Complement Resistance Research Articles

The Capsular Serotype ofStreptococcus pneumoniaeIs 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.

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.

Characterization of the unique complement resistance mechanisms of the periodontal pathogen, Treponema denticola: Insight into the survival strategies within the periodontal pocket

Characterization of the unique complement resistance mechanisms of the periodontal pathogen, Treponema denticola: Insight into the survival strategies within the periodontal pocket

Caveolae- and lipid rafts-mediated endocytosis of the complement C5b-9 is an essential mechanism for complement resistance

Caveolae- and lipid rafts-mediated endocytosis of the complement C5b-9 is an essential mechanism for complement resistance

Downregulation of an Entamoeba histolytica Rhomboid Protease Reveals Roles in Regulating Parasite Adhesion and Phagocytosis

Entamoeba histolytica is a deep-branching eukaryotic pathogen. Rhomboid proteases are intramembrane serine proteases, which cleave transmembrane proteins in, or in close proximity to, their transmembrane domain. We have previously shown that E. histolytica contains a single functional rhomboid protease (EhROM1) and has unique substrate specificity. EhROM1 is present on the trophozoite surface and relocalizes to internal vesicles during erythrophagocytosis and to the base of the cap during surface receptor capping. In order to further examine the biological function of EhROM1 we downregulated EhROM1 expression by >95% by utilizing the epigenetic silencing mechanism of the G3 parasite strain. Despite the observation that EhROM1 relocalized to the cap during surface receptor capping, EhROM1 knockdown [ROM(KD)] parasites had no gross changes in cap formation or complement resistance. However, ROM(KD) parasites demonstrated decreased host cell adhesion, a result recapitulated by treatment of wild-type parasites with DCI, a serine protease inhibitor with activity against rhomboid proteases. The reduced adhesion phenotype of ROM(KD) parasites was noted exclusively with healthy cells, and not with apoptotic cells. Additionally, ROM(KD) parasites had decreased phagocytic ability with reduced ingestion of healthy cells, apoptotic cells, and rice starch. Decreased phagocytic ability is thus independent of the reduced adhesion phenotype, since phagocytosis of apoptotic cells was reduced despite normal adhesion levels. The defect in host cell adhesion was not explained by altered expression or localization of the heavy subunit of the Gal/GalNAc surface lectin. These results suggest no significant role of EhROM1 in complement resistance but unexpected roles in parasite adhesion and phagocytosis.

Screening of Complement Inhibitors: Shielded Baculoviruses Increase the Safety and Efficacy of Gene Delivery

One of the major obstacles in the use of baculovirus vectors for in vivo gene transfer is the virus inactivation by serum complement. In this study, we investigated the effect of decay-accelerating factor (DAF), factor H (FH)-like protein-1 (FHL-1), C4b-binding protein (C4BP), and membrane cofactor protein (MCP) on protection of baculovirus vectors from the complement-mediated inactivation. Complement regulatory proteins were displayed on baculovirus surface as fusions to membrane anchor of the vesicular stomatitis virus-G (VSV-G) protein. This strategy resulted in abundant expression of recombinant proteins on the viral envelope while viral titers comparable to control virus were reached. The surface-modified vectors exhibited complement resistance in vitro, DAF showing the highest level of protection. Intraportal delivery of DAF-displaying baculovirus resulted in increased survival and enhanced gene expression in immunocompetent mice. Mice receiving DAF-displaying baculovirus also exhibited lower level of liver inflammation as evidenced by aspartate aminotransferase (AST). In line with this, macrophages treated with DAF baculovirus produced lower levels of inflammatory cytokines IL-1beta, IL-6, and IL-12p40 compared to control virus. These results suggest that DAF-display can protect the vector against complement inactivation but also reduce complement-mediated inflammation injury. In conclusion, complement shielded baculovirus vectors represent attractive tools for effective in vivo gene delivery.

Trimer Stability of YadA Is Critical for Virulence of Yersinia enterocolitica

Yersinia adhesin A (YadA) is a trimeric autotransporter adhesin with multiple functions in host-pathogen interactions. The aim of this study was to dissect the virulence functions promoted by YadA in vitro and in vivo. To accomplish this, we generated Yersinia enterocolitica O:8 mutants expressing point mutations in YadA G389, a highly conserved residue in the membrane anchor of YadA, and analyzed their impact on YadA expression and virulence functions. We found that point mutations of YadA G389 led to impaired transport, stability, and surface display of YadA. YadA G389A and G389S mutants showed comparable YadA surface expression, autoagglutination, and adhesion to those of wild-type YadA but displayed reduced trimer stability and complement resistance in vitro and were 10- to 1,000-fold attenuated in experimental Y. enterocolitica infection in mice. The G389T, G389N, and G389H mutants lost trimer stability, exhibited strongly reduced surface display, autoagglutination, adhesion properties, and complement resistance, and were avirulent (>10,000-fold attenuation) in mice. Our data demonstrate that G389 is a critical residue of YadA, required for optimal trimer stability, transport, surface display, and serum resistance. We also show that stable trimeric YadA protein is essential for virulence of Y. enterocolitica.

