Serotyping of Campylobacter jejuni based on heat stable antigens: relevance, molecular basis and implications in pathogenesis (1999)

It has been recognized since the 1970s that Campylobacter jejuni, previously named Vibrio fetus and Camp. fetus subsp. jejuni, is a major cause of enteric infections in humans and animals ( Skirrow 1977; Penner 1988). Campylobacter jejuni is the most common bacterial cause of diarrhoea in many industrialized countries and is consequently responsible for a major public health and economic burden ( Ackerley & Jones 1985; Anon 1995; Buzby et al. 1997 ). Also, some strains of the species are associated with the development of diseases of the peripheral nervous system in humans ( Kuroki et al. 1991 ; Mishu & Blaser 1993; Rees et al. 1995 ) which further adds to economic burden ( Buzby et al. 1997 ). Studies on the pathogenesis of Camp. jejuni have yielded insights into the molecular mechanisms by which this bacterium causes disease ( Walker et al. 1986 ; Penner 1988; Ketley 1997), and may lead to the development of new methods for detecting and controlling the spread of this bacterium. Numerous studies of Camp. jejuni infections have been carried out and the epidemiology is now well understood, due, in large measure, to the application of one or both of two systems for serotyping clinical isolates ( Penner & Hennessy 1980; Lior et al. 1982 ). Other strain typing methods have been developed and have met with more limited success. Phage typing has been developed, but used in only a limited number of studies on Camp. jejuni ( Grajewski et al. 1985 ; Salama et al. 1990 ; Patton et al. 1991 ). Similarly, DNA typing, based on total genomic digest patterns from conventional ( Owen & Beck 1987; Bruce et al. 1988 ; Owen et al. 1989 ; Hernandez et al. 1991b ; Patton et al. 1991 ) and pulsed-field gel electrophoresis ( Yan et al. 1991 ; Suzuki et al. 1994 ), ribosomal RNA gene restriction digest patterns ( Geilhausen et al. 1990 ; Owen et al. 1990 ; Hernandez et al. 1991a , b; Patton et al. 1991 ; Fayos et al. 1992, 1993 ; Tee et al. 1992 ) and plasmid profiling ( Tenover et al. 1984 ; Fayos et al. 1993 ) have been applied to a limited extent. The polymerase chain reaction has been used to obtain randomly amplified polymorphic DNA (RAPD) profiles, which provide an additional and apparently discriminatory method of typing Camp. jejuni strains ( Mazurier et al. 1992 ; Fayos et al. 1993 ; Giesendorf et al. 1994 ; Fujimoto et al. 1997 ), and to perform restriction fragment length polymorphism analysis ( Nishimura et al. 1996 ; Fujimoto et al. 1997 ). The serotyping system which differentiates strains on the basis of heat-labile (HL) antigens was developed according to classical methods used for identifying flagellar antigens of Salmonella, Escherichia coli and other Gram-negative species ( Lior et al. 1982 ). This system depends on the absorption of antisera with heated bacterial suspensions of the homologous strains and the heterologous cross-reacting strains to remove antibodies directed against HS antigens. The traditional methods of bacterial cell agglutination are used to identify the specificities of the HL antigens. Progress in elucidating the biochemical characteristics of these antigens has been stimulated by interest in mechanisms of pathogenesis and host cell invasion. The antigens have been asserted to be flagellar proteins and studies on the genetics of flagella are underway to determine if this is the case ( Guerry et al. 1992 ; Ketley 1997). In a departure from the traditional approach, the passive haemagglutination (PHA) technique, formerly used primarily for research on endotoxins ( Neter et al. 1956 ), was introduced in 1980 as the procedure for routine serotyping ( Penner & Hennessy 1980). The system differentiates strains on the basis of soluble heat-stable (HS) antigens and has been used successfully in numerous laboratories for epidemiological studies. In some investigations it was the only system used and in other studies it was used in conjunction with the HL typing system (e.g. Penner et al. 1983 ; Jones et al. 1985 ; Kaijser & Sjögren 