Review TRENDS in Molecular Medicine Vol.10 No.9 September 2004

CD46 in Neisseria pathogenesis

Darcy B. Gill and John P. Atkinson

Washington University School of Medicine, Department of Medicine, Rheumatology Division, Campus Box 8045, 660 S. Euclid, St Louis, MO 63110, USA

CD46 is a complement regulator that serves as a cellular and the genitourinary tract (N. gonorrhoeae) are essential pilus for the human pathogens Neisseria for the survival of these pathogens. Recent studies of gonorrhoeae and Neisseria meningitidis. The role of colonization by the pathogenic Neisseriae have revealed a CD46 in N. gonorrhoeae infection has been further multistep process of adhesion involving interactions characterized through the identification of domains between multiple bacterial proteins and host-cell recep- that are required for bacterial adhesion, as well as the tors (reviewed in [2–7]). delineation of CD46-dependent signaling responses that The Type IV pilus is an important colonization factor promote this adhesion. In addition, the adherence of for both N. gonorrhoeae and N. meningitidis that N. gonorrhoeae to human epithelial cell lines results in facilitates the initial attachment of the bacteria to host cell-surface downregulation followed by the liberation cells (Figure 1). This organelle has been most intensely of CD46 into the surrounding milieu. Recently, trans- studied in gonococci, in which it has been characterized as genic mice expressing human CD46 were used in a a multifunctional filamentous appendage that mediates mouse model of meningitis, in which CD46 enhanced bacterial autoagglutination, competency for DNA trans- meningococcal traversal of the blood–brain barrier. formation, a mode of surface translocation known as Although the role of CD46 in infection by the pathogenic twitching motility and adhesion to host cells (reviewed in Neisseria remains incompletely defined, these advances [2]). Here, we review recent studies investigating the role indicate a dynamic and important contribution of CD46 of the human cell-surface glycoprotein CD46 in pilus- to Neisseria pathogenesis. mediated adhesion of Neisseria and the subsequent host-cell response. The human complement regulator CD46 is a cellular receptor for multiple human pathogens, including Neisseria gonorrhoeae (gonococcus) and Neisseria CD46 function, structure and microbial interactions meningitidis (meningococcus) [1]. The Type IV pilus of CD46, or membrane cofactor protein (MCP), is a type I Neisseria mediates the initial bacterial attachment to the transmembrane glycoprotein (Figures 2 and 3). It is a human epithelium, initiating a multistep cascade that member of the regulators of complement activation (RCA) results in tight bacterial adhesion [2].Undersome protein family, a group of structurally, functionally and experimental conditions, CD46 has a role in this inter- genetically related proteins that protect host cells action between the host and the pathogen by serving as against complement attack and serve as receptors for a receptor for the Type IV pilus and the subsequent complement-coated antigens. Following the deposition of signaling events. C3bandC4bonhostcells,CD46preventsfurther complement activation by serving as a cofactor in the The pathogenic Neisseriae limited proteolysis of and C4b by the plasma serine N. gonorrhoeae and N. meningitidis are the causative protease factor I (Figure 2) (reviewed in [8]). agents of gonorrhea and meningitis, respectively. These CD46 is expressed by nearly every human cell type infections are responsible for substantial morbidity with the exception of erythrocytes [8]. Four major isoforms and mortality worldwide. Individuals infected with (BC1, BC2, C1 and C2) are expressed through the alter- N. gonorrhoeae typically present with inflammation of native splicing of a 46 kb gene. Isoforms vary in their the urethra or cervix. The clinical manifestations vary, quantity of O-glycosylation, as a result of the presence or ranging from asymptomatic carriage to pelvic inflamma- absence of the B module, and in the expression of one of tory disease and sepsis. N. meningitidis is a part of the two possible cytoplasmic tails (Cyt-1 or Cyt-2) (Figure 3). normal nasopharyngeal flora of 3–30% of the human All four isoforms are expressed in most cells, with the population. Under circumstances that are poorly under- exceptions being the kidney, which expresses mainly the stood, this organism traverses the mucosal epithelium, BC2 isoform, and the brain and spermatozoa, which resulting in bacteraemia and, occasionally, sepsis. Once in predominantly express the C2 isoform [9]. Apart from the bloodstream, meningococci can then cross the blood– these exceptions, tissues within a given individual express brain barrier to cause a fulminant meningitis. the same ratio of BC to C isoforms. This is an inherited The colonization of and persistence in the human trait, with 66% of individuals expressing predominantly mucosal epithelium of the nasopharynx (N. meningitidis) the BC isoforms, 29% expressing equal quantities of the

