Molecular and biological characterization of Streptococcal SpyA-mediated ADP-ribosylation of intermediate filament protein vimentin

The Gram-positive bacterial pathogen Streptococcus pyogenes produces a C3 family ADP-ribosyltransferase designated SpyA (S. pyogenes ADP-ribosyltransferase). Our laboratory has identified a number of eukaryotic protein targets for SpyA, prominent among which are the cytoskeletal proteins actin and vimentin. Because vimentin is an unusual target for modification by bacterial ADP-ribosyltransferases, we quantitatively compared the activity of SpyA on vimentin and actin. Vimentin was the preferred substrate for SpyA (k(cat), 58.5 ± 3.4 min(-1)) relative to actin (k(cat), 10.1 ± 0.6 min(-1)), and vimentin was modified at a rate 9.48 ± 1.95-fold greater than actin. We employed tandem mass spectrometry analysis to identify sites of ADP-ribosylation on vimentin. The primary sites of modification were Arg-44 and -49 in the head domain, with several additional secondary sites identified. Because the primary sites are located in a domain of vimentin known to be important for the regulation of polymerization by phosphorylation, we investigated the effects of SpyA activity on vimentin polymerization, utilizing an in vitro NaCl-induced filamentation assay. SpyA inhibited vimentin filamentation, whereas a catalytic site mutant of SpyA had no effect. Additionally, we demonstrated that expression of SpyA in HeLa cells resulted in collapse of the vimentin cytoskeleton, whereas expression in RAW 264.7 cells impeded vimentin reorganization upon stimulation of this macrophage-like cell line with LPS. We conclude that SpyA modification of vimentin occurs in an important regulatory region of the head domain and has significant functional effects on vimentin assembly.     

Pineal peptide with an inhibiting effect on growth of HeLa S3 tumor cells

The aim of the present study was to assess whether a peptide fraction isolated from calf pineal glands has an effect on proliferation and morphology of HeLa S3 tumor cells. Under the experimental conditions adopted, the results showed that the peptide has a marked inhibitory effect on proliferation of HeLa S3 cells and that permeabilization with calcium phosphate of the plasmatic membrane increases this effect. Moreover, the pineal peptide affects the cytoskeletal morphology of HeLa cells by modifying the distribution of actin. The peptide is probably internalized by the cells and irreversibly modifies the cytoskeletal morphology with consequent inhibition of cellular proliferation.     

Two distinct pathways for the invasion of Streptococcus pyogenes in non-phagocytic cells

Adherence to and invasion of epithelial cells represent important pathogenic mechanisms of Streptococcus pyogenes . A fibronectin-binding surface protein of S. pyogenes , SfbI protein, has been implicated in both adherence and invasion processes. Invasion of SfbI-containing strains has been suspected to be responsible for the failure of antibiotics treatment to eradicate S. pyogenes . In this study, we tested the adherence and invasion properties of two well-characterized clinical isolates: A40, which expresses SfbI; and A8, which is SfbI negative and is unable to bind fibronectin. In strain A40, SfbI was the main factor required for attachment and invasion by using fibronectin as a bridging molecule and the α₅β₁ integrin as cellular receptor. The uptake process was characterized by the generation of large membrane invaginations at the bacteria–cell interface without evidence of actin recruitment or cellular injury. A40 cells were located in phagosomes and, only 24 h after infection, a consistent part of the bacterial population reached the cytoplasm. In contrast, uptake of strain A8 required major rearrangements of cytoskeletal proteins underneath attached bacteria. In A8, a proteinaceous moiety was involved, which does not interact with α₅β₁ or need any known bridging molecule. Bacterial attachment stimulated elongation and massive recruitment of neighbouring microvilli, which fused to surround streptococcal chains. They led to the generation of large pseudopod-like structures, which engulfed bacteria that were rapidly released and replicated in the cytoplasm. The identification of two completely different uptake pathways reported here provided further evidence regarding the diversity of S. pyogenes isolates and might contribute towards understanding the pathogenesis and persistence of S. pyogenes .     