1985; Kuroki et al. 1993 ; Patton et al. 1993 ; Lastovica et al. 1997 ). A renewed interest in the use of the HS antigen typing system has arisen since HS antigens of particular serotypes have been implicated as possible pathogenic factors in the development of Guillain–Barré syndrome (GBS) and Miller–Fisher syndrome (MFS) ( Kuroki et al. 1993 ; Yuki et al. 1994 ; Moran et al. 1996a ; Salloway et al. 1996 ; Lastovica et al. 1997 ). The HS antigens have been referred to by such names as ‘Pen’ (for Penner) and ‘O’ to reflect the assumption that they are lipopolysaccharides (LPSs), but in this brief review, the original designation of ‘HS’ will be used. The discovery of neuropathic strains of Camp. jejuni has given urgency to research into determining the molecular structures of the HS antigens, particularly those of strains implicated in neuropathogenesis. Progress to date indicates that the HS antigen is of a novel type, incorporating features of both enterobacterial high molecular weight (-Mr) LPSs and the non-enterobacterial low-Mr lipooligosaccharides (LOSs) as shown in Fig. 1 ( Rietschel et al. 1990 ; Moran 1995a; Preston et al. 1996 ). Rapid progress has been made in gaining new insights into the nature of the unique HS antigen and in this review, attention is focused on the present state of knowledge of the biochemistry and structure of the HS antigens and their possible role in pathogenesis. Schematic representation of the general structure of (a) high-M▵ LPS and (b) low-M▵ LOS In the first attempts to classify strains of Campylobacter species using both slide and tube agglutination tests, three antigens, believed to be O antigens (designated O serotypes A, B, and C), were recognized ( Berg et al. 1971 ). With these procedures, 10 isolates that would currently be classified as either Camp. jejuni or Camp. coli were all found to belong to serotype C. Other investigators of Campylobacter serotyping ( Ristic & Brandly 1959; Winter 1966; Newsam & St. George 1967; Bokkenheuser 1972) used the PHA technique which is known to be effective for identifying enterobacterial LPS antigens ( Neter et al. 1956 ). In an initial trial to define HS antigens of the bacteria then known as Camp. fetus subsp. jejuni, only weak cross-reactive and non-reproducible agglutinations were observed in bacterial agglutination tests and, therefore, the PHA technique was evaluated for serotyping ( Penner & Hennessy 1980). As in the earlier studies ( Ristic & Brandly 1959; Winter 1966; Newsam & St. George 1967; Bokkenheuser 1972), the use of the PHA technique and typing antisera (produced in rabbits against bacterial cell suspensions), enabled the differentiation of strains on the basis of HS antigens. In the initial study, 23 HS antigens were identified in Camp. fetus subsp. jejuni (Penner and Hennessy l980). With the advantage of the hippurate hydrolysis test ( Harvey 1980) for distinguishing Camp. jejuni from Camp. coli, the serotype reference strains (serostrains) were separated to produce a system for each species. The systems have been extended to include 42 serostrains for Camp. jejuni and 18 for Camp. coli (Penner et al. l983; Mills et al. l991, 1992). Tests using hybridization of DNA confirmed the separation of the species on the basis of the hippurate hydrolysis test and other bacteriological tests ( Leaper & Owen 1982). These serotyping systems have been used in reference laboratories to investigate the epidemiology of enteric infections and, in particular, the system for Camp. jejuni has also become useful in studying strains associated with the development of GBS and MFS ( Kuroki et al. 1993 ; Yuki et al. 1994 ; Lastovica et al. 1997 ; Nachamkin 1997; Penner & Aspinall 1997), diseases of the peripheral nervous system ( Fisher 1956; Hughes 1990). Passive haemagglutination (PHA) has been known for decades as a highly sensitive technique for examining the specificities of LPS ( Neter et al. 1956 ). Briefly, extracted LPS molecules adhere to the surface of mammalian erythrocytes, a process referred to as sensitization, and antibodies specific for LPS cause agglutination of the sensitized erythrocytes. As