- Open Access
Brucella spp noncanonical LPS: structure, biosynthesis, and interaction with host immune system
© Cardoso et al; licensee BioMed Central Ltd. 2006
- Received: 30 December 2005
- Accepted: 23 March 2006
- Published: 23 March 2006
Brucella spp. are facultative intracellular pathogens that have the ability to survive and multiply in professional and non-professional phagocytes, and cause abortion in domestic animals and undulant fever in humans. Several species are recognized within the genus Brucella and this classification is mainly based on the difference in pathogenicity and in host preference. Brucella strains may occur as either smooth or rough, expressing smooth LPS (S-LPS) or rough LPS (R-LPS) as major surface antigen. This bacterium possesses an unconventional non-endotoxic lipopolysaccharide that confers resistance to anti-microbial attacks and modulates the host immune response. The strains that are pathogenic for humans (B. abortus, B. suis, B. melitensis) carry a smooth LPS involved in the virulence of these bacteria. The LPS O-chain protects the bacteria from cellular cationic peptides, oxygen metabolites and complement-mediated lysis and it is a key molecule for Brucella survival and replication in the host. Here, we review i) Brucella LPS structure; ii) Brucella genome, iii) genes involved in LPS biosynthesis; iv) the interaction between LPS and innate immunity.
- Brucella Species
- Brucella Melitensis
- Brucella Infection
- Feral Swine
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 . 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 . 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 . 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 . 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 [5, 6]. 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 .
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 . 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.
Recently, the genome sequences of the B. melitensis, B. suis and B. abortus became available [18–20]. 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 . 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 . Using the complete genome sequence of Brucella melitensis, Dricot et al.  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 . Screens for transpositional mutants attenuated in infection models yielded 184 mutants, suggesting that these genes have a function in the infection process . The B. suis 1330 genome consists of two circular chromosomes of 2,107,792 bp and 1,207,381 bp . 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 . 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) . 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.  to potentially affect phenotypes of brucellae, such as host preference.
Genes encoding O-antigen biosynthesis in Brucella spp.
O-antigen export permease
no similarity to known genes
Methionyl tRNA formyltransferase
Below, we describe the main genes involved in LPS biosynthesis and the role of their encoding products.
Godfroid et al.  described molecular analysis of the genes required for the synthesis of the O-antigen of Brucella melitensis 16 M. The perosamine synthetase gene was cloned and sequenced. In V. cholerae O1, perosamine is synthesized from fructose 6-phosphate via four intermediates: mannose 6-phosphate, mannose 1-phosphate, GDP-mannose, and 4-keto-6-dideoxymannose. Ultimately, this final product is converted to GDP-perosamine by the perosamine synthetase . Because the last step of the perosamine synthesis pathway is identical for V. cholerae and B. melitensis, it was assumed that the earlier steps might be similar or identical for these two organisms. In Brucella, the GDP-perosamine would then serve as a substrate for the addition of a formyl group and could then be polymerized into the O-antigen, translocated to the periplasm, transferred to the lipid A-core oligosaccharide, and exported to the cell surface. The disruption of per (B3B2 mutant) totally disabled the O-side chain biosynthesis of B. melitensis 16 M. The mutation was recreated by gene replacement, indicating that the mutant phenotype was due to the transposon insertion rather than to spontaneous mutation . Indeed, such a disruption prevented any O-side chain production, not only at the surface but also in the cytoplasm of the bacteria indicating that the mutation does not affect the transport of the O-side chain to the outer membrane but does affect an earlier stage of biosynthesis.
Phosphomannomutase (pmm or manB)
Allen et al.  to better characterize the role of O-antigen in virulence and survival used transposon mutagenesis to generate B. abortus rough mutants defective in O-antigen presentation. A mutant strain was characterized by a truncated rough LPS and DNA sequence analysis of this mutant revealed a transposon interruption in the gene encoding phosphomannomutase (pmm or manB), suggesting that this activity may be required for the synthesis of a full-length core polysaccharide in addition to O-antigen. This gene is responsible for the interconversion of mannose-6-phosphate and mannose-1-phosphate. In Brucella, mannose is both an important precursor in the O-antigen biosynthetic pathway and in the production of the inner core moiety of LPS .