Corresponding author: John P. Atkinson ( [email protected]). B and C isoforms and 6% expressing predominantly the Available online 11 August 2004 C isoform [10,11]. www.sciencedirect.com 1471-4914/$ - see front matter Q 2004 Published by Elsevier Ltd. doi:10.1016/j.molmed.2004.07.002 460 Review TRENDS in Molecular Medicine Vol.10 No.9 September 2004

(a) (b) (c) PilV? PilC

PilE

OM

IM

PilT

TRENDS in Molecular Medicine

Figure 1. The type IV pilus of Neisseria mediates interactions with host cells. (a) Electron micrograph of a diplococcus showing pili (filamentous appendages radiating outward from the bacterial surface). Magnification ! 39 000. Electron micrograph reproduced with permission from [54]. (b) Confocal image showing microcolonies of piliated gonococci (yellow arrows) adhering to an ME-180 human cervical epithelial cell after a 4 h incubation. Following formaldehyde fixation, adherent bacteria were stained with propidium iodide. (c) Diagram depicting gonococcal pilus components. The pilus fiber itself is composed of the subunit PilE (blue). Pilin subunits originate on the inner membrane (IM) and, upon assembly, extrude through the outer membrane (OM) via the PilQ secretin (grey). PilC (green), a pilus-associated protein and putative adhesin, has been described to localize to the tip of the fibrillum and to the outer membrane of the bacterium [55,56]. PilV (yellow), a minor pilus-associated protein, has yet to be conclusively localized to the pilus, but has been suggested to function in the proper presentation of PilC [57]. PilT (red), an ATPase, is necessary for pilus retraction and has been demonstrated on the inner membrane of the bacterium [58]. Other pilus components are not shown. Although several pilus components of Neisseria exhibit significant variation in sequence and/or glycosylation among strains and species, the overall architecture and general function of the pilus is believed to be similar in Neisseria gonorrhoeae and Neisseria meningitidis.

The crucial role of this protein for preventing excessive CD46 as a cellular pilus receptor for pathogenic Neisseria complement activation was recently highlighted by the Based on three lines of experimentation in a report by description of CD46 mutations in patients with hemolytic Kallstrom et al. [1], CD46 was identified as a cellular uremic syndrome (HUS) [12–14]. In addition, CD46 has receptor for the Neisseria pilus (Figure 4A). First, in an been identified as a key component in fertilization (reviewed in [15]), T regulatory cell differentiation [16] and host-cell interactions with a growing list of human pathogens, includ- 1 1 ing the measles virus [17,18], Streptococcus pyogenes [19], 1 1 the pathogenic Neisseriae [1], human herpesvirus 6 [20] and 2 2 group B and D adenoviruses [21–23]. 2 2 3 3 3 3 4 4 O O O O 4 4 (a) O B O O BB O O O O O O O O O O O O O O C O O C O O C O O C O Factor I Factor I Membrane

C3f Cytoplasm C3bi C3b C3b C3b CD46 CD46 CD46 CD46

BC1 BC2 C1 C2

Cyt-1: TYLTDETHREVKFTSL Cyt-2: KADGGAEYATYQTKSTTPAEQRG (b) Casein kinase2 Casein kinase 2 Protein kinase C Src kinase Factor I Factor I TRENDS in Molecular Medicine C4c C4b C4b C4b Figure 3. Diagram of CD46 structure. CD46 is a type I transmembrane glycoprotein CD46 CD46 CD46 CD46

C4d that is expressed on most tissues as four major isoforms (BC1, BC2, C1 and C2) that arise from alternative splicing of a single gene. All four isoforms have identical N-terminal regions consisting of four complement control protein repeats (CCPs 1–4, ovals). CCPs 1, 2 and 4 have N-linked sugar moieties (forks). The serine– threonine–proline rich (STP) region (triangles) is the site of alternative splicing. All TRENDS in Molecular Medicine four major isoforms use the C region, whereas the B exon is alternatively spliced, resulting in the BC or C STP regions. Following the STP domain is a 12 amino acid Figure 2. Cofactor activity of CD46. CD46 binds efficiently to C3b or C4b that are region of unknown function, a hydrophobic transmembrane domain, a charged deposited on the surface of the cell on which it is expressed. Upon the binding of a cytoplasmic anchor and a C-terminal cytoplasmic tail. The C-terminus is also plasma serine protease (factor I) to this complex, the cleavage of C3b (a) to C3bi and alternatively spliced, giving rise to expression of either Cyt-1 (of isoforms BC1 C3f and the cleavage of C4b (b) to C4c and C4d occurs. The cleavage products C3f and C1) or Cyt-2 (of isoforms BC2 and C2). Cyt-1 is a 16 amino acid peptide and has and C4c are released into the surrounding milieu and the resulting target-bound phosphorylation sites for casein kinase II and protein kinase C. Cyt-2 is a 23 amino cleavage products (C3bi and C4d) are inactive relative to further complement acid peptide and also has a casein kinase II site as well as a src kinase site. Regions activation. Once cleavage occurs, CD46 and factor I dissociate from the complex of CD46 that have been identified as important for attachment of Neisseria are and are available to continue the process on additional C3b and C4b molecules. All indicated in green in the BC1 isoform. These regions were identified by deletion four commonly expressed isoforms of CD46 exhibit cofactor activity. mutagenesis of the BC1 isoform. www.sciencedirect.com Review TRENDS in Molecular Medicine Vol.10 No.9 September 2004 461

