Role of microbial-derived extracellular vesicles in pathophysiology

Background of in vitro - Role of microbial-derived extracellular vesicles in pathophysiology

• Like mammalian cells, in addition to soluble factors, most Gram-negative and -positive bacteria release extracellular vesicles (EVs) which can be involved in the intercellular communication between living organisms.

• Acne vulgaris is a common inflammatory disorder affecting more than 80% of young adolescents. Cutibacterium acnes plays a role in the pathogenesis of acne lesions, although the mechanisms are poorly understood.

Objectives of the model

• To understand the impact of C. acnes’ extracellular vesicles on keratinocytes or sebocytes, two skin cell types in close contact with the colonizing bacterium.

Our approach at Vibiosphen

• After culture in 1.5L of broth, C. acnes cells were pelleted at 4,500 rpm for 20 min at 4°C and the supernatant was filtered through 0.45 and 0.22 µm vacuum filters. The filtrates were concentrated approximately 20-fold through tangential ultrafiltration using a 100 kDa membrane (Vivaflow system, Sartorius), down to 50 mL. The 50 mL were concentrated through centrifugation on amicon columns using a 50 kDa membrane down to approximately 1 mL.
• The concentration of EVs was estimated using protein concentration determination, using the Bradford assay.
• A fraction of the EVs were labeled with the fluorescent dye Vybrant DiI.
• EVs were added to the culture medium of primary human keratinocytes or sebocytes.

Outcomes of in vitro -Role of microbial-derived extracellular vesicles in pathophysiology

This experimental approach may help to elucidate the role of microbial extracellular vesicles in the pathophysiology of various infections.