In vivo, Brucella rapidly propagate over the mucosal epithelial layer (Rossetti et al., 2013) and are taken up by dendritic cells (DCs) and mucosal macrophages. Through cellular tropism, Brucella is able to live and multiply inside competent phagocytic cells, avoid detection by the host immune system, and spread to target organs, such as the reproductive tract, fetal lungs, reticuloendothelial system, and placental trophoblasts in pregnant women (Adams, 2002). To comprehend the adhesion, internalization, intracellular trafficking, survival, and replication of Brucella in vulnerable hosts, in vitro investigations were employed as models. In order to internalize itself, Brucella initiate a zipper-like process on the surface of mucosal epithelial cells (Rossetti et al., 2012). Brucella binds to sialic acid and sulfated residue-containing binding molecules on the surface of epithelial cells, which are activated before and/or upon contact. However, these binding molecules are still not fully understood.

Binding stimulates small GTPase activity, which initiates a signaling cascade that reorganizes the actin cytoskeleton and induces a rearrangement of the host cell membrane along the pathogen’s surface, thereby enhancing invasion. Shortly after contact, entry occurs, necessitating the complete activation of a mitogen-activated protein kinase signaling cascade. Brucella can live and multiply among inactivated phagocytic cells for up to 72 hours in vitro. In vivo, they can traverse the epithelium by undermining the function of the mucosal epithelial barrier, which enables Brucella to undergo transepithelial migration. This connection simultaneously triggers a minor innate immune response and modest proinflammatory activity (Barquero-Calvo et al., 2007). After being transferred across the epithelium, Brucella are taken up by mucosal phagocytic cells, where less than 10% of the phagocytized bacteria make it through a period of adaptation.

Brucella reduce, modify, or hide their pathogen-associated molecular patterns to evade immune recognition and trigger an immune response. On the other hand, some Toll-like receptors (TLRs; primarily TLR2, TLR4, and TLR9) start a limited intracellular signaling that activates the transcription factor NF-κB to control the expression of inflammatory cytokine genes (Oliveira et al., 2008) albeit at a 10-fold lower level than that seen with enterobacteria. Brucella live in a unique vacuole called the Brucella-containing vacuole (BCV) inside mononuclear phagocytic cells. They alter intracellular trafficking in this vacuole and convert it into a replicative compartment called a brucellosome. Research suggests that the BCV’s microenvironment has a restricted supply of nutrients12, to which Brucella quickly adjusts upon invasion (Köhler et al., 2002).

In order to adapt to low oxygen tension, the pathogen first increases amino acid catabolism, switches to alternative energy sources, and quantitatively reduces gene expression and protein synthesis involved in anabolic metabolism (Lamontagne et al., 2009). The development of a type IV secretion system (T4SS) early after infection is crucial for intracellular survival and multiplication inside mammalian cells in an in vitro brucellosis infection model. However, in vivo research has shown that while the T4SS is required for prolonged persistence, it is not necessary for invasion, systemic dispersion, or the development of the initial infection (Roux et al., 2007).

Invading Brucella that survive the adaptation phase during infection progressively restore the expression of important genes encoded by metabolic processes. This transcription-translation reactivation primarily affects cell membranes, transporters, and iron metabolism (Lamontagne et al., 2009). Brucella reproduce in tandem with the restoration of essential processes, such as the expression of virulence genes, which are occasionally strictly regulated by quorum-sensing molecules (Rambow-Larsen et al., 2008; Weeks et al., 2010). When an infection occurs, infected mononuclear phagocytic cells undergo significant transcriptional alterations during the adaptation stage. After 12 hours, when Brucella replication starts, the modifications return to normal. After adapting to the intramacrophage environment, Brucella prolongs its intracellular persistence indefinitely. This leads to the infection of desired targeted cells or tissues, including the reticuloendothelial system, endothelium, male genitalia, fetal lungs, skeletal tissues, and placental trophoblasts, as well as systemic metastasis. In order to give a more comprehensive systems biology description of the pathogenesis of brucellosis at the level of the entire host organism, there is currently a dearth of evidence describing the interaction of Brucella with these target cells and tissues (Carvalho Neta et al., 2008; Delpino et al., 2009).

The type IV secretion system (T4SS) of Brucella has been the most extensively investigated factor influencing its virulence (DelVecchio et al., 2002). The 11 proteins that comprise the Brucella T4SS are a lytic transglycosylase (VirB1) that remodels the bacterial cell peptidoglycan layer during T4SS assembly, two ATPases (VirB4 and VirB11) that supply energy to drive effector secretion and eight proteins that comprise the core of the transporter (VirB2, VirB3, and VirB5 through VirB10) (Watarai et al., 2002c; Höppner et al., 2004; Carle et al., 2006). The operon that contains the T4SS genes is conserved in all strains of Brucella, and the virB mutants of B. abortus, B. melitensis, B. suis, B. canis, B. ovis, B. microti, and B. neotomae are significantly reduced in both natural and cultured hosts (Sieira et al., 2000; Patey et al., 2006; Sivanesan et al., 2010; Kang et al., 2019). Genes located outside of the virB operon also encode Brucella proteins that aid in the assembly and functionality of the T4SS. One such protein, VirJ, is a periplasmic protein whose exact role is still unknown, but it is necessary for the correct assembly of the T4SS and interacts directly with T4SS substrates that have a periplasmic intermediate during their export (Del Giudice et al., 2016). One of the main functions of the T4SS is to regulate the intracellular trafficking of the Brucella-containing vacuoles in host macrophages, preventing the bacteria from being killed and degraded in phagolysosomes (Comerci et al., 2001; Watarai et al., 2002a; Celli et al., 2003).

Initially, Brucella was identified as a facultative internal bacterial parasite that could multiply in phagocytes, such as granulocytes, macrophages, and dendritic cells (DC), as well as epithelial, fibroblastic, and trophoblastic cells (Copin et al., 2012). Through lipid rafts, Brucella interacts with macrophage cell membranes and enters the host cell to produce Brucella-containing vacuoles (BCV), which are encircled by phagocytic vesicles (Köhler et al., 2003). Eight to twelve hours after entry, BCV matures into endosomes within the membrane-bound vacuoles, acidifies, and acquires host marker molecules through contact with lysosomes (Lys) and endosomes. Currently, the term “endosomal Brucella-containing vacuole” (eBCV) is used to refer to a BCV. The Type IV secretory system (T4SS) receives membranes derived from the Golgi apparatus and the endoplasmic reticulum (ER) by mediating the connection between the effector protein and the ER exit site as BCV grows and matures. The eBCV acquired Lys marker molecules (such as Rab7, LAMP-1, etc.) after losing the early host marker molecules (Von Bargen et al., 2012). When the BCV avoids Lys degradation, it will eventually reach the ER and fuse there in a way that depends on Rab2 and Sar1 (Celli et al., 2003). At this stage, the BCV is referred to as a replicative Brucella-containing vacuole, or rBCV. rBCV will change into autophagic Brucella-containing vacuole (aBCV) at a later stage of infection ( Figure 2 ). The aBCV will not continue to develop and destroy cells at this time. At this point, Brucella has finished the intracellular circulation, and the organism uses lysis and nonlysis methods to release pathogens (Starr et al., 2012).

