BopB is a type III secreted protein in Bordetella bronchiseptica and is required for cytotoxicity against cultured mammalian cells

Abstract

SummaryThe cytotoxicity of Bordetella bronchiseptica to infected cells is known to be dependent on a B. bronchiseptica type III secretion system. Although the precise mechanism of the type III secretion system is unknown, BopN, BopD and Bsp22 have been identified as type III secreted proteins. In order to identify other proteins secreted via the type III secretion machinery in Bordetella , a type III mutant was generated, and its secretion profile was compared with that of the wild-type strain. The results showed that the wild-type strain, but not the type III mutant, secreted a 40-kDa protein into the culture supernatant. This protein was identified as BopB by the analysis of its N-terminal amino acid sequence. Severe cytotoxicity such as necrosis was induced in L2 cells by infection with the wild-type B. bronchiseptica . In contrast, this effect was not observed by the BopB mutant infection. The haemolytic activity of the BopB mutant was greatly impaired compared with that of the wild-type strain. The results of a digitonin assay strongly suggested that BopB was translocated into HeLa cells infected with the wild-type strain. Taken together, our results demonstrate that Bordetella secretes BopB via a type III secretion system during infection. BopB may play a role in the formation of pores in the host plasma membrane which serve as a conduit for the translocation of effector proteins into host cells.

The Type III Secreted Protein BopD in Bordetella bronchiseptica Is Complexed with BopB for Pore Formation on the Host Plasma Membrane

“…The wild-type strain used in this study was B. bronchiseptica S798 (23). Both the BopB and type III (disrupted bscN gene) mutants were derived from S798 (23).…”

“…The wild-type strain used in this study was B. bronchiseptica S798 (23). Both the BopB and type III (disrupted bscN gene) mutants were derived from S798 (23). Bordetella strains were cultured in Stainer-Scholte liquid medium with a starting optical density at 600 nm of 0.2, and the inoculum was prepared from colonies grown on Bordet-Gengou agar as described previously (10,11,28).…”

“…Purified His-BopD was inoculated into rabbits as an immunogen, and the resulting antisera were incubated with His-BopD immobilized on an Affi-Gel column (Bio-Rad) to obtain specific immunoglobulin (Ig) fractions. The anti-BopB and anti-BcrH2 antibodies used in this study have been described previously (23).…”

“…Both YopB and YopD have been reported as pore-forming factors in yersiniae (18,32). Recently, we have identified a Bordetella type III secreted protein, BopB, that functions as the poreforming factor in B. bronchiseptica (23). In addition, three type III secreted proteins, i.e., BopN, BopD, and Bsp22, have been reported in B. bronchiseptica (50).…”

N-terminal residues of SipB are required for its surface localization on Salmonella enterica serovar Typhimurium

“…The IpaB and IpaC proteins in Shigella flexneri, BipB and BipD in Burkholderia pseudomallei, YopB and YopD in Yersinia enterocolitica, EspB and EspD in enteropathogenic Escherichia coli (EPEC), BopB and BopD in Bordetella bronchiseptica, and PopB and PopD in Pseudomonas aeruginosa are thought to be homologous components of the translocation apparatus formed by SipB and SipC in Salmonella. All of these secreted proteins have been shown to be delivered to the host-cell membrane, where they form pore complexes (Daniell et al, 2001;Davis et al, 1998;Hayward et al, 2000;Ide et al, 2001;Kuwae et al, 2003;Neyt & Cornelis, 1999;Nogawa et al, 2004;Scherer et al, 2000;Suparak et al, 2005;Faudry et al, 2006;Johnson et al 2006). In addition to its role as a translocon, SipB can also reportedly be phagocytized by macrophages and subsequently translocated via SPI-1 T3SS into the cytoplasm of the macrophage, where it induces apoptosis of the host cell by associating with the proapoptotic protease caspase-1 (Hersh et al, 1999).…”