The Human‐Specific Action of Intermedilysin, a Homolog of Streptolysin O, Is Dictated by Domain 4 of the Protein

Nagamune, Hideaki; Ohkura, Kazuto; Sukeno, Akiko; Cowan, Gillian; Mitchell, Tim; Ito, Wataru; Ohnishi, O.; Hattori, Kanako; Yamato, Munehisa; Hirota, Kaoru; Miyake, Yoichiro; Maeda, Takuya; Kourai, Hiroki

doi:10.1111/j.1348-0421.2004.tb03479.x

Cited by 33 publications

(29 citation statements)

References 38 publications

(29 reference statements)

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“…The role of cholesterol appears to be complex, as preincubation of the CD59-dependent cytolysins VLY (this study) and ILY (31) with cholesterol can inhibit lytic activity and is thought to affect the prepore-to-pore transition. Domain swap experiments have indicated that the region dictating species-specific CD59 binding by ILY resides in domain 4, outside of the undecapeptide (15,28). Alignment of VLY, ILY, and three speciesnonspecific CDCs did not reveal obvious candidate amino acids in domain 4 that might confer human CD59 binding (see the supplemental material).…”

Section: Discussionmentioning

confidence: 99%

Functional and Phylogenetic Characterization of Vaginolysin, the Human-Specific Cytolysin fromGardnerella vaginalis

et al. 2008

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Pore-forming toxins are essential to the virulence of a wide variety of pathogenic bacteria. Gardnerella vaginalis is a bacterial species associated with bacterial vaginosis (BV) and its significant adverse sequelae, including preterm birth and acquisition of human immunodeficiency virus. G. vaginalis makes a protein toxin that generates host immune responses and has been hypothesized to be involved in the pathogenesis of BV. We demonstrate that G. vaginalis produces a toxin (vaginolysin [VLY]) that is a member of the cholesteroldependent cytolysin (CDC) family, most closely related to intermedilysin from Streptococcus intermedius. Consistent with this predicted relationship, VLY lyses target cells in a species-specific manner, dependent upon the complement regulatory molecule CD59. In addition to causing erythrocyte lysis, VLY activates the conserved epithelial p38 mitogen-activated protein kinase pathway and induces interleukin-8 production by human epithelial cells. Transfection of human CD59 into nonsusceptible cells renders them sensitive to VLY-mediated lysis. In addition, a single amino acid substitution in the VLY undecapeptide [VLY(P480W)] generates a toxoid that does not form pores, and introduction of the analogous proline residue into another CDC, pneumolysin, significantly decreases its cytolytic activity. Further investigation of the mechanism of action of VLY may improve understanding of the functions of the CDC family as well as diagnosis and therapy for BV.Bacterial vaginosis (BV) is a common and incompletely understood condition associated with adverse outcomes including preterm birth and acquisition of sexually transmitted diseases (10). Alterations of both local host immunity and the genital tract microflora appear to contribute to the pathogenesis of BV (39), and BV can be difficult to eradicate even in the setting of targeted antimicrobial therapy (4). In addition, randomized trials of antibiotics for the prevention of BV-associated preterm birth have not shown consistently beneficial effects, suggesting that host inflammatory responses set in motion early in the course of disease may contribute significantly to the consequences of infection (27).In the 1950s, Leopold (25) and then Gardner and Dukes (14) observed abundant small, pleomorphic gram-variable rods in the genital tract of women with BV. This organism, first called Haemophilus vaginalis (13) and repeatedly renamed as more information about its characteristics became available (reviewed in reference 5), is now classified as Gardnerella vaginalis, the sole member of the genus Gardnerella (16, 30). Phylogenetic analysis based on 16S rRNA places Gardnerella in the gram-positive family Bifidobacteriales. An abundance of G. vaginalis and a paucity of Lactobacillus species are characteristic of a BV-associated microflora, but the relative contribution of G. vaginalis to the pathogenesis of BV is not clear. G. vaginalis is present in essentially all cases of BV but can also be detected in a minority of asymptomatic women (1). Likewise, us...

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Section: Discussionmentioning

confidence: 99%

Functional and Phylogenetic Characterization of Vaginolysin, the Human-Specific Cytolysin fromGardnerella vaginalis

et al. 2008

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“…4, ILY is not only secreted into the medium but is also bound to the bacterial surface presumably due to the interaction of the strongly basic (isoelectric point: 9.82) ILY and negatively charged teichoic acid in the cell wall of Gram positive bacteria. Judging from the enhancement of S. aureus intake into host cells by ILY and the binding ability of ILY to human cell membrane (22,24), ILY on the bacterial surface is likely to function as a receptor for host cells in the adhesion step of infection. Indeed, treatment of the bacterial surface of ILY-producing strains with ILY-neutralizing antibody prior to binding dramatically reduced the adherence of bacteria to the host cells and reduced proliferation during the subsequent incubation period.…”

Section: Discussionmentioning

confidence: 99%

Intermedilysin Is Essential for the Invasion of Hepatoma HepG2 Cells by Streptococcus intermedius

