Current Issues in Intestinal Microbiology 2001. 2(2): 55-71.
1School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-4233, USA
2Departments of Veterinary Science and Microbiology, University of Arizona, Tucson, AZ 85721, USA
Abstract
Campylobacter jejuni, a gram-negative spiral shaped bacterium, is a frequent cause of gastrointestinal food-borne illness in humans throughout the world. Illness with C. jejuni ranges from mild to severe diarrheal disease. This article focuses on Campylobacter virulence determinants and their potential role in the development of C. jejuni-mediated enteritis. A model is presented that diagrams the interactions of C. jejuni with the intestinal epithelium. Additional work to identify and characterize C. jejuni virulence determinants is certain to provide novel insights into the diversity of strategies employed by bacterial pathogens to cause disease.
Introduction
The genus Campylobacter contains 14 species of which C. jejuni, C. coli and C. fetus are the most frequently isolated from humans. Campylobacter species are gram-negative rods that have curved or spiral morphology and are motile by means of unipolar or bipolar flagella. They grow best in microaerophilic atmosphere at temperatures ranging from 37 to 42°C. The genome of Campylobacter jejuni is roughly 1.6 to 1.7 Mbp with a GC ratio of approximately 30% (Owen and Leaper, 1981). Extrachromosomal elements including plasmids and bacteriophages have also been detected in Campylobacter sp. (Bradbury et al., 1983; Bacon et al., 2000).
The illness associated with Campylobacter infections ranges from mild to severe diarrheal disease, with stool specimens often containing blood and leukocytes. Other symptoms include fever, nausea and abdominal pain. The incubation period of Campylobacter enteritis ranges from two to seven days and the illness is often self-limiting. In severe cases, patients are treated with erythromycin. Experimental C. jejuni infections in humans have revealed that a low dose of organisms (500-800) is sufficient to cause diarrhea and that the numbers of individuals who incur the disease increases with higher doses (Black et al., 1988). The most notable complication of C. jejuni infections is the development of Guillain-Barré syndrome (GBS), an acute demyelinating polyneuropathy. Development of GBS follows gastrointestinal disease and is characterized by flaccid paralysis. O-side chain serotyping studies have revealed that certain Campylobacter serotypes are linked to GBS. Most GBS cases in the United States occur after an infection with serotype O:19. However, other C. jejuni serotypes have also been identified in association with GBS including O:1, O:2, O:2/44, O:4 complex, O:5, O:10, O:16, O:23, O:37, O:41, O:44 and O:64 (Reviewed in (Nachamkin et al., 1998); (Prendergast et al., 1998)).
The ecologic cycle of C. jejuni involves water, animals and food (Figure 1).
Infection with C. jejuni is most frequently acquired from the consumption and handling of chicken. Infections also occur from drinking unpasteurized milk and contaminated water. The majority of C. jejuni infections are sporadic in nature (Friedman et al., 2000). Despite this, C. jejuni is the leading cause of bacterial gastroenteritis in the United States with an estimated 2.4 million cases per year (Allos and Blaser, 1995; Altekruse et al., 1999; Allos, 2001). The incidence of C. jejuni infections in the United States is higher during the late summer and early fall with few cases occurring throughout the year (Allos and Blaser, 1995). C. jejuni affects all age groups but infants and young adults have the highest reported rates of infection (Allos and Blaser, 1995).
In this article, we focus on C. jejuni virulence determinants and their potential role in the development of C. jejuni-mediated enteritis. In the past decade, there has been an explosion of new information as researchers have begun to study the molecular basis of C. jejuni pathogenesis. While a clear picture of C. jejuni virulence determinants and their role in disease has yet to emerge, research in this area is intensifying with the development of new tools to genetically manipulate the organism (Golden et al., 2000) and with the availability of the genome sequence (Parkhill et al., 2000). Perhaps one of the most interesting questions that researchers are just beginning to address is whether C. jejuni isolates possess a repertoire of virulence genes that can be expressed discordantly or are comprised of a mosaic of virulence-associated genes. Surely a picture is likely to evolve that involves both of these possibilities. In this article, we attempt to present a global view of C. jejuni pathogenesis. In most instances, the contribution of a given gene to the organism's virulence is tenuous due to the lack of availability of a simple and inexpensive animal model to researchers. We also present a working model that diagrams the interactions of C. jejuni with the intestinal epithelium. While our current working model is based on speculation, we hope that it will serve to stimulate additional discussion and research in this area. Finally, many fine review articles have been published that focus on only one of the topics discussed below in much greater detail (Ketley, 1997; Wassenaar, 1997; Wooldridge and Ketley, 1997; Nachamkin et al., 1998; Altekruse et al., 1999; Pickett and Whitehouse, 1999; Allos, 2001). We encourage you to revisit the articles as we only briefly discuss some of this organism's virulence attributes due to space constraints.
Motility and Flagella
The role of motility in C. jejuni colonization and subsequent disease production has been intensely studied. Research in this area has been greatly aided by availability of simple and inexpensive models to study C. jejuni colonization and the ease of isolating C. jejuni nonmotile mutants by several methods including treatment of the bacteria with chemical mutagens. The flagellum of C. jejuni is composed of a basal body, hook, and filament. In C. jejuni and Campylobacter coli, the flagellar filament is comprised of two proteins termed FlaA and FlaB. Both C. jejuni flagellin proteins are synthesized concomitantly, but flaA, which is regulated by s28 (Nuijten et al., 1991), is expressed at much greater levels than flaB, which is regulated by s54 (Hendrixson et al., 2001). In contrast to C. jejuni flaA+flaB- mutants in which full-length filaments are produced, C. jejuni flaA-flaB+ mutants produce filaments that are truncated (Guerry et al., 1991; Wassenaar et al., 1991). In Campylobacter organisms, motility correlates with the synthesis of the FlaA protein. However, C. jejuni flaA-flaB+ mutants have been isolated that produce full-length flagella and are motile (Wassenaar et al., 1994).
In 1985, Morooka et al. (Morooka et al., 1985) treated C. jejuni with N-methyl-N'-nitro-N-nitrosoguanidine and methyl methane sulphonate and isolated bacteria with motility defects as judged by the hanging drop method. Each of the C. jejuni nonmotile isolates tested, of which one was flagellated and two were nonflagellated, were unable to colonize suckling mice and were cleared from the intestinal tract 2 d post-challenge. The investigators hypothesized that motility was required for C. jejuni to swim through the viscous mucus (Morooka et al., 1985). Subsequently, Newell (Newell, 1986) reported that a C. jejuni nonflagellated isolate termed SF-2 poorly colonized mice and was cleared from the intestinal tract 7 d post-challenge. In the same study, Newell (Newell, 1986) found that a C. jejuni nonmotile, flagellated isolate termed SF-1 colonized the intestinal tract of mice as successfully as the wild-type strain. It was latter established that the SF-1 isolate was indeed motile, although not to the same extent as that of the C. jejuni wild-type isolate, thus clarifying the discrepancy between their findings and those published by Morooka et al. (Morooka et al., 1985; Wassenaar et al., 1993). Wassenaar et al. (Wassenaar et al., 1993) used genetically defined C. jejuni flaA and flaB mutants to determine the importance of each of the flagellin proteins in the colonization of 1-d-old chicks. The investigators found that neither flagellin expression nor motility were essential for colonization in this model, but that a C. jejuni flaA+flaB- mutant colonized chicks at a level 1000-fold greater than the wild-type isolate. Based on their results, Wassenaar et al. (Wassenaar et al., 1993) concluded that bacteria expressing the flaA+ gene promotes maximal colonization. Consistent with these findings, others have reported that motility is important in promoting the colonization of animals by C. jejuni (Pavlovskis et al., 1991; Nachamkin et al., 1993).
