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.     

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.     

BibA: a novel immunogenic bacterial adhesin contributing to group B Streptococcus survival in human blood

By the analysis of the recently sequenced genomes of Group B Streptococcus (GBS) we have identified a novel immunogenic adhesin with anti-phagocytic activity, named BibA. The bibA gene is present in 100% of the 24 GBS strains analysed. BibA-specific IgG were found in human sera from normal healthy donors. The putative protein product is a polypeptide of 630 amino acids containing a helix-rich N-terminal domain, a proline-rich region and a canonical LPXTG cell wall-anchoring domain. BibA is expressed on the surface of several GBS strains, but is also recovered in GBS culture supernatants. BibA specifically binds to human C4-binding protein, a regulator of the classic complement pathway. Deletion of the bibA gene severely reduced the capacity of GBS to survive in human blood and to resist opsonophagocytic killing by human neutrophils. In addition, BibA expression increased the virulence of GBS in a mouse infection model. The role of BibA in GBS adhesion was demonstrated by the impaired ability of a bibA knockout mutant strain to adhere to both human cervical and lung epithelial cells. Furthermore, we calculated that recombinant BibA bound to human epithelial cells of distinct origin with an affinity constant of approximately 10(-8) M for cervical epithelial cells. Hence BibA is a novel multifunctional protein involved in both resistance to phagocytic killing and adhesion to host cells. The identification of this potential new virulence factor represents an important step in the development of strategies to combat GBS-associated infections.     

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.     

Productive infection of a cervical epithelial cell line with human immunodeficiency virus: implications for sexual transmission.

The human cervix-derived epithelial cell line (ME180) used in this study displays a characteristics epithelial morphology, including numerous desmosomes, tonofilaments, and epidermal filaments. When T-cell lines infected with human immunodeficiency virus (HIV) are added to epithelial cultures, they rapidly adhere to the epithelial monolayer. Within a few minutes, the T cells shed numerous virions into narrow spaces formed between the epithelial cell and the adherent T cells. Virions subsequently enter the ME180 cells via large vesicles. A few days after infection, cytopathic effects and syncytium formation were observed. Infected clones of ME180 cells have remained infected for 8 months. p24 enzyme-linked immunosorbent assay and infectivity assays show that one subclone of the cell line produces virus titers equivalent to those of high-secreting HIV-infected T-cell lines. Electron microscopy reveals numerous virions budding from both the basal and apical surfaces of the epithelium. These observations suggest that cervical epithelium has the potential to serve as a site of HIV infection.     

Helicobacter pylori CagA-mediated IL-8 induction in gastric epithelial cells is cholesterol-dependent and requires the C-terminal tyrosine phosphorylation-containing domain

Upon infection of the gastric epithelial cells, the Helicobacter pylori cytotoxin-associated gene A (CagA) virulence protein is injected into the epithelial cells via the type IV secretion system (TFSS), which is dependent on cholesterol. Translocated CagA is targeted by the membrane-recruited c-Src family kinases in which a tyrosine residue in the Glu-Pro-Ile-Tyr-Ala (EPIYA)-repeat region, which can be phosphorylated, induces cellular responses, including interleukin-8 (IL-8) secretion and hummingbird phenotype formation. In this study, we explored the role of EPIYA-containing C-terminal domain (CTD) in CagA tethering to the membrane lipid rafts and in IL-8 activity. We found that disruption of the lipid rafts reduced the level of CagA translocation/phosphorylation as well as CagA-mediated IL-8 secretion. By CagA truncated mutagenesis, we identified that the CTD, rather than the N-terminal domain, was responsible for CagA tethering to the plasma membrane and association with detergent-resistant membranes, leading to CagA-induced IL-8 promoter activity. Our results suggest that CagA CTD-containing EPIYAs directly interact with cholesterol-rich microdomains that induce efficient IL-8 secretion in the epithelial cells.     

An SH3 binding region in the epithelial Na+ channel (alpha rENaC) mediates its localization at the apical membrane.