By interacting with lipid rafts on the plasma membrane, Brucella can mediate its internalization into phagocytes and to facilitate interaction with host cells. Glycosphingolipids and cholesterol found in lipid rafts have the ability to stimulate biological processes associated to membranes, including membrane fusion, transmembrane signaling, and the production of polybasic membrane complexes (Cutler et al., 2005). LPS can stop complement-mediated bacterial lysis and host cell apoptosis, and it is a crucial molecule in the interaction between Brucella and lipid rafts in the cell (Jiménez De Bagüés et al., 2004). Brucella invades cells through lipid rafts, and it has been demonstrated that the class A scavenger receptor (SR-A) and the prion protein (PrPc) are involved in this process (Watarai et al., 2003; Kim et al., 2004).

Specific lipid rafts contain prion proteins and SR-A, which are receptors for heat shock protein 60 (HSP60) and LPS. The early survival of Brucella in macrophages can be efficiently reduced by lipid raft destruction, suggesting that lipid raft introduction is a prerequisite for the early survival of bacteria (Naroeni and Porte, 2002). Brucella enters the cell to form a phagosome and take part in the endocytosis pathway, but it can be easily detached from the phagosome, suggesting that the lipid raft-mediated signaling pathway plays a role in Brucella’s early survival (Porte et al., 2003). aBCV is required by Brucella to finish its intracellular life cycle and cell-to-cell dissemination during intracellular circulation (Starr et al., 2008). The conversion of rBCV to aBCV starts when the ER Beclin1 and PI3K form a complex, although this process gradually slows down as ATG14L is consumed. The effector protein BspB interacts with the conserved oligomeric Golgi (COG) to regulate COG-dependent transport, reorient Golgi-generated vesicles to BCV, boost rBCV production, and enhance Brucella intracellular proliferation (Jiao et al., 2021).

Modulating the host immune response is another mechanism that the Brucella T4SS increases virulence (Rolán and Tsolis, 2007; Roux et al., 2007; Rolán and Tsolis, 2008). For example, the host cell ER chaperone BiP interacts with the T4SS effector VceC (De Jong et al., 2013). In Brucella-infected cells, this interaction results in ER stress and an unfolded protein response (UPR), which in turn promotes the production of the inflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α). The synthesis of these cytokines by macrophages in response to VceC leads to the formation of granulomas, which promotes the persistence of infection. Researchers hypothesize that this T4SS effector may be crucial to transmission in natural hosts because VceC-mediated inflammatory cytokine release by placental trophoblasts also causes host cell death and fetal disease in the pregnant mice model (Keestra-Gounder et al., 2016; Byndloss et al., 2019).

The T4SS effectors BtpA and BtpB, in contrast to VceC, block dendritic cells’ ability to produce inflammatory cytokines by disrupting the TLR-Myd88-MAL signaling pathway (Salcedo et al., 2008; Sengupta et al., 2010; Alaidarous et al., 2014). It has been suggested that the T4SS effectors’ dual ability to stimulate and inhibit host immune responses enables Brucella to induce a response in the host that is beneficial to their long-term intracellular persistence. This balance ensures enough immunopathology to aid in their spread to other hosts, but not strong enough to cause sterilizing immunity and end the infection (De Jong et al., 2010).

Brucella strains, like most Gram-negative bacteria, produce lipopolysaccharide (LPS), which is essential for maintaining the integrity of their cell envelope (Roop et al., 2021). They have a smooth LPS (S-LPS) composed of a polysaccharide O-chain, core, and lipid A, with two major exceptions: B. ovis and B. canis, which naturally produce a crude LPS lacking the O-chain. There is ample evidence to support the significance of the O-chain for the pathogenicity of naturally occurring smooth Brucella strains (Monreal et al., 2003; Roop et al., 2021). By shielding smooth Brucella strains from the complement’s bactericidal effects (González et al., 2008; Ouahrani-Bettache et al., 2019) and the antimicrobial peptides they encounter when interacting with host phagocytes, the LPS O-chain is one way that it contributes to virulence or by acting as an adhesin. Smooth Brucella strains can enter mammalian cells via an endocytic pathway that circumvents the broad fusion of BCVs with lysosomes, thanks to the interaction of the O-chain with lipid rafts on the surface of these cells (Porte et al., 2003). Before the involvement of the T4SS effectors, this entry point is the first necessary step in the replication of BCV by smooth strains (Naroeni and Porte, 2002; Watarai et al., 2002b).

Because this pathway of entry promotes modest levels of proinflammatory cytokine production in macrophages and dendritic cells, O-chain-mediated uptake of smooth Brucella strains also plays a significant role in immune evasion (Jiménez De Bagüés et al., 2004; Billard et al., 2007). In addition to being resistant to being broken down by macrophages, smooth LPS secreted by Brucella strains into BCVs also forms complexes with major histocompatibility complex class II (MHC-II), which prevents these phagocytes from presenting antigens to T lymphocytes (Forestier et al., 2000; Lapaque et al., 2006a). Inhibiting caspase 2-mediated cell death in these phagocytes is yet another mechanism whereby the O-chain-mediated entrance of smooth Brucella strains into macrophages adds to virulence (Gross et al., 2000; Fernandez-Prada et al., 2003; He et al., 2006). Although the exact processes underlying this suppression are unknown, smooth strains’ ability to prolong macrophage life expectancy probably improves the macrophages’ resistance to immunological clearance and ability to spread to many organs within their mammalian hosts (Pei et al., 2006; Chen and He, 2009).

Since lipid A is the part of LPS that the host pattern recognition receptor Toll-like receptor 4 (TLR4) recognizes, and since the lipid A of many Gram-negative bacteria generates potent inflammatory reactions, lipid A’s are sometimes referred to as “endotoxins” (Bryant et al., 2010). The production of low endotoxin activity lipopolysaccharides (LPS) by Brucella strains has long been reported (Duenas, 2004; Tumurkhuu et al., 2006). The fact that Brucella lipid A contains very-long-chain fatty acids (VLCFAs), in contrast to its enteric counterparts, may be one reason for its reduced inflammatory response in certain strains of the bacteria (Velasco et al., 2000; Ferguson et al., 2004). These VLCFAs most likely stop the Brucella lipid A from interacting with TLR4 in the same potent ways as other bacterial lipid A molecules (Lapaque et al., 2006b). Remarkably, human neutrophils are similarly susceptible to early cell death caused by the Brucella lipid A. Macrophages and dendritic cells then consume dead neutrophils that harbor intracellular BrucellaBrucella through non-inflammatory mechanism. This has been suggested as one further tactic that Brucella strains may use to evade the host immune system’s recognition in the early phases of infection (Barquero-Calvo et al., 2015).

The primary structural function of the LPS core in many bacteria is to connect lipid A with the O-chain. It serves the same purpose in Brucella, but more recently, research has demonstrated that the LPS core is crucial in helping these bacteria evade recognition by the host immune system (Fontana et al., 2016; Salvador-Bescós et al., 2018). To be more precise, the Brucella generates a core structure with a side chain of lateral oligosaccharides that sterically covers lipid A and prevents it from binding to TLR4 on host dendritic cells and macrophages ( Figure 3 ). The resistance of both smooth and rough Brucella strains to complement and bactericidal peptide killing appears to be influenced by this lateral side chain and the positive charge it provides on the LPS core (Soler-Lloréns et al., 2014).

Brucella species that infect domestic animals, along with many strains that infect wildlife, share a highly conserved organization and composition of LPS biosynthesis genes. The absence of the O-chain from the LPS of B. ovis and B. canis is caused by well-documented genomic deletions (Zygmunt et al., 2009). However, some of the so-called early diverging Brucella strains that were isolated from human diseases and found in amphibians use the operon rmlABCD, which consists of four genes, to produce an LPS with an O-chain based on rhamnose rather than the perosamine O-side chain found in all other smooth Brucella strains (Wattam et al., 2012). It has been hypothesized that the acquisition of the genes encoding this latter kind of LPS O-chain was crucial to the evolution of Brucella strains as mammalian pathogens, given the proven significance of the perosamine O-chain in virulence (Wattam et al., 2009). It is interesting to note that serologic research indicates that the LPS cores of some early divergent strains might differ from those of the 200 typical Brucella strains.

