Influence of Mannan and Glucan on Complement Activation and C3 Binding by Candida albicans

The complement system is important for host resistance to hematogenously disseminated candidiasis. However, modulation of complement activation by cell wall components of Candida albicans has not been characterized. Although intact yeast display mannan on the surface, glucan, typically located in the interior, becomes exposed during C. albicans infection. We show here the distinct effects of mannan and glucan on complement activation and opsonophagocytosis. Previous studies showed that intact cells are resistant to initiation of complement activation through the alternative pathway, and antimannan antibody reverses this resistance via an Fc-independent mechanism. The present study shows that this mannan-dependent resistance can be overcome by periodate-borohydride conversion of mannose polysaccharides to polyalcohols; cells treated with periodate-borohydride initiate the alternative pathway without the need for antibody. These observations identify an inhibitory role for intact mannan in complement activation. Next, removal of the surface-displayed mannan by acid treatment of periodate-borohydride cells exposes glucan. Glucan-displaying cells or purified β-glucan initiate the alternative pathway when incubated with the purified proteins of the alternative pathway alone, suggesting that C. albicans glucan is a natural activator of the alternative pathway. Finally, ingestion of mannan-displaying cells by human neutrophils requires anti-mannan antibody, whereas ingestion of glucan-displaying cells requires complement. These results demonstrate a contrasting requirement of natural antibody and complement for opsonophagocytosis of C. albicans cells displaying mannan or glucan. Thus, differential surface expression of mannan and glucan may influence recognition of C. albicans by the complement system.Mannan is predominant (39) on the surface of intact Candida albicans cells and masks β-glucan and chitin in the interior (7). However, recent studies found that glucan may become exposed during C. albicans infection (45) or by treatment with caspofungin (44, 45). The phenomenon of glucan unmasking during infection was initially suggested by studies from the Cassone group. They found that the fraction of murine immune serum reactive with C. albicans β-glucan was protective in a mouse model of hematogenously disseminated candidiasis (6). This anti-glucan antibody-mediated protection was confirmed with both antiserum produced by a β-1,3 glucan conjugate vaccine and a monoclonal antibody (MAb) specific for β-glucan (40). Subsequently, Wheeler et al. (45) demonstrated expression of glucan on the surface of C. albicans cells retrieved from the kidneys of infected mice with anti-glucan antibody. They also reported exposure of glucan on C. albicans following treatment with caspofungin at subinhibitory doses both in vivo and in vitro (44, 45). These studies illustrate dynamics in the display of mannan and glucan on the cell surface. They also raise the possibility that variability in surface expression of mannan and glucan might have other biological consequences, e.g., activation of the complement system.The complement system has an essential role in host innate clearance of initial infections and influences the effector functions of induced immunity. Activation of the complement cascade leads to production of chemotactic agents for recruitment of phagocytes and to deposition of opsonic C3 fragments on the surface of microbes targeted for clearance by phagocytes. Complement activation may occur through the classical pathway, the alternative pathway, or the lectin pathway. Although initiation of the classical pathway begins with C1q recognition of the Fc region of antibody-microbe complex, initiation of the alternative pathway begins with binding of metastable fluid-phase C3b or C3(H₂O) to the microbial surface in an antibody independent manner (35). Thus, alternative pathway activation of complement represents an innate defense, independent of the induced immunity; strategies for evasion of alternative pathway-mediated initiation of complement activation are common in microbes (52). An important role for the complement system in host resistance to systemic candidiasis has been well established with experimental animals deficient in C3 (13, 42), mannan binding lectin A/C (20), or factors B and C2 (20). Furthermore, protection by a murine anti-mannan IgM antibody or its IgG3 variant requires an intact complement system in a mouse model of hematogenously disseminated candidiasis (17).Our previous studies found that intact yeast cells of serotypes A and B of C. albicans are resistant to complement activation and that anti-mannan antibody is required for initiation of both the classical and alternative pathways (3, 26, 50, 51). The intrinsic resistance of intact C. albicans yeast cells to alternative pathway activation was demonstrated in a serum-free assay that consisted of the six alternative pathway proteins (3, 50). Further studies revealed that anti-mannan antibody facilitates alternative pathway activation in an Fc-independent manner (3).The role of Candida glucan in complement activation has not been studied. Glucan of nonencapsulated Cryptococcus neoformans (46) or Blastomyces dermatitidis (48, 49) is cell surface displayed and contributes to initiation of complement activation. Therefore, C. albicans glucan that is exposed during infection may influence the outcome of the interaction of Candida with the human complement system.The objective of the present study was to evaluate the relative contributions of mannan and glucan to complement activation and C3 binding by C. albicans. The results show that mannan contributes to the resistance of C. albicans to complement activation and that glucan is a natural activator of the complement system. Thus, masking glucan by mannan may represent a strategy for evasion of complement recognition.     

Secreted Aspergillus fumigatus Protease Alp1 Degrades Human Complement Proteins C3, C4, and C5

The opportunistic human pathogenic fungus Aspergillus fumigatus is a major cause of fungal infections in immunocompromised patients. Innate immunity plays an important role in the defense against infections. The complement system represents an essential part of the innate immune system. This cascade system is activated on the surface of A. fumigatus conidia and hyphae and enhances phagocytosis of conidia. A. fumigatus conidia but not hyphae bind to their surface host complement regulators factor H, FHL-1, and CFHR1, which control complement activation. Here, we show that A. fumigatus hyphae possess an additional endogenous activity to control complement activation. A. fumigatus culture supernatant efficiently cleaved complement components C3, C4, C5, and C1q as well as immunoglobulin G. Secretome analysis and protease inhibitor studies identified the secreted alkaline protease Alp1, which is present in large amounts in the culture supernatant, as the central molecule responsible for this cleavage. An alp1 deletion strain was generated, and the culture supernatant possessed minimal complement-degrading activity. Moreover, protein extract derived from an Escherichia coli strain overproducing Alp1 cleaved C3b, C4b, and C5. Thus, the protease Alp1 is responsible for the observed cleavage and degrades a broad range of different substrates. In summary, we identified a novel mechanism in A. fumigatus that contributes to evasion from the host complement attack.Aspergillus fumigatus is the most important airborne fungal pathogen. The frequency of invasive mycoses due to this opportunistic fungal pathogen has increased significantly during the last 2 decades (reviewed in references 7 and 42). In healthy individuals, A. fumigatus conidia are inhaled but the establishment of disease is prevented by the host immune system. Inhaled A. fumigatus conidia are immediately confronted with the host complement system and phagocytic cells. The complement system is activated on the conidial and hyphal surface (26), and this activation results in the cleavage of C3. Cleavage products of this central component of the complement cascade act as opsonins on the surfaces of pathogens and enhance phagocytosis by neutrophils, macrophages, and eosinophils (69). Opsonization with complement proteins is important for phagocytosis of A. fumigatus conidia, the key process in the defense against this pathogen (59).Activation of the complement system occurs via three pathways: the alternative pathway (AP), the lectin pathway, and the classical pathway. The AP is activated on microbial surfaces and plays a pivotal role in the clearance of microorganisms (70). Progression of the cascade leads to generation of a C5 convertase, which produces inflammatory C5a anaphylatoxins, and also to the formation of terminal complement complexes (TCC), which can form membrane attack complexes (MAC) and hence pores on target surfaces. C3b surface deposition and MAC formation are important for clearance of bacteria but appear to play a minor role in the defense against fungi. The complement activation system is controlled by fluid-phase and cell surface-bound regulators. We and others showed before (2, 62) that A. fumigatus conidia bind factor H (the central human regulator of the AP), FHL-1, and CFHR1. Factor H acts as a cofactor for the plasma serine protease factor I, which mediates the cleavage of C3b (21, 35, 41). This blocks C3 convertase formation and leads to downregulation or termination of the complement cascade. In contrast to conidia, A. fumigatus hyphae do not bind factor H (2). Instead, they activate complement on their surface (26). Until now a single, nonprotein activity in culture supernatant that inhibits opsonization of the fungal surface by complement proteins has been described (65). The nature of this molecule has not been discovered yet. A. fumigatus inhibition of complement activation is important, since activation of the complement cascade causes toxic and damaging effector functions. Although hyphae are too big to become phagocytosed by macrophages, attraction and activation of neutrophils by C3a and C5a still lead to the destruction of hyphae. Therefore, here we analyzed whether A. fumigatus hyphae use additional strategies to interfere with the human complement system.     

Complement C3 Plays an Essential Role in the Control of Opportunistic Fungal Infections

The innate recognition of fungal pathogens is a crucial first step in the induction of protective antifungal immunity. Complement is thought to be one key component in this process, facilitating fungal recognition and inducing early inflammation. However, the roles of the individual complement components have not been examined extensively. Here we have used mice lacking C3 to examine its role in immunity to opportunistic fungal pathogens and show that this complement component is essential for resistance to infections with Candida albicans and Candida glabrata. We demonstrate that the absence of C3 impairs fungal clearance but does not affect inflammatory responses. We also show that the presence of C3 contributes to mortality in mice challenged with very high doses of Saccharomyces cerevisiae, although these effects were found to be mouse strain dependent.Over the last few decades, modern medical practice and acquired immunodeficiencies have contributed to a substantial increase in the incidence of infections with normally commensal or nonpathogenic fungi (22), prompting a renewed interest in expanding our understanding of the mechanisms underlying protective host immunity. Identification and characterization of the receptors involved in the innate recognition of these organisms are of particular importance, as these “pattern recognition receptors” (14) not only are responsible for mediating the recognition and uptake of fungi by phagocytes but also initiate and direct the resultant immune response (17). While numerous nonopsonic pattern recognition receptors for fungi have been well characterized, including several members of the C-type lectin and Toll-like receptor families (for recent reviews, see references 20, 21, and 32), less is known about the opsonic mechanisms of fungal recognition, such as those mediated by complement, despite the essential role of these systems in antifungal immunity (24).The complement system consists of more than 30 serum and cellular surface proteins and is activated through three main routes: the classical, alternative, and lectin pathways. Triggering of these three pathways initiates enzymatic cascades which converge on the third complement component, C3, whose activation leads ultimately to microbial opsonization, the release of chemotactic factors, including C3a and C5a, and the generation of a membrane attack complex (MAC) (18). Activation of the classical pathway occurs primarily following the binding of C1q to antibody-antigen complexes, and although involved in the adaptive arm of the immune system, this pathway is also triggered by natural antibodies, thereby contributing to the innate recognition of fungi (16). Initiation of the alternative pathway occurs through the spontaneous activation of C3 on microbial surfaces, and the lectin pathway is initiated by the binding of the mannose-binding lectin to carbohydrates on microbial surfaces (24).All three of these pathways are induced by opportunistic fungal pathogens, including Candida albicans (13, 15, 24), although the alternative pathway may be the most critical (7, 15). The protective role of complement in immunity to these pathogens has largely been determined in mice treated with cobra venom factor (CVF), which depletes serum complement by forming a potent C3 convertase, or in C5-deficient mouse strains, such as A/J or DBA2, where fungal opsonization remains intact but the animals lack the ability to generate C5a or the MAC (6, 7, 9, 10, 19). Why C5-deficient mice are susceptible is still unclear, but is likely to be due to aberrant inflammatory responses, as the MAC has little effect on the viability of fungal pathogens (15, 19). Although the roles of each of the pathways of complement activation have been relatively poorly examined, recent studies have started to address this issue, looking at the role of factor B, C2, C1q and mannose-binding lectin in the control of systemic candidiasis (11).C3 is a central component in all of the complement pathways, as described above, but it has not been studied in isolation with respect to fungal infection. Studying the role of this complement component in isolation is particularly relevant, given the alternative route of C5 activation by thrombin, which can substitute for C3-dependent C5 convertase activity (12, 30). Therefore, we undertook to specifically examine the role of C3 in the control of opportunistic fungal infections using C3-sufficient and C3-deficient mice.     

