Functional significance of factor H binding to Neisseria meningitidis

Neisseria meningitidis is an important cause of septicemia and meningitis. To cause disease, the bacterium must successfully survive in the bloodstream where it has to avoid being killed by host innate immune mechanisms, particularly the complement system. A number of pathogenic microbes bind factor H (fH), the negative regulator of the alternative pathway of complement activation, to promote their survival in vivo. In this study, we show that N. meningitidis binds fH to its surface. Binding to serogroups A, B, and C N. meningitidis strains was detected by FACS and Far Western blot analysis, and occurred in the absence of other serum factors such as C3b. Unlike Neisseria gonorrhoeae, binding of fH to N. meningitidis was independent of sialic acid on the bacterium, either as a component of its LPS or its capsule. Characterization of the major fH binding partner demonstrated that it is a 33-kDa protein; examination of insertion mutants showed that porins A and B, outer membrane porins expressed by N. meningitidis, do not contribute significantly to fH binding. We examined the physiological consequences of fH bound to the bacterial surface. We found that fH retains its activity as a cofactor of factor I when bound to the bacterium and contributes to the ability of N. meningitidis to avoid complement-mediated killing in the presence of human serum. Therefore, the recruitment of fH provides another mechanism by which this important human pathogen evades host innate immunity.     

Phase variation of lipopolysaccharide directs interconversion of invasive and immuno-resistant phenotypes of Neisseria gonorrhoeae.

Phase variation of Neisseria gonorrhoeae lipopolysaccharide (LPS) controls both bacterial entry into human mucosal cells, and bacterial susceptibility to killing by antibodies and complement. The basis for this function is a differential sialylation of the variable oligosaccharide moiety of the LPS. LPS variants that incorporate low amounts of sialic acid enter human mucosal epithelial cells very efficiently, but are susceptible to complement-mediated killing. Phase transition to a highly sialylated LPS phenotype results in equally adhesive but entry deficient bacteria which, however, resist killing by antibodies and complement because of dysfunctional complement activation. Phase variation of N. gonorrhoeae LPS thus functions as an adaptive mechanism enabling bacterial translocation across the mucosal barrier, and, at a later stage of infection, escape from the host immune defence.     

Meningococcal factor H-binding protein: implications for disease susceptibility,virulence, and vaccines

Neisseria meningitidis is a human-adapted pathogen that causes meningitis and sepsis worldwide. N. meningitidis factor H-binding protein (fHbp) provides a mechanism for immune evasion by binding human complement factor H (CFH) to protect it from complement-mediated killing. Here, we discuss features of fHbp which enable it to engage human CFH (hCFH), and the regulation of fHbp expression. Studies of host susceptibility and bacterial genome-wide association studies (GWAS) highlight the importance of the interaction between fHbp and CFH and other complement factors, such as CFHR3, on the development of invasive meningococcal disease (IMD). Understanding the basis of fHbp:CFH interactions has also informed the design of next-generation vaccines as fHbp is a protective antigen. Structure-informed refinement of fHbp vaccines will help to combat the threat posed by the meningococcus, and accelerate the elimination of IMD.     

Inefficient Complement System Clearance of Trypanosoma cruzi Metacyclic Trypomastigotes Enables Resistant Strains to Invade Eukaryotic Cells

The complement system is the main arm of the vertebrate innate immune system against pathogen infection. For the protozoan Trypanosoma cruzi, the causative agent of Chagas disease, subverting the complement system and invading the host cells is crucial to succeed in infection. However, little attention has focused on whether the complement system can effectively control T. cruzi infection. To address this question, we decided to analyse: 1) which complement pathways are activated by T. cruzi using strains isolated from different hosts, 2) the capacity of these strains to resist the complement-mediated killing at nearly physiological conditions, and 3) whether the complement system could limit or control T. cruzi invasion of eukaryotic cells. The complement activating molecules C1q, C3, mannan-binding lectin and ficolins bound to all strains analysed; however, C3b and C4b deposition assays revealed that T. cruzi activates mainly the lectin and alternative complement pathways in non-immune human serum. Strikingly, we detected that metacyclic trypomastigotes of some T. cruzi strains were highly susceptible to complement-mediated killing in non-immune serum, while other strains were resistant. Furthermore, the rate of parasite invasion in eukaryotic cells was decreased by non-immune serum. Altogether, these results establish that the complement system recognizes T. cruzi metacyclic trypomastigotes, resulting in killing of susceptible strains. The complement system, therefore, acts as a physiological barrier which resistant strains have to evade for successful host infection.     