(a) CD46Ðpilus interaction (b) Calcium flux (c)Tyrosine (d) Aggregation (e) Release of CD46 phosphorylation of CD46 of CD46

CD46

?

Pilus

-Y--P Ca2+ flux

c-yes Y-PO4 TRENDS in Molecular Medicine

Figure 4. CD46-dependent host cell responses to Neisseria gonorrhoeae adherence. (a) Neisseria (black) adheres to the host cell through an interaction between pilus- associated adhesins and CD46 [1]. Other surface molecules might be involved in this process. (b) Purified pili of Neisseria induce an adhesion-promoting transient release of calcium from intracellular stores [45]. (c) Neisseria attachment leads to tyrosine phosphorylation of CD46 and clustering of the src kinase c-yes at the site of microcolony attachment [46]. Both (b) and (c) promote bacterial adhesion and probably facilitate downstream signaling responses in the host. (d) CD46 clusters at the site of microcolony adhesion in a manner similar to other components of the cortical actin plaque described by Merz et al. [48,49,†]. (e) Following incubation with Neisseria, CD46 is lost from the cell surface and accumulates in the supernatant [37,†]. The mechanism by which CD46 is released is unknown. overlay assay, labeled outer membrane preparations of PilC1 gene (which results in piliated meningococci that do piliated N. gonorrhoeae bound to a protein from ME-180 not adhere to host cells) fail to adhere to CD46 in the human cervical epithelial-cell lysates with an electro- overlay assay that was used for the initial identification of phoretic mobility that was consistent with CD46. Second, the CD46–pilus interaction [1]. the preincubation of cultured epithelial cells with mono- CD46 is an attractive candidate as a pilus receptor clonal antibodies (mAbs) against CD46 or with the because its expression has been demonstrated in tissues purified recombinant CD46 ectodomain inhibited gono- that are relevant to Neisseria infection, including female coccal adherence. Third, piliated N. gonorrhoeae and genital tissues [36], as well as neurons and blood vessel N. meningitidis adhered to Chinese hamster ovary endothelial cells of the brain [9,27]. Additionally, an (CHO) cells that were transfected with human CD46, increased expression of CD46 has been described in but not to untransfected cells. These adherence studies oviductal tissue explants of one group of women with also demonstrated that the expression of the BC1 and BC2 increased susceptibility to N. gonorrhoeae due to use of the isoforms of CD46 in CHO cells conferred higher adherence subdermal contraceptive device Norplant [28]. Although than did the C1 or C2 isoforms [1]. CD46 expression is thought to be predominantly baso- In a subsequent study, COS-7 cells that expressed lateral in epithelial cells [29,30], immunostaining data deletion constructs of the BC1 isoform of CD46 were used presented in several reports indicate that CD46 expres- to assess which domains fo CD46 are required for gono- sion is not limited to the basolateral surface [26,28,31], coccal adhesion (Figure 3) [24]. Reduced gonococcal adhe- suggesting that CD46 might be present on the apical sur- sion was observed in cells expressing constructs lacking face of tissues that are targeted by the pathogenic Neisseria. complement control protein repeat (CCP)-3, the serine– threonine–proline rich (STP) region and the cytoplasmic Controversies surrounding CD46–Neisseria interactions tail. A puzzling observation was that, although the dele- However, the identity of CD46 as a cellular pilus receptor tion of CCP-4 had no effect, gonococcal adhesion was almost is associated with some controversy. Two independent eliminated by loss of the N-glycosylation site in this domain. groups assessed gonococcal adhesion relative to CD46 CD46 interacts with the Neisseria pilus components expression in several human cell lines [24,32].Both PilE (the major pilus subunit) and PilC (a pilus-associated studies demonstrated that the highest levels of bacterial protein that influences pilus fiber assembly) (Figure 1). adherence did not correspond to highest levels of CD46 Both of these proteins have been identified as pilus- expression, as might be expected if CD46 were acting associated adhesins (reviewed in [2]). A truncated, solely as a ‘classical’ pilus receptor. Variations in the secreted form of PilE known as soluble pilin (S-pilin) of isoform distribution pattern among these cell lines did not N. gonorrhoeae adheres to a protein with an SDS–PAGE account for these results. In considering these studies, the electrophoretic pattern similar to that of CD46 in an limitations of cancer cell models must be considered. It is overlay assay and can inhibit the adhesion of gonococci known that cancer cells and cultured cancer cell lines to ME-180 cells [25]. A direct interaction has also been exhibit altered protein expression, including increased reported between CD46 and purified PilC from amounts of CD46 compared with the non-malignant N. meningitidis (unpublished data*). Supporting the tissues of the same origin [33–35]. It is, therefore, of evidence for a CD46–PilC interaction, outer membrane concern that in a primary human cervical cell model [36], preparations from meningococci with mutations in the as well as a model using human epithelial cell lines [37],

