Human C4b-binding protein, structural basis for interaction with streptococcal M protein, a major bacterial virulence factor

Human C4b-binding protein (C4BP) protects host tissue, and those pathogens able to hijack this plasma glycoprotein, from complement-mediated destruction. We now show that the first two complement control protein (CCP) modules of the C4BP alpha-chain, plus the four residues connecting them, are necessary and sufficient for binding a bacterial virulence factor, the Streptococcus pyogenes M4 (Arp4) protein. Structure determination by NMR reveals two tightly coupled CCP modules in an elongated arrangement within this region of C4BP. Chemical shift perturbation studies demonstrate that the N-terminal, hypervariable region of M4 binds to a site including strand 1 of CCP module 2. This interaction is accompanied by an intermodular reorientation within C4BP. We thus provide a detailed picture of an interaction whereby a pathogen evades complement.     

Evidence that polygalacturonase is a virulence determinant in Erwinia carotovora.

Polygalacturonase (PG) was purified from Erwinia carotovora EC. A hybrid cosmid, pSH711, that encodes PG activity but not pectate lyase activity was identified from an E. carotovora genomic library by an immunological screening method. A cell extract of Escherichia coli cells containing pSH711 was able to produce plant tissue maceration when spotted on carrot, potato, or turnip slices. In addition, the E. coli strain containing this plasmid was able to macerate carrot, potato, and turnip slices. Our results suggest that PG plays an important role in soft-rot disease.     

Serum opacity factor is a major fibronectin-binding protein and a virulence determinant of M type 2 Streptococcus pyogenes

Serum opacity factor (SOF) is a fibronectin-binding protein of group A streptococci that opacifies mammalian sera and is expressed by some strains that cause impetigo, pharyngitis and acute glomerulonephritis. Although SOF is expressed by approximately 35% of known serotypes, its role in the pathogenesis of group A streptococcal infections has not been previously investigated. The sof genes from M types 2, 28 and 49 Streptococcus pyogenes were cloned, sequenced, and their deduced amino acid sequences were compared. The gene for FnBA, a fibronectin-binding protein from Streptococcus dysgalactiae, was also cloned and found to express an opacity factor. The leader sequences, the fibronectin-binding domains, and the membrane anchor regions of these proteins were highly conserved. Short spans of conserved sequences were interspersed throughout the remaining parts of the proteins. The sof2 gene was insertionally inactivated in an M type 2 S. pyogenes strain, T2MR. The resultant SOF-negative mutant (YL3) did not express SOF or opacify serum, and exhibited a 71% reduction in binding fibronectin. Complementation of the SOF-negative defect with sof28 in the recombinant strain YL3(pNZ28) fully restored fibronectin-binding activity and the ability to opacify serum. To determine whether sof plays a role in virulence, mice were challenged intraperitoneally with these strains. None of the 10 mice infected with YL3(pNZ28) survived and only 1 out of 15 mice challenged with T2MR survived, whereas 12 out of 15 mice infected with YL3 survived. These data clearly indicate that SOF is a virulence factor, and they provide the first direct evidence that a fibronectin-binding protein contributes to the pathogenesis of group A streptococcal infections in vivo.     

Human fibrinogen bound to Streptococcus pyogenes M protein inhibits complement deposition via the classical pathway

Human fibrinogen (Fg) binds to surface proteins expressed by many pathogenic bacteria and has been implicated in different host-pathogen interactions, but the role of bound Fg remains unclear. Here, we analyse the role of Fg bound to Streptococcus pyogenes M protein, a major virulence factor that confers resistance to phagocytosis. Studies of the M5 system showed that a chromosomal mutant lacking the Fg-binding region was completely unable to resist phagocytosis, indicating that bound Fg plays a key role in virulence. Deposition of complement on S. pyogenes occurred via the classical pathway even under non-immune conditions, but was blocked by M5-bound Fg, which reduced the amount of classical pathway C3 convertase on the bacterial surface. This property of M protein-bound Fg may explain its role in phagocytosis resistance. Previous studies have shown that many M proteins do not bind Fg, but interfere with complement deposition and phagocytosis by recruiting human C4b-binding protein (C4BP), an inhibitor of the classical pathway. Thus, all M proteins may share ability to recruit a human plasma protein, Fg or C4BP, which inhibits complement deposition via the classical pathway. Our data identify a novel function for surface-bound Fg and allow us to propose a unifying mechanism by which M proteins interfere with innate immunity.     

