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Infection and Immunity, April 2002, p. 1761-1771, Vol. 70, No. 4
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.4.1761-1771.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Identification of Motility and Autoagglutination Campylobacter jejuni Mutants by Random Transposon Mutagenesis

Neal J. Golden1 and David W. K. Acheson2*

Department of Epidemiology and Preventative Medicine, University of Maryland School of Medicine, Baltimore, Maryland 21201,2 Department of Immunology and Pathology, Tufts University, Boston, Massachusetts 021111

Received 23 July 2001/ Returned for modification 17 September 2001/ Accepted 15 December 2001


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Campylobacter jejuni has been identified as the leading cause of acute bacterial diarrhea in the United States, yet compared with other enteric pathogens, considerably less is understood concerning the virulence factors of this human pathogen. A random in vivo transposon mutagenesis system was recently developed for the purpose of creating a library of C. jejuni transformants. A total of 1,065 C. jejuni transposon mutants were screened for their ability to swarm on motility agar plates and autoagglutinate in liquid cultures; 28 mutants were subsequently identified. The transposon insertion sites were obtained by using random-primed PCR, and the putative genes responsible for these phenotypes were identified. Of these mutants, all 28 were found to have diminished motility (0 to 86% that of the control). Seventeen motility mutants had insertions in genes with strong homology to functionally known motility and chemotaxis genes; however, 11 insertions were in genes of unknown function. Twenty motility mutants were unable to autoagglutinate, suggesting that the expression of flagella is correlated with autoagglutination (AAG). However, four mutants expressed wild-type levels of surface FlaA, as indicated by Western blot analysis, yet were unable to autoagglutinate (Cj1318, Cj1333, Cj1340c, and Cj1062). These results suggest that FlaA is necessary but not sufficient to mediate the AAG phenotype. Furthermore, two of the four AAG mutants (Cj1333 and Cj1062) were unable to invade INT-407 intestinal epithelial cells, as determined by a gentamicin treatment assay. These data identify novel genes important for motility, chemotaxis, and AAG and demonstrate their potential role in virulence.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Campylobacter jejuni is the most common bacterial cause of food-borne disease in the United States, with an estimated 2.5 million cases per year (8). Infection with this organism leads to a significant amount of acute illness as well as serious life-threatening consequences, such as Guillain-Barré syndrome (1, 30). Even though infection with C. jejuni is more frequent than that with Salmonella, understanding of C. jejuni pathogenesis and the critical virulence factors needed is only just beginning to emerge.

Although the mechanisms by which C. jejuni causes disease are unclear, the roles of motility, chemotaxis, and invasion have been partially established. The C. jejuni flagella, which impart motility to the cell, appear essential in the causation of diarrheal disease. The flagella are thought to be critical in the colonization of the mucous lining of the gastrointestinal tract as well as important for C. jejuni invasion of gastrointestinal epithelial cells (49). Furthermore, previous studies with uncharacterized nonmotile mutants and defined mutants demonstrated the importance of motility and/or the presence of flagella for invasion of intestinal epithelial cells (IECs) by this pathogen (10, 32, 46, 49). Defined mutations in the major flagellum subunit gene, flaA, resulting in bacteria with truncated flagella composed of the minor flagellin subunit, FlaB, produce a mutant that has diminished motility and is unable to invade IECs in vitro (11, 46, 49). Furthermore, a defined mutation in pflA resulting in bacteria with paralyzed flagella leads to a mutant that is still able to adhere but is not capable of invasion in vitro (49). Therefore, motility and not just the expression of the FlaA protein was determined to be critical for invasion by this pathogen in vitro. The role of flagella in adherence is somewhat less clear, with evidence suggesting that flagella can act as an adhesin; however, the degree to which this function plays a role in vivo is unknown (49). Additionally, other adhesins (encoded by the peb1, cadF, and jlpA genes) have been identified as important for C. jejuni adherence in vitro and colonization in vivo (17, 20, 38).

A number of genes have been associated with C. jejuni chemotaxis, including cheY, cetA, and cetB. The association of chemotaxis and virulence has been established by using uncharacterized and defined nonchemotactic mutants. Uncharacterized chemotactic mutants were shown to be unable to colonize in vivo (44), and a defined mutation in the chemotactic regulatory gene, cheY, resulted in reduced colonization and disease in an animal model (50). The reduced virulence of this chemotactic mutant was likely due to an inability to move toward the chemoattractant mucin (15). Furthermore, a recent publication identified two genes, cetA and cetB, involved in energy chemotaxis in C. jejuni (13).

To date, the role of autoagglutination (AAG) in C. jejuni pathogenesis has not been determined. The importance of AAG in virulence has been strongly implicated for other pathogenic bacteria, including Yersinia enterocolitica, enteropathogenic Escherichia coli, and Vibrio cholerae (5, 19, 39). This activity has been associated with pilins and/or outer membrane proteins that have been demonstrated to be critical for pathogenesis. A recent study examined AAG in C. jejuni strain 81-176 and strongly correlated this property with flagellar expression (29). However, the genes responsible for C. jejuni AAG have not been identified.

