Complement inhibition by gram-positive pathogens: molecular mechanisms and therapeutic implications

The plasma proteins of the complement system are essential in the innate immune response against bacteria. Complement labels bacteria with opsonins to support phagocytosis and generates chemoattractants to attract phagocytes to the site of infection. In turn, bacterial human pathogens have evolved different strategies to specifically impair the complement response. Here, we review the large arsenal of complement inhibitors produced by the gram-positive pathogens Staphylococcus aureus and Group A Streptococcus. We discuss how these bacterial molecules provide us with new tools to treat both infectious and inflammatory disease conditions in humans.     

Riemerella anatipestifer extracellular protease S blocks complement activation via the classical and lectin pathways

Riemerella anatipestifer (RA) is the causative agent of infectious serositis in ducklings and other avian species. It is difficult to control the disease due to its 21 serotypes, poor cross-protection, and bacterial resistance to antimicrobial agents. The complement system is an important component of the innate immune system. However, bacterial pathogens exploit several strategies to evade detection by the complement system. Here, we purified and identified a 59-kDa RA extracellular protease S (EcpS) consisting of a gelatinase. In this study, we aimed to determine how EcpS interferes with complement activation and whether it could block complement-dependent neutrophil function. We found that EcpS potently blocked RA phagocytosis and killing by duck neutrophils. Furthermore, EcpS inhibited the opsonization of bacteria by complement 3b. EcpS specifically blocked complement 3b and complement 4b deposition via the classical and lectin pathways, whereas the alternative pathway was not affected. In summary, we show that RA can survive the bactericidal activity of the duck complement system. These results indicate that RA has evolved mechanisms to evade the duck complement system that may increase the efficiency by which this pathogen can gain access and colonize the inner tissues where it may cause severe infections.     

Bacterial DNA Induces the <Emphasis Type="Italic">Complement</Emphasis> System Activation in Serum and Ascitic Fluid from Patients with Advanced Cirrhosis

Translocation of intestinal bacteria to ascitic fluid is, probably, the first step in the development of spontaneous bacterial peritonitis in patients with cirrhosis. Proteins of the complement system are soluble mediators implicated in the host immune response to bacterial infections and its activation has been traditionally considered to be an endotoxin-induced phenomenon. The aim of this study was to compare the modulation of these proteins in response to the presence of bacterial DNA and/or endotoxin in patients with advanced cirrhosis and ascites in different clinical conditions. Groups I and II consisted of patients without/with bacterial DNA. Group III included patients with spontaneous bacterial peritonitis and Group IV with patients receiving norfloxacin as secondary long-term prophylaxis of spontaneous bacterial peritonitis. Serum and ascitic fluid levels of endotoxin and truncated residues of the complement system were measured by ELISA. The complement system is triggered in response to bacterial DNA, as evidenced by significantly increased levels of C3b, membrane attack complex, and C5a in patients from Groups II and III compared with patients without bacterial DNA (Group I) and those receiving norfloxacin (Group IV). Gram classification did not further differentiate the immune response between patients within groups II and III, even though endotoxin levels were, as expected, significantly higher in patients with bacterial DNA from gram-negative microorganisms. The complement protein activation observed in patients with bacterial DNA in blood and ascitic fluid is indistinguishable from that observed in patients with spontaneous bacterial peritonitis and may occur in an endotoxin-independent manner.     

Activation and control of complement, inflammation, and infection associated with the use of biomedical polymers