The E2 glycoprotein of classical swine fever virus is a virulence determinant in swine

To identify genetic determinants of classical swine fever virus (CSFV) virulence and host range, chimeras of the highly pathogenic Brescia strain and the attenuated vaccine strain CS were constructed and evaluated for viral virulence in swine. Upon initial screening, only chimeras 138.8v and 337.14v, the only chimeras containing the E2 glycoprotein of CS, were attenuated in swine despite exhibiting unaltered growth characteristics in primary porcine macrophage cell cultures. Additional viral chimeras were constructed to confirm the role of E2 in virulence. Chimeric virus 319.1v, which contained only the CS E2 glycoprotein in the Brescia background, was markedly attenuated in pigs, exhibiting significantly decreased virus replication in tonsils, a transient viremia, limited generalization of infection, and decreased virus shedding. Chimeras encoding all Brescia structural proteins in a CS genetic background remained attenuated, indicating that additional mutations outside the structural region are important for CS vaccine virus attenuation. These results demonstrate that CS E2 alone is sufficient for attenuating Brescia, indicating a significant role for the CSFV E2 glycoprotein in swine virulence.     

Synthesis and biological evaluation of reversible inhibitors of IdeS,a bacterial cysteine protease and virulence determinant

Analogues of the irreversible protease inhibitors TPCK and TLCK have been synthesized and tested as inhibitors of the bacterial cysteine protease IdeS excreted by Streptococcus pyogenes. Eight compounds were identified as inhibitors of IdeS in an in vitro assay. The most potent compounds contained an aldehyde function, thus acting as efficient reversible inhibitors, nitrile and azide derivatives showed moderate activity.     

The acute myeloid leukemia-associated protein, DEK, forms a splicing-dependent interaction with exon-product complexes

DEK is an approximately 45-kD phosphoprotein that is fused to the nucleoporin CAN as a result of a (6;9) chromosomal translocation in a subset of acute myeloid leukemias (AMLs). It has also been identified as an autoimmune antigen in juvenile rheumatoid arthritis and other rheumatic diseases. Despite the association of DEK with several human diseases, its function is not known. In this study, we demonstrate that DEK, together with SR proteins, associates with the SRm160 splicing coactivator in vitro. DEK is recruited to splicing factor-containing nuclear speckles upon concentration of SRm160 in these structures, indicating that DEK and SRm160 associate in vivo. We further demonstrate that DEK associates with splicing complexes through interactions mediated by SR proteins. Significantly, DEK remains bound to the exon-product RNA after splicing, and this association requires the prior formation of a spliceosome. Thus, DEK is a candidate factor for controlling postsplicing steps in gene expression that are influenced by the prior removal of an intron from pre-mRNA.     

SseG, a virulence protein that targets Salmonella to the Golgi network

Intracellular replication of the bacterial pathogen Salmonella enterica occurs in membrane-bound compartments called Salmonella-containing vacuoles (SCVs). Maturation of the SCV has been shown to occur by selective interactions with the endocytic pathway. We show here that after invasion of epithelial cells and migration to a perinuclear location, the majority of SCVs become surrounded by membranes of the Golgi network. This process is dependent on the Salmonella pathogenicity island 2 type III secretion system effector SseG. In infected cells, SseG was associated with the SCV and peripheral punctate structures. Only bacterial cells closely associated with the Golgi network were able to multiply; furthermore, mutation of sseG or disruption of the Golgi network inhibited intracellular bacterial growth. When expressed in epithelial cells, SseG co-localized extensively with markers of the trans-Golgi network. We identify a Golgi-targeting domain within SseG, and other regions of the protein that are required for localization of bacteria to the Golgi network. Therefore, replication of Salmonella in epithelial cells is dependent on simultaneous and selective interactions with both endocytic and secretory pathways.     

