Host Cell Binding Research Articles

An Immunologically Cryptic Epitope of Plasmodium falciparum Circumsporozoite Protein Facilitates Liver Cell Recognition and Induces Protective Antibodies That Block Liver Cell Invasion

Circumsporozoite, a predominant surface protein, is involved in invasion of liver cells by Plasmodium sporozoites, which leads to malaria. We have previously reported that the amino terminus region (amino acids 27-117) of P. falciparum circumsporozoite protein plays a critical role in the invasion of liver cells by the parasite. Here we show that invasion-blocking antibodies are induced by a polypeptide encoding these 91 amino acids, only when it is presented in the absence of the rest of the protein. This suggests that when present in the whole protein, the amino terminus remains immunologically cryptic. A single reactive epitope was identified and mapped to a stretch of 21 amino acids from position 93 to 113. The epitope is configurational in nature, since its recognition was affected by deleting as little as 3 amino acids from either end of the 21-residue peptide. Lysine 104, the only known polymorphic position in the epitope, affected its recognition by the antibodies, and its conversion to leucine in the protein led to a substantial loss of binding activity of the protein to the hepatocytes. This indicated that in the protein, the epitope serves as a binding ligand and facilitates the interaction between sporozoite and hepatic cells. When considered along with the observation that in its native state this motif is immunologically unresponsive, we suggest that hiding functional moieties of the protein from the immune system is an evasion strategy to preserve liver cell binding function and may be of importance in designing anti-sporozoite vaccines.

Host cell binding of GRA10, a novel, constitutively secreted dense granular protein from Toxoplasma gondii

Monoclonal antibodies (mAbs) against Toxoplasma gondii, Tg378 and Tg556 clones, are specifically observed to localize to the dense granules of tachyzoites by immunofluorescence microscopy. mAb Tg556 is directed against GRA3, a previously described 30 kDa dense granular protein. mAb Tg378 is directed against a novel 36 kDa dense granular protein, which we refer to as GRA10. These are major proteins in the excretory/secretory proteins from T. gondii before the parasite’s entry into host cells, and they are released into the parasitophorous vacuole (PV) during or shortly after invasion to be associated with the PV membrane. GRA10 binds to the membrane of the host cells regardless of its anchorage-dependence or -independence. The cDNA sequence encoding GRA10 was determined by screening a T. gondii cDNA expression library with mAb Tg378. The deduced amino acid sequence of GRA10 consists of a polypeptide of 364 amino acids, and it has no significant homology to any other known proteins. The sequence contains amino terminal signal peptides and two potential transmembrane domains in the middle of sequence that are not near the carboxy terminus. GRA10 has a RGD motif between the two potential transmembrane domains.

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.

Tissue specificity of enhancer and promoter activities of a HERV-K(HML-2) LTR

Transient expression of a luciferase reporter gene was used to evaluate tissue-specific promoter and enhancer activities of a solitary extraviral long terminal repeat (LTR) of the human endogenous retrovirus K (HERV-K) in several human and CHO cell lines. The promoter activity of the LTR varied from virtually not detectable (GS and Jurkat cells) to as high as that of the SV40 early promoter (Tera-1 human testicular embryonal carcinoma cells). The negative regulatory element (NRE) of the LTR retained its activity in all cell lines where the LTR could act as a promoter, and was also capable of binding host cell nuclear proteins. The enhancer activity of the LTR towards the SV40 early promoter was detected only in Tera-1 cells and was not observed in a closely related human testicular embryonal carcinoma cell line of different origin, NT2/D1. A comparison of proteins bound to central part of the LTR in nuclear extracts from Tera-1 and NT2/D1 by electrophoretic mobility shift assay revealed striking differences that could be determined by different LTR enhancer activities in these cells. Tissue specificity of the SV40 early promoter activity was also revealed.

Toxoplasma gondii major surface antigen (SAG1): in vitro analysis of host cell binding.