It is generally acknowledged that artificial biomaterials are much less immunologically active than transplants or tissue derived biomaterials. However, activation of both the coagulation cascade and the complement system is a common occurrence when human blood is exposed to biomaterial surfaces during extracorporeal procedures, such as renal hemodialysis or cardiopulmonary bypass. Both individual and collective activation of these cascades often produce local and systemic effects. A number of complement activation products function as the mediators of inflammation. They serve as ligands for specific receptors on polymorphonuclear leukocytes, monocytes, macrophages, mast cells, and other cells. Such an interaction leads to induction of cellular responses in adhered cells, including release of oxidative products, lysosomal enzymes, or both, which often contribute to a number of pathologic conditions. Most pathogens invading the human body are attacked by the immune system directly following entry, especially when they are in contact with blood. However, bacteria and parasites have developed a large number of specific strategies to overcome immune defense among others by avoiding either recognition or eradication by complement. In this aspect, of concern are several microorganisms responsible for formation of antibiotic resistant biofilms on biomaterial surfaces, namely Staphylococcus epidermidis, Staphylococcus aureus, and Pseudomonas aeruginosa.     

Involvement of complement pathways in patients with bacterial septicemia

The complement system is a major humoral portion of the innate immune system, playing a significant role in host defence against microorganisms. The biological importance of this system is underlined by the fact that at least three different pathways for its activation exist, the classical, the MBL and the alternative pathway. To elucidate the involvement of the classical and/or the MBL pathway during bacterial septicemia, 32 patients with gram-positive and 30 patients with gram-negative bacterial infections were investigated. In patients with gram-positive bacteria, a significant consumption of C1q (p=0.005) but not of mannose-binding lectin (MBL) (p=0.2) was found during the acute phase of infection. In contrast, in patients with gram-negative bacterial infections, a significant reduction of MBL (p=0.002) and only a moderate, less significant reduction of C1q (p=0.03) were observed. As a model for the binding of MBL to gram-negative bacteria, Salmonella strains with defined mutations in their lipopolysaccharide (LPS) structure were used. The comparison of the binding of MBL to these Salmonella strains with that of the corresponding isolated LPS forms bound to microtiter plates revealed a similar binding pattern, supporting the interpretation that LPS on the surface of gram-negative bacteria is the major acceptor molecule for MBL on these bacteria, which according to our results obviously also takes place during gram-negative bacterial septicaemia. Furthermore, we were able to demonstrate that MBL bound to LPS was able to initiate activation of the complement cascade as measured by the occurrence of the cleavage product C4c.     

Complement escape of human pathogenic bacteria by acquisition of complement regulators

Pathogenic micro-organisms employ a broad range of strategies to survive in and to persistently infect the human host. Far from being completely understood by which highly sophisticated means invading pathogens overcome the host's destructive immune defence, there is a growing body of evidence on particular mechanisms which play a pivotal role for immune evasion. This review focuses on evasion of medically and scientifically important bacteria by acquisition of host derived fluid-phase complement regulatory proteins, in particular factor H, FHL-1, and C4b binding protein. Expression of microbial surface molecules binding to human complement regulators and thus fixing them in a functionally active state allows pathogens to inhibit and finely regulate complement activation directly on their surface. Further studies on the utilization of host complement regulatory proteins will likely have a marked impact on a more efficient and specific clinical treatment.     

Evasion of host cell defense mechanisms by pathogenic bacteria

From an immunological viewpoint, invasion of pathogenic bacteria into a susceptible host poses a potential life-threatening situation and thus has to be met with all weapons that are available. A crucial component of host defense mechanisms is the macrophage. The scavenger activity of this cell ensures the uptake and destruction of bacteria in phagolysosomes, on the one hand, and activation of the adaptive component of the immune system through presentation of bacterial antigens, on the other hand. From the bacterial perspective, entry into a phagolysosome is usually fatal and many pathogens have developed strategies that circumvent the destructive environment of this organelle. Such evasion strategies often exploit normal host cell function. Understanding these survival strategies will deepen our insight of the pathogenesis of infection as well as host cell biology.     

