factor complex comprising RelA (p65) and NFB1 (p50) (Figure 15.3). The founding member of this family of transcription factors is v-rel (p58), a viral gene responsible for reticuloendotheliosis in chickens. Mammalian REL-containing proteins include RelB (p60), c-Rel (p66) (involved in lymphopoiesis), and NFB2 (p100/p52).

Dorsal and NF- B also share the characteristic of being sequestered in the cytoplasm by inhibitory proteins, cactus in Drosophila and I B in mammals. These inhibitors are destroyed by an ubiquitylation-based process that permits their translocation to the nucleus where they bind to the B sequence motif (see page 493).

Lipopolysaccharides: shield and signal

Bacteria with no outer membrane have cell walls composed of a thick layer of peptidoglycan, lipoteichoic acid and S-layer proteins. Bacteria with a second, outer membrane have a thin peptidoglycan layer between the two (Fig 15.4). The outermost membrane surface is almost entirely lipopolysaccharide (LPS). The thicker peptidoglycan layers may be detected by the Gram test (staining with crystal violet and iodine), initially devised to discriminate between pneumococci (gram positive) and Klebsiella pneumoniae (gram negative).26 Mycobacteria form a separate group. They have a cell wall made of glycolipids and mycolic acid and only a thin peptidoglycan layer. They are able to survive because, inhabiting the intracellular environment, they are subject to little osmotic stress.

Because the innate immune system lacks any means of instigating receptor diversity, all its targets are highly conserved structural components of micro-organisms. One such is lipopolysaccacharide.27 LPS, shed from the outer membrane of gram-negative bacteria (Table 15.3), are detected at picomolar concentrations and serve to alert the host (Figure 15.4). Of the Tolllike receptors, TLR4 has emerged as a specific conduit for the innate immune response to bacterial LPS.28,29 We focus on LPS-mediated signalling because it is one of the most potent immunostimulants.

LPS and endotoxin. At the start of the 20th century, long before its chemical composition was realized, Richard Pfeiffer identified LPS as endotoxin. It represents the major fever-evoking component of gram-negative bacteria and, when applied systemically, it causes shock.29 We now recognize the lipid A-KDO2 moiety of LPS as being responsible for most of the toxic effects of gram-negative organisms (see Figure 15.4). The immediate pyrogenic effects of endotoxin are due to signalling through TLR-4 that elevates the expression of PLA2 and cyclo-oxygenase2 (COX2) and the production of prostaglandin E2 (PGE2).31

The cross-linking of the peptidoglycan is targeted by penicillin, which causes rapid lysis of microbes.

Virulence of Yersinia pestis and acylation of LPS. On entering a host having a body temperature of 37°C, the plague bacterium

Yersinia pestis switches production of LPS to a form that lacks secondary acylations. This fails

to activate TLR4 and antagonizes the binding of highly acylated LPS. Forced expression of an E. coli gene responsible for secondary acylations renders Y. pestis completely avirulent. Resistance to disease requires TLR4 and the adaptor protein MyD88. Both innate and adaptive responses are required for immunity against the modified strain. By evading TLR4 activation in this way, the altered

LPS may contribute to the virulence of various gramnegative bacteria.30

The outer leaflet of the outer membrane of gram-negative bacteria is made up almost entirely of LPS. These have a basic structure composed of lipid A, consisting of 4–6 acyl chains covalently bound to two diaminodideoxy- d-glucose residues,

and a hydrophilic heteropolysaccharide component. The inner component of the polysaccharide, the inner core, is composed of heptose and keto- 3-deoxyoctonic acid (KDO). To this, a long chain of polysaccharides is attached, the outer core, of which, the O-antigen, is repeated many times. The long polysaccharide chain is not essential for bacterial survival, but the lipid-

A moiety is vital. The polysaccharides play a role in pathogenesis

and serve as an antigen for the generation of protective antibodies.