* Albiger, B and Jonsson, A-B. (2000) 12th International Pathogenic Neisseria mAbs against CD46 failed to inhibit the adherence of Conference, Galveston, TX, USA (Abstract 156) N. gonorrhoeae. www.sciencedirect.com 462 Review TRENDS in Molecular Medicine Vol.10 No.9 September 2004

An additional observation that complicates the inter- was only observed if the mice were pretreated with pretation of CD46 as a cellular pilus receptor involves the antibiotics to reduce the natural flora. The low mortality agglutination of human erythrocytes (which lack CD46 rate that was observed via the natural (intranasal) expression [38]) by piliated gonococci [39]. However, this inoculation route and the unexpected virulence exhibited might be explained by the studies of Rudel et al. [40] by non-piliated meningococci following intraperitoneal and Scheuerpflug et al. [41], which indicate that pilus- inoculation emphasize the complexity of the role of CD46 mediated adhesion of N. gonorrhoeae and N. meningitidis in Neisseria disease progression. Nevertheless, these to epithelial cells is mediated by different adhesins to results illustrate an important role for CD46 in the those that are involved in hemagglutination. These development of lethal meningococcal disease, particularly studies argue that CD46-dependent adhesion to epithelial in traversing the blood–brain barrier. Future experiments cells is facilitated by an interaction with PilC, whereas using this model to assess the role of pilus components of agglutination of the CD46-negative red blood cells occurs N. meningitidis, such as PilC and PilT, should further through interactions that are facilitated by PilE. define the contribution of these proteins to meningococcal Interestingly, controversy also exists regarding the disease progression. use of CD46 as a receptor for the measles virus (MV) hemagglutinin. Many reports have established that CD46 as a mediator of Neisseria-induced cell signaling vaccine strains of MV bind to CD46 with high affinity The signaling capability of CD46 in microbial pathogene- whereas wild-type strains preferentially use an alterna- sis has been characterized for MV infection, in which tive receptor known as SLAM (CDw150) [42]. Studies of CD46-dependent responses contribute both to infectivity the MV–CD46 interaction have defined a site on CD46 and to the host response. CD46 might be similarly exploited that is capable of low affinity and constitutive interaction by Neisseria, especially in processes that are related to with both vaccine and wild-type strains of MV. This is adherence. One study demonstrated a CD46-dependent distinct from the high-affinity site that is used almost calcium flux that promoted adherence of gonococci to exclusively by vaccine strains [43].CD46usageby human cells (Figure 4b) [45]. This rapid transient release Neisseria might involve a similar scenario, in which of calcium from intracellular stores was observed after a CD46 contributes to adhesion through low-affinity inter- w10 min incubation of ME-180 cells with outer membrane actions that require the coordinate engagement of a preparations of piliated N. gonorrhoeae or N. meningitidis. second (unidentified) receptor. Additionally, the pre-incubation of cells with an adhesion- blocking mAb directed against CD46 [1] inhibited the CD46 transgenic mouse model of Neisseria infection N. gonorrhoeae-induced calcium flux [45]. The pharmaco- Recent publications indicate a greater involvement of logical depletion of intracellular calcium stores before the CD46 in the Neisseria–host encounter than a static addition of bacteria resulted in decreased gonococcal interaction between CD46 and the pilus. Using transgenic adhesion. Accordingly, a direct correlation was observed mice that expressed human CD46, a recent study high- between the free cytosolic Ca2C concentration and gono- lighted the importance of CD46 in N. meningitidis coccal adhesion. Interestingly, the selective inhibition of infection. These mice, which exhibit a human-like tissue casein kinase II (CKII, for which there is a site on both distribution of CD46 [44], display enhanced susceptibility cytoplasmic tails of CD46) decreased gonococcal adhesion to meningococcal disease following experimental inocu- but did not inhibit the calcium flux, suggesting that CKII lation via either the intraperitoneal or intranasal route activation contributes to N. gonorrhoeae adhesion but [30]. Following intraperitoneal infection, there was an is downstream of the pilus-induced calcium flux [45].In increase in bacteraemia and mortality of the mice expres- further support for a role of the cytoplasmic domains of sing CD46. Furthermore, bacteria were present in the CD46 in bacterial attachment, COS-7 cells that were trans- cerebrospinal fluid, meninges and choriod plexus, suggest- fected with CD46 deletion mutants lacking portions of the ing that CD46 facilitated the passage of the meningococci cytoplasmic tail had decreased bacterial adhesion [24]. across the blood–brain barrier. The role of the pilus was A later study by Lee et al. [46] indicated that the evaluated by infecting the transgenic mice with isogenic tyrosine phosphorylation of the cytoplasmic tail of CD46 strains of N. meningitidis, one expressing pili and one by the src kinase c-yes promotes bacterial adhesion lacking pili. Following intraperitoneal inoculation, non- (Figure 4c). Tyrosine 354 on Cyt-2 was phosphorylated piliated bacteria were more virulent than the piliated within five minutes following infection with gonococci. strain, as evidenced by an increased mortality rate: a Treatment with an inhibitor of src family kinases reduced surprise given the purported role of CD46 as a pilus both tyrosine phosphorylation and gonococcal adhesion. receptor. Intranasal inoculation, which more closely In addition, c-yes was observed to aggregate at the site of mimics the natural route of infection, resulted in no gonococcal microcolony attachment, co-immunoprecipi- mortality among non-transgenic animals and only 15% tate with CD46, and, following purification, phosphorylate lethality among CD46-transgenic mice that were infected CD46 in vitro [46]. with the piliated strain. Furthermore, in contrast to the In addition to inducing CD46-dependent signaling increased virulence that was observed following intra- pathways, the adherence of N. gonorrhoeae to human peritoneal inoculation, non-piliated meningococci were epithelial cells alters the