In search of Brucella abortus Type IV secretion substrates: screening and identification of four proteins translocated into host cells through VirB system

Abstract

Type IV secretion systems (T4SS) are specialized protein complexes used by many bacterial pathogens for the delivery of effector molecules that subvert varied host cellular processes. Brucella spp. are Gram-negative a-proteobacteria that cause a zoonotic disease called brucellosis. They are facultative intracellular pathogens capable of survival and replication inside mammalian cells. Brucella T4SS (VirB) is essential to subvert lysosome fusion and to create an organelle permissive for replication. One possible role for VirB system is to translocate effector proteins that modulate host cellular functions for the biogenesis of the replicative organelle. In order to identify these effector proteins, we performed an in silico screen and searched the genome of B. abortus S2308 looking for proteins with distinctive features. We hypothesized that proteins with eukaryotic domains or protein-protein interaction domains, among others, would be good candidates for modulation of host cell functions. In this study, all B. abortus S2308 open reading frames were scanned using web-based programs and translocation of 84 potential substrates into host cells was assayed using Bordetella pertussis adenylate cyclase reporter (CyaA). By this approach, we identified six proteins that are delivered to the eukaryotic cytoplasm upon infection of macrophagic-like cells and we could determine that four of them require the presence of a functional T4SS for their delivery. For one of the VirB substrates, a hypothetical protein without a predicted function, we confirmed VirB-mediated protein translocation into phagocytic cells by immunofluorescence confocal microscopy, and we found that VirB substrate recognition involves an N-terminal translocation signal.