One of the limitations in determining critical genes in C. jejuni pathogenesis has been the lack of genetic tools and a method for random transposon mutagenesis. An in vivo random transposon mutagenesis system for C. jejuni was previously described (9), and a library of mutants of an invasive C. jejuni clinical isolate (strain 480) has now been created (47). In this study, we examined 1,065 mutants for motility and ability to autoagglutinate in liquid cultures. Twenty-eight mutants had diminished motility and/or AAG phenotype and were further characterized to determine their adhesive and invasive phenotypes with IECs, the presence of surface FlaA protein, and the site of the disrupted gene. We correlate AAG with flagellar expression, correlate motility with attenuated invasion, and associate the loss of the ability to autoagglutinate with decreased invasion of IECs.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial strains, media, and growth conditions. C. jejuni 480 is an invasive and highly electrocompetent strain isolated during an outbreak of campylobacteriosis; it was provided by B. A. M. van der Zeijst (47). Strain 480 was grown routinely on Mueller-Hinton (MH) agar supplemented with 5% sheep blood (SB), vancomycin at 10 µg/ml, polymyxin B at 2.6 U/ml, and trimethoprim at 5 µg/ml at 42°C (Sigma) under microaerobic conditions (5% O2, 10% CO2, 85% N2 gas). For some experiments, plates were incubated at 11% CO2 and 37°C in a standard cell culture incubator. The fetal human intestinal epithelial cell line INT-407 and the human cervix epithelial cell line HeLa were obtained from the American Type Culture Collection. INT-407 and HeLa cells were typically maintained in minimal essential medium (GibcoBRL) and McCoy's 5A medium modified with L-glutamine (GibcoBRL), respectively. Media were supplemented with 5% fetal bovine serum (GibcoBRL) and 1% each penicillin and streptomycin (GibcoBRL) and incubated at 5% CO2 and 37°C.

Mutant library. A C. jejuni mutant library was created by using random transposon mutagenesis as previously described (9). In brief, C. jejuni strain 480 was electroporated with 5 µg of the transposon mutagenesis vector pOTHM.1 and allowed to recover for 4 to 5 h at 37°C under microaerobic conditions. The bacteria were harvested from the plate surface, and transformants were selected at 37°C for 72 h on MH agar supplemented with 5% SB, chloramphenicol at 5 µg/ml, vancomycin at 10 µg/ml, polymyxin B at 2.6 U/ml, and trimethoprim at 5 µg/ml. This procedure was repeated several times, and a library of 1,251 mutants was obtained. Individual colonies were picked and streaked on the previously described plates to confirm their resistance to chloramphenicol. Transformants were then stored at -70°C in MH broth supplemented with 15% glycerol.

Identification and sequencing of junctional insertions. DNA flanking the transposon was identified by using random-primed PCR (34) as modified by Andrew Camilli (personal communication). To identify chromosomal DNA flanking the 5' end of the transposon, nested primers were used. The first set of primers consists of one primer that is capable of binding randomly to chromosomal DNA flanking the transposon (ARB1; 5'-GGCCACGCGTGCACTAGTACNNNNNNNNNNACNG-3') and a second primer that is complementary to the 5' end of the transposon (mag2F3; 5'-GGAATCATTTGAAGGTTGGTA-3'). ARB1-mag2F3 will create variously sized, low-occurrence PCR products depending on where ARB1 binds to the flanking DNA. The second set of PCR primers is designed to amplify products created by ARB1-mag2F3. Primer mag2F4 (5'-ACTAGCGACGCCATCTATGTG-3') is complementary to a region nested within the transposon amplified by mag2F3. The second primer, ARB2 (5'-GGCCACGCGTGCACTAGTAC-3'), is complementary to a known region of ARB1. The final products include a small portion of the transposon and typically 100 to 600 bp of junctional sequence. Identification of the junctional site 3' of the transposon insertion was used to confirm the results obtained with the first two sets of primers. Primers mag2F5 (5'-AGGAACTAAAGGGCGCAACGCGT-3') and ARB1 were used in the primary reaction, and primers mag2F6 (5'-ACTGATAAAAACCCTTTAGGA-3') and ARB2 were used in the nested reaction.

Primary PCR with a 50-µl reaction mixture was conducted as follows: 95°C for 8 min; 6 cycles of 95°C for 30 s, 30°C for 30 s, and 72°C for 1.5 min plus 5 s/cycle; and 30 cycles of 95°C for 30 s, 45°C for 30 s, and 72°C for 2 min plus 5 s/cycle. Nested PCR with a 50-µl reaction mixture was conducted as follows: 95°C for 8 min and 35 cycles of 95°C for 30 s, 55°C for 45 s, and 72°C for 1.5 min plus 5 s/cycle. Ampli TaqGold (Applied Biosystems) was used at 0.5 µl per reaction. Following PCR, the entire reaction mixture was purified (Qiagen QIAquick PRC purification kit), and the junctional insertion site was identified following primer walking sequencing reactions (9). Template DNA was prepared by boiling a bacterial colony for 5 min in Tris-EDTA. Cell debris was removed by centrifugation, and 8 µl of the supernatant was used in the primary reaction.

Motility assay. A total of 1,065 transposon mutants were screened for motility by measuring swarming on motility agar plates. Transformants were grown biphasically with MH agar and MH broth (see below) and then diluted to an optical density at 600 nm (OD600) of 0.025. Approximately 1 µl of this suspension was then stabbed into a 0.4% MH agar plate. The low density of the agar allows the bacteria to move within the agar, forming a halo of growth around the point of inoculation. Following microaerobic growth at 37°C for approximately 18 h, the radius of the ring was measured relative to that of the ring produced by the control strain. Control strain 480 was grown on each plate to account for plate-to-plate variation.