Omp25, Omp25b, Omp25c, Omp25d, Omp31, Omb31p, and Omp22 are a family of highly conserved outer membrane proteins (OMPs) produced by Brucella strains that are crucial for preserving the integrity of their cell envelope (Cloeckaert et al., 2002). These β-barrel OMPs protect the bacteria against complement and other antimicrobial peptides found in the host when they function in tandem with the LPS O-chain. Their virulence contributions seem to be particularly significant for naturally occurring rough strains such as B. ovis (Edmonds et al., 2002; Martín-Martín et al., 2008). It has been demonstrated that B. abortus and B. melitensis omp25 mutants are attenuated in mice (Edmonds et al., 2002) and natural hosts (Edmonds et al., 2001), and that a B. melitensisomp31 mutant is attenuated in both mice and cultured mammalian cells (Verdiguel-Fernández et al., 2017; Fernández et al., 2020). The Brucella Omp25/Omp31 proteins have been shown to facilitate direct contacts between the Brucella and mammalian cells that are crucial for virulence, in addition to their structural roles in preserving cell envelope integrity. Some of the divergent phenotypes observed in virulence experiments for Brucella omp25 and omp31 mutants may be explained by these latter roles. In smooth strains (Manterola et al., 2007), where the LPS O-chain appears to be the predominant determinant in mammalian cell entry, there is no evidence that Omp25d and Omp22 perform this function. In contrast, Omp25d and Omp22 play significant roles in B. ovis entry into mammalian cells (Martín-Martín et al., 2008). The smooth strain B. abortus’s Omp25 protein also directly interacts with dendritic cells’ SLAMF1 protein on their surface, preventing them from maturing and releasing inflammatory cytokines (Degos et al., 2020) ( Figure 4 ). Jubier-Maurin et al. (2001a) initially documented Omp25’s ability to suppress TNF-α production during Brucella infection; however, the molecular mechanisms of this function have only recently been determined. It is unclear exactly how these other hypothesized Omp25/Omp31 functions contribute to virulence, but there is evidence that the Brucella Omp25 and Omp31 proteins have the ability to modify other elements of host cell function during infection (Jubier-Maurin et al., 2001a; Zhang et al., 2016; Cui et al., 2017; Luo et al., 2018). The great degree of conservation of the Omp25/Omp31 proteins throughout the Rhizobiaceae family is one of its intriguing characteristics. The Omp25/Omp31 orthologs are also crucial for the productive relationships of other alphaproteobacteria with their respective eukaryotic hosts. For example, Agrobacterium tumefaciens’ wild-type pathogenicity in plants requires the Omp25 ortholog AopB (Jia et al., 2002). Bartonella henselae hbp knockdown strains are attenuated in endothelial cell cultures (Liu et al., 2012), and the Bartonella heme-binding proteins (Hbps) are likewise Omp25/Omp31 orthologs (Minnick et al., 2003). Remarkably, it has also been demonstrated that the Brucella Omp31b binds hemin in vitro, and the B. suis gene that codes for this protein is iron-controlled (Jubier-Maurin et al., 2001a).

It has not been demonstrated that any one Brucella species can synthesize all seven Omp25/Omp31 proteins (Martín-Martín et al., 2009). Large genomic deletions have resulted in the loss of genes encoding distinct Omp25/Omp31 proteins in some species, such as B. abortus and B. ovis (Pei et al., 2006). Other Brucella species have different patterns of Omp25/Omp31 production, which seem to be caused by smaller genetic abnormalities (Martín-Martín et al., 2009). The tight coordination of the expression of the omp25 and omp31 genes in response to environmental stimuli and physiological cues by the global regulators BvrRS, VjbR, and CtrA (Viadas et al., 2010) is consistent with the important function that this family of OMPs plays in the fundamental physiology and pathogenicity of Brucella strains.

Three outer membrane lipoproteins (Omp10, Omp16, and Omp19) are produced by different strains of Brucella (Tibor et al., 1999). The peptidoglycan-associated lipoprotein (Pal), which is largely conserved in Gram-negative bacteria, has a homolog in Omp16 (Godlewska et al., 2009). These proteins interact with the constituents of the Tol complex and are essential for maintaining the structural integrity and functionality of the outer membrane. Omp16’s role as a pal homolog is consistent with the fact that it is an important gene in Brucella (Sidhu-Muñoz et al., 2016; Sternon et al., 2018; Zhi et al., 2020). Conversely, Omp19 is the most well-studied lipoprotein produced by Brucella. Through its interactions with TLR2, purified Omp19 has strong immunomodulatory activities that affect a wide range of host cells. These interactions have been linked to Brucella’s ability to evade host immune responses as well as their ability to induce immunopathology in specific tissues, including bone and the central nervous system (Coria et al., 2016; Velásquez et al., 2017) ( Figures 3 , 4 ). Furthermore, phenotypic analysis of an omp19 mutant B. abortus reveals that Omp19 shields the parental strain against lysosomal proteases encountered during intracellular residence in host macrophages and those encountered in the intestinal tract following oral infection (Pasquevich et al., 2019). Omp19 also shares significant amino acid homology with bacterial protease inhibitors (Ibañez et al., 2015), and it prevents proteases from degrading Omp25, another immunomodulatory protein. Omp10 homologs are only found in Brucella and a small number of other alphaproteobacteria (Cloeckaert et al., 1999), in contrast to Omp16 and Omp19, which exhibit homology to proteins from other bacteria. The biological role of this Omp is uncertain. Interestingly, the equivalent B. ovis mutants do not exhibit significant attenuation in mice (De Souza Filho et al., 2015), although B. abortus omp19 and omp10 mutants do (Sidhu-Muñoz et al., 2016). However, the inability to create double mutants of B. ovis omp10 omp19 raises the possibility that these proteins share a physiological role (Sidhu-Muñoz et al., 2016).

In addition to the previously mentioned proteins, Brucella species contains more proteins including Omp2a/2b, and BP26. The omp2a and omp2b gene products are homologous outer membrane proteins that function as porins, meaning they form channels in the outer membrane that allow small molecules to pass through. These proteins are crucial for the bacterial outer membrane, playing a role in nutrient and toxin transport. Variation in the omp2 locus, particularly in the omp2b gene, is linked to Brucella species identification and evolutionary adaptations (Paquet et al., 2001). Brucella BP26, also known as Omp28, is a 26 kDa periplasmic protein of Brucella species, a major immunodominant antigen that is widely used in diagnostic and vaccination efforts against brucellosis. BP26 is a target for antibodies in infected animals and humans, and its presence is a key indicator in the diagnosis of the disease (Kim D. et al., 2013).

