Brucellosis, caused by various Brucella species, is a global zoonotic disease affecting over 170 countries, with 1.6–2.1 million new human cases reported annually. In China, it is categorized as a class B infectious disease (Yang H. et al., 2020; Qureshi et al., 2023). Brucella species are host-specific, and include B. abortus in cattle, B. melitensis in goats and camels, B. suis in pigs, B. ovis in sheep, B. abortus in camels, elk, and bison, B. canis in dogs, B. neotomae and B. microti in rodents, B. papionis in monkeys, B. pinnipedialis and B. ceti in marine mammals (e.g., seals, dolphins, and whales), and B. inopinata in amphibians. Brucella can infect a variety of terrestrial and marine mammals, among which B. melitensis is the most pathogenic and invasive species for humans, followed by B. abortus, B. suis, and B. canis (Qin et al., 2008; Celli, 2019; Bialer et al., 2020; Gunes and Dogan, 2013; Waktole et al., 2022).

Brucella infects humans through several routes: (1) mucous membranes of the gastrointestinal tract, primarily following consumption of contaminated unpasteurized dairy or undercooked meats; (2) mucous membranes of the respiratory tract, primarily following inhalation of aerosolized bacteria; and (3) other mucous membranes and damaged skin, primarily following exposure to infected animals (Atluri et al., 2011; Yu et al., 2024). For humans, Brucella infection can be either asymptomatic or symptomatic, with an incubation period of 1–5 weeks, depending on the virulence of the pathogen. Based on the duration of symptoms, brucellosis is typically classified as acute (≤ 8 weeks), subacute (8–52 weeks), or chronic (> 1 year) (Deng Y. et al., 2019). Because of its intracellular parasitism and immune evasion features, Brucella infection can easily change from an acute condition to a chronic, refractory disease with multiple complications. These include the following: (1) skeletal infections that cause arthritis and joint pain, which can potentially lead to disability; (2) genitourinary system infections, leading to epididymis-orchitis in males and miscarriage in females; and (3) infections of other systems, with symptoms that include weakness, muscle pain, flu-like symptoms, undulant fever, endocarditis, hepatitis, meningitis, and neurological issues (Atluri et al., 2011; Yu et al., 2024; Głowacka et al., 2018; Khairullah et al., 2024). People of all ages and both sexes are at risk for brucellosis, with unreported symptomatic cases estimated to be 10 times higher than the reported cases (Khairullah et al., 2024). Despite its low case-fatality rate, brucellosis can significantly impact quality of life. In animal hosts, Brucella primarily invades reproductive systems (e.g., placenta, mammary glands, and epididymis), causing infertility, miscarriage, weight loss, and reduced milk production, seriously impacting animal husbandry (Bialer et al., 2020; Atluri et al., 2011).

Due to its multiple features, Brucella is considered as a biological weapon. As a Gram-negative facultative intracellular bacterium, it requires as few as 10–100 organisms to efficiently infect a host via aerosol transmission. It can also spread through various routes, including contact with infected animals and ingestion of contaminated food. Brucella is easy to cultivate, store, and transport from biological samples, and it remains stable in environments such as dairy products and soil. Infection can lead to chronic brucellosis, which is characterized by prolonged fever and organ damage. Although the direct mortality rate is low, the disease can place a heavy burden on healthcare systems and cause widespread public panic. As a zoonotic pathogen, it also poses a threat to livestock and may lead to agricultural security crises. In the past, Brucella was considered as a candidate for biological weapon development. Due to its high infectivity, ease of acquisition, and potential socio-economic impact, the U.S. Centers for Disease Control and Prevention (CDC) has classified it as a category B bioterrorism agent (Doganay and Doganay, 2013).