Electrophoretic pattern and distribution of cytoskeletal proteins in flat-epitheloid and stellate process-bearing astrocytes in primary culture

One- and two-dimensional electrophoresis patterns and distribution of major cytoskeletal proteins were studied in primary astrocytes with either flat-epitheloid or stellate appearance. No major differences in the electrophoretic patterns of actin, tubulin, glial fibrillary acidic protein (GFAP) and vimentin were detected between flat-epitheloid and stellate process-bearing astrocytes produced by the exposure of cultures to dibutyryl cyclic AMP (dBcAMP). However the morphological changes of astrocytes were accompanied by marked changes in the quantitative distribution of cytoskeletal proteins. The most prominent change was a large and specific decrease in the amount of actin, detected by [³⁵S]methionine incorporation, densitometric scanning of one-dimensional gels and DNase inhibition assay. In stellate astrocytes produced by a 4 day treatment with dibutyryl cyclic AMP, the amount of actin decreased by 50%. This decrease was not apparently related to the depolymerization of actin.     

SpyA is a membrane-bound ADP-ribosyltransferase of Streptococcus pyogenes which modifies a streptococcal peptide, SpyB

All sequenced genomes of Streptococcus pyogenes (Group A Streptococcus, GAS) encode a protein, SpyA, with homology to C3-like ADP-ribosyltransferase toxins. SpyA is a novel virulence factor which plays a role in pathogenesis in a mouse model of soft-tissue infection. In this study we demonstrate that SpyA is a surface-exposed membrane protein which is anchored to the streptococcal membrane by an N-terminal transmembrane sequence. We identified a small gene upstream of spyA, designated spyB, which encodes a peptide of 35 amino acids, and is co-transcribed with spyA. Expression of spyBA is strongly influenced by translational coupling: mutational inactivation of spyB translation completely abolishes translation of spyA. spyB expression increases with increasing cell density and reaches its maximum at late exponential growth phase. The SpyB N-terminus is predicted to fold into an amphipathic α-helix, a structural motif that targets a protein to the cytoplasmic membrane. Consistent with the prediction, we found that a SpyB fusion with peptide affinity tags is located in the streptococcal membrane. An ADP-ribosylation assay with recombinant SpyA demonstrated that SpyA modifies SpyB. Thus, our study suggests that ADP-ribosylation of SpyB may be an important function of SpyA.     

Actin can act as a cofactor for a viral proteinase in the cleavage of the cytoskeleton

Cytoskeletal proteins are exploited by many viruses during infection. We report a novel finding that actin can act as a cofactor for the adenovirus proteinase (AVP) in the degradation of cytoskeletal proteins. Transfection studies in HeLa cells revealed AVP localized with cytokeratin 18, and this was followed by destruction of the cytokeratin network. For AVP to cleave cytokeratin 18, a cellular cofactor was shown to be required, consistent with AVP being synthesized as an inactive proteinase. Actin was considered a cellular cofactor for AVP, because the C terminus of actin is homologous to a viral cofactor for AVP. AVP was shown to bind to the C terminus of actin, and in doing so AVP exhibited full enzymatic activity. In vitro, actin was a cofactor in the cleavage of cytokeratin 18 by AVP. The proteinase alone could not cleave cytokeratin 18, but in the presence of actin, AVP cleaved cytokeratin 18. Indeed, actin itself was shown to be a cofactor and a substrate for its own destruction in that it was cleaved by AVP in vitro. Cleavage of cytoskeletal proteins weakens the structure of the cell, and therefore, actin as a cofactor may play a role in cell lysis and release of nascent virions.     

