20 research outputs found
Staphylococcal complement evasion by various convertase-blocking molecules
To combat the human immune response, bacteria should be able to divert the effectiveness of the complement system. We identify four potent complement inhibitors in Staphylococcus aureus that are part of a new immune evasion cluster. Two are homologues of the C3 convertase modulator staphylococcal complement inhibitor (SCIN) and function in a similar way as SCIN. Extracellular fibrinogen-binding protein (Efb) and its homologue extracellular complement-binding protein (Ecb) are identified as potent complement evasion molecules, and their inhibitory mechanism was pinpointed to blocking C3b-containing convertases: the alternative pathway C3 convertase C3bBb and the C5 convertases C4b2aC3b and C3b2Bb. The potency of Efb and Ecb to block C5 convertase activity was demonstrated by their ability to block C5a generation and C5a-mediated neutrophil activation in vitro. Further, Ecb blocks C5a-dependent neutrophil recruitment into the peritoneal cavity in a mouse model of immune complex peritonitis. The strong antiinflammatory properties of these novel S. aureus–derived convertase inhibitors make these compounds interesting drug candidates for complement-mediated diseases
Soluble MAC is primarily released from MAC-resistant bacteria that potently convert complement component C5
The membrane attack complex (MAC or C5b-9) is an important effector of the immune system to kill invading microbes. MAC formation is initiated when complement enzymes on the bacterial surface convert complement component C5 into C5b. Although the MAC is a membrane-inserted complex, soluble forms of MAC (sMAC), or terminal complement complex (TCC), are often detected in sera of patients suffering from infections. Consequently, sMAC has been proposed as a biomarker, but it remains unclear when and how it is formed during infections. Here, we studied mechanisms of MAC formation on different Gram-negative and Gram-positive bacteria and found that sMAC is primarily formed in human serum by bacteria resistant to MAC-dependent killing. Surprisingly, C5 was converted into C5b more potently by MAC-resistant compared to MAC-sensitive Escherichia coli strains. In addition, we found that MAC precursors are released from the surface of MAC-resistant bacteria during MAC assembly. Although release of MAC precursors from bacteria induced lysis of bystander human erythrocytes, serum regulators vitronectin (Vn) and clusterin (Clu) can prevent this. Combining size exclusion chromatography with mass spectrom-etry profiling, we show that sMAC released from bacteria in serum is a heterogeneous mixture of complexes composed of C5b-8, up to three copies of C9 and multiple copies of Vn and Clu. Alto-gether, our data provide molecular insight into how sMAC is generated during bacterial infections. This fundamental knowledge could form the basis for exploring the use of sMAC as biomarker
Multifaceted Activities of Seven Nanobodies against Complement C4b
Cleavage of the mammalian plasma protein C4 into C4b initiates opsonization, lysis, and clearance of microbes and damaged host cells by the classical and lectin pathways of the complement system. Dysregulated activation of C4 and other initial components of the classical pathway may cause or aggravate pathologies, such as systemic lupus erythematosus, Alzheimer disease, and schizophrenia. Modulating the activity of C4b by small-molecule or protein-based inhibitors may represent a promising therapeutic approach for preventing excessive inflammation and damage to host cells and tissue. Here, we present seven nanobodies, derived from llama (Lama glama) immunization, that bind to human C4b (Homo sapiens) with high affinities ranging from 3.2 nM to 14 pM. The activity of the nanobodies varies from no to complete inhibition of the classical pathway. The inhibiting nanobodies affect different steps in complement activation, in line with blocking sites for proconvertase formation, C3 substrate binding to the convertase, and regulator-mediated inactivation of C4b. For four nanobodies, we determined singleparticle cryo-electron microscopy structures in complex with C4b at 3.4-4 Ă… resolution. The structures rationalize the observed functional effects of the nanobodies and define their mode of action during complement activation. Thus, we characterized seven anti-C4b nanobodies with diverse effects on the classical pathway of complement activation that may be explored for imaging, diagnostic, or therapeutic applications