AAG assay. A total of 1,065 C. jejuni mutants were initially screened for their ability to autoagglutinate in 96-well plates. Transformants were grown biphasically with MH agar and MH broth (see below) at 11% CO2 and then transferred to a standard 96-well plate. This plate was allowed to remain undisturbed for 24 h at room temperature (RT). Each well was then visually inspected by using an inverted microscope and scored for AAG compared to that of wild-type strain 480. AAG mutants from this initial screen were examined further as follows. Samples were grown for approximately 16 h on MH agar plates supplemented with SB. A 1-ml suspension of the bacteria in MH broth (OD600, 0.5) was prepared and kept undisturbed at RT. Transformants capable of AAG fell to the bottom of the tube, leaving a clear supernatant. The degree of AAG was quantitated by removal of the top 100 µl of the suspension and measurement of the OD600. To examine AAG under conditions similar to those of the invasion assay (see below), the AAG assay was conducted with minimal essential medium supplemented with 5% fetal bovine serum at 37°C and 5% CO2.

Invasion and adherence assays. The procedure used for the invasion assay was based on the work of Oelschlaeger et al. (33). Transformants were grown in 96-well plates by using a biphasic culture. To each well of a 96-well plate was added 60 µl of MH agar supplemented with SB. To each well was added 200 µl of MH broth supplemented with vancomycin at 10 µg/ml, polymyxin B at 2.6 U/ml, and trimethoprim at 5 µg/ml. Transformants were removed from the freezer and inoculated directly into each well. The biphasic plate was allowed to grow under microaerobic conditions overnight for 16 to 20 h at 42°C. Preparation of INT-407 cells was begun at this time. INT-407 cells were plated at a concentration of 5.5 x 105 cells/ml in a 96-well microtiter plate (antibiotics were not used). To decrease well-to-well variation, INT-407 cells were grown for 2 days. Following overnight growth of mutants, 150 µl of bacterial suspension was removed from each well and placed in a standard microtiter plate. This suspension was then examined in a spectrophotometer.

A new microtiter plate containing the same elements as the first plate was prepared for a second round of biphasic growth; however, SB was excluded from the agar. Transformants were added to the second biphasic plate at a final OD600 of 0.025 and allowed to grow biphasically under microaerobic conditions for 18 to 20 h at 37°C. On the following morning, INT-407 cells were washed once with Hanks balanced salt solution to remove cell debris and resupplied with 200 µl of medium. Overnight growth for the transformants typically resulted in an OD600 of 0.2 to 0.4. Transformants were inoculated onto INT-407 monolayers at a concentration of ~105 cells/well. The number of bacteria added to each well was determined by plating on MH agar plates. The contents of the 96-well plate were then gently mixed and centrifuged for 5 min at 750 x g, a process that facilitated the movement of C. jejuni onto the surface of INT-407 monolayers. Invasion was allowed to occur for 2 h at 37°C and 5% CO2. Following incubation, the cells were washed twice. The solution was replaced with 200 µl of medium supplemented with gentamicin at 100 µg/ml per well to kill any remaining external bacteria. Killing was allowed to proceed for 2 h and was followed by washing (three times). Subsequent to the final wash, 200 µl of 0.1% Triton in phosphate-buffered saline was added to each well to lyse cells and release bacteria. Bacteria were enumerated on MH agar.

For the adherence assay, adherence was allowed to occur for 35 min. Maximal adherence for wild-type strain 480 occurred in this time range, as determined by a kinetic adherence assay. Furthermore, wild-type strain 480 was not able to invade IECs in this time range (data not shown). Wells were subsequently washed four times each with 200 µl of Hanks balanced salt solution, and agitation with a microtiter mixer was performed for 10 s following each wash. Lysis and enumeration were carried out as indicated above.

Construction of complementation vectors. Forward and reverse primer pairs were designed based on the C. jejuni NCTC11168 sequence (Table 1) (36) and were used to amplify and clone full-length genes into vector pCR2.1 (Invitrogen). Occasionally, an E. coli ribosome binding site (RBS) (28) was designed into the primer, if a clear C. jejuni RBS was not identified in the published sequence preceding the gene of interest. These constructs were digested with EcoRI and ligated into EcoRI-digested vector pWM1015 (28). This vector contains a constitutive C. jejuni consensus promoter sequence (48) upstream of a single EcoRI site. Proper orientation of the insert was determined by restriction digestion or, where possible, by using primer pWMp1 (5'-TCCGTTATTTTAAGTCTTAGTTTAGTT-3') (internal to the vector) and the corresponding 3'-end primer of the gene of interest. The final construct and pWM1015 were electroporated into their respective mutants in two separate experiments as previously described (47). Complementation of motility and AAG defects was carried out in the presence of kanamycin at 15 µg/ml.


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  		TABLE 1. Primers used for the construction of complementing vectors

 
Electrophoresis and Western blotting. Isolation of surface FlaA protein was conducted by glycine extraction (24). The sample concentration was determined by measuring the OD280, and equal concentrations of protein were loaded in lanes. Samples were electrophoresed using a 12% sodium dodecyl sulfate-polyacrylamide gel (22). Following electrophoresis, samples were stained with Coomassie blue to confirm similar protein loading (data not shown). The gel was transferred to nitrocellulose (Millipore) and blotted with anti-FlaA antiserum (1:10,000), generously provided by P. Guerry. FlaA protein was visualized by using antimouse antibodies conjugated to horseradish peroxidase (Pierce) and chemiluminescence (Pierce).