Autotransporter (AT) adhesins are of significant importance in facilitating the attachment of numerous bacterial pathogens to mammalian cells (Henderson et al., 2004). Brucella is known to possess five distinct AT adhesins. Among these, OmaA and BmaC are categorized as type I monomeric ATs (Posadas et al., 2012), whereas BtaE and BtaF fall under type II trimeric ATs (Muñoz González et al., 2019). Furthermore, BigA (264) showcases shared structural domains with the Escherichia coli adhesin intimin, representing an inverse AT adhesin (265). BmaC specifically binds to fibronectin present on the surface of host cells (260), while BtaE and BtaF have an affinity for hyaluronic acid (Ruiz-Ranwez et al., 2013). However, the specific receptors for OmaA and BigA remain unidentified. Interestingly, Brucella mutants lacking these AT adhesins demonstrate decreased attachment to epithelial cells, while still displaying wild-type intracellular replication in cultured macrophages. Moreover, these mutants exhibit attenuated virulence in mice when administered via intragastric or nasal routes, as opposed to intraperitoneal delivery. These experimental findings suggest that the primary role of AT adhesins is to enhance the attachment of Brucella to the host at mucosal surfaces during the initial phases of infection. Notably, BigA exhibits a preference for eukaryotic cell junctions (Leo et al., 2015; Czibener et al., 2016), a mechanism also employed by various bacterial pathogens to traverse mucosal barriers within the host (Ruch and Engel, 2017). Additionally, a double mutant of B. suis lacking btaE and btaF displays significantly higher attenuation in mice compared to single mutants of btaE or btaF, highlighting the complementary roles these adhesins play in virulence (Ruiz-Ranwez et al., 2013). Besides their attachment function, there is evidence suggesting that BtaF aids in protecting B. suis from the bactericidal effects of serum (Ruiz-Ranwez et al., 2013).

BmaC, BtaE, and BtaF are predominantly localized at a specific pole of the bacterial cell (Posadas et al., 2012). The concentration of these AT adhesins at the new pole, in conjunction with the observation that Brucella cells in the G1 phase of the cell cycle are the predominant infectious form (Deghelt et al., 2014), has led researchers to propose that these adhesins collectively form an adhesive pole on the Brucella cell (Van Der Henst et al., 2013; De Bolle et al., 2015). Only a fraction of Brucella cells in planktonic cultures express these adhesins, indicating that the corresponding genes may only be optimally expressed upon exposure to a host-specific stimulus, such as interaction with mammalian cells. This observation aligns with the regulatory control of several AT-encoding genes by the quorum-sensing regulator VjbR (Uzureau et al., 2010) and the global regulator MucR (Pirone et al., 2018), as well as the intricate regulatory network known to govern btaE expression in B. abortus (Kleinman et al., 2017). The variability in the genes encoding AT-type adhesins within Brucella (Sieira et al., 2017) suggests functional overlap among these adhesins, as previously reported (Ruiz-Ranwez et al., 2013). Therefore, a comprehensive exploration of the roles of Brucella AT adhesins across different species and strains, utilizing mutants with multiple disrupted genes, is crucial for a precise evaluation of their contributions to virulence.

Numerous Gram-negative bacteria synthesize polysaccharide polymers and release them into their periplasmic space, where they execute various physiological functions (Bontemps-Gallo and Lacroix, 2015). Brucella spp. and other alphaproteobacteria discharge a cyclic polymer comprised of 17 to 20 glucose units, referred to as cyclic β-1,2-glucan (CβG), into their periplasmic compartment (Iñón De Iannino et al., 1998). Within Sinorhizobium and Agrobacterium, the synthesis of CβG is controlled by osmotic conditions, indicating a potential function of this compound in osmotic protection (Breedveld and Miller, 1994). However, in Brucella, the production of CβG is not influenced by osmotic factors (De Iannino et al., 2000), and experimental data indicate that this polysaccharide has a minor impact as an osmoprotectant in these bacteria (Roset et al., 2014). Nonetheless, CβG plays a crucial function in the virulence of Brucella (Briones et al., 2001). Research utilizing B. abortus CβG synthase mutants and purified CβG has shown that this compound disrupts lipid rafts on the vacuoles containing Brucella, thereby impeding their interactions with lysosomes (Arellano-Reynoso et al., 2005) ( Figure 5 ). Furthermore, CβG has been demonstrated to significantly affect the ability of macrophages and dendritic cells to generate both proinflammatory and anti-inflammatory cytokines (Martirosyan et al., 2012; Roset et al., 2014; Degos et al., 2015; Guidolin et al., 2018) ( Figures 3 , 4 ). The mechanism by which CβG is released from its periplasmic site in Brucella cells for these biological functions in vivo remains unclear. Nevertheless, CβG appears to possess dual roles in virulence. It is pivotal in the intracellular transportation of Brucella to their replicative environment within host macrophages, and it modulates the host immune response to facilitate their prolonged intracellular survival. Considering the suggested polar nature of the interaction of Brucella cells with their mammalian hosts (Van Der Henst et al., 2013; De Bolle et al., 2015), it is noteworthy that the CβG synthase (Cgs) and transporter (Cgt) exhibit polar distribution on the bacterial cell (Guidolin et al., 2015). The potential contribution of this polar distribution of the CβG biosynthesis and transport apparatus to virulence remains to be elucidated.

In 1998, Halling reported the identification of flagellar biosynthetic genes in B. abortus, even though the majority of Brucella strains are nonmotile (Halling, 1998). Subsequent research revealed that the majority of Brucella strains, if not all, have the genetic potential to create flagella (Fretin et al., 2005). However, only a small percentage of strains in the BO2 clade appear to be able to employ the flagella for motility, and they lack chemotaxis genes. Nevertheless, the discovery of B. melitensisfliF and flgF mutants in signature-tagged mutagenesis screens for attenuation in pregnant goats (Zygmunt et al., 2006) and mice (Lestrate et al., 2003) raised the possibility that these genes are involved in virulence. Studies have confirmed that flagellar biosynthesis genes are necessary for the wild-type virulence of B. melitensis and B. abortus strains in the mouse model. The Letesson group demonstrated that B. melitensis16M does, in fact, produce a single sheathed polar flagellum that is covered by an extension of the outer membrane (Al Dahouk et al., 2017). It is interesting to note that B. ovis mutants devoid of genes involved in flagellar biosynthesis exhibit full virulence in mice, indicating that the role played by flagella in pathogenesis may vary depending on the strain and, perhaps, the host (Sidhu-Muñoz et al., 2020).

Modulating the host immune response is one way that the flagella seem to contribute to pathogenicity (Terwagne et al., 2013). The Brucella flagellin is not detected by TLR5, in contrast to flagella from many other Gram-negative pathogens. This adds to the alleged stealthiness of infections caused by Brucella. However, data from experiments suggest that Brucella flagellin enters the cytoplasm of infected host cells and triggers an inflammatory response mediated by an inflammasome, which is crucial for “limiting” the extent of Brucella reproduction. Therefore, it has been suggested that the flagellum is an additional virulence factor that enables Brucella to modify the host immune response in a manner that facilitates the formation and persistence of chronic infections. It is also possible that these appendages, like those of other closely related alphaproteobacteria, have unidentified functions in pathogenesis, such as adhesins or surface attachment sensors (Hug et al., 2017). Furthermore, studies have revealed that sheathed flagella are involved in the release of outer membrane vesicles (OMVs) (Aschtgen et al., 2016), which is an intriguing relationship given the roles that OMVs have been suggested to play in host-pathogen interactions during Brucella infections (Pollak et al., 2012). Furthermore, sheathed flagella are relatively uncommon in bacteria.

