- Email: [email protected]
Biosynthesis of lipopolysaccharide O antigens
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46 Young, D., Samuels, J. and Clarke, J.K. (1973) Arch. Gesamte 47 48 49 50 51 52 53
Virusforsch. 42,228-234 Cameron, K.R., Birchall, S.M. and Moses, M.A. (1978) Lancet ii, 796 Westarp, M.E. et al. (1992) Neural. Psychiatry Brain Res. l, l-4 Gow, J.W. et al. (1992) J. Clin. Pathol. 45, 1058-1061 Fliigel, R.M. et al. (1992) Clin. Infect. Dis. 14, 623-624 Lycke, J. et al. (1994) J. Neural. 241,204-209 Svenningsson, A. et al. (1992) Ann. Neural. 32,711-714 Pyykko, I. et al. (1994) Acta Oto-laryngol. 114,224
Simonsen, L. et al. (1994) Acta Oto-laryngol. 114,223-224 Stancek, D. and Gressnerova, M. (1974) Acta Virol. 18,365 Werner, J. and Gelderblom, H.R. (1979) Lancet ii, 258-259 Debons-Guillemin, M.C. et al. (1992) AIDS Res. Hum. Retroviruses 8,1547 58 Wick, G. et al. (1993) lntewirology 35,101-107 59 Lagaye, S. et al. (1992) Proc. Nat1 Acad. Sci. USA 89, 10070-10074 60 Schweizer,M. et al. (1994) AIDS Res. Hum. Retrouiruses 10, 601-605 54 55 56 57
Biosynthesis of lipopolysaccharide 0 antigens Chris Whitfield
T
and covalently linked (ligated) Lipopolysaccharide 0 antigens are amphipathic glyhe to a preformed lipid-A-core acimportant virulence determinants for many coconjugates, lipopolyceptor at the periplasmic face bacteria. O-antigen synthesis is an (LPS), are saccharides of the plasma membrane1q2. interesting problem in cell-surface essential and characteristic Ligation therefore effectively assembly. There are two known assembly components of the outer memterminates O-antigen synthesis. pathways, which differ in the cellular branes of Gram-negative bacDetails of the ligation reaction location of their polymerization steps and teria. The hydrophobic domain have not been resolved, and in the direction of chain polymerization. of LPS, lipid A, forms the outer the only ligase component that Some reactions are shared with those for leaflet of the outer membrane. has been characterized is enother surface polymers, such as capsular In enteric bacteria, the core coded by rfuL. RfaL homologs polysaccharides, and may be potential oligosaccharide links lipid A from Salmonella enterica serotargets for therapeutic intervention. to the 0-antigenic side-chain var Typhimurium and Escherpolysaccharide. The structural C. Whitfield is in the Dept of Microbiology, ichiu coli K-12 have little diversity of the 0 antigens University of Guelph, Guelph, Ontario, primary sequence similarity, but stems from variations in sugar Canada Nl G 2 WI. tel: +I 519 824 4120 x3478, have similar hydropathy procomposition, the sequences of fax: +l 519 837 1802, files3. This, together with the sugars and linkages, and the e-mail: [email protected] observation that heterologous substitution of monomers with 0 antigens are expressed (aleither sugars or non-sugar resibeit with variable efficiency) in these bacteria, suggests dues (Fig. 1). These differences give rise to 0-serotype that the mechanism and cellular location of ligation specificity within bacterial species. Much of the traare conserved. After ligation, the completed LPS molditional interest in LPS molecules originates in their ecule is translocated to the cell surface by unknown complex interaction with host defenses and their conmechanisms4. tribution to virulence in pathogenic bacteria. 0 antiThe assembly of 0 antigens is a complex problem in gens form hydrophilic surface layers that often promembrane biogenesis. The principal issues are: (1) the tect the cell from complement-mediated serum killing, maintenance of the structural fidelity of O-repeating and they are therefore essential virulence determinants. units (0 units) during synthesis; (2) the establishment of strain-dependent preferred O-antigen chain lengths; Overview of LPS assembly and (3) the organization in the plasma membrane of a Sugar nucleotides are the activated precursors for system that transfers sugars from cytoplasmic precurcell-surface polysaccharides. To assemble 0 antigens, sors to a membrane-bound lipid (und?), but makes monomers are not transferred directly to a growing hydrophilic polysaccharides available for ligation at LPS molecule. Instead, 0 antigens are synthesized septhe periplasmic face of the plasma membrane. arately on a lipid carrier by enzymes encoded by the rfb This review summarizes current understanding of gene cluster. As in the synthesis of cell-wall peptidothe mechanisms involved in the synthesis and export glycan and capsular polysaccharides, the lipid carrier is across the plasma membrane of LPS 0 polysaccharthe C,,-polyisoprenoid derivative, undecaprenol phosides. The two model pathways described here have phate (und-P). Once complete, 0 antigen is transferred 0 TRENDS
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common precursors and intermediates, and are initiated by similar reactions. However, the cellular location of the polymerization reactions and the direction of polymer growth differ in each pathway. Prototypical systems are drawn from the Enterobacteriaceae, but I attempt to highlight those aspects of synthesis mechanisms for surface polysaccharides that might be conserved in diverse bacteria. Broader coverage of this area is given in several recent reviews3-b.
(a) Salmonella enterica serogroups A, BkD, E
1
3 +2)-a/P-D-Manp(l+4)-a-L-Rhap(
1-t3)-a-D-Galp(l+
Escherichia co/i K-l 2 a-D-Glcp
t2)-o-D-Galf-(l-+6)-a-D-Glcp(1
1 O-YY 2 6 -+3)-a-L-Rhap(l-+3)-a-D-GlcpNAc-(14
Shigella dysenteriae type 1 -+3)-a-L-Rhap(l+3)-a-L-Rhap( 1+2)-a-D-Galp(l-+3)-a-D-GlcpNAc-(l-+ Initiation reactions Reactions involved in the biosynthesis Shigella flexneri of 0 antigens were first described in -+2)-a-L-Rhap( 1-+2)-a-L-Rhap(l-+3)-a-L-Rhap(l+3)-~-D-GlcpNAc-(l+ S. enterica serogroups B and E. The heteropolysaccharide 0 antigens of these bacteria (Fig. 1) are synthesized W by essentially identical mechanisms. Escherichia co/i 08 and Klebsiella pneumoniae 05 The galactosyltransferase RfbP,, (Ref. 7) (see Box 1) catalyzes the -3).B-D-Manp(l+2)-a-D-Manp(l-+2)-a-D-Manp(l+ reversible transfer of galactose lphosphate (Galp-1-P) from the preEscherichia co/i 09 and Klebsiella pneumoniae 03 cursor uridine diphosphogalactose -t3)-a-D-Manp( l-+3)-a-D-Manp( 1+2)-a-D-Manp( l-2)-a-D-Manp( 1-Q.a-D-Manp( 1.+ (UDP-Galp) to und-P (Ref. 8) as the initial synthetic reaction for each of Klebsielia pneumoniae 01 these 0 antigens. Although 0 antigens do not all con-13)~p-D-Galf-(l-3)-a-D-Galp(l + D-Galactan I tain galactose, the only other known +3)-a-D-Galp(l+3)-p-D-Galp(l+ D-Galactan II initiating enzyme is Rfe, an N-acetylglucosamine-1-phosphatetransferase Fig. 1. Structures of representative 0 antigens polymerized by growth at (a) the reducing terminus and (b) the non-reducingterminus. In Salmonella enterica serogroups A. I3 and D, the (GlcpNAc-l-phosphatetransferase)9. main-chain mannose (a-o-Manp) residues are substituted by a 3,6dideoxyhexose residue (R). Rfe transfers GlcpNAc residues found R is a-paratose, a-abequose and a-tyvelose in serogroups A, B and D, respectively. Serogroup in the heteropolysaccharide 0 units E has no 3,6dideoxyhexose residue and has P-WManp instead of a+Manp. Additional 0 factors of several E. coli serotypes’O, Shigella in S. enterica and Shigella species arise by variable (nonstoichiometric) substitution of the base structures with a-glucopyranose and 0-acetyl residues. In Klebsiella pneumoniae 01, dysenteriae type 1 (Ref. 11) and there are two polysaccharide structures in the 0 antigen. o-Galactan I is linked directly to the Shigella flexneri 12.The cryptic 0 antilipid-A core and is synthesized by the products of genes at the rfb (O-antigen biosynthesis) gen of E. coli K-12, recently reconlocus. BGalactan II domains are found in high-molecular-mass 0 antigens and are covalently structed in Reeves’ laboratory’ j, has attached to the distal end of pgalactan I chains. An additional but unknown gene locus participates a similar requirement for Rfe (Ref. with Rfb proteins in formation of c-galactan II. The mannose-containing homopolymers of Escherichia coli 08 and 09, and K. pneumoniae 05 and 03, provide examples of situations 14). Interestingly, Rfe also initiates where different bacteria can produce the same or very similar O-antigen structures. The sugars formation of homopolymeric 0 antiare mannose (oManp). rhamnose (L-Rhap). galactose in the pyranose (Galp) and furanose gens in E. coli 08 and 09 (Refs (Galf) forms, and N-acetylglucosamine (GlcpNAc). 15,16) and in Klebsiella pneumoniae 01 (Ref. 17), even though the 0 units of these homopolysaccharides do not contain phosphate (ECA,,) or, alternatively, to lipid-A core GlcpNAc residues (Fig. l), and their polymerization (ECA,.,,) in a form resembling 0 antigens”O. involves a totally different process from that of heteroInitiating enzymes must interact with the und-P polysaccharides. Pathways for synthesis of O-antigen acceptor and are therefore probably active in a hydrohomopolymers and heteropolymers were formerly phobic environment. Rfe (Refs 9,21) and RfbP,, called ufe dependent and rfe independent, respectively. (Ref. 7) are both predicted to be integral membrane It is now clear that the requirement for Rfe cannot be proteins and, although they differ in size, they have used to distinguish the mechanisms, and this terminsimilar hydropathy profilesl’. It has been suggested ology should be discontinued. that Rfe and RfbP,, are structural homologs, as well While RfbP,, function is confined to O-antigen synas functional homologs in O-antigen initiation”. thesis, Rfe is required for the synthesis of additional surface polysaccharides, which can be coexpressed Polymerization by growth at the reducing terminus with 0 antigens. These include the Tl antigen of SalRol(Cld)- and Rfc-dependent polymerization reactions monella species’* and the enterobacterial common 0 antigens from S. enterica serogroups B and E are antigen (ECA)19. ECA can be linked to diacylglycerol elongated by the addition of 0 units to the reducing
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(or 0 units). Several Rol homologs have been identified and the predicted proteins are quite similar. Escherichiu co/i K-12 strains contain a Rol that can function with heterologous cloned Rfb products (Fig. 4); however, the homologous Rol and Rfb components are required for optimal (strain-dependent) Rol activity3. In contrast, individual Rfc enzymes only polymerize polysaccharides with closely related structures (reviewed in Refs 3,4).