Table 15.3  Examples of Gram-negative bacteria

Signalling through the TLR4 receptor

The TLR4 differs from other TLRs because it requires the collaboration of other receptors, most notably CD14 and MD-2, though how these interact with LPS and how they interact with each other remains uncertain. LPS, bound

to LPS-binding protein (LBP, a trace ‘acute phase’ plasma protein involved in the early stages of septic shock)32 interacts with CD14.33 This presents LPS to MD-2, linked to the membrane via TLR4 (Figure 15.5).34 Two major signalling pathways are activated. A signalling complex is formed with a

Discovery of TLR4, the LPS receptor. The earliest indication that LPS might interact with specific receptors came from the discovery of LPS-resistant mice.35 The search for the receptor then commenced

in earnest with the realization of two substrains both having defective responses to LPS. Four years of fine mapping of the Lps locus by several research groups culminated in the discovery of LTR4

as the sole candidate.29 Although LPS interacts directly with TLR4, the interaction also requires one or more coreceptors, among which CD14, a glycosylphosphoinositolanchored membrane protein expressed on leukocytes.18,33

LPS and mucosa. The form of LPS having secondary acylations (Figure 15.5) allows bacteria to persist in the host mucosa.36–38 They are tolerated in this external environment but as

soon as they traverse the epithelial barrier, as

occurs in trauma, they are effectively eliminated. 

number of adaptors and assembly then proceeds through the association of proteins via the TIR domain. Many components of the pathway also operate downstream of the TNF, Toll, or TGF -receptors (which explains why so many of the acronyms begin with the letter T). One downstream pathway of TLR4 requires the adaptors TIRAP and MyD88 and culminates in the production of pro-inflammatory cytokines (IL-1 , IL-12, TNF- , IL-6). These signal to the transcription factor complexes NF- B, ATF2/c-Jun (AP-1), and IRF-5 (Fig15.5 and pages 464 and 525). The other pathway requires the adaptors TRAM and

TRIF and culminates in the production of IFN- , IL-10, and RANTES. It signals to the transcription factor IRF-3 and acts in cooperation with NK- B.

The TIRAP/MyD88 pathway

Oligomerization of the TLR4 receptor provokes the formation of an adaptor complex through the assembly of components TIRAP and MyD88, carrying TIR domains. How the oligomerization creates homophilic TIR-TIR binding sites remains to be elucidated. The death domain of MyD88 recruits the kinase IRAK4 which phosphorylates IRAK139,40 (see Figure 15.6). This draws TRAF6 to the receptor complex which now acts as a docking platform on to which another large signalling complex develops. The new docking sites are created, not by phosphorylation as is the case for growth factor receptors, but by polyubiquitylation of TRAF6, in the so-called K63-type reaction (i.e. they are interconnected through lysine-63 (see Figure 15.13 and page 468). TRAF6 is an E3-ubiquitin ligase that by association with other factors (Ubc13/ Mms2) constitutes the conjugation system that supplies activated ubiquitin

monomers. Importantly, TRAF6, decorated with its polyubiquitin chains, brings together two kinase complexes that act in series to ensure phosphorylation of I B (inhibitor of NF- B) and activation of the JNK and p38 pathways.

The significance of IRAK4 is clearly illustrated in patients who either fail to express it, or have a mutation in its kinase domain which renders the enzyme inactive. Because they have ineffective innate immunity, these people are particularly susceptible to infection with Streptococcus pneumoniae, suffering recurrent pyrogenic infections and bacteraemia.41

From TRAF6 to activation of NF- B

The first complex recruited by TRAF6 is composed of the serine/threonine kinase TAK1 with its associated adaptor TAB2 (or TAB3) and its regulatory subunit TAB1 (both TAK1/TAB1/TAB2 and TAK1/TAB1/TAB3 complexes have been detected).42,43 It is the TAB2 (or TAB3) that binds K63-ubiquitylated TRAF6 through a CUE domain44 (Figure 15.7 illustrates the domain architecture of these proteins). This activates TAK1 through autophosphorylation.45 The