Identification of a new metalloproteinase inhibitor that forms tight-binding complexes with collagenase.

Connective-tissue cells produce a family of metalloproteinases which, once activated, can degrade all the components of the extracellular matrix. These potent enzymes are all inhibited by the tissue inhibitor of metalloproteinases (TIMP), and it was thought that the levels of this inhibitor controlled the extracellular activity of these enzymes. We recently detected a new metalloproteinase inhibitor present in culture media of WI-38 fibroblasts. The inhibitor, named 'large inhibitor of metalloproteinases' (LIMP), can be separated from TIMP by gel filtration on Ultrogel AcA 44, where it is eluted with an apparent Mr of 76,000. A portion of this inhibitor-containing peak binds to concanavalin A-Sepharose, indicating that at least some of the inhibitor contains carbohydrate. LIMP inhibits collagenase (MMP-1), stromelysin (MMP-3) and gelatinase (MMP-2) in a dose-dependent fashion. Collagenase forms tight-binding complexes with LIMP, which can be separated from free collagenase on gel-filtration columns. The complex is eluted with Mr 81,600 (AcA 44) or Mr 60,000 (Superose 12). This complex is larger than that formed between collagenase and TIMP, which has Mr 52,800 (Aca 44) or 41,000 (Superose 12). Polyclonal antibody to TIMP does not recognize LIMP by immunoblotting, and will not block the inhibition of collagenase by LIMP, showing that LIMP is not a multimeric form of TIMP. The role of this new inhibitor in connective-tissue breakdown studies and its relationship to previously described inhibitors of metalloproteinases is discussed.     

The yersinia virulence factor YopM forms a novel protein complex with two cellular kinases

Pathogenic Yersinia contain a virulence plasmid that encodes genes for intracellular effectors, which neutralize the host immune response. One effector, YopM, is necessary for Yersinia virulence, but its function in host cells is unknown. To identify potential cellular pathways affected by YopM, proteins that co-immunoprecipitate with YopM in mammalian cells were isolated and identified by mass spectrometry. Results demonstrate that two kinases, protein kinase C-like 2 (PRK2) and ribosomal S6 protein kinase 1 (RSK1), interact directly with YopM. These two kinases associate only when YopM is present, and expression of YopM in cells stimulates the activity of both kinases. RSK1 is activated directly by interaction with YopM, and RSK1 kinase activity is required for YopM-stimulated PRK2 activity. YopM activation of RSK1 occurs independently of the actions of YopJ on the MAPK pathway. YopM is also required for Yersinia-induced changes in RSK1 mobility in infected macrophage cells. These results identify the first intracellular targets of YopM and suggest YopM acts to stimulate the activity of PRK2 and RSK1.     

Prion protein aggresomes are poly(A)+ ribonucleoprotein complexes that induce a PKR-mediated deficient cell stress response

In mammalian cells, cytoplasmic protein aggregates generally coalesce to form aggresomal particles. Recent studies indicate that prion-infected cells produce prion protein (PrP) aggresomes, and that such aggregates may be present in the brain of infected mice. The molecular activity of PrP aggresomes has not been fully investigated. We report that PrP aggresomes initiate a cell stress response by activating the RNA-dependent protein kinase (PKR). Activated PKR phosphorylates the translation initiation factor eIF2alpha, resulting in protein synthesis shut-off. However, other components of the stress response, including the assembly of poly(A)+ RNA-containing stress granules and the synthesis of heat shock protein 70, are repressed. In situ hybridization experiments and affinity chromatography on oligo(dT)-cellulose showed that PrP aggresomes bind poly(A)+ RNA, and are therefore poly(A)+ ribonucleoprotein complexes. These findings support a model in which PrP aggresomes send neuronal cells into untimely demise by modifying the cell stress response, and by inducing the aggregation of poly(A)+ RNA.     

