Abstract: This study reports the effects of Textured Soy Protein (TSP) incorporation at different concentrations (0, 5, 10 and 20% of total mass) on physicochemical properties of pan-fried beef Kofte (traditional Turkish meatball). The results indicated that the incorporation of TSP increased pH, a* values and total Unsaturated Fatty Acid (UFA) contents and decreased L* values, cooking loss and lipid oxidation in meatball samples (p<0.05). Addition of TSP also significantly affected some of the sensory attributes of meatballs (p<0.05). It was concluded that addition of TSP up to 10% into meatball formulation may be applied to improve the quality of meatballs without any adverse effects on the final product.

INTRODUCTION

Campylobacter jejuni (C. jejuni) is a gram negative motile bacterium that emerged in the 1970s as an important zoonotic pathogen. They are considered to be a leading cause of diarrheal disease throughout the world (Harris et al., 1986; Tauxe et al., 1985; Van Vliet and Ketley, 2001).

The motility by the flagella of C. jejuni is critical for intestinal colonization and for invasion into intestinal epithelial cells (Aguero-Rosenfeld et al., 1990; Grant et al., 1993; Szymanksi et al., 1995; Van Vliet and Ketley, 2001; Wassenaar et al., 1991). The flagellum of C. jejuni is composed of a basal body, hook and filament. To produce functional flagella, bacteria must coordinate both the temporal expression of over 40 flagella genes and secretion of the encoded protein (Caldwell et al., 1985; Fernando et al., 2007). Flagella filament is comprised of two proteins termed FlaA and FlaB. Sigma factor σ²⁸ encoded by fliA gene is involved in the expression of flaA, encoding the major flagellin that comprises a large proportion of the flagellar filament (Fernando et al., 2007).

Sigma factor σ⁵⁴ encoded by rpoN gene is required for the expression of almost all genes that encode components of the flagella basal body, hook and filament (Hendrixson and Dirita, 2003; Jagannathan et al., 2001). Flagellin is the immunodominant antigen during human and animal infection and is absolutely required for colonization in vivo (Konkel et al., 2001; Nuijten et al., 1991). In this study, C. jejuni rpoN and fliA mutants were constructed by allelic exchange with genes inactivated by deletion and insertion. The mutants obtained were tested for the characteristics of flagella and invasiveness to the epithelial cell in vitro and their ability to colonize the chicken in vivo.

MATERIALS AND METHODS

Bacterial strains and growth conditions: The C. jejuni 04011 (Cj04011) strain was isolated from a chicken carcass and virulence-associated properties in vasion (ciaB, iamA), lipopolysaccharide (wlaN and cgtB) adhesion (cadF, peblA, jlpA and porA), chemotaxis (docB and docC) and cytotoxin (cdtA, cdtB and cdtC) were confirmed by PCR (Muller et al., 2006). C. jejuni was cultured on Mueller Hinton (MH) agar (Difco) supplemented with 5% bovine blood under microaerobic conditions at 42°C. Escherichia coli DH5α used as hosts for the cloning experiments was grown in Luria-Bertani (LB) medium (Sigma) at 37°C. All strains were stored at -80°C in a 85% MH broth or LB medium with 15% glycerol solution.

Construction of C. jejuni rpoN and fliA mutant: For construction of the rpoN and fliA mutant, C. jejuni NCTC11168 sequences were obtained from the Sanger Center website (http://www.sanger.ac.uk/ProjectsC.jejuni) and primers for this study were shown in Table 1. Cj04011 chromosomal DNA was used for the amplification of DNA regions flanking rpoN and fliA with primer pairs of R1-R2 and F1-F2, respectively. The reaction conditions were 94°C for 2 min (1 cycle); 94°C for 45 sec, 65°C (-1°C per cycle) for 30 sec and 72°C for 3 min (5 cycles); 94°C for 15 sec, 60°C for 30 sec and 70°C for 4 min (30 cycles) and 72°C for 5 min (1 cycle). The PCR products were purified by using the QIAquick PCR Purification kit (Qiagen), digested with BamHI and then cloned into the BamHI site of pBS (SK+) (Invitrogen). Clones were selected on ampicillin-contaning blue-white plates and screened by restriction analysis.