Acquisition of Complement Resistance through Incorporation of CD55/Decay-Accelerating Factor into Viral Particles Bearing Baculovirus GP64

A major obstacle to gene transduction by viral vectors is inactivation by human complement in vivo. One way to overcome this is to incorporate complement regulatory proteins, such as CD55/decay accelerating factor (DAF), into viral particles. Lentivirus vectors pseudotyped with the baculovirus envelope protein GP64 have been shown to acquire more potent resistance to serum inactivation and longer transgene expression than those pseudotyped with the vesicular stomatitis virus (VSV) envelope protein G. However, the molecular mechanisms underlying resistance to serum inactivation in pseudotype particles bearing the GP64 have not been precisely elucidated. In this study, we generated pseudotype and recombinant VSVs bearing the GP64. Recombinant VSVs generated in human cell lines exhibited the incorporation of human DAF in viral particles and were resistant to serum inactivation, whereas those generated in insect cells exhibited no incorporation of human DAF and were sensitive to complement inactivation. The GP64 and human DAF were detected on the detergent-resistant membrane and were coprecipitated by immunoprecipitation analysis. A pseudotype VSV bearing GP64 produced in human DAF knockdown cells reduced resistance to serum inactivation. In contrast, recombinant baculoviruses generated in insect cells expressing human DAF or carrying the human DAF gene exhibited resistance to complement inactivation. These results suggest that the incorporation of human DAF into viral particles by interacting with baculovirus GP64 is involved in the acquisition of resistance to serum inactivation.

Vibrio cholerae O139 capsular polysaccharide confers complement resistance in the absence or presence of antibody yet presents a productive target for cell lysis: implications for detection of bactericidal antibodies

Vibrio cholerae O139 variants with different surface phenotypes have been compared for their resistance to complement and also for their susceptibility to antibody-dependent, complement-mediated bacteriolysis (ACB). While both capsular polysaccharide (CPS) and lipopolysaccharide (LPS) contribute to complement resistance in the absence of antibody, the relative survival of variants expressing only one of these surface polysaccharides reveals CPS to be of much greater significance in this respect. Variants with LPS+/CPS− or LPS−/CPS+ surface phenotypes were both susceptible to ACB, showing that antibody binding to either of the O139 polysaccharides can initiate ACB. Demonstration of the lytic potential of antibodies bound to the capsule is novel and necessitates a revision of the suggested mechanism by which CPS confers partial protection of wild-type V. cholerae O139 against ACB. This finding also raised the possibility that there may be CPS-restricted epitopes which can function as substrates for ACB, which would invalidate the common practice of using unencapsulated O139 variants for estimating bactericidal responses. Since our data did not support this scenario, we evaluated several unencapsulated strains - including variants producing normal or elongated polysaccharide chains, and a hybrid O1/O139 strain - for their utility as indicators of bactericidal antibodies to V. cholerae O139. Our findings support the use of the recently-isolated CIRS134, which ensures reproducible titrations and allows assignment of high lytic titres in assays requiring only 2% complement. However low-level, cross-serogroup lytic activity was detected when CIRS134 was used to assay responses of mice injected with O1 serogroup V. cholerae, suggesting that this indicator should be used with caution when evaluating the immunogenicity of bivalent O1/O139 vaccines.