Previous studies have indicated that SAG1, the major surface molecule of the protozoan parasite Toxoplasma gondii, is an important attachment ligand for the host cell. However, the research data that supports this claim comes largely from studies investigating tachyzoite binding, and not SAG1 binding per se. In this study we successfully developed an in vitro attachment assay to directly evaluate the mechanism of SAG1-host cell binding. Competition experiments were then performed using SAG1 that had been pre-treated with the neoglycoprotein BSA-glucosamide or with antibody. Soluble BSA-glucosamide blocked SAG1 attachment to MDBK cells in a dose-dependent manner, implying that SAGI binding is mediated, in part, via attachment to host cell surface glucosamine. Interestingly, pre-incubation of SAG1 in polyclonal sera from chronically infected mice failed to block binding. This challenges the assumption that anti-SAG1 antibodies block parasite attachment through the masking of SAG1 host cell binding domains. Taken together, this evidence presents new strategies for understanding SAG1-mediated attachment.

Fucosylated oligosaccharides in human milk in relation to gestational age and stage of lactation.

Breastfeeding is known to offer significant protection against infectious diarrhea. Oligosaccharides, the third largest solid component of human milk, constitute a major mechanism of innate defense of breastfed infants against infectious diarrhea (Morrow et al. 2004; Jiang et al. 2004; Newburg et al. 2004; Ruiz-Palacios et al. 2003). The fucosyl-ated oligosaccharides of human milk comprise the majority of milk oligosaccharides and have been shown to inhibit host cell binding to specific enteric pathogens (Crane et al. 1994; Newburg et al. 1990; Ruiz-Palacios et al. 2003). Mothers vary in the quantity and type of oligosaccharides found in their milk, which may influence the degree to which their milk offers protection of their infants against specific pathogens. Human milk fucosylated oligosaccharide synthesis is controlled by the same fucosyltransferase genes (FUT2 and FUT3) that control secretor and Lewis blood group types (Newburg et al. 2004; Viverge et al. 1990). Oligosaccharide expression in human milk also may be affected by gestational age and stage of lactation.KeywordsMilk SampleHuman MilkBreastfed InfantInfectious DiarrheaMilk OligosaccharideThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Effects of iron limitation on adherence and cell surface carbohydrates of Corynebacterium diphtheriae strains.

Iron limitation may cause bacterial pathogens to grow more slowly; however, it may also stimulate these microorganisms to produce greater tissue damage, given that many virulence factors are controlled by the iron supply in the environment. The present study investigated the influence of low iron availability on the expression of proteins and surface sugar residues of two toxigenic strains of Corynebacterium diphtheriae subsp. mitis and evaluated their adherence to human group B erythrocytes and HEp-2 cells. A comparison was made between bacteria grown in (i) Trypticase soy broth (TSB), (ii) TSB treated with dipyridyl to deplete free iron, and (iii) TSB enriched with FeCl(3). The effects of iron concentration on adhesive properties were different for strains 241 and CDC-E8392, of the sucrose-fermenting and non-sucrose-fermenting biotypes, respectively. Iron-limited conditions enhanced interaction of strain 241 with erythrocytes and HEp-2 cells. Inhibition assays suggested the involvement of nonfimbrial protein combination 67-72p on hemagglutination of diphtheria bacilli grown under iron-limited conditions. Conversely, iron limitation inhibited adherence to glass and expression of electron-dense material on the bacterial surface. Lectin binding assays demonstrated a reduction in the number of sialic acid residues and an increase in D-mannose and D-galactose residues on the surfaces of both strains. Thus, iron exerts a regulatory role on adhesive properties of diphtheria bacilli, and low iron availability modulates the expression of C. diphtheriae surface carbohydrate moieties. The significant changes in the degree of lectin binding specific for D-mannose, D-galactose and sialic acid residues may have an effect on binding of host cells. The expression of dissimilar microbial virulence determinants may be coordinately controlled by common regulatory systems. For C. diphtheriae, the present results imply regulation of adherence and slime production as part of a global response to iron-limited environmental conditions that includes derepression of genes for the synthesis of cytotoxin and siderophores and for transport of the Fe(III)-siderophore complexes.

Decorin-binding proteins A and B confer distinct mammalian cell type-specific attachment by Borrelia burgdorferi, the Lyme disease spirochete.