The mutation construct was verified by DNA sequencing. Primer pairs R1I-R2I and F1I-F2I were designated to introduce a unique NheI restriction site and deletions of 998 and 509 bp within the cloned rpoN and fliA genes, respectively. The primers were oriented such that amplification of template DNA extended in opposite directions around the cloning vector with 20 ng of plasmid DNA as the template. The PCR cycling was the same with the condition for cloning of the rpoN and fliA genes. The products were digested with NheI enzyme, purified, ligated, transformed into competent E. coli DH5α cells and selected for ampicillin resistance. The 2.3 kbp Tet^r cassette was inserted into the NheI site of rpoN and fliA cloned into pBS (SK+) vector. Antibiotic-resistant colonies carrying the teracycline-resistance cassette in the same orientation with the cloned gene were identified by restriction analyses of the plasmid DNA and confirmed by DNA sequencing. These Tet^r modified constructs were electroporated into Cj04011. The target gene in Cj04011 was disrupted by allelic replacement via a double-crossover event between the target chromosomal gene and a plasmid-borne copy of the target gene containing an internal deletion.

Complementation of C. jejuni rpoN and fliA mutant: The rpoN and fliA complement strains were constructed for this study. Promoter genes for rpoN and fliA were amplified with primer pairs Rp1-Rp2 and Fp1-Fp2, respectively. The entire rpoN and fliA genes were amplified with primer pairs Rc1-Rc2 and Fc1-Rc2. Promoter and each target genes were amplified from Cj04011 by PCR. Following an intermediate cloning step into pCR2.1 (Invitrogen), the gel-purified insert was ligated into the NdeI site of pRY111. These plasmids were introduced into a Cj04011 rpoN and fliA mutant by conjugation.

Phenotypic analysis of the C. jejuni rpoN and fliA mutants: Motility assays were performed with Mueller Hinton broth supplemented with 0.4% Bacto agar (Difco, USA). A 10 μL suspension of each bacterial isolates was spotted on the surface of semisolid medium. Motility plates were incubated at 37°C under microaerophilic conditions for 48 h. For Transmission Electron Microscopy (TEM), bacterial suspensions were prepared from MH agar plates with phosphate-buffered saline and were added dropwise to Formvar coated copper grids. Bacteria were stained with 1% phosphotungstic acid. Samples were analyzed with a JOEL 1200 EX transmission electron microscope.

Table 1:	Characteristics of the primers used in this study

Adherence and invasion assay: Adherence and internalization assays were preformed with INT 407 as previously described (Grant et al., 1993). Briefly, approximately 1x10⁷ cfu of bacteria was inoculated into a semi-confluent INT 407 cell monolayer (10⁵ cells well^-1) on 24 well tissue culture tray. For binding, the infected monolayers were incubated for 3 h in a humidified, 5% CO₂ incubator at 37°C, rinsed 3 times with EMEM without FBS and lysed with a solution of 0.1% (v/v) Triton X-100 (Calbiochem, USA).

The suspensions were serially diluted and the number of adherent bacteria was determined by counting the colonies on MH-blood plates. The number of viable adherent bacteria was determined by counting the colonies. To measure bacterial internalization, the infected monolayer was washed three times with EMEM and reincubated for another 3 h in fresh EMEM containing 250 μg mL^-1 of gentamicin (Sigma). The number of internalized bacteria was determined as described above. Significance of differences among samples was determined with Student’s t-test following log₁₀ transformation of the data. Two-tailed p-values were determined for each sample and a p-value of <0.0005 was considered significant. The experiments were conducted a minimum of three times.

Chick colonization assays: Colonization of day old chicks by C. jejuni was done as described by Carrillo et al. (2004). Briefly, sixty 1 day old Hyline brown commercial chicks were obtained and divided into six groups. Water and a commercial chick starter feed were provided ad libitum.

The chicks were orally inoculated with 0.5 mL of a bacterial suspension strains, 10⁷ CFU cultured in Bolton’s broth at 42°C for 16 h under microaerobic conditions prior to inoculation of the birds. One group of 10 chicks was kept as the uninoculated control group. The remaining groups of chicks were inoculated with the wild-type strain, rpoN mutant, fliA mutant, rpoN complemented strain and fliA complemented strain. After 7 days, chicks were euthanized and their ceca were removed. Cecal contents were weighed, diluted 1:10 (wt/vol) in Bolton’s broth medium (Oxoid), thoroughly stomached and plated onto MH agar after serial dilution. C. jejuni colonies were counted after incubation in a microaerobic environment at 37°C for 72 h.