Effect of parathyroid hormone and forskolin on cytoskeletal protein synthesis in cultured mouse osteoblastic cells

Parathyroid hormone (PTH) has been shown to cause transient cell shape changes in bone cells. We have examined the effects of parathyroid hormone and forskolin on the organization and expression of cytoskeletal proteins in cultured mouse endosteal osteoblastic cells. Analysis of [35S]methionine-labeled cytoskeletal proteins isolated on two-dimensional gel electrophoresis showed that PTH treatment (24 h) stimulated the de novo biosynthesis of actin, vimentin and tubulins in confluent cells, whereas forskolin had a minor effect despite a huge stimulation of cAMP production. This PTH-induced stimulation was associated with cell respreading following a mild and transitory cell retraction. PTH increased the synthesis of monomeric subunits of actin and beta-tubulins in subconfluent bone cells, whereas both monomeric and polymeric levels of beta-tubulins were increased in confluent osteoblasts. Under conditions reducing cell spreading, osteoblastic cells had initially high levels of unpolymerized subunits. In these poorly spread cells, parathyroid hormone or forskolin had no effect on the de novo synthesis of cytoskeletal proteins despite a marked elevation in intracellular cAMP levels. It is concluded that PTH affects the biosynthesis of cytoskeletal proteins in osteoblastic cells and that cAMP production does not seem to be directly involved. In addition, the effect of PTH is modulated by cell spreading and by the initial pool of cytoskeletal subunits.     

Adherence and internalization of Streptococcus gordonii by epithelial cells involves beta1 integrin recognition by SspA and SspB (antigen I/II family) polypeptides

Streptococcus gordonii is a commensal bacterium that colonizes the hard and soft tissues present in the human mouth and nasopharynx. The cell wall-anchored polypeptides SspA and SspB expressed by S. gordonii mediate a wide range of interactions with host proteins and other bacteria. In this article we have determined the role of SspA and SspB proteins, which are members of the streptococcal antigen I/II (AgI/II) adhesin family, in S. gordonii adherence and internalization by epithelial cells. Wild-type S. gordonii DL1 expressing AgI/II polypeptides attached to and was internalized by HEp-2 cells, whereas an isogenic AgI/II- mutant was reduced in adherence and was not internalized. Association of S. gordonii DL1 with HEp-2 cells triggered protein tyrosine phosphorylation but no significant actin rearrangement. By contrast, Streptococcus pyogenes A40 showed 50-fold higher levels of internalization and this was associated with actin polymerization and interleukin-8 upregulation. Adherence and internalization of S. gordonii by HEp-2 cells involved beta1 integrin recognition but was not fibronectin-dependent. Recombinant SspA and SspB polypeptides bound to purified human alpha5beta1 integrin through sequences present within the NAV (N-terminal) region of AgI/II polypeptide. AgI/II polypeptides blocked interactions of S. gordonii and S. pyogenes with HEp-2 cells, and S. gordonii DL1 cells expressing AgI/II proteins inhibited adherence and internalization of S. pyogenes by HEp-2 cells. Conversely, S. gordonii AgI/II- mutant cells did not inhibit internalization of S. pyogenes. The results suggest that AgI/II proteins not only promote integrin-mediated internalization of oral commensal streptococci by host cells, but also potentially influence susceptibility of host tissues to more pathogenic bacteria.     

Cytoskeletal bundle mechanics

The mechanical properties of cytoskeletal actin bundles play an essential role in numerous physiological processes, including hearing, fertilization, cell migration, and growth. Cells employ a multitude of actin-binding proteins to actively regulate bundle dimensions and cross-linking properties to suit biological function. The mechanical properties of actin bundles vary by orders of magnitude depending on diameter and length, cross-linking protein type and concentration, and constituent filament properties. Despite their importance to cell function, the molecular design principles responsible for this mechanical behavior remain unknown. Here, we examine the mechanics of cytoskeletal bundles using a molecular-based model that accounts for the discrete nature of constituent actin filaments and their distinct cross-linking proteins. A generic competition between filament stretching and cross-link shearing determines three markedly different regimes of mechanical response that are delineated by the relative values of two simple design parameters, revealing the universal nature of bundle-bending mechanics. In each regime, bundle-bending stiffness displays distinct scaling behavior with respect to bundle dimensions and molecular composition, as observed in reconstituted actin bundles in vitro. This mechanical behavior has direct implications on the physiological bending, buckling, and entropic stretching behavior of cytoskeletal processes, as well as reconstituted actin systems. Results are used to predict the bending regimes of various in vivo cytoskeletal bundles that are not easily accessible to experiment and to generate hypotheses regarding implications of the isolated behavior on in vivo bundle function.     

