Brucella lipopolysaccharide acts as a virulence factor

Brucella lipopolysaccharide acts as a virulence factor Nicolas Lapaque1, Ignacio Moriyon2, Edgardo Moreno3 and Jean-Pierre Gorvel1 Brucella is a facultative intracellular bacterium responsible for brucellosis. Virulence factors involved in Brucella replication and Brucella’s strategies to circumvent the immune response are under investigation. VirB proteins that form the type IV secretion system and that are involved in intracellular replication are considered as one of Brucella’s virulence factors. In addition to this secretion system, bacterial outer membrane components have also been described as being implicated in Brucella survival in the host. For example, this bacterium possesses an unconventional non-endotoxic lipopolysaccharide that confers resistance to anti-microbial attacks and modulates the host immune response. These properties make lipopolysaccharide an important virulence factor for Brucella survival and replication in the host. Addresses 1 Centre d’Immunologie INSERM-CNRS-Universite´ de la Me´diterrane´e, Parc Scientifique de Luminy, Case 906, 13288 Marseille Cedex 9, France 2 Department of Microbiology, University of Navarra, c/Irunlarrea 1, 31008 Pamplona, Spain 3 Programa de Investigacio´n en Enfermedades Tropicales (PIET), Escuela de Medicina Veterinaria, Universidad Nacional, Aptdo 304-3000 Heredia, Costa Rica Corresponding author: Gorvel, Jean-Pierre ([email protected])

Current Opinion in Microbiology 2005, 8:60–66 This review comes from a themed issue on Host–microbe interactions: bacteria Edited by Pascale Cossart and Jorge Gala´n Available online 8th January 2005 1369-5274/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2004.12.003

been proposed that the lipid-raft-dependent entry occurs via a type IV secretion system, VirB, through the binding of the bacterial exposed protein HSP60 with the cellular prion protein PrPc and the VirB-dependent recruitment of the host class A scavenger receptor (SR-A) to lipid-rafts [4,5,6]. However, this model has been criticized because other studies have shown that the VirB secretion system is not involved in early stages of Brucella trafficking [7] and because surface proteins are not accessible in smooth Brucella strains (discussed by Celli and Gorvel [8]). More studies are needed to resolve this question. It is well established that the bacteria–lipid-raft interaction allows wild-type Brucella to escape from the degradative endocytic pathway and further favors its intracellular replication [2,3,5]. In early stages after entry, wild-type Brucella resides in an acidified vacuole, the Brucella-containing vacuole (BCV), which transiently interacts with early endosomes [7]. The BCV matures in a VirB-dependent manner to form the Brucella replicative niche by stabilizing interactions with the endoplasmic reticulum [7,8]. It is clear that VirB proteins forming the type IV secretion system are involved in Brucella virulence and intracellular replication [9–11]. However, even though type IV secretion systems from other Gram-negative bacteria have been described to deliver effectors proteins into host cells [12,13], no effectors have been identified in Brucella up to now and further studies are needed. In addition to the VirB apparatus, bacterial external membrane components [14], especially lipopolysaccharides (LPSs), have also been described as virulence factors and are important for Brucella replication and survival [15–17].

Introduction Members of the bacterial genus Brucella are facultative intracellular pathogens responsible for brucellosis, a worldwide zoonosis. This pathogen is able to infect a wide variety of mammals, causing abortion and infertility in domestic herds and wild animals. B. melitensis, B. abortus and B. suis cause brucellosis in humans, leading to chronic stages of the disease that can be manifested as orchitis, spondylitis, arthritis and a debilitating illness known as undulant fever (reviewed in [1]). These symptoms have been correlated with Brucella replication and persistence in host cells [1], where its growth is directly linked to its intracellular replication and capacity to circumvent innate and adaptive immunity. Upon entry Brucella hijacks lipid-rafts [2,3]. In macrophages, it has Current Opinion in Microbiology 2005, 8:60–66

In this review, we focus on the importance of an unconventional LPS in Brucella virulence. The LPSs of intracellular Proteobacteria such as Brucella have structures and properties distinct from enterobacterial LPSs [18,19]. For Brucella, these properties include a low endotoxicity, high resistance to macrophage degradation and protection against immune responses [20–22]. Taken together, these properties may constitute key virulence mechanisms for intracellular survival and replication of Brucella.

