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shown to activate IRF7 in response to LPS, linking the MyD88-independent pathway to IFN-α expression7.

Based on these data, Jiang et al. propose two modes of TLR4-MD2 signaling (Fig. 1). The first mode is induced by rough LPS but not by smooth LPS, which engages the TLR4-MD2 complex in the absence of CD14 and initi-ates solely MyD88-dependent responses. In the second mode, which is CD14 dependent, either smooth or rough LPS bound to TLR4-MD2 initiates both MyD88-dependent and MyD88-independent responses. The obser-vation that VSV can induce TLR4-MD2 in a CD14-dependent way to activate IRF-3 but not NF-κB suggests a third mode of receptor signaling. In this mode, TLR4-MD2 activates only MyD88-independent responses. At this point, the details of this mode remain specu-lative, because the VSV product that activates TLR4 is still a mystery and the requirement for TRIF or TRAM has yet to be formally tested.

Exactly how specific LPS chemotypes and CD14 dictate the intracellular responses of TLR4-MD2 signaling is unclear. TLR4-MD2 makes a distinction between rough and smooth LPS, because only the former is bound in the absence of CD14. But this does not explain why rough LPS bound by TLR4-MD2 with or without the aid of CD14 initiates differ-ent intracellular signaling pathways. Ligand-dependent TLR4-MD2 formation of oligomers is a key step in receptor signaling8, but CD14 has not been detected in those complexes. LPS-stimulated TLR4-MD2 has been reported to associate in complexes that include CD11b-CD18, CD55, CD81, hsp70, hsp90, GDF5 and CXCR4 (ref. 9). Moreover, LPS analogs that are

competitive antagonists induce receptor com-plexes of a composition different from those induced by agonists. Although the function of CD14 was not examined in these studies9, the data suggest that the ‘shape’ of the lipid A portion of LPS can influence the nature of the signaling complex. Are the details of the inter-action between rough LPS and TLR4-MD2 influenced by CD14? Crystallographic stud-ies have shown that the leucine-rich array of CD14 folds into a horseshoe-shaped structure with a deep hydrophobic pocket near its N ter-minus10. The top of the pocket is surrounded by a hydrophilic rim and many deep grooves cascade off the lip of the rim and down the outside of the pocket. CD14 probably forms a dimer, and LPS may be presented to TLR4-MD2 with the lipid portion bound within the hydrophobic cup, with the long carbo-hydrate chains that distinguish smooth from rough LPS wrapped over the rim and into the external grooves. Modeling studies predict an immunoglobulin domain fold for MD2 that, analogous to the binding by CD14, may bind LPS with a least some of the acyl side chains of lipid A buried inside a hydrophobic pocket. Two models have been proposed to explain how LPS activates the TLR4-MD2 forma-tion of oligomers11. In one model, the lipid A acyl side chains are shared by two MD2 proteins, thereby linking neighboring TLR4-MD2 complexes. A second model proposes that LPS induces a conformational change in TLR4-MD2, leading to the recruitment of a second receptor complex. Detailed structural studies of TLR4-MD2 bound to lipid A with or without CD14 may shed light on how different modes of signaling can be established.

Craig R. Roy is with the Section of Microbial

Pathogenesis, Yale University School of Medicine,

Boyer Center for Molecular Medicine, New Haven,

Connecticut 06536, USA.

e-mail: craig.roy@yale.edu

The data presented here have additional implications for the nature of host responses to LPS. Although TLR4 is widely expressed, CD14 expression is limited to myeloid cells. This suggests that rough LPS will activate a broader range of cells than will smooth LPS. Furthermore, the nature of the LPS response of the nonmyeloid cells may change depend-ing on the availability of soluble CD14 released from myeloid cells, which at high concentra-tions can substitute for membrane-bound CD14. Finally, the structure and proportions of smooth and rough LPS vary among different species of Gram-negative bacteria. Thus, cel-lular responses to these bacteria or to isolated LPS preparations may vary depending on the proportion of smooth and rough LPS chemo-types. The demonstration TLR4-MD2 can dis-criminate between LPS chemotypes and that the nature of the biological response can be dictated by CD14 provides an additional layer of complexity regarding the innate response to pathogens.

1. Jiang, Z. et al. Nat. Immunol. 6, 565–570 (2005).2. Beutler, B. & Rietschel, E. Nat. Rev. Immunol. 3,

169–176 (2003).3. Hajjar, A. et al. Nat. Immunol. 3, 354–359 (2002).4. Palsson-McDermott, E. & O’Neill, L. Immunology 113,

153–162 (2004).5. Karaghiosoff, M. et al. Nat. Immunol. 4, 471-477

(2003).6. Lund, J. et al. Proc. Natl. Acad. Sci. USA 101, 5598–

5603 (2004).7. Fitzgerald, K.A. et al. J. Exp. Med. 198, 1043–1055

(2003)8. Akashi, S. et al. J. Exp. Med. 198, 1035–1042

(2003).9. Triantafilou, et al. Biochem. J. 381, 527–536

(2004).10. Kim, J.-I. et al. J. Biol. Chem. 280, 11347–11351

(2005).11. Gangloff, M. & Gay, N. Trends Biochem. Sci. 29, 294–

300 (2004).