In the early 1990s, several studies were undertaken to further dissect the importance of motility versus the actual flagellum in the interaction of C. jejuni with cultured epithelial cells (Wassenaar et al., 1991; Grant et al., 1993; Yao et al., 1994). Motility, conferred by the expression of the flaA+ gene, was found necessary for the maximal invasion of eukaryotic cells and for the translocation of polarized cell monolayers by C. jejuni (Wassenaar et al., 1991; Grant et al., 1993). However, differences were noted in the invasive potential of the C. jejuni flaA- flaB+ and C. jejuni flaA- flaB- isolates, with the former being more invasive (Wassenaar et al., 1991; Grant et al., 1993). Given the differences observed in invasive potential of the C. jejuni mutants, Grant et al. (Grant et al., 1993) concluded that the flagellar structure played a role in the internalization process of C. jejuni that was independent of motility.
Of interest is the finding that C. jejuni flagella undergo phase variation (Caldwell et al., 1985). In a study in which human volunteers were challenged with a mixture of a C. jejuni motile isolate and a non-motile phase-variant of the same isolate, only the motile phase variant was recovered from stool samples (Black et al., 1988). Consistent with this study and the importance of motility and flagella in bacteria-host cell interactions, Wassenaar et al. (Wassenaar et al., 1994) isolated a motile variant of a C. jejuni flaA-flaB+ mutant after performing an invasion assay. As discussed above, flaA is normally expressed at greater levels than flaB. Given this fact, Wassenaar et al. (Wassenaar et al., 1994) hypothesized that the co-cultivation of C. jejuni with intestinal cells leads to the expression of flaB and the shutdown of flaA transcription. Collectively, previous work indicates that motility contributes significantly to the colonization of animals by C. jejuni and subsequently in the development of disease in susceptible hosts.
Chemotaxis
Chemotaxis is the movement of an organism towards or away from a chemical stimulus. To determine whether a particular chemical acts as an attractant or repellent, investigators have commonly used a plate assay in which the bacteria are mixed in a PBS-solution containing 0.35 to 0.4% agar (Hugdahl et al., 1988). After the addition of test chemicals to plates by either hard-agar plugs (HAP) or filter discs, the plates can be incubated under a variety of environmental conditions and chemotactic response assessed. A zone of turbidity in an agar plate reflects an organism's migration toward the substrate, and is indicative of a positive chemoattractant response. Hazeleger et al. (Hazeleger et al., 1998) examined the chemotactic behavior of C. jejuni to a variety of chemical stimuli. In their study, C. jejuni was found to exhibit a positive chemotactic response to the carbohydrate L-fucose, the amino acids L-aspartate, L-cysteine, L-glutamate, and L-serine, and the organic acids pyruvate, succinate, fumarate, citrate, malate, and a-ketoglutarate (Hugdahl et al., 1988). The investigators also found that mucin, a glycoprotein of high molecular weight that contains L-fucose as its terminal sugar, acts as chemoattractant for C. jejuni.
The contribution of chemotaxis in colonization has been noted to be important for other pathogenic bacteria including V. cholerae, S. typhimurium, and E. coli (Allweiss et al., 1977). Several reports also suggest that chemotaxis is an important C. jejuni virulence determinant. In 1992, Takata et al. (Takata et al., 1992) found that mice were colonized when challenged orally with 110 CFU of a C. jejuni wild-type isolate, but not when challenged with as many as 5 x 107 CFU of two-independently isolated C. jejuni chemotaxis mutants. The C. jejuni nonchemotactic mutants used in the study were isolated using the HAP assay, and judged to possess flagella of the same length and width as that of the wild-type isolate. Yao et al. (Yao et al., 1997) explored the in vitro and in vivo role of chemotaxis using a set of defined C. jejuni mutants. Here, a C. jejuni cheY null mutant was generated, and found to display a nonchemotactic but motile phenotype. This C. jejuni mutant exhibited a threefold increase in adherence and invasion of INT 407 cells when compared to the wild-type isolate, but was unable to colonize mice or cause symptoms in infected ferrets. A possible explanation for these findings is that the motility of a cheY mutant is altered such that the organism makes longer runs, resulting in increased host cell contacts that promote irreversible cell adherence and invasion. However, the increase in the lengths of the runs in vivo, without chemotaxis providing appropriate directionality towards mucus, could lead to the organism's expulsion from the host by fluid flow and peristaltic activity. In the same study, Yao et al. (Yao et al., 1997) performed similar experiments with a C. jejuni isolate containing two copies of cheY. Of interest was the finding that the C. jejuni cheY diploid isolate displayed remarkably different behavior than the cheY null mutant. While the C. jejuni cheY diploid isolate demonstrated chemotactic behavior as expected, it also exhibited a decrease in its in vitro adherence and invasion capabilities and colonized mice. Similar to the cheY null mutant, the cheY diploid isolate was unable to cause disease in the ferret model. In the in vivo experiments, it is possible that the organism migrated towards the mucus within the crypts, but was unable to produce runs of sufficient lengths to penetrate the mucus due to its viscosity. Thus, the chemotactic response of C. jejuni appears important in directing the organism to specific sites in the host's intestinal tract.
Translocation
Investigators have utilized a unique cell culture system to assess the ability of pathogens to translocate across cell barriers. Briefly, the cells are cultured on a permeable membrane, and fresh media is added to the apical and basolateral chambers to promote cell growth and differentiation. Cell differentiation results in the establishment of distinct apical and basolateral cell surfaces with their own set of surface markers. The apical cell surfaces are further characterized by well-developed microvilli and brush borders. Monolayer integrity can be monitored throughout the course of the assay by measuring transepithelial electrical resistance (TER), and a decrease, or loss of TER, indicates disruption of the tight junctions.
The HT29, Caco-2, and T84 human adenocarcinoma cell lines have been most commonly used to examine the ability of enteric pathogens to translocate across a cell barrier. This capability is considered an important virulence attribute for some pathogens as it permits them access to underlying tissues and could promote their dissemination throughout the host. Nevertheless, the degree to which a pathogen translocates across a cell barrier and the organism's fate beyond the local environment differs greatly among pathogens. For example, S. typhi rapidly translocates across a polarized monolayer, causing cellular destruction and extrusion that leads to a complete loss of monolayer integrity. In contrast, the translocation of S. typhimurium across polarized cells causes minimal damage to the cell monolayer early in the process (Kops et al., 1996). Presumably these data are reflective of in vivo disease presentations where an infection with S. typhi is commonly septic in nature and an infection with S. typhimurium is generally localized to the intestinal mucosa.
In 1992, Everest et al. (Everest et al., 1992) noted that 86% of Campylobacter isolates from individuals with colitis were able to translocate across polarized Caco-2 cells versus 48% of strains isolated from individual with noninflammatory disease. The translocation of C. jejuni across polarized Caco-2 cell monolayers was determined by plating serial dilutions of the basolateral chamber media on agar plates. Interestingly, six C. jejuni isolates characterized as being "non-invasive" were able to translocate across the polarized monolayers. To further define the interactions of C. jejuni with polarized cells, Harvey et al. (Harvey et al., 1999) compared the ability of 4 clinical C. jejuni isolates to translocate across polarized Caco-2 cells with their ability to invade both polarized and non-polarized cells. The authors found that an organism's invasiveness does not quantitatively correlate with its ability to translocate across a cell monolayer. While the investigators also detected fluctuations in the measurable TER with the different C. jejuni isolates over the course of the assay (6 hr), monolayer integrity was maintained and final TER values were comparable to starting baseline values. Maintenance of monolayer integrity, at least over a relatively short period of time (8 h), has also been reported by others (Konkel et al., 1992c; Brás and Ketley, 1999). Noteworthy is that Bras et al. (Brás and Ketley, 1999) detected a loss in TER of Caco-2 cells inoculated with C. jejuni after 24 h, indicating an eventual disruption of cellular tight junctions. The investigators proposed that the loss in monolayer integrity was either a result of long-term effects of translocation and/or invasion or the accumulation of a bacterial toxin(s). The aforementioned studies suggest that the genes that encode the products responsible for invasion in C. jejuni are distinct from those that confer translocation ability.