Human complement activation by smooth and rough Proteus mirabilis lipopolysaccharides

Proteus mirabilis bacilli play an important role in human urinary tract infections, bacteremia, and rheumatoid arthritis. The authors previously studied human complement C3 conversion by smooth-form P. mirabilis O10, O23, O30, and O43 lipopolysaccharides (LPSs) and showed that smooth Proteus LPSs fragmented C3 in a dose- and time-dependent manner. In the present study, one smooth P. mirabilis S1959 and its two polysaccharide-truncated LPSs isolated from an R mutant strain were used to study the C3 conversion. The conversion of C3 to C3c by smooth and rough P. mirabilis LPSs was studied by capture ELISA and crossed immunoelectrophoresis. Proteins isolated from the outer membrane were analyzed by discontinuous sodium dodecyl sulfate gel electrophoresis. The smooth P. mirabilis S1959 (O3) strain was resistant to the bactericidal activity of human serum, in contrast to the Ra and Re mutant strains. The presence of an exposed core oligosaccharide in R110 LPS was not sufficient to protect the strain from serum-dependent killing. In addition to LPS structure, the outer-membrane proteins may also play roles in protecting the smooth P. mirabilis S1959 (O3) strain from the bactericidal action of serum. It was shown that the Ra P. mirabilis R110 and the Re P. mirabilis R45 mutants possess very different OMP compositions from that of the P. mirabilis S 1959 strain. Regardless of the complement resistance of the P. mirabilis strains, the S1959, R110, and R45 LPSs fragmented C3 and induced C3c neo-antigen exposure. The use of complement-deficient human serum allows the conclusion that the Re-type P. mirabilis R45 LPS fragmented C3 by the antibody-independent classical pathway.

Biochemical and Structural Analysis of Bacterial O-antigen Chain Length Regulator Proteins Reveals a Conserved Quaternary Structure

Lipopolysaccharide (LPS) is a major component of the Gram-negative outer membrane and is an important virulence determinant. The O-antigen polysaccharide of the LPS molecule provides protection from host defenses, and the length of O-antigen chains plays a pivotal role. In the Wzy-dependent O-antigen biosynthesis pathway, the integral inner membrane protein Wzz determines the O-antigen chain length. How these proteins function is currently unknown, but the hypothesis includes activities such as a "molecular ruler" or a "molecular stopwatch," and other possibilities may exist. Wzz homologs are membrane proteins with two transmembrane helices that flank a large periplasmic domain. Recent x-ray crystallographic studies of the periplasmic portions of Wzz proteins found multiple oligomeric forms, with quaternary structures favoring the "molecular ruler" interpretation. Here, we have studied full-length Wzz proteins with the transmembrane portions embedded in lipid membranes. Using electron microscopy and image analysis we find a unique hexameric state rather than differing oligomeric forms. The data suggest that in vivo Wzz proteins determine O-antigen chain length via subtle structure-function relationships at the level of primary, secondary, or tertiary structure within the context of a hexameric complex.

Characterization in vivo and in vitro of a strain of Leishmania (Viannia) shawi from the Amazon Region

The present study analyses complement resistance, cell surface carbohydrates expression, lipidic composition and morphology in vivo and in vitro, of Leishmania (Viannia) shawi, a parasite identified in the Amazon region, Pará state, in 1989. We demonstrated that promastigotes in the stationary (STAT) growth phase are more resistant to complement lysis than in the logarithimic (LOG) growth phase. Ultrastructural analyses and imidazol technique showed accumulation of lipids in STAT growth phase promastigotes, which was confirmed by biochemical approach. Light and electron microscopy of skin lesion in hamster footpads caused by promastigotes in STAT growth phase, 90 days post inoculation, showed amastigotes inside of macrophage and free in the tissue surrounded by collagen fibers as well as extensive inflammatory reaction with tissue destruction. We also demonstrated, using lectins by agglutination assays and flow cytometry, the presence of fucose, mannose and/or glucose carbohydrate residues on the surface of LOG and STAT promastigotes. The results constitute the first characterization essay combining biochemical and morphological approaches dedicated to LOG and STAT growth phase promastigotes of L. (V) shawi contributing for a better knowledge of this poorly studied species of the New World.

Polymicrobial interactions stimulate resistance to host innate immunity through metabolite perception

Bacteria in the human oral cavity often grow in an attached multispecies biofilm community. Members of this community display defined interactions that have an impact on the physiology of the individual and the group. Here, we show that during coculture growth with streptococci, the oral pathogen Aggregatibacter actinomycetemcomitans displays enhanced resistance to killing by host innate immunity. The mechanism of resistance involves sensing of the streptococcal metabolite hydrogen peroxide by A. actinomycetemcomitans, which stimulates a genetic program resulting in enhanced expression of the complement resistance protein ApiA. The oxidative stress response regulator OxyR mediates induction of apiA transcription, and this induction is required for coculture resistance to killing by human serum. These findings provide evidence that interaction between community members mediates prokaryotic resistance to host innate immunity and reinforce the need to understand how polymicrobial growth affects interaction with the host immune system.

