Pathogenicity and virulence of Brucella: Strategies for metabolic adaptation and immune evasion

ABSTRACT

Brucella species are facultative intracellular pathogens that have evolved sophisticated mechanisms to evade host immune responses and establish chronic infections. This review provides an analysis of Brucella virulence and pathogenicity, with particular emphasis on the intricate interactions between the pathogen and its host. We examine the molecular and cellular mechanisms underlying Brucella pathogenesis, detailing the processes by which the bacterium invades, survives, and replicates within host cells. An in-depth discussion of the key Brucella virulence factors and their roles in immune evasion is provided, including strategies that enable the pathogen to circumvent immune detection, subvert host immune signaling pathways, and manipulate intracellular trafficking. Furthermore, we explore Brucella ability to modulate host cellular functions, particularly through the induction of the unfolded protein response and its implications for bacterial persistence. The review also highlights the influence of type I interferon responses on host susceptibility to Brucella infection, shedding light on their role in disease progression. Additionally, we discuss Brucella metabolic adaptations, including its ability to exploit host-derived nutrients and reprogram metabolic pathways to sustain long-term persistence. Finally, we address emerging research directions and future perspectives in the field, emphasizing the need for novel therapeutic and vaccine strategies. A deeper understanding of these complex host-pathogen interactions will be instrumental in developing more effective approaches for the control, prevention, and treatment of brucellosis.

Introduction

Brucellosis is a highly prevalent yet often neglected bacterial zoonosis, causing an estimated annual economic loss of US$ 600 million in Latin America alone [1]. The disease is endemic in resource-limited regions such as the Middle East, Mediterranean, and Latin America [2]. Brucella spp, the causative agent of brucellosis, consists of Gram-negative facultative intracellular Alphaproteobacteria, that can persist and invade host cells, leading to chronic granulomatous infections [3,4]. Among the various Brucella species, Brucella abortus is the primary etiological agent of human brucellosis and is the most widely distributed globally [5]. This bacterium has a particular tropism for the reproductive organs of bovines, often resulting in spontaneous abortion in ruminants [6]. Zoonotic transmission of B. abortus typically occurs through the oral route via consumption of animal-derived products, such as unpasteurized dairy and raw meat from infected animals. However, infection can also occur through inhalation or subcutaneous pathways [7].

Recent reports estimate that brucellosis results in 1.6 to 2.1 million new human cases annually [8]. Although rarely fatal, human brucellosis is a chronic illness that can be disabling, affecting various organs and tissues with variable incidence [9]. Common symptoms include fever, fatigue, arthralgia, sweating, lymphadenopathy, hepatomegaly, and splenomegaly [10,11]. Moreover, Brucella infection can lead to a range of complications, such as undulant fever due to episodes of bacteremia, followed by the development of new infection foci, as well as arthritis, endocarditis, osteomyelitis, and neurological complications [12]. Patients undergoing antibiotic treatment often experience high relapse rates, and no vaccines are currently available for humans [13,14].

The immune evasion mechanisms and virulence factors of Brucella play a crucial role in the pathogenesis of brucellosis. This pathogen can proliferate and survive inside phagocytic cells, such as macrophages and dendritic cells (DCs), which function as replicative niches and vectors for systemic dissemination to other organs. Upon entering the host cell, B. abortus is enclosed within a double-membrane compartment called Brucella-containing vacuole (BCV) [15,16]. During intracellular establishment and proliferation, the BCV undergoes various molecular changes as it interacts with distinct host trafficking pathways [17,18]. Firstly, the BCV actively interacts with the endocytic pathway, acquiring early endosomal molecules (eBCV), which are later replaced by late endosomal markers. Meanwhile, controlled fusion with lysosomes leads to vacuole acidification, which kills most bacteria but is essential for the expression of the Type IV Secretion System (T4SS) VirB, a key Brucella virulence factor [16,18]. The T4SS modifies the primary secretory pathway, inducing the biogenesis of the replicative BCV (rBCV), via fusion of mature eBCVs with endoplasmic reticulum (ER) [18,19]. Brucella effectively replicates in the ER, and rBCVs are subsequently incorporated into autophagosome-like multi-membrane structures (aBCV), which facilitate bacterial egress and cell-to-cell spread within the host [20]. The ability of Brucella to grow and survive within professional phagocytes prompts its systemic spread and infection of other myeloid cells, such as liver and spleen macrophages, where Brucella can persist in granulomatous lesions [9].

Brucella major virulence factors

Virulence factors are specialized molecules or structures produced by pathogens to enhance their ability to infect and persist within a host. Brucella, unlike many other pathogens, does not rely on classical virulence factors such as exotoxins, cytolysins, or exoenzymes. Instead, its pathogenicity is driven by unique factors, including LPS, the T4SS and the BvrR/BvrS system. This section explores these key virulence factors and their critical roles in Brucella ability to infect, evade immunity, and cause a persistent infection.

Lipopolysaccharide

Brucella strains have evolved unique mechanisms of virulence compared to other well-described bacteria that infect mammals. One example is the presence of an unusual smooth lipopolysaccharide (S-LPS) in the outer membrane of classical virulent Brucella species, such as B. abortus. The S-LPS comprises three main domains: a polysaccharide O-chain, a core, and lipid A [21]. Compared to other Gram-negative bacteria, Brucella LPS contains longer chains of lipid A, primarily consisting of C16, C18, C28, and other very long-chain fatty acids. As a result, Brucella LPS requires concentrations 100 times higher than those of Enterobacteriaceae LPS to kill the host [22]. Additionally, the core and O-chain of Brucella LPS play multiple roles in immune evasion: (1) protect against the bactericidal actions of complement proteins and antimicrobial peptides produced by host phagocytes, (2) interact with lipid rafts, facilitating entry into host cells via endocytosis and preventing extensive fusion of BCV with lysosomes, (3) inhibit antigen presentation by forming complexes with major histocompatibility complex class II (MHC-II), and (4) prevent caspase-2 mediated apoptosis in phagocytes [23–27]. Studies have identified several glycosyltransferases as essential for the biosynthesis and structural integrity of the LPS core. Among them, WadC, WadB, and WadD function as key enzymes that incorporate specific sugar residues into the LPS core region [28,29]. In particular, mutations in wadC prevent the incorporation of the mannose residue that anchors the lateral branch, leading to significant consequences: wadC mutants display increased sensitivity to host antimicrobial defenses, impaired intracellular replication, enhanced recognition by the CD14-MD2-TLR4 receptor complex, heightened production of inflammatory cytokines, and accelerated dendritic cell maturation [28,30]. WadB also contributes to LPS core assembly, and its disruption alters LPS composition and surface properties, resulting in increased sensitivity to cationic antimicrobial peptides and reduced persistence in host cells [31]. Likewise, WadD has been characterized as a novel LPS core glycosyltransferase, and its inactivation disrupts LPS structure, reducing resistance to polycationic peptides [29].

