David Cue

High‐frequency intracellular invasion of epithelial cells by serotype M1 group A streptococci: M1 protein‐mediated invasion and cytoskeletal rearrangements

A clonal variant of serotype M1 group A streptococcus (designated M1inv+) has been linked to severe and invasive infections, including sepsis, necrotizing fasciitis and toxic shock. High frequency internalization of cultured epithelial cells by the M1inv+ strain 90‐226 is dependent upon the M1 protein. Invasion of HeLa cells was blocked by an anti‐M1 antibody, invasion by an M1− strain (90‐226 emm1::km) was greatly reduced, and latex beads bound to M1 protein were readily internalized by HeLa cells. Beads coated with a truncated M1 protein were internalized far less frequently. Scanning electron microscopy indicated that streptococci invade by a zipper‐like mechanism, that may be mediated by interactions with host cell microvilli. Initially, internalized streptococci and streptococci undergoing endocytosis are associated with polymerized actin. Later in the internalization process, streptococcal‐containing vacuoles are associated with the lysosomal membrane glycoprotein, LAMP‐1.

Recruitment of Complement Factor H-Like Protein 1 Promotes Intracellular Invasion by Group A Streptococci

ABSTRACT Numerous microbial pathogens exploit complement regulatory proteins such as factor H (FH) and factor H-like protein 1 (FHL-1) for immune evasion. Fba is an FHL-1 and FH binding protein expressed on the surface of the human pathogenic bacterium, Streptococcus pyogenes, a common agent of pharyngeal, skin, and soft-tissue infections. In the present study, we demonstrate that Fba and FHL-1 work in concert to promote invasion of epithelial cells by S. pyogenes. Fba fragments were expressed as recombinant proteins and assayed for binding of FHL-1 and FH by Western blotting, enzyme-linked immunosorbent assay, and surface plasmon resonance. A binding site for FHL-1 and FH was localized to the N-terminal half of Fba, a region predicted to contain a coiled-coil domain. Deletion of this coiled-coil domain greatly reduced FHL-1 and FH binding. PepSpot analyses identified a 16-amino-acid segment of Fba which overlaps the coiled-coil domain that binds both FHL-1 and FH. To localize the Fba binding site in FHL-1 and FH, surface plasmon resonance was used to assess the interactions between the streptococcal protein and a series of recombinant FH deletion constructs. The Fba binding site was localized to short consensus repeat 7 (SCR 7), a domain common to FHL-1 and FH. SCR 7 contains a heparin binding site, and heparin was found to inhibit FHL-1 binding to Fba. FHL-1 promoted entry of Fba+ group A streptococci into epithelial cells in a dose-dependent manner but did not affect invasion by an isogenic fba mutant. To our knowledge, this is the first report of a bacterial pathogen exploiting a soluble complement regulatory protein for entry into host cells.

High‐frequency intracellular infection and erythrogenic toxin A expression undergo phase variation in M1 group A streptococci

A clonal variant of serotype M1 group A streptococcus, strain 90‐131, disseminated to several continents, where it was associated with severe systemic infections and toxic shock. Although this strain harbours the speA gene and is efficiently internalized by human epithelial cells, clinical isolates often fail to express the erythrogenic toxin under laboratory growth conditions. Cultures of strain 90‐131 were observed to phase vary between small, dry, compact and larger, more mucoid colonies. The former were shown to be poorly internalized by epithelial cells. Analysis of RNA by Northern hybridization demonstrated that the emm1, hasA and speA genes were weakly transcribed in cultures derived from the small colonies and highly transcribed in those derived from the large colonies. An insertion mutation in mga (the multigene activator) downregulated the invasion of epithelial cells and the transcription of emm1 and hasA, but had little impact on the transcription of speA. These are the first data to suggest the existence of a common regulatory circuit linking intracellular invasion, M protein, hyaluronic acid capsule and erythrogenic toxin expression by group A streptococcus. Moreover, the genetic instability of toxin expression exhibited by this serotype may impact on laboratory studies that attempt to associate toxin production with toxic shock.

Impact of the SpeB Protease on Binding of the Complement Regulatory Proteins Factor H and Factor H-Like Protein 1 by Streptococcus pyogenes

ABSTRACT 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 soft-tissue infections. Fba has been shown to contribute to phagocytosis resistance, intracellular invasion, and virulence in mice. Here, we look at the role of Fba in recruitment of FH and FHL-1 by five serotype M1 isolates of streptococci. Inactivation of fba greatly inhibited binding of FH and FHL-1 by all isolates, indicating that Fba is a major FH and FHL-1 binding factor of serotype M1 streptococci. For three isolates, FH binding was significantly reduced in stationary-phase cultures and correlated with high levels of protease activity and SpeB (an extracellular cysteine protease) protein in culture supernatants. Analysis of a speB mutant confirmed that SpeB accounts for the loss of Fba from the cell surface, suggesting that the protease may modulate FH and FHL-1 recruitment during infection. Comparisons of fba DNA sequences revealed that the FH and FHL-1 binding site in Fba is conserved among the M1 isolates. Although the ligand binding site is not strictly conserved in Fba from a serotype M49 isolate, the M49 Fba protein was found to bind both FH and FHL-1. Collectively, these data indicate that binding of FH and FHL-1 is a conserved function of Fba while modulation of Fba function by SpeB is variable.

High Frequency Invasion of Mammalian Cells by β Hemolytic Streptococci

The impact of intracellular invasion on the virulence of S. pyogenes (group A streptococci) and S. agalactiae (group B streptococci) is a rapidly expanding field of investigation. The discovery that members of these species are internalized by a variety of mammalian cells at frequencies equal to or beyond those of the more classical bacterial pathogens has piqued that interest. Investigators are attempting to relate this newfound potential to the pathophysiology of streptococcal disease. This chapter will focus on S. pyogenes, but where possible comparisons will be made to another important streptococcal pathogen, S. agalactiae. Knowledge of bacterial adhesions, is of course, essential for understanding the mechanisms by which bacteria are ingested by non-professional phagocytes; however, studies of S. pyogenes and S. agalactiae adherence mechanisms are a large body of work that is littered with debate and uncertainty and will therefore not be directly addressed here. Reviews of S. pyogenes (Hasty et al., 1992) and S. agalactiae (Tamura and Rubens, 1994) adherence phenomena are cited.