Oral Presentation The Prato Conference on the Pathogenesis of Bacterial Infections of Animals 2016

Streptococcus equi subsp. zooepidemicus as an intracellular pathogen (#28)

Bolette Skive 1 , Manfred Rohde 2 , Gabriella Molinari 2 , Lisa Glenton 3 , Natali Krekeler 3 , Mette Christoffersen 4 , Line Nymann Thomsen 5 , Anders Miki Bojesen 1
  1. Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg C, Denmark
  2. Central Facility for Microscopy, Helmholtz Centre for Infection Research, Braunschweig, Germany
  3. Faculty of Veterinary and Agricultural Science, University of Melbourne, Melbourne, Victoria, Australia
  4. Department of Large Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Taastrup, Denmark, Danmark
  5. Dako, Agilent, Glostrup, Denmark

Streptococcus equi subsp. zooepidemicus (SEZ) is an opportunistic pathogen of several animal species and humans. SEZ is a commensal on the mucus membranes of horses, but it is a common cause of both acute and chronic endometritis when entering the uterus. In this study we investigated if SEZ was able to invade and survive inside epithelial cells. An intracellular state could be an important part of SEZ pathogenicity when causing acute infections, as well as a way of residing in certain niches, such as the endometrium.

Infection assay: HeLa cells were infected with SEZ strain 1-4a and S31A1, both originating from the uterus of mares with endometritis. The infection was stopped by fixation at various time points. The coverslips were examined with FESEM (field emission scanning electron microscopy) evaluating adhesion and invasion at the cell surface, and immunofluorescence stained to show intra- and extracellular bacteria, and quantify these. Furthermore we stained for lysosome associated membrane protein (LAMP-1) to visualize intracellular trafficking. Lactococcus lactis was included as a non-invasive control. Controls of the immunofluorescence antibodies and a negative control were included.

SEZ was able to invade the non-professional phagocytic human epithelial cell lines. Preliminary data suggest intracellular survival and a substantial cross-talk between the epithelial cell and the bacteria, with changes in the appearance of the bacterial cell wall and pili-like protrusions. SEZ was in some stages of infection found in lysosomes.

The invasion of epithelial cells gives new understanding into SEZ’s way of establishing infection. If some bacteria survive for prolonged periods, these could be a source of recurrent/persistent infections as well. This first study was done in cell lines. The ability of SEZ to internalize and survive in eukaryotic cells will be further investigated using primary equine endometrial cells, isolated from the uterus of mares.