expression and cell-surface avirulent following an intranasal challenge of the trans- distribution of CD46 (Figure 4d). The adherence of genic animals. Notably, the pilus-dependent lethality in N. gonorrhoeae to ME-180 cervical epithelial cells causes the CD46-transgenic mice following intranasal inoculation up to an 80% loss of CD46 from the cell; this is www.sciencedirect.com Review TRENDS in Molecular Medicine Vol.10 No.9 September 2004 463 accompanied by the accumulation of apparently intact CD46 as protection against complement CD46 in the cell culture media [37]. N. gonorrhoeae The is crucial to the control of mutants exhibiting reduced attachment because of the Neisseria infections, as evidenced by the increased deletion of the adhesion-promoting pilus constituents susceptibility to Neisserial infections of individuals PilE, PilV and PilC were unable to induce CD46 down- with complement deficiencies [50]. Neisseria is also regulation, suggesting that bacterial attachment is a known to usurp the protective functions of other human requisite for this response. Interestingly, in this study, complement regulators, such as and C4 binding the preincubation of host cells with mAbs against CD46 protein [51]. It is therefore tempting to speculate that (including some that have been shown to inhibit gono- Neisseria benefit from the complement regulatory activity coccal adhesion [1]) inhibited neither CD46 downregu- of CD46. However, complement deposition on bacterial lation nor the adhesion of gonococci [37]. However, surfaces probably occurs in the extracellular milieu, N. gonorrhoeae mutants lacking the expression of the before interaction with the host cell. The cofactor activity pilus retraction protein PilT are hyperadherent, yet of CD46 is intrinsic: it is primarily active on C3b and C4b incapable of inducing CD46 downregulation [37]. The that are deposited to the same surface on which CD46 is induction of PilT expression in this mutant restored CD46 expressed. Therefore, (C3b and C4b) on the downregulation, confirming that the downregulation of bacterialsurfacemightbeinaccessibletoCD46.In CD46 requires PilT. addition, the N. gonorrhoeae-induced loss of CD46 from CD46 downregulation is also influenced by alterations the host cell probably renders the infected cell more in pilE expression that arise from the passage of susceptible to complement attack, as is observed following N. gonorrhoeae through the human host. Following experi- CD46 downregulation in MV-infected cells [52,53]. Alter- mental intraurethral inoculation of human male volunteers natively, the N. gonorrhoeae-induced aggregation of CD46 with N. gonorrhoeae, gonococci were re-isolated from the at the site of adhesion and release of CD46 might be a urine and pilE (pilin) sequences characterized. Five vari- protective scheme orchestrated by the pathogen to concen- ants were chosen that displayed pilin sequences repre- trate CD46 in close proximity to adherent N. gonorrhoeae sentative of those that arise with high frequency in vivo and thereby inhibit complement activation at the site. and become predominant [47]. The inoculating variant did not exhibit CD46 downregulation, yet three of the five variants re-isolated from the volunteers did induce this Concluding remarks response. Additionally, of the two non-downregulating The importance of CD46 in Neisseria infection has been variants, one was isolated from the urine early in infection demonstrated in vivo through the analysis of CD46 trans- (days 1 and 2) but not later (days 3 and 4), suggesting a genic mice and in vitro by experimentation with primary explants and cultured human cell lines. However, the role reduced level of in vivo fitness. The other exhibited low of CD46 in gonococcal and meningococcal pathogenesis levels of adherence to the ME-180 cells used in the study, remains incompletely defined. A direct interaction has which would account for the lack of CD46 downregulation. been described between CD46 and the Neisseria pilus but These results point to selective in vivo pressures favor- there are also studies indicating that this is not a classical ing N. gonorrhoeae that are capable of inducing CD46 receptor–ligand interaction. Therefore, it is likely that downregulation. other receptors are also involved. CD46 has been related In addition to its downregulation, the cell-surface to several N. gonorrhoeae-induced cellular responses, such distribution of CD46 changes following the attachment as alterations in the cell-surface distribution and cellular of N. gonorrhoeae to host cells. The aggregation of CD46 at levels of CD46, a calcium flux and CD46 tyrosine phos- the site of N. gonorrhoeae microcolony adhesion following phorylation. These CD46-dependent signaling responses a 4–6 h incubation with gonococci has been observed in result in changes in the microenvironment of the host– polarized ectocervical and endocervical cell monolayers pathogen interface that promote strong adhesion and [48] and in cultured cervical epithelial cells (unpublished possibly protect the bacteria from complement activation. data†). This observation might be related to the enrich- The encounter between Neisseria and human cells repre- ment of cytoskeleton and integral membrane proteins in sents a fascinating and complex interplay between bac- the form of a cortical actin plaque that occurs at the site of terial and host factors. A more precise understanding of gonococcal attachment [49]. Interestingly, CD46 down- these events will provide valuable insights into the regulation, cortical actin plaque formation and aggrega- pathogenesis of N. gonorrhoeae and N. meningitidis and tion at the site of gonococcal adherence are all dependent the biology of CD46. Such understanding might reveal upon the expression of PilT. These processes might, there- novel treatment and prevention strategies that interfere fore, be interrelated and dependent upon the expression of either with the CD46–pilus interaction or with CD46- retractile pili. It has been suggested that tensile forces dependent host cell responses to bacterial adhesion that exerted on the cell surface by the retraction of the pili of promote infectivity. Because of the involvement of CD46 in adherent gonococci activate mechanosensory pathways in the pathogenesis of an ever-growing and diverse list of the host cell [2]. human pathogens, the insight gained through further study of the role of CD46 in Neisseria infection will