Many pathogenic microbes have the ability to survive in macrophages. A common

strategy used by bacterial pathogens to avoid being killed by macrophages is to short-circuit

the membrane transport process that leads to fusion of the phagosome with lysosomes1,2. In this issue of Nature Immunology, Arellano-Reynoso and colleagues have identified a mechanism by which the bacterial pathogen Brucella abortus is able to disrupt phagosome transport to avoid fusion with lysosomes3. They show that cyclic β-1,2 glucans (CβG) produced by B. abortus has a biochemical activity that can disturb the formation of lipid rafts and that CβG production is necessary

for the B. abortus-containing vacuole (BCV) to avoid fusion with lysosomes. These data provide new insight into a tactic often used for bacterial immune evasion.

A fundamental aspect of innate immunity is the extraordinary ability of macrophages to ingest microbes and rapidly transport them to lysosomes, where they are usually destroyed. ‘Phagocytosis’ is the term often used to describe the process whereby macrophages ingest invading microbes, and the resulting

Trimming the fat: a Brucella abortus survival strategyCraig R Roy

Some microbes can circumvent phagocytosis by interfering with membrane transport systems. Brucella do this by making cyclic glucan molecules that interfere with lipid raft–mediated vacuole maturation.

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membrane-bound organelle in which the organism is contained is known as a ‘phago-some’4.

Phagosome maturation is a complex pro-cess that involves membrane fusion and organelle transport5. Most phagosomes con-taining a bacterial cell fuse sequential with early, then late, endocytic compartments and lysosomes. These fusion reactions facilitate membrane exchange, which alters the identity of the plasma membrane–derived vacuole. The delivery of new membrane components to the phagosome provides additional signal-ing molecules important for the next series of fusion and transport reactions. Membrane fusion is also critical for the delivery of anti-microbial proteins into the lumen of the phagosome.

The recruitment of cytosolic proteins that regulate vesicle fusion events is determined by the protein and phospholipid composition of the vacuole membrane. Notably, interac-tions between plasma membrane components and factors on the surface of a particle being internalized by a macrophages will influence the composition of the vacuole membrane. Given that surfaces of different bacterial cells vary considerably because of the tremendous diversity in the types of lipids, proteins, oli-gosaccharides and proteinaceous ‘appendages’ on display, it is remarkable that macrophages have the ability to deliver most bacteria to lysosomes after phagocytosis.

Many intracellular pathogens have the ability to alter phagosome transport to avoid delivery to lysosomes. Mycobacterium tuber-culosis, for example, maintains residence in a vacuole that shows properties of early endo-cytic compartments but resists maturation into an acidified phagolysosomal organelle6,7. The mechanism by which M. tuberculosis is able to affect the phagosome maturation process is multifactorial; however, it is well established that glycolipids on the surface of M. tuberculosis are released8 and modulate the recruitment of host factors required for transport and fusion of early endocytic com-partments9,10. Those studies using M. tuber-culosis have demonstrated that production of bioactive lipids is one mechanism by which bacteria can alter phagosome maturation and illustrate how the lipid composition of the phagosome membrane is important for trafficking of this organelle.

In addition to the composition of the phagosome membrane, the spatial organi-zation of signaling molecules into discrete microdomains also influences the phago-some maturation process. How signaling networks might be established on the mem-brane of phagosomes has been discussed11.

In that model, interactions between proteins and lipids govern these networks and estab-lish functional domains that mediate distinct phagosomal maturation processes.

Cholesterol is important in the formation of specialized microdomains in membranes sometimes called ‘lipid rafts’12,13. These cho-lesterol-rich microdomains participate in the organization of receptors and signaling molecules at the plasma membrane and can serve as ‘concentration platforms’ for proteins involved in vesicular transport. Although there is no clearly defined function for lipid rafts in phagosome maturation, it is reasonable to surmise that these microdomains serve impor-tant functions in organizing factors that con-trol transport and fusion of these organelles (Fig. 1a).

