Glycosaminoglycan binding facilitates entry of a bacterial pathogen into central nervous systems.

Certain microbes invade brain microvascular endothelial cells (BMECs) to breach the blood-brain barrier (BBB) and establish central nervous system (CNS) infection. Here we use the leading meningitis pathogen group B Streptococcus (GBS) together with insect and mammalian infection models to probe a potential role of glycosaminoglycan (GAG) interactions in the pathogenesis of CNS entry. Site-directed mutagenesis of a GAG-binding domain of the surface GBS alpha C protein impeded GBS penetration of the Drosophila BBB in vivo and diminished GBS adherence to and invasion of human BMECs in vitro. Conversely, genetic impairment of GAG expression in flies or mice reduced GBS dissemination into the brain. These complementary approaches identify a role for bacterial-GAG interactions in the pathogenesis of CNS infection. Our results also highlight how the simpler yet genetically conserved Drosophila GAG pathways can provide a model organism to screen candidate molecules that can interrupt pathogen-GAG interactions for future therapeutic applications.

Host and pathogen glycosaminoglycan-binding proteins modulate antimicrobial peptide responses in Drosophila melanogaster.

During group B streptococcal infection, the alpha C protein (ACP) on the bacterial surface binds to host cell surface heparan sulfate proteoglycans (HSPGs) and facilitates entry of bacteria into human epithelial cells. Previous studies in a Drosophila melanogaster model showed that binding of ACP to the sulfated polysaccharide chains (glycosaminoglycans) of HSPGs promotes host death and is associated with higher bacterial burdens. We hypothesized that ACP-glycosaminoglycan binding might determine infection outcome by altering host responses to infection, such as expression of antimicrobial peptides. As glycosaminoglycans/HSPGs also interact with a number of endogenous secreted signaling molecules in Drosophila, we examined the effects of host and pathogen glycosaminoglycan/HSPG-binding structures in host survival of infection and antimicrobial peptide expression. Strikingly, host survival after infection with wild-type streptococci was enhanced among flies overexpressing the endogenous glycosaminoglycan/HSPG-binding morphogen Decapentaplegic-a transforming growth factor ß-like Drosophila homolog of mammalian bone morphogenetic proteins-but not by flies overexpressing a mutant, non-glycosaminoglycan-binding Decapentaplegic, or the other endogenous glycosaminoglycan/HSPG-binding morphogens, Hedgehog and Wingless. While ACP-glycosaminoglycan binding was associated with enhanced transcription of peptidoglycan recognition proteins and antimicrobial peptides, Decapentaplegic overexpression suppressed transcription of these genes during streptococcal infection. Further, the glycosaminoglycan-binding domain of ACP competed with Decapentaplegic for binding to the soluble glycosaminoglycan heparin in an in vitro assay. These data suggest that, in addition to promoting bacterial entry into host cells, ACP competes with Decapentaplegic for binding to glycosaminoglycans/HSPGs during infection and that these bacterial and endogenous glycosaminoglycan-binding structures determine host survival and regulate antimicrobial peptide transcription.

Host glycosaminoglycan confers susceptibility to bacterial infection in Drosophila melanogaster.

Many pathogens engage host cell surface glycosaminoglycans, but redundancy in pathogen adhesins and host glycosaminoglycan-anchoring proteins (heparan sulfate proteoglycans) has limited the understanding of the importance of glycosaminoglycan binding during infection. The alpha C protein of group B streptococcus, a virulence determinant for this neonatal human pathogen, binds to host glycosaminoglycan and mediates the entry of bacteria into human cells. We studied alpha C protein-glycosaminoglycan binding in Drosophila melanogaster, whose glycosaminoglycan repertoire resembles that of humans but whose genome includes only three characterized membrane heparan sulfate proteoglycan genes. The knockdown of glycosaminoglycan polymerases or of heparan sulfate proteoglycans reduced the cellular binding of alpha C protein. The interruption of alpha C protein-glycosaminoglycan binding was associated with longer host survival and a lower bacterial burden. These data indicate that the glycosaminoglycan-alpha C protein interaction involves multiple heparan sulfate proteoglycans and impairs bacterial killing. Host glycosaminoglycans, anchored by multiple proteoglycans, thereby determine susceptibility to infection. Because there is homology between Drosophila and human glycosaminoglycan/proteoglycan structures and many pathogens express glycosaminoglycan-binding structures, our data suggest that interfering with glycosaminoglycan binding may protect against infections in humans.