The amiloride-sensitive Na+ channel constitutes the rate-limiting step for Na+ transport in epithelia. Immunolocalization and electrophysiological studies have demonstrated that this channel is localized at the apical membrane of polarized epithelial cells. This localization is essential for proper channel function in Na+ transporting epithelia. In addition, the channel has been shown to associate with the cytoskeletal proteins ankyrin and alpha-spectrin in renal epithelia. However, the molecular mechanisms underlying the cytoskeletal interactions and apical membrane localization of this channel are largely unknown. In this study we show that the putative pore forming subunit of the rat epithelial (amiloride-sensitive) Na+ channel (alpha ENaC) binds to alpha-spectrin in vivo, as determined by co-immunoprecipitation. This binding is mediated by the SH3 domain of alpha-spectrin which binds to a unique proline-rich sequence within the C-terminal region of alpha rENaC. Accordingly, the C-terminal region is sufficient to mediate binding to intact alpha-spectrin from alveolar epithelial cell lysate. When microinjected into the cytoplasm of polarized primary rat alveolar epithelial cells, a recombinant fusion protein containing the C-terminal proline-rich region of alpha rENaC localized exclusively to the apical area of the plasma membrane, as determined by confocal microscopy. This localization paralleled that of alpha-spectrin. In contrast, microinjected fusion protein containing the N-terminal (control) protein of alpha rENaC remained diffuse within the cytoplasm. These results suggest that an SH3 binding region in alpha rENaC mediates the apical localization of the Na+ channel. Thus, cytoskeletal interactions via SH3 domains may provide a novel mechanism for retaining proteins in specific membranes of polarized epithelial cells.     

Regulation and function of pilus island 1 in group B streptococcus

Group B streptococcus (GBS) pili may enhance colonization and infection by mediating bacterial adhesion to host cells, invasion across endothelial and epithelial barriers, and resistance to bacterial ingestion and killing by host phagocytes. However, it remains unclear how pilus expression is regulated and how modulation of pilus production affects GBS interactions with the human host. We investigated the regulation and function of pilus island 1 (PI-1) pili in GBS strain 2603. We found that PI-1 gene expression was controlled by the CsrRS two-component system, by Ape1, an AraC-type regulator encoded by a divergently transcribed gene immediately upstream of PI-1, and by environmental pH. The response regulator CsrR repressed expression of Ape1, which is an activator of PI-1 gene expression. In addition, CsrR repressed PI-1 gene expression directly, independent of its regulation of Ape1. In vitro assays demonstrated specific binding of both CsrR and Ape1 to chromosomal DNA sequences upstream of PI-1. Pilus gene expression was activated by acidic pH, and this effect was independent of CsrRS and Ape1. Unexpectedly, characterization of PI-1 deletion mutants revealed that PI-1 pili do not mediate adhesion of strain 2603 to A549 respiratory epithelial cells, ME180 cervical cells, or VK2 vaginal cells in vitro. PI-1 pili reduced internalization and intracellular killing of GBS by human monocyte-derived macrophages, by approximately 50%, but did not influence complement-mediated opsonophagocytic killing by human neutrophils. These findings shed new light on the complex nature of pilus regulation and function in modulating GBS interactions with the human host.     

Bacterial protein domains with a novel Ig‐like fold target human CEACAM receptors

Streptococcus agalactiae, also known as group B Streptococcus (GBS), is the major cause of neonatal sepsis in humans. A critical step to infection is adhesion of bacteria to epithelial surfaces. GBS adhesins have been identified to bind extracellular matrix components and cellular receptors. However, several putative adhesins have no host binding partner characterised. We report here that surface‐expressed β protein of GBS binds to human CEACAM1 and CEACAM5 receptors. A crystal structure of the complex showed that an IgSF domain in β represents a novel Ig‐fold subtype called IgI3, in which unique features allow binding to CEACAM1. Bioinformatic assessment revealed that this newly identified IgI3 fold is not exclusively present in GBS but is predicted to be present in adhesins from other clinically important human pathogens. In agreement with this prediction, we found that CEACAM1 binds to an IgI3 domain found in an adhesin from a different streptococcal species. Overall, our results indicate that the IgI3 fold could provide a broadly applied mechanism for bacteria to target CEACAMs.     