Loa loa Microfilariae Evade Complement Attack In Vivo by Acquiring Regulatory Proteins from Host Plasma

Loa loa is a filarial nematode that infects humans. The adults live in subcutaneous tissues and produce microfilariae that live for several weeks in the blood circulation in order to be transmitted to another person via blood meals of a dipterian vector. As microfilariae live in continuous contact with plasma, it is obvious that they evade the complement system. We studied markers of complement activation and signs of complement regulation on Loa loa microfilariae in vivo. The microfilariae were isolated from anticoagulated blood samples of a Loa loa-infected Caucasian patient. C1q and some mannose-binding lectin but only a limited amount of C3b or C4b fragments and practically no C5 or C5b-9 were present on the microfilariae. The covalently microfilaria-bound C3 and C4 depositions were mainly inactive iC3b, C3c, and iC4b fragments indicating that microfilariae had regulated complement activation in vivo. Also, in vitro deposition of C3b onto the microfilariae upon serum exposure was limited. The patient-isolated microfilariae were found to carry the host complement regulators factor H and C4b-binding protein on the outermost layer, so called sheath. The microfilaria-bound factor H was functionally active. Binding of the complement regulators to the microfilariae was confirmed in vitro using ¹²⁵I-labeled factor H and C4b-binding protein. In conclusion, our study shows that Loa loa microfilariae block complement activation and acquire the host complement regulators factor H and C4b-binding protein in blood circulation. This is the first time that binding of complement regulators onto nonviral pathogens has been demonstrated to occur in humans in vivo.Loa loa is a filarial parasite and the causative agent of human loiasis. This nematode has adapted well to its human host, as the adults can migrate in subcutaneous tissues for at least 15 years (15). During its whole adult life this helminth can produce microfilariae (MF) in peripheral blood, and these can circulate in blood for several weeks before transmission to the vector. Loiasis is transmitted by a dipteran vector (Chrysops spp.). The infective larvae can enter human subcutaneous tissues through a bite wound when the vector feeds again after the first blood meal. The larvae develop into adults and mate in subcutaneous tissues, resulting in production of sheathed MF after a minimum of 5 months. The MF are found mainly in peripheral blood (34).The prevalence of loiasis is high in the regions of endemicity in western and central Africa, where 20 to 40% of the population is microfilaremic (10). The majority of the infected individuals are asymptomatic, but a significant proportion of patients have symptoms such as calabar swellings, pruritis, secondary dermal lesions, and a subconjunctival eye passage of the adult worm (33, 37). In addition, serious sequelae such as endomyocardial fibrosis, renal complications, and encephalitis have also been reported (1).The main functions of the complement system are to eliminate foreign organisms that come in contact with plasma or other body fluids, either by direct effects or by enhancement of the acquired humoral immune response. Depending on the activator, complement can be activated through three pathways: the classical pathway (CP), the alternative pathway (AP), and the lectin pathway (LP). Upon activation, these pathways lead to the terminal pathway and formation of membrane attack complexes on the target cell (31).Complement activation is regulated by a variety of complement regulatory proteins. Most of the regulators are membrane bound, while two major regulators, complement factor H (CFH) and C4b-binding protein (C4BP), are plasma proteins found in high concentrations (12, 17, 47). All the three pathways lead to activation of C3, the central molecule of the complement cascade (8). Activation of C3 to C3b results in release of an anaphylatoxin, C3a (20, 21), while the C3b fragment can attach covalently to the target surface to start the AP amplification or to promote activation of the CP or LP (32). Inactivation of C3b to inactive C3b (iC3b) is carried out by serine protease factor I (FI), which needs a cofactor such as CFH (6, 8). In addition to the cofactor activity, CFH can also downregulate generation of C3b by two other means (11, 46).In the CP and LP, activation of the component C4 is essential. Upon activation, C4b is attached covalently to the target surface (27, 45) and can form an active C3-convertase, C4b2a, which is essential for propagation of the CP and LP (5). This step is regulated in plasma by complement regulatory protein C4BP, which acts as a cofactor for FI in degradation of C4b to inactive C4b (iC4b) or as an accelerator of the decay of C4b2a (23, 40, 44).Since MF of Loa loa are able to live and migrate in blood for weeks, they are obviously able to resist elimination by complement, but the mechanisms are largely unknown. One immune evasion mechanism of Loa loa is known to be induction of T-cell anergy (25), but nothing is known about evasion of innate or humoral immunity. A few complement resistance mechanisms have been reported for other helminths. Most of these are associated with physical barriers of macroscopic worms, but some helminths are known to have specific molecules mediating complement evasion or ligands that acquire host complement regulators on their surfaces (22). So far, two human helminth parasite structures have been shown to acquire host CFH on their surfaces, the echinococcal cyst wall and MF of a filarial nematode, Onchocerca volvulus (7, 30). So far there are no reports of acquisition of C4BP onto any pathogenic helminth. Several pathogenic bacteria and yeasts and a few viruses are known to utilize acquisition of host CFH or C4BP to evade complement attack (49). Four of these microbes, Streptococcus pyogenes, Borrelia burgdorferi, relapsing fever Borrelia, and Candida albicans, can acquire both CFH and C4BP on their surface (3, 28, 29). So far, all the reports where CFH or C4BP acquisition on microbes has been reported have been based on in vitro experiments only.The aim of our study was to analyze whether patient-derived Loa loa MF carry any markers of complement attack or signs of cessation of the complement cascade. The MF showed C1q deposits, as was expected since the patient had antifilarial antibodies. Despite that, however, only limited amounts of C3 or C4 fragments and practically no C5 or C5b-9 could be detected on MF. The covalently bound C3 or C4 fragments were mainly iC3b, C3c, or iC4b. Most importantly we show that MF had acquired CFH and C4BP on their surfaces in vivo. In conclusion, for the first time we show acquisition of soluble complement regulators on pathogenic microbes in the human body. Our results suggest that acquisition of complement regulators CFH and C4BP from human plasma on MF could at least partially explain the prolonged survival of MF in circulation.     

Immune Evasion of Leptospira Species by Acquisition of Human Complement Regulator C4BP

Leptospirosis is a spirochetal zoonotic disease of global distribution with a high incidence in tropical regions. In the last 15 years it has been recognized as an important emerging infectious disease due to the occurrence of large outbreaks in warm-climate countries and, occasionally, in temperate regions. Pathogenic leptospires efficiently colonize target organs after penetrating the host. Their invasiveness is attributed to the ability to multiply in blood, adhere to host cells, and penetrate into tissues. Therefore, they must be able to evade the innate host defense. The main purpose of the present study was to evaluate how several Leptospira strains evade the protective function of the complement system. The serum resistance of six Leptospira strains was analyzed. We demonstrate that the pathogenic strain isolated from infected hamsters avoids serum bactericidal activity more efficiently than the culture-attenuated or the nonpathogenic Leptospira strains. Moreover, both the alternative and the classical pathways of complement seem to be responsible for the killing of leptospires. Serum-resistant and serum-intermediate strains are able to bind C4BP, whereas the serum-sensitive strain Patoc I is not. Surface-bound C4BP promotes factor I-mediated cleavage of C4b. Accordingly, we found that pathogenic strains displayed reduced deposition of the late complement components C5 to C9 upon exposure to serum. We conclude that binding of C4BP contributes to leptospiral serum resistance against host complement.Leptospirosis is a spirochetal disorder caused by pathogenic species belonging to the genus Leptospira and is regarded as the most widespread zoonotic disease due to the large spectrum of animal species that serve as reservoirs (18). The infection is transmitted by exposure to flood waters, moist soil, or vegetation contaminated with urine from animals infected with Leptospira species. Clinical manifestations range from a mild febrile illness to severe disease forms such as Weil''s syndrome. Leptospirosis-associated severe pulmonary hemorrhagic syndrome has increasingly become recognized as an important manifestation of leptospiral infection (3, 8, 19, 27, 34, 41).After penetrating the host, pathogenic leptospires efficiently colonize target organs, and their invasiveness is attributed to the ability to multiply in blood, adhere to host cells, and penetrate into tissues (22). Therefore, it is clear that these pathogens have evolved strategies to circumvent the immune defense systems of a variety of hosts. Complement is a major component of the innate immune system and is involved in protection against invading microorganisms due to its opsonic, inflammatory, and lytic activities (4). One strategy adopted by pathogens to avoid clearance and destruction by complement is to acquire host fluid-phase regulators, notably factor H and C4b-binding protein (C4BP), the soluble proteins of the alternative and classical pathways, respectively. Factor H, a 150-kDa plasma protein composed of 20 globular domains termed short consensus repeats (SCRs), inhibits the alternative pathway of complement by preventing binding of factor B to C3b, accelerating decay of the C3-convertase C3bBb and acting as a cofactor for the cleavage of C3b by factor I (28, 38, 39). C4BP, composed of seven α-chains (each with eight SCRs) and one β-chain (with three SCRs) linked together by a central core, is a 570-kDa plasma glycoprotein that displays a spiderlike shape (6, 10, 31). It inhibits the classical pathway of complement by interfering with the assembly and decay of the C3-convertase C4bC2a and acts as a cofactor for factor I in the proteolytic inactivation of C4b (7, 31). Therefore, as a consequence of the acquisition of fluid-phase regulators on the surface of a given pathogen, complement activation is downregulated, preventing opsonization and the formation of the lytic membrane attack complex on its surface.Several human pathogens use this strategy to survive, including spirochetes. In the case of Borrelia spp., the causative agent of Lyme disease and relapsing fever, the expression of outer surface lipoproteins known as complement regulatory-acquiring surface proteins (CRASP) which bind factor H and/or factor H-like protein 1 (FHL-1), is restricted to serum-resistant strains (9, 12-17, 37). It has been reported that Borrelia recurrentis and Borrelia duttonii also acquire C4BP on their surfaces, which may contribute to evasion of antibody-mediated clearance from blood circulation (21). Treponema denticola, a spirochete that contributes to the development of periodontal disease, specifically binds FHL-1 via a 14-kDa, surface-exposed protein termed FhbB (FHL-1 binding protein B). This interaction seems to facilitate adhesion, biofilm formation, and possibly tissue penetration (20). Binding of factor H and factor H-related protein 1 (FHR-1) has also been demonstrated for serum-resistant and serum-intermediate strains of Leptospira (22). Moreover, the outer membrane proteins LenA (leptospiral endostatinlike protein A), formerly called LfhA (for leptospiral factor H-binding protein A) (35) and Lsa24 (for leptospiral surface adhesin, 24 kDa) (2), and LenB (for leptospiral endostatinlike protein B) seem to contribute to serum resistance of pathogenic leptospires by interacting with factor H (33, 35).The main purpose of the present study was to evaluate complement evasion by Leptospira spp. Our data indicate that both the alternative and the classical pathways seem to contribute to leptospire killing. Both serum-resistant and serum-intermediate strains are able to bind C4BP, whereas the serum-sensitive strain Patoc I is not. Pathogen-bound C4BP retained its cofactor activity, indicating that acquisition of this complement regulator may contribute to leptospiral serum resistance.     