Mannosyltransferases (wbkA, WbdA, B and C)
B. abortus genes involved in chronic infection were identified by assessing the ability of 178 signature-tagged mutants to establish and maintain persistent infection in mice . Each mutant was screened for its ability to colonize the spleens of mice at 2 and 8 weeks after inoculation. A mutant with defects in establishing chronic infection which carried a transposon insertion in a B. abortus homologue of Brucella melitensis wbkA, encoding a N-formyl-perosaminyltransferase that functions in the biosynthesis of O-antigen, was the most highly attenuated. In E. coli O9a polysaccharide is polymerised by the action of three different mannosyltransferases WbdA, B and C . In this scheme, WbdC transfers a mannose to the endogenous acceptor (GlcNAc-pyrophosphoundecaprenol). This reaction initiates the growth of the polysaccharide chain and provides the acceptor for subsequent progressive chain elongation by the sequential activities of WbdB, transferring successive mannosyl units into the 3 position, and of WbdA, transferring successive mannosyl units at the 2 position of the previous mannose. On the basis of this scheme, the presence of α-1,2 and α-1,3 linkages in the LPS O-side-chain of Brucella suggests the existence of at least two N-formyl-perosaminyltransferases, WbkA and WboA. The WbkA could interact with WboA to elongate the Brucella LPS O-side-chain by α-1,2 and α-1,3 links. The wboA gene that encodes a glycosyltransferase, an enzyme also essential for the biosynthesis of the O-side chain in B. abortus was characterized by McQuiston et al. . The disruption of the wboA gene in smooth strains B. abortus 2308, B. melitensis 16 M and B. suis biovar 4 resulted in conversion to a rough phenotype and attenuated . Vemulapalli et al.  discovered that the wboA gene is interrupted by an IS711 element in B abortus vaccine strain RB51. The complementation of RB51 with a functional wboA gene resulted in O-antigen production but did not result in reversion to the smooth phenotype and did not affect attenuation, suggesting that RB51 contains an additional genetic mutation(s) that probably affects either the export of O-antigen to the bacterial surface, the coupling of O-antigen to core lipopolysaccharide, or both . Two rough mutant strains RA1 and VTRM1 derived from virulent B. abortus 2308 or B. melitensis 16 M, respectively have identical mutations wboA gene. RA1 strain was more sensitive to the bactericidal action of nonimmune human serum and more complement components were deposited on its surface than on strain VTRM1 . Similar species-specific differences in both complement deposition and complement-mediated killing were also observed when strain RA1 was compared with another rough mutant of B. melitensis, WRR51. Strain WRR51 was derived from B. melitensis strain 16 M by replacement of the internal region of the wboA gene with an antibiotic resistance cassette instead of having a transposon insertion on this gene, as in the case of VTRM1 or RA1. There were no significant differences in either complement deposition or killing between VTRM1 and WRR51 . Both strains were less susceptible than RA1 to the deposition of complement and complement-mediated killing. The LPS of strains RA1 and RB51 with the LPS of strain 2308 were compared and silver staining indicated that no O-side chain was associated with LPS of strains RA1 or RB51, and compositional analysis of smooth and rough B. abortus LPS revealed that 2-keto-3-deoxy-D-manno-2-octulosonic acid (KDO) was the predominant glycose in the rough LPS . Vizcaíno et al.  studying DNA polymorphism in the omp25/omp31 family of Brucella spp., identified a 15.1 kb fragment absent in Brucella ovis. The region absent from B. ovis suggests that this DNA fragment is a genomic island acquired by the Brucella ancestor by horizontal transfer and later deleted from B. ovis. This deletion includes wboA and two other genes that might be involved in the LPS synthesis. Absence of these genes in B. ovis may explain, at least in part, the rough phenotype naturally displayed by this Brucella species. The complementation of rough B. ovis PA with plasmids bearing wboA, bearing wboA and the downstream gene potentially encoding a mannosyltransferase or bearing almost the entire 15.1 kb DNA fragment deleted in B. ovis strains did not confer a smooth phenotype, as shown by the lack of reactivity with a MAb specific for the Brucella spp S-LPS. This finding suggests that other genes required for the synthesis of S-LPS located at other chromosomal loci are affected in B. ovis. It seems clear that removal of the 15.1 kb genomic island from the smooth Brucella strains would reduce their virulence, since it was shown that the Tn5-disruption of wboA reduces survival of B. abortus in mice . It would be interesting to determine how the complementation of B. ovis with the deleted genomic island would affect the virulence of this Brucella species.