The mechanism by which C. jejuni translocates across polarized cells is presently unclear but could be accomplished by either a transcellular (through a cell) or a paracellular (between cells) route. Evidence supporting the transcellular route of passage is the presence of intracellular bacteria and the fact that C. jejuni-cellular translocation is reduced at 20°C (Konkel et al., 1992c). Temperatures of 18-22°C preferentially inhibit eukaryotic endocytic and phagocytic processes (Silverstein et al., 1977). Evidence supporting the paracellular route of passage is that the kinetics of C. jejuni translocation and internalization significantly differ (Konkel et al., 1992c; Konkel et al., 1993). In addition, the invasiveness of C. jejuni isolates does not quantitatively correlate with translocation efficiency (Harvey et al., 1999). It is possible that C. jejuni organisms utilize the paracellular route of passage based on work indicating that cellular tight junctions can reseal following bacterial penetration (Takeuchi, 1967). Previous studies have also demonstrated that tight junctions temporally relax to allow regulated passage of both solutes and neutrophils (Madara, 1998).
The consensus among investigators is that C. jejuni initially colonizes the jejunum and ileum, and then the colon (Allos and Blaser, 1995; Skirrow and Blaser, 2000). However, histological examination of C. jejuni-infected humans and animals has revealed pathology primarily in the colon (Black et al., 1988; Babakhani et al., 1993; Russell et al., 1993). Advantages for C. jejuni reaching the underlying tissue and submucosa include access to a different set of cellular molecules that serve as receptors and the fact that the organisms are no longer subject to the peristaltic action of the intestine. If cellular translocation is associated with the development of C. jejuni-mediated enteritis, then several mechanisms of translocation may occur depending on the target site. In the intestinal tract, access to the submucosa could be achieved via translocation of the intestinal epithelia by either the transcellular or paracellular routes discussed above. Alternatively, C. jejuni may gain access to the submucosa via uptake by M cells (Walker et al., 1988). Not known is whether C. jejuni can translocate across the cells in the colon. Noteworthy is that the incidence of C. jejuni septicemia is low (0.4% cases) (Allos and Blaser, 1995), suggesting that C. jejuni organisms are not well equipped to survive and proliferate following dissemination from the intestine.
Adhesins and the Role of Adherence
Adhesins are surface-exposed molecules that facilitate a pathogen's attachment to host cell receptor molecules. In vitro adherence assays have been used extensively to characterize the interactions of C. jejuni with host cells and to attempt to identify the bacterial proteins that mediate binding. Although C. jejuni are capable of binding to cells of human (INT 407, HEp-2, HeLa, and 293) and non-human origin (Vero, CHO-K1, and MDCK) with equal efficiency, the binding of C. jejuni to INT 407 (Henle, a human intestinal epithelial cell line) and Caco-2 (a human colonic cell line) cells has been most extensively studied as these cells are thought to be more reflective of those that C. jejuni encounters in vivo. To date, the molecules proposed to act as adhesins have been found to be synthesized constitutively by C. jejuni. This fact is consistent with early studies, in which metabolically inactive (heat-killed and sodium azide-killed) C. jejuni were found to bind to cultured cells at levels equivalent to metabolically active organisms (Konkel and Cieplak, 1992). In addition, treatment of C. jejuni with chloramphenicol, a specific inhibitor of bacterial protein synthesis, has no effect on adherence (Konkel and Cieplak, 1992).
Prior to the identification of C. jejuni adhesins, the importance of C. jejuni binding to host target cells was questionable. Lee et al. (Lee et al., 1986) observed C. jejuni specifically associated with the intestinal mucus-blanket and mucus-filled crypts of BALB/c mice. This association involved highly motile organisms with no apparent adhesion to epithelial cells of the gut mucosa. However, mucus association was only studied over the course of several days due to experimental difficulties in maintaining depletion of normal surface-associated bacteria. In addition, the relevance of the model is debatable because C. jejuni-infected mice do not develop disease. The investigators hypothesized that the lack of pathology in the mouse model was a result of the host cells lacking the appropriate receptors for bacterial products. The interaction of C. jejuni with mucin was later investigated by Szymanski et al. (Szymanski et al., 1995) using non-polarized Caco-2 cells and carboxymethylcellulose. The addition of carboxymethylcellulose to the cells was used to mimic an in vivo mucus layer. The investigators observed increases in the binding and entry of C. jejuni to the carboxymethylcellulose-treated Caco-2 cells. While it is unclear whether the increase in binding was a result of C. jejuni specifically binding to host cell receptors or to components of the carboxymethylcellulose, an increase in C. jejuni entry into the Caco-2 cells was also noted. The investigators also found that the increased viscosity imparted by the carboxymethylcellulose resulted in longer runs by C. jejuni. The longer runs were proposed to result in an increase in the frequency of contacts between C. jejuni and host cells, thus leading to increases in host cell adherence and invasion. Based on these observations, the association of C. jejuni with the mucus in the crypts was proposed to be essential for cell invasion.
De Melo and Pechère (De Melo and Pechère, 1990) identified four outer membrane proteins (omps) with apparent molecular masses of 28, 32, 36 and 42 kDa that may play a role in mediating C. jejuni binding to host cells using a ligand-binding assay. By screening a C. jejuni genomic - lgt11 library with a hyperimmune antibody raised against the 28 kDa protein, Pei and Blaser (Pei and Blaser, 1993) cloned a gene encoding a protein with a calculated molecular mass of 28,181 Da. More recent evidence suggests that the 28 kDa protein, termed PEB1, mediates the binding of C. jejuni to epithelial cells (Pei et al., 1998). PEB1 is homologous with membrane proteins from other gram-negative bacteria that function in amino acid transport. We have cloned and partially characterized a 37 kDa omp from C. jejuni, termed CadF, that mediates the binding of C. jejuni to fibronectin (Fn) (Konkel et al., 1997). CadF is conserved among all C. jejuni and C. coli isolates tested to date (Konkel et al., 1999a). Whether the 36 kDa protein identified by De Melo and Pechère (De Melo and Pechère, 1990) is the CadF protein is not known. Jin et al. (Jin et al., 2001) identified a 42.3 kDa lipoprotein (JlpA = jejuni lipoprotein A) that mediates the binding of C. jejuni to HEp-2 cells. A mutation in the jlpA gene resulted in an 18 to 19.4% reduction in adherence when compared to the C. jejuni wild-type isolate, but had no effect on C. jejuni invasion. In addition, pretreatment of Hep-2 cells with recombinant JlpA reduced the binding of C. jejuni to the cells in a dose-dependent fashion. It is not known whether JlpA is the same protein as that identified by De Melo and Pechère (De Melo and Pechère, 1990). Investigators have yet to follow up on the adhesive properties of the 32 kDa protein.