Type IV secretion system

The VirB T4SS apparatus, is encoded by the virB1- virB12 genes on Chromosome II. Initially identified in B. suis, the virB operon has since been found to be highly conserved across all Brucella species [32]. Although the precise molecular mechanisms initiating T4SS assembly remain unclear, intracellular signals are thought to play a crucial role in inducing its assembly. One such signal is the acidic pH environment within the BCV, resulting from lysosomal fusion and acidification, which likely triggers the upregulation of the virB operon [33]. Several regulatory factors, including the LuxR-type regulator VjbR [34], the integration host factor (IHF) [35], the Brucella LuxR-like regulator (BlxR) [36], and the Brucella quorum-sensing regulator (BabR) [37], modulate the expression of the virB operon. However, their precise roles in T4SS induction during infection are not fully understood. A functional T4SS is essential for Brucella survival and persistence within host cells as mutants lacking any VirB genes are unable to replicate intracellularly [38,39]. One mechanism by which the T4SS promotes intracellular survival is by regulating the trafficking of BCV within macrophages, ensuring that the bacteria avoid degradation in phagolysosomes [19,40]. Brucella replicates exclusively within the ER after the formation of rBCVs. The acquisition of ER membranes, which is crucial for establishing a replication niche, is dependent on a functional T4SS system [40]. In contrast, virB mutants are persistently trafficked to lysosomes, leading to bacterial degradation. T4SS effectors interact with ER exit sites (ERES) to establish replication niches, preventing lysosomal fusion. Interestingly, the VirB apparatus appear to be more important for late BCV maturation stages than for early survival events [41]. Furthermore, a functional T4SS is necessary for B. melitensis and B. abortus to elicit a robust innate immune response during late stages of infection in mice [42]. virB mutants induce lower IFN-γ production by CD4 T cells, resulting in a diminished Th1-polarized immune response [43]. Notably, it remains unclear whether these immune effects arise directly from T4SS activity or indirectly from lysosomal degradation of virB-deficient mutants. Moreover, Li et al [44]., demonstrated that recombinant T4SS effectors can stimulate both Th1 and Th2-associated cytokines in macrophages, highlighting their direct immunomodulatory activity. More recently, RS15060 was identified as a novel T4SS effector required for Brucella virulence, thereby expanding the known repertoire of bacterial factors that subvert host cellular pathways [45]. Another important effector, NpeA, is actively translocated into host cells and plays a critical role in intracellular replication. Notably, NpeA harbors a conserved short linear motif that binds directly to N-WASP, a central regulator of Arp2/3-dependent actin nucleation. This interaction promotes cytoskeletal remodeling, which facilitates the maturation of the BCV into a replication-permissive niche [46].

VceA and VceC

VirB-co-regulated effector (Vce) A and VceC are two effector proteins translocated into macrophages via the VirB T4SS [47]. The deletion of the VceA effector enhances autophagy in infected human trophoblast cells, suggesting that VceA plays a crucial role in suppressing autophagy to facilitate intracellular survival during infection [48]. Additionally, VceA-deleted mutants stimulate the secretion of tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-1β, which are involved in inhibiting apoptosis, likely as a strategy to preserve the host cell and maintain the intracellular niche [48]. VceC is a conserved effector that translocates to the ER and interacts with the ER chaperone GRP78/BiP, triggering ER stress. This interaction activates the unfolded protein response (UPR), ultimately leading to an inflammatory response through the inositol-requiring enzyme-1-alpha (IRE1α) pathway [47,49]. In addition to its role in ER stress, VceC regulates apoptosis in a cell type-dependent manner. In placental trophoblasts, VceC promotes apoptosis, potentially dampening immune responses and facilitating bacterial escape [50]. Conversely, in goat trophoblast cells, VceC inhibits apoptosis, preserving the intracellular niche and promoting Brucella proliferation [51].

TcpB/BtpA and BtpB

The T4SS effectors Toll/Interleukin 1 like receptor domain containing protein (TcpB/BtpA) and BtpB contain a Toll-interleukin-1 receptor (TIR) domain, which structurally mimics those found in host innate immune receptors, allowing Brucella to subvert the host immune response [52–55]. Although TcpB and BtpB target similar pathways [56], the role of BtpB in modulating Brucella-induced inflammatory responses and bacterial persistence remains unclear. Recent studies have demonstrated that deletion of BtpB increases microtubule-associated protein 1 light chain 3B (LC3-II) expression, decreases p62 levels, and leads to the accumulation of autophagic lysosomes, suggesting that BtpB participates in autophagy inhibition [57]. Beyond Toll-like receptor (TLR) signaling suppression, TcpB interacts with phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) or PI(3,4,5)P3. This interaction suppresses the activation of antigen-presenting cells, impeding CD8 T cells from targeting infected cells and further weakening host immune defenses [58]. Additionally, TcpB modulates microtubule dynamics by acting as a stabilizing factor [59]. This modulation influences B. abortus intracellular survival, the maturation of pathogen-containing vacuoles, and the production of pro-inflammatory cytokines in infected macrophages [60]. TcpB has also been shown to induce the UPR in infected macrophages, although the exact mechanisms underlying this effect remain undefined [61]. Moreover, TcpB and BtpB exhibit NAD⁺ hydrolase activity, enabling Brucella to deplete NAD⁺ levels in host cells during infection. This enzymatic activity leads to actin depolymerization, endocytic block, and a reduction in kinase activity [62].

Two-component system BvrR/Bvrs

The two-component system BvrR/BvrS is a master gene regulator conserved across the Alphaproteobacteria class. BvrS is a sensor histidine kinase protein located in the inner membrane, while BvrR is a cytoplasmic response regulator [63]. BvrRS transcriptionally regulates numerous genes implicated in B. abortus virulence, carbon and nitrogen metabolism, and cell envelope homeostasis. As Brucella migrates to an intracellular lifestyle, its finely tuned gene expression enables adaptation to the stresses of the intracellular environment [64]. In this context, BvrRS is induced under acidic nutrient-limited conditions, helping Brucella bypassing host immune mechanisms during its initial interactions with eukaryotic cells [65]. An example of this regulatory system is the BvrRS/VjbR/VirB circuit [65]. During the early intracellular cycle of B. abortus, BvrS detects pH changes and nutrient starvation, leading to autophosphorylation. The phosphate group is transferred to BvrR (BvrR-P), which binds to target promoters, including one that regulates the expression of the transcriptional factor VjbR. Both VjbR and BvrR subsequently activate the virB operon, driving the expression of genes encoding the T4SS [65]. Moreover, the BvrRS/VjbR/VirB circuit plays a role in sensing and coordinating bacterial release and interactions with new target host cells during the late stages of infection, when B. abortus migrates from ER to the aBCV [66]. Regarding cell envelope regulation, BvrRS controls the expression of genes encoding for outer membrane proteins (Omps), such as Omp25, as well as genes involved in lipid A acylation [67]. B. abortus mutants in bvrR and bvrS are avirulent, exhibiting reduced invasiveness and an inability to replicate in cells and a mouse model [63].

Outer membrane proteins

On the cellular surface of Brucella, the outer membrane proteins (Omp25/Omp31) and lipoproteins (Omp10/Omp16 and Omp19) further contribute to bacterial pathogenicity [68]. Omp16 is a conserved peptidoglycan-associated lipoprotein that plays a crucial role in bacterial virulence. This protein contributes to the structural integrity of the bacterial cell, enhancing resistance to environmental stresses. Deletion or downregulation of the omp16 gene significantly reduces the bacterium’s ability to survive and replicate intracellularly, highlighting its importance in maintaining infection [69,70]. Omp19, the best-characterized lipoprotein from Brucella, presents strong immunomodulatory effects. Omp19 inhibits interferon-γ (IFN-γ)-induced MHC-II expression in macrophages during B. abortus infection by binding to the TLR2 receptor for prolonged periods and disrupts antigen processing and presentation by macrophages to T CD4 lymphocytes, significantly impairing the adaptive immune response in patients with brucellosis [71,72]. Evidence also indicates that Omp19 has notable protease inhibitor activity, avoiding lysosome proteases in host macrophages and intestinal proteases during oral infections [73]. The importance of these molecules as virulence factors is highlighted by the attenuation of B. abortus Omp19 and Omp25 mutants in mice and natural hosts [68,74]. Given the pivotal role of these Omps in host-pathogen interactions, Brucella must ensure their proper folding and insertion into the outer membrane to maintain virulence. These essential processes are facilitated by conserved molecular systems, such as the β-barrel assembly machinery (BAM) complex. The Brucella genome encodes homologs of BamA, BamD, and BamE, but lacks BamB and BamC, which are present in Escherichia coli and other Gammaproteobacteria [75,76]. Complementing BAM complex’s function, EipB is a conserved periplasmic protein essential for maintaining outer membrane integrity. Although not directly linked with individual Omps, mutants deficient in EipB exhibit increased outer membrane fragility, heightened sensitivity to environmental stress, and significant attenuation in vivo. Structural and genetic evidence further suggests that EipB may act synergistically with the BAM complex to support Omp biogenesis [77].

