Brucellae are Gram-negative cocccobacilli, facultative intracellular bacterial pathogens of both humans and animals. The bacteria penetrate the mucosa of the nasal, oral, or pharyngeal cavities and are phagocytized by host macrophages, where survival and replication occurs. Brucellosis is a zoonotic disease that is difficult to diagnose and treat that causes heavy economic losses and human suffering, characterized by undulant fever that, if untreated, can develop into a chronic infection with symptoms persisting for several months. Chronic infections may result in infection of secondary tissues, including heart and brain. Symptoms may also recur years after the original infection. The pathological manifestations of brucellosis are diverse and include arthritis, endocarditis, and meningitis in humans, while animal brucellosis is characterized by spontaneous abortion 1. Six species are recognized within the genus Brucella: B. abortus, B. melitensis, B. suis, B. ovis, B. canis, and B. neotomae. This classification is mainly based on the difference in pathogenicity and in host preference 2. The main pathogenic species worldwide are B. abortus, responsible for bovine brucellosis; B. melitensis, the main etiologic agent of ovine and caprine brucellosis, a disease that causes abortion in ewes and goats resulting in huge economic losses, particularly in Mediterranean countries and B. suis responsible for swine brucellosis. B. abortus infection is acquired by humans through contact with infected livestock and consumption of unpasteurized dairy products. B. ovis and B. canis are responsible for ram epididymitis and canine brucellosis, respectively 3. For B. neotomae only strains isolated from desert rats have been reported. Brucella strains have also been isolated from a great variety of wildlife species such as bison, elk, feral swine, foxes, hares, African Buffalo, reindeer, and caribou 4. Recently, two new species have been proposed to be added to this genus, Brucella cetaceae and Brucella pinnipediae isolated from marine mammals, cetaceans and pinnipeds, respectively 56. Distinction between species and biovars is currently performed by differential tests based on phenotypic characterization of lipopolysaccharide antigens, phage typing, dye sensitivity, CO₂ requirement, H₂S production, and metabolic properties 7.

Additionally, Brucella can be used as a biological weapon since transmission through a spray is possible, as has been reported with human contamination during abortion of infected animals or bacterial spraying in laboratories 8. The bacteria is highly contagious and it is suggested that 10 to 100 bacteria would be sufficient to produce a contaminating spray for humans. Several countries have been suspected of studying the agent as a biological weapon, but to date, no use of Brucella in a bioterrorist attack has been reported.

In this article, we review the importance of the lipopolysaccharide in Brucella virulence, discussing the LPS chemical composition, the Brucella genome, the genes involved in LPS biosynthesis, and the interaction between LPS and innate immunity.

Several studies on virulence factors were directed at the main components of the outer membrane. The outer membrane contains the lipopolysacharide (LPS) that is the Brucella major virulence factor. Lipopolysaccharide is vital to both the structural and functional integrity of the Gram-negative bacterial outer membrane. Ubiquitously expressed by all Gram-negative bacteria, and containing several well-conserved domains, LPS also serves as one of the primary targets of the innate arm of the mammalian immune system. Brucella possesses a peculiar non-classical LPS as compared with classical LPS from enterobacteria such as Escherichia coli 9. The LPS was identified as a major virulence determinant of Brucella recognized for its role in virulence when naturally occurring isolates lacking LPS showed reduced survival. LPS has three domains: lipid A, the core oligosaccharide, and the O-antigen or O-side chain (Figure 1). The O-polysaccharide of smooth-type Brucella LPS (S-LPS) is an unbranched homopolymer of 1,2-linked4,6-dideoxy-4-formamido-α-D-mannopyranosyl usually with an average chain length of 96 to 100 glycosyl subunits 10. The O-polysaccharide is linked to a core oligosaccharide composed of mannose, glucose, 2-amino-2,6-dideoxy-D-glucose (quinovosamine), 2-amino-2-deoxy-D-glucose (glucosamine), 3-deoxy-D-manno-2-octulosonic acid (KDO) and unidentified sugars. The lipid A, linked to the core oligosaccharide, contains 2,3-diamino-2,3-dideoxy-D-glucose (diaminoglucose) as backbone, amide and ester-linked long chain saturated (C_16:0 to C_18:0) and hydroxylated (3-OH-C_12:0 to 29-OH-C_30:0) fatty acids 11. The hydrophobic lipid A region constitutes mostly the outer coating of the outer membrane and is responsible for many of the endotoxic properties attributed to LPS 12. Thermotropic phase behaviour 13 and immunochemical analysis 14 of B. abortus and B. melitensis lipid A suggest a disaccharide backbone molecule linked in a β1–6 configuration. Ethanolamine, neutral sugars and ester-linked acyl-oxyacyl fatty acids are not found, and phosphate is absent or present in reduced quantities 11. Brucella lipid A contains strongly bound outer membrane protein fragments that are not removed by conventional procedures used to release the lipid-A-associated protein of enterobacterial LPS 1114. The heterogeneity of enterobacterial LPS is well known to be related to the length of its O-polysaccharide and different chemical substitutions in the core oligosaccharide and lipid A 15. In enterobacterial lipid A, the degree of heterogeneity depends upon the different combinations in which amide and ester-linked fatty acids, phosphate, neutral sugars, ethanolamine and different types of backbone amino sugars occur in the molecule 16. In Brucellae lipid A, the degree of heterogeneity may depend mainly on the various fatty acid substitutions. The absence of backbone constituents (other than lipids) and ester-linked acyl-oxyacyl residues in Brucella lipid A might account for the restricted number of variants as compared to enterobacterial lipid A. Determination of the intrinsic heterogeneity in Brucella LPS is important to explain in a more realistic perspective its chemical nature and biological behavior. For practical purposes, this is significant, since LPS is the most relevant antigen during infection and vaccination. In addition, LPS and LPS-related molecules are extensively used in immunological studies and in the diagnosis of brucellosis 17.