B. abortus LPS: a non-classical endotoxin The LPS of Gram-negative bacteria is composed of a hydrophobic lipid A linked to a charged, densely compact oligosaccharidic core associated (smooth-LPS) or not www.sciencedirect.com

Brucella lipopolysaccharide acts as a virulence factor Lapaque et al. 61

Figure 1

R

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Brucella LPS structure. B. abortus lipid A possesses a diaminoglucose backbone linked by amide bonds to long acyl groups (C18–C19, C28), which differs from the classical LPS of E. coli. The O-chain is an N-formyl-perosamine homopolymer (top-right). The oligosaccharide core is composed of 3-deoxy-D-manno-2 octulosonic acid (Kdo), mannose and glucosamine (bottom-right).

(rough-LPS) with a hydrophilic O-polysaccharide chain (O-chain). Brucella possesses a peculiar LPS that we call non-classical LPS as compared with the so-called classical LPS from enterobacteria such as Escherichia coli. B. abortus lipid A possesses a diaminoglucose backbone (rather than glucosamine), and acyl groups are longer (C18–C19, C28 rather than C12 and C14) and are only linked to the core by amide bounds (rather than ester and amide bonds) (reviewed in [23]; see Figure 1). This non-classical structure confers to B. abortus LPS peculiar characteristics that make it a virulence factor because it alters the LPS pathogen-associated molecular pattern (PAMP) and reduces the endotoxin-related properties typical of LPSs. In contrast to enterobacterial LPSs, Brucella LPS is several-hundred-times less active and toxic than E. coli LPS [15,20,23–25]. It has been well described that LPSs from most Gram-negative bacteria induce reactive oxygen intermediates, bactericidal nitrogen intermediates, p-47 GTPase expression and cytokine (IFN, TNF) production [26–28], all known to be implicated in the clearance of intracellular pathogens, including Brucella [29–31]. However, highly purified B. abortus LPS is a poor inducer of respiratory burst, bactericidal nitrogen intermediates and lysozyme secretion [15,32]. Furthermore, even though B. abortus LPS signals through the well-described LPS–TLR4 pathway [33] (N Lapaque www.sciencedirect.com

et al., unpublished), it has little influence on the production of cytokines such as TNF-a, type-I and type-II IFNs as well as anti-microbial proteins such as the GTPases of the p-47 family [24,25,34] (N Lapaque et al., unpublished). Consistent with the hypothesis that this is an evolutionary adaptation to an intracellular lifestyle, low endotoxic activity is shared by other intracellular pathogens such as Bartonella and Legionella [18–20].

The role of Brucella LPS in resistance to innate-immunity anti-bacterial responses Independently from the weak endotoxicity of its LPS, Brucella virulence is also linked to the chemical structure of this molecule, which permits these bacteria to become highly resistant to anti-bacterial effectors of the innate immune system. Brucella LPS has been shown to impair anti-microbial responses by inhibiting complement and anti-bacterial-peptide attacks and by preventing the synthesis of immune mediators. Currently, three complement pathways leading to bacterial killing have been elucidated: the classical, the alternative and the lectin pathways (reviewed in [35]). It has been documented that enterobacterial surfaces and especially their LPS trigger both the classical and alternative Current Opinion in Microbiology 2005, 8:60–66

62 Host–microbe interactions: bacteria

pathways (respectively by lipid A and O-chain LPS sections) of complement activation [36–38]. B. abortus has the particular ability to resist to the bactericidal activity of non-immune serum in part by a low activation of the alternative complement pathway by its LPS [20,39]. Using whole bacteria, the B. abortus LPS Ochain has been shown to be involved in this resistance because Brucella rough mutants (which bear a rough-LPS) are more sensitive to normal serum and complement attack [40–42], by the classical and lectin pathways [43]. The Brucella LPS O-chain prevents the deposition of complement at the bacterial surface [43,44]. It has been clearly shown that, as with many LPSs possessing long O-chains [45–47], the smooth LPS of Brucella impairs host complement-mediated killing of bacteria [40,42–44]. The B. abortus O-chain blocks access of C1q to the outer membrane protein targets, and this observation led to the hypothesis that O-chain length and proportion at the bacterial membrane could form a ‘shield’ from complement-mediated attack [20,44]. The virulence of different pathogens such as Trypanosoma cruzi, Yersinia enterocolitica, Salmonella spp. and Helicobacter pylori has been shown to be related to the impairment in binding of complement components [46,48–50]. The presence of O-chains at the surface of Brucella limits complement attack and might help in Brucella extracellular survival. In addition to blocking complement activation, Brucella LPS allows the survival of these bacteria by efficiently protecting against bactericidal cationic peptides. It has been shown that Brucella is resistant to a large variety of such peptides, including defensin NP-2, lactoferrin, cecropines, lysozyme, bactenecin-derived peptides, and the defensin-like antibiotic polymyxin B, as well as to crude lysosomal extracts from polymorphonuclear leukocytes [16,17,32,51]. This resistance has been proposed to be directly correlated to a low number of anionic groups in the core lipid A part of B. abortus LPS, especially charged phosphate groups. The reduced number of phosphate groups could both remove anionic targets and facilitate a tighter aggregation of LPS molecules via their hydrophobic fatty acids, leading to less binding and penetration of antibacterial cationic peptides [16,51]. A contribution of the O-chain has also been postulated in protection against these bactericidal products, as supported by a higher resistance of the smooth strain compared with the rough one. Long O-chains at the bacterial surface should provoke a steric hindrance leading to the formation of a protecting barrier [16]. The O-chains from B. suis and B. melitensis also appear to impair cytokine expression in infected human and mouse macrophages [52,53]. Contrary to what is observed with smooth strains of B. suis and B. melitensis, rough strains induce inflammatory cytokines and inducible nitric oxide synthase (iNOS) expression at a level similar to that Current Opinion in Microbiology 2005, 8:60–66