† Gill, D.B. et al. (2004) Piliated Neisseria gonorrhoeae induce shedding and increase our understanding of the pathogenesis of surface redistribution of CD46. Molecular Immunology 41, 236 multiple infectious diseases. www.sciencedirect.com 464 Review TRENDS in Molecular Medicine Vol.10 No.9 September 2004

References of Neisseria gonorrhoeae and changes in the expression of epithelial 1 Kallstrom, H. et al. (1997) Membrane cofactor protein (MCP or CD46) gonococcal receptors in the Fallopian tube of copper T and Norplant is a cellular pilus receptor for pathogenic Neisseria. Mol. Microbiol. 25, users. Hum. Reprod. 16, 463–468 639–647 28 Maisner, A. et al. (1996) Two different cytoplasmic tails direct isoforms 2 Merz, A.J. and So, M. (2000) Interactions of pathogenic Neisseriae of the membrane cofactor protein (MCP; CD46) to the basolateral with epithelial cell membranes. Annu. Rev. Cell Dev. Biol. 16, 423–457 surface of Madin–Darby canine kidney (MDCK) cells. J. Biol. Chem. 3 Plant, L. and Jonsson, A-B. (2003) Contacting the host: insights and 271, 18853–18858 implications of pathogenic Neisseria cell interactions. Scand. J. Infect. 29 Teuchert, M. et al. (1999) Importance of the carboxyl-terminal FTSL Dis. 35, 608–613 motif of membrane cofactor protein for basolateral sorting and 4 Naumann, M. et al. (1999) Host cell interactions and signalling with endocytosis. Positive and negative modulation by signals inside and Neisseria gonorrhoeae. Curr. Opin. Microbiol. 2, 62–70 outside the cytoplasmic tail. J. Biol. Chem. 274, 19979–19984 5 Gray-Owen, S.D. (2003) Neisserial Opa proteins: impact on coloniz- 30 Johansson, L. et al. (2003) CD46 in meningococcal disease. Science ation, dissemination and immunity. Scand. J. Infect. Dis. 35, 614–618 301, 373–375 6 Hauck, C.R. and Meyer, T.F. (2003) ‘Small’ talk: Opa proteins as 31 Oglesby, T.J. et al. (1996) Human complement regulator expression by mediators of Neisseria–host-cell communication. Curr. Opin. Microbiol. the normal female reproductive tract. Anat. Rec. 246, 78–86 6, 43–49 32 Tobiason, D.M. and Seifert, H.S. (2001) Inverse relationship between 7 Massari, P. et al. (2003) The role of porins in neisserial pathogenesis pilus-mediated gonococcal adherence and surface expression of the and immunity. Trends Microbiol. 11, 87–93 pilus receptor, CD46. Microbiology 147, 2333–2340 8 Hourcade, D. et al. (2000) Functional domains, structural variations 33 Murray, K.P. et al. (2000) Expression of complement regulatory and pathogen interactions of MCP, DAF and CR1. Immunopharma- proteins – CD 35, CD 46, CD 55, and CD 59 – in benign and malignant cology 49, 103–116 endometrial tissue. Gynecol. Oncol. 76, 176–182 9 Johnstone, R.W. et al. (1993) Identification and quantification of 34 Seya, T. et al. (1990) Quantitative analysis of membrane cofactor complement regulator CD46 on normal human tissues. Immunology protein (MCP) of complement. J. Immunol. 145, 238–245 35 Hara, T. et al. (1995) High expression of membrane cofactor protein 79, 341–347 of complement (CD46) in human leukaemia cell lines: implication of 10 Ballard, L. et al. (1987) A polymorphism of the complement regulatory an alternatively spliced form containing the STA domain in CD46 protein MCP (membrane cofactor protein or gp 45–70). J. Immunol. up-regulation. Scand. J. Immunol. 42, 581–590 138, 3850–3855 36 Edwards, J.L. et al. (2002) A co-operative interaction between 11 Bora, N.S. et al. (1991) Membrane cofactor protein of the complement Neisseria gonorrhoeae and complement receptor 3 mediates infection system: A Hind III restriction fragment length polymorphism that of primary cervical epithelial cells. Cell. Microbiol. 4, 571–584 correlates with the expression polymorphism. J. Immunol. 146, 37 Gill, D.B. et al. (2003) Down-regulation of CD46 by piliated Neisseria 2821–2825 gonorrhoeae. J. Exp. Med. 198, 1313–1322 12 Richards, A. et al. (2003) Mutations in human complement regulator, 38 McNearney, T.M. et al. (1989) Membrane cofactor protein of membrane cofactor protein (CD46), predispose to development of complement is present on human fibroblast, epithelial and endothelial familial hemolytic uremic syndrome. Proc. Natl. Acad. Sci. U. S. A. cells. J. Clin. Invest. 