The gene encoding for phosphoglucomutase (pgm) is involved in O-antigen biosynthesis in B. abortus . This gene is absolutely necessary for the biosynthesis of ADP-glucose, UDP-glucose, and UDP-galactose, the donors of glucose or galactose for the biosynthesis of molecules containing these sugars. The predicted protein is 74.7% identical to its homologue in Agrobacterium tumefaciens but is not part of the glycogen operon as it is in Agrobacterium. B. abortus LPS O-antigen is a homopolymer of perosamine, a derivative of mannose that is synthesized through GDP-mannose, thus, a pgm mutant of this species would not be impaired in the synthesis of GDP-perosamine, the sugar donor of O-antigen subunits. Insertional mutagenesis of pgm was carried out introducing a gentamicin-resistant gene within the B. abortus pgm gene and the electrophoretic profile of the LPS extracted from this mutant strain indicated lack of the O-antigen. This mutant was unable to survive in mice but replicates in HeLa cells, indicating that the complete LPS is not essential either for invasion or for intracellular multiplication. This behavior suggests that the LPS may play a role in extracellular survival in the animal, probably protecting the bacteria against complement-mediated lysis, but is not involved in intracellular survival. The fact that the mutant replicates at a lower rate is not necessarily a consequence of the rough phenotype, since the absence of pgm affects many other components of the cell wall, such as, for example, the synthesis of β (1,2) cyclic glucan . Sequence analysis of the regions upstream and downstream of Brucella pgm revealed no significant homology to any gene in the database, which was surprising since in A. tumefaciens and Rhizobium loti, pmg is part of the glycogen operon.
ABC type transporters (Wzm and Wzt)
Godfroid et al.  identified, sequenced and characterized a chromosomal locus of a 14-kb, wbk biosynthesis gene cluster, involved in the LPS O-side-chain biosynthesis of B. melitensis 16 M. Analysis of the nucleotide sequence revealed the presence of seven open reading frames (ORFs), with six of them showing homology with genes involved in LPS O-side-chain biosynthesis from other organisms, and surrounded by four entire and one partial insertion sequences (IS). The seven ORFs were named according to the bacterial polysaccharide gene nomenclature proposed by Reeves et al. . The GCG Gap program (Wisconsin package version 9.1, Genetic Computer Group, Madison, WI) was used to compare the deduced gene products and their similarity to various protein homologues. Seven genes of the wbk locus of Brucella melitensis 16 M were wbkA, gmd, per, wzm, wzt, wbkB, and wbkC, coding, respectively, for proteins homologous to N-formyl-perosaminyltransferase, GDP-mannose 4,6 dehydratase, perosamine synthetase, ABC-type transporter (integral membrane protein), ABC-type transporter (ATPase domain), a hypothetical protein of unknown function, and a putative formyl transferase . The wzm and wzt (putative the integral membrane component of ABC transporters) mutation resulted in a rough phenotype of B. melitensis 16 M colonies as shown by crystal violet colony staining . The wzm/wzt mutant also failed to react in ELISA on whole cells with MAbs directed against the S-LPS O-side-chain of Brucella species (anti-S-LPS MAbs), confirming the absence of the O-side-chain on the bacterial surface. Three complete and one incomplete insertion sequences in close association with the wbk gene cluster were found (ISBm1, ISBm, ISBm3, ISBm 4). The presence of several ISs in intimate association with the wbk locus is intriguing. Since IS elements are suggested to play an important evolutionary role in mediating chromosomal rearrangements, it seems likely that they might have contributed to the structural evolution and probably horizontal acquisition of the O-antigen biosynthesis gene cluster in Brucella spp.