Box 1. Terminology of the rfb genes The 1771 gene locus encodes the enzymes required for the formation of 0 antigens. These include enzymes that catalyze the synthesis of the sugar-nucleotide precursors that are unique to 0 antigens, the glycosyltransferases and polymerases needed for the assembly of the 0 polysaccharides, and the components required for the transfer of O-antigen polymers, or 0 units, across the plasma membrane. The terminology for rfb genes is confusing because different functions are often encoded by rfb genes from different species, or even from different 0 serotypes of the same species. Thus, a gene designated rf?~Acan have different functions depending on the bacterial species and serotype from which it originates. In an attempt to overcome ambiguity in rfb nomenclature, I indicate in a subscript the bacterial species and, where necessary, the 0 serotype. For example, r&P,, is the gene encoding RfbP in Salmonella enterica, r&A,,, is the gene encoding RfbA from Klebsiella pneumoniae serotype 01, and ffbEcK_12 refers to the rfb gene cluster from Escherichia co/iK-12.
erminus of the growing polymer. After formation of und-P-P-Galp, other Rfbs, enzymes catalyze the transfer of successive sugars to form the lipid-linked 0 unit (Fig. 2a). Each step was first identified in vitro, and many activities have now been assigned to specific 0 antigens are then Rfbs, products 6,22. Lipid-linked polymerized in a blockwise fashion (Fig. 2a) in a reaction requiring rfcSe (Ref. 23). A defect in rfcse results in semi-rough LPS (SR-LPS) (Fig. 3). The rfc genes have now been described in several S. enterica serogroups23-zs, Shigellu species1’r26 and E. cofi K-12 (Refs 12,14). Rfc homologs are predicted to be hydrophobic integral membrane proteins with 11-13 transmembrane segments; Rfc primary sequences are not conservedz6. Sequential assembly of repeating units and a blockwise polymerization process provides a mechanism by which the fidelity of the repeating unit structure can be maintained in complex heteropolysaccharides, and is probably not confined to O-antigen biosynthesis. Morona and colleagues have identified an Rfc analog in the rff gene cluster for ECA biosynthesi@, and biochemical data support the operation of the same general pathway in the polymerization of the capsular polysaccharide of Enterobacter (Aerobacter) aerogenes DD45 (Ref. 27). An interesting feature of Rfc-mediated polymerization is the control of the distribution of O-antigen chain lengths (modal distribution). The process involves the protein known as either Rol (regulator of O-chain length28) or Cld (O-chain length determinator29) and its effect is shown in Fig. 4. Bastin et A1.29 have proposed a model in which Rol(Cld) modulates the activity of Rfc to alternate between polymerization and the transfer of lipid-linked 0 antigens to RfaL for ligation. Recently, Morona et A/.” have elaborated on this model, suggesting that Rol may act as a molecular chaperone to regulate the interaction of either RfaL or Rfc with und-P-P-linked 0 antigen
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Site of Rfc-Rol-dependent O-antigen assembly O-antigen biosynthesis in S. enterica serogroups B and E involves a transmembrane-assembly pathway (Fig. 5) in which RfbP,,, Rfc,, RfbX,,, Rol,, and RfaL,, are all integral membrane proteins. Transferase enzymes acting after RfbPs, in O-unit formation are peripheral proteins. It seems reasonable to assume that the enzymes are arranged in a complex, given their sequential action, high specificity and high specific activity. As biosynthetic activity is located in the membrane fraction (where und-P is available) and the precursors are cytoplasmic, the complex must be located at the cytoplasmic face of the plasma membrane. However, evidence available indicates that polymerization reactions involving Rfcs, occur at the periplasmic face of the plasma membrane’, so the assembly system must transport lipid-linked 0 units across the membrane. A group of rfbP,, mutants (formerly designated rfb7’,,) accumulate putative lipid-linked 0 units, but cannot transfer the intermediates to the lipid-A core. These mutants were originally thought to have a defect in ligation, but are now thought to be unable to ‘flip’ und-P-P-O units across the plasma membrane to the active site of Rfcs,. This has led to the suggestion that RfbPs, has an additional role in this transport process31. While these results can be explained by a dual function for RfbPs, as both a galactosyltransferase and a ‘flippase’, the phenotype could alternatively reflect altered protein-protein interactions between RfbP,, and an adjacent flippase component of the complex. The most likely alternative candidate for the flippase is RfbX. Homologs of rfbX have been identified in rfb clusters from several S. enterica serovars6, S. dysenteriae type 1 (Ref. 11) and E. coli K-12 (Refs 12,14). The function of RfbX is at present unknown. RfbX has been suggested to be involved in ligation3. While no data directly preclude this, RfaL efficiently ligates 0 antigens and 0 units synthesized by this pathway, and 0 antigens can be formed by a second pathway lacking RfbX (see below), both of which suggest that an essential role for RfbX in ligation is unlikely. As several r/%X homologs have been cloned, its role can now be addressed directly. Polymerization by growth at the non-reducing terminus Rol- and Rfc-independent polymerization reactions A second, substantially different pathway exists for the synthesis of some 0 antigens. Details of the pathway are only now being elucidated, and it is currently only known to be involved in the synthesis of homopolymer 0 antigens of E. coli 08 and 09 and K. pneumoniue
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(a) Reaction
1
und-@
+ NDPa
=
undm
Reaction
2
undm’+
Reaction
3
undm
+ NDPa
Reaction
4
undm
t
undm
Reaction
5
undm
t
undm
Reaction
1
und@
Reaction
2
undm
NDP-@
+
+ NMP
undm
+
+ NDP
undm
+ NDP
--+
t
und-
undm
+ und@@
@I
Reaction3
undm
Reaction4
undm
Reaction 5
und m
+ NDPa
;t
undm
t NDPa
-
t NDP-@
t
undm
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t
t NMP
t NDP
undm
-+
NDPan
t NDP
unde
t
t NDP
NDP*
n
-----$ und
t
nNDP
Fig. 2. Polymerization of 0 antigens. O-antigen synthesis involves two distinct polymerization models distinguished by the direction of chain elongation. Both models have an analogous reversible initiating step (reaction l), in which a sugar l-phosphate residue is transferred from the cognate nucleotide diphosphate (NDP) derivative to undecaprenol phosphate (und-P). The pathways then diverge. (a) Growth at the reducing terminus. The figure shows the formation of a hypothetical 0 antigen with a trisaccharide repeating unit. After the initiating reaction, subsequent steps (reactions 2 and 3) involve sequential transfer of each sugar to form an und-P-P-linked 0 unit. Preformed und-P-P-linked 0 units are the substrates for polymerization in the absence of de novo synthesis of repeating units (reactions 4 and 5). Polymerization involves the addition of nascent 0 polymer from one und-P-P carrier to the nonreducing terminus of a newly synthesized single 0 unit attached to a second und-P-P carrier 52.The solid-shaded sugars of the 0 units indicate those 0 units added most recently. Growth of the polymer therefore occurs at the reducing terminus, or the end closest to the und-P-P carrier. In Salmonella enterica, addition of glucosyl and 0-acetyl substituents (Fig. 1) occurs after polymerization, which accounts for the nonstoichiometric nature of such modifications 3.4.18.(b) Growth at the non-reducing terminus. The figure shows the synthesis of a hypothetical 0 antigen with a disaccharide repeating unit. The pathway is initiated by the transfer of sugar l-phosphate to und-P. Polymerization involves the sequential processive transfer of sugars to the non-reducing terminus of the nascent polymer (reactions 2 and 3). There is no blockwise polymerization reaction, and the polymer is elongated by processive sugartransferase reactions (reactions 4 and 5). Sugars are transferred directly from NDP-sugar precursors during polymerization. Growth of the polymer occurs at the end of the polymer furthest from the und-P-P carrier.