Host cell binding is an essential step in colonization by many bacterial pathogens, and the Lyme disease agent, Borrelia burgdorferi, which colonizes multiple tissues, is capable of attachment to diverse cell types. Glycosaminoglycans (GAGs) are ubiquitously expressed on mammalian cells and are recognized by multiple B. burgdorferi surface proteins. We previously showed that B. burgdorferi strains differ in the particular spectrum of GAGs that they recognize, leading to differences in the cultured mammalian cell types that they efficiently bind. The molecular basis of these binding specificities remains undefined, due to the difficulty of analyzing multiple, potentially redundant cell attachment pathways and to the paucity of genetic tools for this pathogen. In the current study, we show that the expression of decorin-binding protein (Dbp) A and/or DbpB, two B. burgdorferi surface proteins that bind GAGs, is sufficient to convert a high-passage nonadherent B. burgdorferi strain into one that efficiently binds 293 epithelial cells. Epithelial cell attachment was mediated by dermatan sulfate, and, consistent with this GAG-binding specificity, these recombinant strains did not bind EA-Hy926 endothelial cells. The GAG-binding properties of bacteria expressing DbpB or DbpA were distinguishable, and DbpB but not DbpA promoted spirochetal attachment to C6 glial cells. Thus, DbpA and DbpB may each play central but distinct roles in cell type-specific binding by Lyme disease spirochetes. This study illustrates that transformation of high-passage B. burgdorferi strains may provide a relatively simple genetic approach to analyze virulence-associated phenotypes conferred by multiple bacterial factors.

Contribution of Efa1/LifA to the adherence of enteropathogenic Escherichia coli to epithelial cells

Enteropathogenic E. coli(EPEC) is an important diarrhoeal pathogen that induces characteristic lesions on the host intestine termed attaching and effacing (A/E) lesions. In this study we have examined the contribution of a large gene, efa1, which is present in all A/E pathogens, to the adherence phenotype of EPEC. An efa- derivative of EPEC JPN15 was constructed and this mutant was significantly less adherent to epithelial cells than the parent strain. The JPN15 efa- derivative was FAS-positive, produced EspA filaments and showed comparable levels of EspA secretion to JPN15. In addition, polyclonal antibodies raised to Efa1 partially inhibited the adherence of JPN15 to cultured epithelial cells. In further work, we showed that human and rabbit hosts infected with an A/E pathogen produced antibodies to Efa1 and we observed that the truncated form of efa1 present in EHEC O157:H7 was specific to that serotype. Generally efa1 was present in its entirety in the genomes of other A/E pathogens. Overall our data suggest that Efa1 has host cell binding activity, at least in tissue culture, and that it is produced during infection. These findings suggest that Efa1 may play a direct role in the pathogenesis of infections caused by A/E pathogens.

A C-terminal EGF-like domain governs BAD1 localization to the yeast surface and fungal adherence to phagocytes, but is dispensable in immune modulation and pathogenicity of Blastomyces dermatitidis.

BAD1, an adhesin and immune modulator of Blastomyces dermatitidis, is an essential virulence factor that is released extracellularly before association with the yeast surface. Here, deletion of the C-terminal EGF-like domain profoundly affected BAD1 function, leading to non-association with yeast, extracellular accumulation and impaired yeast adherence to macrophages. In equilibrium binding assays, DeltaC-term BAD1, lacking an EGF-like domain, bound poorly to BAD1 null yeast, yielding a low affinity (Kd, 3 x 10(-7) M versus 5 x 10(-8) M) and Bmax (1.9 x 10(5) versus 7.9 x 10(5)) compared with BAD1. Similar protein binding profiles were observed using chitin particles, reinforcing the notion that chitin fibrils are a receptor for BAD1, and that the EGF-like domain is critical for BAD1 interactions with chitin on yeast. DeltaC-term strains bound poorly to macrophages, compared with parental or BAD1-reconstituted null strains. However, DeltaC-term strains and the purified protein itself sharply suppressed tumour necrosis factor (TNF)-alpha release by phagocytes in vitro and in lung in vivo, and the strains retained pathogenicity in a murine model of blastomycosis. Our results illustrate the previously undefined role of the EGF-like domain for BAD1 localization to yeast surfaces during cell wall biogenesis. They also demonstrate that the requirements for host cell binding and immune modulation by BAD1 can be dissociated from one another, and that the former is unexpectedly dispensable in the requisite role of BAD1 in pathogenesis.