Mannose (manA, B, C)
Monreal et al.  showed that several LPS genes flank the seven genes described by Godfroid et al.. These genes include manA, manB, and manC, and their position strongly suggests that they act coordinately with gmd and per and independently of other mannose genes. Polymyxin B sensitive mutants were isolated by transposon mutagenesis of B. abortus 2308 and screening for viability loss after a controlled exposure to an excess of this antibiotic . Since the O-polysaccharide plays a role in protection against polymyxin B, these mutants were further screened for O-polysaccharide defects by agglutination with anti-S-LPS antibodies. Four mutants negative in this test were then chosen on the basis of their different polymyxin B sensitivities. Computer database analysis revealed that the mini-Tn5 was inserted in the per gene, wbkA, manB and open reading frame provisionally named wa**. This gene product was a membrane protein of the glycosyltransferase family involved in LPS biosynthesis, but it was different from other putative glycosyltransferases described before as involved in LPS synthesis in Brucella. Monreal et al.  did a search in the complete genome sequence of B. melitensis 16 M and B. suis 1330 and revealed a single homologous genes for per and wbkA, both located in the wbk region. The gene homologous to wa** was also in chromosome I, although in a different region. On the other hand, the B. melitensis and B. suis manB homologues were in chromosome II, along with a manC gene putatively coding for both mannose-6-P-isomerase and mannose-1-P-guanylyltransferase activities. Since phenotypic analysis revealed a severe core defect in the mutant, the gene was designated manB core . The genes wa** and manB core are involved in the biosynthesis of the B. abortus LPS core. In contrast to the parental strain, per, wbkA, wa**, and manB core mutants were resistant to the S-Brucella-specific phages and sensitive to the R-Brucella-specific phage R/C. Moreover, it was observed that the manB core mutant showed the lowest R/C phage sensitivity and the highest polymyxin B resistance and that, conversely, the wa** mutant had the highest R/C phage sensitivity and the lowest polymyxin B resistance.
Genes encoding lipid A biosynthesis in Brucella melitensis 16 M.
Acyl-(acyl carrier protein) UDP-N-acetylglucosamine-O acyltransferase
856881 – 857729/I
UDP-3-O-(3Hydroxymyristoyl) N-acetylglucosamine diacetylase
608267 – 609127/I
UDP-3-O-(3Hydroxymyristoyl) glucosamine N-acetyltransferase
855363 – 856418/I
858609 – 859796/I
1067291 – 1068316/II
876651 – 877448/I
3-deoxy-manno octulosanate cytidylyl transferase
1959311 – 19601981/I
3-deoxy-D-manno octulosonic-acid transferase
1068319 – 1069659/II
1159853 – 1160776/I
Genes encoding lipid A biosynthesis in Brucella suis.
Acyl-(acyl carrier protein) UDP-N-acetylglucosamine-O acyltransferase
1130902 – 1131738/I
1379039 – 1379899/I
UDP-3-O-(3Hydroxymyristoyl) glucosamine N-acetyltransferase
1132213 – 1133268/I
1128835 – 1130022/I
202326 – 203351/II
1111145 – 1111978/I
3-deoxy-manno octulosanate cytidylyl transferase
43162 – 43917/I
3-deoxy-D-manno octulosonic-acid transferase
200983 – 202323/II
824862 – 825788/I
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 . 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 . 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 [50, 51]. 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 . 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 .
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 . 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.
This work was supported by CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and FAPEMIG (Fundação de Amparo a Pesquisa no Estado de Minas Gerais).
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