Repression of the PDCD2 gene by BCL6 and the implications for the pathogenesis of human B and T cell lymphomas.

The human BCL6 gene on chromosome 3 band q27, which encodes a transcriptional repressor, is implicated in the pathogenesis of human lymphomas, especially the diffuse large B-cell type. We previously identified the human PDCD2 (programmed cell death-2) gene as a target of BCL6 repression. PDCD2 encodes a protein that is expressed in many human tissues, including lymphocytes, and is known to interact with corepressor complexes. We now show that BCL6 can bind directly to the PDCD2 promoter, repressing its transcription. Knockdown of endogenous BCL6 in a human B cell lymphoma line by introduction of small interfering RNA duplexes increases PDCD2 protein expression. Furthermore, there is an inverse relationship between the expression levels of the BCL6 and PDCD2 proteins in the lymphoid tissues of mice overexpressing human BCL6 (high BCL6 levels, minimal PDCD2) and controls (minimal BCL6, high PDCD2) as well as in tissues examined from some human B and T cell lymphomas. These data confirm PDCD2 as a target of BCL6 and support the concept that repression of PDCD2 by BCL6 is likely important in the pathogenesis of certain human lymphomas.

Identification of a glycosaminoglycan binding region of the alpha C protein that mediates entry of group B Streptococci into host cells.

Group B Streptococcus (GBS) frequently colonizes the human gastrointestinal and gynecological tracts and less frequently causes deep tissue infections. The transition between colonization and infection depends upon the ability of the organism to cross epithelial barriers. The alpha C protein (ACP) on the surface of GBS contributes to this process. A virulence factor in mouse models of infection, and prototype for a family of Gram-positive bacterial surface proteins, ACP facilitates GBS entry into human cervical epithelial cells and movement across cell layers. ACP binds to host cell surface glycosaminoglycan (GAG). From crystallography, we have identified a cluster of basic residues (BR2) that is a putative GAG binding area in Domain 2, near the junction of the N-terminal domain of ACP and the first of a series of tandem amino acid repeats. D2-R, a protein construct including this region, binds to cells similarly to full-length ACP. We now demonstrate that the predicted charged BR2 residues confer GAG binding; site-directed mutagenesis of these residues (Arg(172), Arg(185), or Lys(196)) eliminates cell-binding activity of construct D2-R. In addition, we have constructed a GBS strain expressing a variant ACP with a charge-neutralizing substitution at residue 185. This strain enters host cells less effectively than does the wild-type strain and similarly to an ACP null mutant strain. The point mutant strain transcytoses similarly to the wild-type strain. These data indicate that GAG-binding activity underlies ACP-mediated cellular entry of GBS. GBS entry into host cells and transcytosis of host cells may occur by distinct mechanisms.

Anchors away: contribution of a glycolipid anchor to bacterial invasion of host cells.

Group B Streptococcus (GBS) is an important cause of infections, including meningitis. The molecular events underlying its pathogenesis are poorly understood. A study in this issue of the JCI reports that the GBS invasion-associated gene (iagA) contributes to meningeal infection and virulence by facilitating invasion of the cells that compose the blood-brain barrier and of other host cells. The mechanism involved most likely relates to the gene product's role in synthesis of a glycolipid anchor for a bacterial cell-surface entity that interacts directly with host cells.

Crystal structure of the N-terminal domain of the group B streptococcus alpha C protein.