01 (Fig. 1). Initiation of this pathway involves the transfer of a ‘non-O-antigen residue’ to und-P, and this intermediate is the acceptor for monomers of the 0 antigen. So far, Rfe is the only initiating enzyme identified in this pathway, but there is some confusion about the precise identity of the residue that is transferred to und-P. In E. coli 09, the acceptor has been characterized in in vitro analyses as a glucose-containing lipid intermediate, und-P-P-Glcp (Ref. 32). However, recent in vivo analyses of E. coli 08-K-12 hybrids16 show that the acceptor is und-P-P-GlcpNAc, which is consistent with the activity of Rfe in the synthesis of other 0 antigens and of ECA. In’this path-
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way, the role of Rfe is confined to initiating O-antigen synthesis; GlcpNAc is not found in the 0 units. During the formation of the E. cok’ 09 antigen, mannosyl residues are rapidly added to the nonreducing terminus of the acceptor one residue at a time, in a processive mechanism (Fig. 2b)32. Intermediates containing individual 0 units have not been identified. In K. pneumoniue 01 (Ref. 33), four Rfb proteins and Rfe are required to form D-galactan I 0 antigen (Fig. 1). There are no equivalents of either Rfc or RfbX (Ref. 33) and SR-LPS mutants have not been isolated in K. pneumoniae 01, as predicted by a processive-assembly mechanism that does not involve
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Fig. 3. Activity of the Rfc O-antigen polymerase in assembly of lipopolysaccharide (LPS) in Salmonella enterica serovar Typhimurium LT2. In SDSPAGE, smooth O-substituted LPS (S-LPS) is characterized by a ‘ladder’ of heterogeneous molecules with increasing molecular mass (lane 1). Each ascending ‘rung’ of the ladder is a population of LPS molecules with an increase of one repeating unit in the 0 antigen. Each bacterial strain has a preferred distribution of O-antigen chain lengths, resulting in a characteristic ladder pattern of 1‘“11.11__11.1SLPS molecules. Fast-migrating molecules contain only lipid-A core, as in lane 3, which contains the rough (O-deficient) LPS (R-LPS) from an HaLdeficient (O-antigen ligase-deficient) mutant. An tic mutant is unable to polymerize 0 antigen and ligates a single 0 unit to lipid-A core to form semi-rough LPS (SR-LPS) (lane 2). 1
2
3
blockwise polymerization of 0 units. Completed 0 antigen is transferred to the lipid-A core together with the initiating reducing terminal sugar34. So far, no heteropolysaccharide 0 antigens are known to be synthesized by this pathway. However, growth at the nonreducing terminus is certainly not unique to homopolysaccharides; biosynthesis of heparin in mammalian 1234
5
Fig. 4. Effect of Rol(Cld) on O-antigen chain length. The SDS-PAGE lipopolysaccharide profiles (LPS) show Escherichia co/i K-12 strains expressingthe Roldependent 0 antigen from Shigella dysenteriae type 1 (lanes 2-3) and the Rol-indepenKlebsiella dent pneumoniae 01 polymer (lanes 45). Synthesis is directed by cloned rfb gene clusters. fscherichiaco/iK-12 contains functional Rol(Cld), but mutations in the cryptic rf&cK_12gene cluster result in O-deficient rough LPS (R-LPS) (lane 1). In a Rol+ K-12 strain, S. dysenteriae type 1 O-substituted smooth LPS (S-LPS) molecules show a modal distribution: most O-antigen chains fall within a limited highmolecular-mass range and fewer low-molecular-mass 0 chains are ligated to the lipid-A core (lane 3). In a K-12 strain deleted in n7~and ro/(lane 2), type 1 SLPS has an unregulated appearance, with more low-molecular-mass O-antigen chains and no modal distribution. In contrast, the K. pneumoniae 01 (r>galactan I) has an identical profile in the absence (lane 4) or presence (lane 5) of host Rol. The E. co/i K-12 host strain deleted in ml and r&therefore provides a rapid and valuable first indication of the mechanism of synthesis. The cloned rfb genes for S. dysenteriae type 1 (Refs 11,53) were kindly provided by C. Schnaitman and J. Klena.
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cells3j, hyaluronic- acid capsular polysaccharides in streptococci36 and some group II capsular polysaccharides of E. coli3’J8 all involve rapid and processive transfer of monomers to a growing non-reducing terminus. Maintenance of the fidelity of the structure is more complicated in processive polymerization. Clues as to how this might be achieved have been found in work on the biosynthesis of heparan sulfate, bacterial hyaluronic-acid capsular polysaccharides36 and some group II capsular polysaccharides of E. coli37,38. In these examples, a single enzyme with dual specificity forms the disaccharide repeating units, ensuring the production of a strictly repeating unit structure. The synthetase that forms authentic high-molecular-mass hyaluronic acid is a single polypeptide36. For heparansulfate biosynthesis, a distinct GlcNActransferase initiates assembly39 and an additional single bifunctional polypeptide with both GlcNAc- and glucuronic-acidtransferase activities then catalyzes the polymerizenzyme is also ation step 35*40 . A distinct initiating required for the biosynthesis of the polysialic-acidcontaining group II capsular polysaccharides of E. coli Kl and K92, where the polysialyltransferase enzyme is sufficient for polymerization, but cannot initiate polymer formation by itself3*. It is conceivable that a repeating unit structure more complex than a disaccharide might require additional enzymes to catalyze polymerization. Klebsiella pneumoniae D-galactan I chain-length distribution is identical when the cloned rfbKpo,genes (see Box 1) are expressed in E. coli K-12 hosts with or without rol (Fig. 4). Furthermore, the distribution of chain lengths in authentic E. coli 09 (Ref. 41) and K. pneumoniae D-galactan I (Ref. 17) 0 antigens in wild-type strains cannot be distinguished from that of cloned 0 antigen from E. coli K-12. Together, these observations indicate that a different process determines the strain-dependent distribution of O-antigen chain lengths in this pathway, and are consistent with one or more Rfb proteins being involved. This would explain the aberrant O-antigen chain lengths resulting from some deletions in the rfb region of E. coli 09 (Ref. 42). In E. coli 09 and Klebsiella 05 (Fig. l), O-antigen chains are terminated by 3-O-methyl-Dmannose43, and the addition of this residue may act as a termination reaction and may be involved in chainlength determination. Interestingly, small amounts of 2-O-methyl groups are detected in the perosaminecontaining homopolymeric 01 antigen of Vibrio cholerae44. Loss of the 2-O-methyl groups does not affect the ability to form 0 antigen, but results in seroconversion from Ogawa to Inaba type. Seroconversion is associated with defects in one rfb gene4’. Whether methylation occurs in all homopolymer 0 antigens remains to be established. Site of Rfc-Rol-independent O-antigen assembly How can growth at the non-reducing terminus be accommodated if the immediate donors of monomers (sugar nucleotides) are cytoplasmic, yet ligation of polymer occurs at the periplasmic face of the plasma membrane? Polymerization of the K. pneumoniae 01
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Periplasm
0))
NDP Fig. 5. Model of the plasma-membrane topology of a biosynthesis complex that synthesizes an 0 antigen that is elongated at the reducing terminus. (a) The initiation reaction involves an integral membrane protein (either RfbP, or Rfe), and the formation of an undecaprenol-pyrophosphatelinked sugar (und-P-P-sugar). The precursor is a nucleotidediphosphate-sugar (NDP-sugar) and a nucleotide monophosphate residue (NMP) is released. Additional transferases (shown here as a single component for clarity) are peripheral membrane proteins; they act sequentially to form the lipid-linked repeating unit. (b) Lipid-linked repeating units are translocated (‘flipped’) to the periplasmic face of the plasma membrane by a process that may involve RfbX. (c) Polymerization reactions occur at the periplasmic face of the plasma membrane, through a process involving Rfc and Rol (see Figs 3 and 4), and culminating in an und-P-P-linked polymer. (d) RfaL ligates the nascent polymer to a lipid-Acore molecule at the periplasmic face of the membrane.
