aspergillus species are ascomycetes that are classified in the form subdivision deuteromycotina , as many of them do not show a sexual reproductive phase . generally , they are common ubiquitous saprophytes in soil and on dead organic substrates . being classic opportunistic pathogens , invasive infections by aspergillus species almost exclusively develop in immunocompromised patients , while localized infections and allergic bronchopulmonary aspergillosis occur in individuals without immunosuppression . generally , the species aspergillus fumigatus represents the most common inducer of invasive and allergic manifestations , followed by a. terreus , a. flavus , and a. niger [ 1 , 2 ] . invasive aspergillosis ( ia ) considerably contributes to the morbidity and mortality among immunocompromised individuals , including patients with haematological malignancies , recipients of haematological stem cell and solid organ transplants , aids patients , and patients treated with immunosuppressive regimens due to autoimmune diseases . the most important single risk factor is prolonged and profound neutropenia ( < 500 neutrophils/l for more than 10 days ) [ 1 , 46 ] . over the last decades , invasive fungal infections , particularly aspergillosis , have become more frequent due to a higher number of immunocompromised patients ( new chemotherapy regimens , increasing number of solid organ transplant recipients , and immunosuppressive regimens ) and extended survival time in hiv patients ( haart therapy ) [ 1 , 79 ] . on the side of the pathogen , several characteristics and various putative virulence factors that may facilitate the infection have been described for a. fumigatus . it differs from nonpathogenic species by its growth at 37c ; furthermore , it is rapidly growing and has very small conidiospores ( 35  m ) . these include melanin and a hydrophobic protein - coat layer on the surface of conidia that may help to protect them against recognition , ingestion and/or elimination by complement and phagocytes [ 1014 ] . various proteases that may help to pass tissue barriers and to degrade proteins of the immune response are secreted by the fungus [ 1517 ] , and mycotoxins like gliotoxin might also contribute to undermine the host defence [ 1820 ] . the most important path of aspergillus infections is via inhalation of the conidia into the respiratory tract . as conidia of pathogenic aspergillus species are very small , they can be inhaled deeply into the lung and even into the pulmonary alveoli . in immunocompetent individuals , conidia are effectively phagocytosed and eliminated by alveolar macrophages and infiltrating neutrophils [ 12 , 21 , 22 ] , but in the case of immunologic deficits , they are able to germinate and to penetrate the lung tissue , thus causing an invasive pulmonary aspergillosis . infections of the lung are the by far most frequent type of ia . by penetration of blood vessels , aspergillus can disseminate and invade other organs , including the heart , the liver , and the central nervous system ( cns ) . cerebral aspergillosis occurs in 10%20% of all cases of ia and thus is the most common extrapulmonary form . neuropathologic features include hemorrhagic infarcts and/or necrosis , vascular thrombosis , meningitis , granuloma , and formation of solitary as well as multiple abscesses [ 6 , 2325 ] . according to the division of bacterial and mycotic diseases ( dbmd ) , the incidence of aspergillosis is 1 - 2 per 100,000 per year . incidence rates of ia in high - risk populations depend on the respective group and rise up to 24% in patients with prolonged and profound neutropenia . furthermore , ia is the most expensive opportunistic infection in immunosuppressed patients , with annual treating costs in europe of approximately  200 million . in - hospital stays complicated by ia cause additional costs of  75,000 per patient .   despite antimycotic therapy and surgical interventions , the fatality of ia is high and depends on the degree of immunosuppression and on the affected organs . without treatment , the mortality is nearly 100% , while under treatment the overall case - fatality rate is nearby 60% and rises to more than 90% in cases of cns aspergillosis [ 1 , 6 , 26 ] . complement consists of approximately 30 fluid - phase and membrane - bound proteins that cooperate to form the cascade . regulatory factors control and modulate its activity , and cellular receptors mediate the interaction between complement factors and immune cells . representing a potent component of the innate host defence and an interface to adaptive immunity the most outstanding roles are the direct and indirect defence against infections , the stimulation and regulation of b- and t - cell response , and the disposal of debris [ 2731 ] . hepatocytes are the main producers of complement factors ; however , several other cell types participate in the synthesis . activation of the complement system is triggered by a multiplicity of  