Cyclic β-1,2-glucans

Cyclic β-1,2-glucans (CbG) are ring-shaped molecules consisting of 17–25 glucose units linked by β‐1,2-glycosidic bonds [78]. In many bacterial species, CbG production is regulated by osmotic conditions, highlighting its role in osmoprotection [79]. However, in Brucella, CbG synthesis is not osmoregulated [80], and experimental evidence suggests that this polysaccharide plays a minimal role in Brucella osmoprotection [81]. Nevertheless, CbG is critical for Brucella intracellular survival. Brucella mutants that cannot synthesize or transport CbG to the periplasmic space exhibit reduced virulence [81–83]. Recent structural and functional analyses have underscored the importance of CbG export via the Cgt ABC transporter, as Δcgt mutants retain CbG intracellularly but fail to deliver it to the periplasm, resulting in defective intracellular replication and significant attenuation in murine infection models [84]. CbG interacts with lipid rafts on host cell membranes, disrupting their organization and preventing phagosome-lysosome fusion. This interference allows Brucella to evade degradation and replicate within the ER [82]. Furthermore, CbG modulates the production of both proinflammatory and anti-inflammatory cytokines in macrophages and DCs [81,85,86]. In murine models, Brucella CbG has been linked to spleen inflammation due to the recruitment of monocytes, DCs and neutrophils, driven by the induction of cytokines, such as IL‐12 and TNF‐α [81]. Additionally, CbG acts as a potent activator of DCs, triggering antigen-specific CD8 T cell responses in vivo and enhancing antigen-specific CD4 and CD8 T cell responses [86].

BspA, BspB, BspF, BspG, BspJ and BspL

BspA inhibits the host ER-associated degradation (ERAD) pathway by targeting the E3 ubiquitin ligase membrane-associated RING-CH-type finger 6 (MARCH6). This inhibition disrupts the degradation of ERAD substrates, facilitating the intracellular proliferation of B. abortus [87]. Moreover, BspA, along with other effectors like BspB and BspF, contribute to the inhibition of host protein secretion. This combined interference allows Brucella to modulate host secretory pathways, creating a favorable environment for its replication [88]. BspB specifically targets the conserved oligomeric Golgi (COG) tethering complex, altering Golgi membrane trafficking and redirecting Golgi-derived vesicles to the BCV. This process is vital for the biogenesis of rBCVs and optimal replication within host cells [89]. Additionally, BspF regulates host cell apoptosis by attenuating the crotonylation modification of the tumor suppressor protein p53, leading to reduced p53 expression. This suppression in turn inhibits the transcription of downstream apoptotic genes, thus preventing apoptosis [90]. BspF also promotes Brucella replication within rBCVs by interfering with vesicular transport between the trans-Golgi network and the plasma membrane. This interference occurs through modulation of the Arf6-Rab8a GTPase cascade, a key player in vesicular trafficking [91]. Furthermore, BspG interacts with host proteins involved in mitochondrial respiratory pathways to promote anti-apoptotic mechanisms and enhance the intracellular survival of B. abortus [92]. BspJ has been identified as a nucleomodulin that plays a pivotal role in regulating host energy synthesis, metabolism, and the inhibition of apoptosis signaling pathways. These functions collectively support the intracellular survival of Brucella. BspJ deletion significantly impairs the survival and proliferation of B. abortus during the rBCV phase and affects the secretion of inflammatory factors in both host cells and mice [93,94]. BspL has been characterized as a Brucella effector that specifically targets the host ERAD machinery. By enhancing ERAD activity during the late stages of infection, BspL delays the formation of aBCVs and prevents premature bacterial release from host cells, likely ensuring optimal intracellular replication and promoting efficient cell-to-cell dissemination [95].

RicA

Rab2-interacting conserved protein A (RicA) targets host cellular pathways by specifically interacting with Rab2, a small GTPase involved in vesicular trafficking between the ER and the Golgi apparatus, which is crucial for B. abortus intracellular infection [96]. By hijacking Rab2-mediated trafficking, RicA alters the normal kinetics of BCV maturation, preventing its fusion with lysosomes [40,97]. This interference allows Brucella to avoid lysosomal degradation and supports its establishment within a replicative niche. Mutants lacking RicA exhibit a decrease recruitment of Rab2 to BCVs and significant defects in BCV maturation, underscoring its involvement in modulating host vesicular trafficking [40,96]. However, RicA-deficient mutants display no significant reduction in virulence in infected mice or HeLa cells [98]. Surprisingly, RicA mutants lose the late endosomal marker LAMP1 earlier than wild-type bacteria, indicating accelerated escape from late lysosomes. This earlier arrival at the ER allows RicA mutants to establish their replicative niche faster than wild-type Brucella [97]. This phenomenon appears to be further modulated by the effector BspB, which attenuates the negative impact of RicA on Rab2 function [99]. The interplay between RicA and BspB highlights the intricate regulatory network employed by Brucella to fine-tune host vesicular trafficking for optimal infection outcomes.

Antioxidant activity

Brucella spp. exhibit remarkable resistance to the bactericidal activity of professional phagocytes, allowing them to persist in high numbers within neutrophils and evade oxidative killing mechanisms [100]. The antimicrobial activity of these cells relies heavily on the generation of reactive oxygen species (ROS), such as superoxide anion (O₂⁻) and hydrogen peroxide (H₂O₂). Given that H₂O₂ can freely diffuse across biological membranes, Brucella detoxifies it using both the periplasmic catalase KatE and the cytoplasmic peroxiredoxin AhpC, which work together to neutralize intracellular H₂O₂ [101,102]. Notably, either KatE or AhpC alone is sufficient to maintain chronic infection in murine models. However, a B. abortus double mutant lacking both enzymes shows significant attenuation in IFN-γ-activated macrophages, underlining their complementary roles [102]. Beyond H₂O₂ detoxification, Brucella also employs superoxide dismutases (SODs) to protect against superoxide radicals generated by both host cells and its own metabolism. Cu/Zn-cofactored SOD (SodC) is localized in the periplasm and primarily counters extracellular superoxide. The SodC mutant shows increased susceptibility to macrophage killing following IFN-g stimulation, a phenotype partially reversed by inhibition of NADPH oxidase using apocynin, supporting its role in defending against the respiratory burst [103]. In contrast, Mn-cofactored SOD (SodA) is cytoplasmic and targets superoxide species generated endogenously by the bacterial metabolism, which are less likely to cross membranes due to their charge. Disruption of sodA similarly impairs B. abortus survival in macrophages and during the early stages of infection in mice [104], further emphasizing the importance of these complementary antioxidant systems. Recent studies have revealed that B. melitensis also manipulates host cell ferroptosis pathways to enhance intracellular survival and promote dissemination. Specifically, during the early phase of infection, Brucella activates the GTP cyclohydrolase 1 (GCH1)-tetrahydrobiopterin (BH₄) axis, a GPX4-independent antioxidant mechanism that suppresses lipid peroxidation and ferroptotic cell death in macrophages, thereby creating a permissive niche for bacterial replication. In contrast, during the late phase of infection, the pathogen inhibits the classical GPX4-glutathione (GSH) axis, facilitating ferroptosis and allowing bacterial egress from host cells. This temporal regulation illustrates a sophisticated immune evasion strategy in which Brucella balances intracellular persistence with intercellular spread [105]. OxyR is a central transcriptional regulator that coordinates Brucella’s antioxidant defenses during infection. Deletion of oxyR impairs bacterial growth and reduces tolerance to multiple stressors, including oxidative agents, acidic conditions, and antimicrobial peptides, underscoring its role in stress adaptation. Loss of OxyR also downregulates key antioxidant enzymes such as peroxidases, catalase, and superoxide dismutase, indicating that OxyR orchestrates a coordinated antioxidant response. Notably, intracellular survival within macrophages remains largely unaffected, suggesting that OxyR primarily protects Brucella from environmental stress rather than hot-mediated intracellular killing. Additionally, oxyR deletion suppresses inflammatory cytokines expression, indicating a potential role in modulating host immune responses [106].