The Streptococcus pneumoniae Capsule Inhibits Complement Activity and Neutrophil Phagocytosis by Multiple Mechanisms

The Streptococcus pneumoniae capsule is vital for virulence and may inhibit complement activity and phagocytosis. However, there are only limited data on the mechanisms by which the capsule affects complement and the consequences for S. pneumoniae interactions with phagocytes. Using unencapsulated serotype 2 and 4 S. pneumoniae mutants, we have confirmed that the capsule has several effects on complement activity. The capsule impaired bacterial opsonization with C3b/iC3b by both the alternative and classical complement pathways and also inhibited conversion of C3b bound to the bacterial surface to iC3b. There was increased binding of the classical pathway mediators immunoglobulin G (IgG) and C-reactive protein (CRP) to unencapsulated S. pneumoniae, indicating that the capsule could inhibit classical pathway complement activity by masking antibody recognition of subcapsular antigens, as well as by inhibiting CRP binding. Cleavage of serum IgG by the enzyme IdeS reduced C3b/iC3b deposition on all of the strains, but there were still marked increases in C3b/iC3b deposition on unencapsulated TIGR4 and D39 strains compared to encapsulated strains, suggesting that the capsule inhibits both IgG-mediated and IgG-independent complement activity against S. pneumoniae. Unencapsulated strains were more susceptible to neutrophil phagocytosis after incubation in normal serum, normal serum treated with IdeS, complement-deficient serum, and complement-deficient serum treated with IdeS or in buffer alone, suggesting that the capsule inhibits phagocytosis mediated by Fcγ receptors, complement receptors, and nonopsonic receptors. Overall, these data show that the S. pneumoniae capsule affects multiple aspects of complement- and neutrophil-mediated immunity, resulting in a profound inhibition of opsonophagocytosis.The Gram-positive pathogen Streptococcus pneumoniae is one of the most common causes of pneumonia, septicemia, and meningitis in children and adults in both industrialized and developing parts of the world (10). This large burden of disease is compounded by the increased incidence of S. pneumoniae infections associated with HIV and by increasing antibiotic resistance among clinical isolates, and there is a strong need to understand the molecular pathogenesis of S. pneumoniae infections to assist the development of new therapeutic targets. Probably the most important virulence factor for S. pneumoniae is the extracellular capsule, a layer consisting of chains of monosaccharides that surrounds the bacteria. For S. pneumoniae strains, there are 91 antigenically distinct capsular serotypes, dictated by the order and type of the monosaccharide units within the polysaccharide chain and by different side branches (5, 27). The importance of the S. pneumoniae capsule for virulence is demonstrated by the facts that (i) all clinical isolates causing invasive disease are encapsulated; (ii) loss of the capsule by either genetic mutation or enzymatic degradation dramatically reduces S. pneumoniae virulence in animal models of infection (6, 28, 29, 43, 49); (iii) different capsular serotypes vary in the ability to cause invasive disease (9), and swapping capsular serotypes between strains affects virulence in animal models (21); and (iv) S. pneumoniae opaque-phase variants (which express a thicker capsule than transparent-phase variants) predominate during invasive infection (35). Furthermore, the capsule is the target for existing S. pneumoniae vaccines and widespread vaccination has led to the evolution of vaccine escape mutants expressing nonvaccine capsular serotypes, increasing the importance of a better understanding of how the capsule can affect virulence.One component of the immune system that is likely to be affected by the S. pneumoniae capsule is the complement system. Clinical and experimental evidence has shown the vital role of complement for host immunity to S. pneumoniae and that neutrophil phagocytosis of S. pneumoniae is largely dependent on complement activity (8, 15, 19, 22, 39, 52, 53). The complement system is organized into three enzyme cascades termed the classical, alternative, and mannan binding lectin (MBL) pathways (42). The classical complement pathway is activated by specific immunoglobulin G (IgG) and was generally considered an effector of the adaptive immune response, but recent data have demonstrated an important role for the classical pathway as part of the innate immune response to S. pneumoniae. S. pneumoniae cell wall phosphorylcholine (PC) is recognized by the serum proteins C-reactive protein (CRP) and serum amyloid P (SAP) (collectively termed pentraxins due to their structurally similarity) (40) and also by natural IgM (4). In addition, the cell surface lectin SIGN-R1 binds to the S. pneumoniae capsule (20). Recognition of S. pneumoniae by the pentraxins, natural IgM, and SIGN-R1 results in binding of the first component of the classical pathway, C1q, to the bacterial surface and complement activation. The MBL pathway is activated by binding of MBL to certain sugar residues found on the surface of pathogens. However, MBL binds poorly to S. pneumoniae and seems to have little effect on complement deposition on S. pneumoniae (8, 31), although MBL or other ficolins may directly opsonize microorganisms independent of complement activity. The alternative pathway is spontaneously activated unless the target cell is coated in sialic acid or complement-inhibitory proteins such as factor H (FH) (42) and is therefore a component of the innate immune response to S. pneumoniae. The alternative pathway probably also amplifies the amount of C3b/iC3b deposited on the bacterial surface once complement activation has been initiated by the classical or MBL pathway (8, 42). Each pathway leads to the formation of a C3 convertase that cleaves the central complement component C3, resulting in deposition of C3b on the surface of the pathogen that is further processed to iC3b. C3b and iC3b are opsonins mediating phagocytosis mainly through the complement receptor CR1 and CR3 receptor, respectively. As well as opsonizing bacteria, complement activation aids the inflammatory response through release of anaphylaxins such as C5a (42) and improves the adaptive immune response to S. pneumoniae through direct stimulation of B cells by the C3 breakdown product C3d (13).The external position of the capsule means it is ideally situated to modulate interactions between S. pneumoniae and host proteins and cells. Unencapsulated mutants have been shown to be more susceptible to phagocytosis, and there are limited data showing increased levels of complement deposition on their surface (1, 32, 47), but despite the importance of the capsule for S. pneumoniae virulence, there are few data on the mechanisms involved (32). Data obtained for other pathogens have shown a variety of mechanisms by which polysaccharide capsules can inhibit complement activity. The group B Streptococcus (GBS) and Neisseria meningitidis capsules contain sialic acid, which is thought to prevent alternative complement activity by creating a nonactivating surface and by binding to FH (23, 25, 46). Alternatively, the capsule may inhibit recognition of surface antigens by specific IgG, thereby preventing classical pathway activation or directly prevent binding of complement components to subcapsular targets of complement activity (37). In contrast, the capsule of Cryptococcus neoformans is a potent activator of alternative pathway activity and this is thought to aid immunity by depleting complement (50). For S. pneumoniae, whether the capsule prevents complement deposition indirectly through impairing recognition of the bacteria by IgG or has direct effects on bacterial interactions with non-IgG complement activators or other aspects of complement activity is not known. Given the importance of complement for neutrophil phagocytosis of S. pneumoniae (53), inhibition of opsonization with C3b/iC3b by the capsule could account for all of the effects of the capsule on phagocytosis. However, IgG bound to the bacterial surface and nonopsonic phagocytic molecules such as the mannose and scavenger receptors can mediate phagocytosis independently of complement, and these mechanisms of phagocytosis potentially could also be affected by the S. pneumoniae capsule. Indeed, recent data showing increased phagocytosis of an unopsonized, unencapsulated serotype 6B strain suggest that there can be a capsular effect on nonopsonic phagocytosis (44). Given the importance of the capsule for S. pneumoniae virulence and as a vaccine candidate, a more detailed understanding of the interactions of the capsule with complement and neutrophils would be beneficial.Using unencapsulated mutants from serotype 2 and 4 S. pneumoniae strains that are otherwise isogenic to the encapsulated parental strain, we have investigated the effect of the capsule on IgG-dependent and -independent complement deposition on the bacterial cell surface and on the binding of various complement mediators. We have also assessed the effects of the capsule on complement-dependent and complement-independent neutrophil phagocytosis.     

Functional Characterization of LcpA,a Surface-Exposed Protein of Leptospira spp. That Binds the Human Complement Regulator C4BP

We have previously shown that pathogenic leptospiral strains are able to bind C4b binding protein (C4BP). Surface-bound C4BP retains its cofactor activity, indicating that acquisition of this complement regulator may contribute to leptospiral serum resistance. In the present study, the abilities of seven recombinant putative leptospiral outer membrane proteins to interact with C4BP were evaluated. The protein encoded by LIC11947 interacted with this human complement regulator in a dose-dependent manner. The cofactor activity of C4BP bound to immobilized recombinant LIC11947 (rLIC11947) was confirmed by detecting factor I-mediated cleavage of C4b. rLIC11947 was therefore named LcpA (for leptospiral complement regulator-acquiring protein A). LcpA was shown to be an outer membrane protein by using immunoelectron microscopy, cell surface proteolysis, and Triton X-114 fractionation. The gene coding for LcpA is conserved among pathogenic leptospiral strains. This is the first characterization of a Leptospira surface protein that binds to the human complement regulator C4BP in a manner that allows this important regulator to control complement system activation mediated either by the classical pathway or by the lectin pathway. This newly identified protein may play a role in immune evasion by Leptospira spp. and may therefore represent a target for the development of a human vaccine against leptospirosis.Leptospirosis, an emerging global infectious disease, is caused by spirochetes belonging to different pathogenic species of the genus Leptospira. In recent decades, large outbreaks of leptospirosis have occurred in many countries, particularly in Southeast Asia and in Central and South America (56). In Brazil, more than 10,000 cases of severe leptospirosis are reported each year due to outbreaks in urban centers (46). Clinical manifestations range from a mild febrile illness to severe disease forms, such as Weil''s syndrome, characterized by jaundice, renal failure, and hemorrhage. Leptospirosis-associated severe pulmonary hemorrhagic syndrome has increasingly become recognized as an important manifestation of leptospiral infection (5, 17, 29, 39, 54, 59) and may be considered a relevant prognostic factor associated with fatal outcomes in severe leptospirosis (51).Pathogenic Leptospira spp. have the ability to disseminate and to trigger a specific immune response after penetrating the host. Like other pathogens, they have evolved strategies to evade innate immune defense systems, thereby causing severe disease. The complement system plays a crucial role in innate immunity, strongly contributing to the elimination of invading microorganisms. During evolution, pathogens have developed several strategies to control the host complement response and evade complement attack, including multiphasic antigenic variation, cleavage of complement activation products by proteases, acquisition of fluid-phase complement regulators, and production of human complement-regulatory analogs (for a review, see reference 60). Moreover, some pathogens are known to exploit multiple mechanisms to inactivate complement attack.The acquisition of fluid-phase regulators on the surface of a given pathogen normally results in the downregulation of complement activation. Binding of the negative complement regulators factor H (FH) and factor H-related protein 1 (FHR-1) has been demonstrated for serum-resistant and serum-intermediate Leptospira strains (31). Recently, we have shown that pathogenic leptospiral strains also acquire C4b binding protein (C4BP) from the host. Surface-bound C4BP retains its cofactor activity, indicating that acquisition of this complement regulator may contribute to leptospiral serum resistance (3). C4BP is a 570-kDa plasma glycoprotein that displays a spiderlike quaternary structure composed of seven identical α chains and a unique β chain linked together by a central core (11, 22, 48). The α chains each comprise eight complement control protein (CCP) domains (also termed short consensus repeats), while the β chain has three CCP domains (8). C4BP is involved in the downregulation of the classical and/or the lectin pathway of the complement system by interfering with the assembly and decay of the C3 convertase C4bC2a and acting as a cofactor for factor I in the proteolytic inactivation of C4b (15, 48).In the present study, we investigated whether putative surface proteins encoded by pathogenic Leptospira spp. could bind to C4BP. A novel 20-kDa outer membrane protein (OMP), encoded by the LIC11947 gene, was shown to interact with this complement regulator. To our knowledge, this is the first report describing a Leptospira surface protein that binds the human complement regulator C4BP.     