Currently, several live-attenuated vaccines have been approved for the prevention of brucellosis in animals, including B. melitensis Rev. 1, B. abortus RB51, and B. abortus S19. B. melitensis Rev. 1 and B. abortus S19 are both smooth live-attenuated vaccines and are considered the two traditional vaccines for the prevention and control of animal brucellosis. They also serve as important reference standards for evaluating novel vaccine candidates. B. melitensis Rev. 1 is currently the most effective vaccine for goats and sheep. B. abortus S19 is used to prevent Brucella infection in cattle. B. abortus RB51 is a genetically engineered, rough-type live-attenuated vaccine developed for the control of bovine brucellosis. It is considered a promising candidate for replacing the traditional S19 vaccine, particularly in situations where its lack of O antigen helps avoid serological interference (Heidary et al., 2022). Although these vaccines have shown some effectiveness in controlling the spread of brucellosis among animals, their efficacy varies depending on host species, vaccine dosage, and the route of administration (Yang et al., 2013; Wang and Wu, 2013). Meanwhile, they have clear limitations. In some cases, they may induce abortion in vaccinated animals, and due to their relatively high virulence, they are not suitable for human use (Perkins et al., 2010). To date, no brucellosis vaccine has been approved for use in humans (Wells et al., 2022). Vaccine development remains challenging, and most current strategies have achieved limited success. The pathogenesis of Brucella, including its mechanisms of immune evasion and persistent infection, is still not fully understood. Scare pathogen-associated molecular patterns (PAMPs) with strong immunogenicity in Brucella significantly reduces the availability of effective targets for vaccine development. Moreover, the high genetic variability and diversity of Brucella strains make it difficult for vaccines based on a single strain or antigen to provide broad and effective protection against multiple variants. In practical vaccine development, traditional live attenuated vaccines can induce long-lasting antibody responses and provide relatively strong protection. However, they also present notable challenges, including interference with serological diagnostics, potential induction of antibiotic resistance. Recently, various novel vaccine strategies have emerged. Subunit vaccines offer advantages such as non-infectivity and potential cross-protection, but they often suffer from weak antigenicity, poor stability, and a short half-life. DNA vaccines can elicit immune responses but generally fail to confer long-term protection. Vector-based vaccines, which introduce antigen-encoding genes into attenuated bacteria or viruses to enhance immunogenicity, may carry unforeseen risks due to differences from native proteins. Notably, live attenuated vaccines with virulence gene deletions have shown significantly improved safety profiles. These vaccines can promote T cell proliferation, pro-inflammatory cytokine expression, and antibody production, making them strong candidates for human use. However, concerns about possible reversion to virulence and the development of antibiotic resistance still need to be addressed (Yang et al., 2024). Currently, human brucellosis is treated with a combination of doxycycline and streptomycin (Zheng et al., 2024). However, the failure and recurrence rates of brucellosis treatment remain high. Therefore, gaining a deeper understanding of the pathogenesis and host immune responses to brucellosis is crucial.

Brucella, as a Gram-negative facultative intracellular bacterium, can cross mucosal barriers (e.g., in the gastrointestinal, respiratory, and genital tracts, and the conjunctiva), penetrate damaged skin, invade immune cells (e.g., macrophages and dendritic cells [DCs]), and disseminate systemically via the lymphatic and blood circulation. Subsequently, Brucella colonizes non-phagocytic and phagocytic cells in a variety of tissues and organs, enabling the bacteria to evade the immune system and replicate within cells. Ultimately, the pathogen exits these cells and establishes persistent infections in bodily tissues (de Figueiredo et al., 2015; Atluri et al., 2011; Khairullah et al., 2024; Myeni et al., 2013). Initially, most studies on Brucella were conducted using murine macrophage cell lines, bovine macrophages, human monocytes, and widely used non-phagocytic cell lines, such as HeLa and Vero cells. However, further in vitro research has shown that Brucella can infect and replicate in a variety of human cell types, including osteoblasts, synovial cells, trophoblasts, endothelial cells, lung epithelial cells, dendritic cells, and hepatocytes (Bialer et al., 2020). Additionally, Brucella can also infect and replicate in murine alveolar macrophages, canine trophoblasts and phagocytes, as well as in ovine testicular cells (Bialer et al., 2020). Notably, macrophages, DCs, and trophoblasts have been identified as the primary targets for Brucella infections (Jansen et al., 2020). The ability of Brucella to survive and replicate within host cells is a key pathogenic feature, as mutant strains that lack this capability are typically avirulent (Celli et al., 2003). Brucella mainly achieves immune evasion by manipulating monocytes/macrophages and neutrophils, utilizing virulence factors to regulate host signaling pathways, and employing a unique intracellular parasitic strategy.

