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W2040468286 abstract "1. Summary, 25S 2. Introduction, 25S 3. Overview of surface structures, 25S 4. The outer membrane, 26S 4.1 Methodological approaches, 26S 4.2 Polysaccharides, 26S 4.3 Outer membrane proteins, 27S 4.3.1 Campylobacter jejuni, 27S 4.3.2 Helicobacter pylori, 28S 5. S-layer proteins in campylobacters, 29S 5.1 Campylobacter fetus, 29S 5.2 Campylobacter rectus, 29S 6. Pili, 30S 7. Flagella, 30S 7.1 Genetics and biogenesis of flagella, 30S 7.2 Flagella as virulence factors, 31S 7.3 Flagella as antigens, 32S 7.4 Flagellin glycosylation, 32S 8. Conclusions, 33S 9. References, 33S The major components of the surfaces of Helicobacter pylori and Campylobacter jejuni are considered in turn, comparing and contrasting where possible the key features of each organism. The components considered are the outer membrane, including protein as well as polysaccharide components; the S-layer proteins of Campylobacter fetus and Campylobacter rectus; and the flagella of both organisms including the regulation of flagellar gene expression. Proteins secreted by these organisms are also considered. In conclusion, it is clear that the unique pathogenic properties of these closely related organisms are dependent to a large extent on key differences in their surface components. In the absence of any detailed information about the surface characteristics of Arcobacter, the following review deals only with those of Campylobacter spp. and H. pylori. The availability of genomic sequence data for both H. pylori and C. jejuni, their close phylogenetic relationship yet very distinct natural histories, and the wealth of published detail on their surface components, provides for an interesting comparative study to be made and this is a strong thread in the present review. In each of the major categories of surface structure, comparisons will be made where possible in order to gain insight into the significance of these structures in the host–pathogen context. For simplicity, I shall refer generally only to C. jejuni in this review, embracing observations made in Campylobacter coli, which in most cases appears to be essentially identical to C. jejuni in its surface organization. As Gram-negative organisms, helicobacters and campylobacters appear to possess quite typical surface components in their outer membranes with protein, lipoprotein and lipopolysaccharide macromolecular components. The outer membrane generally appears to be the principal surface-exposed component, although the recent description of capsule-like macromolecules (11; Wren, this volume; 32) requires re-examination of the exact functions of surface polysaccharides. Another major structure external to the outer membrane is the S-layer protein, observed and explored to date only in C. fetus and C. rectus and not present in C. jejuni, C. coli or H. pylori. Both proteins and lipopolysaccharides of the outer membranes have been explored in some detail. There is very little evidence of fimbrial structures. A major area of research in both organisms has been the important surface antigen and mediator of motility, the flagellum, and constructive comparison of H. pylori and C. jejuni can be made here; in addition, comparison with the well understood paradigm of flagellar biogenesis in Salmonella typhimurium is fruitful. Finally, the discovery of glycosylation of the flagellin will be covered briefly. A variety of experimental approaches has been used in the exploration of outer membrane components. For Campylobacter, some workers have adopted the acid glycine extraction described for example by 57. The implication that proteins found in these extracts represent surface components must be treated with caution; for example the periplasmic protein P19 (30) was originally identified as an outer membrane protein based on use of this method. Another widely adopted method for outer membrane preparation has been differential solubilization of cytoplasmic membrane with sarkosyl, which appears to work well with Campylobacter although not with all Gram-negative bacteria. A more recent development in surface fractionation (65) has shown that distinctive periplasmic and outer membrane fractions can be produced by osmotic shock to release periplasmic material, followed by cell disruption, for example by sonication. Recovery of a particulate and membrane fraction, and differential solubilization of cytoplasmic membrane components leaves a residual particulate ‘outer membrane’ fraction which includes at least some outer membrane proteins, flagellar fragments and, presumably, peptidoglycan fragments. The latter are not solubilized by Laemmli SDS–PAGE buffer and are generally neglected. Nevertheless a useful representation of the important surface proteins is obtained. Also significant in the study of outer membrane in C. jejuni is direct observation by electron microscopy of negatively stained material (3). This has revealed a clear ordered array architecture at high magnification, apparently