Critical Roles of Complement and Antibodies in Host Defense Mechanisms against Neisseria meningitidis as Revealed by Human Complement Genetic Deficiencies

Certain complement defects are associated with an increased propensity to contract Neisseria meningitidis infections. We performed detailed analyses of complement-mediated defense mechanisms against N. meningitidis 44/76 with whole blood and serum from two adult patients who were completely C2 or C5 deficient. The C5-deficient patient and the matched control were also deficient in mannose-binding lectin (MBL). The proliferation of meningococci incubated in freshly drawn whole blood was estimated by CFU and quantitative DNA real-time PCR. The serum bactericidal activity and opsonophagocytic activity by granulocytes were investigated, including heat-inactivated postvaccination sera, to examine the influence of antimeningococcal antibodies. The meningococci proliferated equally in C2- and C5-deficient blood, with a 2 log₁₀ increase of CFU and 4- to 5-log₁₀ increase in DNA copies. Proliferation was modestly decreased in reconstituted C2-deficient and control blood. After reconstitution of C5-deficient blood, all meningococci were killed, which is consistent with high antibody titers being present. The opsonophagocytic activity was strictly C2 dependent, appeared with normal serum, and increased with postvaccination serum. Serum bactericidal activity was strictly dependent on C2, C5, and high antibody titers. MBL did not influence any of the parameters observed. Complement-mediated defense against meningococci was thus dependent on the classical pathway. Some opsonophagocytic activity occurred despite low levels of antimeningococcal antibodies but was more efficient with immune sera. Serum bactericidal activity was dependent on C2, C5, and immune sera. MBL did not influence any of the parameters observed.Systemic meningococcal disease evolves when pathogenic Neisseria meningitidis breach the pharyngeal mucosa and start proliferating in the circulation (36, 44). The majority of the patients develops low-grade bacteremia leading to meningitis with a comparatively low case-fatality rate if adequate antibiotic treatment is given early (44). A minority develops fulminant sepsis caused by massive bacterial proliferation in the circulation, resulting in a very high case-fatality rate (44). A number of genetic disorders and polymorphisms in the host that influence the clinical presentation and outcome have been implicated in the response to intruding meningococci (4, 9).The complement system plays a crucial part in the host defense against systemic meningococcal disease (39). Acquisition of serum bactericidal antibodies correlates with protection (14, 16), whereas other mechanisms, primarily opsonophagocytosis, may also be important (1, 47). Deficiencies of the complement system affecting the alternative pathway, C3, and the terminal pathway have for a long time predominantly been associated with increased susceptibility to meningococcal disease (12, 13). Also, the rather common deficiency of mannose-binding lectin (MBL) has been associated with meningococcal disease, but only in early childhood (8, 11, 15, 19, 45). C2 deficiency, which apart from MBL deficiency is the most common inherited complement deficiency affecting about 1/20,000 of Caucasians (41), appears to be associated with a wide range of infections with encapsulated bacteria of which Streptococcus pneumoniae is the most frequent causative agent, whereas infections due to N. meningitidis occur less frequently (12, 25).In the present study blood samples from two individuals being genetically completely deficient in complement factor 2 (C2) or complement factor 5 (C5) and MBL were used to examine details regarding the specific roles of different parts of the complement system in the protection against serogroup B meningococcal disease. Bacterial survival and proliferation was examined in freshly drawn whole blood. Opsonophagocytic activity (OPA) and serum bactericidal activity (SBA), as well as the role of antimeningococcal antibodies, were studied separately. Functionally active and highly purified complement components were used for reconstitution experiments both of whole blood and of serum in order to confirm the specific roles of these components.     

The Capsular Serotype of Streptococcus pneumoniae Is More Important than the Genetic Background for Resistance to Complement

The polysaccharide capsule of Streptococcus pneumoniae inhibits phagocytic killing by innate immune mechanisms. Certain serotypes are associated with invasive disease while others with a nasopharyngeal carriage. The invasiveness of serotypes may partly be explained by ability to resist deposition of complement (C3) on the bacterial surface and consequent opsonophagocytic killing. In our previous studies, we observed that clinical isolates of serotypes 1 and 5, which are rarely detected in asymptomatic carriage, were resistant to complement deposition and opsonophagocytosis, whereas serotypes 6B and 23F, both common in carriage, were more sensitive to deposition of C3 and opsonophagocytic killing. However, presence of significant variation in C3 deposition between isolates of the same serotype indicated that factors other than the capsule also affect complement resistance. To distinguish the relative effect of the capsular serotype and other virulence factors on C3 deposition, we compared capsule-switched mutants prepared in genetic backgrounds of pneumococcal strains TIGR4, 603, and 618. Clinical isolates which had the same multilocus sequence type but expressed different serotypes were also compared. We found that the serotype had a significant impact on complement resistance and that the more resistant the strain was to complement, the higher was the concentration of polysaccharide-specific antibodies required for opsonophagocytic killing. Comparison of strains expressing the same capsular polysaccharides in the different genetic backgrounds and various capsular mutants of the same strain suggests that while the genotype affects complement resistance, the serotype is the most important determinant. Differences between serotypes were more significant than the differences between strains.Streptococcus pneumoniae is a major global pathogen responsible for a wide range of diseases from otitis media to pneumonia, sepsis, and meningitis. Under normal circumstances, pneumococcus enjoys a commensal relationship with its host, and the frequency of invasive disease among individuals colonized by the organism is very low (15). The polysaccharide capsule is considered the major determinant of virulence, because isolates that lack the capsule hardly ever cause invasive disease. The chemical composition of the polysaccharide is also important since only a few of the more than 90 known pneumococcal serotypes are responsible for the majority of invasive infections (14).The ability of nasopharyngeal carriage to progress into invasive disease or the risk of invasive disease after acquisition of the pathogen varies by serotype (4, 13). The relative contribution of the capsular type compared to other virulence factors in pneumococcal diseases is still unclear, but the extent of virulence cannot be predicted from the capsular type only. Clones belonging to the same serotype can have different abilities to cause invasive disease (4, 13, 43).The complement system is an essential element of host defense against pneumococci (3, 42). Activation of the complement leads to opsonization of the bacterial surface with C3 activation products C3b and iC3b, which are recognized by complement receptors of phagocytic cells (10, 41). The pneumococcal capsule impairs clearance by preventing access of phagocytic cells to opsonins deposited on the bacterial cell wall (2). Several pneumococcal proteins have also been shown to interact with complement (19). Pneumococcal surface protein A (PspA) inhibits C3 deposition (49) by interfering with the C1q initiation step of the classical pathway (24), which is the dominant complement activation pathway in innate host defense against pneumococci (3). Pneumolysin depletes complement by activating the classical pathway at a distance from the bacterium (54). The pneumococcal surface protein C (PspC) inhibits the activation of complement by, e.g., binding factor H (7, 8), a serum protein that efficiently modulates the function of the complement (17, 18, 38). The genetic background is likely to affect the relative importance of a surface protein to the complement resistance of the strain. Loss of PspC had variable effects on C3 deposition depending largely on the strain background and less on the serotype (55). Binding of complement inhibitor C4b-binding protein is restricted to certain serotypes, which possess a particular PspC allele (9). Pneumococcal histidine triad proteins may also play a role in complement evasion (35), but the impact they have on complement deposition seems to depend on the genetic background (29).We have previously found that pneumococcal isolates of certain serotypes, such as 1 and 5, associated with invasive disease were particularly resistant to complement deposition and opsonophagocytic killing, while serotypes such as 6B and 23F, associated with carriage, were more sensitive to deposition of C3 and opsonophagocytosis (30, 31). We found significant variation in the magnitude of complement deposition between isolates expressing the same capsular serotype, suggesting a role of serotype-independent factors in the outcome of this particular interaction. In a recent study by Hyams et al. (16) comparing C3 deposition on TIGR4 capsule-switch mutants, the resistance of pneumococcus to complement-mediated immunity was found to vary with the capsular serotype independently of capsular thickness or antibody binding. In the present study, we assessed the influence of the capsular serotype on complement deposition and opsonophagocytic killing by comparing several isogenic capsule-switched mutants and clonally related clinical isolates sharing the same sequence type but expressing different capsular polysaccharides (natural capsule switch variants of the same pneumococcal strain).     

Antibody to the Type 3 Capsule Facilitates Immune Adherence of Pneumococci to Erythrocytes and Augments Their Transfer to Macrophages