the erythrocyte is much larger than the bacterial cell, the detection of an antigen-antibody reaction is more sensitive with the PHA technique than with techniques employing bacterial cell agglutination. The basis for the sensitization is the presence in the erythrocyte membrane of a 228 kDa lipoglycoprotein receptor that binds LPS but not homopolysaccharides, heteropolysaccharides, monosaccharides or protein antigens of either Gram-negative or Gram-positive bacteria ( Springer et al. 1973 ). Like LPS molecules, the HS antigens of Camp. jejuni and Camp. coli that sensitize erythrocytes may be extracted by the hot phenol-water method ( Westphal & Jann 1965) by treating bacterial suspensions with ethylenediaminetetraacetate (EDTA) according to the method of Leive et al. (1968) , or by heating saline suspensions of the bacteria ( Neter et al. 1956 ). Although the titres of the PHA reactions observed with products of the various extraction procedures vary from one product to the next, the antigenic specificities are virtually identical, indicating that the different extraction procedures yield essentially the same antigenic material ( Penner & Hennessy 1980; Preston & Penner 1987). For routine serotyping, the method of heating cell suspensions is used primarily because it is the least labour-intensive method of extracting antigenic material and is more practical in the general microbiology laboratory. The sensitized erythrocytes are washed three times to remove any less firmly bound heat-labile bacterial debris. Thus, the washings serve as a purification step to ensure that the serological reaction is restricted to that between the HS antigen and the antibodies against the HS antigen. Conversely, as only the antibodies directed against the HS antigen participate in the reaction, there is no requirement for costly and labour-intensive absorptions of antisera to remove antibodies against other bacterial components. There continues to be interest in simplifying the serotyping system by eliminating the PHA step. In adopting alternative techniques, such as coagglutination ( Kosunen et al. 1982 ; Wong et al. 1985 ; Fricker et al. 1986 ; Illingsworth & Fricker 1987) or agglutination in microtitration plates ( Frost et al. 1998 ), it is necessary to emphasize that in these procedures there is no equivalent of the erythrocyte LPS receptor to select HS antigen preferentially from the antigenic preparation. In these alternative systems, measures must be taken to provide either purified HS antigens or purified immune reagents, such as monoclonal antibodies, or multiply absorbed polyclonal antisera to achieve reaction specificity ( Moran & Kosunen 1989). As routinely performed in most laboratories, the serotyping procedure consists of dispensing suspensions of heat-extracted, antigen-sensitized sheep or human erythrocytes into wells of microtitration plates containing antiserum in twofold dilutions. The plates are incubated overnight at 37 °C and the highest dilution of the antiserum in which haemagglutination occurs is recorded as the titre ( Penner & Hennessy 1980; Penner et al. 1983 ). Dilution of the antisera may be performed manually or with automated titration equipment. The titration step is omitted in the rapid slide agglutination technique; in this technique, the antiserum is added directly to sensitized and thrice-washed erythrocytes on a slide, which is subsequently gently rocked for 5 min, to observe the occurrence of haemagglutination ( Mills et al. 1991 ). A major advantage of these procedures is that only the heat-extracted antigenic material is required for serotyping by the reference laboratory. The shipment of extracts eliminates the problem of viability loss during transport and reduces the hazard associated with shipment of live cultures ( Lastovica et al. 1986 ). The observation that the HS antigens of Camp. jejuni adhere to erythrocyte membranes and can be extracted by procedures specific for LPS ( Penner & Hennessy 1980) indicates that they are similar to the LPSs of Enterobacteriaceae or the LOSs of Neisseria and Haemophilus ( Rietschel et al. 1990 ; Moran 