Cytokine induction by Gram-positive bacteria

Despite similar clinical relevance of Gram-positive and Gram-negative infections, immune activation by Gram-positive bacteria is by far less well understood than immune activation by Gram-negative bacteria. Our group has made available highly purified lipoteichoic acids (LTA) as a key Gram-positive immunostimulatory component. We have characterized the reasons for lower potency of LTA compared to Gram-negative lipopolysaccharide (LPS), identifying lack of IL-12/IFNgamma induction as a general characteristic of TLR2 agonists, and need for presentation of LTA on surfaces for enhanced immunostimulatory potency, as major aspects. Aspects of chemokine induction, where LTA is more potent than LPS, have been addressed. Furthermore, novel complement and plant defence activation, as well as CD36 as a new LTA receptor, were identified. The bacterial costimuli and modulators of LTA inducible responses are being investigated: LTA isolated from so far 16 bacterial species, although different in structure, behave remarkably similar while whole live and killed bacteria differ with regard to the pattern of induced responses. The purification and characterization of the respective components of the bacterial cell wall has begun.     

Anaphylatoxins

Activation of the complement system plays a crucial role in the pathogenesis of infection and inflammation. Especially the complement activation products C3a and C5a, known as the anaphylatoxins, are potent proinflammatory mediators. In addition to their evident role in innate immunity, it is clear that the anaphylatoxins also play a role in regulation of adaptive immune responses. The anaphylatoxins play a role in a variety of infectious and inflammatory diseases like sepsis, ischemia-reperfusion injury, immune complex diseases, and hypersensitivity diseases like asthma. In this review we discuss the role of anaphylatoxins in infection and inflammation. Furthermore, we focus on bacterial complement evasion strategies that can provide tools for further research on pathogenesis of infectious diseases and a better understanding of the role of complement and anaphylatoxins in infection and inflammation.     

Stages of meningococcal sepsis simulated in vitro, with emphasis on complement and Toll-like receptor activation

The clinical presentation of meningococcal disease is closely related to the number of meningococci in the circulation. This study aimed to examine the activation of the innate immune system after being exposed to increasing and clinically relevant concentrations of meningococci. We incubated representative Neisseria meningitidis serogroup B (ST-32) and serogroup C (ST-11) strains and a lipopolysaccharide (LPS)-deficient mutant (the 44/76 lpxA mutant) in human serum and whole blood and measured complement activation and cytokine secretion and the effect of blocking these systems. HEK293 cells transfected with Toll-like receptors (TLRs) were examined for activation of NF-kappaB. The threshold for cytokine secretion and activation of NF-kappaB was 10(3) to 10(4) meningococci/ml. LPS was the sole inflammation-inducing molecule at concentrations up to 10(5) to 10(6) meningococci/ml. The activation was dependent on TLR4-MD2-CD14. Complement contributed to the inflammatory response at >or=10(5) to 10(6) meningococci/ml, and complement activation increased exponentially at >or=10(7) bacteria/ml. Non-LPS components initiated TLR2-mediated activation at >or=10(7) bacteria/ml. As the bacterial concentration exceeded 10(7)/ml, TLR4 and TLR2 were increasingly activated, independent of CD14. In this model mimicking human disease, the inflammatory response to N. meningitidis was closely associated with the bacterial concentration. Therapeutically, CD14 inhibition alone was most efficient at a low bacterial concentration, whereas addition of a complement inhibitor may be beneficial when the bacterial load increases.     

Contribution of the complement Membrane Attack Complex to the bactericidal activity of human serum

Direct killing of Gram-negative bacteria by serum is usually attributed to the Membrane Attack Complex (MAC) that is assembled upon activation of the complement system. In serum bactericidal assays, the activity of the MAC is usually blocked by a relatively unspecific method in which certain heat-labile complement components are inactivated at 56 °C. The goal of this study was to re-evaluate MAC-driven lysis towards various Gram-negative bacteria. Instead of using heat-treatment, we included the highly specific C5 cleavage inhibitor OmCI to specifically block the formation of the MAC. Using a C5 conversion analysis tool, we monitored the efficacy of the inhibitor during the incubations. Our findings indicate that ‘serum-sensitive’ bacteria are not necessarily killed by the MAC. Other heat-labile serum factors can contribute to serum bactericidal activity. These unidentified factors are most potent at serum concentrations of 10% and higher. Furthermore, we also find that some bacteria can be killed by the MAC at a slower rate. Our data demonstrate the requirement for the use of specific inhibitors in serum bactericidal assays and revealed that the classification of serum-sensitive and resistant strains needs re-evaluation. Moreover, it is important to determine bacterial viability at multiple time intervals to differentiate serum susceptibility between bacterial species. In conclusion, these data provide new insights into bacterial killing by the humoral immune system and may guide future vaccine development studies for the treatment of pathogenic serum-resistant bacteria.     