Other molecules proposed to act as adhesins include the flagellum, lipopolysaccharide (McSweegan and Walker, 1986; Moser et al., 1992), major outer membrane protein (MOMP, also called OmpE) (Moser et al., 1997; Schröder and Moser, 1997), and P95 (Kelle et al., 1998). These molecules are listed as putative adhesins because their adhesive properties are less well characterized. While McSweegan and Walker (McSweegan and Walker, 1986) and Moser et al. (Moser et al., 1992) both reported that purified flagellin is capable of binding to host cells and INT 407 cell membrane fractions, Wassenaar et al. (Wassenaar et al., 1991) found that the addition of purified flagellin did not competitively inhibit the binding of C. jejuni to cultured cells. Thus, the role of the flagellum as an adhesin remains ill-defined. Noteworthy is that the examination of C. jejuni-infected INT 407 cells by scanning electron microscopy has shown the flagella in contact with host cells (Konkel et al., 1992a). In 1986, McSweegan and Walker (McSweegan and Walker, 1986) proposed that the binding of C. jejuni to INT 407 cells was mediated by LPS. This proposal was based on observations that radioactively labeled LPS bound to INT 407 cells and that pretreatment of INT 407 cells with LPS concentrations of 250 µg per well and greater reduced the binding of C. jejuni to cultured cells. Alterations in LPS have been shown to affect binding, and in certain instances internalization, of a number of enteric bacteria including E. coli (Bradley et al., 1991), S. typhi (Mroczenski-Wildey et al., 1989), and N. gonorrhoeae (Schwan et al., 1995) to host cells. These investigators hypothesized that LPS structural changes could affect the pathogen's binding potential by altering the organism's surface charge, masking the specific adhesins, and changing the integrity of the outer membrane. Because the extraction and labeling protocol used by McSweegan and Walker (McSweegan and Walker, 1986) is likely to have resulted in the use of material that is rich in both LPS and capsular polysaccharide, their data with respect to the role of C. jejuni LPS as an adhesive molecule is difficult to interpret. In 1997, Schroder et al. (Schröder and Moser, 1997) proposed that the MOMP of C. jejuni serves as an adhesin because it was found to bind to INT 407 cell membranes. In their study, the MOMP was prepared from crude outer membrane preparations using sarcosyl extraction and further purified using SDS-polyacrylamide gel electrophoresis (PAGE) or native gel electrophoresis. In contrast to the MOMP that was purified by SDS-PAGE, the MOMP purified by native gel electrophoresis was found to bind to INT 407 membranes as determined by enzyme-linked immunosorbant assays. The authors concluded that in addition to its role as a porin, the MOMP may also serve as an adhesin. However, the specificity of the MOMP binding to the INT 407 cell membranes was not determined. The P95 protein was identified by screening C. jejuni clinical isolates with a degenerative oligonucleotide probe by Southern hybridization analysis (Kelle et al., 1998). The sequence of the probe was based on the nucleotide sequences of adhesins identified in other gram-negative bacteria (Kelle et al., 1998). A hybridizing band was detected in six of thirteen C. jejuni clinical isolates as judged by Southern blot analysis. After cloning and sequencing the hybridizing C. jejuni chromosomal DNA fragment, an ORF was subsequently identified that was capable of encoding a polypeptide of 869 amino acids with an Mr of 95 kDa. The investigators reported that the deduced amino acid sequence of the C. jejuni P95 protein shared similarity with adhesins found in other gram-negative organisms including Bordetella pertussis and Haemophilus influenzae. Not determined was whether the P95 gene was expressed in the C. jejuni isolates examined and the phenotype of a C. jejuni P95 mutant. The role of each of the C. jejuni molecules discussed above in adherence requires additional study.
Adhesive pili or fimbriae have been identified in enteric pathogens including E. coli, Salmonella and Vibrio cholerae (Hultgren et al., 1991; Hultgren et al., 1996; Low et al., 1996). In 1996, Doig et al. (Doig et al., 1996) reported that both C. jejuni and C. coli produced environmentally regulated peritrichous pilus-like appendages. The investigators also reported that a mutation in a gene termed pspA (pilus-synthesis protease) resulted in an isolate that was incapable of synthesizing pili when cultured in the presence of the bile salt deoxycholate as evidenced by examination of the bacteria by transmission electron microscopy. While in vitro assays demonstrated that pili played no role in promoting the organism's adherence or invasion of epithelial cells, in vivo studies revealed that the C. jejuni pspA mutant exhibited reduced pathology in ferrets when compared to animals infected with the C. jejuni wild-type isolate. Subsequent work by Gaynor et al. (Gaynor et al., 2001) found that the pilus-like appendages in these Campylobacter isolates were bacteria-independent artifacts induced by the culture conditions. Whether Campylobacter organisms produce fimbriae that assist in colonization remains uncertain. Also not known is the role of the pspA gene from C. jejuni.
Though several adhesins have been characterized, little is known regarding the host cell surface receptors to which C. jejuni bind. CadF, a 37kDa outer membrane protein, binds to Fn. Fn is a component of the extracellular matrix (ECM). The host cell receptor molecules that JlpA and PEB1 bind remain to be elucidated. In addition to broadening of host range, the advantage of possessing multiple adhesins is that these molecules could act individually or in concert or at different stages of the infection. Certain adhesins might be involved initially in promoting the adherence of C. jejuni to the apical surface of intestinal epithelial cells or M cells, while other adhesins could be involved in promoting the organism's binding to molecules or receptors found on basolateral cell surfaces following translocation. Additional characterization of these interactions may provide a basis to help determine the sequential steps in Campylobacter pathogenesis.
In summary, several reports suggest that the adhesins synthesized by C. jejuni are important in colonization. A correlation has been observed between the clinical symptoms of C. jejuni-infected individuals and the degree to which C. jejuni isolates adhered to cultured cells. Fauchere et al. (Fauchere et al., 1986) found that C. jejuni strains isolated from patients with fever and diarrhea adhered to cultured cells at a greater efficiency than those strains isolated from asymptomatic individuals. The role of the adhesins in enabling successful colonization is supported by the experimental inoculation of animals with C. jejuni mutants. For example, the C. jejuni peb1A null mutant exhibits a reduction in the duration of mouse intestinal colonization when compared to the C. jejuni wild-type isolate (Pei et al., 1998). In addition, a C. jejuni cadF mutant lacks the ability to colonize the cecum of newly hatched leghorn chickens (Ziprin et al., 1999). However, studies are lacking to demonstrate that the colonization-impaired phenotype displayed by the peb1 and cadF mutants is due to the specific mutation introduced. In the case of the CadF protein, we have not been able to construct a Campylobacter shuttle vector harboring the cadF gene because expression of the gene from its endogenous promoter appears toxic in a heterologous host such as E. coli.
Invasion
The ability of C. jejuni to enter, survive, and replicate in mammalian cells has been studied extensively using tissue culture models (Newell et al., 1985; De Melo et al., 1989; Konkel and Joens, 1989; Konkel et al., 1990; Wassenaar et al., 1991; Everest et al., 1992; Konkel et al., 1992a; Konkel et al., 1992b; Grant et al., 1993; Oelschlaeger et al., 1993; Russell and Blake, 1994; Yao et al., 1994; Doig et al., 1996; Pei et al., 1998; Konkel et al., 1999b; Rivera-Amill et al., 2001). Typically such model systems involve determination of the number of intracellular C. jejuni by assaying bacterial protection from an antibiotic, such as gentamicin, that does not penetrate eukaryotic cell membranes (Hale et al., 1979). The relative ability of C. jejuni to invade cultured cells appears to be strain-dependent (Newell et al., 1985; Konkel and Joens, 1989; Everest et al., 1992). Newell et al. (Newell et al., 1985) found that environmental isolates were much less invasive for HeLa cells than clinical isolates as determined by immunofluorescence and electron microscopy examination of C. jejuni-infected cells. Everest et al. (Everest et al., 1992) observed a statistically significant difference in the level of invasion between C. jejuni strains isolated from individuals with colitis versus those isolated from individuals with noninflammatory diarrhea. Also of interest is that the ability of C. jejuni to invade cells has been noted to decrease after extensive in vitro passage (Konkel et al., 1990). The percent of the inoculum internalized for C. jejuni 81-176, a strain isolated from a milk-borne outbreak of diarrheal illness, has been reported to range between 0.8 to 1.8% (Yao et al., 1994; Doig et al., 1996; Yao et al., 1997). Biswas et al. (Biswas et al., 2000) and Hu and Kopecko (Hu and Kopecko, 1999) found that C. jejuni invasion is optimal when mammalian cells are inoculated at low MOIs. However, Biswas et al. (Biswas et al., 2000) also noted that the maximal number of internalized bacteria occurs at higher MOIs. Once internalized, C. jejuni organisms can survive for extended periods of time within epithelial cells and ultimately induce a cytotoxic response (Konkel et al., 1992b).
