TheBordetellatype III secretion system effector BteA contains a conserved N-terminal motif that guides bacterial virulence factors to lipid rafts

Abstract

The Bordetella type III secretion system (T3SS) effector protein BteA is necessary and sufficient for rapid cytotoxicity in a wide range of mammalian cells. We show that BteA is highly conserved and functionally interchangeable between Bordetella bronchiseptica, Bordetella pertussis and Bordetella parapertussis. The identification of BteA sequences required for cytotoxicity allowed the construction of non-cytotoxic mutants for localization studies. BteA derivatives were targeted to lipid rafts and showed clear colocalization with cortical actin, ezrin and the lipid raft marker GM1. We hypothesized that BteA associates with the cytoplasmic face of lipid rafts to locally modulate host cell responses to Bordetella attachment. B. bronchiseptica adhered to host cells almost exclusively to GM1-enriched lipid raft microdomains and BteA colocalized to these same sites following T3SS-mediated translocation. Disruption of lipid rafts with methyl-β-cyclodextrin protected cells from T3SS-induced cytotoxicity. Localization to lipid rafts was mediated by a 130-amino-acid lipid raft targeting domain at the N-terminus of BteA, and homologous domains were identified in virulence factors from other bacterial species. Lipid raft targeting sequences from a T3SS effector (Plu4750) and an RTX-type toxin (Plu3217) from Photorhabdus luminescens directed fusion proteins to lipid rafts in a manner identical to the N-terminus of BteA.

Differential regulation of type III secretion and virulence genes inBordetella pertussisandBordetella bronchisepticaby a secreted anti-σ factor

“…B. bronchiseptica isolates have T3SS-dependent cytotoxic effects on a broad range of cell types (14,16,20,30). Most respond like HeLa cells (Fig.…”

“…B. bronchiseptica isolates from diverse hosts, including humans, readily display T3SS activity in vitro (20). In contrast, there are no reports of T3SS-dependent cytotoxicity by B. pertussis, despite the fact that T3SS loci are highly conserved, are functionally interchangeable, and bear the hallmarks of positive selection (3,16,25). Recent studies are beginning to shed light on this paradox.…”

“…In vivo studies with B. bronchiseptica show that T3SS Bsc plays an immunomodulatory role that facilitates persistence in the lower respiratory tract (21,22). Remarkably, only a single effector, BteA, has been definitively identified as a translocated substrate (15,16). In B. bronchiseptica, BteA is necessary and sufficient for cytotoxicity in vitro, and mutations in bteA recapitulate phenotypes associated with deleting the T3SS ATPase gene, bscN (14)(15)(16).…”

“…As a result of their close evolutionary relationship, comparative studies exploiting the experimental tractability of B. bronchiseptica are relevant for understanding B. pertussis (3,4,7,16,(23)(24)(25). B. bronchiseptica isolates from diverse hosts, including humans, readily display T3SS activity in vitro (20).…”

“…BvgAS establishes a spectrum of phenotypic phases in response to environmental cues, functioning as a rheostat that controls the infectious cycle (7,8). The Bvg + phase is characterized by high level BvgAS activity, expression of virulence factors [including pertussis toxin (9), adenylate cyclase toxin (10,11), filamentous hemagglutinin (12), fimbriae (13), the Bordetella secretion complex (Bsc) type III secretion system (T3SS Bsc ) (14)(15)(16)], and the ability to survive and replicate on respiratory epithelia (17). In the Bvg phase, the BvgAS phosphorelay is quiescent, Bvg + phase genes are silent, and "virulence-repressed genes" are maximally transcribed (4,17,18).…”