C. jejuni fluorescence microscopy. HeLa cells (18, 38) were trypsinized and an inoculum suspension was prepared. Cells were added to an eight-well chamber slide (Lab-Tek) at a final concentration of 105 cells/ml and grown overnight under the conditions stated above (without antibiotics). This procedure typically resulted in 80% monolayer confluency. C. jejuni wild-type strain 480 and the isogenic flhA mutant harboring the gfp plasmid pWM1015 (28) were grown overnight by using standard conditions. Bacterial cells were harvested on the following morning and added directly to wells at a final concentration of 2 x 107 cells/ml. Bacterial cells were allowed to adhere for 1.5 h, followed by washing four times with phosphate-buffered saline. Images were observed at a magnification of x400 by using a Labophot biological fluorescence microscope (Nikon) equipped with an N2000 camera (Nikon).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Screening of the C. jejuni mutant library for motility and agglutination mutants. A C. jejuni mutant library was constructed with strain 480 by use of random transposon mutagenesis as described previously (9). A total of 1,065 Cmr mutants were screened on semisolid MH motility agar plates to determine their ability to swarm. An initial screening was performed (n = 1 [per mutant]), and mutants with <=70% the motility of the wild-type strain were selected for further characterization. A total of 168 mutants met this criterion and were rescreened on motility agar (n = 3 [per mutant]). Twenty-four mutants with motility between 0 and 75% that of the control were chosen for further study. Concurrently, the 1,065 mutants were also screened for the loss of AAG as described in Materials and Methods. Four mutants that had lost the ability to autoagglutinate and had motility between 64 and 86% that of the control were also chosen for analysis. Therefore, 28 motility mutants (0 to 86% the motility of the control) were chosen for further characterization (Table 2).


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  		TABLE 2. List of mutants identified by motility and AAG screens

 
Identification of junctional insertion sites of motility and AAG mutants. To identify the DNA flanking the transposon, we used random-primed PCR (34). This nested PCR strategy allowed for the identification of the gene disrupted by the mutagenesis process. The open reading frame (ORF) in which the transposon had inserted was identified by comparing the junctional sequence to the C. jejuni NCTC11168 chromosome sequence (http://www.sanger.ac.uk/Projects/C_jejuni/blast_server.shtml). The position of each gene was identified along the C. jejuni NCTC11168 chromosome (Fig. 1) (36). Complete C. jejuni ORFs were then subjected to a BLAST search analysis to identify homology to known genes. Table 2 presents a list of the genes that were identified by this process. As previously described for C. jejuni (36), genes with putative similar functions did not tend to organize into operons, unlike that observed for many other gram-negative bacteria.



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  		FIG. 1. Location of transposon insertion sites within the C. jejuni genome. The nucleotide sequence flanking the insertion sites of 28 transformants was determined, and the insertion site positions within the C. jejuni NCTC11168 chromosome (36) were mapped. Transformants implicated in chemotaxis are indicated by bold type, while transformants implicated in AAG are indicated by underlining.

 
Based on the analysis of the insertion sites, the 28 motility mutants organized into several clear groups (Table 2). Eight mutants with insertions in putative flagellum structural genes demonstrated greatly attenuated motility compared to the wild type. Furthermore, all eight also had lost the ability to autoagglutinate. Three mutants with mutations in putative flagellum biosynthesis genes were nonmotile, and all three were nonautoagglutinating. Disruptions in two flagellum switch genes were identified; the fliY mutant was nonmotile and nonaggregative, while the fliM mutant had 32% motility and autoagglutinated normally. Insertion into the rpoN regulatory gene resulted in a nonmotile and nonautoaggluinating phenotype. Transposon insertions were also found in three chemotaxis genes. Two of the three (cheA and cheY) mutants were nonmotile, while the third (cetB) mutant had reduced motility (41%). The cheY and cetB mutants were autoagglutinating, while the cheA mutant was nonautoagglutinating. Finally, unlike the previous 17 mutants whose genes showed strong homology to functionally known genes, we identified 11 mutants whose genes had little or no homology to functionally known genes. Three mutants with greatly attenuated motility (0 to 30%) and four mutants with reduced motility (51 to 75%) were found to be nonautoagglutinating and autoagglutinating, respectively. Additionally, four mutants were unable to autoagglutinate yet retained some motility (64 to 86%). We therefore identified 28 mutants with attenuated motility and various AAG phenotypes and assigned putative functions to several previously uncharacterized C. jejuni genes.

Characterization of motility and AAG mutants for surface FlaA. To begin to understand the relationship among the presence of flagella, motility, and AAG, we performed Western blotting by using an anti-FlaA antibody against outer membrane proteins of all 28 mutants (Fig. 2). As indicated above, motility agar was used to determine the presence of functional flagella; however, this assay could not differentiate between mutants with absent flagella and those with nonfunctioning flagella. Therefore, analysis of the surface major flagellin subunit FlaA was conducted to distinguish these two conditions. Figure 2 demonstrates the presence or absence of FlaA on all 28 mutants and correlates this information with motility and AAG. With regard to Western blotting, the mutants fell into three clear groups: those that retained wild-type levels of FlaA, those that had a marked loss of FlaA but for which a band was still present, and those for which no band was present (Fig. 2).



