Involvement of a Plasmid in Virulence of Campylobacter jejuni 81-176

Abstract

Campylobacter jejuni strain 81-176 contains two, previously undescribed plasmids, each of which is approximately 35 kb in size. Although one of the plasmids, termed pTet, carries a tetO gene, conjugative transfer of tetracycline resistance to another strain of C. jejuni could not be demonstrated. Partial sequence analysis of the second plasmid, pVir, revealed the presence of four open reading frames which encode proteins with significant sequence similarity to Helicobacter pylori proteins, including one encoded by the cag pathogenicity island. All four of these plasmid-encoded proteins show some level of homology to components of type IV secretion systems. Mutation of one of these plasmid genes, comB3, reduced both adherence to and invasion of INT407 cells to approximately one-third that seen with wild-type strain 81-176. Mutation of comB3 also reduced the natural transformation frequency. A mutation in a second plasmid gene, a virB11 homolog, resulted in a 6-fold reduction in adherence and an 11-fold reduction in invasion compared to the wild type. The isogenic virB11 mutant of strain 81-176 also demonstrated significantly reduced virulence in the ferret diarrheal disease model. The virB11 homolog was detected on plasmids in 6 out of 58 fresh clinical isolates of C. jejuni, suggesting that plasmids are involved in the virulence of a subset of C. jejuni pathogens.Although Campylobacter jejuni is one of the major causes of bacterial diarrhea worldwide (51, 59), the details of its molecular pathogenesis are not well understood. The clinical symptoms of campylobacter infection can range from a mild, watery diarrhea to a dysentery-like illness with fecal blood and leukocytes (2). Although there are reports of numerous cytotoxins, only the cytolethal distending toxin, which arrests eukaryotic cells at the G 2 phase of the cell cycle (64), has been characterized in detail. There are numerous reports that C. jejuni strains can invade intestinal epithelial cells in vitro (20,21,24,29,30,38), although levels of invasion by different strains vary considerably (20,28,38,52). Strain 81-176, originally isolated from a diarrheal outbreak associated with raw-milk consumption (31), is one of the best-characterized strains of C. jejuni. This strain has been shown to cause an inflammatory diarrhea in two human feeding studies (8; D. Tribble, unpublished data) and to cause disease in experimental models using primates (40) and ferrets (19,67). Further, C. jejuni strain 81-176 invades INT407 cells at levels higher than those of most other C. jejuni strains (28, 38).Plasmids have been found in between 19 and 53% of C. jejuni strains (5, 9-11, 41, 53-58), and many of these have been reported to be R plasmids that are transmissible among Campylobacter spp. but not to Escherichia coli (53)(54)(55)(56)(57)(58)60). Despite the importance of plasmids to virulence in numerous other pathogens, it is generally believed that plasmids play no role in Campylobacter pathogenicity. This paradigm is based on the rather low level at which plasmids ...

Campylobacter

“…has been shown to vary considerably between 44 and 91% for clinical and poultry isolates respectively in one study [75], compared with a plasmid isolation rate of 19% reported in a separate study [14]. As shown in Table II, tetracycline, kanamycin and chloramphenicol resistances are primarily plasmid-mediated.…”

“…Emergence of resistance to these agents however, has since made their efficacy less certain. Resistance was reported to develop among patients after treatment with fluoroquinolones [14], and was also found to coincide with the introduction of these agents in veterinary medicine [1,2,38]. However, an increasing number of Campylobacter isolates resistant to these drugs are now being cultured from both clinical and food samples in several European countries, Canada and the United States.…”

Lack of response of INT-407 cells to the presence of non-culturableCampylobacter jejuni

“…As sublethally injured C. jejuni are sensitive to selective agents, selective enrichment will negatively influence the culturability [34] and the presence of a few culturable C. jejuni might be not detected. Another explanation might be the use of in vivo instead of in vitro models, although previous studies showed a correlation between the adhesion and invasion properties and IL-8 values in vitro to the infectivity in vivo [35][36][37].…”

The versatile bacterial type IV secretion systems

“…Instead of mediating DNA transfer between bacteria, these systems deliver DNA or protein substrates, known as effectors, to eukaryotic target cells during infection [3][4][5] . More recently, several new T4S systems have been described that are also ancestrally related to the conjugation machines, but these systems mediate the exchange of DNA with the extracellular milieu [6][7][8] . Collectively, this diversity of function in the face of a common ancestry makes the T4S machines attractive subjects for comparative studies that explore the dynamics of organelle assembly and action.…”

“…1; Table 1). This recently discovered subfamily presently comprises two COMPETENCE (DNA-uptake) systems -the Campylobacter jejuni Cjp/VirB system and the Helicobacter pylori ComB system 6,7,17,18 -and one DNArelease system, an F-plasmid Tra-like system that is carried on the gonococcal genetic island (GGI) of Neisseria gonorrhoeae 8,19 . As with the conjugation machines, these systems promote genetic exchange and therefore also represent potential mechanisms for the transfer of survival traits during infection 20 .…”