"Molekulare Charakterisierung eines neuen Adhäsions- und Invasionsfaktors des bedeutenden lebensmittelbedingten Pathogens Campylobacter jejuni"
Zusammenfassung der Projektergebnisse
Campylobacter species including C. jejuni are progressively emerging food-borne causes of bacterial gastroenteritis responsible for significant healthcare and socioeconomic burdens worldwide. An important feature of foodborne pathogens is their ability to bind to and invade eukaryotic cells. Enteropathogenic invasion into intestinal epithelial cells is the crucial initial step during the infection process and provide advantages to the bacteria such as avoiding attacks of the host’s immune system. In order to gain access to deeper tissue sites, the pathogen needs to overcome the epithelial barrier by the paracellular or transcellular route. Using the paracellular route, C. jejuni enter between two neighboring cells by cleaving tight- and adherence junction proteins to reach the basolateral side of the host cells. Therefore, distinct pathogenic proteases such as the serine protease HtrA (high temperature requirement A) are needed. One of its major functions is the prevention of protein damage, protein quality control as well as the degradation of misfolded proteins to avoid cytotoxic effects. For this purpose, HtrA exhibits protease and chaperone activities. The HtrA protein acts as an intracellular chaperone during stress resistance, such as oxygen or heat and in addition as an extracellular protease enabling transmigration and invasion of C. jejuni to intestinal epithelial cells. Our research on C. jejuni HtrA revealed that the deletion of HtrA led to a reduction of adherence to and invasion into human epithelial cells in vitro which indicates that the protease plays an important role during infection and in particular, for invading deeper tissues. Based on these findings, HtrA might be an important factor that is responsible for the differences in pathogenicity between humans and chicken. Recently, we could show a HtrA-dependent adherence and invasion of C. jejuni in human versus avian cells. HtrA is assumed to be involved in this scenario, but apparently the protease does not seem to be responsible for the differences of virulence in human versus avian hosts. Our in vivo experiments applying a reliable infection and inflammation model mimicking key features of acute human campylobacteriosis (i.e., the secondary abiotic IL-10-/- mouse model) revealed that HrtA constitutes a remarkable novel virulence determinant of C. jejuni, whose protease activity, however, is not required for intestinal colonization and establishment of disease, but aggravates campylobacteriosis by triggering apoptosis and proinflammatory immune responses in intestinal as well as extra-intestinal including even systemic compartments. We were further able to show that C. jejuni HtrA exhibits a RGD motif that is able to bind cellular surface receptors such as integrins and facilitates invasion into and persistence within host cells. However, adhesion, invasion and transmigration properties were more severely compromised when applying a C. jejuni ∆htrA deletion mutant as compared to a strain with a mutated RGD motif (i.e., ∆htrA/HtrA RGD/AAA mutant strain) in our in vitro analyses. In fact, the RGD motif appears to be crucial for the adhesion of C. jejuni to intestinal epithelial cells irrespective whether of human or chicken origin. Moreover, our in vivo analyses revealed that the RGD motif within the C. jejuni htrA gene plays a pivotal role in the amplification of intestinal and extra-intestinal including systemic immunopathological responses upon peroral C. jejuni infection of secondary abiotic IL-10-/- mice. We further analyzed the immunopathological role of another major adhesin, namely the Campylobacter adhesion to fibronectin (CadF) in our in vivo experiments and included a C. jejuni strain that was deficient in the flagellins A and B as a control given that bacterial motility constitutes an important prerequisite for subsequent immunopathology. Our study provides evidence for the first time, that C. jejuni FlaA/B as opposed to CadF are pivotally involved in inducing acute campylobacterioisis upon peroral infection of the vertebrate host, whereas both deletion mutant strains (i.e., ∆flaA/B and ∆cadF) were able to colonize the intestinal tract of secondary abiotic IL-10-/- mice with comparable loads like the parental wildtype strain. To date, more than 45 peptidase-related proteins have been identified by in silico analysis of the C. jejuni NCTC11168 genome, although their possible roles in pathogenesis remain to be established experimentally. During a screen for proteases expressed by C. jejuni we identified a peptidase of the M24 family, constructed a deletion mutant (i.e., ∆pepP) and analyzed its immunopathological impact during acute murine campylobacteriosis. Following peroral infection, secondary abiotic IL-10-/- mice could be stably infected with ∆pepP strain but suffered less distinctly from C. jejuni induced gross disease as compared to parental and complemented strain infection which was accompanied by less pronounced immunopathological sequelae in the intestinal tract and beyond (namely, the liver, kidney, lungs and systemic compartments). Hence, the peptidase PepP constitutes a novel virulence factor of C. jejuni contributing to acute campylobacteriosis in the vertebrate host. We hypothesize that more bacterial factors including proteases need to be identified and analyzed to further dissect the orchestrated interplay during C. jejuni-host interactions in order to pave the way for novel therapeutic and preventive measured in the combat of campylobacteriosis.
Projektbezogene Publikationen (Auswahl)
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Expression of Helicobacter pylori serine protease HtrA in Campylobacter jejuni reveals a crucial function in oxygen stress resistance, heat tolerance and epithelial barrier disruption. Cel. Immunol Serum Biol. 2017; 3:2
Boehm M, Tegtmeyer N, Harrer A, Skórko-Glonek J, Backert S
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Function of Serine Protease HtrA in the Lifecycle of the Foodborne Pathogen Campylobacter jejuni. Eur J Microbiol Immunol. 2018; 8:70-77
Boehm M, Simson D, Escher U, Schmidt AM, Bereswill S, Tegtmeyer N, Backert S, Heimesaat MM
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HtrA-dependent adherence and invasion of Campylobacter jejuni in human vs. avian cells. Letters in Applied Microbiology. 2019; 70: 326-330
Simson D, Boehm M, Backert S
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Immunopathological properties of the Campylobacter jejuni flagellins and the adhesin CadF as assessed in a clinical murine infection model. Gut Pathog. 2019; 11:24
Schmidt AM, Escher U, Mousavi S, Tegtmeyer N, Boehm M, Backert S, Bereswill S, Heimesaat MM
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Protease Activity of Campylobacter jejuni HtrA Modulates Distinct Intestinal and Systemic Immune Responses in Infected Secondary Abiotic IL-10 Deficient Mice. Front Cell Infect Microbiol. 2019; 9:79
Schmidt AM, Escher U, Mousavi S, Boehm M, Backert S, Bereswill S, Heimesaat MM
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Peptidase PepP is a novel virulence factor of Campylobacter jejuni contributing to murine campylobacteriosis. Gut Microbes. 2020; 12: 1770017
Heimesaat MM, Schmidt AM, Mousavi S, Escher U, Tegtmeyer N, Wessler S, Gadermaier G, Briza P, Hofreuter D, Bereswill S, Backert S
