 (p < 0.05), whereas increased the T-AOC content and the activities of CAT and GPx antioxidant enzymes after 2 h of incubation at 37°C (p < 0.05). Further, the metabolomic analysis revealed that the addition of arbutin principally influenced lipid metabolism, and the Western blot analysis demonstrated that arbutin increased the sperm quality and the antioxidant capacity via the NRF2/GPX4 signalling. Together, arbutin preserves boar sperm during storage at 17°C by enhancing the antioxidant capacity via the NRF2/GPX4 signalling, laying the theoretical foundation for optimisation of the boar semen preservation diluent therefore facilitating the dissemination of superior porcine germplasm resources and improving the economic value.

Brucella is a successful pathogen that employs a plethora of immune evasion mechanisms. This contributes to pathogenesis and persistence and limits the efficacy of available treatments. An increasing understanding of host‒pathogen interactions suggests that integrating host-directed strategies with existing anti-Brucella treatments could lead to more effective bacterial clearance and a reduction in drug-resistant strains. SIRT2 is a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase found in mammals. It can deacetylate various transcription factors and regulatory proteins, playing crucial roles in host‒pathogen interactions and pathogen infection-induced apoptosis. In this study, we investigated the role of SIRT2 in Brucella-induced cell apoptosis using bovine placental trophoblast cells. Our results indicate that B. abortus A19 infection upregulates SIRT2 protein expression and significantly induces mitochondrial apoptosis in these cells. Furthermore, inhibition of SIRT2 exacerbates B. abortus A19-induced mitochondrial apoptosis and markedly inhibits intracellular bacterial survival. These results prove the role of SIRT2 in Brucella pathogenesis and the mechanism of action.

Biofilms are complex adhesive structures that establish chronic infection and allow robust protection from external stressors such as antibiotics. Cellulose as one of the compositions of bacteria biofilm which protect bacteria from stress, host immune responses and resistance to antibiotics. Bacterial stress responses are regulated via guanosine pentaphosphate and tetraphosphate (p)ppGpp. This molecule has been a target of research efforts to counteract biofilm formation in pathogenic bacteria. However, a role for (p)ppGpp synthetase Rel influencing in biofilms and its cellulose formation has not been identified in Brucella. Firstly, rel mutant significantly decreased biofilm biomass and rendered biofilms more susceptible to most antibiotics. The rel mutant also showed greatly decreased biofilm architectures including exopolysaccharide, extracellular DNA, and lipid. Remarkably, we found rel mutant significantly decreased biofilm cellulose formation. We further combined proteomic analysis to explore the key proteins involved in cellulose regulation of Rel in Brucella biofilm formation. 287 differentially expressed proteins (DEPs) were identified and enriched in diverse metabolic pathway between WT and Δrel strains including purine and sulfur metabolism, transcription factors and glycosyltransferases which may be related to cellulose formation. The Q-PCR showed that mRNA levels of only glycosyltransferase (WP_006161578.1) of the 12 down-DFPs had significantly upregulated in rel mutant contrast to WT strain and β-galactosidase assay showed a negative regulatory in rel mutant. Furthermore, Rel-dependent biofilms cellulose was also restored and accompanied by an increase in glycosyltransferase (WP_006161578.1) when glucose was added in TSB medium. Overall, this work expands the role of (p)ppGpp synthetase Rel as an important regulator in biofilm and cellulose formation that is tightly linked with pathogenicity and chronic persistent infections in Brucella.

As an RNA-binding protein, Quaking (QKI) plays a pivotal role in regulating RNA metabolism, including mRNA transcription, pre-mRNA splicing, RNA localization, and RNA stability. To further investigate its specific role in mammalian cells, a QKI-knockout NIH3T3 cell line was generated using CRISPR/Cas9 technology in this study. RNA sequencing analysis showed that QKI deficiency alters several biological processes in NIH3T3 cells, including the Wnt signaling pathway, regulation of epithelial cell proliferation, epithelial cell and tissue migration. Functional analyses revealed that QKI deficiency potently suppressed cell proliferation and migration in NIH3T3 cells. In addition, SFRP1, a negative regulator of the Wnt signaling pathway, was significantly upregulated in QKI knockout NIH3T3 cells. Notably, the expression of several Wnt signaling pathway-related factors (WNT5A, FZD8, β-catenin, CCND1 and CCN4) was significantly downregulated in QKI knockout NIH3T3 cells. CLIP-seq analysis further identified a fragment with the 3'UTR of Sfrp1 mRNA that interacts with QKI and contains two canonical QKI response elements (QKI-REs). Dual-luciferase reporter assays verified that QKI exerts its inhibitory effect by binding to QKI-RE1 within the 3'UTR of Sfrp1 mRNA. Additionally, the actinomycin D assay demonstrated that QKI regulates Sfrp1 expression by suppressing Sfrp1 mRNA stability. Moreover, SFRP1 overexpression inhibited the cell proliferation and migration of NIH3T3 cells, mirroring the effects of QKI knockout. In summary, these findings demonstrate that QKI inhibits Sfrp1 expression by binding to QKI-RE1 within its 3'UTR, leading to activation of the Wnt signaling pathway and subsequent promotion of cell proliferation and cell migration. This study identifies a QKI-SFRP1-Wnt regulatory axis that expands the functional repertoire of QKI in post-transcriptional control of cell dynamics.