Small regulatory RNAs

Small regulatory RNAs (sRNAs) have emerged as central players in bacterial adaptation and pathogenesis. These non-coding RNAs, typically 50 to 300 nucleotides long, modulate gene expression post-transcriptionally through base pairing with target mRNAs, thereby influencing mRNA stability and translation [107]. Many sRNAs function in concert with the RNA chaperone Hfq, which facilitates RNA-RNA interactions and stabilizes both sRNAs and their mRNA targets In Brucella, Hfq is essential for full virulence, and its deletion leads to pleiotropic effects, including enhanced sensitivity to oxidative stress, reduced intracellular survival, and downregulation of key virulence determinants such as the quorum sensing regulator BabR and the VirB system [37]. Among the best-characterized sRNAs in B. abortus are AbcR1 and AbcR2. These trans-encoded sRNAs exhibit partial sequence homology and regulate overlapping but distinct sets of targets, primarily mRNAs encoding amino acid and polyamine ABC transporters. Functional studies show that a double mutant lacking both abcR1 and abcR2 is significantly attenuated in murine models and exhibits reduced intracellular replication, whereas single deletions have minimal impact on virulence [108,109]. Their regulatory activity relies on two conserved seed sequences (M1 and M2), with M2 being essential for in vivo virulence. Mutation of this motif leads to dysregulation of key targets and impairs splenic colonization [109]. High-throughput RNA sequencing has expanded the known repertoire of sRNAs in Brucella, predicting numerous candidates, several of which have been experimentally validated, including BSR0602, BSR0441, BASI74, and Bmsr1 [110,111]. BSR0602 is strongly upregulated during stress and infection and represses the transcriptional regulator gntR via Hfq-dependent pairing, compromising intracellular survival and reducing bacterial burden in the spleen, suggesting a role in negatively regulating virulence [112]. In contrast, Bmsr1 appears to enhance pathogenicity: its expression is induced during macrophage infection, and deletion of bmsr1 leads diminishes survival and splenic colonization. Transcriptomic profiling of the bmsr1 mutant revealed downregulation of key virulence genes, including virB2–virB11 and VjbR, indicating that Bmsr1 modulates virulence programs in response to intracellular cues [113]. A more recently described sRNA, MavR (MurF- and virulence-regulating RNA), is essential for persistent infection in mice. MavR specifically regulates murF, which encodes an enzyme involved in the cytoplasmic steps of peptidoglycan biosynthesis, through a six-nucleotide seed motif. Disruption of this interaction leads to dysregulated murF expression, impaired replication, and reduced colonization during chronic infection, highlining MavR as a bona fide virulence-associated sRNA [114].

Quorum sensing systems (QS)

QS is key regulatory mechanism that allows bacteria to coordinate gene expression in response to population density and environmental cues [115]. In Brucella, QS plays a significant, albeit non-canonical, role in modulating virulence. A hallmark of this system is the detection of N-dodecanoyl homoserine lactone (C₁₂-HSL), a prototypical QS molecule, in the supernatant of B. melitensis cultures. Exogenous C₁₂-HSL represses the expression of critical virulence genes, including components of the T4SS [116]. At the core of QS regulation are the LuxR-type regulators VjbR and BabR (also known as BlxR). VjbR is indispensable for full virulence as vjbR-deficient strains exhibit marked attenuation in both cellular and murine infection models [117,118]. BabR modulates a subset of VjbR-regulated genes, exerting both synergistic and antagonistic effects, though its deletion alone does not significantly impair virulence [36,119]. Recent evidence indicates that VjbR and BabR act cooperatively during chronic infection, with BabR autoregulating its own expression while VjbR mildly represses BabR, highlighting a complex regulatory interplay [120]. Further layers of regulation involve MucR, which directly represses babR expression, indirectly facilitating upregulation of VirB. This dual-layered control underscores the finely tuned regulatory network that governs virulence gene expression [121]. QS is also integrated with environmental sensing systems: the two-component system BvrR/BvrS activates vjbR during early host cell invasion [65], while transcriptional regulators GntR10 and GntR17 positively regulate both vjbR and babR, influencing the production of T4SS effectors such as BspE and BspF [122,123]. Despite these advances, several knowledge gaps remain. The endogenous biosynthetic pathway for C₁₂-HSL in Brucella is still unknown, and while a lactonase capable of degrading C₁₂-HSL has been characterized [124], upstream metabolic processes remain elusive. Additionally, VjbR function at certain promoters appears to require co-regulators that have yet to be fully defined [125].

Moonlighting metabolic enzymes

B. abortus repurposes both bacterial and hots metabolic enzymes to enhance intracellular survival and modulate host-pathogen interactions. One prominent example involves the interaction between the T4SS effector BPE123 and host α-enolase (ENO-1). Marchesi et al [126]., demonstrated that ENO-1 localizes to BCVs in a BPE123-dependent manner, knockdown of ENO-1 in HeLa cells significantly impairs intracellular replication. In parallel, bacterial enolase and elongation factor Tu (EF-Tu) have been detected on the B. abortus surface during biofilm formation, suggesting additional roles in adhesion, immune interaction, or biofilm maintenance [127]. Biochemical studies further show that B. abortus enolase retains its canonical glycolytic activity while also binding host fibronectin and eliciting recognition by sera from infected cattle [128]. Another example of moonlighting behavior is the chaperonin GroEL, a highly conserved protein primarily involved in folding nascent and stress-denatured proteins. Beyond this canonical role, GroEL is surface exposed during infection and interacts directly with host immune components, functioning as an immunodominant antigen. This extracellular localization allows GroEL to influence host immune responses, potentially promoting bacterial persistence and immune evasion [129].

Brucella evades host innate immune activation and signaling mechanisms

The innate immune system relies on its capacity to quickly identify invading pathogenic microbes as foreign and initiate actions to neutralize the threat [130]. This defense mechanism depends on the coordinated interaction between immune cells and invading pathogens to initiate an effective immune response [131]. However, Brucella has developed sophisticated strategies to evade host innate immune receptors, facilitating its survival and replication within host cells [132].

One of the first identified members of pathogen recognition receptors (PRRs), the TLR4 receptor, plays an important role in the detection of bacterial LPS [133]. LPS, an integral component of the outer membrane of Gram-negative bacteria, is a powerful immune stimulant and a major contributor to the onset of septic shock [134]. The identification of TLR4 as the receptor for LPS marked a significant breakthrough in understanding how the innate immune system recognizes and responds to microbial infections. However, several characteristics of Brucella LPS contribute to its ability to evade detection by the innate immune system. Notably, the numerous attenuated mutants with structural defects in their LPS underscore the critical role of this molecule in Brucella virulence [135]. A vital adaptation involves the modification of the lipid A moiety, which enables Brucella to avoid recognition by TLR4. Unlike the lipid A of many bacterial pathogens that typically contains short-chain fatty acids (C12-C16), Brucella lipid A is distinguished by its incorporation of significantly longer fatty acid chains (C28). This structural difference greatly diminishes the agonist activity of TLR4 and reduces the endotoxic potential of Brucella LPS, facilitating its evasion of immune detection [136]. Another characteristic of Brucella LPS that prevents recognition by TLR4 is the unique glycosylation pattern of its core oligosaccharide component [137]. The B. abortus wadC glycosyltransferase mutant, which possesses a disrupted LPS core but an intact O-polysaccharide and lipid A, induces robust inflammatory responses in mice and fail to replicate in DCs, leading to its targeting to lysosomal compartments [30]. Unlike wild-type B. abortus, the wadC mutant triggers DCs maturation and cytokine secretion through TLR4. The LPS core of the mutated wadC strain display increased binding to myeloid differentiation-2 (MD-2), the TLR4 co-receptor, enhancing intracellular signaling [30]. Notably, caspase-11 has been shown to detect cytoplasmic LPS and induce septic shock through a mechanism independent of TLR4 [138,139]. During B. abortus systemic infection, caspase-11 knockout mice were found to be more susceptible than wild-type animals, with fewer immune cells, such as neutrophils, macrophages, and DCs cells, recruited to the spleens. Furthermore, guanylate-binding proteins (GBPs) located on mouse chromosome 3 contribute to LPS recognition by caspase-11, promoting non-canonical inflammasome activation [140]. This mechanism, on the other hand, demonstrates the non-redundant immune system processes that detect LPS derived from pathogenic bacteria, contributing to infection control. Nevertheless, TcpB, induces the ubiquitination and degradation of inflammatory caspases −1 and −11. Hence, TcpB inhibits LPS-induced non-canonical inflammasome activation, suppressing pyroptosis and IL-1β secretion [141].