a ntigen occurs not at the periplasmic face of the membrane, but rather in the cytoplasm (Fig. 6). Although the proteins RfbCDEF,,o, are sufficient for the polymerization of D-galactan I, nascent 0 antigen accumulates in the cytoplasm33. The remaining components of the system, RfbABKpol, form a transporter that is required to transfer the 01 polymer to the ligation site. The transporter is a member of the ‘traffic ATPase’ (Ref. 46) or ATP-binding cassette ‘ABC’ transporter family47, and specifically is of the ABC-2 subfamily48. A similar system has been identified to be needed for the formation of the homopolymeric 0 antigen of Yersinia enterocolitica 0:3 (Ref. 49), and functionally analogous transporters are required for export across the plasma membrane of polymerized group-II-type
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capsular polysaccharides in E. coli, Haemophilus influenzae and Neisseria meningitidis (for further references to these systems, see Refs 430). ABC-2 transporters contain an integral membrane protein with multiple membrane-spanning domains, and a hydrophilic protein containing the consensus ATP-binding motif (Walker box). The organization of similar systems that are involved in nutrient uptake has been studied extensively, but less is known about the polysaccharide exporters. The functional transporter is thought to consist of two subunits of each protein component. In the model illustrated in Fig. 6, export of 0 antigen is shown occurring through the membrane component of the ABC-2 transporter. While this is an attractive
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Fig. 6. Model of the plasma-membrane topology of a biosynthesis complex that synthesizes an 0 antigen elongated at the non-reducing terminus. (a) As in Fig. 5, an integral membrane protein initiates the polymer. So far, only Rfe has been implicated in this process. This enzyme transfers an N-acetylglucosamine l-phosphate (GlcNAc-1-P) from uridine diphospho-N-acetylglucosamine (UDP-GlcNAc) to undecaprenol phosphate (und-P), with the release of a uridine monophosphate (UMP) residue. (b) Polymerization of the polymer involves one or more peripheral proteins and the undecaprenol-pyrophosphate-linked (und-P-P-linked) polymer is completed in the cytoplasm. (c) Translocation of the nascent polymer across the plasma membrane requires the presence of a dedicated ATP-binding cassette (ABC-2) transporter. While export of polymer through the transporter (as shown here) is an attractive hypothesis, such direct involvement has not been demonstrated experimentally. It is also unknown whether transport requires the polymer to be attached to und-P, occurs on an alternative carrier molecule or involves the free polymer. (d) Ligation of the nascent 0 antigen to lipid-A core requires RfaL and the reaction is identical to the equivalent step in Pig. 5.
hypothesis, it must be emphasized that the transporter is only known to be required for export, and its precise role may be less direct. Binding of ATP by the peripheral component is necessary for export of the capsuleSi, although this has not been shown directly for O-antigen export. The shared direction of chain growth and location of polymerization, and the requirement for an ABC-2 transporter in the synthesis of some 0 antigens and group II capsular polysaccharides, suggest that this is a general mechanism for the assembly of cell-surface polymers. The involvement of ABC-2 transporters explains the absence of Rfc, RfbX and Rol activity in this pathway for O-antigen synthesis. Nevertheless, many other important aspects of this pathway are currently unresolved at present. It is not known whether this path-
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way is confined to homopolysaccharide 0 antigens, although similarities with group II capsular heteropolysaccharides suggest that this is not necessarily the case. It is also not known whether Rfe is the only initiating enzyme for this pathway. At the most basic level, the precise role of the ABC-2 transporter is itself unknown. Is the 0 antigen transported while attached to und-P, is an additional unidentified lipid carrier involved, or does export occur without a lipid component at all? At least some of these questions can now be analyzed. Conclusions
The key criteria that distinguish the two known pathways for O-antigen synthesis are: (1) the cellular location of the polymerization reactions; (2) the direction
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of chain growth during polymerization; and (3) the requirement for Rfc, Rol and RfbX proteins in one pathway and for an ABC-2 transporter in the other. The Rfc-Rol-dependent pathway provides a mechanism that allows stringent control of the heteropolysaccharide structure, particularly when an essential side-chain residue is added during synthesis, as occurs in S. enterica serogroup B. The ABC-2-transporter-dependent pathway is well suited to the synthesis of linear polymers and, considering 0 antigens, is currently (but not necessarily) limited to the synthesis of homopolymers. There are relationships between the pathways for the synthesis of 0 antigens and those for other cellsurface polysaccharides. Key differences are probably confined to the later stages of assembly, such as the nature of the linkage moiety or the steps involved in translocation from the periplasmic face of the plasma membrane to the cell surface4. Because surface polymers have essential roles in the survival of bacteria, the pathways involved in their formation are critical to virulence and potentially provide novel targets for therapeutic intervention. Currently available moleculargenetic and biochemical approaches should allow the elucidation of these important and complex systems. Acknowledgements Many of the ideas presented here were developedthrough stimulating discussionswith members(past and present) of the author’slaboratory and with several other colleagues; their contributions are gratefully acknowledged.Researchin the author’slaboratory has been generously supported by funding from the Natural Sciences and Engineering Research Council of Canada, the Medical Research Council of Canada and the Canadian Bacterial Diseases Network (Canadian Networks of Centers of Excellence program).
References 1 McGrath, B.C. and Osborn, M.J. (1991)J.Bacterial. 173, 649454 2 Mulford, CA. and Osborn, M.J. (1983) Proc. Nut[ Acad. Sci. USA 80,1159-1163 3 Schnaitman, C.A. and Kiena, J.D. (1993)Microbioi. Rev. 57, 655-682 4 Whitfield, C. and Valvano, M.A. (1993) Adv. Microb. Physiol. 35,135-246 5 Raetz, C.R.H. (1990) Annu. Rev. Biochem. 59,129-170 6 Reeves, P.R. (1994) in Bacterial Cell Wall (Ghuysen, J-M. and Hakenbeck, R., eds), pp. 281-314, Elsevier Science Publishers 7 Jiang, X-M. et al. (1991) Mol. Microbial. 5,695-713 8 Osborn, M.J. et al. (1972) Methods Enzymol. 28,583-601 9 Meier-Dieter, U. et al. f 19921 1. Biol. Chem. 267.746-753 10 Alexander, d.C. and VaIvanoj M.A. (1994)J. Bicterrol. 176, 7079-7084 11 Klena, J.D. and Schnaitman, CA. (1993) Mol. Microbial. 9, 393-402 12 Yao, Z. and Valvano, M.A. (1994) 1. Bucteriol. 176,4133-4143 13 Liu, D. and Reeves, P.R. (1994) Microbiology 140,49-57 14 Stevenson, G. et al. (1994) 1. Bucteriol. 176,4144-4156 15 lann, K. et al. 119821Eur. 1. Biochem. 127.157-164 16 Rick; P.D., Hubbard, G.L.‘and Barr, K. (1994) /. &cteriol. 176, 2877-2884 17Clarke, B.R. and Whitfield, C. (1992) 1. Bucteriol. 174, 4614-4621 18 Mikeli, P.H. and Stocker, B.A.D. (1984) in Handbook of Endotoxin (Rietschel, E.T., ed.), pp. 59-137, Elsevier Science Publishers
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19Meier-Dieter, U. et al. (1990) 1. Biol. Chem. 265, 13490-13497 Kuhn, H-M. et al. (1988)FEMS Microbial. Rev. 54, 195-222
20 21 22 23 24