Now support for the idea of participation of lipid rafts in phagosome maturation events comes from the work of Arellano-Reynoso and colleagues3. They have been investigat-ing the intracellular pathogen B. abortus, which has the ability to modulate phagosome transport to avoid fusion with lysosomes. B. abortus produces copious amounts of CβG, a cyclic oligosaccharide molecule that is struc-turally similar to cyclodextrins and is capable of extracting cholesterol from cellular mem-branes. When tested, CβG purified from B. abortus was able to disrupt lipid rafts in vitro; however, a purified CβG preparation had 2% the activity of the synthetic molecule methyl-β-cyclodextrin, which is often used to extract cholesterol from cellular membranes.

To test whether CβG is involved in pre-venting fusion of the BCV with lysosomes, the authors analyzed vacuoles containing a cgs mutant strain of B. abortus defective in an

enzyme required for CβG synthesis. Vacuoles containing the cgs mutant fused rapidly with lysosomes, suggesting that CβG produced by B. abortus may disrupt phagosome matura-tion. An extracellular function for CβG was demonstrated by experiments showing that vacuoles containing the cgs mutant strain of B. abortus could avoid lysosome fusion when purified CβG was added exogenously during infection of host cells. These data demonstrate that surface-exposed CβG affects transport of the BCV to prevent fusion of this compart-ment with lysosomes.

The precise mechanism by which CβG molecules modulate phagosome maturation remains to be determined. CβG added exo-genously did not prevent maturation of phagosomes containing latex bead particles3. This suggests that CβG inhibits lysosome fusion by acting in conjunction with other factors produced by B. abortus. Somewhat unexpectedly, CβG production by B. abor-tus did not destroy lipid rafts on the vacuole membrane3. The protein flotillin-1, a marker often used to identify cholesterol-rich micro-domains, was present on vacuoles containing either wild-type B. abortus or cgs mutants3.

The work of Arellano-Reynoso has identi-fied involvement of CβG in controlling host functions to prevent fusion of the BCV with lysosomes. According to these data, if CβG prevents fusion of the BCV with lysosomes by acting directly on lipid rafts, it is most likely affecting the capacity of these microdomains to function as signaling platforms (Fig. 1b). Given that very little is known concerning the function of lipid rafts in controlling phago-some maturation, B. abortus may provide a useful system for testing whether components

Figure 1 A potential function for lipid rafts as signaling platforms important for phagosome maturation. (a) Phagosomes containing avirulent bacteria or the B. abortus cgs mutant have lipid rafts on the membrane surface. These lipid rafts can serve as signaling platforms and recruit a protein complex important for fusion of the phagosome with lysosomes. (b) Lipid rafts on the membrane of phagosomes containing virulent B. abortus have been modified by CβG released into the lumen of the vacuole. These lipid rafts are nonfunctional and may not be able to recruit a signaling complex important for fusion of the phagosome with lysosomes.

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recruited to lipid rafts are involved in signal-ing events leading to the fusion of phagosomes with lysosomes and the eventual destruction of bacteria by macrophages.

It will be useful to determine whether fac-tors secreted by other intracellular pathogens mimic the activities of CβG and similarly disturb the functioning of lipid microdo-mains both on the phagosome and at the plasma membrane. Given the importance of cholesterol-rich microdomains in many different signaling processes14,15, it is likely

that interference of lipid raft function is a conserved strategy used by microbial patho-gens for avoiding a variety of host immune responses.

1. Duclos, S. & Desjardins, M. Cell Microbiol. 2, 365–377 (2000).

2. Scott, C.C., Botelho, R.J. & Grinstein, S. J. Membr. Biol. 193, 137–152 (2003).

3. Arellano-Reynoso, B. et al. Nat. Immunol. 6, 616–623 (2005).

4. Allen, L.A. & Aderem, A. Curr. Opin. Immunol. 8, 36–40 (1996).

5. Vieira, O.V., Botelho, R.J. & Grinstein, S. Biochem. J. 366, 689–704 (2002).

6. Russell, D.G. Nat. Cell. Biol. 5, 776–778 (2003).7. Vergne, I., Chua, J., Singh, S.B. & Deretic, V. Annu.

Rev. Cell. Dev. Biol. 20, 367–394 (2004).8. Beatty, W.L. et al. Traffic 1, 235–247 (2000).9. Fratti, R.A., Chua, J., Vergne, I. & Deretic, V. Proc. Natl.

Acad. Sci. USA 100, 5437–5442 (2003).10. Vergne, I., Chua, J. & Deretic, V. J. Exp. Med. 198,

653–659 (2003).11. Griffiths, G. Trends Cell Biol. 14, 343–351 (2004).12. Helms, J.B. & Zurzolo, C. Traffic 5, 247–254 (2004).13. Simons, K. & Ikonen, E. Nature 387, 569–572

(1997).14. Simons, K. & Toomre, D. Nat. Rev. Mol. Cell. Biol. 1,

31–39 (2000).15. Lafont, F., Abrami, L. & van der Goot, F.G. Curr. Opin.

Microbiol. 7, 4–10 (2004).

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