1995a; Preston et al. 1996 ). The HS antigens are unlikely to be proteins as these are generally thermolabile and would not withstand the heat treatment of the HS antigen extraction procedure. Moreover, the inability to stain phenol-water extracts with Coomassie blue (a protein stain) indicates a lack of protein in the HS preparations ( Preston & Penner 1987). Also, proteins require coupling reagents such as tannic acid, glutaraldehyde or bis-diazotized benzidine to sensitize erythrocytes ( Boyden 1951; Onkelinx et al. 1969 ). Nor are the HS antigens likely to be typical lipid-free capsular polysaccharides which are unable to adsorb to erythrocytes without coupling reagents such as chromium chloride, cyanogen bromide or stearoyl chloride ( Hämmerling & Westphal 1967; Baker et al. 1969 ; Ammann & Pelger 1972; Eriksen 1973; Richter & Kågedal 1974). However, Chart et al. (1996) could not demonstrate the reaction of antisera against serostrains with homologous high-Mr LPS in immunoblotting and suggested that capsular polysaccharides were the HS antigens involved in serotyping. The inability to demonstrate a reaction with LPS in the former study may have been due to technical problems as Jones et al. (1984) inhibited PHA reactions of HS antigens with purified LPSs. Furthermore, these latter investigators showed, using eluates from electrophoresis gels of heat-extracted antigen preparations, that LPSs are involved in serotyping Camp. jejuni by PHA, whereas other bacterial cell surface components had no activity. Neveretheless, to test the possibility that polysaccharide antigens are involved in the PHA system of serotyping Camp. jejuni, extracts were prepared by methods used to isolate lipid-containing capsular polysaccharides of Neisseria meningitidis ( Liu et al. 1971 ; Gotschlich et al. 1981 ). The presence of polysaccharides was demonstrated, but they were found to have the same antigenic specificities as the HS antigen and purified LPS, and to contain 2-keto-3-deoxyoctulosonic acid (Kdo), an 8-carbon acidic sugar unique to LPSs and LOSs ( Moran & Kosunen 1989). The authors concluded that the polysaccharides were derived from LPS as a result of the polysaccharide extraction process employed, and evidence for capsular components like those of N. meningitidis was not obtained. Further evidence that the HS antigens of Camp. jejuni are of the LPS or LOS type molecule was obtained in experiments in which both phenol-water extracts ( Westphal & Jann 1965) and proteinase K-treated whole-cell lysates ( Hitchcock & Brown 1983) of serostrains were examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) with LPS-specific silver staining ( Tsai & Frasch 1982; Mills et al. 1985 ; Preston & Penner 1987, 1989). With these procedures, only low-M▵ components, ranging in M▵ from 4500 to 5000, were detected for each of the 38 strains examined ( Preston & Penner 1987). Similar results were obtained by other investigators (Logan and Trust l984; Perez-Perez & Blaser 1985). The presence of only low-M▵ components implied that the HS antigens are OSs that resemble either the LOSs that occur in Haemophilus and Neisseria ( Inzana et al. 1985 ; Mandrell et al. 1986 ; Moran et al. 1996b ), or the cores of rough enterobacterial mutant strains ( Rietschel et al. 1990 ). However, the observation that these short molecules display a wide diversity of antigenic specificities, a feature uncharacteristic of enterobacterial rough strains, indicated that they are more closely related to the LOSs. Investigators studying the chemical composition of the HS antigens suggested that they were LOSs ( Adeyeye et al. 1989 ; Conrad & Galanos 1990). Further evidence supportive of this suggestion was the finding that three Camp. jejuni serostrains for serotypes HS:1, HS:2 and HS:4 possess sialic acid (N-acetylneuraminic acid, Neu5Ac)–containing oligosaccharides (OS) in the low-M▵ molecules ( Moran et al. 1991a ). As terminally linked Neu5Ac is a feature characteristic of the LOSs of Neisseria and Haemophilus ( Moran et al. 1996b ; Preston et al. 1996 ), the view that the HS antigens of Camp. jejuni are LOSs has gained