Entamoeba histolytica: Oxygen resistance and virulence

Entamoeba histolytica virulence has been attributed to several amoebic molecules such as adhesins, amoebapores and cysteine proteinases, but supporting evidence is either partial or indirect. In this work we compared several in vitro and in vivo features of both virulent E. histolytica (v Eh) and non-virulent E. histolytica (nv Eh) axenic HM-1 IMSS strains, such as complement resistance, proteinase activity, haemolytic, phagocytic and cytotoxic capacities, survival in mice caecum, and susceptibility to O 2. The only difference observed was a higher in vitro susceptibility of nv Eh to O 2. The molecular mechanism of that difference was analyzed in both groups of amoebae after high O 2 exposure. v Eh O 2 resistance correlated with: (i) higher O 2 reduction ( O 2 - and H 2O 2 production); (ii) increased H 2O 2 resistance and thiol peroxidase activity, and (iii) reversible pyruvate: ferredoxin oxidoreductase (PFOR) inhibition. Despite the high level of carbonylated proteins in nv Eh after O 2 exposure, membrane oxidation by reactive oxygen species was not observed. These results suggest that the virulent phenotype of E. histolytica is related to the greater ability to reduce O 2 and H 2O 2 as well as PFOR reactivation, whereas nv Eh undergoes irreversible PFOR inhibition resulting in metabolic failure and amoebic death.

Characterization of DNA Sequences that Confer Complement Resistance in Leishmania chagasi

Serial passage of axenically cultured Leishmania chagasi promastigotes results in a progressive diminution in resistance to complement-mediated lysis (CML), whereas high CML resistance is seen in infectious metacyclic promastigotes from the sandfly vector as well as metacyclic-like promastigotes within low-passage cultures at stationary growth phase. As we previously reported, in a screen seeking to identify novel genes involved in CML resistance: (1) a genomic cosmid library derived from DNA of CML-resistant L. chagasi promastigotes was transfected into high-passage (constitutively CML-sensitive) L. chagasi promastigotes; (2) transformants were screened for acquisition of CML-resistance; (3) multiple cosmid-transfectants exhibited partial CML resistance; and (4) the sequence for one of the cosmids (Cosmid 51) was determined. This report extends the analysis of Cosmid 51, and identifies by deletion analysis a subregion of the cosmid insert that is critical to the CML-resistance phenotype of Cosmid 51 transformants. We also report the sequence determination and initial CML-resistance activity of another cosmid that also confers partial resistance to CML.

Enhanced complement resistance in drug-selected P-glycoprotein expressing multi-drug-resistant ovarian carcinoma cells

Multi-drug resistance (MDR) is a major obstacle in cancer chemotherapy. There are contrasting data on a possible correlation between the level of expression of the drug transporter P-glycoprotein (P-gp) and susceptibility to complement-dependent cytotoxicity (CDC). We therefore investigated the sensitivity of human ovarian carcinoma cells and their P-gp expressing MDR variants to complement. Chemoselected P-gp expressing MDR cells showed increased resistance to CDC associated with overexpression of membrane-bound complement regulatory proteins (mCRP) and increased release of the soluble inhibitors C1 inhibitor and factor I. MDR1 gene transfection alone did not alter the susceptibility of P-gp expressing A2780-MDR and SKOV3-MDR cells to CDC. However, subsequent vincristine treatment conferred an even higher resistance to complement to these cells, again associated with increased expression of mCRP. Blocking the function of P-gp with verapamil, cyclosporine A or the anti-P-gp-antibody MRK16 had no impact on their complement resistance, whereas blocking of mCRP enhanced their susceptibility to complement. These results suggest that enhanced resistance of chemoselected MDR ovarian carcinoma cells to CDC is not conferred by P-gp, but is due at least partly to overexpression of mCRP, probably induced by treatment with the chemotherapeutic agents.

The Neglected Role of Antibody in Protection Against Bacteremia Caused by Nontyphoidal Strains of Salmonella in African Children