representing the packing of major outer membrane protein molecules in trimeric complexes. These probably represent porin structures, at a very high density so that they virtually cover the outer membrane surface, contacting neighbouring complexes in a close packed repeating pattern. In appearance this resembles S-layer arrays. In the case of H. pylori, different approaches to identification of surface proteins have been adopted. Monoclonal antibodies selected for surface reactivity with intact cells revealed proteins of probable porin activity, and sucrose density gradient centrifugation with Triton X-100 extraction was shown to separate an uncontaminated outer membrane fraction (17). The physical property of heat-modifiability of porin proteins was exploited, using a two-dimensional gel electrophoresis technique, in order to differentiate them from other cellular proteins (23). This led to definition of a family of Helicobacter outer membrane porins (Hops, see section 4.3.2) which in turn has enabled predictions from the genome sequence of further outer membrane proteins totalling more than 30 (64). This gene family repesents a substantial proportion, approximately 4% (14), of the total coding capacity of the genome and clearly these proteins are likely to be highly significant in the natural history of this organism. Curiously, no homologues of this extensive protein family are found in C. jejuni, suggesting their specific importance in the gastrointestinal environment of H. pylori. The outer membrane polysaccharides of C. jejuni have recently been the subject of major new insights, arising in part but not exclusively from genome sequence information. The majority of strains were long known to express side chain-deficient, low molecular weight outer membrane antigens of the lipo-oligosaccharide (LOS) type. In a minority (about one-third) of serotype strains a high molecular weight, protease-stable antigen capable of ladder formation on gel electrophoresis and Western blotting, characteristic of the presence of extended side chain or O-antigen of smooth lipopolysaccharide (LPS), was found (58). The chemistry of the core structures of a number of these antigens has been explored extensively (reviewed by 24 and 46), indicating that the LOS type of structure has potential for expression of antigenic diversity. Briefly, the basic structure comprises a lipid A moiety fairly typical of those of the Enterobacteriaceae, with a fatty acid-linked and phosphorylated glucosamine or diaminoglucose disaccharide (46) to which is linked the inner core polysaccharide. This comprises first a 3-deoxy-D-manno-octulosonic acid residue, then two heptoses to which combinations of glucose, phosphoethanolamine or phosphate are linked as side residues. An outer core structure follows, comprising two or three residues usually of galactose or N-acetyl galactosamine, which may also be branched. Crucially, the galactose residues in this outer core region are often sialylated, to create structures of great significance in molecular mimicry of host gangliosides, which is believed to play a key part in the presumed autoimmune processes in Guillain–Barré and Miller–Fisher neuropathies, reviewed elsewhere in this volume. The presence of these sialic acid residues is of interest also in providing for evasion of host defence systems. This may parallel the role of sialic acid-containing capsules in organisms such as group B meningococci and the K1 capsular serotype of Escherichia coli, in which the presence of sialic acids inhibits, for example, complement activation. Recent work by 38 indicates that a specific chromosomal copy of the neuB gene, neuB1, involved in N-acetyl neuraminic acid (or sialic acid) synthesis is active in LOS sialylation, while another copy, neuB2, appears to be specific for flagellin modification (see below). In H. pylori, lipopolysaccharide synthesis has been less well explored. The main feature of interest is the presence in the O-side chain of fucosylated disaccharides of galactose and N-acetyl galactosamine, mimicking host structures and with potential to cause autoimmune tissue damage (14). Genetic data on LOS biosynthesis can be derived largely from the genome sequence data (55). In summary, many of the genes for lipid A and inner core synthesis are scattered in the genome, while a cluster of genes involved in outer core LOS synthesis and assembly (at that time designated lipopolysaccharide) was discovered independently by 24 and 68. This region of the chromosome also contains genes that may be involved in protein glycosylation but is discrete from the region determining capsule synthesis and assembly described below. The exact nature of the higher molecular weight, putative LPS structures mentioned above remains controversial. A convincing case has been made by 32 that this is, at least in some strains including several serotype strains, a capsular structure distinct from LOS and not containing lipid A or LOS core structures. This observation arose from a genome sample sequencing