Group B Streptococcus (GBS) is the leading cause of bacterial pneumonia, sepsis, and meningitis among neonates and an important cause of morbidity among pregnant women and immunocompromised adults. Invasive diseases due to GBS are attributed to the ability of the pathogen to translocate across human epithelial surfaces. The alpha C protein (ACP) has been identified as an invasin that plays a role in internalization and translocation of GBS across epithelial cells. The soluble N-terminal domain of ACP (NtACP) blocks the internalization of GBS. We determined the 1.86-A resolution crystal structure of NtACP comprising residues Ser(52) through Leu(225) of the full-length ACP. NtACP has two domains, an N-terminal beta-sandwich and a C-terminal three-helix bundle. Structural and topological alignments reveal that the beta-sandwich shares structural elements with the type III fibronectin fold (FnIII), but includes structural elaborations that make it unique. We have identified a potential integrin-binding motif consisting of Lys-Thr-Asp(146), Arg(110), and Asp(118). A similar arrangement of charged residues has been described in other invasins. ACP shows a heparin binding activity that requires NtACP. We propose a possible heparin-binding site, including one surface of the three-helix bundle, and nearby portions of the sandwich and repeat domains. We have validated this prediction using assays of the heparin binding and cell-adhesion properties of engineered fragments of ACP. This is the first crystal structure of a member of the highly conserved Gram-positive surface alpha-like protein family, and it will enable the internalization mechanism of GBS to be dissected at the atomic level.

Alpha C protein of group B Streptococcus binds host cell surface glycosaminoglycan and enters cells by an actin-dependent mechanism.

Group B Streptococcus (GBS) colonizes mucosal surfaces of the human gastrointestinal and gynecological tracts and causes disease in a wide range of patients. Invasive illness occurs after organisms traverse an epithelial boundary and enter deeper tissues. Previously we have reported that the alpha C protein (ACP) on the surface of GBS mediates GBS entry into ME180 cervical epithelial cells and GBS translocation across layers of these cells. We now demonstrate that ACP interacts with host cell glycosaminoglycan (GAG); the interaction of ACP with ME180 cells is inhibited if cells are pretreated with sodium chlorate, an inhibitor of sulfate incorporation, or with heparitinases. The interaction is also inhibited in the presence of soluble heparin or heparan sulfate or host cell-derived GAG. In addition, ACP binds soluble heparin specifically in inhibition and dot blot assays. After interaction with host GAG, soluble ACP enters ME180 cells and fractionates to the eukaryotic cell cytosol. These events are inhibited in cells pretreated with cytochalasin D or with Clostridium difficile toxin B. These data indicate that full-length ACP interacts with ME180 cell GAG and enters the eukaryotic cell cytosol by a mechanism that involves Rho GTPase-dependent actin rearrangements. We suggest that these molecular interactions drive ACP-mediated translocation of GBS across epithelial barriers, thereby facilitating invasive GBS infection.

Quantitative determination of immunoglobulin G specific for group B streptococcal beta C protein in human maternal serum.

The beta C protein of group B streptococci (GBS) elicits antibody that is protective against GBS challenge in animals and is considered to be a potential component of a GBS conjugate vaccine. We developed a quantitative enzyme-linked immunosorbent assay to measure beta-specific serum immunoglobulin G (IgG) levels and used it to compare beta-specific IgG in a group of mothers of neonates with invasive type Ib/beta GBS disease and a group of mothers colonized with Ib/beta strains whose neonates remained well. beta-Specific IgG concentrations from these 2 groups were similar. To investigate differences in beta-specific antibody in animals and humans, protein fragments were generated that corresponded to major regions within the beta C protein. A single major region was predominantly recognized in human and rabbit serum samples. Thus, in contrast to immunized animals, no relationship was seen between levels of naturally acquired human beta-specific IgG and protection from neonatal disease. This difference was not explained by a major difference in epitope specificity.