Data published by our group and others, show Bordetella pertussis, B. bronchiseptica and B. parapertussis, can survive and grow intracellular in macrophages and lung epithelial cells. These two aspects have great clinical implications. Intracellular survival could explain vaccine failure (evasion of immune recognition) as well as the long persistence period of clinical disease (disease is reported in adolescent and young adults). This project involves an exceptional opportunity to study all the molecular basis and mechanisms involved in the intracellular survival and replication of Bordetella spp. that can be responsible of the re-emergence, dissemination, lethargy periods, evasion of the immune system, vaccine failure and even transmission.
To understand intracellular survival/growth of Bordetella spp., we propose the following aims:
Aim 1: Identify genes required for specific stages of intracellular survival and replication (intracellular stage).
We will create and screen a transposon library to identify all genes required for survival and replication within macrophages.
Student role: The student will do the transposon library for Bordetella pertussis. Also, the student role in this aim it will be to screen the transposon library of Bordetella bronchiseptica and Bordetella pertussis, using the gentamicin assay and variations of it in order to identify mutations that promote survival / growth within mcarophages.
Aim 2: Define the mechanisms involved during in vitro Bordetella spp. intracellular survival and replication.
Intracellular survival and replication requires to perform several steps; a) adhere, b) invade, c) survive intracellularly, d) obtain nutrients to replicate, and e) avoid killing the host cell (i.e. inducing its apoptosis or targeting it for killing by immune cells). In this specific aim we will examine which of these aspects each mutation affects.
Student role: The student will do immunofluorescence and western blot in order to identify the localization of Bordetella spp. within macrophages, in order to determine if Bordetella can growth within macrophages and to reveal how many of the Bordetella that are engulf are death or alive within macrophages (this techniques will require microscopy and/or flow cytometer). The student also will perform and develop western blot techniques that will identify the mechanism that Bordetella uses in order to kill macrophages.
Understanding how Bordetella species survive and grow within host cells is likely to shed light on the current problem of the high rates of vaccine failure as well as its re-emergence, as current vaccines do not induce Th1 immunity and are not effective against intracellular bacteria. This work will also reveal key genes contributing to intracellular growth/survival that can provide targets for the next generation of vaccines and therapeutics.
Host Lab: Eric Harvill
Type of project: Empirical/Laboratory-based\