While production of the polar flagellum in B. melitensis16M has only been observed in vitro, in bacterial cells grown to an early exponential phase in a rich medium, the genes involved in flagellar biosynthesis are tightly regulated in Brucella. These genes are readily expressed when this strain is replicating in mammalian cells (Fretin et al., 2005). A portion of the genes involved in flagellar biosynthesis in Brucella are expressed in phases; however, the regulatory networks governing the systematic temporal expression of these genes differ from those in other bacteria (Ferooz et al., 2011). Brucella flagellar gene expression is additionally regulated by the quorum-sensing regulators VjbR and BabR (also referred to as BlxR), the light-sensing regulator LovhK, the general stress response regulator RpoE1, and cyclic di-GMP-mediated signaling (Delrue et al., 2005; Kim HS. et al., 2013). Nearly all bacteria that use flagella for movement have chemotaxis genes, which enable them to regulate the direction of their flagellar rotation in response to environmental stimuli that vary in intensity (Wuichet and Zhulin, 2010). This enables them to swim away from harmful substances and toward nutrients. Given that the majority of Brucella strains do not appear to utilize their flagella for movement, the absence of chemotaxis genes in these bacteria is not particularly surprising. However, it does bring up some intriguing issues regarding how the motile BO2 strains navigate their native habitats.

Prokaryotes typically lack the phospholipid phosphatidylcholine (PC), despite being a significant component of eukaryotic cell membranes (Wuichet and Zhulin, 2010). The discovery that some Brucella strains had PC in their cell envelopes occurred about 50 years ago (Thiele and Schwinn, 1973), which raised the possibility that the virulence of the outer membrane (OM) could be influenced by the presence of this “eukaryotic” phospholipid. Brucella strains use two distinct metabolic processes to produce PC: the Pcs system, which converts choline directly into PC, and the Pmt pathway, which methylates the phospholipid phosphatidylethanolamine to form PC (Herrmann et al., 2013). Independent research conducted in two distinct labs has verified that PC is crucial to Brucella pathogenicity. To obtain a comprehensive picture of how PC contributes to virulence, more research will be required as these investigations yielded inconsistent data about the relative contributions of the Pcs and Pmt pathways to virulence. However, the available experimental data generally indicates that the PC/PE ratio in Brucella strains’ outer membrane affects the bacteria’s resistance to complement and antimicrobial peptide killing, and that PC may also be involved in regulating the host’s response to infection (Bukata et al., 2008). It is also noteworthy that some other members of this group, like Agrobacterium and Rhizobia, interact with their respective plant hosts in a significant way due to PC’s incorporation into the outer membrane, which is a characteristic of alphaproteobacteria in general (Aktas et al., 2010).

Exopolysaccharides (EPSs) are polysaccharide polymers released by bacteria that are loosely linked with bacterial cells, generating an amorphous “slime layer” (Cuthbertson et al., 2009), or securely bound to the cell surface to form a capsule. EPSs are involved in several aspects of bacterial pathogenesis. They can act as adhesins and promote bacterial adhesion to eukaryotic cells (Tomlinson and Fuqua, 2009). Additionally, they can help bacteria avoid being recognized by the host’s innate and acquired immune responses (Muñoz et al., 2018), as well as shield them from the bactericidal effects of complement, neutrophils, and macrophages (Mishra et al., 2012). Furthermore, these polymers enable the formation of biofilms by bacteria, which adds to their ability to persist in the environment and mammalian hosts (Jones and Wozniak, 2017).

Genetically, Brucella strains are capable of producing extracellular polymeric substances (EPS) (Caswell et al., 2013), although experimental data indicate that the associated genes are highly regulated. For example, B. melitensis16M does not readily produce an EPS during routine in vitro cultivation; however, this strain produces an apparent EPS detected by calcofluor staining and forms “biofilm-like” bacterial cell aggregates in liquid culture upon disruption of a putative quorum-sensing pathway (Uzureau et al., 2007). Furthermore, a B. melitensisvirB mutant (Wang et al., 2010) and a B. abortus strain that overexpresses the glycosyltransferase WbkA (Dabral et al., 2015) have been reported to produce EPS and exhibit cellular aggregation. EPS production is also supported by reports of Brucella strains (Tang et al., 2019) forming “biofilms” and a B. melitensismucR mutant (Mirabella et al., 2013) exhibiting improved Congo red staining.

It is currently uncertain whether EPS is crucial for Brucella pathogenicity as the precise genes needed for EPS synthesis are still unclear. However, EPS synthesis is necessary for both the symbiotic and pathogenic relationships between rhizobia and agrobacteria and their respective plant hosts (Marczak et al., 2017; Thompson et al., 2018). The conservation of techniques used by alphaproteobacteria to maintain successful relationships with their eukaryotic hosts (Batut et al., 2004) raises the question of whether EPS synthesis contributes significantly to Brucella pathogenicity.

The incidence rate of brucellosis in developing Asian and African nations indicates that the disease is still a significant threat to both animal and human health in these areas. The prevalence of brucellosis in both Asia and African countries is 8% while it is 12% in the Indian livestock population (Suresh et al., 2022). Between 2010 and 2019, the prevalence of brucellosis in livestock ranged from 0.2% to 43.8% in cattle, 0.0% to 20.0% in goats, and 0.0% to 13.8% in sheep in many regions of the world, including sub-Saharan Africa (Djangwani et al., 2021). In Latin America, the seroprevalence of Brucella in bovine was 4%, with Venezuela having the highest prevalence (16%). Among regions, the highest seroprevalence is in Central America and the Caribbean islands (8% and 3%–15%, respectively) (Bonilla-Aldana et al., 2023).

B. canis primarily infects dogs and wild canids, but humans can also become infected. Globally, dog seroprevalence rates range from less than 1% to 15% or higher; greater rates are typically associated with stray dogs and poorer areas, most likely as a result of these communities’ higher numbers of intact canines and uncontrolled mating. In the US, B. suis biovars 1, 3, and 4 are detected. Biovars 1 through 3 have swine as their reservoir host; however, infections can also affect humans, dogs, cattle, and occasionally other species. Caribou and reindeer that inhabit Arctic locations, including Alaska, are infected by B. suis biovar 4. The eradication of B. suis from US commercial swine was achieved in 2011; however, the bacteria still exist in feral swine, especially in the Southern and Western Parts of the country, and it continues to pose a threat to commercial swine operations (Sandfoss et al., 2012).

Although endemic throughout Asia, the Middle East, South America, and Africa, B. melitensisis is not found in the United States. Three biovars and a smooth colony phenotype characterize B. melitensis. The reservoir hosts are sheep and goats, but it has also been reported in a wide range of other species. Similar to Brucella species in other host species, B. melitensis in small ruminants has similar clinical symptoms and modes of transmission (Garin-Bastuji et al., 2014).

Although B. abortus primarily affects cattle and bison, it has also been documented to affect yaks, as well as animals such as antelope, elk, sika deer, African buffalo, horses, camels, and South American camelids that encounter infected ruminants. B abortus biovar one has been eliminated from US cattle and domestic bison, but it was formerly thought to be the most serious livestock disease in the US, resulting in high rates of human infection through contact with sick animals or consumption of unpasteurized dairy products.

In parts of Europe, Africa, Asia, Central and South America, and Asia, B abortus is still enzootic. These locations also have high populations of cows, fewer economic resources, and lower implementation of control and surveillance measures (Pinn-Woodcock et al., 2023).

According to recent studies, there are 1.6–2.1 million new human cases worldwide each year, which is more significant than previously thought (Laine et al., 2023). High incidence rates are reported in areas with limited resources, including the Mediterranean, Middle East, Central Asia, and some parts of Africa. Among the nations with the highest documented rates of brucellosis are Iran, Kyrgyzstan, Tajikistan, Kazakhstan, Azerbaijan, Turkmenistan, Armenia, and Uzbekistan (Pal et al., 2017; Khurana et al., 2021).

Mexico and Peru have reported numerous occurrences in Latin America (Bano and Ahmad Lone, 2015). A study on the epidemiology of brucellosis in California by Fritz et al. found that the disease is most common among older Latino men and is significantly associated with the consumption of unpasteurized Mexican-style soft cheese. The most common species found in cases was B. melitensis. The 492 documented instances in California between 1993 and 2017 highlight the risks posed by brucellosis to human health (Fritz et al., 2021).