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  		FIG. 2. Western blot analysis of surface FlaA from 28 motility mutants. C. jejuni outer membrane proteins were extracted with glycine-HCl and electrophoresed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After transfer of the gel, immunodetection was carried out with a polyclonal anti-FlaA antibody. Motility (Mot) is indicated as follows: +, 67 to 100% control motility; ±, 31 to 66% control motility; -, 0 to 32% control motility. AAG was determined in MH broth at RT for 3 h and is indicated as follows: +, AAG; ±, altered AAG kinetics; -, no AAG. flhAa is a defined flhA mutant of C. jejuni strain 81-176 (27), used as a negative control; flhAb is a putative 480 flhA insertion mutant.

 
All mutants that retained >20% control motility had normal levels of surface FlaA, indicating that a strong correlation exists between the presence of surface FlaA and the ability of a mutant to swarm on motility agar. This result extends and confirms previous reports that the presence of flagella is critical for the normal motility of Campylobacter spp (32, 46, 49). A diminished but not absent FlaA band was likely due to FlaA antibody cross-reactivity to FlaB, the minor flagellin subunit, as previously demonstrated (11, 46). This notion is supported by the band observed in the flaA mutant (Fig. 2), since this mutant was characterized by a complete loss of surface FlaA. Furthermore, flaA mutants are known to export FlaB, accounting for a low level of motility (18%) (11, 46). These mutants did not autoagglutinate, confirming a report indicating that FlaB is not sufficient to mediate this phenotype (Table 2) (29). Interestingly, we identified three mutants that retained wild-type levels of surface FlaA yet had attenuated motility (fliM [35% motility and FlaA+ AAG+], cheY [0% motility and FlaA+ AAG+], and cetB [41% motility and FlaA+ AAG+]). Most likely, these mutants possessed nonfunctioning flagella (fliM) or were defective in chemotaxis (cheY and cetB). Furthermore, wet-mount analysis demonstrated that these three mutants were motile compared to a defined flhA motility mutant (27). Of the motility mutants with absent FlaA, none was found to swarm or autoagglutinate (Fig. 2 and Table 2), extending previous reports that FlaA is necessary for complete motility and AAG (29) and no other flagellin subunit exists that can confer full motility in the absence of FlaA (46). Interestingly, we identified four mutants with that had an altered ability to autoagglutinate yet retained normal FlaA levels (Fig. 3). Mutants Cj1318, Cj1333, and Cj1062 failed to autoagglutinate in MH broth, and mutant Cj1340c was shown to have altered kinetics over a 6-h AAG assay. These observations suggests that flagella (FlaA) are required but not sufficient for AAG.



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  		FIG. 3. Kinetics of AAG for 28 motility mutants. C. jejuni motility mutants were harvested following overnight growth and suspended in MH broth at RT. A 100-µl sample of the suspension was carefully removed at the time points indicated, and the OD600 was determined. All 28 mutants were analyzed; however, lines were drawn for selected mutants for clarity. Cj1318, Cj1333, Cj1340c, and Cj1062 represent AAG mutants. A defined flhA mutant (0% motility and FlaA-) (Fig. 2) was used as a negative control (27). Strains 480 and 81-176 were used as positive controls.

 
Interestingly, the AAG phenotype can be observed microscopically within minutes (data not shown), suggesting that de novo transcription is not necessary for the AAG phenotype. Curiously, Misawa and Blaser (29) found very different kinetics for AAG in their assessment of C. jejuni AAG. The reason for their finding is unclear, as we have assayed C. jejuni strain 81-176 (Fig. 3), a strain used in their report, and shown it to autoagglutinate with kinetics similar to those of strain 480.

Characterization of motility and AAG mutants for adherence to and invasion of IECs. The ability of the 28 motility mutants to adhere to and invade IECs was examined by use of a 96-well invasion assay. As indicated in Table 2, all mutants with <=41% motility, with one exception (cheY mutant), demonstrated a reduced ability to invade INT-407 cells. This mutant was hyperinvasive (476% the control value), a result that reflects the fact that the cheY mutant is still capable of flagellum-based movement but is no longer directional (50). Overall, these findings confirm the work of other researchers, indicating a strong correlation between the presence of functional flagella and efficient uptake into IECs in vitro (10, 46, 49).

Interestingly, three of the four nonautoagglutinating mutants, despite retaining wild-type surface FlaA levels and motility (65 to 86%), showed reduced invasion (14 to 65% that of the control; Table 2). To better study the role of AAG in C. jejuni pathogenesis, an invasion assay was performed to correlate levels of invasion with AAG (Fig. 4). During the invasion experiments, the AAG assay was performed concurrently with the same conditions and media to rule out medium-specific effects on AAG (29). Mutants Cj1333 and Cj1062 showed abrogated AAG and invasion at all time points. Mutant Cj1340c was invasive; however, with the medium used in the invasion assay, Cj1340c autoagglutinated, even though it failed to autoagglutinate in MH broth. Therefore, we believe that this mutant was behaving very much like the wild type in this assay and therefore was invasive. Interestingly, mutant Cj1318 was found to be invasive at levels similar to those of the wild type at all time points yet was still unable to autoagglutinate. Based on the various invasion and AAG results, C. jejuni AAG appears to be a multifactorial process that we are currently investigating to understand more clearly.



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  		FIG. 4. Invasion assay of AAG mutants. Invasion is indicated as a percentage of control invasion. Strain 480 was used as a positive control, and flaA and flhA strains were used as negative controls. Error bars indicate the standard error for three experiments.