Unusual characteristics of ciliate actins

Actin is a cytoskeletal protein that is ubiquitous in eukaryotes, hence the corresponding genes and proteins have been isolated from numerous organisms as different as animals, plants, fungi and protozoa. Several atomic models are available for the monomeric as well as the filamentous form, and more than 70 proteins that bind actin and control filament dynamics have been isolated from diverse eukaryotes. Moreover, the function and dynamics of the actin cytoskeleton in several eukaryotic systems have been depicted in depth. Unlike other protozoa, such as amoeba, actin is not an abundant protein in ciliates, whose cytoskeleton is mainly composed of microtubular arrays. Ciliate actin has been studied in several species, and it was established early on that this ciliate protein is very different from that of other eukaryotes. Similarly, the actin-binding proteins studied in ciliates display great differences with those of other eukaryotes. Consequently, ciliate actin has been considered as "unconventional," and this review focuses on molecular data leading to this conclusion.     

Arachidonic acid signaling to the cytoskeleton: the role of cyclooxygenase and cyclic AMP-dependent protein kinase in actin bundling

Cell adhesion to the extracellular matrix via integrins is a primary regulatory mechanism for numerous aspects of normal cellular function. However, disruption of this interaction can result in pathology. For example, one characteristic of transformed cells is loss of adhesion dependence for viability. Adhesion also is a necessary step in tumor metastasis. It has been shown previously, in HeLa cells, that cell attachment to a gelatin-coated substrate results in the release of arachidonic acid, which is metabolized by lipoxygenase. A subsequent cascade of lipid second messengers activates protein kinase C, which triggers actin polymerization leading to cell spreading. We now demonstrate by inhibitor studies and biochemical analysis, a parallel branch of arachidonic acid signaling that reorganizes the actin cytoskeleton into small bundles. This branch of the pathway is initiated by cyclooxygenase, which generates prostaglandins and causes the downstream activation of cyclic AMP-dependent protein kinase. This work elucidates a system of interacting signals in which arachidonic acid functions at a branch point in cytoskeletal signaling. The lipoxygenase branch provides polymerized actin; these actin filaments act as a substrate for the cylooxygenase branch to generate actin bundles.     

Glycation of Brain Actin in Experimental Diabetes

Abstract: Actin is a neuronal protein involved in axonal transport and nerve regeneration, both of which are known to be impaired in experimental diabetes. To determine if actin is subject to glycation, we rendered rats diabetic by injection of streptozotocin. Two or 6 weeks later brains were removed and a preparation of cytoskeletal proteins was analyzed by two-dimensional polyacrylamide gel electrophoresis. Brains from diabetic animals contained an extra polypeptide that migrated close to actin and reacted with monoclonal antibody C4 against actin. It was also found in a preparation of soluble synaptic proteins from diabetic rat brain, indicating that it was at least partly neuronal in origin. This polypeptide could be produced by incubation of cytoskeletal proteins from brains of nondiabetic rats with glucose-6-phosphate in vitro. The appearance of this glycated actin in diabetic animals was prevented by administration of insulin for a period of 6 weeks. We could not detect any effect of glycation in vitro on the ability of muscle G-actin to form F-actin filaments and its significance for the function of actin remains to be determined. The finding that glycation of platelet-derived actin from diabetic patients was significantly increased implies that the abnormality may also occur in clinical diabetes.     

Identification and characterization of two temperature-induced surface-associated proteins of Streptococcus suis with high homologies to members of the Arginine Deiminase system of Streptococcus pyogenes