Ohta, M. et al. (1991) Mol. Microbial. 5, 1853-1862 Liu, D. etal. (1993)l. Bucteriol. 175,3408-3413 Collins, L.V. and Hackett, J. (1991) I. Bucteriol. 173,2521-2529 Brown, P.K., Romana, L.K. and Reeves, P.R. (1992) Mol. Microbrol. 6,1385-1394 25 Wang, L. et al. (1992) Genetics 130,429-443 26 Morona. R. rt ul. (1994) 1. Bucteriol. 176, 733-747 27 Troy, F.A., Frerman, F.E. and Reeves, P.R. (1971) 1. Biol. Chew. 246,118-133 28 Batchelor, R.A. et al. (1991) J. Bucteriol. 173,5699-5704 29 Bastin, D.A. et al. (1993) Mol. Microbial. 7, 725-734 30 Morona, R., Van Den Bosch, L. and Manning, P.A. (1995) 1. Bucteriol. 177, 1059-1068 31 Wang, L. and Reeves, P.R. (1994)]. Bucteriol. 176,4348-4356 32 Weisgerber, C. and Jann, K. (1982) Eur. 1. Biochem. 127, 165-168 33 Bronner, D., Clarke, B.R. and Whitfield, C. I 1994) Mol. Microbial. 14,505-519 34 Weisgerber, C., Jann, B. and Jann, K. (1984) Eur. J. Biochem. 140,553-556 35 Lind, T., Lindahl, U. and Lidholt, K. (1993) J. Biol. Chem. 268, 20705-20708 36 DeAngelis, P.L. and Weigel, P.H. (1994) Biochemrstry 33, 9033-9039 37 Lidholt, K. et al. (1994) Curbohydr. Res. 255, 87-101 38 Steenbergen, SM., Wrona, T.J. and Vimr, E.R. (1992) J. Bucteriol. 174, 1099-1108 39 Fritz, T.A. et al. (1994) 1. Biol. Chem. 269,28808-28814 40 Lidholt, K. et ul. (1992) Proc. Nut1Acud. Sci. USA 89, 2267-2271 41 Jayaratne, P. et al. (1994)]. Bucteriol. 176,3126-3139 42 Kido, N. et al. (1989) 1. Bucteriol. 171, 3629-3633 43 Jansson, P-E., LGnngren, J. and Widmalm, G. (1985) Carbohydr. Res. 145,59-66 44 Ito, T. et al. (1994) Curbohydr. Res. 256, 113-128 45 Stroeher, U.H. et al. (1992) hoc. Nat1Acad. Sci. USA 89, 2566-2570 46 Doige, C.A. and Ames, G.F-L. (1993) Annu. Rev. Microbial. 47, 291-319 47 Higgins, CF. (1992) Annu. Rev. Cell Biol. 8,67-l 13 48 Reizer, J., Reizer, A. and Saier, M.H.J. (1992) Protein Sci. 1, 1326-1332 49 Zhang, L. et al. (1993) Mol. Microbial. 9, 309-321 50 Fath, M.J. and Kolter, R. (1993) Microbial. Rev. 57, 995-10 17 51 Pavelka, M.S.J., Hayes, S.F. and Silver, R.P. (1994) J. Biol. Chem. 269,20149-20158 52 Robbins, P.W. et al. (1967) Science 158, 1536-1542 53 Sturm, S. and Timmis, K.N. (1986) Microb. Puthog. 1,289-297
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Cmningsoon
in7ktzd~ in Microbiology
*Herpes simplex virus vaccines as lmmunotherapeutic agents, by L.R. Stanberry lUnconstrained bacterial promiscuity: the Tn916--Tnl545 family of conjugative transposons, by D.B. Ckwell, SE. Ftannwn and D.D. Jaworski 44oving thmugh the membrane with filamentous phage, by M. Russel *The epidemiology of HIV infection and AIDS in Africa, by P. Van de Perra ----I_
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46 Young, D., Samuels, J. and Clarke, J.K. (1973) Arch. Gesamte 47 48 49 50 51 52 53
Virusforsch. 42,228-234 Cameron, K.R., Birchall, S.M. and Moses, M.A. (1978) Lancet ii, 796 Westarp, M.E. et al. (1992) Neural. Psychiatry Brain Res. l, l-4 Gow, J.W. et al. (1992) J. Clin. Pathol. 45, 1058-1061 Fliigel, R.M. et al. (1992) Clin. Infect. Dis. 14, 623-624 Lycke, J. et al. (1994) J. Neural. 241,204-209 Svenningsson, A. et al. (1992) Ann. Neural. 32,711-714 Pyykko, I. et al. (1994) Acta Oto-laryngol. 114,224
Simonsen, L. et al. (1994) Acta Oto-laryngol. 114,223-224 Stancek, D. and Gressnerova, M. (1974) Acta Virol. 18,365 Werner, J. and Gelderblom, H.R. (1979) Lancet ii, 258-259 Debons-Guillemin, M.C. et al. (1992) AIDS Res. Hum. Retroviruses 8,1547 58 Wick, G. et al. (1993) lntewirology 35,101-107 59 Lagaye, S. et al. (1992) Proc. Nat1 Acad. Sci. USA 89, 10070-10074 60 Schweizer,M. et al. (1994) AIDS Res. Hum. Retrouiruses 10, 601-605 54 55 56 57
Biosynthesis of lipopolysaccharide 0 antigens Chris Whitfield
T
and covalently linked (ligated) Lipopolysaccharide 0 antigens are amphipathic glyhe to a preformed lipid-A-core acimportant virulence determinants for many coconjugates, lipopolyceptor at the periplasmic face bacteria. O-antigen synthesis is an (LPS), are saccharides of the plasma membrane1q2. interesting problem in cell-surface essential and characteristic Ligation therefore effectively assembly. There are two known assembly components of the outer memterminates O-antigen synthesis. pathways, which differ in the cellular branes of Gram-negative bacDetails of the ligation reaction location of their polymerization steps and teria. The hydrophobic domain have not been resolved, and in the direction of chain polymerization. of LPS, lipid A, forms the outer the only ligase component that Some reactions are shared with those for leaflet of the outer membrane. has been characterized is enother surface polymers, such as capsular In enteric bacteria, the core coded by rfuL. RfaL homologs polysaccharides, and may be potential oligosaccharide links lipid A from Salmonella enterica serotargets for therapeutic intervention. to the 0-antigenic side-chain var Typhimurium and Escherpolysaccharide. The structural C. Whitfield is in the Dept of Microbiology, ichiu coli K-12 have little diversity of the 0 antigens University of Guelph, Guelph, Ontario, primary sequence similarity, but stems from variations in sugar Canada Nl G 2 WI. tel: +I 519 824 4120 x3478, have similar hydropathy procomposition, the sequences of fax: +l 519 837 1802, files3. This, together with the sugars and linkages, and the e-mail: [email protected] observation that heterologous substitution of monomers with 0 antigens are expressed (aleither sugars or non-sugar resibeit with variable efficiency) in these bacteria, suggests dues (Fig. 1). These differences give rise to 0-serotype that the mechanism and cellular location of ligation specificity within bacterial species. Much of the traare conserved. After ligation, the completed LPS molditional interest in LPS molecules originates in their ecule is translocated to the cell surface by unknown complex interaction with host defenses and their conmechanisms4. tribution to virulence in pathogenic bacteria. 0 antiThe assembly of 0 antigens is a complex problem in gens form hydrophilic surface layers that often promembrane biogenesis. The principal issues are: (1) the tect the cell from complement-mediated serum killing, maintenance of the structural fidelity of O-repeating and they are therefore essential virulence determinants. units (0 units) during synthesis; (2) the establishment of strain-dependent preferred O-antigen chain lengths; Overview of LPS assembly and (3) the organization in the plasma membrane of a Sugar nucleotides are the activated precursors for system that transfers sugars from cytoplasmic precurcell-surface polysaccharides. To assemble 0 antigens, sors to a membrane-bound lipid (und?), but makes monomers are not transferred directly to a growing hydrophilic polysaccharides available for ligation at LPS molecule. Instead, 0 antigens are synthesized septhe periplasmic face of the plasma membrane. arately on a lipid carrier by enzymes encoded by the rfb This review summarizes current understanding of gene cluster. As in the synthesis of cell-wall peptidothe mechanisms involved in the synthesis and export glycan and capsular polysaccharides, the lipid carrier is across the plasma membrane of LPS 0 polysaccharthe C,,-polyisoprenoid derivative, undecaprenol phosides. The two model pathways described here have phate (und-P). Once complete, 0 antigen is transferred 0 TRENDS
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common precursors and intermediates, and are initiated by similar reactions. However, the cellular location of the polymerization reactions and the direction of polymer growth differ in each pathway. Prototypical systems are drawn from the Enterobacteriaceae, but I attempt to highlight those aspects of synthesis mechanisms for surface polysaccharides that might be conserved in diverse bacteria. Broader coverage of this area is given in several recent reviews3-b.
