Opsonization of bacteria by complement proteins is an important component of the immune response. The pathogenic bacterium Streptococcus pyogenes has evolved multiple mechanisms for the evasion of complementmediated opsonization. One mechanism involves the binding of human regulators of complement activation such as factor H (FH) and FH-like protein 1 (FHL-1). Acquisition of these regulatory proteins can limit deposition of the opsonin C3b on bacteria, thus decreasing the pathogen's susceptibility to phagocytosis. Binding of complement regulatory proteins by S. pyogenes has previously been attributed to the streptococcal M and M-like proteins. Here, we report that the S. pyogenes cell surface protein Fba can mediate binding of FH and FHL-1. We constructed mutant derivatives of S. pyogenes that lack Fba, M1 protein, or both proteins and assayed the strains for FH binding, susceptibility to phagocytosis, and C3 deposition. Fba expression was found to be sufficient for binding of purified FH as well as for binding of FH and FHL-1 from human plasma. Plasma adsorption experiments also revealed that M1 ؉ Fba ؉ streptococci preferentially bind FHL-1, whereas M1 ؊ Fba ؉ streptococci have similar affinities for FH and FHL-1. Fba was found to contribute to the survival of streptococci incubated with human blood and to inhibit C3 deposition on bacterial cells. Streptococci harvested from log-phase cultures readily bound FH, but binding was greatly reduced for bacteria obtained from stationary-phase cultures. Bacteria cultured in the presence of the protease inhibitor E64 maintained FH binding activity in stationary phase, suggesting that Fba is removed from the cell surface via proteolysis. Western analyses confirmed that E64 stabilizes cell surface expression of Fba. These data indicate that Fba is an antiopsonic, antiphagocytic protein that may be regulated by cell surface proteolysis.The gram-positive bacterium Streptococcus pyogenes, or group A streptococcus (GAS), is an important human pathogen. Most streptococcal infections (e.g., pharyngitis, impetigo) are relatively mild and readily treated with antibiotics. However, GAS cause a variety of severe and invasive diseases as well (12). For example, rheumatic fever, a poststreptococcal sequela, is endemic in the third world, where it is a major cause of cardiovascular disease and mortality in persons under 50 years of age (36). Also, since the mid-1980s, Western countries have reported significant increases in severe GAS infections (e.g., sepsis, necrotizing fasciitis, streptococcal toxic shock syndrome) with high mortality rates (7,17,35).GAS express an array of cell surface molecules that contribute to pathogenesis. Among the best studied of these are the M proteins (20). M proteins are multifunctional proteins that contribute to GAS pathogenesis in a number of ways, including by adherence to host tissues, intracellular invasion, and autoaggregation of bacterial cells. Perhaps the most important function of M protein, however, is to confer bacterial resistance to phag...

Activation of the complement system in response to infectious agents is a key component of the innate immune response, resulting in opsonization and/or lysis of invading pathogens. Uncontrolled complement activation, however, damages host tissues and results in complement depletion (38). Therefore, complement activity is tightly regulated by nearly a dozen plasma and cell membrane regulatory proteins. Two fluid-phase regulators of complement activation (RCAs) are factor H (FH) and factor H-like protein 1 (FHL-1). The two proteins are encoded by the same gene and their transcripts are derived by alternative splicing (11,15,57). FH is a 150-kDa protein composed of 20 repeat elements known as short consensus repeats (SCRs). Each SCR constitutes an independently folded domain of approximately 60 amino acid residues and contains two invariant disulfide bonds that form the framework of the domain. FHL-1 is a 42-kDa protein composed of seven SCRs that are identical, with the exception of four amino acids at the C terminus, to SCRs 1 to 7 of FH (57).A number of bacterial pathogens, including Streptococcus pyogenes (21), Streptococcus pneumoniae (9, 23, 49), Streptococcus agalactiae (1), Neisseria gonorrhoeae (48), and Borrelia burgdorferi (30), exploit FHL-1 and FH for host colonization. In the absence of an RCA, C3b deposited on the surface of a pathogen serves as a nucleus for formation of the alternative complement pathway C3 convertase C3bBb. Once formed, C3 convertase cleaves additional C3, thereby promoting further C3b deposition and C3bBb formation. Recruitment of FH or FHL-1 by microbes facilitates factor I-mediated cleavage of cell surface-bound C3b to iC3b. Because iC3b does not participate in C3bBb formation, C3b cleavage results in decreased deposition of C3 fragments on bacterial cells. Because C3 fragments can be bound by receptors on phagocytes, limiting the amount of C3 deposited on cells can lessen the probability that a pathogen will be phagocytized (21, 57).The group A streptococcus (GAS) S. pyogenes is an important human pathogen that exploits FHL-1 and FH to inhibit opsonization by C3, a mechanism that contributes to GAS resistance to phagocytosis (21). GAS also utilize the cell membrane-bound RCA CD46 for adherence to human keratinocytes (41, 44). Numerous laboratories have reported that RCA binding by GAS is mediated by M proteins, a family of cell surface antiphagocytic proteins (13). We recently identified a novel GAS cell surface protein, Fba, that mediates binding of both FHL-1 and FH. Fba is the first non-M-like protein of GAS shown to bind human RCAs (43). The same protein was identified by Terao et al. (55) as a fibronectin binding protein involved in invasion of Hep-2 cells. Although Fba was initially identified in M1 serotype isolates, fba is present in at least 18 GAS serotypes (46,55). The conservation of fba suggests that the gene product contributes to survival of GAS during infection. We previously demonstrated that Fba contributes to GAS resistance to phagocytosis and that M1 ϩ Fba...

