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Bordetella pertussisuses a type III secretion system (T3SS) to inject virulence proteins into host cells. Although theB. pertussisT3SS was presumed to be involved in host colonization, efficient secretion of type III secreted proteins fromB. pertussishas not been observed. To investigate the roles of type III secreted proteins during infection, we attempted to optimize culture conditions for the production and secretion of a type III secreted protein, BteA, inB. pertussis. We observed thatB. pertussisefficiently secretes BteA in ascorbic acid-depleted (AsA−) medium. When L2 cells, a rat lung epithelial cell line, were infected withB. pertussiscultured in the AsA−medium, BteA-dependent cytotoxicity was observed. We also performed an immunofluorescence assay of L2 cells infected withB. pertussis. Clear fluorescence signals of Bsp22, a needle structure of T3SS, were detected on the bacterial surface ofB. pertussiscultured in the AsA−medium. Since ascorbic acid is known as a reducing agent, we culturedB. pertussisin liquid medium containing other reducing agents such as 2-mercaptoethanol and dithioerythritol. Under these reducing conditions, the production of type III secreted proteins was repressed. These results suggest that inB. pertussis, the production and secretion of type III secreted proteins are downregulated under reducing conditions.IMPORTANCEThe type III secretion system (T3SS) ofBordetella pertussisforms a needlelike structure that protrudes from the bacterial cell surface.B. pertussisuses a T3SS to translocate virulence proteins called effectors into host cells. The culture conditions for effector production inB. pertussishave not been investigated. We attempted to optimize culture medium compositions for producing and secreting type III secreted proteins. We found thatB. pertussissecretes type III secreted proteins in reducing agent-deprived liquid medium and that BteA-secretingB. pertussisprovokes cytotoxicity against cultured mammalian cells. These results suggest that redox signaling is involved in the regulation ofB. pertussisT3SS.

Enteropathogenic Escherichia coli (EPEC) is a major cause of diarrhea in young children (12). This pathogen induces a characteristic histopathological lesion referred to as an attaching/effacing lesion, which is defined by the intimate attachment of bacteria to the epithelial surface and the effacement of host cell microvilli (25). Factors responsible for the formation of attaching/effacing lesions are encoded by a 35-kbp locus designated LEE (24), which encodes the following components: (i) the type III secretion (TTS) apparatus (15), (ii) E. coli-secreted proteins (EspA, EspB, and EspD) and several effectors, including Tir, (iii) type III-specific chaperones, and (iv) regulators (9). LEE is highly conserved among enterohemorrhagic E. coli, rabbit EPEC, and Citrobacter rodentium. TTS systems are found in many other gram-negative bacterial species, and they constitute a strategy for the delivery of effectors into host cells, a process which in turn disrupts host physiology and thereby contributes to the disease process (14). Unlike the sec-dependent secreted proteins, the effector does not have a typical signal sequence at its N terminus, and it is directly delivered via the TTS system without N-terminal processing in the bacterial periplasm. Analysis by transmission electron microscopy (TEM) revealed that the supermolecular structures of the TTS apparatus in EPEC (8, 31), Salmonella enterica serovar Typhimurium (18, 20-22), and Shigella flexneri (2, 32) are highly conserved with respect to each other and that their shapes are similar to that of the flagellar basal body complex. The core TTS apparatus, which is referred to as a needle complex (NC), is composed of two distinct portions: (i) a needle structure that extrudes from the bacterial outer membrane and functions as an injector of effectors into host cells and (ii) a cylindrical basal body that is similar to the flagellar basal body and functions as a channel that spans the outer and inner membranes of the bacterium as well as the periplasmic region. The basal body is further divided into three major portions: (i) an outer ring, (ii) an inner ring, and (iii) a presumed central rod (22) that can connect the outer and inner rings to build a channel. Although the supermolecular structure of the EPEC NC is similar in shape to that of the NCs of both Salmonella and Shigella, a unique extracellular appendage in the needle structure of the EPEC NC was discovered previously (31). The EPEC needle structure is composed of a thin needle (neck portion) and an expandable sheath-like structure (31). EPEC EscF is predicted to be an 8-kDa protein and may polymerize to form the thin needle in the needle structure. EscF shows homology to PrgI (24% identity) in the Salmonella pathogenicity island 1, Shigella MxiH (25% identity), and Yersinia enterocolitica YscF (20% identity), which are major components of the thin and stiff needle structures of the Salmonella, Shigella, and Yersinia NCs, respectively (21, 26, 32). EscF is required for NC formation and the secretion of the Esp proteins (31); this observation agrees with findings of secretion-defective phenotypes of Salmonella prgI and Shigella mxiH mutant strains (21, 32). EspA is predicted to be a 20-kDa protein and is secreted via the EPEC TTS system (17). We previously demonstrated that EspA is directly associated with the tip of the putative EscF needle and polymerizes into an expandable filamentous structure, referred to as the sheath-like structure (31). The