Brucella infection of host cells involves several steps, including adhesion, internalization, intracellular replication/survival, and transport (Yu et al., 2024). The adhesins of Brucella facilitate the binding of Brucella to host cells and/or extracellular matrix (ECM). These adhesins include the following: sialic acid-binding proteins SP29 and SP41, for binding to erythrocytes and epithelial cells; BigA and BigB proteins, which contain Ig-like domains for binding to adhesion molecules in epithelial cells; monomeric autotransporters BmaA, BmaB, and BmaC, for binding to ECM components, epithelial cells, osteoblasts, synovial cells, and trophoblasts; trimeric autotransporters BtaE and BtaF, for binding to ECM components and epithelial cells; Bp26, for binding to ECM components; and the T4SS protein VirB5 (Bialer et al., 2020; Xiong et al., 2021).

Adherence of Brucella to host cells occurs through a variety of receptors on the surface of those cells, resulting in ligand–receptor binding. Brucella enters host cells via either opsonic or non-opsonic pathways. Early in infection, prior to antibody production, Brucella invades cells via non-opsonic mechanisms. Specifically, the class A scavenger receptor (SR-A) on host cells binds to lipid A of the bacterial LPS, enabling the entry of Brucella via clathrin- and dynamin-dependent endocytosis pathways, with the involvement of membrane-bound lipid rafts, actin, and the cytoskeletal regulator Rho small GTPase. This route is applicable throughout infection, from early to late stages (Lee et al., 2013; Jimenez de Bagues et al., 2005; Guzmán-Verri et al., 2001; Atluri et al., 2011; Oliveira et al., 2008; Kim et al., 2004). However, as antibody levels increase during the intermediate and late stages of infection, Brucella also enters host cells via opsonization, wherein Fc receptors (FcRs) expressed on the phagocyte surface bind to antibodies, thereby facilitating bacterial entry (Atluri et al., 2011). Non-phagocytes have low susceptibility to Brucella uptake and weaker phagocytic ability (Chaves-Olarte et al., 2002). Professional phagocytes possess Fc and complement receptors, exhibit very high phagocytic efficiency. Research has shown that in the process of Brucella infection in phagocytes, using immune serum containing antibodies specific to Brucella to opsonize B. suis significantly enhances the phagocytosis process. Although the initial bacterial load inside the cells is higher after opsonization compared to untreated cells, bacterial proliferation within the cells is markedly reduced in opsonized infections. the Fc receptor (FcR) is associated with MAPKs. During opsonized Brucella infection, the FcR binds to its ligands, possibly influencing post-transcriptional expression of iNOS. The NO produced by iNOS can participate in bacterial killing. This suggests that opsonization, via receptors on macrophage surfaces, promotes bacterial phagocytosis and aids in bacterial destruction within the cells (Gross et al., 1998).

The entry of Brucella into host cells (phagocytes or non-phagocytics) triggers the formation of BCVs (Atluri et al., 2011). In BMDMs, HeLa cells, and NIH3T3 cells, BCVs interact with early endosomes (EE) in the cytoplasm to obtain early endosomal markers such as Rab5 and EEA-1, forming early endosomal BCVs (eBCVs) (Celli, 2019; Celli et al., 2003; Comerci et al., 2001; Chaves-Olarte et al., 2002). In HeLa and NIH3T3 cells, after Brucella infection, the EE containing the bacteria avoids fusion with late endosomes (LE) and lysosomes. The bacteria then alter their transport route and reach the ER through autophagosomes, forming rBCV (Chaves-Olarte et al., 2002; Comerci et al., 2001; Pizarro-Cerdá et al., 1998a; Pizarro-Cerdá et al., 1998b). In BMDMs, early eBCVs interact and partially fuse with LE and lysosomes. During this process, the BCV undergoes acidification, with a pH reaching 4.5–5.0, and acquires markers of LE and lysosomes, such as Rab7, LAMP-1, CD63, and RILP. Additionally, during this process, Brucella is exposed to proteases, ROS, reactive nitrogen species (RNS), and low pH conditions. Under these circumstances, 90% of Brucella will be eliminated by the lysosome (Starr et al., 2008; Celli et al., 2003; Gómez et al., 2020).

