Study of endocytosis, transcytosis and nuclear location of porcine lactoferrin in enteroids and design of smart lactoferrin by nanobody mediated targeting of lactoferrin to intestinal Chlamydia suis and its virulence factors

Chlamydia suis is a gram negative obligate intracellular bacterium. C. suis is an emerging pig pathogen causing significant economic losses in the pork industry. The focus of our proposal is on prevention of intestinal C. suis in swine as: i) the gut functions as a C. suis reservoir, ii) C. suis and especially tetracycline resistant C. suis, causes economic loss and preventive strategies are currently unavailable and iii) C. suis is a zoonotic pathogen. We hypothesize that porcine LF (pLF), a protein of the innate immune system, might prevent C. suis intestinal infection through its antibacterial and immunomodulatory capacity. To support our hypothesis, we will first study receptor-mediated endocytosis and biodistribution of pLF inside porcine enteroids as well as the signaling pathways triggered by pLF in enteroids. We strongly believe that the antibacterial capacity of pLF can even be increased by nanobody-mediated targeting of pLF towards the outer membrane of chlamydia or its Type 3 Secretion System (T3SS). Nanobodies are single-domain antibody fragments originating from the variable part of the heavy chain of camelid heavy chain antibodies. Nanobodies against the chlamydia outer membrane and against components of the T3SS will be coupled to pLF and used against C. suis infections in porcine enteroids. In the end, we will study the effect of these ‘smart’ pLFs on intestinal C. suis infection during small intestinal segment perfusion (SISP) experiments in SPF pigs.