exercise before to the determination of the complete genome sequence of strain NCTC 11168, which revealed genes named kpsM and kpsT, homologues of ATP-binding cassette (ABC) transporters involved in capsular polysaccharide export in other bacteria. Examination of the genome sequence subsequently revealed that numerous predicted genes, homologous to capsular polysaccharide synthesis, assembly and export genes in other bacteria, were clustered in a specific region of the genome. Most of these gene homologues were absent from the H. pylori genome. A critical observation was that mutation in the kpsM, kpsS or kpsC genes led to loss of high molecular weight, protease-stable antigen, and that serotype specificity in the Penner scheme was also lost in such mutants. Hence the serotype-specific antigen previously assumed to comprise a lipopolysaccharide O-antigen appeared to be actually a capsular polysaccharide. This work is, however, unconfirmed by others at the time of writing. The major outer membrane protein of C. jejuni is by far the most highly expressed protein in this species as shown by coomassie blue staining, and shows extensive, albeit apparently stable, polymorphism between isolates, ranging in relative molecular weight from 41 000 to 46 000 (48). Its invariable presence suggests that it is essential for the organism. There is substantial evidence for its function as a porin, shown by conductivity measurements in lipid bilayers incorporating the protein, yet surprisingly it appears to have little significant sequence homology with porins of other Gram-negative bacteria and there is no genetic homologue in H. pylori. The pores are believed to be cation selective, small and voltage sensitive. The native protein retains elements of folded structure unless heated in SDS (71), at first dissociating from a trimeric to a monomeric state which, like the trimer, shows pore-forming ability until completely denatured (13). Antigenically, the protein is difficult to characterize because antibody raised against purified, denatured protein does not recognize the molecule in its native state by immunoblotting, while antibodies to the native molecule produced during infection, whether in birds or mammals, do not recognize the denatured molecule after SDS–PAGE and blotting. The contribution of the molecule to pathogenesis is uncertain, although there is some evidence of its involvement in adhesion to host cells or mucus. It also has toxic properties when complexed with LPS (4). There is little evidence of any other porin-like outer membrane proteins in C. jejuni, which from genome sequence data appears not to possess homologues of the major porins of the Enterobacteriaceae. The large family of ‘Hop’ porin proteins of H. pylori also appears from the genome sequence to be totally absent. It remains to be discovered, however, whether there are additional porins or other outer membrane proteins among the very large number (up to 160) of predicted membrane proteins, including integral membrane proteins and lipoproteins, of unknown function discernible from the genome sequence (55). Perusal of the genome sequence does however, reveal a small number of predicted candidate outer membrane proteins. Cj0129c is homologous with a probable outer membrane protein in H. pylori, Omp85 of Neisseria meningitidis and surface antigen d15 of Haemophilus influenzae. Cj0975 is a homologue of Hxb1, a haemopexin utilization protein of Haem. influenzae and HlyB of Proteus mirabilis. A tandem pair of genes, Cj0628/0629, which are repeats of a second pair Cj1677/1678, offer the interesting possibility of high molecular weight lipoproteins showing weak homology to an S-layer protein of Rickettsia typhi. In addition the first pair shows evidence of high frequency switching or polymorphism based on a homopolymeric base sequence. This sequence is subject to slipped strand mispairing which is predicted to lead to phase variable expression either as a gene fusion or as two separate proteins (55). The significance of these predicted proteins is unknown. Other outer membrane and associated proteins, previously described independently from the genome sequence, include the following. A significant host-interactive protein candidate is putative fibronectin adhesin CadF. First identified by its ability to bind fibronectin in a ligand immunoblot, the 37-kDa protein is believed to reside in the outer membrane (36). Its presence has been demonstrated in every strain tested by immunoblot analysis, and polymerase chain reaction amplifies a gene fragment from 95% of C. jejuni and 83% of C. coli (37), suggesting it is important in the niche occupied by these closely related organisms. Furthermore it appears to be required for colonization of newly hatched chicks, since a knock-out mutant did not colonize (72). No gene homologue is present in H. pylori, suggesting a role unique to campylobacters, and based on sequence analysis the protein shows some homology with features of Pseudomonas aeruginosa porin OprF and to OmpA of