Flagellin, the monomeric building block of the bacterial flagellar filament, functions as a pathogen-associated molecular pattern (PAMP) recognized extracellularly by TLR5 [142]. Upon activation, TLR5 triggers cellular signaling pathways that activate immune cells, leading to the production and release of pro-inflammatory cytokines essential for combating bacterial infections [143]. In that context, bacterial strategies to evade flagellin detection by the innate immune system have also been identified [144]. Brucella evades TLR5 activation by tightly regulating the synthesis and delivery of flagellin into host cells, a key aspect of its stealth strategy to avoid detection by the innate immune system [145].

Beyond its ability to avoid innate immune receptor recognition, Brucella also employs mechanisms to counteract cellular signaling transduction, disrupting the immune response to facilitate its survival within the host. An example of this is the Brucella TIR containing protein BtpA/TcpB, which is translocated into host cells and targets the pathways activated by TLR2 and TLR4, a key host response mechanism involved in bacterial detection [53,137]. TcpB has been shown to share similarities with the TIR domain protein MyD88-adapter – like (MAL) [55]. Mechanistically, TcpB disrupts the MAL-TLR4 interaction, thereby attenuating the cellular signaling cascade [54]. Additionally, TcpB interacts with MAL, promoting its polyubiquitination and subsequent degradation via the proteasome. This process ultimately decreases the amount of phosphorylated MAL available for signaling transduction [55]. Indeed, TcpB-mediated disruption of MAL functionality enhances the survival of Brucella within the host [146]. Similarly, BtpA interferes with TLR2-mediated activation of the nuclear factor-κB (NF-B) pathway, which inhibits DCs maturation and function. Consequently, this impairs the production of inflammatory cytokines and may disrupt antigen presentation to T lymphocytes [53].

The Omps of Brucella are crucial for maintaining the integrity of the bacterial membrane and serve as key virulence factors. These proteins interfere with cellular signaling pathways, aiding in immune evasion and promoting Brucella pathogenesis [147,148]. A notable characteristic of Omp25 is its capacity to suppress TNF-α production in macrophage cell lines [149], a crucial cytokine for protection against persistent Brucella infection [150]. This virulence factor suppresses the NF-κB pathway downregulating TNF-α transcription [148]. Remarkably, Omp25 inhibits TNF-α expression by modulating microRNAs that target TNF receptor associated factor 6 (TRAF6) and Interleukin 1 Receptor Associated Kinase 1 (IRAK1), thereby negatively impacting NF-κB signaling [148]. Brucella Omp25 also induces the expression of the programmed cell death-1 (PD-1) receptor, which modulates microRNAs to target TAK1-binding protein 2 (TAB2) [151]. TAB2, an adaptor protein, mediates the IκB phosphorylation and degradation, leading to NF-κB release and nuclear translocation and in this manner driving inflammatory cytokine expression [152]. By disrupting this pathway, Omp25 inhibits IL-12 production via PD-1 signaling and the upregulation of specific microRNAs [151]. Moreover, Omp25 also binds to the immune receptor SLAMF1, a member of the signaling lymphocyte activation molecule family (SLAMF). This interaction inhibits NF-κB translocation in DCs, thereby reducing inflammatory cytokine secretion and cellular activation, ultimately dampening the immune response during the acute phase of infection [153]. More recently, it was demonstrated that Omp25 inhibits inflammatory cytokine production by promoting the ubiquitination and degradation of TLRs and their adaptor proteins [154]. In addition to modulating inflammatory cytokine production, Omp25 inhibits the activation of the stimulator of interferon genes (STING) pathway by suppressing the phosphorylation and nuclear translocation of interferon regulatory factor 3 (IRF3) in virus-infected macrophages. This mechanism involves the proteasomal degradation of cyclic guanosine monophosphate – adenosine monophosphate synthase (cGAS), leading to reduced cyclic GMP-AMP (cGAMP) production and subsequent suppression of type I IFN (IFN-I) production [155].

Omp31 is another important factor that contributes to maintaining the integrity of the bacterial outer membrane, and its role in B. melitensis virulence has been demonstrated [156]. Interesting, Omp31 is not essential for the virulence of B. abortus, as the gene encoding it is absent in this species due to a genomic deletion [157]. Additionally, Omp31 was associated with autophagosome formation by increasing the levels of LC3B-II and Beclin-1. Furthermore, Omp31-induced autophagy suppresses TNF-α production through the modulation of NF-κB signaling [158]. Notably, Omp31 also reduces apoptotic factors in macrophages induced by TNF-α [159]. The modulation of TNF-α production and its pro-apoptotic effects may enhance the intracellular survival of the bacteria.

Brucella employs a sophisticated immune evasion strategy by manipulating host microRNA pathways to impair STING-mediated innate immune signaling. The pathogen suppresses STING expression at both transcript and protein levels via post-transcriptional regulation. Specifically, Brucella upregulates miR-24–2, which directly targets STING mRNA for degradation. Inhibition of miR-24 or deletion of the mirn23a locus in macrophages restores STING expression and is associated with reduced bacterial replication [160].

Unfolded protein response activation during Brucella infection: implications for host-pathogen interactions

The ER is a vital organelle responsible for the synthesis, folding, and processing of secretory and membrane proteins. Despite providing an optimized environment for protein folding, the ER capacity can be overwhelmed by physiological stressors, including increased protein synthesis, oxidative stress, or nutrient deprivation. This imbalance leads to the accumulation of misfolded or unfolded proteins, a condition referred to as ER stress [161]. In response, the UPR is activated as an adaptative mechanism to restore homeostasis by enhancing the protein-folding capacity, attenuating protein synthesis, and promoting the degradation of misfolded proteins [162]. This response is orchestrated by three key ER-resident sensors: IRE1, PERK, and ATF6, which activate interconnected signaling pathways regulating both adaptive and apoptotic responses to ER stress [163].

Beyond maintaining proteostasis, the UPR has emerged as a critical regulator in host-pathogen interactions [164]. During infections, the UPR acts as a double-edged sword, acting as both a target exploited by pathogens and a defense mechanism for the host. Pathogens such as viruses, bacteria, and parasites exploit the host ER machinery for replication, thereby inducing ER stress and activating the UPR. Conversely, host cells utilize UPR signaling to counteract infections by enhancing antigen presentation, promoting autophagy or inducing apoptosis to limit pathogen spread [165]. This interplay is particularly evident during Brucella infections, as the bacterium directly interacts with the ER to establish its replicative niche, positioning the UPR at the intersection of host-pathogen interactions.

The role of the UPR during Brucella infections was first suggested nearly two decades ago by an RNA interference screen that identified IRE1α as a key factor in Brucella pathogenesis [166]. Subsequent studies confirmed the activation of the UPR in infections caused by different Brucella species. For instance, B. melitensis activates all three primary UPR signaling pathways through the bacterial effector protein TcpB, which reorganizes ER structure and induces UPR target genes, such as BiP and CHOP. Pharmacological inhibition of the UPR reduces B. melitensis replication, emphasizing the role of the UPR in promoting intracellular survival [61]. Similarly, B. abortus triggers the UPR through a mechanism involving the bacterial second messenger cyclic di-GMP (c-di-GMP), which activates the ER-resident protein STING. STING induces the UPR and drives IFN-I production, facilitating bacterial replication [167]. Although both species activate the UPR to facilitate intracellular replication, their distinct mechanisms underscore species-specific strategies Brucella employs to exploit host cellular pathways to its advantage.

Brucella effectors play critical roles in inducing the UPR. While virB mutants can still activate the UPR, TcpB mutants exhibit reduced expression of UPR markers and ER structural disruption [61]. In B. abortus, VceC directly induces ER stress by interacting with the ER chaperone BiP, leading to IRE1α activation and pro-inflammatory responses via UPR-mediated NF-κB signaling [47]. This VceC-mediated ER stress activates the NOD1/NOD2/RIP2 signaling axis, contributing to inflammation, placentitis and abortion in mice [49]. Other effectors, such as BspC, BspG, and BspK, have also been implicated in ER stress induction, though their precise roles in Brucella pathogenesis remain to be fully elucidated [88].