Complement therapeutic strategies in trauma,hemorrhagic shock and systemic inflammation – closing Pandora’s box?

After severe trauma, the immune system is challenged with a multitude of endogenous and exogenous danger molecules. The recognition of released danger patterns is one of the prime tasks of the innate immune system. In the last two decades, numerous studies have established the complement cascade as a major effector system that detects and processes such danger signals. Animal models with engineered deficiencies in certain complement proteins have demonstrated that widespread complement activation after severe injury culminates in complement dysregulation and excessive generation of complement activation fragments. Such exuberant pro-inflammatory signaling evokes systemic inflammation, causes increased susceptibility to infections and is associated with a detrimental course of the disease after injury. We discuss the underlying processes of such complementopathy and recapitulate different intervention strategies within the complement cascade. So far, several orthogonal anti-complement approaches have been tested with varying success in a large number of rodent, in several porcine and few simian studies. We illustrate the different features among those intervention strategies and highlight those that hold the greatest promise to become potential therapeutic options for the intricate disease of traumatic injury.     

Dichotomy between opsonization and serum complement activation by encapsulated staphylococci.

Previous studies have demonstrated that encapsulated Staphylococcus aureus strains are not effectively opsonized by the serum complement system. Encapsulated staphylococci thereby "resist phagocytosis." To test whether this phenomenon might be explained by an inability of encapsulated strains to activate complement, the relationship between staphylococcal opsonization and serum complement activation was studied. Although encapsulation was found to interfere with opsonization by pooled human serum (human polymorphonuclear leukocytes phagocytized significantly fewer encapsulated bacteria than unencapsulated bacteria after incubation in this opsonic source), encapsulated (S. aureus M and Smith diffuse) and unencapsulated (S. aureus M variant and Smith compact) strains had similar capacities for complement activation as measured by C3-C9 consumption. When C2-deficient and immunoglobulin-deficient sera were studied, again C3-C9 consumption was not influenced by the presence or absence of a capsule. In addition, C3 was detected on the surface of both S. aureus M and M variant strains after incubation in pooled serum and staining with fluorescein-conjugated anti-C3 antibody. Thus, encapsulated staphylococci are not effectively opsonized even though complement is activated and C3 is present on the bacterial surface. The exact mechanism by which the capsule interferes with opsonization is still not known; however, inhibition of complement activation appears not to be the explanation of this phenomenon.     

Biofilm Formation Avoids Complement Immunity and Phagocytosis of Streptococcus pneumoniae

Streptococcus pneumoniae is a frequent member of the microbiota of the human nasopharynx. Colonization of the nasopharyngeal tract is a first and necessary step in the infectious process and often involves the formation of sessile microbial communities by this human pathogen. The ability to grow and persist as biofilms is an advantage for many microorganisms, because biofilm-grown bacteria show reduced susceptibility to antimicrobial agents and hinder recognition by the immune system. The extent of host protection against biofilm-related pneumococcal disease has not been determined yet. Using pneumococcal strains growing as planktonic cultures or as biofilms, we have investigated the recognition of S. pneumoniae by the complement system and its interactions with human neutrophils. Deposition of C3b, the key complement component, was impaired on S. pneumoniae biofilms. In addition, binding of C-reactive protein and the complement component C1q to the pneumococcal surface was reduced in biofilm bacteria, demonstrating that pneumococcal biofilms avoid the activation of the classical complement pathway. In addition, recruitment of factor H, the downregulator of the alternative pathway, was enhanced by S. pneumoniae growing as biofilms. Our results also show that biofilm formation diverts the alternative complement pathway activation by a PspC-mediated mechanism. Furthermore, phagocytosis of pneumococcal biofilms was also impaired. The present study confirms that biofilm formation in S. pneumoniae is an efficient means of evading both the classical and the PspC-dependent alternative complement pathways the host immune system.     