Glutamate utilization promotes meningococcal survival in vivo through avoidance of the neutrophil oxidative burst

Polymorphonuclear neutrophil leucocytes (PMNs) are a critical part of innate immune defence against bacterial pathogens, and only a limited subset of microbes can escape killing by these phagocytic cells. Here we show that Neisseria meningitidis, a leading cause of septicaemia and meningitis, can avoid killing by PMNs and this is dependent on the ability of the bacterium to acquire L-glutamate through its GltT uptake system. We demonstrate that the uptake of available L-glutamate promotes N. meningitidis evasion of PMN reactive oxygen species produced by the oxidative burst. In the meningococcus, L-glutamate is converted to glutathione, a key molecule for maintaining intracellular redox potential, which protects the bacterium from reactive oxygen species such as hydrogen peroxide. We show that this mechanism contributes to the ability of N. meningitidis to cause bacteraemia, a critical step in the disease process during infections caused by this important human pathogen.     

A tick mannose-binding lectin inhibitor interferes with the vertebrate complement cascade to enhance transmission of the lyme disease agent

The Lyme disease agent Borrelia burgdorferi is primarily transmitted to vertebrates by Ixodes ticks. The classical and alternative complement pathways are important in Borrelia eradication by the vertebrate host. We recently identified a tick salivary protein, designated P8, which reduced complement-mediated killing of Borrelia. We now discover that P8 interferes with the human lectin complement cascade, resulting in impaired neutrophil phagocytosis and chemotaxis and diminished Borrelia lysis. Therefore, P8 was renamed the tick salivary lectin pathway inhibitor (TSLPI). TSLPI-silenced ticks, or ticks exposed to TSLPI-immune mice, were hampered in Borrelia transmission. Moreover, Borrelia acquisition and persistence in tick midguts was impaired in ticks?feeding on TSLPI-immunized, B.?burgdorferi-infected mice. Together, our findings suggest an essential role for the lectin complement cascade in Borrelia eradication and demonstrate how a vector-borne pathogen co-opts a vector protein to facilitate early mammalian infection and vector colonization.     

Protease-dependent mechanisms of complement evasion by bacterial pathogens

Abstract The human immune system has evolved a variety of mechanisms for the primary task of neutralizing and eliminating microbial intruders. As the first line of defense, the complement system is responsible for rapid recognition and opsonization of bacteria, presentation to phagocytes and bacterial cell killing by direct lysis. All successful human pathogens have mechanisms of circumventing the antibacterial activity of the complement system and escaping this stage of the immune response. One of the ways in which pathogens achieve this is the deployment of proteases. Based on the increasing number of recent publications in this area, it appears that proteolytic inactivation of the antibacterial activities of the complement system is a common strategy of avoiding targeting by this arm of host innate immune defense. In this review, we focus on those bacteria that deploy proteases capable of degrading complement system components into non-functional fragments, thus impairing complement-dependent antibacterial activity and facilitating pathogen survival inside the host.     

The Role of the Exopolyphosphatase PPX in Avoidance by Neisseria meningitidis of Complement-mediated Killing

The complement system is critical for immunity against the important human pathogen Neisseria meningitidis. We describe the isolation of a meningococcal mutant lacking PPX, an exopolyphosphatase responsible for cleaving cellular polyphosphate, a polymer of tens to hundreds of orthophosphate residues found in virtually all living cells. Bacteria lacking PPX exhibit increased resistance to complement-mediated killing. By site directed mutagenesis, we define amino acids necessary for the biochemical activity of meningococcal PPX, including a conserved glutamate (Glu¹¹⁷) and residues in the Walker B box predicted to be involved in binding to phosphate. We show that the biochemical activity of PPX is necessary for interactions with the complement. The relative resistance of the ppx mutant does not result from changes in structures (such as capsule, lipopolysaccharide, and factor H-binding protein), which are known to be required for evasion of this key aspect of host immunity. Instead, expression of PPX modifies the interaction of N. meningitidis with the alternative pathway of complement activation.     