E. coli. Several proteins, PEB1, PEB2, PEB3 and PEB4, present in acid glycine extracts were identified as strongly antigenic polypeptides and purified (57). Independently identified cell binding factors CBF1 and CBF2 were shown to be identical to PEB1 and PEB4 respectively, and evidence for a possible cell surface location for PEB1 was given (34). Sequence homology indicates that PEB1 may in fact be a periplasmic binding protein for an ABC-type amino acid transporter, and that PEB2–PEB4 may also be located in the periplasm (55). Other proteins clearly associated with the outer membrane include a phospholipase A, encoded by pldA (26) of which a homologue is found in H. pylori where it is implicated in virulence (19). Both proteins are homologues of the larger family of conserved outer membrane phospholipases A. Also a member of a widely conserved family is the immunogenic peptidoglycan-associated outer membrane lipoprotein Omp18 (9); this protein probably has a structural role and the gene is designated Cj0113 in the genome. Several other putative outer membrane proteins have been described. A 32–34-kDa protein OmpH 1 with homology to glutamine-binding protein GlnH of Bacillus stearothermophilus showed similarity to PEB1 (44). A 59-kDa protein described by 47 and implicated by ligand immunoblot in binding to INT 407 cell membranes and fibronectin showed N-terminal sequence identity to CadF (see above), although there is a major discrepancy in molecular weights reported for these two proteins. 33 described a 95-kDa surface protein implicated in adhesion to CaCo-2 cells; it was present in some but not all strains of C. jejuni, and showed similarity to the filamentous adhesin of Bordetella pertussis. In addition, two groups have reported intriguing evidence of expression of certain proteins, including putative surface components, only in host-associated organisms. 54 reported expression of proteins of 180, 66, 43 and 35 kDa during growth in rabbit intestinal loops but not in vitro. Chart et al. (10) showed that outer membrane proteins of 55, 35 and 20 kDa were expressed only by bacteria maintained in chickens and not in laboratory-grown organisms. These proteins do not appear to have been investigated further. The most striking element of outer membrane proteins is the large family of Hop porins mentioned above, which were identified from the genome sequence (64) on the basis of conserved sequence features at the N-termini and especially the C-termini of the predicted proteins. The latter showed a characteristic alternation of hydrophilic and hydrophobic amino acid residues. A member of this family, HopE, has been well characterized (15; 23; 6, 7). Although of relatively low abundance it forms large, non-specific porin channels consistent with susceptibility of the organism to large molecular antimicrobial agents. It was shown that the recombinant protein expressed in E. coli was similarly capable of pore formation as when expressed by H. pylori, and the nature of folding was predicted to comprise a beta-barrel with 16 transmembrane amphipathic beta-strands. Another member of the Hop family, HopZ, has also been investigated (56) and shown to be polymorphic owing to a slipped strand mispairing mechanism as described above, based on a series of C–T repeats in the region of the gene encoding the signal peptide. A knock-out mutant in this gene showed reduced adherence to gastric epithelial cells. In similar investigations of related members of this extended family of proteins, two lines of evidence confirm their potential role in pathogenesis. First, identification of mutants of reduced ability to adhere to gastric epithelial cell lines identified the alpAB locus as a critical determinant of adhesion to a receptor distinct from the Lewis receptor involved in interaction with other ligands (52). AlpA and AlpB shared sequence homology although the first is predicted to be a lipoprotein and the second to have a regular signal peptide. Another example of a role in adhesion of an outer membrane protein is that of BabA and BabB, Lewis B blood group antigen-binding factors described by 29. Recently, investigation of knock-out mutations in several members of the porin family for ability to stimulate interleukin 8 secretion in co-culture with gastric cancer cell lines identified HP0638 as a critical determinant of such stimulation. The protein was designated OipA (outer membrane proinflammatory protein A) in recognition of this activity (69) and potentially may have a critical role in stimulation of chronic inflammation during infection. This extended and branching family of porins has now been reviewed in detail (1) and shown to be subdivided into several groups. The Hop group, represented by the first described members HopA–D, possesses a characteristic N-terminal motif and includes OipA or HP0638 as well as BabA (HopS) and BabB (HopT). Related proteins that do not possess the N-terminal motif are designated Hop-related or Hor proteins. Further divisions are distinguished on the basis of the C-terminal amino acid residue, which in many members of the Hop family is phenylalanine but in some, designated Y-Hops, is tyrosine. As in C. jejuni, the outer membrane phospholipase A has been investigated in H. pylori. 