The interplay between the UPR and immune responses during Brucella infection is further highlighted by the critical role of IRE1α in activating the NLRP3 inflammasome. Specifically, IRE1α facilitates ER-mitochondria communication via NLRP3, leading to mitochondrial damage and the release of mitochondrial danger-associated molecular patterns through the caspase-2-Bid signaling axis. This cascade induces mitochondrial damage, further amplifying inflammasome signaling. Remarkably, this pathway amplifies inflammation independently of ASC and involves the interaction between NLRP3 and thioredoxin-interacting protein (TXNIP) [168].

A recent study identified the T4SS effector BspI as a key modulator of the UPR during Brucella infection. Mechanistically, BspI selectively inhibits the kinase activity of IRE1α, thereby attenuating IRE1-mediated proinflammatory signaling cascades. This includes the suppression of NF-κB activation and downstream cytokine production, ultimately leading to a dampened inflammatory response [169].

The UPR also plays a crucial role in regulating intracellular lifecycle of Brucella. Specifically, the host factor YPT-interacting protein 1A (Yip1A) is essential for the biogenesis of rBCVs, a process driven by the activation of IRE1α. Disruption of Yip1A function prevents maturation of B. abortus into rBCVs, and confines BCVs within Lamp2-positive compartments, impairing bacterial replication [170]. Additionally, B. melitensis suppresses Bloc1s1 expression through IRE1-dependent decay (RIDD), disrupting lysosomal trafficking and facilitating ER-BCV fusion [171]. The interplay between the UPR and autophagy pathways also appears to regulate the biogenesis of rBCVs. Yip1A-dependent activation of IRE1α induces the formation of replicative vacuoles, a process that also requires the autophagy proteins ATG9 and WIPI. Silencing this autophagy components disrupts rBCV formation, confining Brucella to early endosomal compartments [170]. Additionally, Brucella subverts the IRE1α-ULK1 signaling to enhance its survival and disruption of ULK1 and Beclin-1 compromises bacterial intracellular replication [172].

IRE1α also plays a crucial role in immunometabolism during B. abortus infection by driving the metabolic shift from oxidative phosphorylation (OXPHOS) to glycolysis. This occurs through the stabilization of hypoxia-inducible factor-1alpha (HIF-1α), a key regulator of cellular metabolism that supports the inflammatory phenotype in Brucella-infected macrophages [173,174]. Additionally, IRE1α enhances the production of mitochondrial reactive oxygen species (mROS), nitric oxide (NO) and the release of IL-1β all hallmarks of inflammatory macrophages. This inflammatory profile is crucial for controlling bacterial replication, as HIF-1α deficiency increases the susceptibility to B. abortus infection in mice [173]. Recent evidence demonstrates that B. abortus modulates host mitochondrial dynamics by inducing dynamin-related protein 1 (DRP1)-dependent mitochondrial fission in infected macrophages. This process is driven by IRE1α activation, effectively linking the UPR to mitochondrial remodeling. DRP1-mediated fission disrupts mitochondrial metabolism, leading to decreased mitochondrial ATP production and impaired bioenergetic capacity [175]. The key insights into the UPR during Brucella infections, as discussed in this review, are summarized in Figure 1.

Figure 1.

Brucella and the unfolded protein response. (a) B. abortus triggers the upr via bacterial cyclic dinucleotides (CDN) that activate sting. sting activation leads to upr induction and IFN-I production. IRE1α drives the metabolic reprograming in macrophages by stabilizing HIF-1α. IRE1α enhances mROS production, as well as NO and IL-1β release. (b) VceC directly activates the upr by interacting with the er chaperone BiP, leading to IRE1α activation. This promotes pro-inflammatory responses through the NOD1/NOD2/RIP2 axis and NF-κB signaling, contributing to inflammation. (c) IRE1α promotes ROS-dependent translocation of NLRP3 to mitochondria, where it activates the caspase-2-bid pathway, leading to mitochondrial damage and release of danger signals that engage the inflammasome via NLRP3-TXNIP interactions. (d) Yip1A facilitates the activation of IRE1α by promoting the high-order assembly of IRE1α molecules at eres under upr conditions. Upon activation, IRE1α drives the biogenesis of ER-derived vacuoles.

Subversion of host autophagy pathways by Brucella

Autophagy is a fundamental stress response that preserves cellular homeostasis by degrading damaged organelles, protein aggregates, and invading pathogens. While many intracellular bacteria evade xenophagy – the selective autophagic clearance of pathogens – Brucella has developed unique mechanisms to subvert this pathway, redirecting components of the autophagic machinery to establish a replication-permissive niche and ensure completion of its intracellular life cycle [176].

Autophagic proteins are engaged at distinct stages of the Brucella intracellular cycle [20]. For example, Brucella activates IRE1α through Yip1A, which upregulates COPII vesicle components and facilitates the conversion of BCVs into rBCVs, thereby promoting bacterial survival [170]. rBCVs formation further requires ATG9 and WIPI proteins [177]. In parallel, IRE1α activation by B. melitensis induces phosphorylation of ULK1, Atg9a, and Beclin-1, supporting BCV maturation while preventing lysosomal degradation [172]. Importantly, the biogenesis of autophagic aBCVs depends on ULK1, Beclin-1, ATG14L, and class III PI3-kinase, but bypasses canonical elongation factors such as ATG5, ATG7, ATG4, ATG16L1, and LC3-II. This selective exploitation of noncanonical pathways exemplifies how Brucella avoids xenophagic clearance while co-opting autophagy to complete its intracellular life cycle and promote intracellular dissemination [17,20].

Multiple evidence further supports the role of autophagy in Brucella survival. In mouse macrophages infected with B. melitensis LC3-II accumulation and autophagosome formation increase, whereas pharmacological inhibition of autophagy markedly reduces bacterial replication [178]. Similarly, in B. suis-infected macrophages, activation of the autophagy-lysosomal pathway enhances bacterial proliferation [179]. Notably, Brucella replicates efficiently in Atg5-deficient fibroblasts, reinforcing that canonical macroautophagy is not strictly required for intracellular survival [180].

VirB T4SS effectors further modulate autophagic flux and apoptosis, reflecting redundant strategies of host manipulation. In hepatic stellate LX-2 cells, wild-type B. abortus induces LC3-II and Beclin-1 accumulation, whereas virB10 mutants fail to do so, confirming that T4SS effectors are required for autophagy induction [181]. Among these, BPE005 promotes autophagy, and deletion of bpe005 abrogates LC3-II accumulation. Pharmacological inhibition of PI3-kinase, lysosomal proteases, or autophagosome-lysosome fusion reverses these effects, demonstrating that effector-driven autophagy is crucial for intracellular persistence [181]. Other effectors display distinct modulatory patterns: BtpB inhibits autophagy and autophagolysosome formation, while VceA selectively promotes autophagy in trophoblasts to prevent host cell death and sustain persistent infection [48,169]. BspL interacts with the ERAD pathway, delaying aBCV formation and fine-tuning replication and dissemination [95]. NyxA and NyxB interfere with the nucleolar protease SENP3, relocalizing it into structures Beclin1/PIAS3-dependent structures to create a replication-permissive environment [182]. Bioinformatics analysis of macrophages infected with ∆Omp25 B. melitensis identified key miRNA-mRNA networks regulating autophagy, indicating that Omp25 modulates host autophagic flux through post-transcriptional mechanisms to influence bacterial survival [183].

Recent studies reveal even more sophisticated layers of autophagy modulation. B. abortus triggers mitochondrial fragmentation and BNIP3L-mediated mitophagy in an iron- and HIF-1α-dependent manner. While dispensable for replication per se, mitophagy is essential for aBCV biogenesis and bacterial egress [184]. Brucella also modulates ferritinophagy, the selective degradation of ferritin. Secretion of the ferritin-like protein Dps sequesters labile iron and suppresses ROS, which in turn triggers compensatory NCOA4-mediated ferritinophagy, releasing iron that supports bacterial replication and ferroptotic cell death [185]. Furthermore, host genetic factors further shape autophagy responses. A CRISPR screen revealed that knockout of DEFB103B reduces LC3-II accumulation and enhances bacterial clearance, suggesting that certain host antimicrobial molecules paradoxically support Brucella persistence by sustaining autophagic processes [186].

