Surface components of Campylobacter and Helicobacter (2001)

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 in this The presence of these acid residues is of also in for of host This may the of in organisms as and the serotype of coli, in which the presence of for work by that a specific of the in N-acetyl acid is in LOS appears to be specific for flagellin In H. pylori, lipopolysaccharide has been well The of is the presence in the chain of of galactose and N-acetyl galactosamine, host structures and with potential to autoimmune data on LOS can be from the genome sequence data In of the for lipid A and inner core are in the a of in outer core LOS and that was by 24 and This region of the also that may be in protein glycosylation but is from the region and described The exact of the molecular weight, structures A case has been made by that this at least in some strains including serotype a structure distinct from LOS and not lipid A or LOS core structures. This observation from a genome to the of the genome sequence of which revealed and homologues of in polysaccharide in other bacteria. of the genome sequence revealed that to polysaccharide and in other were in a specific region of the of these gene homologues were from the H. pylori A observation was that in the or led to of high molecular weight, protease-stable and that serotype in the was also in the antigen to a lipopolysaccharide O-antigen to be a polysaccharide. This work by at the of The major outer membrane protein of C. jejuni is by the most highly protein in this as shown by and apparently in molecular weight from to presence that it is for the organism. There is substantial evidence for its as a shown by in lipid the protein, yet it appears to have little significant sequence with porins of other Gram-negative and is no in H. pylori. The are believed to be and The protein of structure in at first from a trimeric to a the the protein is to protein not the in its by antibodies to the produced in or not the SDS–PAGE and The of the to is although is some evidence of its in to host cells or also has properties with There is little evidence of any other outer membrane proteins in C. jejuni, which from genome sequence data appears not to possess homologues of the major porins of the The large family of porin proteins of H. pylori also appears from the genome sequence to be to be are porins or other outer membrane proteins the very large number to of membrane proteins, including membrane proteins and of from the genome sequence of the genome sequence a number of outer membrane is with a probable outer membrane protein in H. pylori, of and surface antigen of is a of a protein of and of A of which are of a the interesting of high molecular weight to an S-layer protein of In the first evidence of high or based on a This sequence is subject to which is to to expression as a gene or as two separate proteins The significance of these proteins is outer membrane and proteins, described from the genome the A significant protein is identified by its to in a the protein is believed to in the outer membrane presence has been in by and chain a gene from of C. jejuni and of C. coli suggesting it is important in the by these closely related it appears to be for of a not gene is present in H. pylori, suggesting a unique to campylobacters, and based on sequence the protein some with features of porin and to of proteins, and present in acid glycine extracts were identified as antigenic and identified cell and were shown to be identical to and and evidence for a possible cell surface for was that may in be a periplasmic protein for an acid and that may also be in the proteins clearly with the outer membrane a by of which a is found in H. pylori where it is in virulence Both proteins are homologues of the family of outer membrane Also a of a widely family is the outer membrane lipoprotein this protein probably has a and the gene is in the other outer membrane proteins have been A protein with to protein of to A protein described by and by in to cell membranes and sequence to although is a major in molecular for these two described a surface protein in to it was present in some but not all strains of C. jejuni, and to the of In addition, two have evidence of expression of proteins, including surface components, only in expression of proteins of and in but not in that outer membrane proteins of and were only by in and not in These proteins not appear to have been The most of outer membrane proteins is the large family of porins which were identified from the genome sequence on the of sequence features at the and the of the The latter a characteristic of and acid residues. A of this has been well of low it porin with of the to large molecular was shown that the protein in coli was capable of formation as by H. pylori, and the of was to a with Another of the has also been and shown to be to a as described based on a of in the region of the gene the A in this gene to In of related of this extended family of proteins, two of evidence their potential in identification of of to to cell identified the as a of to a distinct from the in with other and sequence although the first is to be a lipoprotein and the to have a Another example of a in of an outer membrane protein is that of and described by of in of the porin family for to in with cell identified as a of The protein was membrane protein in