Protease Activity of Campylobacter jejuni HtrA Modulates Distinct Intestinal and Systemic Immune Responses in Infected Secondary Abiotic IL-10 Deficient Mice.

Anna-Maria Schmidt,Manja Boehm,Steffen Backert,Ulrike Escher,Markus M Heimesaat,Soraya Mousavi,Stefan Bereswill

doi:10.3389/fcimb.2019.00079

Abstract

Even though human Campylobacter jejuni infections are progressively increasing worldwide, the underlying molecular mechanisms of pathogen-host-interactions are still not fully understood. We have recently shown that the secreted serine protease HtrA plays a key role in C. jejuni cellular invasion and transepithelial migration in vitro, and is involved in the onset of intestinal pathology in murine infection models in vivo. In the present study, we investigated whether the protease activity of HtrA had an impact in C. jejuni induced acute enterocolitis. For this purpose, we perorally infected secondary abiotic IL-10−/− mice with wildtype C. jejuni strain NCTC11168 (11168WT) or isogenic bacteria carrying protease-inactive HtrA with a single point mutation at S197A in the active center (11168HtrA−S197A). Irrespective of the applied pathogenic strain, mice harbored similar C. jejuni loads in their feces and exhibited comparably severe macroscopic signs of acute enterocolitis at day 6 postinfection (p.i.). Interestingly, the 11168HtrA−S197A infected mice displayed less pronounced colonic apoptosis and immune cell responses, but enhanced epithelial proliferation as compared to the 11168WT strain infected controls. Furthermore, less distinct microscopic sequelae in 11168HtrA−S197A as compared to parental strain infected mice were accompanied by less distinct colonic secretion of pro-inflammatory cytokines such as MCP-1, IL-6, TNF, and IFN-γ in the former as compared to the latter. Strikingly, the S197A point mutation was additionally associated with less pronounced systemic pro-inflammatory immune responses as assessed in serum samples. In conclusion, HtrA is a remarkable novel virulence determinant of C. jejuni, whose protease activity is not required for intestinal colonization and establishment of disease, but aggravates campylobacteriosis by triggering apoptosis and pro-inflammatory immune responses.

Highlights

Campylobacter jejuni is a Gram-negative, spiral-shaped, flagellated bacterium that grows under microaerobic conditions at a temperature optimum of 37–42◦C (Parkhill et al, 2000; Brøndsted et al, 2005; Young et al, 2007)
The serine protease and chaperone high-temperature requirement A (HtrA) constitutes a remarkable virulence factor that enables C. jejuni to transmigrate across polarized epithelial cells by cleavage of E-cadherin and probably other junctional proteins (Boehm et al, 2012, 2018; Hoy et al, 2012)
We have recently shown in infected mice, that the serine protease HtrA is not required for induction of enterocolitis by C. jejuni per se and plays a role in the modulation of apoptosis and immunopathology during campylobacteriosis (Heimesaat et al, 2014a,b)

Summary

Introduction

Campylobacter jejuni is a Gram-negative, spiral-shaped, flagellated bacterium that grows under microaerobic conditions at a temperature optimum of 37–42◦C (Parkhill et al, 2000; Brøndsted et al, 2005; Young et al, 2007). C. jejuni bacteria enter the host intestine via the oral route and colonize the distal ileum and colon by attaching to epithelial cells (Kist and Bereswill, 2001; Boehm et al, 2018). To reach deeper tissues and cause inflammatory responses during the infection process, C. jejuni need to cross the epithelial barrier, which is accomplished by paracellular transmigration as well as invasion into intestinal epithelial cells (Boehm et al, 2012). After crossing the intestinal epithelial barrier and invasion of the underlying submucosa, C. jejuni can enter the mesenteric lymph nodes (MLN), and may reach different organs such as the spleen or liver via the bloodstream, thereby causing extra-intestinal inflammation (Backert et al, 2013)

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