danger signals ,  such as pathogen - associated molecular patterns ( pamps ) , antigen / antibody complexes , and the presence of transformed cells , apoptotic cells , or cell debris . three different activation pathways start the complement cascade , all of them resulting in the cleavage of the central complement factor c3 by proteolytic enzyme complexes ( c3 convertases ) , and subsequently leading to the common terminal pathway ( figure 1 ) . in the classical pathway , binding of complement factor c1q to immunoglobulin class g or m ( igg , igm ) of antigen - antibody complexes represents the initial step . alternatively , the globular heads of c1q can interact with microbial surfaces that had been covered by pentraxins , a class of soluble pattern recognition molecules . the thereby induced conformational changes of c1q subsequently activate the associated proteases c1r and c1s , which cleave the factors c4 and c2 . the resulting fragments form the c3 convertase c4b2a .   in the lectin pathway , foreign carbohydrate molecules on the surface of pathogens are recognized by mannose - binding lectin ( mbl ) or the related ficolins . mbl - associated serine proteases ( masps ) cleave c4 and c2 , and the fragments build up the c3 convertase c4b2a , which is identically equal to the one of the classical pathway . ficolin-2 can also interact with pentraxin - covered microbes , thus starting the lectin pathway in an alternative manner . interestingly , mbl was recently described to support c3 cleavage by a c2 bypass mechanism , which results in activation of the alternative pathway . the alternative pathway is triggered via activating foreign surfaces and creates an amplification loop by spontaneous reaction of c3 with h2o ( c3(h2o ) ) ; alternatively , c3b generated by the other pathways represents the starting trigger . surface - bound c3b associates with factor b , which is then cleaved by the plasma serine protease factor d. these steps result in the formation of the c3 convertase c3bbb [ 27 , 36 ] . proteolytic cleavage of c3 by one of the c3 convertases is the common and central step of all three activation pathways . this split generates the fragments c3a and c3b , which are two important components that mediate a multitude of complement functions ( see below ) . the product c3b associates with the c3 convertases , thus forming the c5 convertases , which cleave factor c5 into c5a and c5b . this step initiates a chain of assembly processes of the proteins c6 , c7 , c8 , and c9 . the bound and polymerized c9 units create the terminal complement complex ( tcc ) that can form a pore in the target lipid bilayer , called membrane attack complex ( mac ) . targeted cells , bacteria and viruses die or are inactivated by efficient disruption of the membrane integrity [ 31 , 37 ] . beneath the mac formation and direct pathogen destruction , complement displays several additional antimicrobial mechanisms aiming to neutralize invading microbes and to restore body homeostasis . surface - bound c3b undergoes internal cleavage steps ; the derived products ic3b , c3d , and further , coat and label the pathogens for phagocytosis ( opsonization ) . effector cells with specific membrane - bound complement receptors ( crs ) recognize the opsonizing complement fragments , ingest the labeled pathogens , and eliminate them . furthermore , the interaction of the opsonized particles with cr - bearing immune cells results in their activation and their increased proliferation . the receptor cr3 , a heterodimer of cd11b and cd18 , is regarded to be the most important mediator for complement - driven phagocytosis . being expressed on phagocytes like dendritic cells , neutrophils , macrophages , and microglia , it interacts with ic3b on the pathogen [ 27 , 38 ] . the complement receptors cr3 and cr4 allow adhesion of cells to cells of the same and other cell types , respectively , ( homotypic and heterotypic adhesion ) . immune cells can bind via these receptors to their ligands on endothelium of the blood vessels , a prerequisite for penetration through the vessel wall into the tissue and migration to the site of infection and inflammation . surface - bound ic3b can be further cleaved proteolytically to generate the opsonizing fragment c3d . binding of c3d - opsonised pathogens to the corresponding complement receptor cr2 ( cd35 ) on b cells induces cross - linkage with the b cell receptor complex , a process that lowers the threshold for b cell activation by the specific antigen by several orders of magnitude . cleavage of c3 and c5 generate the potent anaphylatoxins c3a and c5a , respectively , which exert several biological functions by binding to their corresponding cellular receptors c3ar , c5ar ( cd88 ) , and c5l2 . they provoke chemotactic attraction of immune cells to the site of infection and an increase of vascular permeability [ 40 , 41 ] . furthermore , c3a and c5a trigger an efficient proinflammatory response by stimulating cytokine