The pathogenic potential of Campylobacter concisus strains associated with chronic intestinal diseases

Abstract

Campylobacter concisus has garnered increasing attention due to its association with intestinal disease, thus, the pathogenic potential of strains isolated from different intestinal diseases was investigated. A method to isolate C. concisus was developed and the ability of eight strains from chronic and acute intestinal diseases to adhere to and invade intestinal epithelial cells was determined. Features associated with bacterial invasion were investigated using comparative genomic analyses and the effect of C. concisus on host protein expression was examined using proteomics. Our isolation method from intestinal biopsies resulted in the isolation of three C. concisus strains from children with Crohn's disease or chronic gastroenteritis. Four C. concisus strains from patients with chronic intestinal diseases can attach to and invade host cells using mechanisms such as chemoattraction to mucin, aggregation, flagellum-mediated attachment, "membrane ruffling", cell penetration and damage. C. concisus strains isolated from patients with chronic intestinal diseases have significantly higher invasive potential than those from acute intestinal diseases. Investigation of the cause of this increased pathogenic potential revealed a plasmid to be responsible. 78 and 47 proteins were upregulated and downregulated in cells infected with C. concisus, respectively. Functional analysis of these proteins showed that C. concisus infection regulated processes related to interleukin-12 production, proteasome activation and NF-κB activation. Infection with all eight C. concisus strains resulted in host cells producing high levels of interleukin-12, however, only strains capable of invading host cells resulted in interferon-γ production as confirmed by ELISA. These findings considerably support the emergence of C. concisus as an intestinal pathogen, but more significantly, provide novel insights into the host immune response and an explanation for the heterogeneity observed in the outcome of C. concisus infection. Moreover, response to infection with invasive strains has substantial similarities to that observed in the inflamed mucosa of Crohn's disease patients.

Figure 1. Scanning electron microscopy of four Campylobacter concisus strains.

C. concisus UNSW2 was observed as spiral curved-shaped bacteria with rounded ends and a single polar flagellum as shown in Panel A (bar = 3 µm). In Panel B (bar = 1.5 µm) C. concisus UNSW3 was observed to be curved-shaped bacteria with rounded ends and a single polar flagellum, while in Panels C (bar = 2 µm) and D (bar = 2.5 µm) C. concisus strains UNSW1 and UNSWCD were shown to be spiral curved-shaped bacterium with rounded ends and a single flagellum.

Figure 2. Scanning electron microscopy of human intestinal cell line Caco-2 infected with Campylobacter concisus strains for six hours.

Panel A shows an overview of uninfected Caco-2 monolayer. The Caco-2 cells expressed differentiating microvilli (Panel A1) and differentiated microvilli (Panel A2). C. concisus was shown to aggregate upon interaction with host cells as shown in Panel B (Panel B1, bar = 1.5 µm and Panel B2, bar = 2 µm). In Panel C, the polar flagellum of C. concisus is shown binding to the tips of host cell microvilli which mediated initial contact with host cells (as indicated by the arrows). Abnormalities in the epithelial host cell structure and microvilli were observed following infection with C. concisus (indicated by a ring in Panel D and arrows in Panel E). Panel F shows the flagellum of C. concisus appeared to wrap itself around the microvilli (as indicated by arrows). Following adherence, C. concisus induced a “membrane ruffling”-like effect on the host cell membrane (indicated by an asterisk in Panel F), and penetrated the host cell membrane from the non-flagellated end (indicated by an arrow in Panel G). C. concisus was observed invading the host cell (indicated by arrows in Panels G, H and I) resulting in irregular shaped membrane protrusions (indicated by asterisks in Panels G, H and I), leading to host cell damage (indicated by “#” in Panels G, H and I).

Figure 3. Scanning electron microscopy of human mucin producing intestinal cell line LS174T infected with Campylobacter concisus strains for six hours.

Panel A shows uninfected LS174T monolayers. LS174T cells expressing microvilli (indicated by a ring) and goblet cells (indicated by a arrow) are shown in Panel B. The mucus layer was found on the monolayer surface of LS174T cells as indicated by an “#” in Panel C. C. concisus appeared to be attracted to the mucus layer of host cells (indicated by “#” in Panel D) using their single polar flagellum (indicated by arrows in Panel E) and upon the interaction with host cells tended to aggregate (Panel F). Panel G shows the polar flagellum (as indicated by an arrow) of C. concisus binding to the tips of host cell microvilli (as indicated by a ring) and goblet cells (as indicated by an arrow in Panel H) which appeared to mediate initial contact with host cells. Following adherence, C. concisus induced a “membrane ruffling”-like effect on the host cell membrane (indicated by an asterisk in Panel I) and penetrated the host cell membrane from the non-flagellated end (indicated by an arrow in Panel I) resulting in cell damage (indicated by “#” in Panel I).

Figure 4. Graphical representation of the genes encoded by the plasmid purified from Campylobacter concisus UNSWCD.

Outer circle (blue) represents the coding sequences within the plasmid; inner circle (black) represents the GC content; inner circle (purple/green) represents the GC skew.

Figure 5. PCR analysis of the exotoxin 9 gene in the eight Campylobacter concisus strains.

Lane 1: FN-1 marker, lane 2: UNSWCD, lane 3: UNSW2, lane 4: UNSW3, lane 5: UNSW1, lane 6: BAA-1457, lane 7: UNSWCS, lane 8: ATCC 51562, lane 9: ATCC 51651 and lane 10: negative control.

Figure 6. Two-dimensional proteomes of (A) non-infected Caco-2 cells (pI 4–7), (B) Caco-2 cells infected with C. concisus UNSWCD (pI 4–7), (C) non-infected Caco-2 cells (pI 7–10), and (D) Caco-2 cells infected with C. concisus UNSWCD (pI 7–10).

Proteins differentially expressed between the two growth conditions are listed in Table 3 and Table 4. Spot numbers correspond to numbers in Table S1 and Table S2.

Figure 7. Levels of interleukin-12 and interferon-γ produced by the human monocytic leukemia cell line THP-1 following infection with Campylobacter concisus strains and Escherichia coli K-12.

* represents P<0.05; ** represents P<0.01. Data of three independent experiments ± standard error of the mean.

Figure 8. Proposed immune response to Campylobacter concisus UNSWCD.

(A) Non-invasive C. concisus strains adhere to the host cell and induce the production of IL-12. (B) Invasive C. concisus strains adhere to and invade the host cell inducing both IL-12 and IFN-γ, which in turn activate the immunoproteosome. The bacterial insult upregulates ubiquitinating and downregulates de-ubiquitinating enzymes which leads to the ubiquitination of NF-κB inhibitors. The immunoproteosome targets these inhibitors which activates NF-κB.