Short Lecture “Antiadhesive natural products against Campylobacter: Recombinant expression of C. jejuni FlpA and JlpA adhesins and development of a screening ELISA”

Different species of the genus Campylobacter are main causes of acute severe bacterial gastroenteritis [1]. Infections by Campylobacter are initiated by recognition of intestinal epithelial cells by the bacteria, followed by adhesion to host cells, invasion, cellular destruction and subsequent strong inflammation. Antiadhesive strategies aim to interfere with the early host-pathogen interaction. C. jejuni has a complex adhesion strategy, using mainly three outer-membrane-proteins CadF (Campylobacter adhesion protein to Fibronectin), FlpA (Fibronectin like protein A) and JlpA (Jejuni like protein A) for adhesion to host cells. For identification of specific adhesion blockers, FlpA and JlpA were chosen for development of ELISA protocols.

FlpA from C. jejuni binds to 45 kDa gelatine binding domain of fibronectin, leading to integrin activation and to invasion [1].

JlpA is a glycosylated lipoprotein, interacting with HsP90α and activating NF-κB-dependent inflammation [2].

FlpA and JlpA from Campylobacter DSM 27 585 were amplified by PCR and cloned into pCOLD I. Recombinant protein was expressed using ArcticExpress E. coli and induction with 1 mM IPTG at 11.5 °C (cold shock). Both His-tagged proteins were purified by Immobilized Metal Affinity Chromatography (IMAC) yielding in 20 mg/L culture. Identity and purity of the proteins was confirmed by MS-sequencing and PAGE.

For screening of FlpA and JlpA inhibitors, specific ELISAs were developed and validated [3]. The plate was coated with FN or HSP90α, coincubated with test compounds and FlpA or JlpA. Bound protein was detected by antibodies, followed by colorimetric readout. Using this system glucosamine-polymers were identified as strong and specific JlpA inhibitors.

Fig. 1 Setup of FlpA ELISA.

Publication History

Article published online:
12 December 2022

• References

• 1 Konkel ME, Talukdar PK, Negretti NM, Klappenbach CM. Taking Control: Campylobacter jejuni Binding to Fibronectin Sets the Stage for Cellular Adherence and Invasion. Front Microbiol 2020; 11: 564
• 2 Jin S, Song YC, Emili A. et al. JlpA of Campylobacter jejuni interacts with surface-exposed heat shock protein 90alpha and triggers signalling pathways leading to the activation of NF-kappaB and p 38 MAP kinase in epithelial cells. Cell Microbiol 2003; 5: 165-174
• 3 Konkel ME, Larson CL, Flanagan RC. Campylobacter jejuni FlpA binds fibronectin and is required for maximal host cell adherence. J Bacteriol 2010; 192: 68-76