A cooperative virulence plasmid imposes a high fitness cost under conditions that induce pathogenesis

Harbouring a plasmid often imposes a fitness cost on the bacterial host. Motivated by implications for public health, the majority of studies on plasmid cost are focused on elements that impart antibiotic resistance. Plasmids, however, can provide a wide range of ecologically important phenotypes to their bacterial hosts-such as virulence, specialized catabolism and metal resistance. The Agrobacterium tumefaciens tumour-inducing (Ti) plasmid confers both the ability to infect dicotyledonous plants and to catabolize the metabolites that plants produce as a result of being infected. We demonstrate that this virulence and catabolic plasmid imposes a measurable fitness cost on host cells under resource-limiting, but not resource replete, environmental conditions. Additionally, we show that the expression of Ti-plasmid-borne pathogenesis genes necessary to initiate cooperative pathogenesis is extremely costly to the host cell. The benefits of agrobacterial pathogenesis stem from the catabolism of public goods produced by infected host plants. Thus, the virulence-plasmid-dependent costs we demonstrate constitute costs of cooperation typically associated with the ability to garner the benefits of cooperation. Interestingly, genotypes that harbour derived opine catabolic plasmids minimize this trade-off, and are thus able to freeload upon the pathogenesis initiated by other individuals.     

Pyruvate oxidase, as a determinant of virulence in Streptococcus pneumoniae

Pneumococcus has been shown to bind to epithelial cells of the nasopharynx and lung, and to endothelial cells of the peripheral vasculature. To characterize bacterial elements required for attachment to these cell types, a library of genetically altered pneumococci with defects in exported proteins was screened for the loss of attachment to glycoconjugates representative of the nasopharyngeal cell receptor, type II lung cells (LC) and human endothelial cells (EC). A mutant was identified which showed a greater than 70% loss in the ability to attach to all cell types. This mutant also showed decreased adherence to the glycoconjugates containing the terminal sugar residues GalNAcβ1-3Gal, GalNAcβ1-4Gal and the carbohydrate GlcNAc, which are proposed components of the pneumococcal receptors specific to the surfaces of LC and EC. Analysis of the locus altered in this mutant revealed a gene, spxB, that encodes a member of the family of bacterial pyruvate oxidases which decarboxylates pyruvate to acetyl phosphate plus H₂O₂ and CO₂. This mutant produced decreased concentrations of H₂O₂ and failed to grow aerobically in a chemically defined medium, unless supplemented with acetate which presumably restores acetyl phosphate levels by the action of acetate kinase, further suggesting that spxB encodes a pyruvate oxidase. The addition of acetate to the growth medium restored the adherence properties of the mutant indicating a link between the enzyme and the expression of bacterial adhesins. A defect in spxB corresponded to impaired virulence of the mutant in vivo. Compared to the parent strain, an spxB mutant showed reduced virulence in animal models for nasopharyngeal colonization, pneumonia, and sepsis. We propose that a mutation in spxB leads to down-regulation of the multiple adhesive properties of pneumococcus which, in turn, may correlate to diminished virulence in vivo     

Escherichia coli single-strand binding protein forms multiple, distinct complexes with single-stranded DNA