Adaptation of the Lyme disease spirochaete to the mammalian host environment results in enhanced glycosaminoglycan and host cell binding.

The Lyme disease spirochaete, Borrelia burgdorferi, is transmitted to mammals by Ixodes ticks and can infect multiple tissues. Host cell attachment may be critical for tissue colonization, and B. burgdorferi cultivated in vitro recognizes heparin- and dermatan sulphate-related glycosaminoglycans (GAGs) on the surface of mammalian cells. To determine whether growth of the spirochaete in the mammalian host alters GAG binding, we assessed the cell attachment activities of B. burgdorferi grown in vitro or in dialysis membrane chambers implanted intraperitoneally in rats. Host-adapted B. burgdorferi exhibited approximately threefold better binding to purified heparin and dermatan sulphate and to GAGs expressed on the surface of cultured endothelial cells. Three B. burgdorferi surface proteins, Bgp, DbpA and DbpB, have been demonstrated previously to bind to GAGs or to GAG-containing molecules, and we show here that recombinant derivatives of each of these proteins were able to bind to purified heparin and dermatan sulphate. Immunofluorescent staining of in vitro-cultivated or host-adapted spirochaetes revealed that DbpA and DbpB were present on the bacterial surface at higher levels after host adaptation. Recombinant Bgp, DbpA and DbpB each partially inhibited attachment of host-adapted B. burgdorferi to cultured mammalian cells, consistent with the hypothesis that these proteins may promote attachment of B. burgdorferi during growth in the mammalian host. Nevertheless, the partial nature of this inhibition suggests that multiple pathways promote mammalian cell attachment by B. burgdorferi in vivo. Given the observed increase in cell attachment activity upon growth in the mammalian host, analysis of host-adapted bacteria will facilitate identification of the cell binding pathways used in vivo.

Involvement of Trypanosoma cruzi metacyclic trypomastigote surface molecule gp82 in adhesion to gastric mucin and invasion of epithelial cells.

Upon oral infection, Trypanosoma cruzi metacyclic trypomastigotes invade and replicate in the gastric mucosal epithelium, being apparently uniquely specialized for adhesion to mucin and mucosal invasion. Here we investigated the involvement of gp82, the metacyclic-stage-specific surface glycoprotein implicated in host cell entry, in both adhesion to gastric mucin and invasion of the mucosal epithelium upon oral challenge. Metacyclic forms, preincubated with a control monoclonal antibody (MAb) or with MAb 3F6 directed to gp82, were administered orally to BALB/c mice, and parasitemia was monitored. Mice that received parasites treated with MAb 3F6 had greatly reduced parasitemia, displaying at the peak a mean number of blood parasites more than 100-fold lower than that of the control group. MAbs directed to other T. cruzi surface glycoproteins had no such effect. gp82, as either a native or a recombinant molecule, but not the metacyclic trypomastigote surface molecule gp90 or gp35/50, bound to gastric mucin in enzyme-linked immunosorbent assays. MAb 3F6 significantly inhibited the penetration of cultured epithelial HeLa cells by metacyclic forms in the absence or in the presence of gastric mucin. Mucin alone did not affect parasite internalization. Parasite infectivity was not altered by treatment of metacyclic forms with pepsin, to which gp82 was resistant, or with proteinase K, which removed the N-terminal portion of gp82 but preserved its host cell binding site. Taken together, these findings suggest that gp82 mediates the interaction of metacyclic trypomastigotes with gastric mucin and the subsequent penetration of underlying epithelial cells.

Recruitment of mammalian cell fibronectin to the surface of Chlamydia trachomatis.

Pathogenic bacteria exploit the presence of various host cell molecules in order to colonize new tissues. Fibronectin is involved in a wide range of cell functions in vivo, and staphylococci, streptococci, and gonococci have evolved mechanisms to utilize this glycoprotein to mediate host cell binding. We show that elementary bodies (EB) from two biovars of Chlamydia trachomatis recruit fibronectin to their surfaces upon lysis of the host cell. We also demonstrate that a heparan sulfate lyase-sensitive molecule on chlamydial EB is responsible for binding at least a portion of this fibronectin.