The classical complement pathway plays a critical role in the opsonisation of uropathogenic Escherichia coli

Urinary tract infection due to uropathogenic Escherichia coli is a common clinical problem. The innate immune system and the uroepithelium are critical in defence against infection. The complement system is both part of the innate immune system and influences the interaction between epithelium and pathogen. We have therefore investigated the mechanism by which uropathogenic E. coli activate complement and the potential for this to occur during clinical infection. The classical pathway is responsible for bacterial opsonisation when complement proteins are present at low concentrations. At higher concentrations the alternative pathway predominates but still requires the classical pathway for its initiation. In contrast the mannose binding lectin pathway is not involved. Early classical pathway components are present in the urine during infection and actively contribute to bacterial opsonisation. The classical pathway could be initiated by anti-E. coli antibodies of IgG or IgM subclasses that are present in urine during infection. Additionally immunoglobulin-independent mechanisms, such as direct C1q binding to bacteria, may be involved. In conclusion, uropathogenic E. coli are readily opsonised by complement in a classical pathway dependent manner. This can occur within the urinary tract during the development of clinical infection.     

Opsonization of Legionella pneumophila in human serum: key roles for specific antibodies and the classical complement pathway

Legionella pneumophila has previously been shown to require serum factors for efficient uptake by phagocytic cells. In this investigation, the roles of specific antibody and complement in phagocytosis of L. pneumophila by human polymorphonuclear leucocytes (PMN) and tissue macrophages were determined. Opsonization was assessed by quantitating the uptake of [3H]-labelled Legionellae. Compared to other Gram-negative and to Gram-positive bacterial species, L. pneumophila was highly resistant to the opsonic activity of normal pooled human serum (PHS). Of 12 donor sera tested, only four promoted significant L. pneumophila uptake when used at full strength. Experiments with immune antibody, and with human sera deficient in immunoglobulins, or the complement components C2, C3, or C5, revealed that L. pneumophila opsonization was dependent on antibody-mediated activation of the classical complement pathway; activation of the alternative pathway could not be detected. At high concentrations, immune antibody alone could adequately opsonize L. pneumophila. Human alveolar and peritoneal macrophages required very similar amounts and types of opsonins for L. pneumophila phagocytosis as did human PMN. Heating L. pneumophila to temperatures greater than or equal to 80 degrees abolished its resistance to opsonization by diluted PHS; however, activation of complement via the alternative pathway or via other antibody-independent routes remained undetectable. These studies show that, in addition to immune antibody, the classical pathway of complement plays an important role in the opsonization of L. pneumophila. The limited ability of these bacteria to interact with human complement provides a likely explanation for their resistance to opsonization and may be partly based on heat-sensitive structures on the surface of L. pneumophila.     

Complement inhibition in cancer therapy

For decades, complement has been recognized as an effector arm of the immune system that contributes to the destruction of tumor cells. In fact, many therapeutic strategies have been proposed that are based on the intensification of complement-mediated responses against tumors. However, recent studies have challenged this paradigm by demonstrating a tumor-promoting role for complement. Cancer cells seem to be able to establish a convenient balance between complement activation and inhibition, taking advantage of complement initiation without suffering its deleterious effects. Complement activation may support chronic inflammation, promote an immunosuppressive microenvironment, induce angiogenesis, and activate cancer-related signaling pathways. In this context, inhibition of complement activation would be a therapeutic option for treating cancer. This concept is relatively new and deserves closer attention. In this article, we summarize the mechanisms of complement activation on cancer cells, the cancer-promoting effect of complement initiation, and the rationale behind the use of complement inhibition as a therapeutic strategy against cancer.     