Parasite-directed endocytosis is a process in which a microorganism synthesizes the proteins required to promote their internalization by host cells. In addition to this basic requirement, investigators also discovered that the maximal uptake into cells occurs with metabolically active Haemophilus influenzae (St. Geme III and Falkow, 1990), Neisseria gonorrhoeae (Richardson and Sadoff, 1988), Rickettsia prowazekii (Walker and Winkler, 1978), Salmonella typhimurium (Finlay et al., 1989; Lee and Falkow, 1990), and Shigella flexneri (Hale and Bonventre, 1979; Headley and Payne, 1990). In the early 1990s, the internalization of C. jejuni was found to be significantly reduced in the presence of chloramphenicol, a specific inhibitor of bacterial protein synthesis (Konkel and Cieplak, 1992). This finding, coupled with the fact that metabolically inactive (heat-killed and sodium azide-killed) C. jejuni are not internalized, suggested that C. jejuni synthesize entry-promoting proteins (Konkel and Cieplak, 1992). One and two-dimensional electrophoretic analyses of metabolically labeled C. jejuni cultured in the presence and absence of epithelial cells revealed that a number of proteins were synthesized exclusively, or preferentially, in the presence of epithelial cells while others were selectively repressed (Konkel and Cieplak, 1992; Konkel et al., 1993). In support of these findings, Panigrahi et al. (Panigrahi et al., 1992) demonstrated that C. jejuni synthesized a number of proteins during growth in rabbit ileal loops that were not synthesized under standard laboratory conditions. Two of the newly synthesized proteins, with apparent molecular masses of 84 and 47 kDa, were detectable using convalescent sera from C. jejuni-infected individuals. Additional work revealed that the de novo proteins synthesized by C. jejuni upon co-cultivation with INT 407 cells were unique from those proteins induced by thermal stress of C. jejuni (Konkel et al., 1998). These findings suggest a coordinated response, whereby C. jejuni expresses certain genes after encountering the epithelial cell microenvironment.
The challenge of Macaca mulatta primates with C. jejuni has provided the most convincing experimental evidence that the primary mechanism of colonic damage and diarrheal disease is related to the organism's ability to invade colonic epithelial cells (Russell et al., 1993). Examination of colonic biopsy specimens from C. jejuni-infected primates revealed organisms, in association with dense concentrations of microfilaments, penetrating epithelial cells. C. jejuni were also observed within membrane bound vacuoles and within the cytoplasm of damaged cells. The investigators concluded that early mucosal damage, occurring prior to any inflammatory response, resulted from C. jejuni penetrating the colonic epithelial cells. Infection of a number of animals (i.e.: newborn piglets (Babakhani et al., 1993), chicken embryos (Field et al., 1986b), newly-hatched chicks (Welkos, 1984), hamsters (Humphrey et al., 1985), and gamma-irradiated mice (Sosula et al., 1988)) has supported the hypothesis that the ability of C. jejuni to cause illness is related to its ability to invade the epithelial cells lining the intestinal tract.
Secretion
Campylobacter jejuni secretes a set of proteins termed the Campylobacter invasion antigens (Cia proteins). The Mr of the Cia proteins range from 12.8 to 108 kDa (Konkel et al., 1999b). In the laboratory, the synthesis of the Cia proteins can be induced by culturing C. jejuni on medium supplemented with bile salts whereas both Cia protein synthesis and secretion are induced by culturing C. jejuni with eukaryotic cells or in serum-supplemented medium (Rivera-Amill et al., 2001). Thus, it appears that C. jejuni Cia protein synthesis and secretion are induced in the laboratory when the organism is cultured using conditions that mimic the in vivo environment.
To date, only one secreted protein termed CiaB has been identified (Konkel et al., 1999b). The C. jejuni ciaB gene encodes a protein of 610 aa with a calculated molecular mass of 73,154 Da. While confocal microscopic examination of C. jejuni-infected cells suggests that CiaB is translocated into the cytoplasm of the host cells, the specific function of CiaB is not known. Perplexing is that C. jejuni ciaB null mutants are deficient in the secretion of the total pool of Cia proteins. One possible explanation for this observation is that the synthesis of a 3'-truncated version of CiaB may obstruct the secretory apparatus. Data suggest that the Cia proteins promote C. jejuni uptake by host cells. C. jejuni ciaB null mutants bind to cultured epithelial cells at levels equal to or slightly greater than C. jejuni wild-type isolates, but exhibit a reduction in INT 407 cell invasion when compared to the wild-type isolates. In addition, preculturing C. jejuni wild-type isolates on plates supplemented with the bile salt deoxycholate retards the inhibitory effect of chloramphenicol on C. jejuni invasion as judged by the gentamicin-protection assay (Rivera-Amill et al., 2001). This finding supports the notion that the Cia proteins promote the organism's uptake because the synthesis of the Cia proteins is induced by deoxycholate prior to incubating the bacteria in chloramphenicol containing media. Infection of newborn piglets with C. jejuni Cia secretion-competent and secretion-deficient isolates has revealed that the secreted Cia proteins contribute to the pathology of C. jejuni-mediated enteritis. Piglets infected with the C. jejuni wild-type and complemented ciaB isolate developed diarrhea 24 h post-infection, whereas diarrhea was not observed in piglets infected with the C. jejuni ciaB mutant until 3 days post-infection. More severe histological lesions were also observed in piglets infected with the C. jejuni complemented ciaB isolate when compared to the C. jejuni ciaB mutant. Additional studies are necessary to determine the roles of the Cia proteins. While some of the Cia proteins may serve as functional components of the secretory apparatus, others likely directly interact with host cell molecules.
Preliminary data has been generated in our laboratory suggesting that the Cia proteins are secreted via the flagellar type III secretion apparatus. A precedent for protein secretion via the flagellar apparatus does exist in that Yersinia secrete proteins termed Fops for flagellar outer proteins from this apparatus (Young et al., 1999). While recent data indicates that the Fops contribute to the pathogenesis of Yersinia (Schmiel et al., 1998), the precise role of these proteins in infection has yet to be defined. If the Cia proteins are indeed secreted via the flagellar apparatus in C. jejuni, it would indicate that motility and virulence are linked in a novel fashion in this organism as Yersinia are nonmotile when cultured at 37°C.
Cytolethal Distending Toxin
A number of Campylobacter strains, including C. jejuni, C. coli, C. lari, C. fetus, and C. upsaliensis, produce cytolethal distending toxin (CDT) (Johnson and Lior, 1988; Mooney et al., 2001). The production of CDT by Campylobacter isolates was first reported by Johnson and Lior in 1988 (Johnson and Lior, 1988). The investigators reported that 41% of the 718 isolates examined produced CDT. Typically, the production of CDT by Campylobacter isolates is assessed by the addition of serially-diluted bacterial whole cell lysates (sonicates) to actively proliferating cells. In susceptible cells, toxin activity is evident from cell distension characterized by both elongation and swelling to nearly 5 times its normal size. Enlargement of nuclei is also common in the distended cells. Ultimately, CDT-treated cells die or disintegrate. Cell lines found to be susceptible to CDT include CHO, Vero, HeLa, HEp-2, Caco-2, COS-1, REF52, and INT 407 cells (Johnson and Lior, 1988; Whitehouse et al., 1998; Pickett and Whitehouse, 1999; Lara-Tejero and Galán, 2000). The sensitivity of different cell lines to CDT is variable, which may be due to differences in their surface receptors (Pickett and Whitehouse, 1999).