 
The ability of the various mutants to adhere to IECs was also determined (Table 2). Adherence was increased for 27 of the motility mutants; however, despite the increased adherence, 25 mutants showed decreased invasion. For the four AAG mutants, adherence was similar to that of the wild type and varied between 121 and 193% that of the control (Table 2). Only mutant Cj0399 had reduced adherence (89% that of the control). Overall, increased adherence inversely correlated with motility, where mutants that retained some level of swarming on motility agar had levels of adherence similar to those of the wild type.

Complementation of motility and AAG defects. To determine if putative ORFs were responsible for several of the motility and AAG defects, complementing vectors were constructed harboring a wild-type copy of the gene of interest as described in Materials and Methods. Motility mutants Cj0256, Cj1062, Cj1075, Cj1497c, and Cj1564 were electroporated with complementing plasmids pWM0256, pWM1062, pWM1075, pWM1497c, and pWM1564, respectively. Restoration of motility and AAG defects was observed for mutants Cj0256, Cj1062, Cj1497c, and Cj1564 (data not shown), strongly indicating that the disrupted gene is responsible for the observed phenotype. Mutant Cj1075 harboring pWM1075 did not show restoration of the motility defect, suggesting that a polar effect accounts for the phenotype.

C. jejuni fluorescence microscopy. To correlate AAG in vitro with an in vivo representation, we performed an AAG assay with HeLa cells (Fig. 5). Wild-type strain 480 cells labeled with gfp could clearly be seen adhering and autoagglutinating on the surface of HeLa cells following 1.5 h of incubation (Fig. 5A and B). In comparison, the 480 flhA mutant strain was unable to form aggregates and could not be observed adhering to HeLa cells. We found that single C. jejuni cells harboring gfp could not be easily observed at the magnification used, a fact that likely accounts for the negative result observed for the flhA mutant strain (Fig. 5D). Additionally, an independent adherence assay confirmed that this mutant adheres to epithelial cells (Table 2). Concurrently, an in vitro AAG assay was performed under analogous conditions, and normal AAG was observed for the wild-type strain but not for the mutant strain (data not shown). These data suggest that AAG occurs on epithelial cells in cultures and may be important for C. jejuni pathogenesis.



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  		FIG. 5. AAG of C. jejuni on HeLa cells. Wild-type strain 480 (control) containing gfp and an isogenic flhA motility mutant containing gfp were allowed to adhere to HeLa cells for 1.5 h and were observed microscopically. (A and B) Strain 480 under phase-contrast and fluorescent conditions, respectively. (C and D) flhA mutant under phase-contrast and fluorescent conditions, respectively.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
C. jejuni is a common cause of bacterial food-borne disease, but its pathogenic mechanisms are poorly understood. However, it is clear from previous studies that both motility and invasion are critical for pathogenesis. By use of in vivo transposon mutagenesis, we identified 28 mutants of C. jejuni strain 480 that show altered motility, AAG, or adherence to or invasion of IECs. During the course of this study, we confirmed the role of a variety of genes that are important in C. jejuni motility and chemotaxis and identified several genes putatively involved in these two phenotypes. Also, we correlated many of the genes with a reduced ability to invade IECs, directly identifying a role in pathogenesis. Furthermore, we have begun to distinguish the role of C. jejuni AAG in pathogenesis by identifying four novel genes putatively important for this phenotype.

Motility is critical to C. jejuni pathogenesis both in vitro and in vivo (46, 49). Seventeen of the 28 motility mutants found in this study can be placed in several groups based on putative functions. Three of the 17 mutants appear to be involved in flagellar biosynthesis—the flhA (13), flhB (13), and flhF mutants—and are likely part of a system that resembles type III secretion (for a review, see reference 25). Only flhA has been well characterized previously for C. jejuni (27). These three mutants were nonmotile and did not express any surface FlaA. Four mutants were likely to comprise the flagellar basal body (the flgB, flgI, fliP, and fliS mutants), a complex structure involved in motor rotation and transmission of torque from the motor to the external flagellar components (25). Only fliP has been described previously for C. jejuni (6). These mutants were nonmotile and lacked surface FlaA protein. Two motility proteins likely to be directly associated with the cytoplasmic surface of the basal body are the motor switch proteins, FliM (3, 13) and FliY (6). Motor switch proteins are responsible for the toggle between the clockwise and counterclockwise rotation of the flagella (25). The fliY mutant was nonmotile and had no FlaA, while the fliM mutant was attenuated for motility and expressed surface FlaA. These phenotypes are consistent with defined mutants identified for Bacillus subtilis and Salmonella enterica serovar Typhimurium, respectively (2, 45). Externally connected to the basal body are the hook and hook-associated proteins. Two organisms with mutations in putative hook-associated proteins, FlgK (3) and FliD, were identified in this screen. The hook-associated proteins connect the flagellar filament to the hook protein that attaches to the basal body, which is critical in the generation and transmission of torque (25). Two mutants were found with disruptions in genes encoding proteins identified as components of the flagellar filament, FlaA (46) and FlaD (6). FlaA, the major flagellin subunit, makes up the filament along with the minor flagellin subunit, FlaB, and has been well studied (11, 46). FlaD has homology to other bacterial flagellins and could therefore serve as a minor flagellin; however, it has never been identified as part of the C. jejuni filament.