84, 538–545 100, 12966–12971 39 Koransky, J. (1975) Bacterial hemagglutination by Neisseria 13 Noris, M. et al. (2003) Familial haemolytic uraemic syndrome and an gonorrhoeae. Infect. Immun. 12, 495–498 MCP mutation. Lancet 362, 1542–1547 40 Rudel, T. et al. (1992) Interaction of two variable proteins (PilE and 14 Goodship, T.H.J. et al. (2004) Mutations in CD46, a complement PilC) required for pilus-mediated adherence of Neisseria gonorrhoeae regulatory protein, predispose to atypical HUS. Trends Mol. Med. 10, to human epithelial cells. Mol. Microbiol. 6, 3439–3450 226–231 41 Scheuerpflug, I. et al. (1999) Roles of PilC and PilE proteins in 15 Riley, R.C. et al. (2002) Characterization of human membrane cofactor pilus-mediated adherence of Neisseria gonorrhoeae and Neisseria protein (MCP; CD46) on spermatozoa. Mol. Reprod. Dev. 62, 534–546 meningitidis to human erythrocytes and endothelial and epithelial 16 Kemper, C. et al. (2003) Activation of human CD4C cells with CD3 and cells. Infect. Immun. 67, 834–843 CD46 induces a T-regulatory cell 1 phenotype. Nature 421, 388–392 42 Schneider-Schaulies, J. et al. (2003) Measles infection of the central 17 Naniche, D. et al. (1993) Measles virus haemagglutinin induces down- nervous system. J. Neurovirol. 9, 247–252 regulation of gp57/67, a molecule involved in virus binding. J. Gen. 43 Masse, N. et al. (2002) Identification of a second major site for CD46 Virol. 74, 1073–1079 binding in the hemagglutinin protein from a laboratory strain of 18 Dorig, R.E. et al. (1993) The human CD46 molecule is a receptor for measles virus (MV): potential consequences for wild-type MV infec- measles virus (Edmonston strain). Cell 75, 295–305 tion. J. Virol. 76, 13034–13038 19 Okada, N. et al. (1995) Membrane cofactor protein (CD46) is a 44 Kemper, C. et al. (2001) Membrane cofactor protein (MCP; CD46) keratinocyte receptor for the M protein of the group A streptococcus. expression in transgenic mice. Clin. Exp. Immunol. 124, 180–189 Proc. Natl. Acad. Sci. U. S. A. 92, 2489–2493 45 Kallstrom, H. et al. (1998) Cell signaling by the type IV pili of 20 Santoro, F. et al. (1999) CD46 is a cellular receptor for human pathogenic Neisseria. J. Biol. Chem. 273, 21777–21782 herpesvirus 6. Cell 99, 817–827 46 Lee, S.W. et al. (2002) CD46 is phosphorylated at tyrosine 354 upon 21 Gaggar, A. et al. (2003) CD46 is a cellular receptor for group B infection of epithelial cells by Neisseria gonorrhoeae. J. Cell Biol. 156, adenoviruses. Nat. Med. 9, 1408–1412 951–957 22 Segerman, A. et al. (2003) Adenovirus type 11 uses CD46 as a cellular 47 Hamrick, T.S. et al. (2001) Antigenic variation of gonococcal pilin receptor. J. Virol. 77, 9183–9191 expression in vivo: analysis of the strain FA1090 pilin repertoire and 23 Wu, E. et al. (2004) Membrane cofactor protein is a receptor for identification of the pilS gene copies recombining with pilE during adenoviruses associated with epidemic keratoconjunctivitis. J. Virol. experimental human infection. Microbiology 147, 839–849 78, 3897–3905 48 Edwards, J.L. et al. (2000) Neisseria gonorrhoeae elicits membrane 24 Kallstrom, H. et al. (2001) Attachment of Neisseria gonorrhoeae to the ruffling and cytoskeletal rearrangements upon infection of primary cellular pilus receptor CD46: identification of domains important for human endocervical and ectocervical cells. Infect. Immun. 68, 5354–5363 bacterial adherence. Cell. Microbiol. 3, 133–143 49 Merz, A.J. et al. (1999) Type IV pili of pathogenic Neisseriae elicit 25 Rytkonen, A. et al. (2001) Soluble pilin of Neisseria gonorrhoeae cortical plaque formation in epithelial cells. Mol. Microbiol. 32, interacts with human target cells and tissue. Infect. Immun. 69, 1316–1332 6419–6426 50 Vogel, U. and Frosch, M. (1999) Mechanisms of neisserial serum 26 Johnstone, R.W. et al. (1993) Polymorphic expression of CD46 protein resistance. Mol. Microbiol. 32, 1133–1139 isoforms due to tissue-specific RNA splicing. Mol. Immunol. 30, 51 Ram, S. et al. (1999) The contrasting mechanisms of serum resistance 1231–1241 of Neisseria gonorrhoeae and group B Neisseria meningitidis. Mol. 27 Fernandez, R. et al. (2001) Increased adhesiveness and internalization Immunol. 36, 915–928 www.sciencedirect.com Review TRENDS in Molecular Medicine Vol.10 No.9 September 2004 465