Phosphoethanolamine Modification of Neisseria gonorrhoeae Lipid A Reduces Autophagy Flux in Macrophages

Autophagy, an ancient homeostasis mechanism for macromolecule degradation, performs an important role in host defense by facilitating pathogen elimination. To counteract this host defense strategy, bacterial pathogens have evolved a variety of mechanisms to avoid or otherwise dysregulate autophagy by phagocytic cells so as to enhance their survival during infection. Neisseria gonorrhoeae is a strictly human pathogen that causes the sexually transmitted infection, gonorrhea. Phosphoethanolamine (PEA) addition to the 4'' position of the lipid A (PEA-lipid A) moiety of the lipooligosaccharide (LOS) produced by gonococci performs a critical role in this pathogen’s ability to evade innate defenses by conferring decreased susceptibility to cationic antimicrobial (or host-defense) peptides, complement-mediated killing by human serum and intraleukocytic killing by human neutrophils compared to strains lacking this PEA decoration. Heretofore, however, it was not known if gonococci can evade autophagy and if so, whether PEA-lipid A contributes to this ability. Accordingly, by using murine macrophages and human macrophage-like phagocytic cell lines we investigated if PEA decoration of gonococcal lipid A modulates autophagy formation. We report that infection with PEA-lipid A-producing gonococci significantly reduced autophagy flux in murine and human macrophages and enhanced gonococcal survival during their association with macrophages compared to a PEA-deficient lipid A mutant. Our results provide further evidence that PEA-lipid A produced by gonococci is a critical component in the ability of this human pathogen to evade host defenses.     

Interactions between encapsulated Neisseria meningitidis and host cells.

A major feature of Neisseria meningitidis is its ability to invade human brain meninges. To access the meninges, the bacteria must cross the blood-brain barrier (BBB), which is one of the tightest barriers in the body. Therefore, N. meningitidis must have evolved some type of sophisticated means to bypass the physical properties of this cellular barrier. As N. meningitidis is encapsulated when present in the bloodstream, this review will focus on the mechanisms that encapsulated N. meningitidis has developed to interact with host cells and will suggest ways in which these mechanisms may be helpful for crossing the BBB.     

Multiple Activities of LigB Potentiate Virulence of Leptospira interrogans: Inhibition of Alternative and Classical Pathways of Complement

Microbial pathogens acquire the immediate imperative to avoid or counteract the formidable defense of innate immunity as soon as they overcome the initial physical barriers of the host. Many have adopted the strategy of directly disrupting the complement system through the capture of its components, using proteins on the pathogen's surface. In leptospirosis, pathogenic Leptospira spp. are resistant to complement-mediated killing, in contrast to the highly vulnerable non-pathogenic strains. Pathogenic L. interrogans uses LenA/LfhA and LcpA to respectively sequester and commandeer the function of two regulators, factor H and C4BP, which in turn bind C3b or C4b to interrupt the alternative or classical pathways of complement activation. LigB, another surface-proximal protein originally characterized as an adhesin binding multiple host proteins, has other activities suggesting its importance early in infection, including binding extracellular matrix, plasma, and cutaneous repair proteins and inhibiting hemostasis. In this study, we used a recent model of ectopic expression of LigB in the saprophyte, L. biflexa, to test the hypothesis that LigB also interacts with complement proteins C3b and C4b to promote the virulence of L. interrogans. The surface expression of LigB partially rescued the non-pathogen from killing by 5% normal human serum, showing 1.3- to 48-fold greater survival 4 to 6 d following exposure to complement than cultures of the non-expressing parental strain. Recombinant LigB7'-12 comprising the LigB-specific immunoglobulin repeats binds directly to human complement proteins, C3b and C4b, with respective K(d)s of 43±26 nM and 69±18 nM. Repeats 9 to 11, previously shown to contain the binding domain for fibronectin and fibrinogen, are also important in LigB-complement interactions, which interfere with the alternative and classical pathways measured by complement-mediated hemolysis of erythrocytes. Thus, LigB is an adaptable interface for L. interrogans to efficiently counteract the multiple homeostatic processes of the host.     

Protection of xenogeneic cells from human complement-mediated lysis by the expression of human DAF, CD59 and MCP

CD59 and membrane cofactor protein (MCP, CD46) are widely expressed cell surface glycoproteins that protect host cells from the effect of homologous complement attack. cDNAs encoding human CD59 and MCP cloned from Chinese human embryo were separately transfected into NIH/3T3 cells resulting in the expression of human CD59 and MCP protein on the cell surface. The functional properties of expressed proteins were studied. When the transfected cells were exposed to human serum as a source of complement and naturally occurring anti-mouse antibody, they were resistant to human complement-mediated cell killing. However, the cells remained sensitive to rabbit and guinea pig complement. Human CD59 and MCP can only protect NIH/3T3 cells from human complement-mediated lysis. These results demonstrated that complement inhibitory activity of these proteins is species-selective. The cDNAs of CD59 and MCP were also separately transfected into the endothelial cells (ECs) of the pigs transgenic for the human DAF gene to investigate a putative synergistic action. The ECs expressing both DAF and MCP proteins or both DAF and CD59 proteins exhibited more protection against cytolysis by human serum compared to the cells with only DAF expressed alone.     