The yearly incidence rate for the 28 EU countries from 2017 to 2018 was 0.09 per 100,000 population (Uzunović et al., 2020). Successful intervention efforts were highlighted by the European Food Safety Authority (EFSA), which reported a decrease in brucellosis cases from 735 in 2008 to 352 in 2011 (Bano and Ahmad Lone, 2015).

In Bosnia and Herzegovina, 263 instances were examined between 2008 and 2018, down from 102 in 2008 to only three in 2018. The evidence from other international research is consistent with the epidemiological characteristics found in this investigation. In particular, there was a noticeable male preponderance; patients were mostly from rural areas or had prior animal contact; the most affected age group was 25–49 years old (Bano and Ahmad Lone, 2015; Ali et al., 2018; Uzunović et al., 2020).

Since 1989, 80,000 instances of brucellosis have been documented annually, and the disease is found across much of Iran. Healthcare personnel in Iran have reportedly come into unintentional contact with Brucella strains while administering standard animal vaccinations (Alavi and Motlagh, 2012).

According to a study by Holt et al. (2021), brucellosis, a zoonotic illness caused by the Brucella species, is prevalent in rural India, with a seroprevalence rate of 15.1%. Due to close human-animal contact, this finding highlights the disease’s prevalence in areas with a high concentration of agriculture and livestock production, which facilitate disease transmission.

According to a study by Nawaz et al. on the epidemiology of brucellosis in Punjab, Pakistan, the seroprevalence was 13.13%, with higher rates in males aged 25 to 40 years (Sero-epidemiology, 2021).

With a national average annual incidence of 3.0 per 100,000, a four-year study found that the incidence of brucellosis varied throughout China. The rate more than doubled in Inner Mongolia, leading to a greater incidence rate in Northern China, while it significantly dropped in Xinjiang. Interestingly, men in this region aged between 45 and 64 are more than twice as likely to be impacted by women (Tao et al., 2021).

In many parts of the world, particularly sub-Saharan Africa, brucellosis is endemic. The prevalence of brucellosis in livestock varied from 0.2% to 43.8% in cattle, 0.0% to 20.0% in goats, and 0.0% to 13.8% in sheep, according to studies published between 2010 and 2019. Prevalence of human brucellosis in sub-Saharan Africa varies from 0% to 55.8%, indicating the substantial frequency of infection in this region (Djangwani et al., 2021).

A study performed in Algeria revealed that 15% of the veterinarians interviewed had contracted brucellosis during their professional practice. Direct, unprotected exposure to infected animals and/or their products, mainly during intervention for placental retention, recurrent encounters with brucellosis-infected farms, and unprotected handling of anti-Brucella vaccine appear to be the most common modes of contamination. The lack of protective equipment worn by veterinarians in their daily practice could be an important risk factor for brucellosis in these professionals. The lack of training in the handling of the antibrucellosis vaccine has made it a potential factor for brucellosis contamination, resulting in several cases of professional contamination (Lounes et al., 2022).

Brucella species can be diagnosed using a variety of techniques, although the most reliable ones are isolation and culture of the organism (Al Dahouk et al., 2003). The use of a selective medium, such as Farrell’s medium, is recommended because all Brucella strains develop somewhat slowly, and the specimens from which isolations are typically conducted or attempted are frequently highly contaminated (Pappas et al., 2006). A negative diagnosis may not be made until after a week of incubation, which typically lasts 72 hours. Fetal stomach fluid, spleen, liver, placenta, lochia, milk (particularly colostrum or milk within a week of calving), semen, and lymph nodes (supramammary for chronic and latent infections, and retropharyngeal for early infections) are among the specimens that can be used for Brucella isolation. Brucella colonies are high, translucent, convex, with complete boundaries, smooth, and radiant surfaces (Bedore and Mustefa, 2019). Under transmitted light, the colonies seem honey-colored. The ideal pH range for culture is between 6.6 and 7.4, while the ideal temperature range is between 20°C and 40°C. CO₂ is necessary for the growth of certain Brucella species. A culture can only be deemed negative if no colonies form after two to three weeks of incubation, while typical colonies emerge after two to thirty days (Ashenafi et al., 2007).

Since the majority of brucellosis control and eradication programs rely on serological testing, these tests are essential for laboratory diagnosis. They are classified into two main categories: screening tests and confirmatory tests. Although a number of serological tests have been utilized for laboratory testing of brucellosis, no single test is practical for all epidemiological studies because of issues with sensitivity and/or specificity (Mert et al., 2003). The Rose Bengal plate test, indirect enzyme-linked immunosorbent assay, and serum agglutination test are the most often utilized serological assays for brucellosis (Luelseged, 2018). Due to its ease of use and apparent readability, the Rose Bengal plate test (RBPT) is the most popular screening method for brucellosis in both humans and animals. Personal experience, however, may influence how the RBPT results are interpreted (Cho et al., 2010).

RBT’s shortcomings include limited specificity in endemic areas, low sensitivity, especially in chronic patients, and prozones that cause strongly positive sera to look negative (Díaz et al., 2011). Another effective screening test for dairy cattle is the Milk RingTest (MRT) (Mohamand et al., 2014). Although MRT is a straightforward and efficient serological technique, it is limited to usage with cow’s milk (Mohamand et al., 2014). In a glass or plastic tube, a drop of hematoxylin-stained antigen is combined with a tiny amount of milk. MRT is highly ambiguous at the individual animal level, but it is applicable to the entire herd and provides a basic picture of the infection status. Its shortcomings include its incapacity to male animals and its decreased dependability in large herds (Luelseged, 2018).

One popular confirmatory test for brucellosis is the complement fixation test (CFT). The Organization for Animal Health (WOAH) recommends CFT as the reference test for international animal transit due to its high accuracy. It is used as a confirmatory test for B. abortus, B. melitensis, and B. ovis infections (Sam et al., 2012). CFT is typically utilized on sera that test positive for RBPT, but like RBPT, it is also heavily impacted by the overuse of the strain 19 vaccine, especially when sexually mature cows and heifers have received recent or repeated immunizations. Strict cutoff readings that indicate infection are nearly impossible to prescribe, especially when S19 vaccine reactions are involved due to their potential for abuse. Occasionally negative results in the early stages of infections, high cost and technical complexity are some of CFT’s drawbacks. Additional limitations include the test’s incapacity to be used with hemolyzed serum samples and the subjectivity of interpreting the sporadic direct activation of complement by serum (anti-complementary activity). Animals infected with species antigenically similar to Brucella can also provide false-positive results. As a common test for brucellosis diagnosis, the enzyme-linked immunosorbent assay (ELISA) has gained popularity (Al Dahouk and Nöckler, 2011). It is a valuable method for detecting Brucella antibodies in large populations and distinguishing between the acute and chronic stages of the illness. All four antibody classes (IgM, IgG1, IgG2, and IgA) can be identified with the ELISA method (Franco et al., 2007). The ELISA is an excellent control test in regions free of brucellosis and for survey testing in areas where vaccination has not been administered. However, it is difficult to use and cannot be used everywhere, particularly in areas where vaccination has been administered and is still not widely standardized (Al Dahouk and Nöckler, 2011).