Streptococcus pneumoniae has been shown to bind to erythrocytes via a process called immune adherence. This adherence and the subsequent transfer of pneumococci from erythrocytes to macrophages are both dependent on complement C3 deposition onto the pneumococcal surface. The observation that anti-capsule antibody increases C3 deposition on the pneumococcal capsule indicated that anti-capsule antibody may also facilitate the clearance of pneumococci through immune adherence. Using pneumococcal strain WU2 (capsule type 3) and its nonencapsulated mutant JD908, we found that monoclonal antibody (MAb) to type 3 capsule increases complement C3, C1q, and C4 deposition on WU2 and enhanced the immune adherence of WU2 to erythrocytes. The MAb to type 3 capsule also enhanced the transfer of WU2 from erythrocytes to macrophages. Moreover, the transfer reaction was inhibited by preincubating macrophages with anti-CR3 or anti-FcγRIII/II MAb, indicating that CR3 and FcγRIII/II on macrophages mediate this process. The transfer reactions of JD908 (opsonized with complement) and WU2 (opsonized with complement plus MAb to type 3 capsule) were similarly inhibited by anti-CR3 MAb, but only the latter was inhibited by anti-FcγRIII/II MAb. This finding indicates that although complement and the macrophage receptor CR3 are essential for the transfer reaction, if antibody is present it can further enhance the transfer reaction through a process dependent on FcγRIII/II. Using pre- and postvaccination sera of people immunized with the 23-valent pneumococcal polysaccharide vaccine, we confirmed that human anti-capsule antibodies are also able to increase the immune adherence of pneumococci and their transfer to macrophages.Streptococcus pneumoniae (pneumococci) is a major human pathogen that causes pneumonia, bacteremia, meningitis, otitis media, and sinusitis, especially in children, the elderly, and immunocompromised patients (36). All of the natural strains of pneumococci are encapsulated by polysaccharide. According to the different constituents of their capsular polysaccharide, 91 serotypes of pneumococci are known (39). Among these, types 14, 6B, 19F, and 18C are most prevalent in small children and types 4, 14, 9V, and 23F are more frequently isolated from adults with invasive pneumococcal diseases (29). The 23-valent polysaccharide vaccine and a protein conjugate vaccine are recommended for adults and children, respectively (3).Pneumococci are able to activate both the classical and alternative pathways of complement (12, 41). The thick and rigid cell wall of pneumococci can protect them from being lysed by the complement membrane attack complex (28), and therefore opsonophagocytosis, mediated by surface-bound C3b, is thought to be essential for the elimination of pneumococci from the bloodstream (5, 9). The ability of complement to effectively opsonize pneumococci is dependent on the location and orientation of C3b bound to the bacterial surface, as this determines the accessibility of C3b to phagocytic cell C3b receptors (10). Although capsular polysaccharide, the outermost layer of pneumococci, is not an efficient activator of complement, the underlying cell wall teichoic acid has been reported to activate complement via the alternative pathway (45). Being sheltered by capsular polysaccharide, however, C3b deposited on the pneumococcal cell wall cannot interact efficiently with complement receptors (CR) on phagocytic cells. As a result, antibody to the pneumococcal cell wall is much less opsonic and less protective than antibody to pneumococcal capsular polysaccharides (6, 7, 10).S. pneumoniae adheres to erythrocytes in a complement- and antibody-dependent process called immune adherence (IA), which enhances the phagocytosis of pneumococci by polymorphonuclear leukocytes (23, 38). Studies using soluble immune complexes have shown that IA is mediated by complement C3b, C1q, C4b, and MBL interacting with CR type 1 (CR1) on human erythrocytes (21, 22, 43). The IA of pneumococci to human erythrocytes, as well as their subsequent transfer from erythrocytes to macrophages for clearance, depends on complement C3 deposition onto the pneumococcal surface (31). The known ability of antibody to pneumococcal capsular polysaccharide to enhance complement activation and C3 deposition led us to hypothesize that anti-capsule antibody might facilitate the IA and transfer reaction of pneumococci.In this study, a capsular type 3 pneumococcal strain and its capsule-negative isogenic mutant were used to investigate the effects mediated by anti-capsule antibody. We found that deposition of complement C3b, C1q, and C4b was associated with elevated IA of pneumococci in the presence of anti-capsule antibody. Moreover, anti-capsule antibody increases the transfer of pneumococci from erythrocytes to macrophages by promoting interaction with both CR3 and Fcγ receptors.     

Role of Complement in Protection against Cryptococcus gattii Infection

Previous studies have shown that the alternative pathway of complement activation plays an important role in protection against infection with Cryptococcus neoformans. Cryptococcus gattii does not activate the alternative pathway as well as C. neoformans in vitro. The role of complement in C. gattii infection in vivo has not been reported. In this study, we used mice deficient in complement components to investigate the role of complement in protection against a C. gattii isolate from an ongoing outbreak in northwestern North America. While factor B-deficient mice showed an enhanced rate of death, complement component C3-deficient mice died even more rapidly, indicating that the alternative pathway was not the only complement pathway contributing to protection against disease. Both C3- and factor B-deficient mice had increased fungal burdens in comparison to wild-type mice. Histopathology revealed an overwhelming fungal burden in the lungs of these complement-deficient mice, which undoubtedly prevented efficient gas exchange, causing death. Following the fate of radiolabeled organisms showed that both factor B- and C3-deficient mice were less effective than wild-type mice in clearing organisms. However, opsonization of C. gattii with complement components was not sufficient to prolong life in mice deficient in complement. Killing of C. gattii by macrophages in vitro was decreased in the presence of serum from factor B- and C3-deficient versus wild-type mice. In conclusion, we have demonstrated that complement activation is crucial for survival in C. gattii infection. Additionally, we have shown that the alternative pathway of complement activation is not the only complement pathway contributing to protection.The complement system consists of a cascade of serum proteins that are involved in opsonization, membrane lysis, and chemotaxis. There are three pathways through which complement can be activated: classical, alternative, and lectin. Complement components C3 to C9 participate in all three complement cascade pathways. C1q is used only in the classical pathway, and factor B is used only in the alternative pathway. While C4 is used in both the classical and lectin pathways, recently it was reported that the lectin pathway can function in the absence of C2 and/or C4 if the alternative pathway is intact (23). Mannose binding lectin (MBL) (24), which is used only in the lectin pathway, exists in two forms in rodents (MBL-A and MBL-C) while only one form is found in humans (8). MBL-A and MBL-C have different levels in serum and differ in their affinity for d-glucose and α-methyl-d-glucose but have redundant function (24).Cryptococcus spp. are fungal pathogens that possess a polysaccharide capsule composed mainly of glucuronoxylomannan (GXM). The capsule is antiphagocytic and anti-inflammatory but is known to activate the alternative complement pathway (11). Previously, Cryptococcus spp. were identified serologically and were all considered as one species, Cryptococcus neoformans. More recently, two species have been designated, C. neoformans (serotypes A and D) and Cryptococcus gattii (serotypes B and C). A key difference between the two species is that C. neoformans tends to infect immunocompromised individuals while C. gattii generally infects apparently immunocompetent people (15). Cryptococcus spp. most commonly cause pulmonary and central nervous system infections in humans (15, 25) and mouse models (3).In 1999, an outbreak of C. gattii began on Vancouver Island, British Columbia, infecting people, companion animals, and porpoises. Strain A1MR265 is the major clinical reference isolate from the Vancouver Island outbreak. Recently, a case of cryptococcosis caused by the strain predominant in Vancouver Island was identified in Puget Sound, WA (27). A total of eight human cases have been reported in Washington State in the past 2 years; four of these individuals had not traveled out of state (29). Nine cases have been reported in Oregon (L. Hoang, presented at the 108th General Meeting of the American Society of Microbiology, Boston, MA, 1 to 5 June 2008). The potential spreading of a strain of Cryptococcus capable of infecting immunocompetent people is cause for concern, and C. gattii infection is now a reportable disease in Washington State (29).The Kozel laboratory and others have shown that Cryptococcus spp. strongly activate the alternative pathway of the complement cascade (5, 14) while the polysaccharide capsule blocks the activation of the classical pathway that occurs at the cell wall in nonencapsulated strains (13). The capsule serves as a site for activation and deposition of C3 fragments, mainly iC3b, which promote phagocytosis of the yeast (12). C. gattii does not appear to activate the alternative pathway as potently as C. neoformans (28, 31). One study found that C. gattii binds fewer C3 molecules than C. neoformans (28). A later report indicated that while the maximum amount of bound C3 did not differ significantly between species, there was more rapid accumulation of C3 on C. neoformans before a steady state was achieved (31). In the absence of C5 in mouse strains B10.D2/oSn, DBA/2, and A/J, C. neoformans infection proceeds to a fatal pneumonia with higher fungal burdens in the blood, brain, lungs, and liver than in complement-sufficient animals (6, 21, 22). Together, these studies indicate a significant role for the alternative pathway of complement in protection against C. neoformans. While C. gattii has been reported to not activate the alternative pathway as vigorously as C. neoformans in vitro, the role of complement in in vivo C. gattii infection has not been determined.In this study, we investigated the role of complement pathways in protecting against infection with C. gattii. Mice deficient in complement components C1q, C4, C3, and factor B have been generated on the C57BL/6J background (2, 16, 30). Using these mice and mice treated with cobra venom factor (CVF), which depletes C3 and prevents cascade progression from C3 to C9, we have now shown that complement activation plays an essential role in delaying disease progression in mice infected with C. gattii.     

Clumping Factor A Interaction with Complement Factor I Increases C3b Cleavage on the Bacterial Surface of Staphylococcus aureus and Decreases Complement-Mediated Phagocytosis

The human complement system is important in the immunological control of Staphylococcus aureus infection. We showed previously that S. aureus surface protein clumping factor A (ClfA), when expressed in recombinant form, bound complement control protein factor I and increased factor I cleavage of C3b to iC3b. In the present study, we show that, compared to the results for the wild type, when isogenic ClfA-deficient S. aureus mutants were incubated in serum, they bound less factor I, generated less iC3b on the bacterial surface, and bound fewer C3 fragments. It has been shown previously that two amino acids in ClfA (P₃₃₆ and Y₃₃₈) are essential for fibrinogen binding. However, S. aureus expressing ClfA(P336A Y338S) was less virulent than ClfA-deficient strains in animal models. This suggested that ClfA contributed to S. aureus virulence by a mechanism different than fibrinogen binding. In the present study, we showed that S. aureus expressing ClfA(P336A Y338S) was more susceptible to complement-mediated phagocytosis than a ClfA-null mutant or the wild type. Unlike ClfA, ClfA(P336A Y338S) did not enhance factor I cleavage of C3b to iC3b and inhibited the cofactor function of factor H. Fibrinogen enhanced factor I binding to ClfA and the S. aureus surface. Twenty clinical S. aureus strains all expressed ClfA and bound factor I. High levels of factor I binding by clinical strains correlated with poor phagocytosis. In summary, our results suggest that the interaction of ClfA with factor I contributes to S. aureus virulence by a complement-mediated mechanism.Staphylococcus aureus is a significant cause of morbidity and mortality; methicillin-resistant S. aureus (MRSA) caused an estimated 18,650 deaths in the United States in 2005 (19). Antibiotic resistance continues to increase among S. aureus isolates, including isolates of community-associated MRSA (CA-MRSA) (7, 30), health care-associated MRSA (12), and S. aureus with reduced susceptibility to vancomycin (20). Understanding how this organism avoids host immune defenses is crucial for the development of new strategies to prevent and treat infections.Complement is a major component of innate immunity and plays a vital role in the control of many bacterial pathogens (28), including S. aureus (15, 21, 33). Indeed, this organism secretes several small soluble proteins that interfere with normal complement host defense mechanisms, including SCIN and Efb (15, 32). We have previously shown that the human complement regulator factor I is captured on the S. aureus cell surface, where it is activated and cleaves the crucial opsonin C3b (22) to iC3b (3). This results in decreased phagocytosis by human neutrophils (2). We subsequently showed that the A domain of clumping factor A (ClfA), an important surface-located fibrinogen-binding protein, bound factor I and acted as a cofactor to trigger cleavage of C3b to iC3b (13).The binding to fibrinogen by ClfA involves the C terminus of the γ-chain binding to a trench located between subdomains N2 and N3 by a “dock-lock-latch” mechanism (18). Residues Pro336 and Tyr338 are located in the trench and are crucial for ligand binding, and a P336S Y338A mutant (ClfAPYII) is completely defective in fibrinogen binding (23).Clumping factor A is covalently anchored to the cell wall of S. aureus and promotes adhesion of the bacterium to fibrin clots and to thrombi created on heart valves in a rat model of endocarditis (25). In addition, ClfA is required for survival of bacteria following injection into the bloodstream of mice (16). This was attributed to the ability of the protein to promote bacterial resistance to phagocytosis by neutrophils. It was proposed that binding to fibrinogen prevented the deposition or recognition of opsonins. However, phagocytosis experiments performed in the absence of fibrinogen demonstrated that expression of ClfA still had an antiphagocytic effect, suggesting that there is another mechanism (14).In mouse models of S. aureus bacteremia and septic arthritis, bacteria expressing the non-fibrinogen-binding mutant of ClfA were less virulent than a null mutant that was devoid of the surface protein (17). It was difficult to explain these effects by the loss of fibrinogen binding alone. In the present study, we analyzed the interaction of ClfA with factor I on the bacterial cell surface and the roles of these proteins in triggering cleavage of C3b to iC3b. In doing so, we developed a novel explanation for the role of ClfA in disrupting opsonophagocytosis.     