Sukeno

Nagamune

Whiley

et al. 2005

Microbiology and Immunology

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Streptococcus intermedius is a member of the Anginosus group streptococci (AGS), which form part of the normal oral, urogenital and gastrointestinal microflora. AGS are recognized as opportunistic pathogens that cause purulent infection and abscess formation (6,16,32,(37)(38)(39). Notably, S. intermedius is an important pathogen of humans frequently causing deep-seated infections including abscesses in the brain and liver (38,39 Abstract: Streptococcus intermedius causes endogenous infections leading to abscesses. This species produces intermedilysin (ILY), a human-specific cytolysin. Because of the significant correlation between higher ILY production levels by S. intermedius and deep-seated abscesses, we constructed ily knockout mutant UNS38 B3 and complementation strain UNS38 B3R1 in order to investigate the role of ILY in deep-seated infections. Strain UNS38 reduced the viability of human liver cell line HepG2 at infection but not of rat liver cell line BRL3A. Isogenic mutant strain UNS38 B3 was not cytotoxic in either cell line. Quantification of S. intermedius revealed that in infected HepG2 cells UNS38 but not UNS38 B3 increased intracellularly concomitantly with increasing cell damage. This difference between UNS38 and UNS38 B3 was not observed with UNS38 B3R1. Invasion and proliferation in BRL3A cells was not observed. Masking UNS38 or UNS38 B3R1 with ILY antibody drastically decreased adherence and invasion of HepG2. Moreover, coating strain UNS38 B3 with ILY partially restored adherence to HepG2 but without subsequent bacterial growth. At 1 day post-infection, many intact UNS38 were detected in the damaged phagosomes of HepG2 with bacterial proliferation observed in the cytoplasm of dead HepG2 after an additional 2 day incubation. These results indicate that surface-bound ILY on S. intermedius is an important factor for invasion of human cells by this bacterium and that secretion of ILY within host cells is essential for subsequent host cell death. These data strongly implicate ILY as an important factor in the pathogenesis of abscesses in vivo by this streptococcus.

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“…It appears that if there is an interaction between monomers of the oligomer via domain 4, it is only transient and is not present in the oligomer (75,99). Domain 4 of the CDCs mediates membrane recognition, whether it is via cholesterol or another receptor, as in the case of ILY (20,53,72,90,99) (Fig. 1).…”

Section: Figmentioning

confidence: 99%

“…The structure of the ILY undecapeptide is not involved in ILY receptor binding (72), but its alteration appears to be necessary to prevent ILY from binding directly to cholesterol-containing membranes. Nagamune et al (53) showed that the substitution of the SLO undecapeptide sequence for the ILY undecapeptide converted ILY into a nonspecific CDC that could lyse both human and animal erythrocytes. These results suggested that the substitution of the consensus undecapeptide from SLO into the ILY enabled the ILY mutant to bind to cholesterol-rich membranes, as evidenced by its ability to lyse both human and rabbit erythrocytes.…”

Section: Figmentioning

confidence: 99%

Cholesterol-Dependent Cytolysins, a Family of Versatile Pore-Forming Toxins

2005

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The cholesterol-dependent cytolysins (CDCs) are a large family of pore-forming toxins that are produced by more than 20 species from the genera Clostridium, Streptococcus, Listeria, Bacillus, and Arcanobacterium. The pore-forming mechanism of these toxins exhibits two hallmark characteristics: an absolute dependence on the presence of membrane cholesterol and the formation of an extraordinarily large pore. Each CDC is produced as a soluble monomeric protein that, with the exception of one member, is secreted by a type II secretion system. Upon encountering a eukaryotic cell, the CDCs undergo a transformation from a soluble monomeric protein to a membrane-embedded supramolecular pore complex. The conversion of the monomers to an oligomeric, membrane-inserted pore complex requires some extraordinary changes in the structure of the monomer, yet the mechanism of pore formation may be only the beginning of the CDC story.Although the CDCs are well known as beta-hemolytic proteins, it has become increasingly apparent that bacterial pathogens use these proteins in much more sophisticated ways than as simple hemolysins or general cell-lytic agents. The CDC structure also exhibits a plasticity that has allowed the evolution of unique features for some CDCs, without compromising the fundamental pore-forming mechanism. Some of these features are reflected in CDCs that activate complement, that utilize a nonsterol receptor, that exhibit a pH-sensitive, poreforming mechanism, or that can function as a protein translocation channel. As will be apparent in this review, our knowledge of how the CDCs are used by pathogens lags behind our understanding of the CDC pore-forming mechanism, although some studies have provided some provocative glimpses into how the bacterial cells use CDCs during an infection. This review will focus on the advancements in our understanding of the CDC pore-forming mechanism, the unique structural and mechanistic features that are exhibited by many of the CDCs, and how these features might contribute to pathogenesis.

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The Human‐Specific Action of Intermedilysin, a Homolog of Streptolysin O, Is Dictated by Domain 4 of the Protein

Cited by 33 publications

References 38 publications

Functional and Phylogenetic Characterization of Vaginolysin, the Human-Specific Cytolysin fromGardnerella vaginalis

Functional and Phylogenetic Characterization of Vaginolysin, the Human-Specific Cytolysin fromGardnerella vaginalis

Intermedilysin Is Essential for the Invasion of Hepatoma HepG2 Cells by Streptococcus intermedius

Cholesterol-Dependent Cytolysins, a Family of Versatile Pore-Forming Toxins

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