One of the common serological assays for brucellosis diagnosis is the serum agglutination test (SAT) (Memish and Balkhy, 2004). It is straightforward to execute and does not require costly tools or extensive training. The total amount of IgM and IgG agglutinating antibodies is measured by SAT. The foundation of this test is the responsiveness of antibodies to Brucella’s smooth lipopolysaccharide. Applying a serial dilution of 1:2 through 1:64 to the blood samples can increase test specificity by removing excess antibodies that cause false-negative results due to the prozone effect (Al Dahouk and Nöckler, 2011). The incapacity to identify B. canis infections and the emergence of IgM immunoglobulin cross-reactions with Salmonella urbana, Escherichia coli O116 and O157, Francisella tularensis, and Yersinia enterocolitica O:9 are disadvantages of the serum agglutination test. By adding EDTA, 2-mercaptoethanol, or antihuman globulin, for example, some of these drawbacks can be addressed (Shemesh and Yagupsky, 2011).

An in vitro method for amplifying nucleic acids, the polymerase chain reaction (PCR) is frequently employed in the diagnosis of infectious disorders. PCR is one of the most widely used tests for brucellosis diagnosis in humans nowadays (Queipo-Ortuño et al., 2008). The most widely used molecular methods for diagnosing brucellosis are PCR and/or its variations, which are based on the amplification of particular genomic sequences of the genus, species, or even biotypes of Brucella species (Dauphin et al., 2009). Compared with traditional PCR, real-time PCR is faster and more sensitive. Because PCR products do not require post-amplification handling, there is a reduced risk of contamination in the lab and false-positive results. Brucella cells can now be tested using real-time PCR techniques, according to recent studies (Al-Nakkas et al., 2005).

Flower-like gold nanoparticles labeled and silver deposition rapid vertical flow technology for highly sensitive detection of Brucella antibodies. The rapid vertical flow technique (RVFT) was discovered to identify brucellosis antibodies. It can successfully avoid the false negative problem in lateral flow assay. It is easy to use, with a quick time of 2–3 minutes visible to the naked eye and no special equipment. LPS were utilized to detect brucellosis antibodies to improve the procedure’s sensitivity. The advantages of the lateral flow immunoassay were kept while a single multipurpose buffer was developed in whole blood and other biological samples to enhance the sensitivity of serum antibody detection (Elrashedy et al., 2022).

Iron is a co-factor for many different proteins because of its chemical flexibility. Iron is presumably necessary for the activity of a wide variety of Brucella proteins. Catalase, aldolase (Roop, 2012), and CobG, an enzyme involved in the production of cobalamin (vitamin B12), are a few examples of which this requirement has been experimentally confirmed (Schroeder et al., 2009).

One high-affinity Mn transporter, MntH, is produced by Brucella strains, and the Mn-responsive transcriptional regulator Mur controls the expression of the related gene (Anderson et al., 2009). Mn-dependent enzymes are essential to Brucella strains’ fundamental physiology and pathogenicity, according to phenotypic examination of a range of mutations. These bacteria may also modify their cellular Mn level as a defense against oxidative stress (Roop Ii et al., 2017c). A B. abortus mntH mutant is significantly attenuated in the mouse model of chronic infection. It is yet unknown what causes this attenuation. The B. abortus mntH mutant has lower Mn superoxide dismutase activity than the parental strain, but an isogenic sodA mutant only shows mild attenuation in mice (Martin et al., 2012), suggesting that the mntH mutant’s severe attenuation is not caused by lower SodA activity. Although the relationship between Mn²⁺ transport and virB expression has not been studied, the B. abortus mntH mutant also shows aberrant expression of the genes encoding the Type IV secretion machinery (Anderson et al., 2009). One possible explanation for this relationship is that orthologs of the (p)ppGpp synthetase/hydrolase known as Rsh (Dozot et al., 2006), Manganese-dependent enzymes are necessary for both the induction of the stringent response in Brucella and the production of VirB (Pappas et al., 2006).

According to recent genetic and biochemical studies, E. coli can replace Fe²⁺ in important metabolic enzymes like ribulose-5-phosphate epimerase (Rpe), a key enzyme in the pentose-phosphate pathway, by increasing the intracellular ratio of Mn²⁺:Fe²⁺. Escherichia coli also shows increased mntH expression in response to exposure to H₂O₂ (Anjem et al., 2009). Since Mn²⁺ does not take part in Fenton reaction like Fe²⁺ does, this substitution shields Rpe from damage caused by H₂O₂. In vitro exposure of B. abortus 2308 to H₂O₂ also increases the expression of mntH, and in vitro experiments show that an isogenic mntH mutant is more sensitive to exposure to H₂O₂ than the original 2308 strain (Anderson et al., 2009). In order to protect these proteins from H₂O₂-mediated damage, it will be crucial to ascertain whether Brucella strains possess the same ability as E. coli to replace Fe²⁺ in metabolic enzymes with Mn²⁺.

Many bacteria, including Brucella species, depend on zinc as a micronutrient. In Brucella strains, zinc is necessary for the correct operation of multiple critical enzymes involved in the production of amino acids and oxidative stress tolerance (Andreini et al., 2006). Zinc is also a crucial cofactor for the type IV secretion system effector protein RicA (Rab2 interacting conserved protein A) and the virulence-associated transcriptional regulatory protein MucR. Brucella strains have a high affinity zinc uptake mechanism termed ZnuABC that preferentially imports zinc because of its significance in their biology. On the other hand, the Brucella also encode a zinc export mechanism, ZntA, which aids in the intracellular detoxification of excess zinc due to the hazardous potential of free zinc cations (Sheehan et al., 2015). Zinc uptake and export systems are tightly regulated by two zinc-responsive regulatory proteins, Zur and ZntR, which regulate transcription of the znuABC and zntA genes, respectively (Clapp et al., 2011). Crucially, effective pathogenesis in animal models of Brucella infection depends on the appropriate homeostasis of zinc levels in the virus. The systems that control zinc uptake and export in Brucella strains will be described in this chapter, along with their function in virulence, the genetic control of zinc homeostasis, and zinc’s involvement as a cofactor for key enzymes in Brucella strains (Caswell, 2017). SodC is necessary for the Brucella’s complete pathogenicity and the bacteria’s high resistance to exogenous O₂. In the periplasmic region of many Gram-negative bacteria, including several dangerous organisms, SodC, a periplasmic superoxide dismutase (SOD), is in charge of detoxifying exogenously produced superoxide (i.e., O₂ ⁻). The SodC protein is frequently referred to as Cu-Zn SOD because bacterial SodC proteins require copper and zinc cofactors for proper enzymatic function, while the zinc cation appears to play a crucial structural role (Caswell, 2017).

It has been demonstrated that urease is one of the few bacterial enzymes that needs nickel as a cofactor (Li and Zamble, 2009). According to Bandara et al. (2007) and Sangari et al. (2007), this enzyme is necessary for the pathogenicity of B. abortus 2308 and B. suis 1330 in mice when these strains are introduced orally, but not when they are given peritoneally. In mammalian cell cultures, B. suis and B. abortus urease mutants display wild-type pathogenicity. Urease is not necessary for intracellular survival in eukaryotic cells, but it helps the Brucella withstand the extremely low pH they experience during passage through the stomach and gastrointestinal tract following ingestion, according to the theory put up to explain these observations. Although NikABCDE and NikKMLQO, two nickel transporters, have been found in Brucella (Jubier-Maurin et al., 2001b; Sangari et al., 2010), how these transporters contribute to virulence is unclear. An isogenic nikA mutant generated from B. suis 1330 exhibits wild-type pathogenicity in the human monocytic cell line THP-1, even though nikA expression is increased in this strain during intracellular replication in J774 cells (Jubier-Maurin et al., 2001a). It will be crucial to evaluate the virulence characteristics of Brucella strains deficient in either the NikABCDE or NikKMLQO transporter, or both, in cultured macrophages and in mice infected by both the intraperitoneal and oral routes to better understand the necessity of nickel transport by Brucella strains in the host.