The present study was performed to identify stress-induced putative virulence proteins of Streptococcus suis. For this, protein expression patterns of streptococci grown at 32, 37, and 42 degrees C were compared by one- and two-dimensional gel electrophoresis. Temperature shifts from 32 and 37 to 42 degrees C induced expression of two cell wall-associated proteins with apparent molecular masses of approximately 47 and 53 kDa. Amino-terminal sequence analysis of the two proteins indicated homologies of the 47-kDa protein with an ornithine carbamoyltransferase (OCT) from Streptococcus pyogenes and of the 53-kDa protein with the streptococcal acid glycoprotein (SAGP) from S. pyogenes, an arginine deiminase (AD) recently proposed as a putative virulence factor. Cloning and sequencing the genes encoding the putative OCT and AD of S. suis, octS and adiS, respectively, revealed that they had 81.2 (octS) and 80.2% (adiS) identity with the respective genes of S. pyogenes. Both genes belong to the AD system, also found in other bacteria. Southern hybridization analysis demonstrated the presence of the adiS gene in all 42 serotype 2 and 9 S. suis strains tested. In 9 of these 42 strains, selected randomly, we confirmed expression of the AdiS protein, homologous to SAGP, by immunoblot analysis using a specific antiserum against the SAGP of S. pyogenes. In all strains AD activity was detected. Furthermore, by immunoelectron microscopy using the anti-S. pyogenes SAGP antiserum we were able to demonstrate that the AdiS protein is expressed on the streptococcal surface in association with the capsular polysaccharides but is not coexpressed with them.     

The spectrin cytoskeleton is crucial for adherent and invasive bacterial pathogenesis

Various enteric bacterial pathogens target the host cell cytoskeletal machinery as a crucial event in their pathogenesis. Despite thorough studies detailing strategies microbes use to exploit these components of the host cell, the role of the spectrin-based cytoskeleton has been largely overlooked. Here we show that the spectrin cytoskeleton is a host system that is hijacked by adherent (Entropathogenic Escherichia coli [EPEC]), invasive triggering (Salmonella enterica serovar Typhimurium [S. Typhimurium]) and invasive zippering (Listeria monocytogenes) bacteria. We demonstrate that spectrin cytoskeletal proteins are recruited to EPEC pedestals, S. Typhimurium membrane ruffles and Salmonella containing vacuoles (SCVs), as well as sites of invasion and comet tail initiation by L. monocytogenes. Spectrin was often seen co-localizing with actin filaments at the cell periphery, however a disconnect between the actin and spectrin cytoskeletons was also observed. During infections with S. Typhimurium ΔsipA, actin-rich membrane ruffles at characteristic sites of bacterial invasion often occurred in the absence of spectrin cytoskeletal proteins. Additionally, early in the formation of L. monocytogenes comet tails, spectrin cytoskeletal elements were recruited to the surface of the internalized bacteria independent of actin filaments. Further studies revealed the presence of the spectrin cytoskeleton during SCV and Listeria comet tail formation, highlighting novel cytoplasmic roles for the spectrin cytoskeleton. SiRNA targeted against spectrin and the spectrin-associated proteins severely diminished EPEC pedestal formation as well as S. Typhimurium and L. monocytogenes invasion. Ultimately, these findings identify the spectrin cytoskeleton as a ubiquitous target of enteric bacterial pathogens and indicate that this cytoskeletal system is critical for these infections to progress.     

Structural insights into bacterial modulation of the host cytoskeleton

Many bacterial pathogens manipulate the host cell cytoskeleton during infection. Such cytoskeletal modulation can occur at several points of contact between the pathogen and the host, and involves extracellular receptors, intracellular signal transduction and cytoskeletal proteins themselves. The field of bacterial pathogenesis has progressed dramatically over the past decade, such that structural knowledge is both timely and essential for a full appreciation of the biology at the pathogen-host interface. Several recent examples involving bacterial proteins that target actin, Rho family GTPases and extracellular receptors have contributed to a structural understanding of eukaryotic cytoskeletal modulation by pathogens.     

