Streptococcus pneumoniae evades complement attack and opsonophagocytosis by expressing the pspC locus-encoded Hic protein that binds to short consensus repeats 8-11 of factor H

Streptococcus pneumoniae is an important cause of upper and lower respiratory tract infections, meningitis, peritonitis, bacterial arthritis, and sepsis. Here we have studied a novel immune evasion mechanism of serotype 3 pneumococci, which are particularly resistant to phagocytosis. On their surfaces the bacteria express the factor H-binding inhibitor of complement (Hic), a protein of the pneumococcal surface protein C family. Using radioligand binding, microtiter plate assays, surface plasmon resonance analysis, and recombinant constructs of factor H, we located the binding site of Hic to short consensus repeats (SCRs) 8-11 in the middle part of factor H. This represents a novel microbial interaction region on factor H. The only other ligand known so far for SCRs 8-11 of factor H is C-reactive protein (CRP), an acute phase protein that binds to the pneumococcal C-polysaccharide. The binding sites of Hic and CRP within the SCR8-11 region were different, however, because CRP did not inhibit the binding of Hic and required calcium for binding. Binding of factor H to Hic-expressing pneumococci promoted factor I-mediated cleavage of C3b and restricted phagocytosis of pneumococci. Thus, virulent pneumococci avoid complement attack and opsonophagocytosis by recruiting functionally active factor H with the Hic surface protein. Hic binds to a previously unrecognized microbial interaction site in the middle part of factor H.     

Induction of normal levels of genetic transformation in a class of endonuclease-defective mutants of Pneumococci.

Pneumococcal mutants were isolated that showed no zones of DNA hydrolysis around the bacterial colonies, even after prolonged incubation on the surface of agar plates containing DNA and methylgreen. Lysates of such mutants contained only very low levels of endonuclease activity; the mutants were also defective in genetic transformation even though they could still bind DNA to their surface. However, the same mutants could be made to undergo normal, high frequency genetic transformation by treatment with the activator protein, under appropriate conditions. The same treatment caused no detectable increase in the endonuclease level. Poor transformability of these mutants seems to be related to an inhibitory factor(s) released into the growth medium. Activation in the presence of this factor(s) leads to an abnormal (non-productive) DNA adsorption-both in mutant and in wild type pneumococci.     

Hic, a novel surface protein of Streptococcus pneumoniae that interferes with complement function

The important human pathogen Streptococcus pneumoniae was found to absorb factor H, an inhibitor of complement, from human plasma. We identified the gene encoding a novel surface protein, factor H-binding inhibitor of complement (Hic), in the pspC locus of type 3 pneumococci. Unlike PspC proteins in other serotypes, Hic is anchored to the cell wall by means of an LPXTG motif, and the overall sequence homology to various PspC proteins is low. However, the NH(2)-terminal region showed significant homology to the NH(2)-terminal region of several PspC proteins. A fragment of Hic, covering this homologous region, was expressed as a glutathione S-transferase (GST) fusion protein. GST:Hic(39-261) bound radiolabeled factor H and inhibited binding of factor H to pneumococci of different serotypes. Interaction kinetics between GST:Hic(39-261) and factor H were studied with surface plasmon resonance and showed a high affinity binding (K(A) = 5 x 10(7), K(D) = 2.3 x 10(-)(8)). Mutant pneumococci lacking Hic showed no absorption of factor H in human plasma and no binding of radiolabeled factor H, suggesting that Hic is responsible for factor H-binding in type 3 pneumococci. Factor H-dependent inhibition of the alternative pathway was not diminished by the presence of GST:Hic(39-261). In addition, an intrinsic inhibitory effect of Hic is suggested.     