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.

Cartilage repair is the key to the treatment of joint-related injury. However, because cartilage lacks vessels and nerves, its self-repair ability is extremely low. Extracellular vesicles (EVs) are bilayer nanovesicles with membranes mainly composed of ceramides, cholesterol, phosphoglycerides, and long-chain free fatty acids, containing DNA, RNA, and proteins (such as integrins and enzymes). For mediating intercellular communication and regulating mechanisms, EVs have been shown by multiple studies to be effective treatment options for cartilage repair. This review summarizes recent findings of different sources (mammals, plants, and bacteria) and uses of EVs in cartilage repair, mechanisms of EVs captured by injured chondrocytes, and quantification and storage of EVs, which may provide scientific guidance for promoting the development of EVs in the field of cartilage injury treatment.

The bacterial flagellum is an elongated filament that protrudes from the cell and is responsible for bacterial motility. It can also be a pathogen-associated molecular pattern (PAMP) that regulates the host immune response and is involved in bacterial pathogenicity. In contrast to motile bacteria, the Brucella flagellum does not serve a motile purpose. Instead, it plays a role in regulating Brucella virulence and the host‘s immune response, similar to other non-motile bacteria. The flagellin protein, FliK, plays a key role in assembly of the flagellum and also as a potential virulence factor involved in the regulation of bacterial virulence and pathogenicity. In this study, we generated a Brucella suis S2 flik gene deletion strain and its complemented strain and found that deletion of the flik gene has no significant effect on the main biological properties of Brucella, but significantly enhanced the inflammatory response induced by Brucella infection of RAW264.7 macrophages. Further experiments demonstrated that the FliK protein was able to inhibit LPS-induced cellular inflammatory responses by down-regulating the expression of MyD88 and NF-κB, and by decreasing p65 phosphorylation in the NF-κB pathway; it also inhibited the expression of NLRP3 and caspase-1 in the NLRP3 inflammasome pathway. In conclusion, our study suggests that Brucella FliK may act as a virulence factor involved in the regulation of Brucella pathogenicity and modulation of the host immune response.

Persister cells are transiently tolerant to antibiotics and are associated with recalcitrant chronic infections due to recolonization of host cells after antibiotic removal. Brucella spp. are facultative pathogens that establish intracellular infection cycles in host cells which results in chronic persistent infections. Brucella abortus forms multi-drug persister cells which are promoted by the (p)ppGpp synthetase Rsh during rifampicin exposure. Here, we confirmed that Rsh promoted persister cells formation in B. abortus stationary phase treated with rifampicin and enrofloxacin. Deletion of the gene for Rsh decreased persister cells level in the presence of these drugs in different growth phases. However, persister cells formation by deletion strain varied in different growth phases in the presence of other antibiotics. Rsh also was involved in persister cells formation during rifampicin treatment under certain stress conditions, including acidic conditions, exposure to PBS, and heat stress. Moreover, Rsh impacted persister cell levels during rifampicin or enrofloxacin treatment in RAW264.7 macrophages. Certain typeIItoxin-antitoxin modules were upregulated under various stress conditions in B. abortus. We established that Rsh positively regulated the type II toxin-antitoxin mbcTA. Moreover, rifampicin-tolerant persister cells formation was elevated and ATP levels were decreased when mbcTA promoter was overexpressed in Rsh deletion background in stationary phase. Our results establish that (p)ppGpp synthetase Rsh plays a key role in B. abortus persistence and may serve as a potent novel target in combination with rifampicin in the development of new therapeutic approaches and prevention strategies to treat chronic infections of Brucella.

Brucella abortus is facultative intracellular pathogen that causes chronic persistent infections and results in abortion and infertility in food animals. Recurrent infections can be one of the results of persister cells formation that transiently displays phenotypic tolerance to high dose of antibiotics treatment. We examined persister cells formation of B. abortus strain A19 in stationary phase and investigated a potential role for the (p)ppGpp synthetase Rsh in this process. We found that B. abortus stationary phase cells can produce higher levels of multi-drugs tolerant persister cells in vitro under high dose of antibiotics (20 × MIC) exposure than do exponential phase cells. Persister cell formation was also induced with environmental stressors pH 4.5, 0.01 M PBS (pH7.0), 2% NaCl and 25 °C, upon exposure to ampicillin, enrofloxacin and rifampicin. Persister cells were not formed following exposure to 1 mM H2O2. The numbers of persister cells were significantly increased following uptake of B. abortus stationary phase cells by RAW264.7 macrophages in contrast with cultures in TSB liquid medium. Environmental stressors to B. abortus significantly increased expression of rsh mRNA level. The rsh null mutant (Δrsh) formed significantly fewer persister cells than the complemented (CΔrsh) and wildtype (WT) strains under high dose of rifampicin in vitro. These data for the first time demonstrate that B. abortus can produce multi-drug tolerant persister cells in stationary phase. The (p)ppGpp synthetase Rsh is necessary for persister cell formation in B. abortus in the presence of rifampicin. On this basis, a new understanding of the recurrent infections of Brucella was advanced, thus provided a new basis for revelation of pathogenic mechanism of the chronic persistent infection in Brucella.