Carbohydrate binding and gene expression by in vitro and in vivo propagated Campylobacter jejuni after Immunomagnetic Separation

Abstract

Campylobacter jejuni is an important human food‐borne intestinal pathogen, however relatively little is known about its mechanisms of pathogenesis or pathogen‐host interactions. To monitor changes in gene expression and glycan binding of C. jejuni within a common avian host, an immunomagnetic separation technique (IMS) was utilised to directly isolate infecting C. jejuni 81116 from a chicken host. An average of 105 cells/g was re‐isolated from chicken caecal samples by IMS technique. The in vivo passaged strains were used successfully in evaluation of carbohydrate binding through the use of a glycan array and were further suitable for transcriptome analysis. The glycan microarray analysis demonstrated differences in binding to negatively charged glycans of laboratory grown strains of C. jejuni compared with strains isolated after in vivo passage. The in vivo passaged strains showed marked up‐regulation of chemotaxis receptors and toxin genes. The optimised Campylobacter IMS technique described in this study allowed isolation directly from an animal host. Changes in gene expression and glycan binding at an in vivo level can also be identified by using this method.

A peculiar case of Campylobacter jejuni attenuated aspartate chemosensory mutant, able to cause pathology and inflammation in avian and murine model animals

“…The cdtA gene expression by ccaA isogenic mutant cells isolated from chickens was up-regulated as was the expression of cdtB in the mutant cells isolated from the mouse. This was in agreement with a previous study showing that in chickens the cdtA/C subunits could be up-regulated 34 . Therefore, while it may be a contributing pathogenicity factor, it is possible to speculate that they are unlikely to account for the observed pathology.…”

“…jejuni 11168-GS and 11168-O isolated from the caecal content of chicks at day 5p.i. by IMS 34 . The binding specificities of 11168-O and 11168-O ΔccaA::cat were compared and only statistically significant differences (p < 0.05) are described in Table 1 .…”

“…jejuni from mouse intestinal content or chicken caecal content was performed as previously described by King et al . 34 .…”

Variation of chemosensory receptor content of Campylobacter jejuni strains and modulation of receptor gene expression under different in vivo and in vitro growth conditions

“…Cultures for RNA analysis were grown under the following conditions: Cultures that mimic environmental conditions were performed as previously described [ 12 ]. Cultures grown for laboratory conditions were grown at either 37 or 42°C as described in Day et al (2009) and processed to minimise effects on RNA expression as per King et al (2012) [ 12 , 21 ].…”

Carbohydrate-binding protein identification by coupling structural similarity searching with binding affinity prediction

“…Moreover, more and more proteins have been sequenced without knowledge of their function(s) due to increasingly inexpensive sequencing techniques. Although there is one high‐throughput technique (glycan arrays) for detecting novel CBPs and investigating their binding specificity, it is challenging to construct a sizeable, diverse glycan array because of difficulty in synthesis and isolation of carbohydrates. Here, we focus on a complementary approach: prediction of CBPs and their binding amino acid residues by computational techniques.…”