Pickett et al. (Pickett et al., 1996) was the first to clone the cdt toxin genes. The CDT toxin was found to be encoded by three adjacent genes termed cdtA, cdtB, and cdtC (Pickett et al., 1996). The cdtA, cdtB, and cdtC genes encode proteins of approximately 30, 29 and 21 kDa, respectively. Not known is whether the expression of the toxin genes is environmentally regulated. Moreover, no differences have been detected in CDT production by C. jejuni in response to modified environmental conditions including alterations in iron, growth phase, and growth temperature (Pickett, 2000). With respect to its biological properties, CDT is both heat-labile and trypsin-sensitive (Johnson and Lior, 1988). All three components of the toxin, which are associated with the bacterial outer membrane, are required for toxin delivery and activity (Hickey et al., 2000; Lara-Tejero and Galán, 2001). Microinjection studies have revealed that CdtB is the active subunit of the toxin (Lara-Tejero and Galán, 2000). The structure of the mature holotoxin is not known, and attempts to purify the holotoxin from membrane fractions have proved inherently difficult due to association of the toxin with outer membrane components (Pickett, 2000). However, Lara-Tejero and Galán (Lara-Tejero and Galán, 2001) recently reported that the purified toxin components can be reconstituted by mixing the recombinant CdtA, CdtB, and CdtC proteins. The reconstituted toxin exhibits biological activity as evidenced by cytoplasmic distension and cell cycle arrest (see below). The investigators concluded that CdtA and CdtC most likely comprise the heterodimeric B subunit of the toxin, and are required for CdtB delivery into a cell. Based on their work, Lara-Tejero and Galán (Lara-Tejero and Galán, 2001) propose that CDT is an AB2 heterodimeric toxin.
CDT causes progressive cell distention by causing cells to irreversibly arrest in the G2/M transition phase of the cell cycle (Whitehouse et al., 1998; Lara-Tejero and Galán, 2000). CDT prevents dephosphorylation of CDC2. CDC2 is the catalytic subunit of the cyclin-dependent kinase and must be activated (dephosphorylated) for cells to enter mitosis. Thus, CDT prevents CDC2 dephosphorylation, which in turn causes cells to arrest in the G2 phase. How this occurs is not yet understood. CDT may cause the G2 block by directing the cell into a DNA damage/incomplete replication checkpoint pathway (Pickett and Whitehouse, 1999). Lara-Tejero and Galán (Lara-Tejero and Galán, 2000) reported that the deduced amino acid sequence of CdtB shares similarity with members of the type I deoxyribonuclease protein family and speculated that DNA damage could occur during the S phase of the cell cycle. In a recent report, Mooney et al. (Mooney et al., 2001) found that extracts prepared from CDT-producing isolates of C. upsaliensis induced cells to undergo programmed cell death (apoptosis) as judged by TUNEL and flow cytometric analyses. However, it is not yet known whether CDT from C. upsaliensis, or C. jejuni, is in itself capable of inducing apoptosis as purified toxin or cdt mutants were not included in their study (Mooney et al., 2001).
The effects of CDT on cultured cells are profound, but little is known regarding the functional role of the toxin in bacterial pathogenesis. To begin to address the contribution of CDT in C. jejuni pathogenesis, Purdy et al. (Purdy et al., 2000) intragastrically challenged severe combined immunodeficient (SCID) mice with 109 cfu of a C. jejuni wild-type isolate and an isogenic cdtB mutant. Blood, liver and spleen samples were acquired at 2, 6, and 24 h post-challenge to assay for the presence of invasive Campylobacter organisms. A total of 30 mice were infected and 5 mice per C. jejuni isolate sacrificed at each timepoint. Wild-type bacteria were readily present in 8 of 15 samples (3 spleens, 4 livers, and 1 blood) at 2 h. However, the C. jejuni cdtB mutant was recovered in only 4 of 15 samples (1 spleen and 3 livers). Later timepoints showed approximately identical results for both the C. jejuni wild-type and cdtB mutant. Colonization levels were also monitored over the course of the assay and found to be identical between the two isolates. Based on these data, the authors suggested a possible role for CDT in invasion (Purdy et al., 2000). The investigators also noted that the sonicates of a cdtB mutant still exhibited some cytopathic effects on HeLa cells and suggested that there may be a second active toxin. Nevertheless, CDT appeared to be the principal toxin that is active in C. jejuni sonicates. Future work involving the characterization of CDT and its role in bacterial invasion or alternative functions in Campylobacter pathogenesis will surely prove interesting.
Lipopolysaccharide and Capsular Polysaccharide
Lipopolysaccharide (LPS) is a major component of the outer membrane in gram-negative bacteria. LPS has three distinct structural components: lipid A, which serves as the membrane anchor; a core composed of heterogeneous glycoses; and the somatic O antigen (O Ag) composed of a repeating unit of one or more glycosyl residues attached covalently to the core. LPS molecules without O-side chains are referred to as lipooligosaccharides. Early reports indicated that the LPS of C. jejuni is similar to that of Haemophilus and Neisseria spp. More specifically, C. jejuni LPS was characterized as being of low molecular weight (Mr) and lacking detectable amounts of O polysaccharide chains (Logan and Trust, 1984). Interestingly, the investigators remarked that it was unusual for the low Mr LPS of C. jejuni to confer such an extensive number of serotypes as identified by the Penner serotyping scheme. The Penner serotyping scheme, based on the presence of soluble heat-stable (HS) antigens that were presumed to be LPS in nature, was introduced in 1980 (Penner and Hennessy, 1980). In 1987, Preston and Penner (Preston and Penner, 1987) reported that approximately one-third of C. jejuni isolates produced a high Mr LPS, characteristic of LPS molecules with O side chains, that could be observed by immunoblotting with serotyping antisera.
An impressive amount of work has been done to determine the structures of LPS molecules from various C. jejuni isolates. Three different lipid A backbones have been identified in serostrain HS:2. Approximately 73% of the LPS molecules in this strain have a disaccharide backbone composed of diaminoglucose and D-glucosamine, 15% contain a backbone with two diaminoglucose residues, and the remaining 12% contain a backbone consisting of two D-glucosamines (Moran et al., 1991; Moran and Penner, 1999). All three backbones are acylated and phosphorylated in a similar manner (Moran et al., 1991; Moran, 1997; Moran and Penner, 1999). Identical backbones are present in other C. jejuni serostrains, but the molar ratios of the disaccharide units vary (Moran, 1997). The C. jejuni core oligosaccharide contains two distinct regions. The inner core is invariably comprised of a trisaccharide of 2-keto-3-deoxy-octulosonic acid (Kdo) and two heptoses (Aspinall et al., 1993). A second common feature is that the heptose adjacent to Kdo is substituted by D- glucose (beta 1-4 linkage) (Moran and Penner, 1999). Variation has been observed in the core region of the C. jejuni HS:1 and HS:2 reference strains, where the second heptose is substituted by glucose. Another variation observed among strains is that the heptose adjacent to Kdo is substituted at position 6 with either a phosphate or a phosphoethanolamine. In contrast to the conserved nature of the inner core, the outer core is more variable consisting of two or three hexoses that are substituted laterally or terminally with sialic acid or quinovosamine residues. Structural variations within this region were proposed to provide the diversity seen among serostrains containing only low Mr components (Aspinall et al., 1992; Aspinall et al., 1993; Moran and Penner, 1999).