of this and may have a in of This extended and family of porins has been reviewed in detail and shown to be into The by the first described a characteristic and includes or as well as and proteins that not possess the are or are on the of the acid residue, which in of the family is but in is As in C. jejuni, the outer membrane A has been in H. pylori. that a in and as well as to although its to to a cell was The exact of this protein in outer membrane proteins of H. pylori with a potential as antigens or in lipoprotein a protein, and outer membrane In the outer membrane proteins of both C. jejuni and H. pylori are a of probable and potential in virulence and of these There is significant evidence of both and suggesting that of these proteins is a key in the of in the of host the close phylogenetic relationship of these organisms, they in outer membrane protein in the of the major outer membrane protein in C. jejuni and of the large family of porins in H. pylori. are ordered surface described as a distinct external to the outer membrane in Gram-negative and usually comprising a highly high molecular weight S-layer proteins have been reviewed recently by S-layer proteins have to be and to have properties of to which to a for the S-layer proteins were first in the Campylobacter in C. fetus, following the observation that organisms the high molecular weight, protein a capsule-like outer were to of and or these proteins were to This of strains to host is significant for the which and of surfaces of with which can to and In extensive on the of expression of these proteins, and explored the of and These for a of antigenic of S-layer protein to be of host (reviewed by The S-layer proteins, in molecular weight from to not appear to be of S-layer proteins in other they are and high of and low of The proteins appear to be secreted by a using a type to that of coli to the outer membrane is and dependent on with and is serotype strains with type A have surface proteins type strains have proteins have been described in the campylobacters, C. jejuni or C. coli, and no to the have been in the C. jejuni genome There an interesting of section that may homologues of S-layer proteins of with potential for no evidence of their expression in the has been Campylobacter rectus is a S-layer proteins in this were first by The proteins strains with molecular from to and some differences were have shown a for the protein in to by and in the absence of specific of a gene an S-layer protein and a for gene homologues that it was a with no evidence of the for expression shown by C. fetus As in the latter was no evidence of an by a The significance of the C. rectus S-layer protein in with some based on with that it may to of This may the to in the strong that be to the organism. for expression in these organisms is and has been published structures, in strains of C. jejuni, C. coli and C. fetus to as The structures were in and to protein be although in C. jejuni a gene to a was and the no these of the genome sequence of C. jejuni that is not linked to any homologues of protein or biogenesis is a of this gene in H. pylori, have been no of structures in the latter organism. There is also some evidence from the C. jejuni genome sequence that may be a type to known for biogenesis of type as well as other cellular functions A gene to a is present although not to any other known gene it is clearly distinct from the described In a of from to includes to type or of these gene homologues is present in the genome of H. pylori. There is no direct evidence of their in C. In both helicobacters and campylobacters, as in and are well virulence factors, and for C. jejuni they are of the The biogenesis and regulation of expression of flagella, their in virulence and their are of great Flagella of the most surface structures of and in these organisms, as in the in which they have been a large and flagellar their expression. A of the biogenesis and of flagella in the typhimurium and coli review see will be useful in important or features in Helicobacter and flagella can be into the structures, including the and which are in the cytoplasmic membrane, and the which the cell the and the which is the external the of the of a flagellar in H. pylori the basic structure is in H. pylori and C. jejuni to those of Gram-negative as In the or so in flagellar expression are in three and and are and of the majority of these are also present in H. pylori and C. jejuni, but are and in the Nevertheless for of comparison the for the be where In to their are also in the into which not with their to their expression the of the flagellar only of a which must be any other flagellar gene can be of the is by including are generally in and and an which is for expression of including the protein the of gene expression is and linked to the of the flagellar structure, which is from the first the cytoplasmic components, then the and and the all the and components are from the an at the cytoplasmic of the which of components a in the of these structures. in flagellar the of the by of is by a and is its is and at this an which that of is to from the cell the of the flagellar structure by the of a protein The of to expression of in work on the of Campylobacter and their was by and recent genome that of the or which flagellar biogenesis in the is present in H. pylori or C. jejuni genome to date that the is to