(a) Salmonella enterica serogroups A, BkD, E
1
3 +2)-a/P-D-Manp(l+4)-a-L-Rhap(
1-t3)-a-D-Galp(l+
Escherichia co/i K-l 2 a-D-Glcp
t2)-o-D-Galf-(l-+6)-a-D-Glcp(1
1 O-YY 2 6 -+3)-a-L-Rhap(l-+3)-a-D-GlcpNAc-(14
Shigella dysenteriae type 1 -+3)-a-L-Rhap(l+3)-a-L-Rhap( 1+2)-a-D-Galp(l-+3)-a-D-GlcpNAc-(l-+ Initiation reactions Reactions involved in the biosynthesis Shigella flexneri of 0 antigens were first described in -+2)-a-L-Rhap( 1-+2)-a-L-Rhap(l-+3)-a-L-Rhap(l+3)-~-D-GlcpNAc-(l+ S. enterica serogroups B and E. The heteropolysaccharide 0 antigens of these bacteria (Fig. 1) are synthesized W by essentially identical mechanisms. Escherichia co/i 08 and Klebsiella pneumoniae 05 The galactosyltransferase RfbP,, (Ref. 7) (see Box 1) catalyzes the -3).B-D-Manp(l+2)-a-D-Manp(l-+2)-a-D-Manp(l+ reversible transfer of galactose lphosphate (Galp-1-P) from the preEscherichia co/i 09 and Klebsiella pneumoniae 03 cursor uridine diphosphogalactose -t3)-a-D-Manp( l-+3)-a-D-Manp( 1+2)-a-D-Manp( l-2)-a-D-Manp( 1-Q.a-D-Manp( 1.+ (UDP-Galp) to und-P (Ref. 8) as the initial synthetic reaction for each of Klebsielia pneumoniae 01 these 0 antigens. Although 0 antigens do not all con-13)~p-D-Galf-(l-3)-a-D-Galp(l + D-Galactan I tain galactose, the only other known +3)-a-D-Galp(l+3)-p-D-Galp(l+ D-Galactan II initiating enzyme is Rfe, an N-acetylglucosamine-1-phosphatetransferase Fig. 1. Structures of representative 0 antigens polymerized by growth at (a) the reducing terminus and (b) the non-reducingterminus. In Salmonella enterica serogroups A. I3 and D, the (GlcpNAc-l-phosphatetransferase)9. main-chain mannose (a-o-Manp) residues are substituted by a 3,6dideoxyhexose residue (R). Rfe transfers GlcpNAc residues found R is a-paratose, a-abequose and a-tyvelose in serogroups A, B and D, respectively. Serogroup in the heteropolysaccharide 0 units E has no 3,6dideoxyhexose residue and has P-WManp instead of a+Manp. Additional 0 factors of several E. coli serotypes’O, Shigella in S. enterica and Shigella species arise by variable (nonstoichiometric) substitution of the base structures with a-glucopyranose and 0-acetyl residues. In Klebsiella pneumoniae 01, dysenteriae type 1 (Ref. 11) and there are two polysaccharide structures in the 0 antigen. o-Galactan I is linked directly to the Shigella flexneri 12.The cryptic 0 antilipid-A core and is synthesized by the products of genes at the rfb (O-antigen biosynthesis) gen of E. coli K-12, recently reconlocus. BGalactan II domains are found in high-molecular-mass 0 antigens and are covalently structed in Reeves’ laboratory’ j, has attached to the distal end of pgalactan I chains. An additional but unknown gene locus participates a similar requirement for Rfe (Ref. with Rfb proteins in formation of c-galactan II. The mannose-containing homopolymers of Escherichia coli 08 and 09, and K. pneumoniae 05 and 03, provide examples of situations 14). Interestingly, Rfe also initiates where different bacteria can produce the same or very similar O-antigen structures. The sugars formation of homopolymeric 0 antiare mannose (oManp). rhamnose (L-Rhap). galactose in the pyranose (Galp) and furanose gens in E. coli 08 and 09 (Refs (Galf) forms, and N-acetylglucosamine (GlcpNAc). 15,16) and in Klebsiella pneumoniae 01 (Ref. 17), even though the 0 units of these homopolysaccharides do not contain phosphate (ECA,,) or, alternatively, to lipid-A core GlcpNAc residues (Fig. l), and their polymerization (ECA,.,,) in a form resembling 0 antigens”O. involves a totally different process from that of heteroInitiating enzymes must interact with the und-P polysaccharides. Pathways for synthesis of O-antigen acceptor and are therefore probably active in a hydrohomopolymers and heteropolymers were formerly phobic environment. Rfe (Refs 9,21) and RfbP,, called ufe dependent and rfe independent, respectively. (Ref. 7) are both predicted to be integral membrane It is now clear that the requirement for Rfe cannot be proteins and, although they differ in size, they have used to distinguish the mechanisms, and this terminsimilar hydropathy profilesl’. It has been suggested ology should be discontinued. that Rfe and RfbP,, are structural homologs, as well While RfbP,, function is confined to O-antigen synas functional homologs in O-antigen initiation”. thesis, Rfe is required for the synthesis of additional surface polysaccharides, which can be coexpressed Polymerization by growth at the reducing terminus with 0 antigens. These include the Tl antigen of SalRol(Cld)- and Rfc-dependent polymerization reactions monella species’* and the enterobacterial common 0 antigens from S. enterica serogroups B and E are antigen (ECA)19. ECA can be linked to diacylglycerol elongated by the addition of 0 units to the reducing
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(or 0 units). Several Rol homologs have been identified and the predicted proteins are quite similar. Escherichiu co/i K-12 strains contain a Rol that can function with heterologous cloned Rfb products (Fig. 4); however, the homologous Rol and Rfb components are required for optimal (strain-dependent) Rol activity3. In contrast, individual Rfc enzymes only polymerize polysaccharides with closely related structures (reviewed in Refs 3,4).
Box 1. Terminology of the rfb genes The 1771 gene locus encodes the enzymes required for the formation of 0 antigens. These include enzymes that catalyze the synthesis of the sugar-nucleotide precursors that are unique to 0 antigens, the glycosyltransferases and polymerases needed for the assembly of the 0 polysaccharides, and the components required for the transfer of O-antigen polymers, or 0 units, across the plasma membrane. The terminology for rfb genes is confusing because different functions are often encoded by rfb genes from different species, or even from different 0 serotypes of the same species. Thus, a gene designated rf?~Acan have different functions depending on the bacterial species and serotype from which it originates. In an attempt to overcome ambiguity in rfb nomenclature, I indicate in a subscript the bacterial species and, where necessary, the 0 serotype. For example, r&P,, is the gene encoding RfbP in Salmonella enterica, r&A,,, is the gene encoding RfbA from Klebsiella pneumoniae serotype 01, and ffbEcK_12 refers to the rfb gene cluster from Escherichia co/iK-12.
erminus of the growing polymer. After formation of und-P-P-Galp, other Rfbs, enzymes catalyze the transfer of successive sugars to form the lipid-linked 0 unit (Fig. 2a). Each step was first identified in vitro, and many activities have now been assigned to specific 0 antigens are then Rfbs, products 6,22. Lipid-linked polymerized in a blockwise fashion (Fig. 2a) in a reaction requiring rfcSe (Ref. 23). A defect in rfcse results in semi-rough LPS (SR-LPS) (Fig. 3). The rfc genes have now been described in several S. enterica serogroups23-zs, Shigellu species1’r26 and E. cofi K-12 (Refs 12,14). Rfc homologs are predicted to be hydrophobic integral membrane proteins with 11-13 transmembrane segments; Rfc primary sequences are not conservedz6. Sequential assembly of repeating units and a blockwise polymerization process provides a mechanism by which the fidelity of the repeating unit structure can be maintained in complex heteropolysaccharides, and is probably not confined to O-antigen biosynthesis. Morona and colleagues have identified an Rfc analog in the rff gene cluster for ECA biosynthesi@, and biochemical data support the operation of the same general pathway in the polymerization of the capsular polysaccharide of Enterobacter (Aerobacter) aerogenes DD45 (Ref. 27). An interesting feature of Rfc-mediated polymerization is the control of the distribution of O-antigen chain lengths (modal distribution). The process involves the protein known as either Rol (regulator of O-chain length28) or Cld (O-chain length determinator29) and its effect is shown in Fig. 4. Bastin et A1.29 have proposed a model in which Rol(Cld) modulates the activity of Rfc to alternate between polymerization and the transfer of lipid-linked 0 antigens to RfaL for ligation. Recently, Morona et A/.” have elaborated on this model, suggesting that Rol may act as a molecular chaperone to regulate the interaction of either RfaL or Rfc with und-P-P-linked 0 antigen
TRENDSINMICROB!OLOGY
