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1.
Infect Immun ; 86(4)2018 04.
Article in English | MEDLINE | ID: mdl-29378798

ABSTRACT

The pneumococcal capsular serotype is an important determinant of complement resistance and invasive disease potential, but other virulence factors have also been found to contribute. Pneumococcal surface protein C (PspC), a highly variable virulence protein that binds complement factor H to evade C3 opsonization, is divided into two subgroups: choline-bound subgroup I and LPxTG-anchored subgroup II. The prevalence of different PspC subgroups in invasive pneumococcal disease (IPD) and functional differences in complement evasion are unknown. The prevalence of PspC subgroups in IPD isolates was determined in a collection of 349 sequenced strains of Streptococcus pneumoniae isolated from adult patients. pspC deletion mutants and isogenic pspC switch mutants were constructed to study differences in factor H binding and complement evasion in relation to capsule thickness. Subgroup I pspC was far more prevalent in IPD isolates than subgroup II pspC The presence of capsule was associated with a greater ability of bound factor H to reduce complement opsonization. Pneumococcal subgroup I PspC bound significantly more factor H and showed more effective complement evasion than subgroup II PspC in isogenic encapsulated pneumococci. We conclude that variation in the PspC subgroups, independent of capsule serotypes, affects pneumococcal factor H binding and its ability to evade complement deposition.


Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/immunology , Complement System Proteins/immunology , Genotype , Pneumococcal Infections/immunology , Pneumococcal Infections/microbiology , Streptococcus pneumoniae/genetics , Streptococcus pneumoniae/immunology , Aged , Complement Factor H/immunology , Complement Factor H/metabolism , Complement System Proteins/metabolism , Female , Humans , Immune Evasion , Male , Middle Aged , Molecular Typing , Mutation , Pneumococcal Infections/epidemiology , Prevalence , Serogroup , Virulence/genetics , Virulence Factors/genetics
2.
Sci Rep ; 7: 42137, 2017 02 08.
Article in English | MEDLINE | ID: mdl-28176849

ABSTRACT

Most bacteria entering the bloodstream will be eliminated through complement activation on the bacterial surface and opsonophagocytosis. However, when these protective innate immune systems do not work optimally, or when bacteria are equipped with immune evasion mechanisms that prevent killing, this can lead to serious infections such as bacteremia and meningitis, which is associated with high morbidity and mortality. In order to study the complement evasion mechanisms of bacteria and the capacity of human blood to opsonize and kill bacteria, we developed a versatile whole blood killing assay wherein both phagocyte function and complement activity can easily be monitored and modulated. In this assay we use a selective thrombin inhibitor hirudin to fully preserve complement activity of whole blood. This assay allows controlled analysis of the requirements for active complement by replacing or heat-inactivating plasma, phagocyte function and bacterial immune evasion mechanisms that contribute to survival in human blood.


Subject(s)
Biological Assay , Complement Activation , Immune Evasion , Leukocytes, Mononuclear/immunology , Phagocytosis/immunology , Adult , Complement C3/chemistry , Escherichia coli/growth & development , Escherichia coli/immunology , Female , Fibrinolytic Agents/chemistry , Fluoresceins/chemistry , Fluorescent Dyes/chemistry , Haemophilus influenzae/growth & development , Haemophilus influenzae/immunology , Hirudins/chemistry , Humans , Immunoglobulin G/chemistry , Immunoglobulin M/chemistry , Klebsiella pneumoniae/growth & development , Klebsiella pneumoniae/immunology , Leukocytes, Mononuclear/microbiology , Male , Neisseria meningitidis/growth & development , Neisseria meningitidis/immunology , Pseudomonas aeruginosa/growth & development , Pseudomonas aeruginosa/immunology , Staphylococcus aureus/growth & development , Staphylococcus aureus/immunology , Streptococcus pneumoniae/growth & development , Streptococcus pneumoniae/immunology , Succinimides/chemistry
3.
Cytokine ; 88: 281-286, 2016 12.
Article in English | MEDLINE | ID: mdl-27721145

ABSTRACT

Bacterial pathogens not only stimulate innate immune receptors, but also activate the complement system. Crosstalk between complement C5a receptor (C5aR) and other innate immune receptors is known to enhance the proinflammatory cytokine response. An important determinant of the magnitude of complement activation is the activity of the alternative pathway, which serves as an amplification mechanism for complement activation. Both alternative pathway activity as well as plasma levels of factor H, a key inhibitor of the alternative pathway, show large variation within the human population. Here, we studied the effect of factor H-mediated regulation of the alternative pathway on bacterial-induced proinflammatory cytokine responses. We used the human pathogen Streptococcus pneumoniae as a model stimulus to induce proinflammatory cytokine responses in human peripheral blood mononuclear cells. Serum containing active complement enhanced pneumococcal induced proinflammatory cytokine production through C5a release and C5aR crosstalk. We found that inhibition of the alternative pathway by factor H, with a concentration equivalent to a high physiological level, strongly reduced C5a levels and decreased proinflammatory cytokine production in human peripheral blood mononuclear cells. This suggests that variation in alternative pathway activity due to variation in factor H plasma levels affects individual cytokine responses during infection.