New insights into the pneumococcal fratricide: relationship to clumping and identification of a novel immunity factor

In 1971, Tomasz and Zanati discovered that competent pneumococci have a tendency to form aggregates when pelleted by centrifugation and resuspended in 0.01 N HCl by brief vortexing. Interestingly, no clumping was observed with parallel cultures of non-competent cells treated in the same way. We set out to elucidate the mechanism behind this striking phenomenon, and were able to show that it depends on extracellular DNA that is presumably released by so-called competence-induced cell lysis. Competence-induced cell lysis, which was first described a few years ago, seems to rely on the concerted action of several murein hydrolases. Our results confirmed and extended previous findings by showing that competence-induced aggregation is abolished in a lytA-lytC double mutant, and absolutely requires CbpD and its N-terminal CHAP amidase domain. Furthermore, we discovered a novel competence stimulating peptide (CSP)-induced immunity protein, encoded by the early competence gene comM (spr1762), which protects competent pneumococci against their own lysins. Together, the murein hydrolases and the immunity protein constitutes a CSP-controlled mechanism that allows competent pneumococci to commit fratricide by killing non-competent pneumococci sharing the same ecological niche. Through such predatory behaviour, pneumococci can get access to transforming DNA and nutrients, promote the release of virulence factors, and at the same time get rid of competitors.     

Functional properties of complement factor H-related proteins FHR-3 and FHR-4: binding to the C3d region of C3b and differential regulation by heparin

The human factor H-related proteins FHR-3 and FHR4 are members of a family of proteins related to the complement factor H. Here, we report that the two proteins bind to the C3d region of complement C3b. The apparent K(A) values for the interactions of FHR-3 and FHR-4 with C3b are 7.5 x 10(6) M(-1) and 2.9 x 10(6) M(-1), respectively. Binding studies performed with C3b-coated pneumococci confirmed the results obtained with the biosensor system. A C-terminal construct of factor H showed similar binding characteristics. The interaction of FHR-3, but not of FHR4, with opsonised pneumococci was inhibited by heparin.     

Enantioselective binding of ofloxacin to B form DNA.

The binding affinity and binding mode of S- and R-ofloxacin, one of the quinolone antibiotics, to B form calf thymus DNA were studied in this work. The binding affinity of S-ofloxacin measured by both Stern-Volmer and Benesi-Hilderbrand methods was greater by a factor of 5 compared to R-enantiomer and the CD spectrum of the former is largely altered while that of the latter remained the same in the presence of DNA, indicating the enantiospecific binding of this drug to DNA. The binding geometry of both S- and R-ofloxacin calculated from the reduced linear dichroism was similar to norfloxacin, which is partially intercalated from the minor groove.     

The host immune regulator factor H interacts via two contact sites with the PspC protein of Streptococcus pneumoniae and mediates adhesion to host epithelial cells

Pneumococcal surface protein C (PspC) of Streptococcus pneumoniae is a key virulence factor that mediates adhesion to host cells and immune evasion of the host complement. PspC binds the host immune and complement regulator factor H, which is composed of 20 short consensus repeats (SCR). This interaction contributes to pneumococcal virulence. In this study, we identified within the factor H protein two separate PspC binding regions, which were localized to SCR8-11 and SCR19-20, by using recombinant factor H deletion constructs for Western blotting assays and surface plasmon resonance studies. A detailed analysis of binding epitopes in these SCR by peptide spot arrays identified several linear binding regions within the sequences of SCR8-11 and SCR19-20. In addition, the factor H binding site was mapped within the pneumococcal PspC protein to a 121-aa-long stretch positioned in the N terminus (residues 38-158). Factor H attached to the surface of pneumococci via PspC significantly enhanced pneumococcal adherence to host epithelial and endothelial cells. This adhesion was specific and was blocked with a truncated N-terminal factor H-binding fragment of PspC. In conclusion, the acquisition of factor H by pneumococci via PspC occurs via two contact sites located in SCR8-11 and SCR19-20, and factor H attached to the surface of the pneumococcus promotes adhesion to both host epithelial and endothelial cells.     

肺炎链球菌磷壁酸胆碱成分介导细菌的侵袭

分析肺炎链球菌细胞壁胆碱成分在其侵袭宿主细胞的过程中的作用。通过肺炎链球菌对人脐静脉内皮细胞的侵袭实验 ,观察血小板活化因子受体 (PAF R)拮抗剂BN 5 2 0 2 1对肺炎链球菌侵袭率的变化 ,以及乙醇胺取代细胞壁胆碱后肺炎链球菌侵袭率的变化。研究发现受体拮抗剂BN 5 2 0 2 1处理活化血管内皮细胞后 ,肺炎链球菌的侵袭率显著降低 (P <0 0 1 ) ,乙醇胺取代肺炎链球菌细胞壁成份中的胆碱同样降低了细菌对活化内皮细胞的侵袭 (P<0 0 1 )。实验表明肺炎链球菌可能是通过脂磷壁酸和磷壁酸的胆碱成分与内皮细胞表面的血小板活化因子受体结合来介导侵袭作用的     

Nucleolytic degradation of homologous and heterologous deoxyribonucleic acid molecules at the surface of competent pneumococci.