Distinct localization of the complement C5b-9 complex on Gram-positive bacteria
The plasma proteins of the complement system fulfil important immune defence functions, including opsonization of bacteria for phagocytosis, generation of chemo-attractants and direct bacterial killing via the Membrane Attack Complex (MAC or C5b-9). The MAC is comprised of C5b, C6, C7, C8, and multiple copies of C9 that generate lytic pores in cellular membranes. Gram-positive bacteria are protected from MAC-dependent lysis by their thick peptidoglycan layer. Paradoxically, several Gram-positive pathogens secrete small proteins that inhibit C5b-9 formation. In this study, we found that complement activation on Gram-positive bacteria in serum results in specific surface deposition of C5b-9 complexes. Immunoblotting revealed that C9 occurs in both monomeric and polymeric (SDS-stable) forms, indicating the presence of ring-structured C5b-9. Surprisingly, confocal microscopy demonstrated that C5b-9 deposition occurs at specialized regions on the bacterial cell. On Streptococcus pyogenes, C5b-9 deposits near the division septum whereas on Bacillus subtilis the complex is located at the poles. This is in contrast to C3b deposition, which occurs randomly on the bacterial surface. Altogether, these results show a previously unrecognized interaction between the C5b-9 complex and Gram-positive bacteria, whichmight ultimately lead to a new model of MAC assembly and functioning
Streptococcal Inhibitor of Complement Promotes Innate Immune Resistance Phenotypes of Invasive M1T1 Group A Streptococcus
Streptococcal inhibitor of complement (SIC) is a highly polymorphic extracellular protein and putative virulence factor secreted by M1 and M57 strains of group A Streptococcus (GAS). The sic gene is highly upregulated in invasive M1T1 GAS isolates following selection of mutations in the covR/S regulatory locus in vivo. Previous work has shown that SIC (allelic form 1.01) binds to and inactivates complement C5b67 and human cathelicidin LL-37. We examined the contribution of SIC to innate immune resistance phenotypes of GAS in the intact organism, using (1) targeted deletion of sic in wild-type and animal-passaged (covS mutant) M1T1 GAS harboring the sic 1.84 allele and (2) heterologous expression of sic in M49 GAS, which does not possess the sic genein its genome. We find that M1T1 SIC production is strongly upregulated upon covS mutation but that the sic gene is not required for generation and selection of covS mutants in vivo. SIC 1.84 bound both human and murine cathelicidins and was necessary and sufficient to promote covS mutant M1T1 GAS resistance to LL-37, growth in human whole blood and virulence in a murine model of systemic infection. Finally, the sic knockout mutant M1T1 GAS strain was deficient in growth in human serum and intracellular macrophage survival. We conclude that SIC contributes to M1T1 GAS immune resistance and virulence phenotypes
Monitoring phage-induced lysis of gram-negatives in real time using a fluorescent DNA dye
Bacteriophages (phages) are viruses that specifically attack bacteria. Their use as therapeutics, which constitutes a promising alternative to antibiotics, heavily relies on selecting effective lytic phages against the pathogen of interest. Current selection techniques are laborious and do not allow for direct visualization of phage infection dynamics. Here, we present a method that circumvents these limitations. It can be scaled for high-throughput and permits monitoring of the phage infection in real time via a fluorescence signal readout. This is achieved through the use of a membrane-impermeant nucleic acid dye that stains the DNA of damaged or lysed bacteria and new phage progeny. We have tested the method on Pseudomonas aeruginosa and Klebsiella pneumoniae and show that an increase in fluorescence reflects phage-mediated killing. This is confirmed by other techniques including spot tests, colony plating, flow cytometry and metabolic activity measurements. Furthermore, we illustrate how our method may be used to compare the activity of different phages and to screen the susceptibility of clinical isolates to phage. Altogether, we present a fast, reliable way of selecting phages against Gram-negative bacteria, which may be valuable in optimizing the process of selecting phages for therapeutic use.</p
