Impact of the SpeB protease on binding of the complement regulatory proteins factor H and factor H-like protein 1 by Streptococcus pyogenes.

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 GAS (61). Additionally, we demonstrated that Fba, via binding of FHL-1, can promote the entry of GAS into human epithelial cells (62). The fba gene is present in GAS of numerous serotypes, suggesting that the protein contributes to GAS survival in its host (70, 78). In fact, Terao et al. (78) reported that Fba contributes to GAS virulence in mice. The N- and C-terminal regions of Fba are well conserved among strains with various alleles, but the amino acids in the central region of the protein can differ between strains (70, 78). It is unknown to what extent these differences in amino acid sequence affect the functions of the protein; however, an earlier study suggested that the Fba protein of a serotype M49 GAS isolate does not bind FH (70). Deletion analysis of Fba from GAS strain 90-226 localized the major binding site for FH and FHL-1 to a putative coiled-coil-forming region in the N-terminal half of the protein. PepSpot analyses confirmed that a linear peptide sequence that overlaps the coiled-coil region (YKQKIKTAPDKDKLLF) directly interacts with FH and FHL-1 (62). Interestingly, this sequence is not strictly conserved in all Fba proteins, suggesting that some proteins may not bind FH or FHL-1 or may bind with a different affinity than that of the protein from strain 90-226 (70, 78). The Fba binding site in FH and FHL-1 has been localized to SCR 7, a domain common to both proteins (83). SCR 7 also contains binding sites for heparin and M proteins. Accordingly, heparin can inhibit binding of FH by Fba or M proteins (46, 62, 76, 83). Although Fba binds both FH and FHL-1, strain 90-226 selectively binds FHL-1, rather than FH, when incubated with human plasma or serum (61). This result was surprising as the concentration of FH in human plasma (400 μg/ml) is significantly higher than that of FHL-1 (10 to 50 μg/ml) (32). An fba mutant of strain 90-226 does not bind appreciable amounts of either ligand from plasma (61). Selective binding of FHL-1 has also been observed for other GAS isolates, but the GAS factor(s) that mediates binding was not identified (43, 63). Fba possesses a conserved amino acid sequence (LPSTG) near the C terminus to anchor the protein to the bacterial cell wall (25). Several lines of experimentation have confirmed that Fba is produced and anchored to the cell surface during the exponential phase of growth (61, 78). In our studies with a single GAS strain (strain 90-226), however, we observed that Fba was not present on the surface of stationary-phase streptococci. GAS grown to stationary phase in media containing E64, a cysteine protease inhibitor, retained Fba on the cell surface (61). While these results suggested that the GAS SpeB protease, an extracellular cysteine protease expressed in the late exponential to early stationary phases of growth (8), was responsible for the loss of Fba from stationary-phase cells, this possibility had not been directly tested. Investigation of a possible role for SpeB in the loss of Fba was warranted, as many GAS isolates do not produce significant amounts of SpeB during growth in vitro (8, 12, 54). Moreover, culture supernatants of some GAS isolates, such as strain 90-226, often contain temperate bacteriophage, suggesting that bacterial cells are subject to lysis during normal growth, a process that could release intracellular proteinases into the culture medium. Thus, it was plausible that strain 90-226 expresses a proteinase other than SpeB that affects cell surface presentation of Fba. Here we extend our previous studies by investigating the roles of Fba and SpeB in the recruitment of FH and FHL-1 by other serotype M1 isolates of GAS. Our results demonstrate that Fba is a major FHL-1 and FH binding factor of this serotype that selectively binds FHL-1 from human plasma. We also confirmed that SpeB is involved in the loss of Fba from stationary-phase streptococci. Perhaps more importantly, we determined that strain 90-226 is neither a unique nor a representative strain with regard to stationary-phase expression of Fba. Finally, we established that the Fba protein of a serotype M49 strain, CS101, can bind both FH and FHL-1, suggesting that this is a conserved function of Fba.