Gonococci in vivo and in vitro. Further studies on the host and bacterial determinants of gonococcal resistance to killing by human serum,and by phagocytes

A small M, heat and acid labile, host inducer(s) of gonococcal resistance to complement mediated killing by fresh human serum (-FHS), being purified from red blood cell (RBC) extracts, produced changed in lipopolysaccharide (LPS) structure, surface antigens and proteins; and acquirement of resistance related to loss of a target antigen for bactericidal IgM, possibly LPS components. A 20 kDalt, lipoprotein with a high content of glutamic acid isolated from outer membranes of a gonococcal strain selected in vivo is a determinant of gonococcal resistance to killing by human phagocytes. Somic extracts of gonococci may contain a cytotoxin for human phagocytes.At the 4th International Pathogenic Neisseriae Conference, we reported (Parsons et al. 1985) that conditions in vivo induced phenotypic change leading to gonococcal resistance to complement-mediated killing by human serum; and, also, selected gonococcal types which showed a greater resistance to intracellular killing by human phagocytes than laboratory strains. Furthermore, evidence was presented that not only was resistance to complement mediated killing important in gonococcal pathogenesis, but also resistance to phagocytic defences. This paper describes the continuance of our studies on the determinants of induced serum resistance and of resistance to killing by phagocytes including toxicity to these cells. Each section begins by summarising previous work that was referenced in Parsons et al. (1985).     

A reexamination of the role of clusterin as a complement regulator.

Clusterin is a highly conserved glycoprotein which has been proposed to protect host cells against complement-mediated cytolysis. We tested the hypothesis that clusterin is a complement regulator using erythrocytes and cells which had been stably transfected with a membrane-anchored form of clusterin as targets for complement-mediated cytolysis. Clusterin gave dose-dependent protection of antibody-coated sheep erythrocytes against complement-mediated lysis by diluted normal human serum. There was a linear relationship between the concentration of clusterin giving 50% protection and the concentration of serum; extrapolation of this to the case of undiluted human serum showed that a clusterin concentration at least two orders of magnitude greater than its physiological plasma concentration would be needed to confer protection against complement-mediated cytolysis under physiological conditions. Physiological concentrations of clusterin did not protect rabbit erythrocytes against alternative complement pathway-mediated lysis using dilute human serum. Exogenous clusterin had no effect on lysis of human erythrocytes triggered by the addition of inulin to autologous human serum. Induction of cell-surface clusterin expression by L929 (murine fibroblast) cells which had been stably transfected with cDNA for human clusterin linked to DNA coding for the 44 C-terminal amino acid residues of CD55 did not protect the cells against complement-mediated lysis by either normal or clusterin-depleted human serum. These data suggest that clusterin may not be a physiologically relevant regulator of complement activation.     

Complement-resistance mechanisms of bacteria

Despite more than a century of parallel research on bacteria and the complement system, relatively little is known of the mechanisms whereby pathogenic bacteria can escape complement-related opsonophagocytosis and direct killing. It is likely that pathogenicity in bacteria has arisen more accidentally than in viruses, and on the basis of selection from natural mutants rather than by outright stealing or copying of genetic codes from the host. In this review we will discuss complement resistance as one of the features that makes a bacterium a pathogen.     

Resistance of fluorescent-labelled Actinobacillus actinomycetemcomitans strains to phagocytosis and killing by human neutrophils

Neutrophils are initially the predominant cells involved in the host defence of bacterial infections, including periodontal disease. Aggressive periodontitis is associated with Actinobacillus actinomycetemcomitans, a Gram-negative capnophilic microorganism. Infections caused by A. actinomycetemcomitans are not resolved by the host immune response despite the accumulation of neutrophils at the site of inflammation. To better understand the role of natural host defence mechanisms in A. actinomycetemcomitans infections, the interaction of phenotypically diverse strains of this pathogen with human neutrophils was assessed directly using techniques such as genetic labelling with the gene for green fluorescent protein, fluorescence-activated cell sorting and fluorescence imaging. The study included clinical isolates of A. actinomycetemcomitans represented by self-aggregating, biofilm-associated and isogenic planktonic variants. Data obtained showed that complement-mediated phagocytosis of A. actinomycetemcomitans was generally inefficient regardless of strain-specific serotype or leukotoxin production. Furthermore, the majority of ingested bacteria remained viable after exposure to neutrophils for 1 h. Interestingly, uptake of antibody-opsonized bacteria resulted in the rapid cell death of neutrophils. This was in contrast to ingestion of complement-opsonized bacteria, which did not affect neutrophil viability. The methods used in this study provided reliable and reproducible results with respect to adherence, phagocytosis and killing of A. actinomycetemcomitans when encountering human neutrophils.     