The formation of early and late eBCVs occurs within the first 8 h post infection (Celli, 2019). The acidic endosomal/lysosomal environment triggers T4SS expression, regulated by various factors at the transcriptional level. These regulatory factors include the vacuolar hijacking Brucella regulator (VjbR), Brucella luxR-like regulator (BlxR), and two-component regulatory system BvrR/BvrS, in addition to host-encoded histidine utilization regulator (HutC), RelA/SpoT homolog (Rsh), MarR-like sodium deoxycholate-responsive activator (MdrA), Brucella quorum-sensing regulator (BabR), and integration host factor (IHF) (Ke et al., 2015). The T4SS secretes various effector proteins that enable Brucella to escape from phagolysosomes and mediate the exclusion of phagolysosomal markers (e.g., LAMP-1) from the eBCV, resulting in the survival of approximately 10% of Brucella (de Jong et al., 2013; Zheng et al., 2024; Gómez et al., 2020). Furthermore, it has been observed in both phagocytic cells (e.g., mouse-derived macrophages) and non-phagocytic cells (e.g., mouse embryonic fibroblasts) that these effector proteins induce the UPR in host cells, activating the RNase of IRE1α and degrading Blocis1 mRNA, resulting in decreased BLOCIS protein production (a process known as regulated IRE1-dependent mRNA decay). BLOCIS is a component of the BLOC-one-related complex (BORC), which is involved in the biosynthesis of lysosome-associated organelles and is reduced through Brucella-mediated downregulation of Blocis1 expression. BORC also mediates the transport of late eBCVs to lysosomes, thus allowing Brucella to inhibit the fusion between late eBCVs and lysosomes (Wells et al., 2022; Celli et al., 2003). Meanwhile, in HeLa cells and macrophages, Brucella-secreted cyclic β-1,2-glucans (CβGs) disrupt the cholesterol-rich lipid rafts on the BCV membrane, impairing BCV maturation and thus preventing BCV fusion with lysosomes (Guo et al., 2023). T4SS-secreted effector proteins mediate the interaction of eBCVs containing viable Brucella with ER exit sites (ERES) and coat protein complex II (COPII) vesicles, a process regulated by the small GTPase Sar1, facilitating the fusion of eBCVs with the ribosome-associated ER membrane to acquire ER markers, including glucose-6-phosphatase, calnexin, calreticulin, Sec61, and PDI (Celli, 2019; Keriel et al., 2015; Starr et al., 2012; Guo et al., 2023; Myeni et al., 2013). During this process, the BCV recruits the small GTPase Rab2 and GAPDH, which are involved in regulating vesicle transport between the ER and the ERGIC. These factors contribute to the formation of the bacterial replicative niche and are essential for Brucella replication (Fugier et al., 2009).

The rBCVs form at 8–12 h post infection, losing the lysosomal component LAMP-1. Host proteins including the small GTPase IRE1α, Yip1A, Atg9, and WD-repeat domain phosphoinositide-interacting protein 1 (WIPI1), as well as the COG complex, are crucial for rBCV biogenesis (Celli, 2019; Keriel et al., 2015; Starr et al., 2012; Guo et al., 2023). Brucella cells in rBCVs proliferate in large numbers from 12 to 48 h post infection (Celli, 2019). Additional studies have shown that the endosomal/lysosomal vesicles fuse with ER-derived autophagic vesicles, leading to the production of rBCVs (as detailed in 5.2 the ER stress pathways subsection below) (Taguchi et al., 2015). In BMDMs from C57BL/6J mice and in HeLa cells, during 48 to 72 h post-infection, the intracellular rBCVs become enveloped by crescent-shaped, autophagosome-like membrane structures, gradually developing into aBCVs. The formation of aBCVs requires the functioning of the classical autophagosome nucleation complex, bypassing that of the elongation complex, and requires the autophagy initiators beclin1, ULK1, Atg14L, and PI3Ks, but not the autophagy elongation proteins ATG5, ATG16L1, ATG4B, ATG7, and LC3B (Starr et al., 2012). Therefore, aBCV formation relies on the molecular mechanisms of autophagy initiation in the host. The fusion of aBCVs and multivesicular bodies (MVBs) forms amphisomes, which then fuse with the plasma membrane for bacterial budding, completing the intracellular Brucella cycle and initiating new infections (Spera et al., 2023). Smith et al. (2016) demonstrated that, in BMDMs, the formation of aBCVs and subsequent bacterial release also rely on the T4SS. In BMDMs and HeLa cells, before aBCVs form, the Brucella-secreted T4SS effector protein BspL engages with the homocysteine-inducible ER stress protein (Herp), a core component of ER-associated degradation (ERAD), stabilizing Herp and preventing its degradation. Once Herp has been hijacked and stabilized by BspL, Beclin-1 activity declines, which slows the formation of aBCVs, the final step of the intracellular cycle. This ultimately gives Brucella more time to proliferate in rBCVs (Luizet et al., 2021). The intracellular survival mode of Brucella limits its exposure to and activation of the innate immune system. Furthermore, because innate immunity is a bridge to adaptive immunity, the latter is also not effectively activated. Additionally, this survival mode undermines the efficacy of some antibiotics, favoring the intracellular parasitism of Brucella (Wells et al., 2022; Figure 3).