Solution Structure of Bacteriophage PRD1 Vertex Complex

Bacteriophage PRD1 is a prototype of viruses with an internal membrane. The icosahedral capsid and major coat protein share structural similarity with the corresponding structures of adenovirus. The present study further explores similarities between these viruses, considering the 5-fold vertex assemblies. The vertex structure of bacteriophage PRD1 consists of proteins P2, P5, and P31. The vertex complex mediates host cell binding and controls double-stranded DNA delivery. Quaternary structures and interactions of purified spike proteins were studied by synchrotron radiation x-ray solution scattering. Low resolution models of the vertex proteins P5, P2, and P31 were reconstructed ab initio from the scattering data. Protein P5 is a long trimer that resembles the adenovirus spike protein pIV. The receptor-binding protein P2 is a 15.5-nm long, thin monomer and does not have an adenovirus counterpart. P31 forms a pentameric base with a maximum diameter of 8.5 nm, which is thinner than the adenovirus penton pIII. P5 further polymerize into a nonameric form ((P5(3))(3)). In the presence of P31, P5 associates into a P5(6):P31 complex. The constructed models of these assemblies provided support for a model of vertex assembly onto the virion. Although similar in overall architecture, clear differences between PRD1 and adenovirus spike assemblies have been revealed.

Protease Activity and Host Cell Binding of the 42-kDa Rhoptry Protein from Toxoplasma gondii after Secretion

Three proteases were identified in the excretory/secretory proteins (ESP) from Toxoplasma gondii by the gelatin acrylamide gel electrophoresis (GAGE), of which the molecular masses were 80, 70, and 42 kDa. One of the proteases with 42 kDa was reactive to a monoclonal antibody (mAb), Tg786 clone, which was localized in the rhoptry of T. gondii by immunohistochemistry. The protease was maximally active at the pH range between 7.5 and 8.5, and was sensitive to inhibition by TPCK and EGTA. The gelatinolytic activity of the protease was dependent on the concentration of calcium ion. The protease was active only in the millimolar ranges of calcium but not in micromolar ranges, implicating that the secretion is critical event for the activation of the protease. The secreted protease was shown to bind to the host cells upon Western blot and immunofluorescence analysis. It is suggested that the protease may target to the plasma membrane of the host cells, which provides appropriate environment for the entry of the parasite into host cells. The mAb (Tg786) of T. gondii also reacted with a protein of the same size and equivalent locality of rhoptry in Neospora caninum, a similar Apicomplexan protozoa, suggesting that secreted protease mediates a common function in the mechanism of entry into host cells.

Biophysical characterization and vector-specific antagonist activity of domain III of the tick-borne flavivirus envelope protein.

The molecular determinants responsible for flavivirus host cell binding and tissue tropism are largely unknown, although domain III of the envelope protein has been implicated in these functions. We examined the solution properties and antagonist activity of Langat virus domain III. Our results suggest that domain III adopts a stably folded structure that can mediate binding of tick-borne flaviviruses but not mosquito-borne flaviviruses to their target cells. Three clusters of phylogenetically conserved residues are identified that may be responsible for the vector-specific antagonist activity of domain III.

Role of anaerobiosis in virulence of Salmonella typhimurium.

Intestinal pathogens are exposed to various stress conditions during their infectious cycle. Anaerobiosis, one of such hostile condition, is offered by the host within gut and intestinal lumen, where survival, multiplication and entry into intestinal epithelial cells is priority for the invading pathogen. In the present study, a virulent strain of S. typhimurium (1402/84) was grown under anaerobic conditions and its virulence characteristics such as host cell binding, penetration and intracellular survival were compared with aerobic S. typhimurium. Anaerobically grown S. typhimurium showed significantly higher binding to immobilized mice enterocytes and intestinal mucus as compared to bacteria grown aerobically. Anaerobic bacteria also showed an early penetration of mucus and subsequent binding to underlying immobilized enterocytes, in vitro. Anaerobic S. typhimurium exhibited increased intracellular survival within spleen macrophages of mice and caused significantly higher fluid accumulation in ligated rabbit ileal loops as compared to aerobic bacteria. LD50 of anaerobic S. typhimurium was also observed to be 2 fold lower when compared to aerobic bacteria. Cell surface hydrophobicity of anaerobic S. typhimurium was also found to be significantly higher than aerobic bacteria. Thus, it appears that exposure of S. typhimurium to anaerobiosis results in its enhanced virulence, adhesion and penetration of host cells.