MacLennan CA, Gondwe EN, Msefula CL, et al. J Clin Invest. 2008;118(4):1553–1562 PURPOSE OF THE STUDY. To investigate whether specific antibody protects against nontyphoidal Salmonella (NTS) bacteremia. POPULATION STUDIED AND METHODS. Admissions for NTS bacteremia during 1 year were reviewed (N = 352). Sera from 65 healthy Malawian children (median age, 24 months: range, 3–107 months) was used for in vitro NTS-killing assays. Purified immunoglobulin G (IgG) from patient sera was used to delineate the role of antibody versus complement. Immunoglobulin and complement deposition on bacteria was also assessed by flow cytometry. Complement dependency was tested by heat inactivation and by using C9-deficient serum. RESULTS. Overall, 82% of the children with NTS bacteremia were <36 months old, but there was a nonuniform distribution with fewer-than-expected cases in children <4 months old (actual: 9.7% vs 16.7%), suggesting a role for passive humoral immunity. From healthy donors, 9 of 25 children <16 months of age had serum with normal in vitro killing, whereas 40 of 40 children >16 months of age did so. NTS killing in vitro was complement and membrane attack complex dependent. Effective killing corresponded to a specific IgG or IgM Salmonella titer of 1.5 U and was associated with the deposition of complement (C3 and C9) measured by flow cytometry. Resistance to killing by alternative complement activation was mediated by long-chain lipopolysaccharide and the rck gene product. CONCLUSIONS. Salmonella-specific antibody that overcomes the complement resistance of NTS develops by 2 years of life in Malawian children and is mediated by specific IgG or IgM antibody. REVIEWER COMMENTS. NTS is the most common cause of bacteremia in Malawi and much of tropical Africa and is associated with high rates of mortality even with appropriate culture facilities and access to antibiotics. The finding that specific antibody is protective against this invasive, facultative intracellular pathogen supports the importance of vaccine development for this pathogen.

Immune Evasion of Enterococcus faecalis by an Extracellular Gelatinase That Cleaves C3 and iC3b

Enterococcus faecalis (Ef) accounts for most cases of enterococcal bacteremia, which is one of the principal causes of nosocomial bloodstream infections (BSI). Among several virulence factors associated with the pathogenesis of Ef, an extracellular gelatinase (GelE) has been known to be the most common factor, although its virulence mechanisms, especially in association with human BSI, have yet to be demonstrated. In this study, we describe the complement resistance mechanism of Ef mediated by GelE. Using purified GelE, we determined that it cleaved the C3 occurring in human serum into a C3b-like molecule, which was inactivated rapidly via reaction with water. This C3 convertase-like activity of GelE was shown to result in a consumption of C3 and thus inhibited the activation of the complement system. Also, GelE was confirmed to degrade an iC3b that was deposited on the Ag surfaces without affecting the bound C3b. This proteolytic effect of GelE against the major complement opsonin resulted in a substantial reduction in Ef phagocytosis by human polymorphonuclear leukocytes. In addition, we verified that the action of GelE against C3, which is a central component of the complement cascade, was human specific. Taken together, it was suggested that GelE may represent a promising molecule for targeting human BSI associated with Ef.

Evasion of Complement-Mediated Lysis and Complement C3 Deposition Are Regulated by Francisella tularensis Lipopolysaccharide O Antigen

The bacterium Francisella tularensis (Ft) is a potential weapon of bioterrorism when aerosolized. Macrophage infection is necessary for disease progression and efficient phagocytosis by human macrophages requires serum opsonization by complement. Microbial complement activation leads to surface deposition of a highly regulated protein complex resulting in opsonization or membrane lysis. The nature of complement component C3 deposition, i.e., C3b (opsonization and lysis) or C3bi (opsonization only) fragment deposition, is central to the outcome of activation. In this study, we examine the mechanisms of Ft resistance to complement-mediated lysis, C3 component deposition on the Ft surface, and complement activation. Upon incubation in fresh nonimmune human serum, Schu S4 (Ft subsp. tularensis), Fn (Ft subsp. novicida), and LVS (Ft subsp. holarctica live vaccine strain) were resistant to complement-mediated lysis, but LVSG and LVSR (LVS strains altered in surface carbohydrate structures) were susceptible. C3 deposition, however, occurred on all strains. Complement-susceptible strains had markedly increased C3 fragment deposition, including the persistent presence of C3b compared with C3bi, which indicates that C3b inactivation results in survival of complement-resistant strains. C1q, an essential component of the classical activation pathway, was necessary for lysis of complement-susceptible strains and optimal C3 deposition on all strains. Finally, use of Francisella LPS mutants confirmed O Ag as a major regulator of complement resistance. These data provide evidence that pathogenic Francisella activate complement, but are resistant to complement-mediated lysis in part due to limited C3 deposition, rapid conversion of surface-bound C3b to C3bi, and the presence of LPS O Ag.

Endocytosis of complement C5b-9 and complement resistance depend on dynamin and lipid rafts

Endocytosis of complement C5b-9 and complement resistance depend on dynamin and lipid rafts