synthesis and secretion [ 40 , 41 ] . various cell types harboring the corresponding anaphylatoxin receptors on their surface react on ligand binding with cell activation , stimulation of cell specific signaling pathways , or of oxidative burst [ 27 , 42 ] . the complement cascade needs a tight control to prevent host damage by cell / tissue lysis and excessive inflammation . a variety of both soluble and membrane - bound regulators can influence all steps of the complement cascade , with the c3/c5 convertases as main control targets [ 27 , 37 , 4345 ] . under normal conditions , these regulators should protect all body cells against auto - attack by the complement system . the serine protease factor i cleaves both c4b and c3b and is thereby supported by various cofactor molecules . c4 binding protein ( c4  bp ) and factor h ( fh ) are fluid - phase proteins that enable the cleavage of c4b and c3b , respectively , moreover , the membrane - anchored molecules complement receptor 1 ( cr1 , cd35 ) and membrane cofactor protein ( mcp , cd46 ) support the degradation of both c3b and c4b . in addition , fh and c4  bp accelerate the decay of assembled c3 convertase ; cr1 affects both c3 and c5 convertases . decay accelerating factor ( daf , cd55 ) is another notable membrane - bound regulator that efficiently prevents the assembly and promotes the disintegration of both c3 and c5 convertases [ 27 , 4345 ] .   in the terminal pathway , the membrane - anchored cd59 ( protectin ) binds to c8 in the c5b-8 complex and thus inhibits further incorporation and polymerization of c9 units to form the mac [ 27 , 4345 ] . the potency of complement represents a valuable tool to attack invading pathogens and to defend the host against penetration and dissemination . one particular advantage of complement lies in the fact that activation can start within seconds after contact with the microbe and ends with a multifaceted spectrum of antimicrobial reactions . however , the fact that microbial infections occur in a considerable proportion , already implicates that the pathogens have developed appropriate counterstrategies to avoid elimination , thus starting a vicious circle of reaction and counterreaction . furthermore , the antimicrobial effector mechanisms of the complement system might also harbour harmful consequences for the affected host . as known from several infectious and noninfectious diseases , chronic or exceeding complement - mediated inflammation putative mechanisms for such complement - induced tissue damage may include a fulminant inflammatory reaction and opsonization of surrounding  bystander  cells with subsequent lysis . aspergillus conidia and hyphae activate the complement system via all three pathways [ 4648 ] ( figure 1 ) . initiation of the complement cascade by resting conidia is mediated predominantly by the alternative pathway . however , when the conidia begin to swell and transform into hyphae , there is a progressive involvement of the classical pathway . these differences in the activation pathways are reflected by different kinetics ; the slowest initiation is seen with resting conidia . furthermore , mbl as a pattern recognition molecule of the lectin pathway is able to bind to carbohydrate structures on the surface of aspergillus and promotes complement activation via the lectin pathway , which results in the deposition of c4 . as mentioned above , mbl can support c3 cleavage by a c2 bypass mechanism after contact with a. fumigatus conidia , resulting in activation of the alternative pathway and avoiding formation of the classical pathway c3 convertase . this mechanism is not restricted to a. fumigatus , but can also take place in the presence of a. terreus , a. niger , and a. flavus . mbl generally seems to be a molecule of high significance for innate defence against a range of pathogens . in several studies , it was shown to bind to various sugars on the surfaces of viruses , bacteria , yeasts , fungi , and protozoa [ 4851 ] . further evidence for the crucial role of mbl arises from findings in patients with chronic necrotizing pulmonary aspergillosis and mouse models of pulmonary aspergillosis [ 52 , 53 ] ; these facts suggest mbl as a promising molecule for prophylaxis and therapeutical treatment ( see below ) . a further mechanism for complement activation driven by aspergillus involves the interaction with the pattern recognition molecule pentraxin-3 ( ptx-3 ) . when a. fumigatus is opsonized with ptx-3 , the complement cascade can be activated either by interaction between ptx-3 and c1q via the classical pathway , or by interaction between ptx-3 and ficolin-2 via the lectin pathway . after seroconversion , anti - aspergillus antibodies in the serum can trigger the start of the classical complement pathway . the thesis that complement represents a central tool in antifungal host defence is supported by several findings . furthermore , recognition by the complement system and activation of the cascade