EPEC needle structure, including the EspA sheath-like structure, extended to a length of more than 600 nm and was 10 times longer than the Shigella needle (45 nm) (31). Three-dimensional structure analysis of the EspA filament revealed that the structure consists of a helical tube with a diameter of 12 nm enclosing a central channel with a diameter of 2.5 nm (7). Recent TEM analysis of purified EspA revealed that EspA alone is able to polymerize into irregular short filaments (33). On the other hand, the widths of the outer and inner membrane rings in the basal body of the EPEC NC are estimated to be 17 and 18 nm, respectively, and the height of the basal body is 31 nm (31). However, the molecular composition of the EPEC NC basal body remains unclear. From the results of a membrane fractionation study (13), yeast two-hybrid analysis (5), whole mutation analyses of LEE (10), and computer modeling predictions, several proteins are thought to be components for the EPEC TTS apparatus. The outer ring protein of the basal body has been suggested to be EscC, according to a membrane fractionation study (13), and this hypothesis received further support from a demonstration of its similarity to the YscC protein (31.1% identity) in the Yersinia TTS system, which forms a ring-shaped oligomeric complex with a diameter of 20 nm in the outer membrane (4, 19). EscC belongs to a member of the secretin superfamily, which participates in the delivery of large molecules through the outer membrane by the formation of a channel. EscC is predicted to be synthesized as a 56-kDa preprotein possessing a signal sequence that is cleavable with type I signal peptidase after amino acid residue 19. Then, the EscC preprotein most likely undergoes signal peptide cleavage after its export across the inner membrane by a sec-dependent secretion pathway, thus generating a mature 54-kDa protein. Indeed, a mature form of Salmonella InvG, an EscC orthologue, starts at amino acid residue 25, suggesting that its preprotein is cleaved by type I signal peptidase in order to reach maturation (20). Recently, the association of EscC with EscD was suggested by use of a yeast two-hybrid system (5). EscD is predicted to be a 45-kDa protein and shows amino acid sequence similarity to Yersinia YscD, a bacterial inner membrane protein (27). In Salmonella, PrgK and PrgH together form the inner membrane ring of the TTS apparatus (18). Although a PrgH orthologue has not been found in EPEC, a PrgK orthologue, EscJ, is thought to form a portion of the inner ring. Molecular modeling using the 1.8-A crystal structure of EscJ suggests that EscJ oligomerizes to form a large 24-subunit ring structure (34). Furthermore, a membrane fraction study indicates that EscJ localizes mainly to the inner membrane (34). These findings suggest that the inner ring of the EPEC TTS apparatus contains the EscJ multimeric complex. EscJ is predicted to be produced as a 21-kDa preprotein with a lipoprotein signal sequence that can be cleaved after amino acid residue 19 by type II signal peptidase. After sec-dependent translocation, the EscJ preprotein may undergo lipid modification and signal peptide cleavage to form a mature 19-kDa lipoprotein, which may be anchored to the inner membrane via its N-terminal lipid moiety (34). Although several Esc proteins, namely, EscR, EscS, EscT, and EscU, are predicted to be components of the basal body, their precise functions and localizations in the EPEC TTS apparatus remain unclear. In this study, we analyzed the structures of NCs isolated from EPEC by using centrifugation techniques, including CsCl density gradient centrifugation. Furthermore, protein components of NCs were examined by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS), Western blotting, and immunoelectron microscopy. In addition, the protein-protein interactions required for assembly of the EPEC TTS apparatus were analyzed using a glutathione S-transferase (GST) pulldown assay, and confirmation of the putative components of the TTS apparatus needed for NC assembly, as well as those needed for the secretion of Esp proteins and the Tir effector, was carried out by the construction of deletion mutants.

The cytotoxicity of Bordetella bronchiseptica to infected cells is known to be dependent on a B . bronchiseptica type III secretion system. Although BopB, BopN, BopD, and Bsp22 have been identified as type III secreted proteins, these proteins remain to be characterized. In this study, in order to clarify the function of BopD during Bordetella infection, a BopD mutant was generated. Although secretion of BopD into the culture supernatant was completely abolished by the bopD mutation, the secretion of other type III secreted proteins was not affected by this mutation. It has been reported that severe cytotoxicity, including cell detachment from the substrata, and release of lactate dehydrogenase (LDH) into the supernatant are induced in L2 cells by wild-type B . bronchiseptica infection, and these phenotypes are dependent on the type III secretion system. In contrast, neither cell detachment nor LDH release was induced in L2 cells infected with the BopD mutant. Furthermore, the hemolytic activity of the BopD mutant was greatly impaired compared with that of the wild-type strain. On the basis of the results of coimmunoprecipitation assays with anti-BopB antibodies, we conclude that BopD has the ability to associate with BopB. Finally, we show that the BopD-BopB complex is responsible for the pore formation in the host plasma membrane that functions as the conduit for the transition of effector proteins into host cells.