Recently, the genome sequences of the B. melitensis, B. suis and B. abortus became available 181920. The genomes of B. suis, B. melitensis, and B. abortus are very similar in sequence, organization, and structure. Few fragments are unique among the genomes 20. Although many aspects of its biology remain to be understood, the sequencing and annotation of its genome paved the way for a highly comprehensive and rapid analysis of its proteome. Comparative genomics provide insights into aspects of Brucella virulence that were only suspected before. The era of post-genomic technology offers new and exciting opportunities to understand the complete biology of different Brucella species. At the proteome level, extensive metabolic differences were found between the B. melitensis reference strain 16 M and its vaccine strain Rev 1. Similarly, laboratory grown B. melitensis can be distinguished from B. abortus by just looking at their proteome 21. Using the complete genome sequence of Brucella melitensis, Dricot et al. 22 generated a database of protein-coding ORFs and constructed an ORFeome library of 3091 Gateway entry clones, each containing a defined ORF. This first version of the Brucella ORF (Version 1.1) provides the coding sequences in a user-friendly format amenable to high-throughput functional genomic and proteomic experiments, as the ORFs are conveniently transferable from the entry clones to various expression vectors by recombinational cloning. The cloning of the Brucella ORFeome v.1.1 should help to provide a better understanding of the molecular mechanisms of virulence, including the identification of bacterial protein-protein interactions, but also interactions between bacterial effectors and their host targets.

The genome of B. melitensis strain 16 M contain 3,294,935 bp distributed over two circular chromosomes of 2,117,144 bp and 1,177,787 bp encoding 3,197 open reading frames (ORFs). The origins of replication of the two chromosomes are similar to those of other proteobacteria. Housekeeping genes, including those involved in DNA replication, transcription, translation, core metabolism, and cell wall biosynthesis, are distributed on both chromosomes 21. Screens for transpositional mutants attenuated in infection models yielded 184 mutants, suggesting that these genes have a function in the infection process 23. The B. suis 1330 genome consists of two circular chromosomes of 2,107,792 bp and 1,207,381 bp 19. A total of 2,185 and 1,203 ORFs were identified on chromosomes I and II, respectively. Comparison of the B. suis genome with that of the B. melitensis genome revealed extensive similarity and gene synteny 21. The majority (>90%) of B. suis and B. melitensis genes share 98–100% identity at the nucleotide level. The more variable genes (<95% identity) consist primarily of hypothetical genes, as well as a UreE urease component, and probable surface-exposed genes such as outer membrane proteins, membrane transporters, a putative invasin, and ShdA-like adhesins. These more variable genes may significantly contribute to the differences in pathogenicity or host preference between these two organisms. The high degree of similarity between the B. suis and B. melitensis genomes at both the gene and nucleotide level is consistent with the proposition that Brucella species should be grouped as biovars of a single species.

The genome of B. abortus biovar 1 (Strain 9-941) have 3.3 Mb and is composed of two circular chromosomes of 2,124,242 (Chr I) and 1,162,780 bp (Chr II) 20. The chromosome sequences of B. abortus 9-941 were assigned the same strand orientation and origin as those of B. suis. The B. abortus genome contains 3,296 ORFs annotated as genes, 2,158 on Chr I and 1,138 an Chr II. This is similar to the annotated ORF counts for B. suis (3,388) and B. melitensis (3,197). The genome of B. abortus shared more fragments with B. suis and B. melitensis than B. suis and B. melitensis did with each other. B. abortus shared more fragments with B. melitensis than B. suis. Two fragments shared by B. suis and B. melitensis were not found in B. abortus. A 2,774-bp fragment encoding a probable surface protein and two partial ORFs with homology to the insertion sequences IS711 and ISBm1 is missing from B. abortus. The second fragment is a 25-kb sequence that may be involved in polysaccharide synthesis and was predicted by Vizcaino et al. 24 to potentially affect phenotypes of brucellae, such as host preference.