RESULTS AND DISCUSSION

Phenotypically, the C. jejuni rpoN mutant exhibited aflagellated filament on TEM and non-motily on MH medium supplemented with 0.4% Bacto agar while the fliA mutant possessed a truncated flagellar filament in most cells and strongly diminished motility (Fig. 1). Flagella sigma factor σ⁵⁴ (rpoN) and sigma factor σ²⁸ (fliA) regulate a large number of genes for the expression and function of C. jejuni flagella. Inactivation of the rpoN regulatory gene abolished flagellar function completely, otherwise inactivation of the fliA gene had a less detrimental effect on the flagella of C. jejuni.

A test of the ability of the rpoN and fliA mutant to invade INT 407 cells was performed to assess the role of these genes in C. jejuni pathogenesis and colonization in vitro (Grant et al., 1993). The rpoN and fliA mutants showed significantly less (p<0.0005) adherence and invasion compared to the wild type C. jejuni. While complemented mutants with entire rpoN and fliA genes showed similar values for adhesion and invasion as the corresponding wild-type strain but complement strains were not totally able to recover the ability of adhesion and invasion.

The investigators reported that C. jejuni flagellum is responsible for motility, protein secretion and invasion of host cells (Guerry et al., 1991; Wassenaar et al., 1991). In vitro adherence and invasion assays have been used extensively to characterize the interactions of C. jejuni with host cell and the binding of C. jejuni to INT 407 cell (a human intestinal epithelial cell) has been most extensively studied to be a more accurate reflection of C. jejuni in vivo.

Wassenaar et al. (1991) and Grant et al. (1993) reported that 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 monolyers by C. jejuni (Fig. 2). However, differences were noted in the invasive potential of the C. jejuni flA^- 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. (1993) concluded that the flagella structure played a role in the internalization process of C. jejuni that was independent of motility.

In parallel with the results of in vitro experiments, the colonization abilities of the rpoN and fliA mutants were tested in a day old chicken model. In this study, both rpoN and fliA mutants were completely attenuated for cecal colonization. But the result of the cecal colonization level of wild type parent strain used in this study was lower than that used in previous studies and the defect in colonization capacity of the present rpoN and fliA mutants were much more severe than used in other studies (Biswas et al., 2005; Fernando et al., 2007).

Fig. 1:	Characterization of the rpoN and fliA mutants. (a) Motility phenotypes on MH medium supplemented with 0.4% Bacto agar after 48 h and (b) Transmission electron microscopy. In panels a and b, 1, 2, 3, 4 and 5 indicate wild-type C. jejuni strain 04011, Cj04011 rpoN mutant, Cj04011 fliA mutant, Cj04011 rpoN complemented strain and Cj04011 fliA complemented strain, respectively

Fig. 2:	Adherence and invasion of INT 407 cell with with-type C. jejuni, rpoN mutant, fliA mutant and complemented strains. The adherence and invasion data are averages of three independent experiments performed in duplicate. As asterisk indicates a statistically significant difference (p<0.0005) aginst the C. jejuni 04011 wild-type isolate as determined by Student’s t-test

Fig. 3:	Cecal colonization levels (cfu g-1) of wild-type C. jejuni, rpoN mutant, fliA mutant and complemented strains. Ten birds were orally infected with 107 cfu of each strain at 1 day of hatch. Cecal samples were collected from at 7 dpi. The median values for each group are indicated by horizontal lines

Moreover, the colonization ability of the wild type C. jejuni is a remarkable contrast between the strains. The relative ability of C. jejuni to invade cells appears to be strain-dependent (Newell et al., 1985) (Fig. 3). 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. (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. It is also well documented that ability of C. jejuni to invade cells has been noted to decrease after extensive in vitro passage (Konkel et al., 2001). In this study, despite the high prevalence of flagella in the wild type parent, cecal colonization shows the dissimilarity with previous reported wild type strains. These findings suggest the difference of unknown gene expression between strains in vitro and in vivo is related to its ability to invade the epithelial cells lining the intestinal tract.

CONCLUSION

In study control of flagellin expression in C. jejuni involves the alternative sigma factor genes rpoN and fliA. The results of this study are consistent with the previous results that both fliA and rpoN genes regulate the process of binding and internalization of C. jejuni to epithelial cells in vitro and in vivo.

ACKNOWLEDGEMENT

This research was supported by the Korea Research Foundation Grant funded by the Korean Government (KRF-2008-1-E00077).