support. Unexpected, however, was the finding that not all of the Camp. jejuni strains have HS antigens that conform completely in structure to the LOS molecule. When electrophoresed extracts of HS antigens were immunoblotted with homologous antisera, 16 of 38 serostrains were shown to have, in addition to the low-M▵ components, a series of high-M▵ components in ladder-like patterns typical of LPS molecules with O chains ( Preston & Penner 1987, 1989). This new evidence indicated that the species consists of two classes of strains; one class with HS antigens consisting only of low-M▵ components, and the second class with HS antigens that have both low-M▵ and high-M▵ components. Examples of reactions of six serostrains are shown in Fig. 2. Although it has been reported that high-M▵ components could be visualized by silver staining under certain conditions ( Blake & Russell 1993), this has not been confirmed by other investigators. Instead, re-electrophoresis of high-M▵ material from LPS gels and LPS radiolabelling experiments have indicated that high-M▵ bands are aggregates of low-M▵ LPS ( Logan & Trust 1984; Mills et al. 1985 ). SDS-PAGE gel of proteinase K digests of six Campylobacter jejuni serostrains were (a) silver-stained and (b) immunoblotted with homologous antisera. Lanes: 1, serostrain HS:17; 2, serostrain HS:44; 3, serostrain HS:52; 4, serostrain HS:12; 5, serostrain HS:21; 6, serostrain HS:33. Note the ladder-like banding in lanes 4, 5 and 6 of panel (b) Immunoblotting of electrophoresed extracts with both homologous and heterologous antisera also showed that both the low-M▵ and high-M▵ components are strain-specific antigens ( Preston & Penner 1987, 1989; Moran & Kosunen 1989). These unanticipated complexities clearly indicated that neither the typical LPS nor LOS molecule constitutes a model that accommodates the structure of the HS antigen of Camp. jejuni. The HS antigens of Camp. jejuni and Camp. coli reflect, in some cases, the two component structure of LOSs composed of lipid A and core OS ( Moran et al. 1996b ; Preston et al. 1996 ) and, in others, the three component LPSs composed of lipid A, core OS and O-polysaccharide chain ( Rietschel et al. 1990 ; Moran 1995a) ( Fig. 1), except for the greater diversity in the structure of their core OS. It is not surprising therefore that much attention has focused on determining the structures of the HS antigens because of the interest in investigating the basis for the unique combination of components from these two distinctly different bacterial glycolipids. Progress to date includes the complete determination of the structure of lipid A of one strain, Camp. jejuni serostrain HS:2 ( Moran et al. 1991b ). The molecular structures of the HS antigens have been determined for serostrains HS:1, HS:2 and HS:3 that show only low-M▵ components in immunoblots ( Aspinall et al. 1993a , b, 1995). The structures of the HS antigens have also been determined for serostrains HS:19, HS:23 and HS:36 that have both high-M▵ and low-M▵ components ( Aspinall et al. 1992a ; 1993b; 1994b,c). Only the low-M▵ component of serostrain HS:4 has been analysed structurally ( Aspinall et al. 1993b ). In addition, the structures of both low-M▵ and high-M▵ molecules of five serotype HS:19 isolates from patients with the GBS have been determined ( Yuki et al. 1993 ; Aspinall et al. 1994b , c; Moran & O’Malley 1995). The structure of the low-M▵ molecules of a serotype HS:41 isolate from a GBS patient ( Prendergast et al. 1998 ), and of a serotype HS:10 isolate and a serotype HS:23 isolate from patients with MFS, have been determined ( Salloway et al. 1996 ; Aspinall et al. 1998 ). Although the structures of only a small number of HS antigens have been the results have to new insights into unique previously in Gram-negative The structures are in the The lipid A of LPS and LOS molecules from bacterial species adhere to a similar in which occur three a or linked to the and acid chains bound to the from one bacterial species to may occur in one or more of these three components, but the is essentially ( Rietschel et al. 1990 ; et al. 1994 ; Moran In