One mutant was identified as a regulatory mutant. RpoN, the RNA polymerase sigma-54 factor, has been recently described to serve as a regulator of C. jejuni motility (16). The rpoN mutant was nonmotile and lacked FlaA protein. Chemotaxis has also been demonstrated to be important for C. jejuni pathogenesis, as exhibited by the attenuated virulence of a cheY mutant in the ferret diarrheal model (50). Three mutants were identified that are likely involved in chemotaxis (the cheA, cheY, and cetB mutants). cheY, a gene involved in chemotaxis regulation, was previously cloned and studied in C. jejuni (50). Our results confirm the previous data. cetB, a gene involved in energy chemotaxis, was also identified in a motility screen. Our results extend this recent report by demonstrating a direct role for this gene in C. jejuni invasion in IECs (19% of the control value) (13). cheA, a chemotaxis histidine kinase gene, was recently identified in C. jejuni in two independent in vitro mutagenesis screens and shown to cause diminished motility (6, 13); however, this was the extent of the characterization. The cheA mutant was nonmotile; however, it demonstrated diminished surface FlaA expression. This was an unexpected finding in the context of the other two chemotaxis mutants, as well as cheA mutants of other gram-negative bacteria (37, 42). Interestingly, this mutant was nonautoagglutinating and did not move when observed microscopically, consistent with our findings for other surface FlaA-negative mutants. At this point, we cannot exclude the possibility that the mutation in cheA is polar. Furthermore, C. jejuni is known to undergo phase variation, resulting in nonmotile, FlaA-negative clones (4, 7). It is possible that this scenario could have occurred in the cheA mutant, for which slipped-strand mispairing during replication in another gene has resulted in a FlaA-negative phenotype (35). Overall, FlaA expression was absent or significantly reduced in all but 10 of our 28 motility mutants. When FlaA was absent or significantly reduced in level, there was loss of AAG. These observations extend previous results indicating that FlaA is critical for wild-type motility (11, 46).

To connect the 28 motility mutants with virulence, adherence and invasion assays were performed as described in Materials and Methods. There was a very strong correlation among normal motility, normal expression of FlaA, and normal ability to invade IECs (Table 2). However, the fliM mutant (35% motility and FlaA+ AAG+) demonstrated an ability to invade IECs (90% of control). This result suggests that in this mutant, the combination of 35% motility and the presence of FlaA and normal AAG confers a phenotype of virtually normal invasion. The cetB mutant (41% motility and FlaA+ AAG+) had a profile similar to that of the fliM mutant yet had reduced invasion (19%), suggesting that this combination plus the inability to engage in chemotaxis leads to reduced invasion. It is unclear why the cheY mutant (0% motility and FlaA+ AAG+) was hyperinvasive (476%); however, this finding has been noted by others (50). Inspection of the fliM, cetB, and cheY mutants by microscopy demonstrates them to be motile but suggests that they are not capable of complete or directional motility. Overall, it is unclear if the motility mutants in our study have lost the capacity to invade as a direct consequence of diminished motility or if other factors, such as a diminished capacity to secrete virulence factors (21), also affect invasion (36, 51).

We found that 18 motility mutants adhered to INT-407 cells to a greater degree (>2-fold) than the wild-type strain (Table 2). However, despite the increased adherence, the level of invasion was reduced in all mutants except the cheY mutant. It is unclear why attenuated motility correlates with increased adherence. However, we believe that this situation may be a reflection of a decreased on/off rate of the bacteria for IECs due to the lack of motility and therefore may be a function of the in vitro assay. This theory is supported by evidence that increasing the viscosity of the medium in this assay leads to greater adherence of C. jejuni to IECs (43). Furthermore, because bacteria are centrifuged to the cell surface, a functional flagellum is not needed to reach the monolayer. Also, it is likely that flagella functioning as an adhesin do not play a strong role in adherence in vitro (11, 26, 46). The presence of increased adherence of motility-defective mutants is supported by the work of Hu and Kopecko. They demonstrated that only 66% of an IEC monolayer is invaded and that two bacteria are maximally taken up by each epithelial cell (14). Therefore, increased adherence is not sufficient for increased invasion, indicating the presence of other invasion-dependent factors besides adherence. Additionally, the loss of AAG clearly did not affect the ability of the mutants to adhere to IECs, suggesting that the ligands responsible for AAG are different from those responsible for IEC adherence.

Aside from the 17 mutants with mutations in genes sharing significant homology with known motility genes, we found 11 genes that did not demonstrate homology to any functionally known gene. Three mutants, Cj0041 (0% motility and FlaA- AAG-), Cj1075 (0% motility and FlaA- AAG-), and Cj1497c (20% motility and FlaA- AAG-), demonstrated strongly attenuated motility. Complementation was successful with mutant Cj1497c, suggesting that the Cj1497c gene was responsible for the observed phenotype. We attempted to complement mutant Cj1075; however, the addition of the full-length Cj1075 gene in trans did not restore the phenotype. Interestingly, the Cj1075 gene exhibits homology to B. subtilis yviF (E value, 2.0 x 10-10). yviF is a gene of unknown function; however, it resides within a B. subtilis flagellar operon (41), suggesting a potential role in C. jejuni motility. We identified four mutants that had a motility of 51 to 75% that of the control, mutants Cj0256 (64% motility and FlaA+ AAG+), Cj0399 (70% motility and FlaA+ AAG+), Cj1011 (51% motility and FlaA+ AAG+), and Cj1564 (75% motility and FlaA+ AAG+). Complementation was successful with mutants Cj0256 and Cj1564, again suggesting that the genes were responsible for the observed phenotype. Interestingly, the Cj1564 gene contains a methyl-accepting chemotaxis domain (E value, 4.1 x 10-30) (http://www.sanger.ac.uk/cgi-bin/yeastpub/get_cj_cds.pl) located within the 3' portion of the gene. The Cj1564 protein is therefore likely to be a methyl-accepting chemotaxis signal transduction protein and the reduced motility likely is due to an inability to properly engage in chemotaxis. Overall, the presence or absence of the FlaA protein corresponded well with the ability of these 7 mutants to autoagglutinate, consistent with surface FlaA being necessary for the AAG phenotype. Furthermore, all seven mutants showed reduced invasion, consistent with motility being critical for invasion. The final four mutants were designated AAG mutants and are discussed below.