The majority of studied genes and their products involved in Brucella LPS biosynthesis are those related to O-chain synthesis (Table 1). The large number of attenuated mutants with a structural defect in their lipopolysaccharide (LPS) confirms the importance of this molecule in Brucella virulence. In spite of the importance of LPS in the Brucella life cycle, very little is known about the metabolic pathways and enzymes required to synthesize it. Certain strains carry mutations in the genes involved in O-chain biosynthesis and attachment and are termed 'rough mutants'(R) strains to differentiate them from the wild-type 'smooth' (S) strains that express O-chain-bearing LPS. Historically, the search for rough mutants was performed in two ways: by a spontaneous phenomenon known as phase variation, in which rough variants appear in culture by a mechanism not well understood 7, or by successive passages in different culture media with searching for spontaneously rough phenotypes. These procedures gave a high number of rough mutants. All of them are avirulent in animal models, but in most cases the parental strain is not available. B. abortus, B. melitensis, and B. suis strains may occur as either smooth or rough, expressing smooth LPS (S-LPS) or rough LPS (R-LPS) as major surface antigen, while B. ovis and B. canis are two naturally R species, expressing R-LPS as major surface antigen. Generally these mutants are less virulent than the wild type, with the exception of those of Brucella ovis and B. canis, which are rough but virulent. It is accepted that rough mutants are more sensitive to lysis mediated by complement, and probably this is the main reason why most rough variants have an avirulent phenotype in animal models. To date the question about the capacity of rough mutants to replicate intracellularly is not solved. Some authors have reported that smooth LPS is essential for intracellular survival 25, for example, the vaccine strain RB51 exhibits loss of virulence and cannot replicate within macrophages 26. On the other hand, there are some reports in which genetically characterized rough mutants did not loose the capacity to replicate intracellularly despite the total absence of the O-antigen 27B. abortus strain RB51 is a stable rough (R) mutant derived from the standard smooth (S) virulent strain 2308. This rough mutant strain that does not contain the O-antigen (O polysaccharide chain of the smooth LPS) is attenuated in its virulence compared to their smooth virulent parental strain 27. Even though the Brucella RB51 rough strain has been widely used as a live vaccine, it induces lower protection compared to the smooth vaccine strain S19 28. At similar concentrations, the Brucella vaccine strain S19 induces higher protection than the R vaccine strain (RB51). The lack of protective antibodies to the O-side chain of the LPS in animals immunized with B. abortus RB51 may explain in part why the R strain induces lower protection against infection 29. A major problem in animal vaccination against brucellosis with currently used live attenuated smooth Brucella strains, such as B. melitensis strain Rev.1 for sheep and goats and B. abortus strain S19 for cattle, is the fact that vaccinated animals cannot be clearly differentiated from infected animals by the current serological tests. Further, these strains may induce abortions when used in pregnant animals and are virulent for humans. These tests are indeed based on detection of antibody to S-LPS that is immunodominant in the serological responses of both infected and vaccinated animals.

Below, we describe the main genes involved in LPS biosynthesis and the role of their encoding products.

In contrast to other intracellular pathogens, Brucella species do not produce exotoxins, antiphagocytic capsules or thick cell walls, resistant forms or fimbriae and do not show antigenic variation 48. A key aspect of the virulence of Brucella is its ability to proliferate within professional and non-professional phagocytic host cells. Therefore, Brucella successfully bypasses the bactericidal effects of phagocytes, and their virulence and chronic infections are thought to be due to their ability to avoid the killing mechanisms of host cells 49. Some studies with non-professional phagocytes have shown that Brucella invades host cells and is contained within early endosome-like vacuoles. These vacuoles rapidly fuse with early autophagosomes that acquire vacuolar H+-ATPase and lysosome-associated membrane proteins (LAMP) maturing into a late autophagosome. These autophagosomes inhibit fusion with lysosomes and finally become a replicating vacuole normally associated with the endoplasmic reticulum 5051. Porte et al. showed that the LPS O-side chain is involved in inhibition of the early fusion between Brucella suis containing phagosomes and lysosomes in murine macrophages at least during the first few hours after phagocytosis 52. In contrast, the phagosomes containing rough mutants, which fail to express the O-antigen, rapidly fuse with lysosomes. The LPS O-chain might be a major factor that governs the early behavior of bacteria inside macrophages.

