Immun. and 30 min) it had to be accompanied by YadA, Ail, and outer core. In normal serum, the bacteria were less resistant to C3b deposition. However, no direct correlation between the C3 deposition pattern and bacterial resistance was observed. The complement system is a key player in defending the host against intruders like microbes. Activation of complement by serum-sensitive microorganisms leads to activation of C3. The cleavage product of C3, C3b, deposits on microbial surface and binds complement component C5. C5b, the cleavage product of C5, subsequently initiates the cascade of interactions that lead to insertion of the membrane attack complex, MAC (C5b-9), into the bacterial membrane. This event results in bacterial lysis and cell death. Many microbes, however, evolved mechanisms to evade this attack. In general, the serum resistance factors of pathogenic bacteria are surface components. The thick peptidoglycan layer of gram-positive bacteria prevents access of the MAC to the cytoplasmatic membrane, thus protecting bacteria (e.g., (45) or (47) is mediated especially by the O-antigen chains. Bacterial capsules, on the other hand, not Clodronate disodium only function as a barrier preventing MAC penetration through the bacterial membrane, but in some cases they also inhibit the alternative pathway activation, e.g., sialilated capsular polysaccharides of type III group B streptococci (22). Finally, some microbes are equipped with membrane proteins conferring complement resistance. For example, TraT protein of inhibits formation of C5b6 complex (31), and M protein of blocks activity of the alternative pathway Clodronate disodium C3 convertase by binding to the complement regulator, factor H. Microbial structures constantly evolve in response to the host immune system, and the above-given examples illustrate only a few multiple-complement evasion strategies. virulence factors encoded Clodronate disodium by genes located on the chromosome, e.g., Ail and lipopolysaccharide (LPS) O-antigen (O-ag) and outer core (OC), and on the 65- to 70-kb virulence plasmid pYV, e.g., YadA and Yop proteins. Expression of YadA and Ail occurs exclusively at 37C; O-side chain is more abundant at room temperature (RT), whereas OC is produced at both RT and 37C. Due to temperature-dependent gene regulation, only bacteria grown at 37C are resistant to killing by alternative pathway (AP), classical pathway (CP), or both (CP/AP). C3b, the key protein of complement activation, binds more strongly to pYV-negative or YadA-negative bacteria than to pYV-positive or YadA-positive bacteria grown at 37C (11, 44). Moreover, pYV-positive strains, which are serum resistant even when opsonized, display much less of the MACs deposited on their surfaces than do the pYV- and YadA-negative strains (30). These results suggest that YadA may be involved in inhibition of complement activation at both the C3b (possibly via factor H binding) and C9 levels. Another outer membrane (OM) protein conferring resistance on complement killing is Ail. Ail-mediated resistance was prominent not only in YadA- or invasin-negative mutants (7) but also in carrying the cloned gene, which displayed resistance to complement about 100 times as high as that of lacking Ail (29). No Ail-depending molecular mechanism has been established. Theoretically, Ail could interact with some host factors and interfere with formation of active attack complexes. In addition to OM proteins, Clodronate disodium serum resistance of depends on the LPS compounds O-ag and OC. O-ag presumably plays a role in inhibition of the early phase of alternative pathway activation (46). As for OC, our previous studies do not indicate any direct involvement of OC in serum resistance (39). On the contrary, OC-positive, YadA- and Ail-negative strains were efficiently killed by complement. In the absence of YadA, however, OC seemed to potentiate Ail-mediated resistance. In this report, we analyzed serum resistance of O:3 using a collection of strains expressing different combinations of YadA, Ail, O-ag, and OC. OC and O-ag are linked to different parts of the Rabbit Polyclonal to HMGB1 inner core (39, 40). This particular feature of serotype O:3 LPS made it possible to construct mutants missing OC and still expressing O-ag. The strains were analyzed for serum resistance in a killing assay in normal and EGTA [ethylene glycol-bis(-aminoethyl ether)-strains????6471/76 (YeO3)Serotype O:3, patient isolate, wild type37????YeO3-Ailstrains????S17-1pir(Am) (gene; AmprThis work????pAil-8.1PvuII deletion derivative of pAil-8; AmprThis work????pAil-8.2EcoRV-NruI deletion derivative of pAil-8.1; AmprThis work????pBR322Cloning vector; Ampr Tetr9????pJM703.1Suicide vector, contains R6K origin of replication and RP4 Mob.