PRRs encompass a variety of components, including TLRs, cyclic GMP-AMP synthase (cGAS), absent in melanoma 2 (AIM2), and nucleotide-binding domain leucine-rich repeats (NLRs). TLRs located on cellular and endosomal membranes detect PAMPs from outside cells and inside endosomal lumens, respectively. In the cytoplasm, cGAS and AIM2 sense cytoplasmic double-stranded DNA. NLRs are also located in the cytoplasm, where they recognize PAMPs and damage-associated molecular patterns (DAMPs) (Kumar et al., 2011; Foley et al., 2015; Bai and Liu, 2019; Tupik et al., 2020). Upon activation of TLR2 by lipoproteins from B. abortus, such as lipidated outer membrane proteins L-Omp16 and L-Omp19, the adaptor protein TIRAP is recruited. TIRAP then binds to MyD88, which subsequently engages IRAK-4 through its death domain. Activated IRAK-4 promotes the oligomerization of TRAF6, leading to the activation of the key signaling molecule TAK1. TAK1 transmits signals through two major pathways: first, it activates the NF-κB pathway by phosphorylating the IKK complex (IKKα/β subunits), which then phosphorylates IκB. This phosphorylation targets IκB for ubiquitination and degradation, thereby releasing the NF-κB to translocate into the nucleus and promote transcription of pro-inflammatory cytokines such as TNF-α and IL-6. Second, TAK1 activates the MAPK pathway, which leads to the activation of AP-1 transcription factors that regulate genes involved in inflammation. TLR4 is activated upon recognition of B. abortus LPS, Brucella lumazine synthase (BLS), and U-Omp16. It signals through two distinct pathways. The first is the MyD88-dependent pathway, which mirrors that of TLR2, leading to activation of NF-κB and MAPKs/AP-1, and the subsequent production of pro-inflammatory cytokines. The second is the MyD88-independent pathway, in which TLR4 recruits TRAM and then TRIF, initiating TRIF-dependent signaling. This results in activation and nuclear translocation of IRF3, which drives transcription of IFN-β (Gomes et al., 2012; Oliveira et al., 2008; Campos et al., 2004). Additionally, Brucella gDNA activates TLR9, triggering NF-κB and MAPK pathways via a MyD88-dependent mechanism, similar to TLR2 signaling (Oliveira et al., 2008; Gomes et al., 2012). TLR9 also recruits IRAK1/4 and TRAF6 through MyD88, which leads to activation of IRF7 and induction of IFN-α/β (Oliveira et al., 2008). Notably, although the non-classical LPS of Brucella elicits a weak immune response, other components such as OMPs and gDNA are more immunostimulatory. In particular, the recognition of Brucella gDNA by host TLR9 has been shown to trigger a strong immune response. gDNA from Brucella activates cGAS, while cyclic di-GMP (c-di-GMP) released by the bacterium activates STING, leading to STING- and IRF3-dependent IFN-β production (Costa Franco et al., 2018; Burdette et al., 2011). Autocrine signaling through the IFN I receptor (IFNAR) leads to activation of signal transducers JAK and STAT1, forming a heterotrimeric transcription factor (STAT1-STAT2-IRF9), which binds to IFN-stimulated response elements (ISREs) in the promoters/enhancers of IFN-stimulated genes (ISGs). This binding upregulates the expression of ISGs such as Mx, OAS, PKR, and ISG15 (Gomes et al., 2012; Freitas et al., 2021; Szczerba et al., 2022; Samsing et al., 2020; Barrat et al., 2019). Activation of IFNAR can also induce the expression of GBPs, which facilitate the rupture of BCVs and promote the formation of inflammasomes, resulting in the release of IL-1β (detailed in the section on 5.3 Inflammasomes and pyroptosis pathways) (Marinho et al., 2024). Upon infection with Brucella, the nucleotide oligomerization domain-like receptor protein 3 (NLRP3) inflammasome is triggered by DAMPs, while the AIM2 inflammasome is activated by Brucella gDNA. Both inflammasomes facilitate the secretion of IL-1β and IL-18, triggering pyroptosis (Tupik et al., 2020; Figure 2).