Staphylococcal Complement Inhibitor Modulates Phagocyte Responses by Dimerization of Convertases
Group IIA-secreted phospholipase A2 in human serum kills commensal but not clinical Enterococcus faecium isolates
Human innate immunity employs cellular and humoral mechanisms to facilitate rapid killing of invading bacteria. The direct killing of bacteria by human serum is attributed mainly to the activity of the complement system, which forms pores in Gram-negative bacteria. Although Gram-positive bacteria are considered resistant to killing by serum, we uncover here that normal human serum effectively kills Enterococcus faecium. Comparison of a well-characterized collection of commensal and clinical E. faecium isolates revealed that human serum specifically kills commensal E. faecium strains isolated from normal gut microbiota but not clinical isolates. Inhibitor studies show that the human group IIA secreted phospholipase A2 (hGIIA), but not complement, is responsible for killing of commensal E. faecium strains in human normal serum. This is remarkable since the hGIIA concentration in "noninflamed" serum was considered too low to be bactericidal against Grampositive bacteria. Mechanistic studies showed that serum hGIIA specifically causes permeabilization of commensal E. faecium membranes. Altogether, we find that a normal concentration of hGIIA in serum effectively kills commensal E. faecium and that resistance of clinical E. faecium to hGIIA could have contributed to the ability of these strains to become opportunistic pathogens in hospitalized patients
Convertase Inhibitory Properties of Staphylococcal Extracellular Complement-binding Protein*
The human pathogen Staphylococcus aureus secretes several complement evasion molecules to combat the human immune response. Extracellular complement-binding protein (Ecb) binds to the C3d domain of C3 and thereby blocks C3 convertases of the alternative pathway and C5 convertases via all complement pathways. Inhibition of C5 convertases results in complete inhibition of C5a generation and subsequent neutrophil migration. Here, we show that binding of Ecb to the C3d domain of C3b is crucial for inhibition of C5 convertases. Ecb does not interfere with substrate binding to convertases but prevents formation of an active convertase enzyme
Evolution of colistin resistance in the klebsiella pneumoniae complex follows multiple evolutionary trajectories with variable effects on fitness and virulence characteristics
The increasing prevalence of multidrug-resistant Klebsiella pneumoniae has led to a resurgence in the use of colistin as a last-resort drug. Colistin is a cationic antibiotic that selectively acts on Gram-negative bacteria through electrostatic interactions with anionic phosphate groups of the lipid A moiety of lipopolysaccharides (LPSs). Colistin resistance in K. pneumoniae is mediated through loss of these phosphate groups, their modification by cationic groups, and by the hydroxylation of acyl groups of lipid A. Here, we study the in vitro evolutionary trajectories toward colistin resistance in four clinical K. pneumoniae complex strains and their impact on fitness and virulence characteristics. Through population sequencing during in vitro evolution, we found that colistin resistance develops through a combination of single nucleotide polymorphisms, insertions and deletions, and the integration of insertion sequence elements, affecting genes associated with LPS biosynthesis and modification and capsule structures. Colistin resistance decreased the maximum growth rate of one K. pneumoniae sensu stricto strain, but not those of the other three K. pneumoniae complex strains. Colistin-resistant strains had lipid A modified through hydroxylation, palmitoylation, and l-Ara4N addition. K. pneumoniae sensu stricto strains exhibited cross-resistance to LL-37, in contrast to the Klebsiella variicola subsp. variicola strain. Virulence, as determined in a Caenorhabditis elegans survival assay, was increased in two colistin-resistant strains. Our study suggests that nosocomial K. pneumoniae complex strains can rapidly develop colistin resistance through diverse evolutionary trajectories upon exposure to colistin. This effectively shortens the life span of this last-resort antibiotic for the treatment of infections with multidrug-resistant Klebsiella