Karlyshev et al. (Karlyshev et al., 2000) recently reported that all C. jejuni isolates, including those isolates previously thought to contain only low Mr polysaccharide, produce high Mr polysaccharide. As previously proposed by Chart et al. (Chart et al., 1996), this high Mr component was found to be biochemically similar to group II capsular polysaccharide and not O-antigen (Karlyshev et al., 2000). The investigators identified kps-like genes showing significant sequence similarity and overall organization to capsular polysaccharide genes of E. coli. Mutagenesis of the kps-like genes in C. jejuni resulted in production loss of capsular material and attributed serotypic determinants. The high Mr LPS material was also demonstrated by immunoblotting to be susceptible to phospholipase treatment presumably due to cleavage of a phospholipid moiety, instead of lipid A, from the polysaccharide. Removal of the lipid moiety hinders the polysaccharide from migrating in SDS-polyacrylamide gels (Tsai and Frasch, 1982). The investigators concluded that the basis of Penner serotyping is the capsular material and not the LOS or O-antigen. Bacon et al. (Bacon et al., 2001) reached similar conclusions, but noted the presence of a possible second glycan structure that could be visualized in a kpsM mutant. These data are intriguing and provide the foundation for future studies to investigate the importance of capsular polysaccharide in the development of GBS and in evasion of the pathogen from the host immune response. Of additional interest will be dissecting the roles of C. jejuni LPS and the capsular polysaccharide in the pathogenesis of C. jejuni.
Iron Acquisition
In the host, free iron is complexed with transferrin and lactoferrin at binding constants of approximately 1020 (Ratledge and Dover, 2000), making it a limiting nutrient for bacterial growth. Therefore, most pathogenic bacteria have developed mechanisms to scavenge iron that enables them to successfully colonize and survive within a host. Siderophore mediated iron uptake (1022 to 1050 dissociation constant) is a common method used by bacterial pathogens to acquire iron in the extracellular environment (Drechsel and Winkelmann, 1997). Iron loaded siderophores are bound via specific outer membrane receptors and are transported to the cytosol. The synthesis of siderophores and corresponding uptake systems is often iron repressed. Field et al. (Field et al., 1986a) found that only 7 of 26 C. jejuni isolates produced siderophores when grown in iron depleted media. However, each of three C. jejuni isolates tested were also found to utilize exogenously supplied enterochelin and ferrichrome siderophores to satisfy their iron requirement (Field et al., 1986a). Genes encoding both the enterochelin (ceuBCDE) and ferrichome (fhuABD) uptake systems have been characterized in Campylobacter isolates (Richardson and Park, 1995; Galindo et al., 2001).
The cfrA gene of C. jejuni encodes a protein that shares similarity with the siderophore receptor BfrA from Bordetella bronchiseptica (Guerry et al., 1997). The siderophore that binds to the CfrA putative receptor is not known. The cfrA gene was identified in 27 of 33 isolates as judged by Southern hybridization analysis (Guerry et al., 1997). A second iron uptake system, the fhuABD operon, was recently found within a C. jejuni putative pathogenicity island. A C. jejuni fhuA putative mutant failed to grow in iron depleted media, was readily killed following internalization in cultured epithelial cells, and elicited increased sensitivity to peroxide killing when compared to the wild-type isolate (L.A. Joens, unpublished). Interestingly, fhuABD was found in only 6 of 11 isolates tested (Galindo et al., 2001). Hence, cfrA and fhuABD are not uniformly present in C. jejuni isolates and it is not known whether C. jejuni contain only one or both of these systems. Noteworthy is the absence of an outer membrane receptor in the ceuBCDE operon and a cytoplasmic ATPase in the fhuABD operon. It is possible that individual components of various iron-uptake systems may complement each other.
Analysis of the C. jejuni NCTC 11168 genome has revealed the presence of additional iron acquisition systems including a hemin uptake operon consisting of the four proteins ChuABCD (Van Vliet et al., 1998). The NCTC 11168 genome also contains genes that encode for: a putative siderophore receptor; a periplasmic binding protein dependent system (Parkhill et al., 2000); and a ferrous uptake protein that shares similarity with the FeoB from E. coli (Van Vliet and Ketley, 2001). The importance of the FeoB protein in the pathogenesis of C. jejuni awaits investigation, however, the synthesis of FeoB affects the ability of H. pylori to colonize the stomach of mice (Velayudhan et al., 2000).
Transport of siderophores and certain host-iron sequestering molecules across the outer membrane requires the activity of the TonB system (Ratledge and Dover, 2000). The system is encoded by the tonB-exbB-exbD genes. TonB couples the proton motive force to actively drive the movement of molecules across the outer membrane and into the periplasm (Larsen et al., 1999). Analysis of the C. jejuni genome has revealed three sets of genes whose deduced amino acid sequences share similarity with TonB, ExbB, and ExbD. Two sets of these genes are located in the proximity of a putative siderophore receptor and cfrA (Parkhill et al., 2000). While most Enterobacteriaceae, including E. coli, only possess one copy of the TonB system, Vibrio cholerae encodes two complete systems (Seliger et al., 2001). Studies are needed to determine whether each of the three sets of genes identified in the C. jejuni genome encode for a functional system, and whether there are particular environmental conditions under which each system operates. Of additional interest will be to determine whether any of the three putative systems displays specificity for a specific siderophore/heme.
Fur is a repressor of iron regulated genes which uses ferrous ion (Fe2+) as a cofactor. In a C. jejuni fur mutant, the transcription of various iron uptake genes including cfrA became derepressed under high iron conditions (Van Vliet et al., 1998). However, oxidative stress response genes such as ahpC and katA maintain iron regulation. Another Fur homolog, PerR, was shown to repress the activity of oxidative stress genes in the presence of iron (Van Vliet et al., 1999). PerR had no effect on the transcription of other iron-regulated genes. Hence, C. jejuni contains two fur homologs that repress the activity of separate iron-regulated genes.
Ferritin, which stores iron intracellularly and prevents oxidative damage, has been purified from C. jejuni (Wai et al., 1995). Ferritin, encoded by cft, prevents oxidative damage by lowering the intracellular concentration of iron which may react to form various oxygen radicals (Wai et al., 1996). A C. jejuni cft mutant not only grows poorly in iron depleted media but is also more sensitive to peroxide killing than a wild-type isolate (Wai et al., 1996). Thus, the presence of the iron storage protein ferritin not only protects against oxidative stress but might also allow the organism to weather a varied range of iron concentrations.
In summary, C. jejuni has a number of iron-uptake systems for both ferric (Fe3+) and ferrous ions (Fe2+). The role of these systems in C. jejuni pathogenesis is not yet clear, however, the redundancy in function suggests that none will be singularly required for virulence.
Intracellular Survival in Mononuclear Phagocytes
In the piglet model, C. jejuni penetrates and proliferates within the intestinal epithelium of the ileum and colon. Cell damage occurs with degeneration of the superficial epithelium leading to the shortening of the villi and the production of an exudate in the lumen of the intestine (Figure 2).