Fibronectin binding protein and host cell tyrosine kinase are required for internalization of Staphylococcus aureus by epithelial cells.

Staphylococcus aureus expresses several surface proteins that promote adherence to host cell extracellular matrix proteins, including fibronectin (Fn). Since this organism has recently been shown to be internalized by nonprofessional phagocytes, a process that typically requires high-affinity binding to host cell receptors, we investigated the role of its Fn binding proteins (FnBPs) and other surface proteins in internalization by the bovine mammary gland epithelial cell line (MAC-T). Efficient internalization of S. aureus 8325-4 required expression of FnBPs; an isogenic mutant (DU5883), not expressing FnBPs, was reduced by more than 95% in its ability to invade MAC-T cells. Moreover, D3, a synthetic peptide derived from the ligand binding domain of FnBP, inhibited the internalization of the 8325-4 strain in a dose-dependent fashion and the efficiency of staphylococcal internalization was partially correlated with Fn binding ability. Interestingly, Fn also inhibited the internalization and adherence of S. aureus 8325-4 in a dose-dependent manner. In contrast to internalization, adherence of DU5883 to MAC-T was reduced by only approximately 40%, suggesting that surface binding proteins, other than FnBPs, can mediate bacterial adherence to cells. Adherence via these proteins, however, does not necessarily result in internalization of the staphylococci. An inhibitor of protein tyrosine kinase, genistein, reduced MAC-T internalization of S. aureus by 95%, indicating a requirement for a host signal transduction system in this process. Taken together, these results indicate that S. aureus invades nonprofessional phagocytes by a mechanism requiring interaction between FnBP and the host cell, leading to signal transduction and subsequent rearrangement of the host cell cytoskeleton.

Receptor/ligand interactions between Cryptosporidium parvum and the surface of the host cell

The ability of membrane antigens on sporozoites of the intestinal pathogen, Cryptosporidium parvum, to bind host cell surface antigens was investigated. A novel membrane-associated protein of approximately 47 kDa, designated CP47, was found to possess significant binding affinity for the surface of both human and animal ileal cells. This protein was purified by a combination of anion-exchange chromatography on FPLC and immunoaffinity chromatography. Purified CP47 demonstrated competitive binding with parasite-associated membrane antigens to membranes of HCT-8 and ileal cells in a dose-dependent manner. Furthermore, the binding activity of CP47 was found to be Mn 2+-sensitive, and was completely inhibited in the presence of 10 mM MnCl 2. These results were consistent with earlier findings demonstrating the inhibitory effect of Mn 2+ ions on Cryptosporidium infection both in vitro and in vivo (Nesterenko et al., Biol. Trace Elem. Res. 56 (1997) 243–253). Immunoelectron microscopy using gold-conjugated antibodies revealed CP47 to be localized at the apical end of the sporozoites. A single protein with an electrophoretic mobility of 57 kDa was purified from host cell membranes using CP47-Affigel. Similarly, affinity purification of this protein was abrogated in the presence of Mn 2+. These data suggest that a novel parasite protein, CP47, may play an important role in sporozoite/host cell attachment.

Two Structural Transitions in Membrane Pore Formation by Pneumolysin, the Pore-Forming Toxin of Streptococcus pneumoniae

The human pathogen Streptococcus pneumoniae produces soluble pneumolysin monomers that bind host cell membranes to form ring-shaped, oligomeric pores. We have determined three-dimensional structures of a helical oligomer of pneumolysin and of a membrane-bound ring form by cryo-electron microscopy. Fitting the four domains from the crystal structure of the closely related perfringolysin reveals major domain rotations during pore assembly. Oligomerization results in the expulsion of domain 3 from its original position in the monomer. However, domain 3 reassociates with the other domains in the membrane pore form. The base of domain 4 contacts the bilayer, possibly along with an extension of domain 3. These results reveal a two-stage mechanism for pore formation by the cholesterol-binding toxins.