Bacterial cells contain large quantities of magnesium (mM). It is a structural and enzymatic co-factor for many cellular proteins and is crucial for preserving the structural integrity of ribosomes and cell membranes (Moomaw and Maguire, 2008). For example, Mg²⁺ is necessary for the activity of erythritol kinase, the enzyme that catalyzes the initial stage of erythritol catabolism in Brucella strains.

Virulence in Brucella strains has been genetically related to homologs of two genes involved in magnesium transport in other bacteria. Salmonella is the greatest example of the activity of the bacterial P-type ATPase MgtB as a magnesium transporter (Smith et al., 1993). During a screening of signature-tagged transposon mutants obtained from B. melitensis16M for attenuation in experimentally infected mice, a B. melitensis mgtB mutant was discovered (Lestrate et al., 2000). Remarkably, when cultivated in magnesium-limited media, this mutant showed no signs of growth defects. This implies that the Brucella have several magnesium transport mechanisms, just like other bacteria and as shown in Figure 10 . A B. suis mgtC mutant does not grow well in a magnesium-restricted medium and exhibits significant attenuation in the murine macrophage-like J774 cell line (Lavigne et al., 2005), although the exact function of MgtC in magnesium transport has not been determined (Günzel et al., 2006; Alix and Blanc-Potard, 2007). Adding MgCl2 to the cell growth media can help mitigate this attenuation to some extent.

Many bacteria, including Brucella, require cobalt as a micronutrient because it is a necessary component of cobalamin (vitamin B12) (Warren et al., 2002). Additionally, cobalamin is necessary for the activity of several key bacterial enzymes, including the ribonucleotide reductase NrdJ (Taga and Walker, 2010) and the methionine synthase MetH (Roop Ii et al., 2017a). In mice and cultured mammalian cells, B. suis and B. melitensis mutants with defects in the cobalamin biosynthesis pathway are attenuated (Delrue et al., 2004). Brucella strains have an intact cobalamin biosynthesis pathway (Lundqvist et al., 2009). These infection models also attenuate a B. melitensis metH mutant (Lestrate et al., 2000). Between the cobalamin biosynthesis genes cobQ and cobU in Brucella are two genes that are anticipated to encode a CbtAB-type Cotransporter. The idea that the Brucella cbtA and B genes express a high-affinity Co transporter is supported by their location and the fact that they are located downstream of a cobalamin-responsive riboswitch (Rodionov et al., 2003), but this function has not been experimentally verified (Roop Ii et al., 2017a).

The prevention of toxicity resulting from the excessive buildup of these crucial micronutrients depends on proteins that directly contribute to metal homeostasis. Three transcriptional regulators—Irr (Martínez et al., 2005; Martínez et al., 2006), DhbR (Anderson et al., 2008), and Mur (Menscher et al., 2012)—have been identified as controlling the expression of Brucella metal acquisition genes. DhbR is an AraC-type transcriptional regulator that activates the transcription of the siderophore biosynthesis genes in B. abortus 2308 in response to Fe³⁺-siderophore levels in the external environment; Mur controls the expression of the gene encoding the Mn²⁺ transporter MntH in response to cellular Mn²⁺ levels; and Irr is an iron-responsive transcriptional regulator that governs the genes involved in iron acquisition and iron metabolism. Bacterioferritin (Bfr), a protein that accumulates and detoxifies intracellular iron, is also produced by certain strains of Brucella (Denoel et al., 1995; Almirón and Ugalde, 2010). Only Irr and Bfr have had their involvement in virulence investigated thus far. Though neither B. abortus nor B. melitensis-bfr mutants show attenuation in cultured human primary explant macrophages (Denoel et al., 1997), J774 or HeLa cells (Almirón and Ugalde, 2010), or experimentally infected mice (Denoel et al., 1997), a B. abortus irr mutant is attenuated in the mouse model (Anderson et al., 2011).

In order to improve safety and immune responses, recent developments have concentrated on modified live attenuated vaccines with removed virulence genes. For instance, a mutant of the B. melitensis TcfSR promoter (Li et al., 2015) and the B. melitensis 16M hfq mutant strain (Zhang et al., 2013) showed considerable protection and minimal interference with serodiagnostic assays. Although they are sensitive to polymyxin B (Wang et al., 2014), other possible vaccines, including the M5-90ΔwboA mutant and 6MΔwzt, have shown decreased pathogenicity and enhanced defense mechanisms. Similar to the B. ovisabcBA (BoabcBA) vaccine (Costa et al., 2020), the 2308DNodVDNodW rough vaccine from the virulent B. abortus 2308 strain provides a strong immune response against the B. melitensis strain 16M. Brucella double gene knock-out vaccine strain MB6 Δbp26ΔwboA (RM6) was constructed and evaluated by Shi et al. the researchers have found that the RM6 strain had good proliferative ability and stable biological characteristics in vivo and in vitro. Moreover, it had a favorable safety profile and elicited specific immune responses in mice and sheep (Shi et al., 2022).

Subunit vaccines exhibit promise in terms of non-infectious, non-viable, and safety. Nevertheless, they are not very good at simulating the spread of genuine illnesses (Ficht et al., 2009). Subunit vaccines have the advantage of being safe, but in order to produce strong immunity and protect cattle from brucellosis, they need to be administered in numerous booster doses and utilizing a variety of antigens, adjuvants, and delivery systems. However, due to associated expenditures, this strategy could not be commercially viable (Perkins et al., 2010). Regretfully, no viable subunit vaccination against brucellosis has been created in spite of countless attempts (Zimmermann and Curtis, 2019).

Oral vaccines containing nanoparticles and the Brucella vaccine produced antibody responses, including IgM, mucosal IgA, and IgG, in animal model studies. In animal investigations, these vaccinations have shown significant benefits, including a stronger Th1–Th17 immune response (Abkar et al., 2017). However, human brucellosis cannot be prevented by nanoparticle-based vaccinations because of the possible danger of disease transmission (Maleki et al., 2019). These vaccines’ primary disadvantages include their toxicity, limits in terms of antigen loading and manufacturing, and their less-than-ideal capacity to activate the immune system (Al-Halifa et al., 2019).

In the fight against brucellosis, DNA-based Brucella vaccines have proven to be both safe and effective. These vaccines’ capacity to produce antigens and integrate CpG patterns results in strong cellular immune responses. Simple storage conditions are another benefit of DNA-based vaccinations. They have important gene sequences that are essential to Brucella species’ intracellular survival (Hu et al., 2009). However, as compared to live-attenuated vaccines, DNA-based vaccines do not offer as much protection. Studies show no discernible changes in the expression of IL-4, IL-10, or IFN-γ, suggesting an immunological response to DNA-based vaccinations (Rosinha et al., 2002).

Using Brucella as a delivery vehicle, live vector-based vaccines have become a successful way to deliver a variety of antigens, both homologous and heterologous. By multiplying inside host cells and creating several copies of the Brucella antigen, these genetically engineered vaccines are designed to elicit an antigen-specific T-cell immune response (Al-Mariri et al., 2002).

One promising strategy for preventing and controlling brucellosis is the use of recombinant peptides as vaccinations. Conventional vaccinations, such as the Rev-1 vaccine, have drawbacks, such as the potential to cause abortion in fetuses and disrupt diagnostic procedures. Recombinant peptides, on the other hand, provide safer and more precise substitutes (Cassataro et al., 2005).