Competent pneumococci can catalyze the rapid and quantitative degradation of extracellular deocyribonucleic acid (DNA) molecules through the activity of surface-located nucleases (endo- and, possibly, exonucleases as well). Both homologous and heterologous DNAs are degraded by a mechanism that seems to involve a cyclic process: (i) attachment of DNA to the cell surface followed by (ii) nucleolytic attack, and (iii) release to the medium. Processes (ii) and (iii) are both inhibited by ethylenediaminetetraacetate. Whereas surface nuclease activity is specific for competent cells, the bulk of this activity is not coupled to irreversible DNA uptake (deoxyribonuclease-resistant binding). Pneumococcal DNA treated with ultraviolet irradiation or nitrous acid (cross-linking?) is selectively impaired in the ability to irreversibly bind to competent cells, whereas reversible binding is normal.     

Functional homology between the sequence-specific DNA-binding proteins nuclear factor I from HeLa cells and the TGGCA protein from chicken liver.

Nuclear factor I from HeLa cells, a protein with enhancing function in adenovirus DNA replication, and the chicken TGGCA protein are specific DNA-binding proteins that were first detected by independent methods and that appeared to have similar DNA sequence specificity. To test whether they are homologous proteins from different species we have compared (i) their DNA binding properties and (ii) their function in reconstituted adenovirus DNA replication systems. Using deletion and substitution mutants derived from the DNA binding site on the adenovirus 2 inverted terminal repeat, it was found that the two proteins protect the same 24-nucleotide region of both strands against DNase I digestion and that they have identical minimal recognition sequences of 15 bp containing dyad symmetry. Like nuclear factor I, the TGGCA protein enhances the initiation reaction of adenovirus 2 DNA replication in vitro in a DNA recognition site-dependent manner.     

Cell surface-located deoxyribonucleic acid receptors in transformable pneumococci.

We studied deoxyribonucleic acid (DNA) binding in transformable pneumococci. The relevant findings are as follows. (i) At least half of the DNA Molecules adsorbed to competent cells in the growth medium are attached to sites on the protoplast membrane. (ii) Most of the DNA bound to live competent cells in the presence of glucose is not released by moderate shear or by autolysin treatment. In contrast, most of the DNA adsorbed to competent cells in the absence of glucose is shear and autolysin sensitive. (iii) The presence of binding sites resembling in properties the sites in live competent cells can be demonstrated in wall-membrane complexes. Most of these sites are lost during preparation of cell walls and protoplasts. It is suggested that the DNA-binding site is a membrane component (protein?) Stabilized by polysaccharide (cell Wall) material. (IV) Mechanical or enzymatic damage to the cell wall or change in the ionic conditions can induce DNA binding (and surface-nuclease activity) in the incompetent pneumococci. However, such cells still show neither genetic transformation nor extensive nuclease-resistant binding of DNA. It is suggested that both competent and incompetent cells contain a large number of sequestered DNA-binding sites that can be unmasked by several experimental conditions. Induction of the competent state by the competence activator protein may involve an endogenous unmasking process.     

Competence-induced cells of Streptococcus pneumoniae lyse competence-deficient cells of the same strain during cocultivation

Several streptococcal species are able to take up naked DNA from the environment and integrate it into their genomes by homologous recombination. This process is called natural transformation. In Streptococcus pneumoniae and related streptococcal species, competence for natural transformation is induced by a peptide pheromone through a quorum-sensing mechanism. Recently we showed that induction of the competent state initiates lysis and release of DNA from a subfraction of the bacterial population and that the efficiency of this process is influenced by cell density. Here we have further investigated the nature of this cell density-dependent release mechanism. Interestingly, we found that competence-induced pneumococci lysed competence-deficient cells of the same strain during cocultivation and that the efficiency of this heterolysis increased as the ratio of competent to noncompetent cells increased. Furthermore, our results indicate that the lysins made by competent pneumococci are not released into the growth medium. More likely, they are anchored to the surface of the competent cells by choline-binding domains and cause lysis of noncompetent pneumococci through cell-to-cell contact.     