Adhesion of Listeria monocytogenes to intestinal endothelial cells is an important initial event in the pathogenesis of infection which is not well understood. The suggestion has been made that some proteins, including internalin and actin polymerisation protein (ActA), and carbohydrate molecules mediate, at least in part, the adhesion of listeria to certain cultured mammalian cells. This study investigated the role of a L. monocytogenes cell-surface protein of 104 kDa (p104) in adhesion to human intestinal enterocyte-like Caco-2 cell lines by transposon (Tn916) mutagenesis and a pl04-specific monoclonal antibody (MAb-H7). Genotypic and phenotypic characteristics of Tn916-transformed L. monocytogenes strains, AAMU530 and AAMU572, revealed that these strains did not express p104, and the transposon had been inserted at a single locus in the structural gene. Strains AAMU530 and AAMU572 yielded only 10 and 6.3% adhesion to Caco-2 cells. Coating of L. monocytogenes and L. innocua wild-type strains with MAb-H7 reduced adhesion to Caco-2 cells from 100% to 50 and 45%, respectively, whereas on isotype control MAb EM-7G1 had no effect. Western blot analysis with MAb-H7 indicated that p104 is present in all Listeria spp. except in L. grayi. Furthermore, p104 is also present in internalin (BUGS) and ActA (LUT12) deficient strains, suggesting that p104 is indeed different from internalin or ActA proteins. Cytotoxicity analysis of strains AAMU530 and AAMU572 demonstrated that these strains, although haemolytic and phospholipase-positive, were avirulent when tested with a hybridoma B-lymphocyte cell line. Loss of virulence could be attributed to the interruption of adhesion of mutant strains to the hybridoma cell line. These results strongly suggest that p104 is an adhesion factor in L. monocytogenes and involved in adhesion to intestinal cells.

Microbial pathogens often exploit human complement regulatory proteins such as factor H (FH) and factor H-like protein 1 (FHL-1) for immune evasion. Fba is an FH and FHL-1 binding protein expressed on the surface of the human pathogenic bacterium Streptococcus pyogenes, a common agent of pharyngeal, skin, and softtissue infections. Fba has been shown to contribute to phagocytosis resistance, intracellular invasion, and virulence in mice. Many infectious agents, including viruses, fungi, and bacteria, utilize host complement regulatory proteins for immune evasion and host colonization (60). Two fluid-phase regulators of complement activation commonly recruited by pathogens are factor H (FH) and factor H-like protein 1 (FHL-1), two proteins encoded by the same gene (22,27,83). FH is a 150-kDa protein composed of 20 repeat elements known as short consensus repeats (SCRs). Each SCR constitutes an independently folded domain of approximately 60 amino acid residues. FHL-1 is a 42-kDa protein composed of seven SCRs that are identical to SCRs 1 to 7 of FH with four additional amino acids at the C terminus (83). The ability of bacterial pathogens to evade complement attack and opsonophagocytosis is often influenced or dictated by a pathogen's ability to bind FH or other complement regulatory proteins (1,19,20,39,40,42,47,73). Recruitment of FH or FHL-1 can facilitate both dissociation of C3 convertase and cleavage of cell surface-bound C3b to iC3b. Because iC3b does not participate in C3bBb formation, C3b cleavage can result in decreased deposition of C3 fragments on cell surfaces (17,28,36,38,59,60).The gram-positive bacterium Streptococcus pyogenes, or group A streptococcus (GAS), is a major human pathogen causing infections ranging from superficial infections of the throat (pharyngitis) or skin (impetigo) to highly invasive and potentially lethal infections such as necrotizing fasciitis and streptococcal toxic shock syndrome (16). Horstmann et al. (36) first proposed that the acquisition of FH by GAS contributes to the bacterium's capacity to evade phagocytosis by polymorphonuclear leukocytes. For many GAS isolates, FH binding is mediated by M proteins, a family of cell surface, antiphagocytic proteins (25, 26). Some types of M proteins have also been shown to bind FHL-1 (42, 46). It should be noted, however, that GAS resistance to phagocytosis is a complex phenotype, resulting from the collective activities of numerous virulence factors, including capsules, secreted proteins, and cell surface proteins (4,13,18,34,49,68,81,82). Because of this, recruitment of complement regulatory factors, or even expression of M protein, is not essential for some GAS isolates to resist phagocytosis (41-43, 45, 64, 68).We recently identified a novel protein expressed by a serotype M1 GAS isolate, 90-226, that mediates binding of both FH and FHL-1 (61). The protein, Fba, is the first non-M-like protein of GAS shown to bind these complement regulatory factors. We previously reported that Fba contributes to the phagocytosis resistance of...