Brucella Interaction with Mucosal Cells

NON-TECHNICAL SUMMARY: Brucellosis is a zoonosis commonly encountered in a number of animals. Brucella infection can be potentially used as a bioterrorism weapon. This work addresses the mechanisms of Brucella infection.

<P>
APPROACH: B. abortus is an intracellular pathogen that needs to interact with the mucosal barriers in order to gain access to deep tissues in the host. Brucella is known to cause infection when acquired by the nasal and respiratory route, as well as the oral and gastrointestinal mucosas. If used as a biological weapon, both for humans and animals, Brucella can be efficiently delivered by aerosol and in food. Therefore, the study of the interaction between B. abortus and the host mucosa is key to increase the knowledge about virulence determinant in the pathogen and mechanisms of host defense. To determine if B. abortus not only is able to invade mucosal cells but also is capable of translocating across the epithelial layer, we propose to employ the polarized model that has been routinely used in the laboratory. Since not all the cells proposed above can polarize, we will concentrate our efforts working with A549 alveolar epithelial cells representing the cells of the respiratory tract and Caco-2 intestinal epithelial cells for the intestinal tract. Previous manuscripts described in detail the establishment of a transwell system using A549 and Caco-2 cells. Once infected the resistance of the monolayer will be monitored daily as described. The percentage of bacteria that cross the membrane will be determined and analyzed as a fraction of the inoculum per hour. We plan to use two different approaches for the examination of the interaction between Brucella and the host cytoskeleton: (1) We will perform confocal microscopy to determine if B. abortus interact with cytoskeleton-related proteins upon uptake into epithelial cells. (2) We will use the mutants of B. abortus that have impaired ability to enter epithelial cells to determine or confirm the interactions with the cytoskeleton. To screen the brucella genome with the aim of identifying virulence genes involved in the interaction between the bacterium and both the intestinal and respiratory mucosal cells, we plan to use the transposon library we are already constructing. The mutant identified using the A549 cell line will then be tested for invasion in the Caco-2 cell line to determine if the gene inactivated also has a role in the invasion of the intestinal mucosa. We believe that many genes will be associated with the invasion of both intestinal and respiratory mucosas but some of those may be unique. Initially, we are going to concentrate on those that are common for the two mucosas. After the sequence have been obtained, we will compare it with both the many Brucella sequences available (B. melitensis and B. suis) as well as the Gene bank database. The G-C content of the gene will be determined to examine if it belongs to B. rucella genera or there will be a chance that the gene was transferred from another organism. We anticipate that this approach will lead to several candidate genes for further analysis.