Binding of Complement Regulators to Invasive Nontypeable Haemophilus influenzae Isolates Is Not Increased Compared to Nasopharyngeal Isolates,but Serum Resistance Is Linked to Disease Severity

The aim of the present study was to analyze the importance of nontypeable Haemophilus influenzae (NTHi) isolated from patients with sepsis (invasive isolates) compared to nasopharyngeal isolates from patients with upper respiratory tract infection for resistance to complement-mediated attack in human serum and to correlate this result with disease severity. We studied and characterized cases of invasive NTHi disease in detail. All patients with invasive NTHi isolates were adults, and 35% had a clinical presentation of severe sepsis according to the ACCP/SCCM classification of sepsis grading. Moreover, 41% of the patients had evidence of immune deficiency. The different isolates were analyzed for survival in human serum and for binding of ¹²⁵I-labeled, purified human complement inhibitors C4b-binding protein (C4BP), factor H, and vitronectin, in addition to binding of regulators directly from serum. No significant differences were found when blood-derived and nasopharyngeal isolates were compared, suggesting that interactions with the complement system are equally important for NTHi strains, irrespective of isolation site. Interestingly, a correlation between serum resistance and invasive disease severity was found. The ability to resist the attack of the complement system seems to be important for NTHi strains infecting the respiratory tract as well as the bloodstream.Haemophilus influenzae is an important human-specific pathogen that can be classified according to the presence of a polysaccharide capsule (20). The encapsulated strains cause invasive diseases, whereas the unencapsulated and hence nontypeable H. influenzae (NTHi) strains are mainly found in local upper and lower respiratory tract infections (2, 35). However, NTHi is, after Streptococcus pneumoniae, the most common microbe found in children with acute otitis media and is the main cause of exacerbations in patients suffering from chronic obstructive pulmonary disease and bronchiectasis (3, 4, 13, 28, 32, 34). NTHi can also cause sinusitis, conjunctivitis, and pneumonia in children (25). Thus, NTHi is a heterogenous species, capable of great variation in virulence, and is found in the airway either as a commensal or as a pathogen with the capacity to invade the airway epithelium.Invasive disease caused by H. influenzae type b (Hib) mainly affects infants and children, causing potentially life-threatening conditions, such as meningitis, epiglottitis, and severe sepsis. After introduction of the conjugate vaccine against Hib in the early 1990s, the incidence of invasive disease caused by Hib has decreased substantially in the Western hemisphere (5). In contrast, it has been suggested that the incidence of NTHi septicemia is increasing (36). Most clinical studies of invasive Haemophilus infections have been about Hib, and less is known about the clinical characteristics of invasive disease caused by NTHi. In relation to its extensive presence in nasopharyngeal and sputum cultures, NTHi is infrequently found in the bloodstream and it seems likely that host factors are equally important as specific bacterial virulence in patients with NTHi sepsis.The complement system is the first line of defense against pathogenic microorganisms (7). Activation of the complement system leads to a cascade of protein activation and deposition on the surface of the pathogen, resulting in formation of the membrane attack complex (MAC) and opsonization of the pathogen, followed by phagocytosis (38). The classical pathway of the complement system is activated by target-bound antibodies and C-reactive protein (37), whereas the alternative pathway is spontaneously activated through direct contact with foreign particles or cells (38). Both pathways lead to the formation of the C3 convertases, with subsequent cleavage of C3 to C3a and C3b. Thereafter, the C5 convertases are formed and the terminal pathway is activated, which results in the formation of the MAC and lysis of the cell. To prevent nonspecific damage from excess complement activation, the complement cascade is tightly regulated. Important regulators of the complement system are C4b-binding protein (C4BP) (governing the classical/lectin pathway) (6), factor H and factor H-like protein 1 (alternative pathway) (41), and vitronectin and complement factor H-related protein 1 (terminal pathway) (18, 33).The complement system is classified as a part of serum, but there are several studies demonstrating the presence of complement in various sites of the body. Reports of the presence of complement components in the respiratory tracts of healthy individuals are scarce. There are several studies, however, indicating the importance of complement in the respiratory tract during infections. The permeability of the mucosa increases during inflammation, and plasma, including complement proteins and immunoglobulins, enters the airway lumen (11, 12, 29). This process, designated plasma exudation, has been suggested to be the first line of the mucosal defense.The pathogenesis of many microorganisms relies on their capacity to avoid, resist, or neutralize the host defense, including the complement system. Therefore, many pathogens have evolved different mechanisms to avoid complement-mediated killing. A frequent strategy used by some pathogens is binding of complement inhibitors such as C4BP, factor H, and vitronectin, which all protect from complement-mediated attacks (7, 22, 31). These inhibitors are captured on the bacterial surface in such a way that they are still functionally active. In previous studies, we have shown that NTHi binds C4BP and factor H and that these interactions significantly contribute to bacterial serum resistance (15, 17). In addition, Haemophilus surface fibrils that can be found solely in Hib, and protein E, which exists in both encapsulated and nontypeable strains (30), interact with vitronectin and thereby prevent complement-induced lysis, resulting in increased bacterial survival in normal human serum (NHS) (14, 16).In the present study, the characteristics of invasive NTHi infections, including evidence of immune deficiency in the individual patient and the clinical presentation of the septic event, were studied. We correlated these findings with the capacity to bind specific complement regulators and the in vitro serum resistance of the individual isolates. The invasive NTHi isolates obtained from patients with sepsis were compared to nasopharyngeal strains from patients with upper respiratory tract infection. There was no clear difference in serum resistance or binding to complement inhibitors between the two groups of NTHi. Our findings also demonstrate that binding of complement regulators and resistance to human serum are important for NTHi isolates from the upper respiratory tract as well as those from blood samples. Furthermore, a significant correlation between disease severity and in vitro serum resistance was identified in cases of NTHi invasive disease.     

The Effects of PspC on Complement-Mediated Immunity to Streptococcus pneumoniae Vary with Strain Background and Capsular Serotype

Streptococcus pneumoniae may evade complement activity by binding of factor H (FH), a negative regulator of the alternative pathway, to the surface protein PspC. However, existing data on the effects of FH binding to PspC on complement activity are conflicting, and there is also considerable allelic variation in PspC structure between S. pneumoniae strains that may influence PspC-dependent effects on complement. We have investigated interactions with complement for several S. pneumoniae strains in which the gene encoding PspC has been deleted. The degree of FH binding varied between strains and was entirely dependent on PspC for seven strains. Data obtained with TIGR4 strains expressing different capsular serotypes suggest that FH binding is affected by capsular serotype. Results of immunoblot analysis for C3 degradation products and iC3b deposition assays suggested that FH bound to PspC retained functional activity, but loss of PspC had strikingly varied effects on C3b/iC3b deposition on S. pneumoniae, with large increases on serotype 4, 6A, 6B, and 9V strains but only small increases or even decreases on serotype 2, 3, 17, and 23F strains. Repeating C3b/iC3b assays with TIGR4 strains expressing different capsular serotypes suggested that differences in the effect of PspC on C3b/iC3b deposition were largely independent of capsular serotype and depend on strain background. However, data obtained from infection in complement-deficient mice demonstrated that differences between strains in the effects of PspC on complement surprisingly did not influence the development of septicemia.Streptococcus pneumoniae is a common cause of invasive diseases such as pneumonia, meningitis, and septicemia even in immunocompetent subjects. One important component of host immunity to S. pneumoniae is the complement system, a series of approximately 30 serum and cell surface proteins organized into three enzyme cascades termed the classical, alternative, and mannan binding lectin (MBL) pathways (44). Infection experiments using complement-deficient mice have demonstrated that both the classical and alternative pathways are important for immunity to S. pneumoniae (2, 11, 15, 23, 43), and the high incidence of S. pneumoniae infections in patients with complement deficiencies confirms the relevance of complement for preventing disease in humans (3, 21). Both pathways lead to the formation of a C3 convertase that cleaves the central complement component C3, resulting in deposition of C3b on the surface of the pathogen that is further processed to iC3b. C3b and iC3b are opsonins, (44), and coating of S. pneumoniae with C3b/iC3b is vital for efficient phagocytosis of this organism by neutrophils (49). The importance of complement for immunity to S. pneumoniae is emphasized by the identification of several mechanisms by which the bacteria can inhibit complement activity. The choline binding surface protein PspA and the capsule prevent C3b/iC3b deposition on S. pneumoniae by mechanisms that remain unclear, whereas the secreted toxin pneumolysin seems to divert classical pathway activity away from the bacteria (1, 30, 32, 35, 39, 43, 48).PspC is another choline binding surface protein that is related to PspA and is important for virulence in models of nasopharyngeal colonization, septicemia, and pneumonia (16, 22, 33, 35, 38). PspC can bind to factor H (FH), a negative regulator of the alternative pathway (8, 13, 18). PspC and the closely related proteins encoded at the same genetic locus in different strains (termed SpsA, CbpA, PbcA, and Hic) could, therefore, prevent alternative pathway complement activity against S. pneumoniae by one of three potential mechanisms (7). First, FH may speed up the degradation of C3b by promoting the factor I-dependent cleavage of C3b bound to the bacterial surface to iC3b; second, FH causes the dissociation of factor B from the alternative pathway C3 convertase C3bBb, decreasing C3b deposition on the bacteria; and third, FH may also inhibit the formation on the bacterial surface of the C3 convertase C3bBb by preferentially binding C3b, thus preventing C3b binding to factor B. However, although the affinity of PspC for FH has been clearly demonstrated and the binding sites have been identified (6, 12, 13, 17, 18, 28, 38), the effects of this interaction on complement-mediated immunity to S. pneumoniae is relatively poorly defined. Loss of PspC can cause reduced iC3b (compatible with reduced processing of C3b to iC3b) (28) and increased C3b/iC3b (compatible with reduced C3 convertase activity) deposition on a capsular serotype 2 (ST2) strain D39 or an unencapsulated mutant derived from a capsular ST3 strain (19, 35). However, in contrast to Quin et al., Lu et al. and Li et al. found little effect on total C3 bound to a ΔpspC mutant derived from the D39 strain compared to the wild type unless this mutation was combined with mutation of pspA (25, 28, 35).The role of PspC on complement-mediated immunity in different strains could also be affected by the marked allelic variation in the structure of PspC. Ianelli et al. sequenced pspC from 43 different strains, and although the derived protein sequences had the same domain organization, each protein had a unique sequence that could be divided into 11 subgroups (17). The majority of PspC alleles contain a C-terminal cell wall choline binding motif (similar to PspA), but 17 of the allelic variants contain a cell wall anchor LPTXG motif instead. FH binding requires only the N-terminal 89 amino acids of PspC from the D39 strain and is dependent on a 12-amino-acid motif which is conserved between PspC alleles from different S. pneumoniae strains (28). However, this domain is not enough for full FH binding capacity and requires additional flanking amino acids that vary with allelic variation of PspC. Indeed, it is known that the ability of S. pneumoniae to bind to FH varies between strains (36). This variation in FH binding between strains seems to be independent of serotype but is increased on strains isolated from the blood or cerebrospinal fluid (CSF), under which conditions PspC expression is increased (29, 34). Overall, the existing data suggest that the effects of PspC on complement deposition on S. pneumoniae is not clear and could vary between strains. Furthermore, several other functions have been ascribed to PspC, including direct binding to C3 (which might counteract any FH-mediated reduction in C3b deposition) (4, 40) and aiding S. pneumoniae adhesion to host cells and translocation across epithelial layers (12, 13, 37, 38, 50), which could also affect virulence independent of any effect of PspC on complement-mediated immunity. Further clarification of the effects of PspC on opsonization of S. pneumoniae with C3b/iC3b is important for a better understanding of how PspC aids S. pneumoniae virulence. To address this question, in this study we have assessed the effects of loss of PspC on C3b/iC3b deposition on a range of S. pneumoniae strains representing different capsular STs.     