Subject(s)
Complement Factor H/immunology , Complement Pathway, Alternative/immunology , Leukocytes, Mononuclear/immunology , Receptor, Anaphylatoxin C5a/immunology , Streptococcus pneumoniae/immunology , Adult , Female , Humans , Male
4.
PLoS One ; 11(2): e0149307, 2016.
Article in English | MEDLINE | ID: mdl-26872035

ABSTRACT

Streptococcus pneumoniae is a common cause of sepsis. Effective complement activation is an important component of host defence against invading pathogens, whilst excessive complement activation has been associated with endothelial dysfunction and organ damage. The alternative pathway amplification loop is important for the enhancement of complement activation. Factor H is a key negative regulator of the alternative pathway amplification loop and contributes to tight control of complement activation. We assessed the effect of inhibition of the alternative pathway on sepsis associated inflammation and disease severity using human factor H treatment in a clinically relevant mice model of pneumococcal sepsis. Mice were infected intravenously with live Streptococcus pneumoniae. At the first clinical signs of infection, 17 hours post-infection, mice were treated with ceftriaxone antibiotic. At the same time purified human factor H or in controls PBS was administered. Treatment with human factor H did not attenuate disease scores, serum pro-inflammatory cytokines, or vascular permeability and did not significantly affect C3 and C3a production at 26 h post-infection. Therefore, we conclude that inhibition of the alternative complement pathway by exogenous human factor H fails to attenuate inflammation and vascular leakage at a clinically relevant intervention time point in pneumococcal sepsis in mice.


Subject(s)
Anti-Bacterial Agents/therapeutic use , Capillary Permeability/drug effects , Ceftriaxone/therapeutic use , Pneumococcal Infections/drug therapy , Sepsis/drug therapy , Streptococcus pneumoniae/drug effects , Animals , Complement Factor H/therapeutic use , Cytokines/blood , Cytokines/immunology , Disease Models, Animal , Female , Humans , Mice , Mice, Inbred C57BL , Pneumococcal Infections/blood , Pneumococcal Infections/complications , Pneumococcal Infections/immunology , Sepsis/blood , Sepsis/complications , Sepsis/immunology , Streptococcus pneumoniae/immunology
5.
J Infect Dis ; 213(11): 1820-7, 2016 06 01.
Article in English | MEDLINE | ID: mdl-26802141

ABSTRACT

Streptococcus pneumoniae is a major cause of life-threatening infections. Complement activation plays a vital role in opsonophagocytic killing of pneumococci in blood. Initial complement activation via the classical and lectin pathways is amplified through the alternative pathway amplification loop. Alternative pathway activity is inhibited by complement factor H (FH). Our study demonstrates the functional consequences of the variability in human serum FH levels on host defense. Using an in vivo mouse model combined with human in vitro assays, we show that the level of serum FH correlates with the efficacy of opsonophagocytic killing of pneumococci. In summary, we found that FH levels determine a delicate balance of alternative pathway activity, thus affecting the resistance to invasive pneumococcal disease. Our results suggest that variation in FH expression levels, naturally occurring in the human population, plays a thus far unrecognized role in the resistance to invasive pneumococcal disease.


Subject(s)
Pneumococcal Infections/immunology , Animals , Complement C3/immunology , Complement Factor H/immunology , Disease Resistance/immunology , Female , Humans , Male , Mice , Mice, Inbred C57BL , Pneumococcal Infections/prevention & control
6.
J Virol ; 87(18): 10394-400, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23864632

ABSTRACT

Alphavirus nonstructural protein 2 (nsP2) has pivotal roles in viral RNA replication, host cell shutoff, and inhibition of antiviral responses. Mutations that individually rendered other alphaviruses noncytopathic were introduced into chikungunya virus nsP2. Results show that (i) nsP2 mutation P718S only in combination with KR649AA or adaptive mutation D711G allowed noncytopathic replicon RNA replication, (ii) prohibiting nsP2 nuclear localization abrogates inhibition of antiviral interferon-induced JAK-STAT signaling, and (iii) nsP2 independently affects RNA replication, cytopathicity, and JAK-STAT signaling.


Subject(s)
Chikungunya virus/physiology , Cytopathogenic Effect, Viral , Interferons/antagonists & inhibitors , Signal Transduction , Viral Nonstructural Proteins/metabolism , Virus Replication , Animals , Chikungunya virus/pathogenicity , Chlorocebus aethiops , Vero Cells
7.
Infect Immun ; 81(2): 505-13, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23208608

ABSTRACT

Streptococcus pneumoniae is a diverse species causing invasive as well as localized infections that result in massive global morbidity and mortality. Strains vary markedly in pathogenic potential, but the molecular basis is obscured by the diversity and plasticity of the pneumococcal genome. In the present study, S. pneumoniae serotype 3 blood (n = 12) or ear (n = 13) isolates were multilocus sequence typed (MLST) and assessed for biofilm formation and virulence phenotype. Blood and ear isolates exhibited similar MLST distributions but differed markedly in phenotype. Blood isolates formed robust biofilms only at pH 7.4, which were enhanced in Fe(III)-supplemented medium. Conversely, ear isolates formed biofilms only at pH 6.8, and Fe(III) was inhibitory. Biofilm formation paralleled luxS expression and genetic competence. In a mouse intranasal challenge model, blood isolates did not stably colonize the nasopharynx but spread to the blood; none spread to the ear. Ear isolates colonized the nasopharynx at higher levels and also spread to the ear compartment in a significant proportion of animals; none caused bacteremia. Thus, pneumococci of the same serotype and MLST exhibit distinct phenotypes in accordance with clinical site of isolation, indicative of stable niche adaptation within a clonal lineage.


Subject(s)
Biofilms/growth & development , Pneumococcal Infections/microbiology , Streptococcus pneumoniae/physiology , Animals , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacterial Typing Techniques/methods , Carbon-Sulfur Lyases/genetics , Carbon-Sulfur Lyases/metabolism , Female , Ferric Compounds/metabolism , Genes, Bacterial , Hydrogen-Ion Concentration , Mice , Nasopharynx/metabolism , Nasopharynx/microbiology , Phenotype , Pneumococcal Infections/metabolism , Serotyping/methods , Streptococcus pneumoniae/genetics , Streptococcus pneumoniae/isolation & purification , Streptococcus pneumoniae/pathogenicity , Virulence/genetics
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