Site of Rfc-Rol-dependent O-antigen assembly O-antigen biosynthesis in S. enterica serogroups B and E involves a transmembrane-assembly pathway (Fig. 5) in which RfbP,,, Rfc,, RfbX,,, Rol,, and RfaL,, are all integral membrane proteins. Transferase enzymes acting after RfbPs, in O-unit formation are peripheral proteins. It seems reasonable to assume that the enzymes are arranged in a complex, given their sequential action, high specificity and high specific activity. As biosynthetic activity is located in the membrane fraction (where und-P is available) and the precursors are cytoplasmic, the complex must be located at the cytoplasmic face of the plasma membrane. However, evidence available indicates that polymerization reactions involving Rfcs, occur at the periplasmic face of the plasma membrane’, so the assembly system must transport lipid-linked 0 units across the membrane. A group of rfbP,, mutants (formerly designated rfb7’,,) accumulate putative lipid-linked 0 units, but cannot transfer the intermediates to the lipid-A core. These mutants were originally thought to have a defect in ligation, but are now thought to be unable to ‘flip’ und-P-P-O units across the plasma membrane to the active site of Rfcs,. This has led to the suggestion that RfbPs, has an additional role in this transport process31. While these results can be explained by a dual function for RfbPs, as both a galactosyltransferase and a ‘flippase’, the phenotype could alternatively reflect altered protein-protein interactions between RfbP,, and an adjacent flippase component of the complex. The most likely alternative candidate for the flippase is RfbX. Homologs of rfbX have been identified in rfb clusters from several S. enterica serovars6, S. dysenteriae type 1 (Ref. 11) and E. coli K-12 (Refs 12,14). The function of RfbX is at present unknown. RfbX has been suggested to be involved in ligation3. While no data directly preclude this, RfaL efficiently ligates 0 antigens and 0 units synthesized by this pathway, and 0 antigens can be formed by a second pathway lacking RfbX (see below), both of which suggest that an essential role for RfbX in ligation is unlikely. As several r/%X homologs have been cloned, its role can now be addressed directly. Polymerization by growth at the non-reducing terminus Rol- and Rfc-independent polymerization reactions A second, substantially different pathway exists for the synthesis of some 0 antigens. Details of the pathway are only now being elucidated, and it is currently only known to be involved in the synthesis of homopolymer 0 antigens of E. coli 08 and 09 and K. pneumoniue
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(a) Reaction
1
und-@
+ NDPa
=
undm
Reaction
2
undm’+
Reaction
3
undm
+ NDPa
Reaction
4
undm
t
undm
Reaction
5
undm
t
undm
Reaction
1
und@
Reaction
2
undm
NDP-@
+
+ NMP
undm
+
+ NDP
undm
+ NDP
--+
t
und-
undm
+ und@@
@I
Reaction3
undm
Reaction4
undm
Reaction 5
und m
+ NDPa
;t
undm
t NDPa
-
t NDP-@
t
undm
+
NDP-@
t
t NMP
t NDP
undm
-+
NDPan
t NDP
unde
t
t NDP
NDP*
n
-----$ und
t
nNDP
Fig. 2. Polymerization of 0 antigens. O-antigen synthesis involves two distinct polymerization models distinguished by the direction of chain elongation. Both models have an analogous reversible initiating step (reaction l), in which a sugar l-phosphate residue is transferred from the cognate nucleotide diphosphate (NDP) derivative to undecaprenol phosphate (und-P). The pathways then diverge. (a) Growth at the reducing terminus. The figure shows the formation of a hypothetical 0 antigen with a trisaccharide repeating unit. After the initiating reaction, subsequent steps (reactions 2 and 3) involve sequential transfer of each sugar to form an und-P-P-linked 0 unit. Preformed und-P-P-linked 0 units are the substrates for polymerization in the absence of de novo synthesis of repeating units (reactions 4 and 5). Polymerization involves the addition of nascent 0 polymer from one und-P-P carrier to the nonreducing terminus of a newly synthesized single 0 unit attached to a second und-P-P carrier 52.The solid-shaded sugars of the 0 units indicate those 0 units added most recently. Growth of the polymer therefore occurs at the reducing terminus, or the end closest to the und-P-P carrier. In Salmonella enterica, addition of glucosyl and 0-acetyl substituents (Fig. 1) occurs after polymerization, which accounts for the nonstoichiometric nature of such modifications 3.4.18.(b) Growth at the non-reducing terminus. The figure shows the synthesis of a hypothetical 0 antigen with a disaccharide repeating unit. The pathway is initiated by the transfer of sugar l-phosphate to und-P. Polymerization involves the sequential processive transfer of sugars to the non-reducing terminus of the nascent polymer (reactions 2 and 3). There is no blockwise polymerization reaction, and the polymer is elongated by processive sugartransferase reactions (reactions 4 and 5). Sugars are transferred directly from NDP-sugar precursors during polymerization. Growth of the polymer occurs at the end of the polymer furthest from the und-P-P carrier.
01 (Fig. 1). Initiation of this pathway involves the transfer of a ‘non-O-antigen residue’ to und-P, and this intermediate is the acceptor for monomers of the 0 antigen. So far, Rfe is the only initiating enzyme identified in this pathway, but there is some confusion about the precise identity of the residue that is transferred to und-P. In E. coli 09, the acceptor has been characterized in in vitro analyses as a glucose-containing lipid intermediate, und-P-P-Glcp (Ref. 32). However, recent in vivo analyses of E. coli 08-K-12 hybrids16 show that the acceptor is und-P-P-GlcpNAc, which is consistent with the activity of Rfe in the synthesis of other 0 antigens and of ECA. In’this path-
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way, the role of Rfe is confined to initiating O-antigen synthesis; GlcpNAc is not found in the 0 units. During the formation of the E. cok’ 09 antigen, mannosyl residues are rapidly added to the nonreducing terminus of the acceptor one residue at a time, in a processive mechanism (Fig. 2b)32. Intermediates containing individual 0 units have not been identified. In K. pneumoniue 01 (Ref. 33), four Rfb proteins and Rfe are required to form D-galactan I 0 antigen (Fig. 1). There are no equivalents of either Rfc or RfbX (Ref. 33) and SR-LPS mutants have not been isolated in K. pneumoniae 01, as predicted by a processive-assembly mechanism that does not involve
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Fig. 3. Activity of the Rfc O-antigen polymerase in assembly of lipopolysaccharide (LPS) in Salmonella enterica serovar Typhimurium LT2. In SDSPAGE, smooth O-substituted LPS (S-LPS) is characterized by a ‘ladder’ of heterogeneous molecules with increasing molecular mass (lane 1). Each ascending ‘rung’ of the ladder is a population of LPS molecules with an increase of one repeating unit in the 0 antigen. Each bacterial strain has a preferred distribution of O-antigen chain lengths, resulting in a characteristic ladder pattern of 1‘“11.11__11.1SLPS molecules. Fast-migrating molecules contain only lipid-A core, as in lane 3, which contains the rough (O-deficient) LPS (R-LPS) from an HaLdeficient (O-antigen ligase-deficient) mutant. An tic mutant is unable to polymerize 0 antigen and ligates a single 0 unit to lipid-A core to form semi-rough LPS (SR-LPS) (lane 2). 1
2
3
blockwise polymerization of 0 units. Completed 0 antigen is transferred to the lipid-A core together with the initiating reducing terminal sugar34. So far, no heteropolysaccharide 0 antigens are known to be synthesized by this pathway. However, growth at the nonreducing terminus is certainly not unique to homopolysaccharides; biosynthesis of heparin in mammalian 1234
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Fig. 4. Effect of Rol(Cld) on O-antigen chain length. The SDS-PAGE lipopolysaccharide profiles (LPS) show Escherichia co/i K-12 strains expressingthe Roldependent 0 antigen from Shigella dysenteriae type 1 (lanes 2-3) and the Rol-indepenKlebsiella dent pneumoniae 01 polymer (lanes 45). Synthesis is directed by cloned rfb gene clusters. fscherichiaco/iK-12 contains functional Rol(Cld), but mutations in the cryptic rf&cK_12gene cluster result in O-deficient rough LPS (R-LPS) (lane 1). In a Rol+ K-12 strain, S. dysenteriae type 1 O-substituted smooth LPS (S-LPS) molecules show a modal distribution: most O-antigen chains fall within a limited highmolecular-mass range and fewer low-molecular-mass 0 chains are ligated to the lipid-A core (lane 3). In a K-12 strain deleted in n7~and ro/(lane 2), type 1 SLPS has an unregulated appearance, with more low-molecular-mass O-antigen chains and no modal distribution. In contrast, the K. pneumoniae 01 (r>galactan I) has an identical profile in the absence (lane 4) or presence (lane 5) of host Rol. The E. co/i K-12 host strain deleted in ml and r&therefore provides a rapid and valuable first indication of the mechanism of synthesis. The cloned rfb genes for S. dysenteriae type 1 (Refs 11,53) were kindly provided by C. Schnaitman and J. Klena.