Exhaustion of cytotoxic effector systems may limit monoclonal antibody-based immunotherapy in cancer patients

The CD20 mAb ofatumumab (OFA) induces complement-mediated lysis of B cells. In an investigator-initiated phase II trial of OFA plus chemotherapy for chronic lymphocytic leukemia (CLL), OFA treatment promoted partial CLL B cell depletion that coincided with reduced complement titers. Remaining CLL B cells circulated with bound OFA and covalently bound complement breakdown product C3d, indicative of ongoing complement activation. Presumably, neither complement- nor effector cell-based mechanisms were sufficiently robust to clear these remaining B cells. Instead, almost all of the bound OFA and CD20 was removed from the cells, in accordance with previous clinical studies that demonstrated comparable loss of CD20 from B cells after treatment of CLL patients with rituximab. In vitro experiments with OFA and rituximab addressing these observations suggest that host effector mechanisms that support mAb-mediated lysis and tumor cell clearance are finite, and they can be saturated or exhausted at high B cell burdens, particularly at high mAb concentrations. Interestingly, only a fraction of available complement was required to kill cells with CD20 mAbs, and killing could be tuned by titrating the mAb concentration. Consequently, maximal B cell killing of an initial and secondary B cell challenge was achieved with intermediate mAb concentrations, whereas high concentrations promoted lower overall killing. Therefore, mAb therapies that rely substantially on effector mechanisms subject to exhaustion, including complement, may benefit from lower, more frequent dosing schemes optimized to sustain and maximize killing by cytotoxic immune effector systems.     

Factor H binding and function in sialylated pathogenic neisseriae is influenced by gonococcal, but not meningococcal, porin

Neisseria gonorrhoeae and Neisseria meningitidis both express the lacto-N-neotetraose (LNT) lipooligosaccharide (LOS) molecule that can be sialylated. Although gonococcal LNT LOS sialylation enhances binding of the alternative pathway complement inhibitor factor H and renders otherwise serum-sensitive bacteria resistant to complement-dependent killing, the role of LOS sialylation in meningococcal serum resistance is less clear. We show that only gonococcal, but not meningococcal, LNT LOS sialylation enhanced factor H binding. Replacing the porin (Por) B molecule of a meningococcal strain (LOS sialylated) that did not bind factor H with gonococcal Por1B augmented factor H binding. Capsule expression did not alter factor H binding to meningococci that express gonococcal Por. Conversely, replacing gonococcal Por1B with meningococcal PorB abrogated factor H binding despite LNT LOS sialylation. Gonococcal Por1B introduced in the background of an unsialylated meningococcus itself bound small amounts of factor H, suggesting a direct factor H-Por1B interaction. Factor H binding to unsialylated meningococci transfected with gonococcal Por1B was similar to the sialylated counterpart only in the presence of higher (20 microg/ml) concentrations of factor H and decreased in a dose-responsive manner by approximately 80% at 1.25 microg/ml. Factor H binding to the sialylated strain remained unchanged over this factor H concentration range however, suggesting that LOS sialylation facilitated optimal factor H-Por1B interactions. The functional counterpart of factor H binding showed that sialylated meningococcal mutants that possessed gonococcal Por1B were resistant to complement-mediated killing by normal human serum. Our data highlight the different mechanisms used by these two related species to evade complement.     

Minimization of bacterial size allows for complement evasion and is overcome by the agglutinating effect of antibody

The complement system, which functions by lysing pathogens directly or by promoting their uptake by phagocytes, is critical for controlling many microbial infections. Here, we show that in Streptococcus pneumoniae, increasing bacterial chain length sensitizes this pathogen to complement deposition and subsequent uptake by human neutrophils. Consistent with this, we show that minimizing chain length provides wild-type bacteria with a competitive advantage in vivo in a model of systemic infection. Investigating how the host overcomes this virulence strategy, we find that antibody promotes complement-dependent opsonophagocytic killing of Streptococcus pneumoniae and lysis of Haemophilus influenzae independent of Fc-mediated effector functions. Consistent with the agglutinating effect of antibody, F(ab')(2) but not Fab could promote this effect. Therefore, increasing pathogen size, whether by natural changes in cellular morphology or via antibody-mediated agglutination, promotes complement-dependent killing. These observations have broad implications for how cell size and morphology can affect virulence among pathogenic microbes.