Type I IFN responses and host susceptibility to infection

Type I interferons (IFN-I), including IFN-α and IFN-β, are crucial cytokines in the innate immune response, playing a central role in defending against viral infections [187]. Their production is triggered by the activation of innate immune system receptors, including TLRs and cytoplasmic receptors that detect nucleic acids, particularly through the MyD88 and TRIF pathways [188]. The main sources of IFN-I are plasmacytoid dendritic cells and interstitial macrophages, activated by cytosolic receptors that recognize double-stranded RNA (dsRNA). In macrophages and DCs, TLR3 and TLR4 also induce IFN-I upon detecting dsRNA and LPS [134].

IFN-I signaling is mediated by the type I interferon receptor (IFNAR), composed of IFNAR1 and IFNAR2 subunits [189]. The binding of IFN-I to IFNAR activates the JAK-STAT pathway, promoting phosphorylation of STAT1 and STAT2, which heterodimerize and recruit IRF9 [190]. This complex translocates to the nucleus to induce the transcription of interferon-stimulated genes (ISGs). These responses include pro-apoptotic genes, chemokines, and other mediators crucial for immunity [191]. While IFN-I is traditionally associated with antiviral immunity, studies have revealed its crucial role in bacterial infections, where its production is triggered by various Gram-negative bacteria during cellular interaction or invasion [192]. The role of IFN-I in bacterial infections was first observed in investigations of Chlamydia spp. infections, which, despite their intracellular cycle, induce IFN-I production through TLR4 and MyD88-dependent signaling pathways [193]. Intracellular pathogens induce IFN-I through TLR4, MyD88, or cytosolic pathways, regulating both protective and detrimental responses [42]. Infections caused by pathogens such as Listeria monocytogenes, Mycobacterium tuberculosis (Mtb), Salmonella Typhimurium (SesT), and B. abortus demonstrate that sensors like STING, cGAS, RIG-I, and NOD2 are involved in IFN-I production. This response, by inducing apoptosis of immune cells, suppressing inflammatory cytokines (such as IL-1β and IL-18), and reducing neutrophil recruitment, ultimately favors bacterial persistence and replication [194–197].

In tuberculosis, active human disease is consistently associated with the induction of IFN-I [198]. Evidence that IFN-I exacerbates tuberculosis in humans comes from observations that viral infections are associated with worsened outcomes in Mtb infections. Recently, Ji et al [199]., identified Sp140 as a transcriptional regulator of IFN-I that controls Mtb susceptibility. This research group suggests a model of tuberculosis pathogenesis in which IFN-I drives an initial loss of bacterial control, possibly by impairing IFN-γ responses, that, in turn, initiates a positive feedback loop of NET production and IFN-I expression by pDCs, leading to uncontrolled bacterial replication and active tuberculosis disease [196].

In the context of Brucella infections, the pioneering study by Huang et al [200]., investigated the relationship between B. abortus and IFN-I, demonstrating that wild-type mice had detectable levels of IFN-α in their serum just three hours after administration of heat-killed B. abortus. In contrast, TLR9-deficient mice showed significantly reduced levels of IFN-α, indicating that IFN-I induction by B. abortus depends on a TLR9-mediated pathway. Additionally, IFNAR-deficient mice showed a reduced bacterial load in the spleen and higher production of inflammatory mediators, such as IFN-γ and NO, compared to wild-type controls.

Our group has been investigating the interaction between IFN-I and B. abortus for several years. We demonstrated that Brucella infection induces IFN-I production in macrophages and splenocytes mediated by MyD88-dependent pathways, IRF3 signaling and intracellular sensors such as STING and ZBP1 [167,201]. We observed that IFN-I has a detrimental effect on the host by suppressing the production of inflammatory cytokines like IL-1β and IFN-γ, while also modulating pro-apoptotic genes such as TRAIL. IFN-I-deficient mice exhibited lower bacterial load and reduced apoptosis, suggesting that IFN-I favors B. abortus survival [197]. Similar findings have been observed with Listeria monocytogenes [202,203].

Recently, we demonstrated that B. abortus-induced UPR has an important role in inducing IFN-β responses and is linked to the production of several molecules associated with the IFN-I pathway [167]. Additionally, macrophages treated with a mouse recombinant IFN-β (rIFN-β) showed enhanced XBP1(s) expression, a downstream target of IRE1α activation, and enhanced number of B. abortus CFU in vitro [167]. Collectively, these findings demonstrate that treatment with rIFN-β enhances B. abortus replication, underscoring the pivotal role of IFN-β in B. abortus replication and survival.

We recently identified a previously unrecognized role for DRP1 in regulating IFN-I production and signaling during Brucella infection. Mechanistically, DRP1-dependent mitochondrial fission facilitates the release of mitochondrial DNA into the cytosol, possibly serving as a potent trigger for IFN-I responses. This process establishes a link between mitochondrial dynamics and innate immune signaling, highlighting how B. abortus can modulate host pathways through targeted remodeling of mitochondrial morphology [175].

Furthermore, Brucella Omp25 specifically targets the cGAS-STING signaling pathway, inhibiting its production of IFN-β by promoting cGAS degradation in macrophages via the ubiquitin-proteasome-dependent pathway upon DNA virus infection or DNA stimulation [155].

IFN-I is unquestionably crucial for the immune response. However, in some cases, it can promote pathogen evasion and reduce the effectiveness of the immune response, compromising bacterial infection control. This ambiguous effect highlights the need for careful regulation of IFN-I signaling to ensure an effective response against a variety of pathogens.

Brucella and host metabolism: a battle for resources

Cellular metabolism is crucial in regulating innate immune activation, as metabolic changes in immune cells often dictate pro-inflammatory or anti-inflammatory responses [204]. Immune cells undergo metabolic reprogramming to meet the energy and biosynthetic demands necessary for their activation and effector functions. During an inflammatory response, macrophages and DCs primarily shift toward glycolysis, enabling the rapid production of ATP and biosynthetic intermediates vital for cytokine secretion and pathogen elimination. Notably, cells may prioritize glycolysis for ATP production even in the presence of sufficient oxygen, a phenomenon known as the Warburg effect (aerobic glycolysis) [205]. This metabolic reprogramming impacts the production of key metabolites such as lactate, succinate, and itaconate, which act as signaling molecules regulating immune responses [206].

Brucella exploits these metabolic adaptations to establish and sustain chronic infection. Key Brucella genes essential for persistence are associated with its ability to utilize diverse nutrient sources, suggesting that the bacterium has evolved mechanisms to exploit both immune responses and limited nutrient availability within host cells [137]. The adaptation of Brucella to the macrophage environment is particularly significant, as it adjusts to distinct metabolic pathways associated with pro-inflammatory (M1) and anti-inflammatory (M2) macrophages. M1 macrophages rely primarily on aerobic glycolysis, producing lactate while utilizing the tricarboxylic acid cycle (TCA) cycle to generate citrate and succinate, which are essential for fatty acid metabolism and HIF-1α stabilization. These processes enhance the transcription of pro-inflammatory and glycolytic genes while promoting the production of NO and ROS. In contrast, M2 macrophages depend on fatty acid oxidation and OXPHOS to meet their metabolic needs [207].

Inflammatory M1 macrophages predominate during the early stages of Brucella infection and are characterized by increased nitric oxide synthase 2 (NOS2) expression and other pro-inflammatory markers, while anti-inflammatory M2 macrophages become more prominent during the chronic phase [173]. Since NOS2 expression and pro-inflammatory cytokines are crucial for controlling B. abortus growth [208,209], M1 macrophages are expected to manage the infection more effectively than the M2 phenotype. Indeed, NF-κB signaling is crucial to promote M1 polarization, which significantly reduces B. abortus survival in a macrophage cell line. In this scenario, the glutaminase (Gls) enzyme, a key NF-κB target, enhances the expression of M1-associated markers, while its inhibition promotes M2 polarization and facilitates bacterial replication [210].