Camp. jejuni, however, there is more than in most Gram-negative three different lipid A In Camp. jejuni serostrain of the lipid A molecules have a of and have in which both in the are and have of two which is the composition most typical of in enterobacterial lipid three are and in the same ( Moran et al. 1991b ; Moran The occurrence of both and is a common of all Camp. jejuni serostrains examined ( Moran et al. 1991a ; Moran analysis of lipid A from different Camp. jejuni serostrains has confirmed the presence of the same type of structures to those in lipid A of Camp. jejuni serostrain but the of the various ( Moran Thus, in Camp. jejuni lipid A may occur ( Moran In examining the structures of the core OSs of Camp. jejuni HS antigens of serostrains HS:1, HS:19, HS:23 and HS:36 ( Fig. it is that two distinctly different in each OS ( Aspinall et al. ; 1995; et al. 1998 ). to lipid A is the OS which includes a of and two The to is by in all OSs examined whereas the second is by only in the OSs of reference strains for serotypes and HS:2 ( Aspinall et al. 1993a , The only other in the OS is the or at 6 of the first of the core oligosaccharides of Campylobacter jejuni serostrains HS:2 HS:3 HS:4 and HS:19 HS:23 and HS:36 acid The of the core OSs are much more in structure ( Fig. consists of either two or three which are terminally or with sialic acid more with the OSs in of the in the of the and in the and provide further These provide the diversity that differentiation of strains that have HS antigens with only low-M▵ components ( Aspinall et al. ). It be that the core OSs of serostrains HS:4 and HS:19 are structurally ( Aspinall et al. 1993b ; Fig. Similarly, the OSs of serostrains HS:23 and HS:36 are ( Aspinall et al. 1993b ) but different from those of serostrains HS:4 and HS:19 ( Aspinall et al. 1993b ; these it is that a wide of core OS structures are present in this bacterial but also that strains of different serotypes may the same OS with OSs are on the basis of their polysaccharide in the high-M▵ components of serotypes HS:19, HS:23 and HS:36 that are in a for the OS of serotype ( Moran et al. 1991a ; Aspinall et al. 1995 ; Moran the OSs of all other serotypes for which the molecular structures have been determined contain one to three of sialic acid linked terminally to of ( Fig. The presence of the sialic acid the structures of the OSs similar to the of human ( Moran et al. 1996a , The OSs of serostrains HS:1, HS:23 and HS:36 ( Fig. have structures that the of the ( Aspinall et al. 1993b ), whereas the OSs of serotypes HS:4 and HS:19 ( Fig. the of ( Aspinall et al. 1993b ; molecules the sialic acid were also found in the low-M▵ components of both serotype HS:4 and These OSs the ( Moran et al. 1996a , The of the OS of the serostrain HS:2 ( Fig. is present as the of and is also present as the in other and ( Aspinall et al. 1993a ; Moran et al. 1996b ). The molecular of human and the for Camp. jejuni pathogenesis have been ( Moran et al. 1996a , In Camp. jejuni LPS can antibodies in animals ( et al. 1995 ; et al. 1996 ), and GBS and MFS patients with Camp. jejuni have to during the of ( Yuki et al. 1990 ; et al. 1995 ; et al. 1995 ; et al. 1997 ). Furthermore, antibodies at the of and ( et al. 1991 ; et al. 1995 ) and can and, with the ( 1995). The OS of serostrain HS:3 ( Fig. is different from those of the other serostrains in that it not contain sialic It also has an linked as a A Camp. jejuni strain of serotype and serostrain HS:3 are the only in the species in which the has been detected to date ( et al. 1986 ; Aspinall et al. 1995 ). Although no high-M▵ components were visualized immunoblotting the electrophoresed phenol-water from serostrain HS:3 with homologous antiserum ( Preston & Penner on chemical the was found to contain a polysaccharide The consists of of but has no which indicates a lack of to the core OS ( Aspinall et al. 1995 ). all the serotypes for which the HS antigen structure has been the core OS of this serotype is the only one that not known structures of human components. Thus, extracts of HS antigen and LPS from this strain can serve as in experiments with