With regard to AAG, we identified four motility mutants (Cj1062, Cj1318, Cj1333, and Cj1340c) that expressed similar levels of wild-type FlaA (Fig. 2) but had either lost the ability to autoagglutinate or demonstrated altered AAG kinetics (Fig. 3). Cj1333 and Cj1062 showed markedly diminished invasion (14% of control), Cj1318 showed intermediate invasion (65% of control), and Cj1340c, with variable AAG kinetics, had a normal to hyperinvasive phenotype. The role of AAG in C. jejuni pathogenesis is unclear, although these data suggest that a multifactorial process is required to mediate the phenotype. Misawa and Blaser (29) reported that the C. jejuni AAG phenotype correlated with the presence of flagella, and we have confirmed that observation. However, we have found that other factors are clearly involved beyond the presence of FlaA. To test the involvement of surface-exposed posttranslational modifications in the agglutination event (31, 40), we acquired two Campylobacter posttranslational modification mutants, ptmA and ptmB mutants (Fig. 6) (12). These mutants were unable to autoagglutinate in liquid broth compared to their isogenic wild-type strain (data not shown), suggesting that posttranslational structures play a role in the AAG phenotype. Also, the rapid AAG of the wild-type strain is consistent with a posttranslational modification structure; however, this notion does not negate the possibility that protein synthesis is needed following the initial interaction.



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  		FIG. 6. C. jejuni NCTC11168 Cj1318 family. Shown is a C. jejuni NCTC11168 segment of DNA representing three of the four AAG mutants (Cj1318, Cj1333, and Cj1340c) as well as other Cj1318 family members, indicated in gray. neuC2, neuB2, neuB3, ptmA, and ptmB are known to affect the addition of sialic acid to C. jejuni proteins (12, 23). flaA and flaB are the genes for the major and minor flagellin subunits, respectively.

 
The identification of the four AAG mutants (motility positive and FlaA+ AAG-) demonstrates that FlaA can be expressed at wild-type levels yet AAG can still be disrupted. Therefore, these data suggest that FlaA is necessary but not sufficient for AAG. Interestingly, three of the four AAG mutants (Cj1318, Cj1333, and Cj1340c) were clustered together within a segment of the published strain NCTC11168 chromosome sequence (Fig. 6) (36). Analysis of the surrounding DNA has identified five genes involved in the sialylation (12, 23) of C. jejuni proteins, including the major flagellin subunit, FlaA. Furthermore, five novel genes (those in Cj1334, Cj1335, Cj1336, Cj1337, and Cj1341c) were also found in this region of DNA and had strong homology to the genes in three of the four AAG mutants (Cj1318, Cj1333, and Cj1340c). This group of seven proteins is known as the 1318 family; the genes reside within the C. jejuni flagellar operon, and the proteins are believed to be hypervariable surface structures (36). These data suggest that the sialylation, flagellum, and Cj1318 families of protein act coordinately to mediate motility and AAG.

In conclusion, we have confirmed the role of a variety of previously described C. jejuni genes that are involved in motility and have generated data to support the role of other specific genes that have significant homology to known flagellar genes in other bacteria. Also, we have assigned a phenotype to four functionally unknown C. jejuni genes by complementation. We have confirmed and extended reports indicating that motility and FlaA are important for C. jejuni invasion into IECs. Furthermore, we have confirmed that AAG is strongly associated with flagellar expression, and we have extended previous results by demonstrating that surface FlaA protein is required but not sufficient for AAG. Additionally, we have shown that for mutants in which AAG is lost but in which motility is largely preserved, there is diminished invasion. This finding suggests that AAG may play a role in the invasive process, and we are currently investigating this possibility.


   ACKNOWLEDGMENTS
 
Research support for this study includes the following grants from the National Institutes of Health (NIH), Bethesda, Md.: HL-55660, AI-16242, and AI-39067 to D.W.K.A. and pilot project funding P 30 DK-34928 for the Center of Gastroenterology Research on Absorptive and Secretory Processes.

We thank P. Guerry for the generous gift of anti-FlaA antibody and ptmA and ptmB mutants, C. L. Pickett for providing 81-176 flhA, and R. E. Mandrell for kindly providing pWM1015. We also thank Anne V. Kane for technical assistance.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Epidemiology and Preventative Medicine, University of Maryland School of Medicine, 10 S. Pine St., MSTF Building, Baltimore, MD 21201. Phone: (410) 706-4583. Fax: (410) 706-4581. E-mail: dacheson{at}epi.umaryland.edu. Back

Editor: A. D. O'Brien


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Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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Infection and Immunity, April 2002, p. 1761-1771, Vol. 70, No. 4
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.4.1761-1771.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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