Quantification of serine protease HtrA molecules secreted by the foodborne pathogen Campylobacter jejuni

Abstract

Campylobacter jejuni is a major food-borne pathogen and a worldwide health threat. Utilizing different virulence factors, C. jejuni invades the host’s intestinal epithelial cell layer. One important factor in this process is the serine protease HtrA, which is secreted into the extracellular space, and helps the bacteria to transmigrate across the gut epithelium by cleaving various cell–cell adhesion proteins. The aim of the present study is to quantify the amount of HtrA molecules secreted per bacterial cell in liquid culture and during infection. HtrA protein purification and quantitative Western blotting were used to determine the number of HtrA molecules secreted by two C. jejuni model strains, 11168 and 81-176, in liquid culture during an 8-h time course. On average, the two strains yielded similar HtrA secretion rates, with strain 11168 secreting 4314 ± 949 molecules and 81-176 secreting 5483 ± 1246 per bacterium after 2 h. After 8 h, both strains showed a decrease in the average amount of HtrA secreted per bacterial cell over time. Secretion of HtrA by strain 11168 reduced to about 1772 ± 520 molecules and only 2151 ± 562 HtrA molecules were secreted by strain 81-176 at this time point. During infection of gut epithelial cells, the secretion of HtrA is slightly higher with a similar secretion pattern over time compared to culturing in vitro. We determined the number of HtrA molecules secreted by single C. jejuni cells over time. The results suggest that HtrA secretion is regulated in a time-dependent fashion, leading to increasing accumulative HtrA concentrations in the extracellular medium.