reached upon E. coli LPS stimulation [52]. Like in other Gram-negative bacteria, the Brucella outer membrane is composed of a multitude of potential PAMPs that could be recognized by immune receptors such as Tolllike receptors (TLRs) in ‘danger’ signaling [33]. The O-chain could prevent a specific recognition of these PAMPs and as a consequence impair expression of any cytokines or iNOS, both of which are known to be involved in the clearance of intracellular Brucella [30,31,54–56]. The fact that smooth Brucella strains prevent the induction of such products may be responsible for its long-term survival. During infection, resistance to complement and cationicpeptide-mediated attacks as well as protection against host recognition of PAMPs enhances Brucella survival before reaching its intracellular niche. Moreover, at the very early stages of infection, complement is the only source of opsonic molecules. Thus, by preventing opsonization, this LPS also contributes to avoid uptake into host cells by unfavorable complement-receptor-dependent routes. This set of resistance properties makes this unconventional LPS a virulence factor.

The role of Brucella LPS in its entry and early stages of trafficking As summarized above, it is accepted that the susceptibility to destruction of the outer membrane in rough strains explains their sensitivity to antibacterial attacks in the host. Moreover, the O-chain and lipid A also seem implicated in the entry and early stages of Brucella trafficking [57,58]. Entry of Brucella, accepted to pass through lipid-rafts [2], is highly complex. This entry-gateway seems to include HSP60–PrPc interaction [6] but also a SR-A–lipid-A interaction [5] and is also dependent on the LPS O-chain [59] (Figure 2). It has been reported that entry and early survival stages of smooth B. suis are lipid-raft-dependent and impair phagosome-lysosome fusion during the first hours of infection (Figure 2). Compared with smooth Brucella, the homologous rough strain (which does not possess O-chains) seems not to enter by lipid-rafts, and fuses rapidly with lysosomes. By contrast, killed smooth B. suis shows delayed fusion with lysosomes, leading to the hypothesis that the O-chain is involved in these early events [59]. It has been postulated that the smooth LPS interacts with lipid-rafts through an unknown receptor on the surface of macrophages, and that this permits B. suis to enter cells via a pathway allowing it to avoid fusion with lysosomes. It seems that smooth LPS, and consequently its O-chain, is important for entry and during early stages of BCV development. Formation and early maintenance of BCV may involve specific unknown O-chain receptors (Figure 2). Another entry process seems to be LPS-dependent. In this second model of entry, SR-A would be recruited to lipid-raft by a VirB-dependent process (Figure 2). www.sciencedirect.com

Brucella lipopolysaccharide acts as a virulence factor Lapaque et al. 63

Figure 2

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Entry and intracellular trafficking of B. abortus in macrophages. Entry of Brucella through lipid-rafts seems to be mediated by HSP60–PrPc and SR-A–LPS interactions and is also dependent on the LPS O-chain. Brucella rapidly escapes the endocytic pathway and is located in an intermediate LAMP-positive compartment before reaching its replicative organelle derived from the ER in a VirB-dependent manner. Rough mutants do not enter via lipid-rafts, do not escape the endocytic pathway and are degraded in lysosomes.