Recognition of the presence of LPS by cells such as monocytes and macrophages has evolved over centuries to provide the mammalian host with a rapid recognition of and reaction towards Gram-negative infection. This rapid, innate response against LPS typically involves the release of a range of pro-inflammatory mediators, such as TNF-α, IL-6, IL-12 and IL-1β, which in local sites of infection and in moderate levels benefit the host greatly by promoting inflammation and otherwise priming the immune system to eliminate the invading organisms. However, in conditions where the body is exposed to LPS excessively or systemically (as when LPS enters the blood stream), a systemic inflammatory reaction can occur, leading to multiple organ failure, shock and potentially death 53.

Recognition of bacterial LPS is mediated by CD14, however, CD14 lacks transmembrane and intracellular domains necessary for signal transduction and thus requires the involvement of molecules belonging to the TLR family. The recent discovery of TLR proteins, a family of mammalian pattern recognition receptors, has provided new insights into our understanding of the mechanisms by which Brucella can elicit cellular responses from innate immune cells. B. abortus induces interleukin (IL)-12 production from human monocytes and this effect was blocked by anti-CD14 antibody, suggesting that the Brucella binding and/or signaling to monocytes was mediated via LPS 54. Additionally, Brucella's ability to elicit IL-12 secretion enables it to drive Th0 cells to differentiate into Th1 effector and memory cells that are a central feature of the potential use of B. abortus as a vaccine carrier and adjuvant.

Our group has investigated the ability of B. abortus and its purified LPS and lipid A to trigger TLR2 and TLR4 and the impact of this activation in innate recognition and elimination of invading bacteria 55. CHO reporter cell lines transfected with CD14 and TLRs showed that B. abortus triggers both TLR2 and TLR4. In contrast, lipopolysaccharide (LPS) and lipid A derived from Brucella rough (R) and smooth (S) strains activate CHO cells only through TLR4. Consistently, macrophages from C3H/HePas mice exposed to R and S strains and their LPS produced higher levels of tumor necrosis factor alpha (TNF-α) and interleukin-12 compared to C3H/HeJ, a TLR4 mutant mouse. The essential role of TLR4 for induction of proinflammatory cytokines was confirmed with diphosphoryl lipid A from Rhodobacter sphaeroides. Furthermore, to determine the contribution of TLR2 and TLR4 in bacterial clearance, numbers of Brucella were monitored in the spleen of C3H/HeJ, C3H/HePas, TLR2 knockout, and wild-type mice at 1, 3, and 6 weeks following B. abortus infection. Interestingly, murine brucellosis was markedly exacerbated at weeks 3 and 6 after infection in animals that lacked functional TLR4 (C3H/HeJ) compared to C3H/HePas that paralleled the reduced gamma interferon production by this mouse strain. By mass spectrometry analysis we found dramatic differences on the lipid A profiles of R and S Brucella strains when compared to E. coli lipid A (Figure 2). As shown in Figure 2, Brucella rough and smooth lipid A possess more heptaacylated species than E. coli lipid A that presents more hexaacylated residues. In conclusion, these results indicate that TLR4 plays a role in resistance to B. abortus infection. In contrast, Giambartolomei et al 56 claimed that lipoproteins Omp19 and Omp16 signaling through TLR2 rather than LPS are the key molecules involved in the proinflammatory response mediated by IL-6 and TNF-α observed during Brucella infection. This phenomenon seems to be related to the lipid moiety of the proteins. Therefore, TLR2 and TLR4 signaling during Brucella infection are mediated by two different ligands, lipoproteins and LPS, respectively. More recently, Weiss et al. 57 found that during infection of macrophages, Brucella avoids activation of TLR4 at six hours but activates TLR4, TLR2, and MyD88 (myeloid differentiation factor 88) at 24 hours postinfection. Interestingly, even though its activation is delayed, among the three MyD88 is the most important molecule for host defense against Brucella infection in vivo, since MyD88 knockout (KO) mice are more susceptible to infection when compared to TLR4, TLR2, TLR4/TLR2 KO mice. These findings open the possibility for the involvement of other TLRs that signal through MyD88 in host resistance against Brucella infection. As a matter of fact, Huang et al. 58 demonstrated that HKBA (heat-killed Brucella abortus) stimulates dendritic cells (DC) via TLR9 to secrete IL-12. Furthermore, this effect can be stimulated by DNA derived from HKBA and can be blocked by addition of suppressive oligodeoxynucleotides (ODNs). The consequence of TLR9 engagement is that Th1-like responses such as IFN-g production takes place in wild-type but not TLR9 KO mice.