seems to interfere with efficient dissemination in the host . this conclusion is strongly indicated by the fact that the level of complement deposition on different aspergillus species correlates inversely with their pathogenicity : highly virulent species like a. fumigatus and a. flavus bind less c3 on their surface than nonpathogenic species like a. glaucus or a. nidulans . the antimicrobial potency of the complement cascade appears to be independent from direct killing via formation of a mac ; presumably , the thick fungal cell wall block the formation of a pore by the c9 polymers and the subsequent lysis of the cells . opsonization of the fungal surface with c3-derived fragments are presumably the most relevant complement - associated weapon , stimulating efficient phagocytosis or release of damaging compounds , oxidative burst and killing by monocytes , bronchoalveolar macrophages , and polymorphonuclear cells [ 46 , 47 , 57 ] . the capacity to opsonize pathogens and to exert antifungal effects strictly depends on the available complement levels in the respective compartment of the body . the complement concentrations in the central nervous system ( cns ) are low and thus only allow a rather weak deposition . consequently , the complement amounts in the cerebrospinal fluid are unable to induce a significant oxidative burst in immune cells and to result in reduced fungal viability , thus making the cns a highly vulnerable organ . however , the brain cells react to the fungal presence with an upregulation of complement synthesis to enable better opsonization and therefore a more efficient clearance of the fungus . mice deficient in complement factor c5 can exert the early opsonization processes with c3 fragments , but are unable to fulfil the complete cascade . when infected with a. fumigatus , these mice show decreased resistance and lower 50% lethal conidia dosage for a disseminated infection [ 59 , 60 ] . since this enhanced susceptibility is unlikely to be due to absent mac formation in the fungal cell membrane , it might be supposed that the inability to form the anaphylatoxin c5a could be the relevant deficit in these mice [ 59 , 60 ] . c5a exerts a wide range of proinflammatory effects ; by binding to its receptor c5ar , c5a recruits inflammatory cells to the site of infection , enhances cellular adhesion , and stimulates oxidative metabolism . in addition , c5a can trigger the release of lysosomal enzymes and of inflammatory mediators such as tumor necrosis factor - alpha ( tnf- ) and interleukin-6 ( il-6 ) [ 61 , 62 ] . furthermore , a higher susceptibility to aspergillosis in c5-deficient mice might be attributed to missing tcc . low doses of this soluble complex were shown to bind to the membrane of a range of cell types , thereby triggering various effects like activation , rescue from apoptosis , and secretion of prostaglandins , which are important regulators of the immune response [ 6367 ] . the potency of aspergillus to cope with the complement system and to undermine its mechanisms for elimination determines how successful the fungus can establish an infection . aspergillus has developed a complex repertoire of effector mechanisms for this purpose ( table 1 ) . single or multiple abscess formation is a characteristic feature of aspergillosis , particularly in the central nervous system ( cns ) . the fungal hyphae are found in brain blood , vessels with invasion through vascular walls into adjacent parenchymal tissue . fungal brain abscesses may arise from these sites of localized parenchymal infection . in this case , white blood cells collect in the affected part of the brain , and fibrous tissue forms around this area , creating a mass . cns abscesses typically present with headache , focal neurological abnormalities , and/or seizure , which is the consequence of local destruction or compression of adjacent brain tissue . mature fungal abscesses exhibit a central necrotic area with fungal hyphae , surrounded by a capsule of newly formed fibrous tissue . the formation of abscesses represents a host mechanism to inhibit further spreading of invading pathogens . however , this  sealing off  not only inhibits fungal dissemination , but also forms some kind of protection shields against the complement attack . immunohistochemical staining revealed that effect : whereas the fibrous surrounding tissue was intensely stained for complement proteins , the central necrotic area contained only minor complement concentrations . no deposition of complement factors on the fungal surface in the abscess was visible , implying that the encapsulation protects the fungus within the abscess from any efficient complement attack . putative complement recognition sites on the conidial surface of a. fumigatus are optimally masked to minimize the stimulus for complement activation . experiments aiming to identify the relevant fungal structure indicated that melanin could play a substantial role for masking ; for this purpose , knock out mutants lacking enzymes of the melanin biosynthesis pathway were used [ 10 , 11 , 76 ] . disruption of the gene alb1 , which encodes a polyketide synthase in the synthesis of melanin , results in increased opsonization of the conidia with c3 and in a better ingestion by human neutrophils . deposition of pigments on the conidial surface might mask the c3 binding sites , and disruption of the alb1 gene might expose these sites and thus allow enhanced c3 binding . a mouse model confirms this function of alb1 in fungal pathogenesis , since the alb1-deficient mutant of a. fumigatus turned out to be less virulent than the wild - type fungus [ 11 , 76 ] . inactivation of the gene for the pigmentation protein arp1 similarly increased the deposition of c3 on conidia . this pigment seems to be a central element of aspergillus against the host defence , as it is also involved in scavenging reactive oxygen species ( ros ) and inhibits the acidification of phagolysosomes of alveolar macrophages , monocyte - derived macrophages , and human neutrophil granulocytes after ingestion of conidia [ 12 , 13 , 77 ] . as mentioned above , the activity of the complement cascade is strictly limited by several fluid - phase inhibitors . immunofluorescence analysis , adsorption assays and flow cytometry studies showed that aspergillus acquires fh , factor h - like protein 1 ( fhl-1 ) , factor h - related protein 1 ( fhr-1 ) , and c4  bp from the host [ 70 , 71 ] . fhl-1 is a splicing product of the fh gene , and fhr-1 is a related protein belonging to the fh family . bound to the conidial surface , fh maintained its regulatory activity and could act as a cofactor for the factor i - mediated cleavage of c3b . as a consequence of covering the fungal surface with these complement inhibitors , all three pathways might be downmodulated . the attachment molecules on aspergillus are not yet known , whereas the corresponding binding regions within fh were recently described . one of them was identified within n - terminal short consensus repeats ( scrs ) 1 to 7 and a second one within c - terminal scr 20 . a. fumigatus not only acquires complement inhibitors from the host , but also produces and releases its own soluble factor that inhibits complement activation and opsonization of the fungus [ 72 , 73 ] . this complement inhibitor ( ci ) , which is also synthesized by a. flavus , selectively abolishes activation of the alternative pathway and interferes with c3b - dependent phagocytosis and killing . the exact chemical composition of ci is as yet unknown ; it contains 15% protein and 5% polysaccharide . recent own results raise the possibility that this described ci or a closely related activity also contributes to the pathogenesis in cerebral aspergillosis , since immunohistochemical studies show deposition of c1q and c4 , but not of c3 , on the fungal hyphae in the cns . studies by sturtevant revealed the synthesis of a proteolytic enzyme that is able to degrade c3 ( , reviewed in : ) . this is confirmed by own experiments showing complement - degrading proteolysis in the supernatant of aspergillus when grown in cerebrospinal fluid ( csf ) . the fungus - induced degradation of complement in csf evoked a drastic reduction of the opsonization of the fungal hyphae . in parallel , the fungal serine protease alp1 might participate in complement degradation and thus be partly responsible for the complement evasion [ 15 , 17 ] . to date , degradation of c1q , c3 , c4 , c5 , mbl , and factor d were shown [ 15 , 17 ] . the high morbidity and lethality of invasive aspergillosis strongly demands for an expansion of the current treatment options . the antimycotic therapy might be completed by new approaches aiming to strengthen the host immune response against the fungus . putative approaches could be to increase the available complement concentrations or to improve the efficiency of complement attack by undermining the fungal evasion strategies . appropriate strategies that target the complement system may aim to the following .   our own studies about cerebral aspergillosis showed a clear correlation between the complement levels in the csf and the capacity of csf to opsonize fungal hyphae and designate them for phagocytic killing . a therapeutic increase of mbl concentrations in invasive aspergillosis might be an appropriate approach , since patients with chronic necrotizing pulmonary aspergillosis show more frequently mbl haplotypes that encode for low levels of the protein than healthy control persons . further support comes from a murine model of invasive pulmonary aspergillosis : those mice with externally administered recombinant mbl reveal better survival , compared to untreated animals . detailed studies confirmed that mbl - treated mice show a significant increase in the levels of the proinflammatory cytokines tnf- and il-1 , together with a marked decrease of anti - inflammatory il-10 and of fungal hyphae in the lung . blocking of the