19 showed that a knock-out mutant in pldA had reduced phospholipase A2 and haemolytic activity as well as decreased ability to colonize mice, although its ability to adhere to a human gastric carcinoma cell line was unaffected. The exact role of this protein in pathogenesis remains unclear. Additional outer membrane proteins of H. pylori with a potential role as vaccinogenic antigens or in pathogenesis include lipoprotein Lpp20, a lactoferrin-binding protein, and iron-repressible outer membrane proteins. In summary, the outer membrane proteins of both C. jejuni and H. pylori are a rich source of probable and potential factors in virulence and antigenicity of these organisms. There is significant evidence of both polymorphism and inducible expression, suggesting that modulation of these proteins is a key process in the maintenance of infection in the face of host responses. Despite the close phylogenetic relationship of these organisms, they differ significantly in outer membrane protein organization, notably in the dominance of the major outer membrane protein in C. jejuni and of the large paralogous family of porins in H. pylori. S-layers are ordered surface arrays, described as paracrystalline lattices, forming a distinct layer external to the outer membrane in Gram-negative bacteria and usually comprising a single, highly expressed high molecular weight protein. S-layer proteins have been reviewed recently by 59. Many S-layer proteins have proven to be glycoproteins and to have several unusual properties of resistance to degradation which presumably relate to a protective function for the cell. S-layer proteins were first noted in the genus Campylobacter in C. fetus, following the observation that wild-type organisms expressing the high molecular weight, abundant protein forming a capsule-like outer layer were resistant to bactericidal effects of antibody and complement (8). Spontaneous or laboratory-selected mutants lacking these proteins were susceptible to serum. This ability of wild-type strains to resist bactericidal host defences is significant for the organism which causes chronic colonization and infection of mucosal surfaces of animals, with invasive tendencies which can lead to fetal infection and abortion. In subsequent extensive studies on the variability of expression of these proteins, Blaser and colleagues explored the role of invertible genetic elements and RecA-dependent recombinational events. These provided for a repertoire of antigenic types of S-layer protein to be expressed, thus allowing evasion of host immune responses (reviewed by 20). The S-layer proteins, ranging in molecular weight from 97 to 149 kDa, do not appear to be glycosylated. Typically of S-layer proteins in other bacteria, they are weakly acidic and contain high levels of hydrophobic and low levels of sulphur-containing amino acids. The proteins appear to be secreted by a Sec-independent mechanism, using a type 1 mechanism analogous to that of E. coli haemolysin (63). Their attachment to the outer membrane is non-covalent and dependent on interaction with LPS, and hence is serotype specific: strains with type A LPS have surface layer proteins SlpA, while type B LPS strains have SlpB proteins. No such proteins have been described in the enteric campylobacters, C. jejuni or C. coli, and no genes homologous to the sap (Slp encoding) genes have been detected in the C. jejuni genome (55). There is, however, an interesting set of genes (see above, section 4.3.1) that may encode distant homologues of S-layer proteins of R. typhi, possibly with potential for phase variable expression, albeit no evidence of their expression in the laboratory has been reported. Campylobacter rectus is a putative periodontal pathogen. S-layer proteins in this organism were first reported by 49. The proteins varied between strains with molecular weights ranging from 150 to 166 kDa, and some structural differences were seen. Subsequent studies have shown a role for the protein in resistance to killing by complement and phagocytes in the absence of specific antibody (53). Cloning of a gene crs encoding an S-layer protein and a search for gene homologues indicated that it was a single-copy gene, with no evidence of the mechanism for variable expression shown by C. fetus (66). As in the latter organism, there was no evidence of an N-terminal signal sequence, implying export by a Sec-independent route. The significance of the C. rectus S-layer protein in pathogenesis remains unclear, with some indication based on studies with defined mutants that it may act to downregulate induction of inflammatory cytokines (67). This may allow the organism to persist in the mouth without exciting strong inflammatory responses that might be disadvantageous to the organism. Evidence for pilus expression in these