Four distinct binding modes for the interaction of Escherichia coli single-strand binding (SSB) protein with single-stranded (ss) DNA have been identified on the basis of quantitative titrations that monitor the quenching of the SSB protein fluorescence upon binding to the homopolynucleotide poly(dT) over a range of MgCl2 and NaCl concentrations at 25 and 37 degrees C. This is the first observation of multiple binding modes for a single protein binding to DNA. These results extend previous studies performed in NaCl (25 degrees C, pH 8.1), in which two distinct SSB-ss DNA binding modes possessing site sizes of 33 and 65 nucleotides per bound SSB tetramer were observed [Lohman, T.M., & Overman, L. B. (1985) J. Biol. Chem. 260, 3594-3603]. Each of these binding modes differs in the number of nucleotides occluded upon interaction with ss DNA (i.e., site size). Along with the previously observed modes with site sizes of 35 +/- 2 and 65 +/- 3 nucleotides per tetramer, a third distinct binding mode, at 25 degrees C, has been identified, possessing a site size of 56 +/- 3 nucleotides per bound SSB tetramer, which is stable over a wide range of MgCl2 concentrations. At 37 degrees C, a fourth binding mode is observed, possessing a site size of 40 +/- 2 nucleotides per tetramer, although this mode is observable only over a small range of salt concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cbp1, a fungal virulence factor under positive selection,forms an effector complex that drives macrophage lysis

Intracellular pathogens secrete effectors to manipulate their host cells. Histoplasma capsulatum (Hc) is a fungal intracellular pathogen of humans that grows in a yeast form in the host. Hc yeasts are phagocytosed by macrophages, where fungal intracellular replication precedes macrophage lysis. The most abundant virulence factor secreted by Hc yeast cells is Calcium Binding Protein 1 (Cbp1), which is absolutely required for macrophage lysis. Here we take an evolutionary, structural, and cell biological approach to understand Cbp1 function. We find that Cbp1 is present only in the genomes of closely related dimorphic fungal species of the Ajellomycetaceae family that lead primarily intracellular lifestyles in their mammalian hosts (Histoplasma, Paracoccidioides, and Emergomyces), but not conserved in the extracellular fungal pathogen Blastomyces dermatitidis. We observe a high rate of fixation of non-synonymous substitutions in the Cbp1 coding sequences, indicating that Cbp1 is under positive selection. We determine the de novo structures of Hc H88 Cbp1 and the Paracoccidioides americana (Pb03) Cbp1, revealing a novel “binocular” fold consisting of a helical dimer arrangement wherein two helices from each monomer contribute to a four-helix bundle. In contrast to Pb03 Cbp1, we show that Emergomyces Cbp1 orthologs are unable to stimulate macrophage lysis when expressed in the Hc cbp1 mutant. Consistent with this result, we find that wild-type Emergomyces africanus yeast are able to grow within primary macrophages but are incapable of lysing them. Finally, we use subcellular fractionation of infected macrophages and indirect immunofluorescence to show that Cbp1 localizes to the macrophage cytosol during Hc infection, making this the first instance of a phagosomal human fungal pathogen directing an effector into the cytosol of the host cell. We additionally show that Cbp1 forms a complex with Yps-3, another known Hc virulence factor that accesses the cytosol. Taken together, these data imply that Cbp1 is a fungal virulence factor under positive selection that localizes to the cytosol to trigger host cell lysis.     

Characterization of membrane-associated Pseudomonas aeruginosa Ras-like protein Pra, a GTP-binding protein that forms complexes with truncated nucleoside diphosphate kinase and pyruvate kinase to modulate GTP synthesis.