Serotype-Related Variation in Susceptibility to Complement Deposition and Opsonophagocytosis among Clinical Isolates of Streptococcus pneumoniae

The polysaccharide capsule is a major virulence factor of Streptococcus pneumoniae; it affects complement resistance and shields the bacterium from phagocytes. Certain capsular serotypes appear to be better able to cause invasive disease than others. Serotypes 1 and 5 are common causes of invasive disease but are rarely isolated from healthy carriers, whereas serotypes 6B and 23F are more frequently isolated from carriage than invasive disease. We have recently shown that serotypes 6B and 19F differ in resistance to complement C3 deposition and opsonophagocytic killing. In this study we assessed the complement resistance and susceptibility to opsonophagocytosis of several other serotypes targeted by the pneumococcal conjugate vaccines. Clinical isolates of serotypes 1, 4, 5, 14, 18C, and 23F were tested along reference strains of corresponding capsular types. The concentration of anticapsular antibodies required for opsonophagocytic killing correlated inversely with C3 deposition on the serotype. Serotype 1 was the most resistant of the clinical isolates to C3 deposition and, along with serotypes 5 and 19F, required the highest concentration of capsule antibodies for opsonophagocytic killing, whereas serotype 23F was the most sensitive to opsonophagocytosis. Sensitivity to C3 deposition and opsonophagocytosis was associated with serotype-specific mortality of invasive pneumococcal disease, suggesting that the primary pathogens, such as serotypes 1 and 5, are more resistant to complement and require a higher concentration of capsule antibodies for opsonophagocytic killing than the opportunistic serotypes such as 6B and 23F, which are associated with a more severe disease outcome.Streptococcus pneumoniae colonizes the nasopharynx (NP) of healthy individuals but occasionally breaks from its carriage habitat and causes diseases ranging from acute otitis media to more severe diseases such as pneumonia, sepsis, and meningitis. Isolates that lack the capsule rarely cause invasive disease in humans, and loss of the capsule greatly attenuates virulence in animal models of disease (31, 54). Each of the more than 90 known pneumococcal serotypes produces a biochemically distinct polysaccharide structure, which is a major determinant of pneumococcal virulence since only a small number of serotypes account for the majority of infections (18). The polysaccharide capsule shields the bacterium from phagocytosis by inhibiting recognition of opsonins adhered to the bacterial cell wall.Certain serotypes are relatively common causes of invasive pneumococcal disease (IPD) with respect to their prevalence in carriage, whereas others are common colonizers of the NP but rarely cause disease (6, 16). The disease potential, or relative invasiveness, is a measure of the ability of pneumococci to progress from nasopharyngeal carriage to invasive disease in humans, similar to the attack rate, which is the risk of disease as a result of pathogen acquisition (6, 50). Most of the invasive property of pneumococci seems to be determined by their capsular serotype rather than genetic background. However, different clones of the same serotype can vary in an ability to cause IPD (6, 16, 45).Variation in disease potential among serotypes has been reported in a number of epidemiological studies. In young children in England, the relative invasiveness of more prevalent carriage serotypes—6B, 19F, and 23F—was low compared to the high disease potential of serotypes 1, 4, 14, 18C, and 7F (6). In another United Kingdom study in infants <2 years old, serotypes 23F, 6A, 19F, 16F, 6B, and 15B/C were associated with low attack rates; serotypes 4, 14, 7F, 9V, and 18C were associated with relatively high attack rates; and serotypes 1, 5, and 9A were only isolated from IPD (50). In a study of Finnish children <2 years of age serotypes 19F and 23F showed a tendency to be more common in carriage and serotypes 14, 18C, 19A, and 6B were significantly more common in IPD (16). Even in populations with a high proportion of disease caused by serotypes 1 and 5, such as in the Gambia (2), serotypes 1 and 5 are rarely detected in nasopharyngeal carriage (19, 29, 47). In a meta-analysis of seven data sets, serogroups 1, 5, and 7 had the highest invasive disease potential (7).The duration of carriage varies by capsular type and is inversely correlated with the attack rate (50). Capsular serotypes carried for a short duration and with a high attack rate, such as serotypes 1, 5, and 7F, behave like “primary pathogens,” which affect previously healthy individuals and are associated with lower mortality. Meanwhile, serotypes that are carried for a long duration, such as 6B, 19F, and 23F, behave like opportunistic pathogens causing disease in patients with an underlying disease and are associated with a more severe disease and higher mortality (49). This observation correlates with the outcome of analysis of the largest ever population-based study on the severity of IPD published recently by Harboe et al. Odds ratios (ORs) for the death as an outcome of IPD were higher for opportunistic serotypes compared to invasive capsular types (17).Comparison of pneumococcal strains isolated from carriage and IPD suggests that carriage isolates are more heterogeneous and invasiveness is associated with clonality (6, 42). Isolates of serogroups 7 and 14 are clonal and rare among carriers, whereas isolates of serogroups 6, 19, and 23 are heterogeneous, suggesting that the clonality may represent an advantage for invasiveness (51). Molecular epidemiology studies reveal that IPD caused by serotype 1 in the Gambia (3) and serotypes 1 and 5 in Israel (37) resulted from expansion of single, virulent clones, in contrast to serotypes 6B and 23F, which showed a large diversity in their genotypic characteristics (37). Studies of serotype 1 disease cases in different geographic regions did not identify clones with distinct virulence properties (8), which could reflect either a high virulence potential of the serotype 1 capsule or selection of genotypes advantageous for invasiveness.Variations in susceptibility to host immune defense mechanisms can contribute to the differences between capsular serotypes in invasiveness. Immune response to pneumococcus strongly depends on opsonization of the bacteria with complement C3 molecules (C3b and iC3b), the deposition of which may be influenced by the capsular polysaccharides (20). We have previously shown that significantly more C3 is deposited on serotype 6B than 19F isolates and that a significantly higher concentration of polysaccharide-specific antibodies is required for opsonophagocytic killing of serotype 19F isolates (33). The fact that less C3 was deposited on serotype 19F pneumococci in the presence or absence of antibodies suggests innate differences between capsular serotypes in resistance to the complement. Comparison of C3 deposition on capsule-switched mutants of TIGR4 demonstrated large differences between serotypes (22), which indicates that the capsular serotype alone can have a significant impact on the complement resistance of strains.Weinberger et al. described recently an association between serotype prevalence in carriage and the amount of capsular polysaccharide produced in vitro by isolates of a particular serotype (55). The association linked capsule chemistry to the success of the serotype as a colonizer. This observation suggested that a similar correlation can be present between capsule and the invasiveness of the serotype. In the present study we compared the resistance to complement and opsonophagocytosis of clinical isolates of several different pneumococcal serotypes. Each serotype was represented by multiple different clones. Isolates from blood or cerebrospinal fluid, from serotypes 1, 4, 5, 14, 18C, and 23F, as well as mucosal isolates from serotypes 1, 14, and 23F, were analyzed, along with reference strains used in the standard opsonophagocytic assay. We analyzed the results in a context of the serotype-specific severity of invasive disease and the chemical structure of the polysaccharides.     

Shiga Toxin 2 Targets the Murine Renal Collecting Duct Epithelium

Hemolytic-uremic syndrome (HUS) caused by Shiga toxin-producing Escherichia coli infection is a leading cause of pediatric acute renal failure. Bacterial toxins produced in the gut enter the circulation and cause a systemic toxemia and targeted cell damage. It had been previously shown that injection of Shiga toxin 2 (Stx2) and lipopolysaccharide (LPS) caused signs and symptoms of HUS in mice, but the mechanism leading to renal failure remained uncharacterized. The current study elucidated that murine cells of the glomerular filtration barrier were unresponsive to Stx2 because they lacked the receptor glycosphingolipid globotriaosylceramide (Gb₃) in vitro and in vivo. In contrast to the analogous human cells, Stx2 did not alter inflammatory kinase activity, cytokine release, or cell viability of the murine glomerular cells. However, murine renal cortical and medullary tubular cells expressed Gb₃ and responded to Stx2 by undergoing apoptosis. Stx2-induced loss of functioning collecting ducts in vivo caused production of increased dilute urine, resulted in dehydration, and contributed to renal failure. Stx2-mediated renal dysfunction was ameliorated by administration of the nonselective caspase inhibitor Q-VD-OPH in vivo. Stx2 therefore targets the murine collecting duct, and this Stx2-induced injury can be blocked by inhibitors of apoptosis in vivo.Shiga toxin-producing Escherichia coli (STEC) is the principal etiologic agent of diarrhea-associated hemolytic-uremic syndrome (HUS) (42, 60, 66). Renal disease is thought to be due to the combined action of Shiga toxins (Shiga toxin 1 [Stx1] and Stx2), the primary virulence factors of STEC, and bacterial lipopolysaccharide (LPS) on the renal glomeruli and tubules (6, 42, 60, 66). Of these, Stx2 is most frequently associated with the development of HUS (45). Shiga toxin enters susceptible cell types after binding to the cell surface receptor glycosphingolipid globotriaosylceramide (Gb₃) and specifically depurinates the 28S rRNA, thereby inhibiting protein synthesis (42, 60, 66). The damage initiates a ribotoxic stress response consisting of mitogen-activated protein (MAP) kinase activation, and this response can be associated with cytokine release and cell death (21, 22, 25-27, 61, 69, 73). This cell death is often caspase-dependent apoptosis (18, 61). Gb₃ is expressed by human glomerular endothelial cells, podocytes, and multiple tubular epithelial cell types, and damage markers for these cells can be detected in urine samples from HUS patients (10-12, 15, 49, 73). Shiga toxin binds to these cells in renal sections from HUS patients, and along with the typical fibrin-rich glomerular microangiopathy, biopsy sections demonstrate apoptosis of both glomerular and tubular cell types (9, 29, 31).Concomitant development of the most prominent features of HUS: anemia, thrombocytopenia, and renal failure, requires both Shiga toxin and LPS in the murine model (30, 33). Nevertheless, our previous work demonstrated that renal failure is mediated exclusively by Stx2 (33). While it is established that Gb₃ is the unique Shiga toxin receptor (46), the current literature regarding the mechanism by which Shiga toxin causes renal dysfunction in mice is inconsistent. Even though Gb₃ has been localized to some murine renal tubules and tubular damage has been observed (19, 23, 46, 53, 65, 68, 72, 74), the specific types of tubules affected have been incompletely characterized. Although multiple groups have been unable to locate the Shiga toxin receptor Gb₃ in glomeruli in murine renal sections (19, 53), one group has reported that murine glomerular podocytes possess Gb₃ and respond to Stx2 in vitro (40), and another group has reported that renal tubular capillaries express the Gb₃ receptor (46). Furthermore, murine glomerular abnormalities, including platelet and fibrin deposition, occur in some murine HUS models (28, 30, 33, 46, 59, 63). We demonstrate here that murine glomerular endothelial cells and podocytes are unresponsive to Stx2 because they do not produce the glycosphingolipid receptor Gb₃ in vitro or in vivo. Further, murine renal tubules, including collecting ducts, express Gb₃ and undergo Stx2-induced apoptosis, resulting in dysfunctional urine production and dehydration.     

