The expression of MD-2, which associates with Toll-like receptor (TLR) 4 on the cell surface, confers LPS and LPS-mimetic Taxol responsiveness on TLR4. Alanine-scanning mutagenesis was performed to identify the mouse MD-2 residues important for conferring LPS and Taxol responsiveness on mouse TLR4, and for forming the cell surface TLR4-MD-2 complex recognized by anti-TLR4-MD-2 Ab MTS510. Single alanine mutations were introduced into mouse MD-2 (residues 17–160), and the mutants were expressed in a human cell line expressing mouse TLR4. Mouse MD-2 mutants, in which a single alanine mutation was introduced at Cys37, Leu71, Leu78, Cys95, Tyr102, Cys105, Glu111, Val113, Ile117, Pro118, Phe119, Glu136, Ile138, Leu146, Cys148, or Thr152, showed dramatically reduced ability to form the cell surface mouse TLR4-mouse MD-2 complex recognized by MTS510, and the mutants also showed reduced ability to confer LPS and Taxol responsiveness. In contrast, mouse MD-2 mutants, in which a single alanine mutation was introduced at Tyr34, Tyr36, Gly59, Val82, Ile85, Phe126, Pro127, Gly129, Ile153, Ile154, and His155 showed normal ability to form the cell surface mouse TLR4-mouse MD-2 complex recognized by MTS510, but their ability to confer LPS and Taxol responsiveness was apparently reduced. These results suggest that the ability of MD-2 to form the cell surface mouse TLR4-mouse MD-2 complex recognized by MTS510 is essential for conferring LPS and Taxol responsiveness on TLR4, but not sufficient. In addition, the required residues at codon numbers 34, 85, 101, 122, and 153 for the ability of mouse MD-2 to confer LPS responsiveness are partly different from those for Taxol responsiveness.

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.Three major pathogenic species in the genus Bordetella are known. Bordetella pertussis and B. parapertussis are the causative agents of whooping cough in humans, and B. bronchiseptica infects the respiratory tracts of a broad range of mammals (17,26). Numerous virulence factors have been identified in Bordetella spp., including toxins such as pertussis toxin (expressed only in B. pertussis) (25,35,48), adenylate cyclase toxin (24), and dermonecrotic toxin (46) and adhesins such as filamentous hemagglutinin (12, 36), pertactin (37), and fimbriae (31). The expression of these virulence factors is coordinately regulated by the Bordetella virulence gene (bvg) locus (5, 49), which encodes the BvgA/BvgS two-component regulatory system (43). Under growth conditions of 37°C in the relative absence of MgSO 4 or nicotinic acid, the BvgAS phosphorelay is activated and bordetellae grow under Bvg ϩ phase conditions (8). The Bvg ϩ phase is characterized by the expression of various virulence factors, such as toxins and adhesins, and is necessary for respiratory tract colonization in rabbits and rats (1, 10). The Bvg Ϫ phase is avirulent and is characterized by loss of expression of both toxin and adhesin genes and by induction of genes that are not expressed under Bvg ϩ phase conditions. In B. bronchiseptica, Bvg Ϫ phase loci include genes and operons that encode the motility apparatus (2, 3). It has been shown that the type III secretion system in bordetellae is activated in the Bvg ϩ phase (51). Wild-type B. bronchiseptica, but not the type III secretion mutant, was shown to induce cytotoxicity against L2 cells th...

These results suggest that carbocisteine could exert anti-inflammatory and anti-oxidant effects through directly scavenging ROS in addition to its previously known mucoregulatory effect.

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