Murine brucellosis is typically divided into four stages: the initial infection stage (≤ 48 h), the acute phase (from day 3 to weeks 2–3 post-infection), the chronic stable phase (weeks 3 to 8–11), and the chronic declining phase (beyond 36 weeks) (Grilló et al., 2012).

During the early stage of infection, the innate immune response is not strongly activated. This is evidenced by normal counts of blood cells and platelets, a lack of pro-inflammatory cell recruitment at the infection site, and minimal serum levels of cytokines such as IL-1β, TNF-α, IL-10, and IL-6. Chemokines like MCP-1 and RANTES also remain at very low concentrations. In addition, there is no evidence of fibrinogen synthesis or degradation, nor are there signs of coagulation abnormalities. At this stage, Brucella can already be detected in macrophages. Brucella is detectable in the liver within just 3 h post-inoculation (Barquero-Calvo et al., 2007; Grilló et al., 2012). Despite their presence, Brucella barely activates the complement system, and macrophages exhibit limited bactericidal activity. Tissue damage caused by the bacteria is also minimal at this point (Barquero-Calvo et al., 2007).

In the acute phase, Brucella rapidly replicate within target cells such as macrophages, DCs, or trophoblasts. Meanwhile, both innate and adaptive immune responses are activated. Innate immune cells, including macrophages, neutrophil, and other cells, participate in the host’s defense. A type IV delayed-type hypersensitivity (DTH) reaction is also triggered, marking the initiation of a Th1-type immune response. By 1 week post-infection, high levels of cytokines such as IFN-γ, IL-12, IL-6, and RANTES can be detected in the serum of susceptible mice. However, some cytokines remain at relatively low levels. For example, in the acute phase, the production of IL-18, which works together with IL-12 to induce the production of IFN-γ, is suppressed in the splenocytes of mice infected with B. abortus. Additionally, the acute phase is marked by significant inflammatory responses, including splenitis, lymphadenitis, and granuloma formation in the liver (Grilló et al., 2012).

During the chronic stable phase, the bacterial load in the target organs reaches its peak and remains at a high level, establishing a stable plateau. In this phase, granulomas in the liver, bone marrow, and spleen are most prominent. The production of IFN-γ in susceptible mice significantly decreases in this phase. Serum levels of IL-6 peak at the end of the acute phase and remain relatively high during the first 2 weeks of the chronic stable phase. In contrast, the secretion of IL-2, IL-10, GM-CSF, and IL-4 is minimal during this period (Grilló et al., 2012).

During the chronic regression phase, the bacterial load in the target organs gradually decreases, and the splenomegaly phenomenon also progressively alleviates. Pathological damage in the liver and spleen gradually disappears. DTH weakens, and the majority of cytokines in the blood decrease, with some even becoming undetectable. However, immune memory seems to be fully consolidated (Grilló et al., 2012).

Notably, murine models are not the natural hosts of Brucella, and the pathogenesis of brucellosis in these models may differ from that in humans in certain respects, such as the pathological features of chronic infection and the dynamics of immune responses. Despite this limitation, murine models remain the most widely used experimental system for studying Brucella immune evasion mechanisms and evaluating vaccine efficacy.

The innate defenses against Brucella include physical barriers on the body surface, PMNs, macrophages, DCs, NK cells, chemokines, PRRs, and the complement system. Later, Brucella infection elicits three primary adaptive immune responses: (1) CD4⁺, CD8⁺, and γδ T cells secrete IFN to activate the bactericidal functions of macrophages, preventing the intracellular survival of the bacteria; (2) cytotoxic CD8⁺ T cells target the infected macrophages; and (3) Th1 antibody subtypes (e.g., IgG2a and IgG3) enhance macrophage phagocytosis via ADCP, aiding in Brucella elimination while also facilitating its invasion of phagocytic cells, which it uses as a base for replication and survival (Guo et al., 2023).

