Characterization and Vaccine Potential of Membrane Vesicles Produced by Francisella noatunensis subsp. orientalis in an Adult Zebrafish Model.

Vaccine development against extracellular bacteria has been important for the sustainability of the aquaculture industry. In contrast, infections with intracellular pathogens remain largely an unresolved problem. subsp. is a Gram-negative, facultative intracellular bacterium that causes the disease francisellosis in fish. Francisellosis is commonly characterized as a chronic granulomatous disease with high morbidity and can result in high mortality depending on the host. In this study, we explored the potential of bacterial membrane vesicles (MVs) as a vaccine agent against subsp. Bacterial MVs are spherical structures naturally released from the membrane of bacteria and are often enriched with selected bacterial components such as toxins and signaling molecules. MVs were isolated from broth-cultured subsp. in the present work, and proteomic analysis by mass spectrometry revealed that MVs contained a variety of immunogenic factors, including the intracellular growth proteins IglC and IglB, known to be part of a pathogenicity island (FPI), as well as outer membrane protein OmpA, chaperonin GroEL, and chaperone ClpB. By using flow cytometry and electron microscopy, we observed that subsp. mainly infects myelomonocytic cells, both and Immunization with MVs isolated from subsp. protects zebrafish from subsequent challenge with a lethal dose of subsp. To determine if MVs induce a typical acute inflammatory response, mRNA expression levels were assessed by quantitative real-time PCR. Expression of , , and , as well as , , and , was upregulated, thus confirming the immunogenic properties of subsp. -derived MVs.