the the absence of which its in the Enterobacteriaceae, is present in both H. pylori and C. jejuni, and is for expression of the major flagellin a to the expression of the flagellin in the the by which in other a variety of functions including is in flagellar expression in both H. pylori and C. In both organisms the protein as well as flagellin are from these two factors, which appear from the genome to be the only to the have to date been only in flagellar gene expression in of these A for in flagellar in the is quite in other including and These organisms all possess flagella and it is to that this is a them from the and differences in flagellar and of expression. flagella a different method of flagellar gene regulation linked to cell new cell requires new found in the are from H. pylori or C. jejuni or These a for of the structure the and which a in flagellar a a or of and of as and are also the other of as which to but flagella, are present in both organisms and in typhimurium A C. jejuni and H. pylori is that the the gene the protein in the only published of regulation of flagellar in these organisms is that of detailed the of the a protein which in with A of known factors, is its on proteins which it to formation by the to the These are dependent activity, and to of the in other in which they have been is known of in H. pylori or C. In some the of are by the of work by that the in H. pylori, which has sequence to is by a that of led to of expression of and identified as gel by this have been to of in turn of and and of flagellar or and identified components of the flagellar and as well as the flagellin with were to of these and, based on of in a it was shown that was for their that of the and is dependent on and also that a on expression of major flagellin although no of protein expression was flagellar expression in H. pylori to be by the of this is not clear expression of the is and expression of the major flagellin is in in which are to the and and that in the described expression is This with and that of is in the of in and its be to be For both H. pylori and C. jejuni of flagella and the to are well for virulence in in and in The evidence is from the of virulence or properties of or in comparison with is widely that and are for of the and so that in both organisms they may in to the gastrointestinal has been that H. pylori requires to be quite the so that it is from the of is not of at the from host in the is little evidence of to the cell surface, although microscopy that organisms to in the The flagellar is of in to this very has been that the may to of the flagellar a property of flagellar in low A antigenic lipoprotein originally and believed to be a surface or outer membrane has more recently been shown to to the flagellar exact in this and evidence of its as an is The specific of flagella and in C. jejuni is is evidence that cells are than it is not clear this is to a specific of flagella in cell is possible that to the in or to close with the cell surface in by motility, may of There is evidence that flagella of both H. pylori and C. jejuni are strong antigens, and flagellin protein in Western of cellular proteins with or by with bacteria. is also well that the flagellar antigen to antigenic strains of C. jejuni, although not in H. pylori. The antigenic in C. jejuni flagella is to be the in the antigens known as the data this flagellin that is in the region of the flagellin proteins, and flagellin gene have also been as a for molecular As in other the and of the protein appear more and are probably in molecular in flagellar appears that in C. jejuni, at least and in only is to any significant although in is clearly some expression of There is also evidence that the flagellar protein in and this may be a significant of antigenic strains In H. pylori, is also in flagellin gene the acid of and their appear to be highly a for of the In this is at a significant comprising or more of the total flagellin present in cells in The exact of and in the flagellar and their but the use of different in their is of of into the at an than at the of the of proteins is and quite recently has been about the of in bacteria. that and S-layer proteins, as well as for are is well that the as well as a number of as yet proteins of C. jejuni are date is no evidence of in H. pylori. For it was known that to be in the flagellar proteins of C. coli, based on the of flagellar antigenic a and on of flagellin molecular weight These observations into two that of both C. coli and C. jejuni were to and and evidence of and other properties of the presence of structures. in and strains based on and specific for structures, as well as to that a variety of proteins are also in this is that are by C. is that a of revealed by genome including at least of a acid gene to that for is in flagellin glycosylation have been in the antigenic of surface components of C. jejuni, in the of development of This work is and to in the of genome sequence and into the and of polysaccharide and are generally the surface components of C. jejuni are in the of this organism. In the case of H. pylori, polysaccharide protein glycosylation appears at present to be in its surface structures. into the of outer membrane proteins more detailed in the latter organism. In both of lipopolysaccharide structures and their potential in autoimmune processes in and in molecular mimicry is