Fluorescence Microscopy, a Key Tool for the Understanding of Bordetella parapertussis Immune Evasion Strategies

LAMBERTI, YANINA; GORGOJO, JUAN; RODRIGUEZ, MARIA EUGENIA

La Plata

Workshop; 105. Imaging Techniques for Biotechnological and Biomedical Applications Workshop; 2016

Secretaría de Ciencia y Técnica, Facultad de Ciencias Exactas, UNLP

Bordetella pertussis and Bordetella parapertussis are the etiological agents of whooping cough, a reemerging disease that remains a threat to human health. Both species are strictly human and persistent pathogens that could not be eradicated despite decades of vaccination. Clinical surveys indicate that B. parapertussis is responsible for a significant proportion of cases of whooping cough, particularly in vaccinated populations, which was intensified after introduction of acellular pertussis vaccines. Current acellular pertussis vaccines formulated only with B. pertussis failed to protect against B. parapertussis infections. This is due to the presence of the O-antigen molecule on B. parapertussis surface membrane that blocks antibody access to the vaccine antigens common to both species. As a result vaccinated populations are not immunized against this pathogen. Given the lack of bacterial recognition by antibodies induced by pertussis vaccines we investigated the innate interaction of phagocytes and B. parapertussis. We have demonstrated for the first time that in the absence of opsonic antibodies B. parapertussis avoids killing and survive intracellularly in macrophages and neutrophils, both constituent of the first line of defense against microbes. By using specific fluorescent probes and fluorescence microscopy (confocal and wide-field epifluorescence microscopy), we could dissect many aspects of the innate encounter of B. parapertusis with these immune cells both at early and late stages during infection. Among them, the preclusion of neutrophil extracellular bactericidal mechanisms know as degranulation and neutrophil extracellular traps, the evasion of Intracellular bactericidal mechanisms of neutrophils both by preventing bacterial uptake and by avoiding phagosome-lysosome fusion if it is phagocytosed. By mean of microscopy tools we were able to dissect the role of the site of entry in the intracellular trafficking of phagocytosed bacteria. As with neutrophils, microscopic techniques allowed us to describe the innate interaction of B. parapertussis with macrophages. We performed a fluorescent in situ hybridization (FISH) assay to image live B. parapertussis inside macrophages. By this technique we demonstrated that solely those bacteria that avoided phagosome-lysosome fusion were alive. Moreover, live intracellular B. parapertussis were found in lipid rafts enriched phagosomes that retained early endosome characteristics until late stages post-infection. Although this bacterium was assumed as extracellular pathogens for a long time, our results challenges this hypothesis suggesting that there might one or more intracellular phases playing a role in the infectious process, the immune evasion, and the persistence of this pathogen within the population. An intracellular phase in these types of cells could allow the pathogen to hide from host anti-microbial defense system, repopulate the extracellular media once the environmental conditions are favorable, manipulate host immune response, or disseminate to other areas in the host or toward other hosts.ORAL PRESENTATION