52 Schnorr, J.J. et al. (1995) Measles virus-induced down-regulation of 55 Rudel, T. et al. (1995) Neisseria PilC protein identified as type-4 pilus CD46 is associated with enhanced sensitivity to complement- tip-located adhesin. Nature 373, 357–359 mediated lysis of infected cells. Eur. J. Immunol. 25, 976–984 56 Rahman, M. et al. (1997) PilC of pathogenic Neisseria is associated 53 Tishon, A. et al. (1996) A model of measles virus-induced immuno- with the bacterial cell surface. Mol. Microbiol. 25, 11–25 suppression: enhanced susceptibility of neonatal human PBLs. Nat. 57 Winther-Larsen, H.C. et al. (2001) Neisseria gonorrhoeae PilV, Med. 2, 1250–1254 a type IV pilus-associated protein essential to human epithelial cell 54 Park, H.S. et al. (2002) Modification of type IV pilus-associated adherence. Proc. Natl. Acad. Sci. U. S. A. 98, 15276–15281 epithelial cell adherence and multicellular behavior by the PilU 58 Brossay, L. et al. (1994) Identification, localization, and distribution of protein of Neisseria gonorrhoeae. Infect. Immun. 70, 3891–3903 the PilT protein in Neisseria gonorrhoeae. Infect. Immun. 62, 2302–2308

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