fungal surface pigments by specific antibodies or peptides might be a hypothetical approach that could help to expose the c3 binding sites and thus improve complement deposition and ingestion of conidia by phagocytes . another approach might target the acquisition of the negative complement regulators fh , fhl-1 , and c4  bp to the fungal surface . for candida albicans , some molecules that bind c4b and fh have recently been identified [ 78 , 79 ] , while the attachment sites on aspergillus are still unknown but might include related molecules . blocking antibodies , designed peptides or other inhibitors against these complement regulator binding molecules might help to make the fungus more vulnerable towards complement attack . a similar approach might be developed for the aspergillus - derived complement inhibitor described by washburn [ 72 , 73 ] . our own results open the possibility to neutralize the fungal protease(s ) that is / are secreted by aspergillus to degrade complement proteins . two different strategies were tested by first in vitro experiments : the neutralization of the protease by specific inhibitors or interference with the production of this proteolytic enzyme . in our studies , we could prevent the complement degrading activity by serine protease inhibitors . however , a therapeutically used protease inhibitor must be highly specific , since a general block of serine proteases might be fatal for the host . alternatively , our experiments exhibited that the secretion of complement - degrading enzymes strictly depends on the availability of nitrogen sources . thus , the supply of amino acids in the infected host might downmodulate the secretion of the relevant fungal protease that cleaves the complement factors of the host . despite new antifungal drugs and improved medical treatment , invasive aspergillosis remains a dangerous threat for immunocompromised patients , as the innate immune defence is the most crucial weapon against this infection . the complement system is of particular importance , as it harbours multiple effects against infectious diseases , bridges the elements of the human defence network by a multitude of factors , and helps to preserve the homeostasis of the body . the presence of fungal pathogens is detected by different pattern recognition molecules ; three pathways guarantee the activation of the complement cascade by resting , swollen , and germinating conidia as well as by hyphae of aspergillus . direct lysis of fungal cells by the membrane attach complex ( mac ) appears to be of minor importance for the antifungal defence . presumably , attraction and activation of immune cells ( monocytes , pulmonary macrophages , and polymorphonuclear neutrophils ) are the most essential mechanisms . anaphylatoxins ( c3a , c5a ) chemotactically recruit immune cells to the site of the infection and induce further inflammatory reactions . opsonization of conidia and hyphae with complement fragments like c3b and ic3b mediate phagocytosis , oxidative burst , and release of damaging compounds by binding to corresponding receptors on immune cells . however , highly virulent aspergillus species have evolved mechanisms to evade the attack by complement . they hide from recognition , acquire complement regulatory molecules from the host , and secrete proteases to degrade complement factors . the multifaceted interactions between complement and aspergillus represent promising approaches for future therapeutic strategies that may help to improve the outcome of invasive aspergillosis .
invasive aspergillosis shows a high mortality rate particularly in immunocompromised patients . perpetually increasing numbers of affected patients highlight the importance of a clearer understanding of interactions between innate immunity and fungi . 
 innate immunity is considered to be the most significant host defence against invasive fungal infections . 
 complement represents a crucial part of this first line defence and comprises direct effects against invading pathogens as well as bridging functions to other parts of the immune network . 
 however , despite the potency of complement to attack foreign pathogens , the prevalence of invasive fungal infections is increasing . two possible reasons 
 may explain that phenomenon : first , complement activation might be insufficient for an effective antifungal defence in risk patients ( due to , e.g. , low complement levels , poor recognition of fungal surface , or missing interplay with other immune elements in immunocompromised patients ) . 
 on the other hand , fungi may have developed evasion strategies to avoid recognition and/or eradication by complement . 
 in this review , we summarize the most important interactions between aspergillus and the complement system . 
 we describe the various ways of complement activation by aspergillus and the antifungal effects of the system , and also show proven and probable mechanisms of aspergillus for complement evasion .