Furthermore, during Brucella infection, STING promotes HIF-1α stabilization, reprogramming macrophage metabolism from OXPHOS to glycolysis, enhancing NO production and inflammasome activation. Likewise, the mitochondrial function shifts in macrophages from ATP synthesis to ROS production. These processes are critical for controlling Brucella persistence [173]. However, during chronic infection, Brucella circumvents these defenses by preferentially replicating in M2-like macrophages, where it benefits from increased glucose accumulation driven by the activity of peroxisome proliferator-activated receptor γ (PPARγ). M2-like macrophages rely on fatty acid β-oxidation for ATP production in a PPARγ-dependent manner, leading to higher intracellular glucose accumulation compared to M1-like macrophages. This glucose accumulation supports Brucella growth, and the inactivation of the bacterial glucose transporter gluP significantly reduces bacterial survival in macrophages [211]. The enhanced survival of B. abortus in M2-like macrophages is also linked to a shift in host arginine metabolism from NO production to polyamines synthesis, driven by arginase-1 expression. Inhibiting polyamine synthesis or inactivating the putrescine transporter (potIHGF) reduces B. abortus persistence in M2-like macrophage, highlighting the importance of polyamine metabolism in sustaining chronic infection [212]. Another example of adaptation to the intracellular niche is observed in B. abortus-infected THP-1 cells. Brucella disrupts mitochondrial function and alters the localization of mitochondria, changing the metabolism of amino acids that feed the TCA. This metabolic shift reduces amino acid catabolism and increases lactate production, allowing the bacteria to utilize host-derived amino acids and lactate as energy sources to support its intracellular persistence [213].

Brucella also regulates glucose metabolism by activating NF-κB to modulate glucose-6-phosphate dehydrogenase (G6PD) expression, which influences the switch to glycolysis by regulating NO levels during Brucella infection [214]. Additionally, MyD88 contributes to macrophage glycolysis in response to B. melitensis, by inducing metabolic changes that control key metabolites, including itaconate, a metabolite with antibacterial effects against Brucella [215]. Moreover, BtpA and BtpB, through their NAD⁺ hydrolase activity, deplete NAD⁺ levels in host cells, modulating host metabolism. Both proteins induce growth arrest in yeast cells, a process associated with actin depolymerization and reduced kinase activity, suggesting impaired energy metabolism linked to decreased ATP and NAD⁺ levels [62]. A summary of the data presented in this section is provided in Figure 2.

Figure 2.

Overview of immunometabolism in Brucella-infected macrophages. Inflammatory M1 macrophages (left) predominate during the early stages of Brucella infection, playing a critical role in infection control. The TLR/MyD88 and NF-κB signaling pathways promote glycolysis and glutaminase-driven expression of pro-inflammatory markers. NF-κB upregulates G6PD, which facilitates the shift to glycolysis by regulating no production. sting activation enhances mROS production, contributing to HIF-1α stabilization, which promotes glycolysis and suppresses OXPHOS. This metabolic reprogramming increases glucose consumption to meet energy demands, resulting in elevated lactate levels, which Brucella can exploit as an energy source. During chronic infection, anti-inflammatory M2 macrophages (right) support Brucella persistence. PPARγ activation promotes fatty acid β-oxidation, reducing host glucose utilization and increasing intracellular glucose availability for bacterial replication. Arginase-1 shifts arginine metabolism from no production to polyamines synthesis, providing additional nutrients for the bacteria. Moreover, reduced no and suppressed pro-inflammatory cytokine production contribute to infection chronicity, allowing the pathogen to establish a long-term intracellular niche.

Conclusions and future perspectives

Brucella species have evolved highly specialized mechanisms to manipulate host immune responses and metabolic pathways, facilitating their adaptation to the host environment. This review has highlighted the intricate strategies by which Brucella modulates immune signaling networks, particularly IFN-I responses and immunometabolism, to evade detection and suppress antimicrobial defenses. Additionally, the ability to exploit the UPR underscores their sophisticated adaptation to intracellular stress, promoting long-term persistence within host cells.

Despite significant progress in understanding Brucella pathogenesis, critical knowledge gaps remain regarding the precise molecular interplay between Brucella effectors and host immune regulators, as well as the metabolic rewiring strategies that sustain chronic infection. Future research should focus on elucidating the regulatory circuits governing Brucella adaptive responses and identifying metabolic vulnerabilities that could be exploited as therapeutic targets. Furthermore, investigating the crosstalk between Brucella-induced UPR activation and immune evasion mechanisms may reveal novel potential targets for host-directed therapies.

Advances in single-cell RNA sequencing (scRNA-seq) have enabled the analysis of transcriptional heterogeneity during infections. In the bacterial context, methods such as MATQ-seq, microSPLiT, and PETRI-seq allow unbiased gene expression profiling in individual prokaryotic cells, revealing phenotypic states and transcriptional programs associated with persistence and immune evasion [216]. These transcriptomic technologies are increasingly being applied to dissect host-pathogen interactions in Brucella infection models. A dual RNA-seq study in murine macrophages simultaneously profiled both B. abortus and host gene expression, revealing how the bacterium adapts its metabolic and virulence gene programs to the intracellular environment while modulating host innate immune pathways [217]. Complementarily, a recent single-cell RNA-seq study in human brucellosis patients identified immune phase-specific signatures, including functional exhaustion and impaired antigen presentation in chronic stages of disease [218]. These findings highlight the value of transcriptomic approaches in uncovering key virulence strategies and host responses, offering new directions for future research and therapeutic development.

Alongside transcriptomic innovations, artificial intelligence (AI) and machine learning (ML) approaches are being progressively explored in Brucella research. Recent studies have applied ML algorithms to tasks such as species classification based on MALDI-TOF MS spectra, with promising levels of accuracy [219,220] and to reverse vaccinology pipelines aimed at optimizing antigen selection for vaccine development [221–223]. In addition to these applications, AI-based methods have shown promise in analyzing multi-omics and functional genomics data in the context of Brucella [224]. Models such as Vaxign ML, for example, have been successfully employed to prioritize multi-epitope vaccine candidates against Brucella, based on genomic and proteomic features [224–226]. Although still in early stages, the integration of AI-guided analyses with experimental techniques such as scRNA-seq and dual RNA-seq holds promise for uncovering novel virulence mechanisms, predicting molecular markers of persistence, and refining the selection of therapeutic and vaccine targets.

Beyond pathogenesis, there is an urgent need for more effective diagnostic tools and next-generation vaccines with improved safety and immunogenicity. Current vaccines, while partially protective, present limitations such as residual virulence and inconsistent efficacy across host species. Integrating advanced approaches in systems biology, genomics, and immunology may facilitate the design of targeted therapeutic interventions and optimized vaccine formulations.

Among next-generation vaccine platforms, mRNA-based vaccines are gaining attention as a promising alternative to overcome the limitations of conventional Brucella vaccines, particularly in the context of persistent infection [222]. In contrast to attenuated or inactivated bacterial vaccines, which retain residual virulence and interfere with standard serological diagnostics [227,228], mRNA vaccines offer several advantages, including rapid development, strong immunogenicity, and flexible epitope targeting [229]. Recent in silico studies have designed multi-epitope mRNA vaccine constructs targeting antigens such as BtuB, LptD, and outer membrane proteins of B. melitensis, showing favorable molecular stability, antigenicity, and MHC-binding affinity [229,230]. These formulations are predicted to induce robust CD4⁺ and CD8⁺ T cell responses with a Th1 profile, which is essential for the clearance of intracellular pathogens [231]. As these candidates progress toward preclinical validation, they are expected to complement systems-level immunological insights and pave the way for more precise therapeutic strategies against chronic Brucella infection.

A deeper understanding of Brucella immunomodulatory strategies, metabolic adaptations, and virulence determinants will be instrumental in advancing targeted intervention strategies. This knowledge will not only enhance our ability to combat Brucella infections but also provide broader insights into host-pathogen interactions and chronic bacterial persistence mechanisms.

Funding Statement

This work was supported by grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (303044/2020–9 and 406974/2023–3), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) [Grants 2022/15358–7, 2023/02577–5, 2024/12744–9, 2023/09226-3 and 2024/13812-8] and National Institutes of Health [Grant R01 AI116453].

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

Data sharing is not applicable to this article as no new data were created or analyzed in this study.