An endoplasmic reticulum protein, p180, is highly expressed in human cytomegalovirus-permissive cells and interacts with the tegument protein encoded by UL48

We have used a virus overlay assay to detect cellular proteins associated with human cytomegalovirus (HCMV) particles. The radiolabeled HCMV particles specifically bound to two host proteins with molecular sizes of 150 and 180 kDa. By a micro-amino-acid sequencing technique, the 180-kDa protein was identified as a human homologue of the ES130/p180 ribosome receptor (p180), which is an integral endoplasmic reticulum (ER) membrane protein possessing a very unique tandem repeat domain at its N-terminal region. The virus overlay assay using truncated p180 polypeptides revealed that HCMV binding to human p180 occurred through the N-terminal region. In HCMV-permissive cells the high level of expression of the human p180 protein was clearly observed regardless of cell type. Furthermore, we showed that p180 binds to the UL48 gene product, which is one of the predominant tegument proteins of HCMV and which is considered to be tightly associated with the capsid. The interaction between the two proteins was assumed to be specific and was observed both in vitro and in vivo. During the late phase of infection, the unique relocation of human p180 was observed, that is, to the juxtanuclear region, which appeared to be in the vicinity of the area where naked virions were frequently observed in an electron-microscopic study. Thus our data suggest that p180 interacts with the HCMV tegument, at least through pUL48, during the HCMV replication process. We discuss the possible role of the interaction between p180 and pUL48 in the intracellular transport of HCMV virions.     

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.     

An Escherichia coli-Enterococcus faecalis shuttle vector as a tool for the construction of a group B Streptococcus heterologous mutant expressing the β antigen (Bac) of the C protein complex

Group B streptococci (GBS) represent a very important group of human pathogens. So far little is known about the mechanisms by which these bacteria can cause disease and the bacterial factors involved. One putative virulence factor is the beta antigen of the C protein complex (Bac), which can bind to the Fc region of human IgA. Its binding function might represent an important virulence mechanism. However, the genetic manipulation of this group of bacteria, necessary to prove involvement of bacterial factors in pathogenesis, is still in its infancy. We therefore tested the pAM401 vector system for its suitability in the construction of a heterologous expression mutant using the Bac protein as a model antigen. The bac gene, including its own promoter, was cloned into the Escherichia coli-Enterococcus faecalis shuttle vector pAM401 and was stably maintained extrachromosomally in the bac-deficient GBS strain 335. Expression of Bac was assessed by extracting the protein from transformed 335(pPJTU1) cells, negative controls (335 wild-type, 335(pAM401)) and other Bac-expressing GBS strains (A909, LA239). Blots of the extracted proteins probed with IgA, polyclonal sera and a monoclonal antibody raised against Bac clearly revealed expression of the 130-kDa protein in the transformed GBS 335(pPJTU1) cells. The correct processing and surface anchoring of the expressed Bac was demonstrated by binding of (125)I-labelled IgA to whole cells. Strain 335(pPJTU1) bound 12 times as much IgA compared to the parental strain LA239 and the GBS 335 negative controls, and a total of 25% compared to the high-level-expressing strain A909. Our studies show that the pAM401 shuttle vector can be used for stable heterologous expression of surface proteins in GBS. Our strategy is also of major importance for the complementation of deletion mutants in GBS and other Gram-positive human pathogens to fulfill Koch's postulates. The Bac mutant constructed in this study, 335(pPJTU1), can be used in animal models to assess the importance of Bac in GBS pathogenesis.     

