Alpha C Protein of Group B Streptococcus Binds Host Cell Surface Glycosaminoglycan and Enters Cells by an Actin-dependent Mechanism

Abstract

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.Streptococcus agalactiae (Group B Streptococcus, GBS) 1 has long been recognized as an important cause of infection in pregnant/peripartum women and neonates. A frequent colonizer of the human gastrointestinal and gynecological tracts, GBS has been noted more recently to cause a range of invasive syndromes in non-pregnant adults. Most commonly these patients have comorbid conditions, including malignancy, diabetes, and renal disease (1), that may predispose to bacterial invasion because of a loss of epithelial barrier protection in a chronically colonized site such as the rectum, vagina, cervix, urethra, skin, or pharynx. The molecular basis of the interaction between GBS and epithelial cells remains poorly understood.We have reported that the alpha C protein (ACP) on the surface of GBS interacts with epithelial cells. Expressed by many serotype Ia, Ib, and II GBS strains, ACP is the prototype for a family of Gram-positive surface proteins, the alpha-like proteins (Alps). Found on most GBS strains and some Enterococcus and group A Streptococcus strains, Alps share considerable sequence homology and common structural elements, including an N-terminal region, a series of tandem repeats of ϳ80 amino acids each, and a C-terminal region containing a cellwall anchor LPXTG motif common to several Gram-positive species. Despite the fact that these proteins may vary in size due to gene truncation within the repeat region (2), Alps elicit protective antibody in both adult and neonatal mouse models of GBS sepsis (3). In a neonatal mouse model of disease, deletion of the gene encoding ACP attenuates the virul...

Crystal Structure of the N-terminal Domain of the Group B Streptococcus Alpha C Protein

“…5). The distribution of positive charges may be important for understanding the structural basis of the ability of ACP to bind heparin (20). One basic cluster (BR1) is located in domain 1.…”

“…NtACP mediates GBS internalization within human epithelial cells (15). NtACP in association with a repeat domain is necessary to bind GAG (20). Moreover, both the alpha C protein and the isolated N-terminal domain are immunogenic and elicit antibodies that protect against GBS infections in experimental animals (21,22).…”

“…Furthermore, the amount of internalized GBS within human cervical epithelial cells in vitro is inhibited by 75% in the presence of the N-terminal domain of ACP (NtACP) (15), suggesting that ACP is a major determinant of virulence. Recently, ACP has been reported to bind glycosaminoglycans (GAG) (20). ACP consists of an Nterminal domain (174 amino acids), a variable number of tandem repeats of 82 amino acids each, and a 45-amino acid C-terminal domain containing a LPXTG peptidoglycan-binding motif.…”

Characterization of heparin–living bacteria interactions by chemiluminescence electrophoretic mobility shift assay

“…This adhesion is, therefore, a promising target for the development of new antimicrobial therapeutics [3]. In the adhesion, heparin or heparin-related oligosaccharides are one of the extracellular matrix molecules of the host recognized by cell surface proteins of bacteria [4][5][6][7][8].Due to the lack of appropriate techniques for the study of the interactions of heparin-living bacteria, most heparin-bacteria binding studies have been conducted with isolated bacterial proteins [9,10]. Although useful information can be obtained from such studies, one potential drawback is the exclusion of membrane phenomena such as ligand-induced receptor oligomerization that can affect the overall binding affinity [11].…”

“…This adhesion is, therefore, a promising target for the development of new antimicrobial therapeutics [3]. In the adhesion, heparin or heparin-related oligosaccharides are one of the extracellular matrix molecules of the host recognized by cell surface proteins of bacteria [4][5][6][7][8].…”