Figure 3

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Intracellular trafficking of B. abortus LPS in macrophages and inhibition of antigen presentation. B. abortus LPS is internalized in macrophages and follows the classical endocytic pathway used by protein antigens but with a slower kinetics. Brucella LPS is found in a compartment (MIIC) that is enriched in MHC-II and is recycled to the cell surface, where it forms dense detergent-resistant clusters with MHC-II that are named macrodomains. B. abortus LPS interferes with the MHC-II presentation of peptides to specific CD4+ T cells.

The LPS of Brucella, a modulator of the immune response B. abortus LPS is internalized by macrophages, in which it resists intracellular degradation [21]. Once inside macrowww.sciencedirect.com

phages, it follows the same classical endocytic pathway as protein antigens do but with slower kinetics [21] (Figure 3). Then, Brucella LPS is found in the MHC class II (MHCII)-enriched compartment (MIIC); the function of the Current Opinion in Microbiology 2005, 8:60–66

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MIIC is the loading of antigenic peptides onto MHC-II molecules. From there, the LPS is subsequently recycled to the cell surface where it forms stable large clusters with MHC-II that have been named macrodomains [21,22,60] (Figure 3). Furthermore, B. abortus LPS interferes with MHC-II presentation of peptides to specific CD4+ T cells but has no effect on MHC class I antigenic presentation [22] (Figure 3). This impairment is not due to antigen processing, to a decrease of MHC-II or co-stimulatory molecules at the cell surface, or to an effect on the dimer formation [22]. In addition, this inhibition is not due to a direct suppressive effect on T cells or prevention of the binding of antigenic peptides in the MHC-II groove [22]. Further characterization of these structures has demonstrated that B. abortus LPS macrodomains are not only enriched in MHC-II but also possess lipid-raft components (Figure 3). Although LPS macrodomains act as detergent-resistant membranes, they possess peculiar properties: they are electron-dense, b-cyclo-resistant structures with reduced membrane fluidity (N Lapaque et al., unpublished). B. abortus could avoid the targeting of MHC-II to the well-characterized functional lipid-raft and segregate MHC-II to these dense structures. The differences between dynamic lipid-rafts and the dense and stable LPS complexes might be related to the inhibition of MHC-II dependent presentation to CD4+ T cells. From a molecular point of view, the stable and dense complexes of LPS and MHC-II at the cell surface could also produce a steric hindrance between the antigenpresenting cells and the T cells, impeding a sufficient recognition between the T cell receptor (TCR) and the MHC-II–peptide complex. These results suggest that LPS–cell-membrane complexes may be important in Brucella pathogenesis to control host immune responses during infections by impairing infected host cells from activating Brucellaantigen-specific CD4+ T cells. It is important to notice that this LPS is not secreted in the external environment after recycling at the cell surface and remains intact for long periods of time without being degraded. As cellular immunity is important for controlling pathogen proliferation in the host, the presence and persistence of LPSmacrodomains at the cellular surface and their immunomodulatory activity may be considered as a strategy used by Brucella to maintain long-term residence in its intracellular niche.

Conclusions Its importance in the surface of bacteria and its presence as a free form during infections explain why Brucella LPS is a major antigen and why it constitutes a virulence factor of this bacterium [61,62]. This peculiar LPS acts as a shield that protects from antimicrobial attacks, not only by the action of its O-chain as Current Opinion in Microbiology 2005, 8:60–66

happens in some other Gram-negative bacteria but also by virtue of the unconventional structure of its core and lipid A. This barrier could hide PAMPs from immunereceptor recognition, leading to an impairment of the danger signals that alert the host to the presence of a pathogen. It is also easy to hypothesize that the low endotoxic activities induced by Brucella LPS might be one factor implicated in the intracellular survival of Brucella. In an infectious process, the low endotoxicity, non-activating and shield-like properties of LPS suggest that host cells are still able to support intracellular pathogen replication even if the cellular immune response is modulated to the Brucella’s advantage. Brucella could be compared to an undercover agent whose strategy is to hide itself in a specific compartment and to disturb the immune system. This could explain the long-term persistence of Brucella in the host and the chronicity of the disease it causes. During evolution Brucella might have taken advantage of an unconventional LPS structure to favor its replication in its hosts. Escape from the difficult extracellular environment to survive intracellularly could also be seen as an efficient evolutionary adaptation to a parasite’s host cells. In this context, Brucella LPS could be considered as a virulence factor that helps bacterial survival by circumventing the immune response. Brucella LPS mutants will certainly provide new targets for future vaccine design in both human and animal brucellosis.

Acknowledgements We are grateful to S Salcedo and S Garvis for critically reading the manuscript.

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