In conclusion, Brucella infection induces weak innate immune responses in humans. Brucella employs a variety of strategies to suppress innate immunity: blockage of TLR/NLR signaling pathways and the complement system; disruption of pyroptosis, apoptosis, and ferroptosis; induction of ER stress; modulation of autophagy; neutralization of antimicrobial peptides, oxidative stress, and nitrosative stress; regulation of vesicle trafficking; reprograming of host nuclear gene expression; and prevention or suppression of BCV-lysosome fusion (Figure 4). The successful implementation of these measures by Brucella significantly suppresses host innate immunity, initiating a cascade of reactions. On the one hand, the decreased secretion of host pro-inflammatory cytokines and chemokines leads to reduced recruitment and activation of macrophages, DCs, and other phagocytes at the infection site; on the other hand, the impaired phagocyte function contributes to inadequate activation of CD8⁺ T lymphocytes (which are responsible for cellular immunity), culminating in adaptive immunosuppression. In such an immunosuppressed environment, Brucella can thrive and smoothly transition to the chronic phase. The intracellular niche favors Brucella replication and survival, while inadequate immune protection leads to prolonged tissue and organ invasion, causing chronic inflammation and jeopardizing the normal functions of the body.

Brucella has co-evolved with its hosts over millions of years, developing a range of virulence strategies that facilitate its survival and ultimately lead to disease onset through the manipulation of host systems. Brucella employs several primary strategies to evade the host immune system, enabling its proliferation and sustained intracellular survival, which leads to chronic infection. These include the following: inhibition of complement and TLR pathways; blockage of maturation and disruption of antigen presentation by DCs; formation of intracellular replication vesicles; and manipulation of autophagy, ER stress, inflammasome activation, pyroptosis, apoptosis, ferroptosis, and the cGAS-STING pathway, among others. Brucella produces virulence factors and effector proteins that manipulate host cell signaling pathways, disrupting normal cellular processes to its advantage. However, the mechanisms by which some effector proteins regulate cellular processes during infection are poorly understood. Autophagy and programed cell death pathways (e.g., pyroptosis, apoptosis, and ferroptosis) serve as host defense strategies, containing infections by eliminating intracellular pathogens from infected cells (Zhang et al., 2024). However, Brucella can exploit programed cell death pathways to evade killing, employing specific molecular mechanisms to enhance its replication, survival, and dissemination within host cells. Therefore, researchers can disrupt the specific molecular mechanisms by which Brucella manipulates autophagy, pyroptosis, apoptosis, and ferroptosis, enabling the corresponding cell death program to proceed as normal and thereby eradicating the bacteria. Doxycycline (Dox) can inhibit CALR protein expression, activate the JNK/p53 signaling pathway, and induce apoptosis in HMC3 cells, thereby achieving the therapeutic goal of treating brucellosis (Wang et al., 2021b). The interaction between Brucella and programed cell death remains unclear. Thus, elucidation of the mechanisms governing these interactions is essential for identifying potential drug targets that could induce programed cell death and eradicate the pathogen.

It is noteworthy that Brucella adopts significantly different survival strategies in various host cells: in non-phagocytic cells, early eBCV can avoid degradation by late endosomes and lysosomes through specific mechanisms; however, in phagocytic cells (such as macrophages), early eBCV briefly fuses with late endosomes and lysosomes. Subsequent studies, however, have revealed two key mechanisms that effectively block the fusion of eBCV with lysosomes: Brucella activates the host RIDD pathway to disrupt BLOS1-mediated immune defense and secretes CβGs. This leads to a paradoxical conclusion in research: in phagocytic cells, it remains unclear whether both the fusion of early eBCV with lysosomes and the anti-fusion mechanisms coexist simultaneously, or if only one of these processes actually occurs. Further studies are needed to clarify the true nature of these seemingly contradictory phenomena in macrophages. In addition, compared with other intracellular bacteria of the Proteobacteria, the number of identified Brucella effector proteins remains relatively limited. This suggests that many effectors may still be undiscovered. Therefore, further in-depth research is needed in this area, as effector proteins are key molecules through which Brucella evades host immune responses.

In summary, future research need to focus on the molecular mechanisms by which Brucella regulates programed cell death, the differences in vesicle trafficking across various cell types, and the identification of novel effector proteins. These efforts will be essential for uncovering the mechanisms underlying its intracellular survival and for developing targeted strategies for prevention and treatment.