Complement Receptor 1 Expression on Mouse Erythrocytes Mediates Clearance of Streptococcus pneumoniae by Immune Adherence

Complement-containing immune complexes can be presented to phagocytes by human erythrocytes bearing complement receptor 1 (CR1). Although this has long been assumed to be a mechanism by which humans are able to protect themselves from “extracellular” bacteria such as pneumococci, there is little direct evidence. In these studies we have investigated this question by comparing results for erythrocytes from transgenic mice expressing human CR1 on their erythrocytes to the results for wild-type mouse erythrocytes that do not express CR1. We demonstrate that human CR1 expression on murine erythrocytes allows immune adherence to beads opsonized with either mouse or human serum as a source of complement. The role of CR1 in immune adherence was supported by studies showing that it was blocked by the addition of antibody to human CR1. Furthermore, human CR1 expression enhances the immune adherence of opsonized pneumococci to erythrocytes in vitro, and the pneumococci attached to erythrocytes via CR1 can be transferred in vitro to live macrophages. Even more importantly, we observed that if complement-opsonized pneumococci are injected intravenously with CR1⁺ mouse erythrocytes into wild-type mice (after a short in vitro incubation), they are cleared faster than opsonized pneumococci similarly injected with wild-type mouse erythrocytes. Finally, we have shown that the intravenous (i.v.) injection of pneumococci into CR1⁺ mice also results in more rapid blood clearance than in wild-type mice. These data support that immune adherence via CR1 on erythrocytes likely plays an important role in the clearance of opsonized bacteria from human blood.Streptococcus pneumoniae (pneumococcus) is a major pathogen causing bacteremia in young children and the elderly (22). Pneumococci in the blood are cleared mainly through complement- and antibody (Ab)-dependent opsonization and phagocytosis (8). Previous studies have shown that pneumococci can attach to erythrocytes through immune adherence (IA), which facilitates the clearance of pneumococci by increasing the transfer of pneumococci from erythrocytes to macrophages (20, 24). IA is mediated by complement receptor 1 (CR1) (or CD35) on erythrocytes interacting with C3b, C1q, C4b, and mannose-binding lectin (MBL) on the immune complexes (13, 14, 28). Factors that influence complement deposition on pneumococci thus also affect the IA of pneumococci.For example, the expression of pneumococcal surface protein A (PspA) and PspC can protect pneumococci from IA. PspA interferes with C1q binding and the classical pathway of complement activation, and PspC interferes with the amplification of the alternative pathway of complement activation that is triggered by the absence of PspA (20, 26). In addition, anticapsule antibody induced by immunization with a 23-valent pneumococcal polysaccharide vaccine can enhance the IA of pneumococci and the subsequent transfer of pneumococci from erythrocytes to macrophages by promoting classical pathway C3 activation (21). Once complement is deposited on pneumococci, through either the absence of PspA and PspC or the presence of antibody to capsule, the pneumococci are able to show IA to CR1 of human erythrocytes and can be readily transferred to macrophages (20, 21).Human CR1 is a single-chain transmembrane protein expressed on erythrocytes, most white blood cells, tissue phagocytes, and glomerular podocytes (17). Levels of CR1 are variable between individuals, ranging from ∼100 to over 1,000 per human erythrocyte (30). The clustered expression of CR1 on human erythrocytes results in the high-affinity binding of immune complexes (11). In contrast to human erythrocytes, those of mice do not express CR1 (18). In the present paper we utilize transgenic mice expressing human CR1 on mouse erythrocytes (27) to examine the role of erythrocyte CR1 in immune adherence. In this work, we first establish that mouse complement C3b supports IA to human CR1 expressed by transgenic mouse erythrocytes, before investigating the role that human CR1 could play in the clearance of pneumococci from the blood.     

Salmonella enterica Serovar Typhimurium Vaccine Strains Expressing a Nontoxic Shiga-Like Toxin 2 Derivative Induce Partial Protective Immunity to the Toxin Expressed by Enterohemorrhagic Escherichia coli

Shiga-like toxin 2 (Stx2)-producing enterohemorrhagic Escherichia coli (referred to as EHEC or STEC) strains are the primary etiologic agents of hemolytic-uremic syndrome (HUS), which leads to renal failure and high mortality rates. Expression of Stx2 is the most relevant virulence-associated factor of EHEC strains, and toxin neutralization by antigen-specific serum antibodies represents the main target for both preventive and therapeutic anti-HUS approaches. In the present report, we describe two Salmonella enterica serovar Typhimurium aroA vaccine strains expressing a nontoxic plasmid-encoded derivative of Stx2 (Stx2ΔAB) containing the complete nontoxic A2 subunit and the receptor binding B subunit. The two S. Typhimurium strains differ in the expression of flagellin, the structural subunit of the flagellar shaft, which exerts strong adjuvant effects. The vaccine strains expressed Stx2ΔAB, either cell bound or secreted into the extracellular environment, and showed enhanced mouse gut colonization and high plasmid stability under both in vitro and in vivo conditions. Oral immunization of mice with three doses of the S. Typhimurium vaccine strains elicited serum anti-Stx2B (IgG) antibodies that neutralized the toxic effects of the native toxin under in vitro conditions (Vero cells) and conferred partial protection under in vivo conditions. No significant differences with respect to gut colonization or the induction of antigen-specific antibody responses were detected in mice vaccinated with flagellated versus nonflagellated bacterial strains. The present results indicate that expression of Stx2ΔAB by attenuated S. Typhimurium strains is an alternative vaccine approach for HUS control, but additional improvements in the immunogenicity of Stx2 toxoids are still required.Shiga-like toxins (Stx) play a crucial role in the pathogenesis of enterohemorrhagic Escherichia coli (EHEC) strains, which may lead to hemorrhagic colitis, central nervous system disturbances, and hemolytic-uremic syndrome (HUS) (27, 33). HUS involves acute renal failure, thrombocytopenia, and microangiopathic hemolytic anemia, with mortality rates ranging from 1% to 4% (45, 50). EHEC strains may express different serotypes, including the widely distributed O157:H7 serotype, and infection correlates with the ingestion of contaminated ground beef and cow manure-contaminated water, vegetables, juices, and other products (13, 18). The incidence of EHEC-associated HUS cases is particularly high in developed countries, and high incidence rates have been recorded in Argentina, where cultural and diverse epidemiological factors contribute to the widespread dissemination of the disease among children and teenagers (38).EHEC strains may express two different Stx types. Stx1 is virtually identical to Stx produced by Shigella dysenteriae, while Stx2 shows only 56% homology to Stx1 at the amino acid sequence level (14, 33, 51). Both toxin types are formed by one A subunit and five B subunits, which bind to glycosphingolipid receptors, such as globotriaosyl ceramide (Gb3), on host cell membranes and promote retrograde toxin transport through the Golgi complex and endoplasmic reticulum. In the cell cytoplasm, Stx2 subunit A is processed into two fragments; one of them (A1) is endowed with N-glycosidase activity, which depurinates a specific adenine residue of the eukaryotic 28S rRNA, inhibits protein synthesis, and induces apoptosis of the target cell (18, 51).After ingestion and gut colonization, Stx molecules are released by the bacterial cells and translocate across the gut epithelium to reach, via the bloodstream, capillary endothelial cells at renal glomeruli, where the most relevant tissue damage occurs (33, 45, 50). Epidemiological data indicate that individuals infected with Stx2-producing bacterial strains, and some closely related variants, have a high probability of developing HUS (45, 50). In addition, Stx2 expression has been shown to increase gut colonization by bacterial cells due to induction of increased receptor expression by enterocytes (39).So far, there is no effective prophylactic or therapeutic approach for the prevention of HUS development among EHEC-infected individuals. The treatments available involve platelet transfusion in cases of severe anemia, hemodialysis, and supportive care (7, 50). A more direct anti-Stx treatment under clinical or preclinical evaluation involves the use of synthetic Stx glycolipid receptor analogs and humanized anti-Stx monoclonal antibodies (44, 52).Attempts to develop prophylactic anti-HUS vaccines are focused on the generation of Stx-neutralizing antibodies or the blockade of gut colonization. The vaccine strategies based on Stx2 that have been tested under experimental conditions have included DNA vaccines (5, 12), protein-conjugated polysaccharides (28), purified recombinant B subunits (24, 25, 29, 30, 47, 53, 55, 58), and B-subunit-derived synthetic peptides (19, 20). Anti-EHEC vaccine approaches based on the blockade of gut colonization have employed intimin and type III secreted proteins, such as EspA and EspB (3, 37, 54).Live bivalent anti-Stx vaccines based on genetically modified, attenuated Vibrio cholerae or Salmonella enterica serovar Typhimurium strains have been reported to induce anti-StxB antibody responses following oral administration to mice or rabbits (1, 10, 49). Attenuated Salmonella strains, used as orally administered vaccine vectors for the expression of heterologous antigens, show several advantages over conventional parenterally delivered cellular or acellular vaccine formulations (15, 16). Attenuated Salmonella strains are safe, are easily administered by untrained personnel, and, more relevantly, may induce systemic and secreted antigen-specific antibody and cell-based immune responses against self and heterologous antigens. In addition, whole bacterial cells carry on their surfaces several molecular structures known to activate both innate and adaptive immune responses. These molecules, such as lipopolysaccharide and flagellin, act as strong adjuvants, both systemically and at mucosal surfaces.Flagellins, the structural subunit of flagellar filaments, contribute both to the virulence of bacterial pathogens and to the activation of inflammatory responses in mammalian hosts. Bacterial flagellins have been shown to bind both extracellular and intracellular receptors of antigen-presenting cells, leading to inflammation and increased adaptive immune responses, including the generation of antigen-specific antibodies and T cells (2, 26). The strong adjuvant effects of Salmonella flagellins, either when admixed with purified antigens or when used as hybrid proteins genetically fused to the target antigens, have been demonstrated recently (4, 8, 22, 23, 36). However, there is no clear evidence that the expression of flagellin affects the immunogenicity of heterologous antigens expressed by attenuated Salmonella vaccine strains.In the present study, we generated new experimental anti-HUS vaccine formulations based on two recombinant attenuated S. Typhimurium aroA vaccine strains differing in the expression of flagellin. The two strains were genetically modified in order to express a nontoxic Stx2 derivative consisting of the whole Stx2 B subunit and a partially deleted A subunit encompassing the first amino acid of the A1 subunit genetically fused to the whole A2 subunit (Stx2ΔAB). The Stx2ΔAB protein was previously tested in mice immunized with a DNA vaccine (5). The results of the present study show that the S. Typhimurium vaccine strains express and secrete the recombinant toxin and induce both systemic and mucosal anti-StxB antibodies with anti-Stx2 neutralization activity, conferring partial protection against intravenous (i.v.) challenge with Stx2.