Among the most common food-borne pathogens affecting the U.S., U.K., and other Western countries, Campylobacter jejuni leads in number of human gastrointestinal illnesses most commonly resulting in diarrhea, vomiting and gastroenteritis abdominal cramping (“Information for Health Professionals | Campylobacter | CDC,” 2018). The majority of cases are sporadic and do not infect large populations. Rarely does this illness lead to an outbreak, even more rarely is it associated with death. Unlike other gastrointestinal pathogens, such as E. coli O157:H7 or Salmonella, which can cause severe symptoms including bloody diarrhea, severe abdominal cramps, vomiting, and even death, Campylobacter is more often a short-lived illness that resolves within days (“Information for Health Professionals | Campylobacter | CDC,” 2018).
As a gram-negative, spiral-shaped bacterium, with a single polar flagella at one or both ends, Campylobacter spp. have been well studied and documented in its relation to poultry in nearly every aspect of growth to virulence to environmental susceptibility. More recently, within the past decade or so, C. jejuni along with other campylobacter have been investigated as to their ability to form biofilms along various surfaces in aquatic environments (Reeser, Medler, Billington, Jost, & Joens, 2007). As in poultry studies, its virulence, growth, and susceptibility have also been documented as well as genetic factors inducing various protein production in response to such conditions. However, the survivability of C. jejuni within vegetables while retaining its virulence is poorly understood. Due to most vegetables being consumed in raw form, any infection stemming from such consumption is considered a result of cross contamination. Incidentally, reports have surfaced whereas clinical cases have been observed and reported with the claim of no possibility of cross contamination with meat. Increasing numbers of these reports prompted a limited number of studies that found C. jejuni in the soil of crops, mainly due to infected fertilization, as well as the surfaces of improperly washed vegetables not associated with cross contamination (Brandl, Haxo, Bates, & Mandrell, 2004). Of the vegetables that enter our markets, the possibilities remain that contamination with C. jejuni may be due to cross contamination with infected meats, contaminated soil adhering to the vegetables, or even internalization into the vegetables. The later possibility has yet to be investigated in any significant manner. This study will investigate the ability for C. jejuni to invade plant systems and assess its virulence thereafter, allowing host infection upon consumption, testing the hypothesis that Campylobacter jejuni will become internalized into plants via contaminated nutrients and remain virulent to human consumption. The strong genetic component affecting virulence of C. jejuni is this literature review’s focus.
GENETICS OF Campylobacter jejuni VIRULENCE
The virulence of C. jejuni is affected by four primary components: flagella, adherence, cellular invasion, and toxin production. Here we will examine the important genetic components of those categories based on the comparison of several strains (NCTC11168 V1, NCTC1168 V26, 81-176, RM1211, RM1221, and RM2228) due to their historical data of past outbreaks and commonality.
Flagellar motility in C. jejuni is seen as a major factor of virulence and is considered the primary mode for intestinal colonization. Highlighting this importance, Carrillo et al. (2004) characterized and constructed the flhA gene by means of whole genome microarray analysis with fluorescently-labeled genomic DNA by competitive hybridization. Identified to transcribe the protein FlhA, the team found it to be a key component of the flagellar apparatus. This export protein had demonstrated to regulate motility and virulence pertaining to colonization while its transcribing flhA gene was found to be highly regulated and conserved across species (Fleiszig, Arora, Van, & Ramphal, 2001; Ghelardi et al., 2002; McGEE et al., 2002). Inactivation of the flhA gene by Carrillo et al. (2004) demonstrated a 10-fold decrease in the pathogen’s overall virulence, motility, and colonization. The genes encoding for proteins that are used for the flagellar assembly in the later stages, along with flagellar glycosylation, were seen to be more prominent in strains showing greater virulence. In a study conducted by Fouts et al. (2005), whole genome sequencing generated a greater amount of information that allowed for resolving differences between closely related organisms. They found that out of 580 conserved open reading frames (ORFs) between Campylobacter and a related Proteobacteria Helicobacter, 27 ORFs involved the flagellar biosynthesis and function, emphasizing its importance.
Bacterial adherence to host cells is an important role in virulence as it allows the act of cellular invasion to take place. Fibronectin (FN) has been demonstrated to be an important factor for bacterial cell adhesion to epithelial cells by various encoded proteins (Fouts et al., 2005). Exposed fibronectin on cellular basolateral membrane of epithelial cells were determined as targets for adherence and internalization. After sequencing the genome of strain RM1221 and comparing it with others, two additional FN-binding proteins had been found to be conserved across 5 strains (Fouts et al., 2005). Aiding in the attachment of Campylobacter to host cells, the expression of phosphorylcholine from a newly discovered licA locus was also found by Fouts et al. (2005), utilizing a shotgun method on several species for comparison. It was noted that regulation of licA is due to alterations in the number of repeats of intragenic tandem tetranucleotide repeats of CAAT, resulting in on/off regulation of production ( Weiser, Shchepetov, & Chong, 1997; Serino & Virji, 2002), suggesting a mutational variance with increased virulence similar to that found in Haemophilus influenza.
After entire nucleotide sequencing for the highly virulent strain C. jejuni 81-176 was completed, several new genetic loci were revealed, along with some deletions, indicating unique pathogenic features when compared to lesser virulent strains (Hofreuter et al., 2006). This team’s study utilized a new high-throughput sequencing of whole-genome comparison that found 37 relevant genes in strain 81-176. Some of these genes, which are absent or are pseudogenes in strains NCTC 11168 and RM1221, allow for an increased and more robust anaerobic respiration system. These added encoding regions are suspected to form a more efficient system, enhancing the ability for colonization. During cellular invasion, C. jejuni uses a variety of different methods for electron transport. Alternate electron acceptors have been determined such as fumerate, nitrate, nitrite, trimethylamine-N-oxide, dimethyl sulfoxide (DMSO), and sulfate ( Sellars, Hall, & Kelly, 2002; Myers & Kelly, 2005; Pittman & Kelly, 2005). Gene clusters in strain 81-176 were found to encode proteins that support the function and signaling of an additional DMSO reductase system, allowing for increased capability of oxygen-restricted respiration (Hofreuter et al., 2006). Allowing Campylobacter to colonize the intestinal tract, a temperature-dependent signaling pathway was discovered (Fouts et al., 2005) and subsequently determined that the ORFs containing this pathway were highly conserved, indicating its importance of pathogenicity.
Toxins are another important factor in the virulence of Campylobacter jejuni in which it plays a key role, among other roles, disrupting host cells from dividing, preventing immune system activation, and allowing it to evade the immune system and survive intracellularly (Parkhill et al., 2000). The shotgun assembly of the genome was reported by Parkhill et al. (2000) to contain a domain coding for a contact-dependent hemolysin, which is an important factor for virulence allowing the disruption and destruction of erythrocytes by means of autolysis, effectively evading the host immune response. In comparison with lesser attenuated strains, genes encoding for virulence factors, such as cytolethal distending toxin, iron binding protein, and a catalase for hydrogen peroxide resistance, were found to be expressed more so in the virulent strain (Carrillo et al., 2004). In addition, there have been 3 cytolethal distending toxins found to be conserved within various strains of Campylobacter jejuni, causing a disruption to the tight junctions of the intestinal epithelial cells ( Muza-Moons, Koutsouris, & Hecht, 2003; Fouts et al., 2005).
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