organisms is sparse and incomplete. Only one report has been published describing pilus-like structures, in strains of C. jejuni, C. coli and C. fetus (18) when exposed to bile salts such as deoxycholate. The structures were 4–7 nm in width and up to 1 μm long. No structural pilus protein could be identified, although in C. jejuni strain 81-176 a gene pspA predicted to encode a protease homologue was deleted and the resulting mutant no longer expressed these pili (18). Examination of the genome sequence of C. jejuni reveals that pspA is not linked to any predicted homologues of pilus protein or biogenesis genes; furthermore there is a homologue of this gene in H. pylori, albeit there have been no reports of pilus-like structures in the latter organism. There is also some independent evidence from the C. jejuni genome sequence that there may be a type II secretory pathway, homologous to known pathways for biogenesis of type 4 pili as well as other cellular functions involving extracellular secretion. A gene predicted to encode a prepilin peptidase, Cj0825, is present although not adjacent to any other known pilus gene homologues; it is clearly distinct from the protease pspA described above. In addition a cluster of genes from Cj1470c to 1474c includes four genes predicted to encode type II or general secretory pathway functions. None of these gene homologues is present in the genome of H. pylori. There is no direct evidence of their role in C. jejuni. In both helicobacters and campylobacters, as in many motile intestinal pathogens, motility and chemotaxis are well established virulence factors, and for C. jejuni they are one of the best proven experimentally. The genetics, biogenesis and regulation of expression of flagella, their role in virulence and their antigenicity are therefore of great interest. Flagella comprise one of the most complex surface structures of bacteria and in these organisms, as in the Enterobacteriaceae in which they have been best characterized, a large and complex flagellar regulon governs their expression. A brief overview of the biogenesis and genetics of flagella in the enteric bacteria S. typhimurium and E. coli (for review see 43) will be useful in highlighting important or novel features in Helicobacter and Campylobacter. Structurally, flagella can be broken down into the basal structures, including the motor and switch which are embedded in the cytoplasmic membrane, and the rod which traverses the cell wall; the hook; and the filament, which is the helical external structure. With the exception of the existence of a flagellar sheath in H. pylori (see below), the basic structure is broadly similar in H. pylori and C. jejuni to those of better characterized Gram-negative bacteria such as S. typhimurium. In the enterics, 50 or so genes involved in flagellar expression are clustered in three main chromosomal regions I, II and III and are named accordingly flg, flh and fli. Homologues of the majority of these genes are also present in H. pylori and C. jejuni, but frequently are differently arranged and scattered in numerous loci around the chromosome. Nevertheless for ease of comparison the nomenclature for the enterics should be retained where possible. In addition to their positional groupings, genes are also grouped in the enterics into classes, which do not fully coincide with their location, according to their hierarchical expression during the sequential assembly of the complex flagellar structure. Class 1 genes consist only of a ‘master operon’flhDC which must be activated before any other flagellar gene can be expressed. Transcription of the master operon is controlled by mechanisms including catabolite repression. Class 2 genes are generally involved in basal body and hook expression, and include an alternative RNA polymerase sigma factor, σ28, which is essential for expression of class 3 genes including the single filament protein FliC. Thus the hierarchy of gene expression is sequential and linked to the growth of the flagellar structure, which is assembled from the inside outwards, first the cytoplasmic membrane-associated components, then the rod and hook, and finally the filament. Nearly all the rod, hook and filament components are exported from the cytoplasm via an export apparatus at the cytoplasmic end of the rod, which initiates transport of distal components through a channel in the centre of these structures. Additional complexities include check points in flagellar assembly whereby, first, the growth of the hook by sequential addition of monomeric subunits is controlled by a hook length sensing and regulatory component FliK. When hook length is sufficient its assembly is halted and at this point an antisigma fac" @default.
W2040468286 created "2016-06-24" @default.
W2040468286 creator A5061733701 @default.
W2040468286 date "2001-06-01" @default.
W2040468286 modified "2023-09-24" @default.
W2040468286 title "Surface components of Campylobacter and Helicobacter" @default.
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