Regulation of Cell Component Production by Growth Rate in the Group B Streptococcus

Group B Streptococcus (GBS) is the leading cause of bacterial sepsis and meningitis among neonates. While the capsular polysaccharide (CPS) is an important virulence factor of GBS, other cell surface components, such as C proteins, may also play a role in GBS disease. CPS production by GBS type III strain M781 was greater when cells were held at a fast (1.4-h mass-doubling time [td]) than at a slow (11-h td) rate of growth. To further investigate growth rate regulation of CPS production and to investigate production of other cell components, different serotypes and strains of GBS were grown in continuous culture in a semidefined and a complex medium. Samples were obtained after at least five generations at the selected growth rate. Cells and cell-free supernatants were processed immediately, and results from all assays were normalized for cell dry weight. All serotypes (Ia, Ib, and III) and strains (one or two strains per serotype) tested produced at least 3.6-fold more CPS at a td of 1. 4 h than at a td of 11 h. Production of beta C protein by GBS type Ia strain A909 and type Ib strain H36B was also shown to increase at least 5.5-fold with increased growth rate (production at a td of 1. 4 h versus 11 h). The production of alpha C protein by the same strains did not significantly change with increased growth rate. The effect of growth rate on other cell components was also investigated. Production of group B antigen did not change with growth rate, while alkaline phosphatase decreased with increased growth rate. Both CAMP factor and beta-hemolysin production increased fourfold with increased growth rate. Growth rate regulation is specific for select cell components in GBS, including beta C protein, alkaline phosphatase, beta-hemolysin, and CPS production.     

Nonimmunodominant regions are effective as building blocks in a streptococcal fusion protein vaccine

Identification of antigens that elicit protective immunity is essential for effective vaccine development. We investigated the related surface proteins of group B Streptococcus, Rib and alpha, as potential vaccine candidates. Paradoxically, nonimmunodominant regions proved to be of particular interest as vaccine components. Mouse antibodies elicited by Rib and alpha were directed almost exclusively against the C-terminal repeats and not against the N-terminal regions. However, a fusion protein derived from the nonimmunodominant N-terminal regions of Rib and alpha was much more immunogenic than one derived from the repeats and was immunogenic even without adjuvant. Moreover, antibodies to the N-terminal fusion protein protected against infection and inhibited bacterial invasion of epithelial cells. Similarly, the N-terminal region of Streptococcus pyogenes M22 protein, which is targeted by opsonic antibodies, is nonimmunodominant. These data indicate that nonimmunodominant regions of bacterial antigens could be valuable for vaccine development.     

Candida albicans Als3, a multifunctional adhesin and invasin

Candida albicans is part of the normal human flora, and it grows on mucosal surfaces in healthy individuals. In susceptible hosts, this organism can cause both mucosal and hematogenously disseminated disease. For C. albicans to persist in the host and induce disease, it must be able to adhere to biotic and abiotic surfaces, invade host cells, and obtain iron. The C. albicans hypha-specific surface protein Als3 is a member of the agglutinin-like sequence (Als) family of proteins and is important in all of these processes. Functioning as an adhesin, Als3 mediates attachment to epithelial cells, endothelial cells, and extracellular matrix proteins. It also plays an important role in biofilm formation on prosthetic surfaces, both alone and in mixed infection with Streptococcus gordonii. Als3 is one of two known C. albicans invasins. It binds to host cell receptors such as E-cadherin and N-cadherin and thereby induces host cells to endocytose the organism. Als3 also binds to host cell ferritin and enables C. albicans to utilize this protein as a source of iron. Because of its multiple functions and its high expression level in vivo, Als3 is a promising target for vaccines that induce protective cell-mediated and antibody responses. This review will summarize the multiple functions of this interesting and multifunctional protein.     