A protein target site in an early replicated human DNA sequence: a highly conserved binding motif

We have previously reported that a human nuclear factor, probably corresponding to the USF/MLTF protein [1,2], is able to bind specifically to a DNA sequence present in DNA replicated at the onset of S-phase [3]. Here we demonstrate that the same factor binds also to several other similar sequences, present in eukaryotic and viral genomes. Mutations or methylation in a CpG dinucleotide, central in the palindromic binding site, completely abolish binding. Furthermore, we present evidence for the existence of at least two other nuclear proteins in human cells with the same DNA binding specificity. The data presented suggest a strong evolutionary conservation, among distantly related organisms, of the binding motif, which is probably the target of a number of nuclear factors that share the same DNA binding specificity albeit in the context of different functions.     

Intraspecies and Interspecies Transformation Reactions in Pneumococcus and Streptococcus

The efficiency of transformation of pneumococcus and a strain of viridans streptococcus (strain D) to streptomycin resistance is influenced by the species in which the mutation to resistance occurred, as well as by the species in which the mutated gene has been replicated. Pneumococcus and streptococcus strain D transform in higher frequency with DNA that has been replicated in bacteria of the same species than with DNA from the heterologous species. However, the difference between the frequencies of interspecific and intraspecific transformation is much greater with pneumococcus as receptor than with streptococcus. In addition pneumococcus transforms in higher frequency with wholly homologous (pneumococcal) DNA than with DNA from pneumococci that have replicated the streptococcal Sm^r gene. Pneumococcus is transformed in lower frequency by wholly heterologous (streptococcal) DNA than by DNA from streptococci that have replicated the pneumococcal Sm^r gene. Streptococcus behaves similarly in that wholly homologous (streptococcal) DNA transforms it more efficiently than when the transforming fragment contains a pneumococcal moiety. Streptococcus is transformed in the same or lower frequency by wholly heterologous (pneumococcal) DNA than by DNA from pneumococci that have replicated the streptococcal Sm^r gene. When erythromycin resistance was used as genetic marker instead of streptomycin resistance, similar results were found.     

Pneumococcal Adhesins PavB and PspC Are Important for the Interplay with Human Thrombospondin-1

The human matricellular glycoprotein thrombospondin-1 (hTSP-1) is released by activated platelets and mediates adhesion of Gram-positive bacteria to various host cells. In staphylococci, the adhesins extracellular adherence protein (Eap) and autolysin (Atl), both surface-exposed proteins containing repeating structures, were shown to be involved in the acquisition of hTSP-1 to the bacterial surface. The interaction partner(s) on the pneumococcal surface was hitherto unknown. Here, we demonstrate for the first time that pneumococcal adherence and virulence factor B (PavB) and pneumococcal surface protein C (PspC) are key players for the interaction of Streptococcus pneumoniae with matricellular hTSP-1. PavB and PspC are pneumococcal surface-exposed adhesins and virulence factors exhibiting repetitive sequences in their core structure. Heterologously expressed fragments of PavB and PspC containing repetitive structures exhibit hTSP-1 binding activity as shown by ELISA and surface plasmon resonance studies. Binding of hTSP-1 is charge-dependent and inhibited by heparin. Importantly, the deficiency in PavB and PspC reduces the recruitment of soluble hTSP-1 by pneumococci and decreases hTSP-1-mediated pneumococcal adherence to human epithelial cells. Platelet activation assays suggested that PavB and PspC are not involved in the activation of purified human platelets by pneumococci. In conclusion, this study indicates a pivotal role of PavB and PspC for pneumococcal recruitment of soluble hTSP-1 to the bacterial surface and binding of pneumococci to host cell-bound hTSP-1 during adhesion.