High molecular/low acetylated chitosans reduce adhesion of Campylobacter jejuni to host cells by blocking JlpA

Abstract

Infections caused by Campylobacter spp. are a major cause of severe enteritis worldwide. Multifactorial prevention strategies are necessary to reduce the prevalence of Campylobacter. In particular, antiadhesive strategies with specific inhibitors of early host-pathogen interaction are promising approaches to reduce the bacterial load. An in vitro flow cytometric adhesion assay was established to study the influence of carbohydrates on the adhesion of C. jejuni to Caco-2 cells. Chitosans with a high degree of polymerization and low degree of acetylation were identified as potent antiadhesive compounds, exerting significant reduction of C. jejuni adhesion to Caco-2 cells at non-toxic concentrations. Antiadhesive and also anti-invasive effects were verified by confocal laser scanning microscopy. For target identification, C. jejuni adhesins FlpA and JlpA were expressed in Escherichia coli ArcticExpress, and the influence of chitosan on binding to fibronectin and HSP90α, respectively, was investigated. While no effects on FlpA binding were found, a strong inhibition of JlpA-HSP90α binding was observed. To simulate real-life conditions, chicken meat was inoculated with C. jejuni, treated with antiadhesive chitosan, and the bacterial load was quantified. A strong reduction of C. jejuni load was observed. Atomic force microscopy revealed morphological changes of C. jejuni after 2 h of chitosan treatment, indicating disturbance of the cell wall and sacculi formation by electrostatic interaction of positively charged chitosan with the negatively charged cell surface. In conclusion, our data indicate promising antiadhesive and anti-invasive potential of high molecular weight, strongly de-acetylated chitosans for reducing C. jejuni load in livestock and food production. KEY POINTS: • Antiadhesive effects of chitosan with high DP/low DA against C. jejuni to host cells • Specific targeting of JlpA/Hsp90α interaction by chitosan • Meat treatment with chitosan reduces C. jejuni load.

Keywords: Adhesion; Atomic force microscopy; Campylobacter jejuni; Chitosan; JlpA; Sacculus.

Fig. 1

Confocal laser scanning microscopy of Caco-2 cells, incubated for 0.5 (A), 2 (B) and 5 h (C) with C. jejuni (BCR 100:1). Quantitative evaluation by pixel counting of green fluorescence CFDA-SE stained bacteria over the time clearly shows strong invasion after 5 h (D). Different z-stack sections reveal representative depth sections of infected Caco-2 cells after 5 h of incubation (E to I) differentiating between adhering and internalized C. jejuni. C. jejuni cells are displayed in green after staining with CFDA-SE, cell nuclei of T24 cells (blue) are stained with DAPI, and wheat germ agglutinin stain host cell membranes with AlexaFluor 594 (red)

Fig. 2

Relative binding affinity of A recombinant JlpA (0.5 µg/well) to Hsp90α and B recombinant FlpA (7.5 μg/well) to FN under coincubation with chitosan 134. Data represent the mean ± SD from n = 3 independent experiments with n = 2 technical replicates

Fig. 3

Atomic force microscopy of C. jejuni cells under ambient conditions in air. Representative ultrastructural morphology of untreated C. jejuni cells (A) compared to chitosan 114 (B) and chitosan 134 (C) treated bacteria at 100 µg/mL. Elongated cells were frequently observed in chitosan-treated individuals at 100 µg/mL, indicating potential cell wall defects induced by chitosan treatment

Fig. 4

Atomic force microscopy of isolated C. jejuni peptidoglycan sacculi under ambient conditions in air. Representative ultrastructural morphology of an isolated peptidoglycan sacculus from an untreated C. jejuni cell. In case of chitosan treatment, peptidoglycan isolation procedures yielded agglomerated specimens and did not allow closer insights into peptidoglycan ultrastructure by AFM. Hence, no representation of a chitosan-treated C. jejuni sacculus is shown