The N terminus of the transmembrane protein BP180 interacts with the N-terminal domain of BP230, thereby mediating keratin cytoskeleton anchorage to the cell surface at the site of the hemidesmosome

In epidermal cells, the keratin cytoskeleton interacts with the elements in the basement membrane via a multimolecular junction called the hemidesmosome. A major component of the hemidesmosome plaque is the 230-kDa bullous pemphigoid autoantigen (BP230/BPAG1), which connects directly to the keratin-containing intermediate filaments of the cytoskeleton via its C terminus. A second bullous pemphigoid antigen of 180 kDa (BP180/BPAG2) is a type II transmembrane component of the hemidesmosome. Using yeast two-hybrid technology and recombinant proteins, we show that an N-terminal fragment of BP230 can bind directly to an N-terminal fragment of BP180. We have also explored the consequences of expression of the BP230 N terminus in 804G cells that assemble hemidesmosomes in vitro. Unexpectedly, this fragment disrupts the distribution of BP180 in transfected cells but has no apparent impact on the organization of endogenous BP230 and alpha6beta4 integrin. We propose that the BP230 N terminus competes with endogenous BP230 protein for BP180 binding and inhibits incorporation of BP180 into the cell surface at the site of the hemidesmosome. These data provide new insight into those interactions of the molecules of the hemidesmosome that are necessary for its function in integrating epithelial and connective tissue types.     

Vitronectin receptor antibodies inhibit infection of HeLa and A549 cells by adenovirus type 12 but not by adenovirus type 2.

The penton base gene from adenovirus type 12 (Ad12) was sequenced and encodes a 497-residue polypeptide, 74 residues shorter than the penton base from Ad2. The Ad2 and Ad12 proteins are highly conserved at the amino- and carboxy-terminal ends but diverge radically in the central region, where 63 residues are missing from the Ad12 sequence. Conserved within this variable region is the sequence Arg-Gly-Asp (RGD), which, in the Ad2 penton base, binds to integrins in the target cell membrane, enhancing the rate or the efficiency of infection. The Ad12 penton base was expressed in Escherichia coli, and the purified refolded protein assembled in vitro with Ad2 fibers. In contrast to the Ad2 penton base, the Ad12 protein failed to cause the rounding of adherent cells or to promote attachment of HeLa S3 suspension cells; however, A549 cells did attach to surfaces coated with either protein and pretreatment of the cells with an integrin alpha v beta 5 monoclonal antibody reduced attachment to background levels. Treatment of HeLa and A549 cells with integrin alpha v beta 3 or alpha v beta 5 monoclonal antibodies or with an RGD-containing fragment of the Ad2 penton base protein inhibited infection by Ad12 but had no effect on and in some cases enhanced infection by Ad2. Purified Ad2 fiber protein reduced the binding of radiolabeled Ad2 and Ad12 virions to HeLa and A549 cells nearly to background levels, but the concentrations of fiber that strongly inhibited infection by Ad2 only weakly inhibited Ad12 infection. These data suggest that alpha v-containing integrins alone may be sufficient to support infection by Ad12 and that this pathway is not efficiently used by Ad2.     

The R28 protein of Streptococcus pyogenes is related to several group B streptococcal surface proteins, confers protective immunity and promotes binding to human epithelial cells

The R28 protein is a surface molecule expressed by some strains of Streptococcus pyogenes (group A streptococcus). Here, we present evidence that R28 may play an important role in virulence. Sequence analysis demonstrated that R28 has an extremely repetitive sequence and can be viewed as a chimera derived from the three surface proteins Rib, alpha and beta of the group B streptococcus (GBS). Thus, the gene encoding R28 may have originated in GBS. The R28 protein promotes adhesion to human epithelial cells, as shown by experiments with an R28-negative mutant and by the demonstration that antibodies to highly purified R28 inhibited adhesion. In a mouse model of lethal intraperitoneal S. pyogenes infection, antibodies to R28 conferred protective immunity. However, the virulence of an R28-negative mutant was similar to that of the parental strain in the intraperitoneal infection model. Together, these data indicate that R28 represents a novel type of adhesin expressed by S. pyogenes and that R28 may also act as a target for protective antibodies at later stages of an infection. We consider the hypothesis that R28 played a pathogenetic role in the well-known epidemics of childbed fever (puerperal fever), which were caused by S. pyogenes. A role for R28 in these epidemics is suggested by epidemiological data.