In some cases, as in human infections (Blaser
et al., 1980), there is deeper tissue involvement resulting in hemorrhagic necrosis in the lamina propria, the formation of crypt abscesses and the influx of inflammatory cell exudate. It is during this
phase of the disease that C. jejuni encounters phagocytic cells of the lamina propria (Duffy
et al., 1980; Blaser et al., 1983) and the blood. Subsequently, the ability of
C. jejuni to survive phagocytosis could exacerbate the disease by enabling the organism to be disseminated in a host.
Although C. jejuni is readily taken up by monocytes and macrophages in vitro (Kiehlbauch et al., 1985; Myszewski and Stern, 1991; Wassenaar et al., 1997; Day et al., 2000), contradictory data have been generated regarding whether this pathogen is capable of surviving in these cells. Myszewski and Stern (Myszewski and Stern, 1991) examined the ability of both a C. jejuni high passage clinical isolate and a C. jejuni chicken isolate to resist killing by macrophages. They found that both C. jejuni isolates were killed by peritoneal macrophages, which were harvested from chickens, within a 6 hr incubation period. A greater number of C. jejuni organisms were phagocytized when serum reactive against the corresponding isolate was included in the assay, however, the bacteria were killed within the 6 hr incubation period regardless of whether the antiserum was included or omitted from the assay. Similar findings were described by Wassenaar et al. (Wassenaar et al., 1997) in examining the survival of 16 isolates of C. jejuni internalized by activated human peripheral monocytes. Survival assays conducted for 72 h demonstrated the killing of C. jejuni by the majority of the donors monocytes within 24 to 48 h. However, approximately 10% of the monocytes demonstrated normal uptake of C. jejuni but failed to kill the bacterium. The authors concluded that C. jejuni infected individuals are prone to develop a bacteremia if their monocytes fail to kill the organism. Contrary to the above work, other investigators have demonstrated intracellular survival of C. jejuni for at least 72 h after internalization by mononuclear phagocytes (Kiehlbauch et al., 1985; Day et al., 2000). Kiehlbauch et al. (Kiehlbauch et al., 1985) examined the survival of a C. jejuni clinical isolate using the J774G8 BALB/c mouse macrophage cell line, BALB/c macrophages, and human monocytes using acridine orange as a vital stain. The researchers were able to recover C. jejuni from all three cell types over a 6 d period. Day et al. (Day et al., 2000) reported similar findings in that a clinical isolate of C. jejuni (M129) was able to survive following phagocytosis by porcine peritoneal macrophages, murine peritoneal macrophages and J774A.1 cells. However, there was a noticeable reduction in the number of C. jejuni recovered from the three phagocytic cell types at 72 h post-inoculation; the greatest survival of C. jejuni was noted with the porcine peritoneal macrophages. It is our belief that the differences noted between laboratories with respect to the ability of C. jejuni to survive within phagocytes reflects the use of phagocyte cells of different origins and various bacterial isolates.
Role of Oxidative Radicals in Phagolysosome Survival
Although intracellular existence provides bacteria an unoccupied niche and shelter from immune surveillance, internalized bacteria must be able to survive a variety of reactive oxygen species, especially in the phagolysosome of professional phagocytes. Bacterial factors such as superoxide dismutase and catalase, which inactivate these products, allow invasive bacteria to persist in host cells and tissues. Experiments have been conducted to examine the effect of oxygen radicals on the survival of Salmonella typhimurium (DeGroote et al., 1997). It was found that a sodC mutant of S. typhimurium was more susceptible to killing by superoxide and nitric oxide than the wild-type isolate. Moreover, greater numbers of the S. typhimurium sodC mutant were recovered when the respiratory burst inhibitor acetovanillone or nitric oxide synthase inhibitor NG-L-monomethyl arginine was added to the culture medium. To address the role of superoxide dismutase in C. jejuni survival in macrophages, assays were performed with a C. jejuni sodB mutant and the J774A.1 murine macrophage-like cell line. No difference was noted in the survival of the C. jejuni sodB mutant in J774A.1 cells when compared to the C. jejuni 81-176 wild-type isolate (L.A. Joens, unpublished data). However, a C. jejuni katA mutant was not recovered from J774A.1 cells 24 h post-inoculation (Day et al., 2000); the katA gene from C. jejuni encodes the enzyme catalase. Also noteworthy is that the C. jejuni katA mutant was recovered when the respiratory burst or production of nitric oxide was inhibited. This finding demonstrates that C. jejuni possesses certain virulence attributes that enable it to survive intracellularly within mononuclear phagocytes. Additional work is required to determine whether C. jejuni is able to alter its intracellular trafficking and the precise role of macrophages in the development of campylobacteriosis.
A Model of C. jejuni Pathogenesis
We conclude this review by presenting a diagram that represents our current perspective of C. jejuni-virulence determinants and their potential role in the development of the histological lesions observed in C. jejuni-infected individuals (Figure 3).
Our model is based on articles discussed above and examination of biopsy specimens from piglets infected with C. jejuni clinical isolates. We believe that the infection of piglets with C. jejuni represents a relevant model for C. jejuni-mediated enteritis as these animals are anatomically similar to humans. Moreover, C. jejuni-infected piglets develop the clinical symptoms (e.g., bloody diarrhea) and histopathological lesions (e.g., epithelial cell degeneration, exudation of fibrin and inflammatory cells in both the small intestine and colon) similar to that of C. jejuni-infected humans.
C. jejuni is proposed to initially colonize the jejunum and ileum, and then the colon, of humans (Allos and Blaser, 1995; Skirrow and Blaser, 2000). However, the precise in vivo target site of C. jejuni is not known because autopsy and surgical material is rare. Motility and chemotaxis likely play critical roles in disease. Upon passage into the small intestine and migration of bacteria toward the mucus-filled crypts, we propose that C. jejuni engage in an adaptive response to the intestinal microenvironment where they synthesize a novel set of proteins that promote their subsequent interaction with host target cells. In vitro data supports the notion that C. jejuni has the ability to migrate across the enterocytes via a paracellular or transcellular route (Everest et al., 1992; Konkel et al., 1992c; Grant et al., 1993; Brás and Ketley, 1999; Harvey et al., 1999). However, not known is the significance of either route of translocation versus the role of M cells in the organism's ability to breach the intestinal barrier. We speculate that adherence plays an early role in the infectious process and that C. jejuni binds specifically to host cell receptors. If studies continue to support the necessity of adhesins in establishing disease, it will prove interesting to determine whether C. jejuni has a predilection for receptors on the apical or basolateral surfaces of the host cells. Following the intimate binding of C. jejuni to host cells, in vivo evidence indicates that C. jejuni is internalized by a host cell. Not clear is the contribution of invasion, versus Cia protein secretion, to the severity of Campylobacter-mediated enteritis. One of the hallmarks of infection in C. jejuni-inoculated piglets is villous atrophy (Babakhani et al., 1993). More specifically, C. jejuni appears to destroy the cells at the tips of the villi that are fully differentiated rather than the undifferentiated cells in the crypt. We propose that the necrosis of the villi is primarily caused by one or more bacterial toxins. We speculate that CDT contributes to villous atrophy by targeting the actively proliferating cells within the crypt. Thus, replacement of differentiated cells at the tips of the villi could be retarded by inhibiting crypt cell hyperplasia. How CDT is delivered to the cells within the crypt is not known. We would be remiss without mentioning that C. jejuni infection is accompanied by an intense inflammatory response that no doubt results from the heightened production of cellular cytokines. While this aspect of C. jejuni infection warrants a review article all to itself, space constraints have not permitted us to discuss this area. We hypothesize that the inflammatory response is responsible for intensifying the symptoms exhibited by C. jejuni-infected individuals, but is not responsible for causing the villous atrophy observed early in infection. We base this statement on the apparent lack of significant numbers of neutrophils and other inflammatory cells at tissue damaged sites. However, detailed studies are required to more closely examine the presence of inflammatory cell infiltrates at sites of C. jejuni infection.
Concluding Comments
A more accurate and comprehensive understanding of C. jejuni-mediated enteritis will emerge as researchers functionally characterize putative virulence genes and discover virulence attributes that are unique among particular C. jejuni isolates. There is little doubt that additional work will unveil that C. jejuni organisms have a repertoire of unique virulence strategies. Moreover, elucidation of C. jejuni virulence determinants and the stages at which they contribute in infection will yield new insights into the diverse mechanisms by which bacteria cause disease.
Acknowledgements
We thank Brian Raphael for assistance in preparation of this manuscript. Work in MEK's laboratory is supported by a grant from the NIH (DK58911) and USDA National Research Initiative Competitive Grants Program (99-35201-8579). Work in LAJ's laboratory is supported by USDA-NRICGP grants (98-35201-6195 and 01-35201-9948).
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