Role of structural variations of polysaccharide antigens in the pathogenicity of Gram-negative bacteria.

Pathogenic bacteria employ many strategies to overcome the host immune system for extended survival and propagation in their hosts. Components of the bacterial outer-membrane play an important role in this process. When invading the host, Gram-negative bacteria often use a strategy, known as phase variation, that involves a reversible change in antigenic determinants, frequently polysaccharides. This means that the genes encoding the outer-membrane antigens undergo reversible changes within repeated simple DNA sequence motifs. The antigenic structure of the bacterial outer-membrane is influenced by the character of the host immune system, as well as by the targets for bacterial invasion. When the selection pressure of the immune system is absent or weak, bacteria can fail to synthesise the outer-membrane antigens, which are not needed at that time. Smooth-to-rough (S-R) mutation, an economical and often irreversible process in some Gram-negative bacteria, involves the gradual shortening of the lipopolysaccharide (LPS) O-chain. Under certain conditions, e.g., propagation in embryonated eggs or cell lines, some bacteria will cease synthesis of the complete LPS O-chain because it is an energy-demanding process. A type of gradual shortening of the LPS O-chain by Coxiella burnetii, traditionally called phase variation, is used in serological tests for the diagnosis of Q fever. This review discusses the role and function of polysaccharides, especially LPS produced by some Gram-negative bacteria, in bacterial survival.     

Bacterial TIR-containing proteins and host innate immune system evasion

The innate immune system provides the first line of host defence against invading pathogens. Key to upregulation of the innate immune response are Toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns (PAMPs) and trigger a signaling pathway culminating in the production of inflammatory mediators. Central to this TLR signaling pathway are heterotypic protein–protein interactions mediated through Toll/interleukin-1 receptor (TIR) domains found in both the cytoplasmic regions of TLRs and adaptor proteins. Pathogenic bacteria have developed a range of ingenuous strategies to evade the host immune mechanisms. Recent work has identified a potentially novel evasion mechanism involving bacterial TIR domain proteins. Such domains have been identified in a wide range of pathogenic bacteria, and there is evidence to suggest that they interfere directly with the TLR signaling pathway and thus inhibit the activation of NF-κB. The individual TIR domains from the pathogenic bacteria Salmonella enterica serovar Enteritidis, Brucella sp, uropathogenic E. coli and Yersinia pestis have been analyzed in detail. The individual bacterial TIR domains from these pathogenic bacteria seem to differ in their modes of action and their roles in virulence. Here, we review the current state of knowledge on the possible roles and mechanisms of action of the bacterial TIR domains.     

Multiple routes of complement activation by Mycobacterium bovis BCG

Mycobacterium tuberculosis is the leading cause of infectious disease in humans in the world. It evades the host immune system by being phagocytosed by macrophages and residing intracellularly. Complement-dependent opsonisation of extracellular mycobacteria may assist them to enter macrophages. This work examines in detail the mechanisms of complement activation by whole mycobacteria using Mycobacterium bovis BCG as a model organism. M. bovis BCG directly activates the classical, lectin and alternative pathways, resulting in fixation of C3b onto macromolecules of the mycobacterial surface. Investigation into the classical pathway has shown direct binding of human C1q to whole mycobacteria in the absence of antibodies. Most human sera contain IgG and IgM-anti-(M. bovis BCG), and pre-incubation with human immunoglobulin enhances C1q binding to the bacteria. Therefore classical pathway activation is both antibody-independent and dependent. The bacteria also activate the alternative pathway in an antibody-independent manner, but Factor H also binds, suggesting some regulation of amplification by this pathway. For the lectin pathway we have demonstrated direct binding of both MBL and L-ficolin from human serum to whole mycobacteria and subsequent MASP2 activation. H-ficolin binding was not observed. No M. bovis BCG cell surface or secreted protease appears likely to influence complement activation. Together, these data provide a more detailed analysis of the mechanisms by which M. bovis BCG interacts with the complement system.