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cells3j, hyaluronic- acid capsular polysaccharides in streptococci36 and some group II capsular polysaccharides of E. coli3’J8 all involve rapid and processive transfer of monomers to a growing non-reducing terminus. Maintenance of the fidelity of the structure is more complicated in processive polymerization. Clues as to how this might be achieved have been found in work on the biosynthesis of heparan sulfate, bacterial hyaluronic-acid capsular polysaccharides36 and some group II capsular polysaccharides of E. coli37,38. In these examples, a single enzyme with dual specificity forms the disaccharide repeating units, ensuring the production of a strictly repeating unit structure. The synthetase that forms authentic high-molecular-mass hyaluronic acid is a single polypeptide36. For heparansulfate biosynthesis, a distinct GlcNActransferase initiates assembly39 and an additional single bifunctional polypeptide with both GlcNAc- and glucuronic-acidtransferase activities then catalyzes the polymerizenzyme is also ation step 35*40 . A distinct initiating required for the biosynthesis of the polysialic-acidcontaining group II capsular polysaccharides of E. coli Kl and K92, where the polysialyltransferase enzyme is sufficient for polymerization, but cannot initiate polymer formation by itself3*. It is conceivable that a repeating unit structure more complex than a disaccharide might require additional enzymes to catalyze polymerization. Klebsiella pneumoniae D-galactan I chain-length distribution is identical when the cloned rfbKpo,genes (see Box 1) are expressed in E. coli K-12 hosts with or without rol (Fig. 4). Furthermore, the distribution of chain lengths in authentic E. coli 09 (Ref. 41) and K. pneumoniae D-galactan I (Ref. 17) 0 antigens in wild-type strains cannot be distinguished from that of cloned 0 antigen from E. coli K-12. Together, these observations indicate that a different process determines the strain-dependent distribution of O-antigen chain lengths in this pathway, and are consistent with one or more Rfb proteins being involved. This would explain the aberrant O-antigen chain lengths resulting from some deletions in the rfb region of E. coli 09 (Ref. 42). In E. coli 09 and Klebsiella 05 (Fig. l), O-antigen chains are terminated by 3-O-methyl-Dmannose43, and the addition of this residue may act as a termination reaction and may be involved in chainlength determination. Interestingly, small amounts of 2-O-methyl groups are detected in the perosaminecontaining homopolymeric 01 antigen of Vibrio cholerae44. Loss of the 2-O-methyl groups does not affect the ability to form 0 antigen, but results in seroconversion from Ogawa to Inaba type. Seroconversion is associated with defects in one rfb gene4’. Whether methylation occurs in all homopolymer 0 antigens remains to be established. Site of Rfc-Rol-independent O-antigen assembly How can growth at the non-reducing terminus be accommodated if the immediate donors of monomers (sugar nucleotides) are cytoplasmic, yet ligation of polymer occurs at the periplasmic face of the plasma membrane? Polymerization of the K. pneumoniae 01
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0))
NDP Fig. 5. Model of the plasma-membrane topology of a biosynthesis complex that synthesizes an 0 antigen that is elongated at the reducing terminus. (a) The initiation reaction involves an integral membrane protein (either RfbP, or Rfe), and the formation of an undecaprenol-pyrophosphatelinked sugar (und-P-P-sugar). The precursor is a nucleotidediphosphate-sugar (NDP-sugar) and a nucleotide monophosphate residue (NMP) is released. Additional transferases (shown here as a single component for clarity) are peripheral membrane proteins; they act sequentially to form the lipid-linked repeating unit. (b) Lipid-linked repeating units are translocated (‘flipped’) to the periplasmic face of the plasma membrane by a process that may involve RfbX. (c) Polymerization reactions occur at the periplasmic face of the plasma membrane, through a process involving Rfc and Rol (see Figs 3 and 4), and culminating in an und-P-P-linked polymer. (d) RfaL ligates the nascent polymer to a lipid-Acore molecule at the periplasmic face of the membrane.
a ntigen occurs not at the periplasmic face of the membrane, but rather in the cytoplasm (Fig. 6). Although the proteins RfbCDEF,,o, are sufficient for the polymerization of D-galactan I, nascent 0 antigen accumulates in the cytoplasm33. The remaining components of the system, RfbABKpol, form a transporter that is required to transfer the 01 polymer to the ligation site. The transporter is a member of the ‘traffic ATPase’ (Ref. 46) or ATP-binding cassette ‘ABC’ transporter family47, and specifically is of the ABC-2 subfamily48. A similar system has been identified to be needed for the formation of the homopolymeric 0 antigen of Yersinia enterocolitica 0:3 (Ref. 49), and functionally analogous transporters are required for export across the plasma membrane of polymerized group-II-type
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capsular polysaccharides in E. coli, Haemophilus influenzae and Neisseria meningitidis (for further references to these systems, see Refs 430). ABC-2 transporters contain an integral membrane protein with multiple membrane-spanning domains, and a hydrophilic protein containing the consensus ATP-binding motif (Walker box). The organization of similar systems that are involved in nutrient uptake has been studied extensively, but less is known about the polysaccharide exporters. The functional transporter is thought to consist of two subunits of each protein component. In the model illustrated in Fig. 6, export of 0 antigen is shown occurring through the membrane component of the ABC-2 transporter. While this is an attractive
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Fig. 6. Model of the plasma-membrane topology of a biosynthesis complex that synthesizes an 0 antigen elongated at the non-reducing terminus. (a) As in Fig. 5, an integral membrane protein initiates the polymer. So far, only Rfe has been implicated in this process. This enzyme transfers an N-acetylglucosamine l-phosphate (GlcNAc-1-P) from uridine diphospho-N-acetylglucosamine (UDP-GlcNAc) to undecaprenol phosphate (und-P), with the release of a uridine monophosphate (UMP) residue. (b) Polymerization of the polymer involves one or more peripheral proteins and the undecaprenol-pyrophosphate-linked (und-P-P-linked) polymer is completed in the cytoplasm. (c) Translocation of the nascent polymer across the plasma membrane requires the presence of a dedicated ATP-binding cassette (ABC-2) transporter. While export of polymer through the transporter (as shown here) is an attractive hypothesis, such direct involvement has not been demonstrated experimentally. It is also unknown whether transport requires the polymer to be attached to und-P, occurs on an alternative carrier molecule or involves the free polymer. (d) Ligation of the nascent 0 antigen to lipid-A core requires RfaL and the reaction is identical to the equivalent step in Pig. 5.
hypothesis, it must be emphasized that the transporter is only known to be required for export, and its precise role may be less direct. Binding of ATP by the peripheral component is necessary for export of the capsuleSi, although this has not been shown directly for O-antigen export. The shared direction of chain growth and location of polymerization, and the requirement for an ABC-2 transporter in the synthesis of some 0 antigens and group II capsular polysaccharides, suggest that this is a general mechanism for the assembly of cell-surface polymers. The involvement of ABC-2 transporters explains the absence of Rfc, RfbX and Rol activity in this pathway for O-antigen synthesis. Nevertheless, many other important aspects of this pathway are currently unresolved at present. It is not known whether this path-
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way is confined to homopolysaccharide 0 antigens, although similarities with group II capsular heteropolysaccharides suggest that this is not necessarily the case. It is also not known whether Rfe is the only initiating enzyme for this pathway. At the most basic level, the precise role of the ABC-2 transporter is itself unknown. Is the 0 antigen transported while attached to und-P, is an additional unidentified lipid carrier involved, or does export occur without a lipid component at all? At least some of these questions can now be analyzed. Conclusions
The key criteria that distinguish the two known pathways for O-antigen synthesis are: (1) the cellular location of the polymerization reactions; (2) the direction
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of chain growth during polymerization; and (3) the requirement for Rfc, Rol and RfbX proteins in one pathway and for an ABC-2 transporter in the other. The Rfc-Rol-dependent pathway provides a mechanism that allows stringent control of the heteropolysaccharide structure, particularly when an essential side-chain residue is added during synthesis, as occurs in S. enterica serogroup B. The ABC-2-transporter-dependent pathway is well suited to the synthesis of linear polymers and, considering 0 antigens, is currently (but not necessarily) limited to the synthesis of homopolymers. There are relationships between the pathways for the synthesis of 0 antigens and those for other cellsurface polysaccharides. Key differences are probably confined to the later stages of assembly, such as the nature of the linkage moiety or the steps involved in translocation from the periplasmic face of the plasma membrane to the cell surface4. Because surface polymers have essential roles in the survival of bacteria, the pathways involved in their formation are critical to virulence and potentially provide novel targets for therapeutic intervention. Currently available moleculargenetic and biochemical approaches should allow the elucidation of these important and complex systems. Acknowledgements Many of the ideas presented here were developedthrough stimulating discussionswith members(past and present) of the author’slaboratory and with several other colleagues; their contributions are gratefully acknowledged.Researchin the author’slaboratory has been generously supported by funding from the Natural Sciences and Engineering Research Council of Canada, the Medical Research Council of Canada and the Canadian Bacterial Diseases Network (Canadian Networks of Centers of Excellence program).
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*Herpes simplex virus vaccines as lmmunotherapeutic agents, by L.R. Stanberry lUnconstrained bacterial promiscuity: the Tn916--Tnl545 family of conjugative transposons, by D.B. Ckwell, SE. Ftannwn and D.D. Jaworski 44oving thmugh the membrane with filamentous phage, by M. Russel *The epidemiology of HIV infection and AIDS in Africa, by P. Van de Perra ----I_
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