We report the purification and characterization of a protein from the membrane fraction of Pseudomonas aeruginosa showing intrinsic guanosine triphosphatase (GTPase) activity. The protein was purified as a 48-kDa polypeptide capable of binding and hydrolyzing GTP. The N-terminal sequence of the purified protein revealed its similarity to the Escherichia coli Ras-like protein (Era), and the protein cross-reacted with anti-Era antibodies. This protein was named Pseudomonas Ras-like protein (Pra). Anti-Pra antibodies also cross-reacted with E. coli Era protein. Pra is autophosphorylated in vitro, with phosphotransfer of the terminal phosphate from [gamma-32P]GTP but not [gamma-32P]ATP. Pra is capable of complex formation with the truncated 12-kDa form of nucleoside diphosphate kinase (Ndk) but not with the 16-kDa form. Purified Pra was also shown to physically interact with pyruvate kinase (Pk); Pk and Pra can form a complex, but when the 12-kDa Ndk, Pk, and Pra are all present, Pk has a higher affinity than Pra for forming a complex with the 12-kDa Ndk. The 12-kDa Ndk-Pra complex catalyzed increased synthesis of GTP and dGTP and diminished synthesis of CTP and UTP or dCTP and dTTP relative to their synthesis by uncomplexed Ndk. Moreover, the complex of Pra with Pk resulted in the specific synthesis of GTP as well when Pra was present in concentrations in excess of that of Pk. Membrane fractions from cells harvested in the mid-log phase demonstrated very little nucleoside triphosphate (NTP)-synthesizing activity and no detectable Ndk. Membranes from cells harvested at late exponential phase showed NTP-synthesizing activity and the physical presence of Ndk but not of Pk or Pra. In contrast, membrane fractions of cells harvested at early to late stationary phase showed predominant GTP synthesis and the presence of increasing amounts of Pk and Pra. It is likely that the association of Pra with Ndk and/or Pk restricts its intrinsic GTPase activity, which may modulate stationary-phase gene expression and the survival of P. aeruginosa by modulating the level of GTP.     

Skp1 forms multiple protein complexes, including RAVE, a regulator of V-ATPase assembly

SCF ubiquitin ligases are composed of Skp1, Cdc53, Hrt1 and one member of a large family of substrate receptors known as F-box proteins (FBPs). Here we report the identification, using sequential rounds of epitope tagging, affinity purification and mass spectrometry, of 16 Skp1 and Cdc53-associated proteins in budding yeast, including all components of SCF, 9 FBPs, Yjr033 (Rav1) and Ydr202 (Rav2). Rav1, Rav2 and Skp1 form a complex that we have named 'regulator of the (H+)-ATPase of the vacuolar and endosomal membranes' (RAVE), which associates with the V1 domain of the vacuolar membrane (H+)-ATPase (V-ATPase). V-ATPases are conserved throughout eukaryotes, and have been implicated in tumour metastasis and multidrug resistance, and here we show that RAVE promotes glucose-triggered assembly of the V-ATPase holoenzyme. Previous systematic genome-wide two-hybrid screens yielded 17 proteins that interact with Skp1 and Cdc53, only 3 of which overlap with those reported here. Thus, our results provide a distinct view of the interactions that link proteins into a comprehensive cellular network.     

Cripto is a noncompetitive activin antagonist that forms analogous signaling complexes with activin and nodal

Cripto plays critical roles during embryogenesis and has been implicated in promoting the growth and spread of tumors. Cripto is required for signaling by certain transforming growth factor-beta superfamily members, such as Nodal, but also antagonizes others, such as activin. The opposing effects of Cripto on Nodal and activin signaling seem contradictory, however, because these closely related ligands utilize the same type I (ALK4) and type II (ActRII/IIB) receptors. Here, we have addressed this apparent paradox by demonstrating that Cripto forms analogous receptor complexes with Nodal and activin and functions as a noncompetitive activin antagonist. Our results show that activin-A and Nodal elicit similar maximal signaling responses in the presence of Cripto that are substantially lower than that of activin-A in the absence of Cripto. In addition, we provide biochemical evidence for complexes containing activin-A, Cripto, and both receptor types and show that the assembly of such complexes is competitively inhibited by Nodal. We further demonstrate that Nodal and activin-A share the same binding site on ActRII and that ALK4 has distinct and separable binding sites for activin-A and Cripto. Finally, we show that ALK4 mutants with disrupted activin-A binding retain Cripto binding and prevent the effects of Cripto on both activin-A and Nodal signaling. Together, our data indicate that Cripto facilitates Nodal signaling and inhibits activin signaling by forming receptor complexes with these ligands that are structurally and functionally similar.