Senescence-associated alterations of cytoskeleton: extraordinary production of vimentin that anchors cytoplasmic p53 in senescent human fibroblasts

The cytoskeleton of senescent cells was systematically studied using senescent and young fibroblasts. In the cell senescence, skin fibroblasts extraordinarily produced vimentin in contrast to actin and tubulin, which were down-regulated. Among the focal adhesion proteins, paxillin and c-Src decreased also. Senescent cells developed a long and dense vimentin network, long and thin actin fibers, and numerous small focal contact sites, which contrasted with young cells with short and thick actin stress fibers and prominently large focal adhesions. Noticeably, senescent fibroblasts markedly produced p53 molecules and anchored them to vimentin-cytoskeleton in the cytoplasm. The vimentin-anchored p53 was detected with antibody PAb240 that specifically recognizes a conformation variant of p53. A GFP-tagged wild type p53 cDNA was expressed by transfection and shown also to be retained in the cytoplasm in senescent cells, suggesting that p53 is structurally modified to be recognized by PAb240 and anchored to vimentin filaments. We discuss the correlation of the marked alteration of cytoskeleton and senescent cells diminished proliferation and migration, as well as the significance of cytoskeletal anchorage of tumor suppressor p53.     

Identification of plant cytoskeleton-interacting proteins by screening for actin stress fiber association in mammalian fibroblasts

Taking advantage of the high conservation of the cytoskeleton building blocks actin and tubulin between plant and animal kingdoms, we developed a functional genomic screen for the isolation of new plant cytoskeleton-binding proteins that uses a mammalian cell expression system. A yellow fluorescent protein (YFP)-fusion cDNA library from Arabidopsis was inserted into rat fibroblasts and screened for fluorescent chimeras localizing to cytoskeletal structures. The high-throughput screen was performed by an automated microscope. An initial set of candidate genes identified in the screen was isolated, sequenced, the full-length cDNAs were synthesized by RT-PCR and tested by biochemical approaches to verify the ability of the genes to bind actin directly. Alternatively, indirect binding via interaction with other actin-binding proteins was studied. The full-length cDNAs were transferred back to plants as YFP chimeras behind the CAMV-35S promoter. We give here two examples of new plant cytoskeletal proteins identified in the pilot screen. ERD10, a member of the dehydrin family of proteins, was localized to actin stress fibers in rat fibroblasts. Its direct binding to actin filaments was confirmed by several biochemical approaches. Touch-induced calmodulin-like protein, TCH2, was also localized to actin stress fibers in fibroblasts, but was unable to bind actin filaments directly in vitro. Nevertheless, it did bind to the IQ domains of Arabidopsis myosin VIII in a calcium-dependent manner. Further evidence for a cytoskeletal function of ERD10 was obtained in planta; GFP-ERD10 was able to protect the actin cytoskeleton from latrunculin-mediated disruption in Nicotiana benthamiana leaves.     

IdeS and SpeB: immunoglobulin-degrading cysteine proteinases of Streptococcus pyogenes

The Gram-positive bacterium Streptococcus pyogenes is a major human pathogen causing substantial morbidity and mortality in society. S. pyogenes has evolved numerous molecular mechanisms to avoid the various actions of the human immune system and has established means to modulate both adaptive and innate immune responses. S. pyogenes produces and secretes proteolytic enzymes, which have an important impact on the ability of the bacteria to survive in the human host. Prominent among these are two immunoglobulin-degrading enzymes: the newly discovered streptococcal cysteine proteinase, IdeS, and the classical cysteine proteinase of S. pyogenes, SpeB.     

Integrating an integrin: a direct route to actin

Integrins were so named for their ability to link the extracellular and intracellular skeletons. Now almost 20 years into integrin research, numerous questions remain as to how this interaction is accomplished and how it is modified to achieve a desired phenotype. As the cell adhesion and actin assembly fields are merging in combined approaches, novel actin assembly mechanisms are being uncovered. Some of the earliest identified cytoplasmic linker molecules, believed to mediate integrin-actin binding, are once again the subject of scrutiny as potential dynamic mediators of cell anchorage. It seems plausible that each unique cellular morphology occurs as the result of activation of distinct actin assembly systems that are either stabilized by unique bundling and linker proteins or modified for progression to a new phenotype. While this research initiative is likely to continue rapidly in a forward fashion, it remains to be clarified how integrins assemble the most stable and basic cytoskeletal phenotype, the adherent cell with prominent stress fibers. Recent investigations point towards a shift in the current model of anchoring at the cell periphery by providing both mechanisms and evidence for de novo actin assembly orchestrated by the adhesion site. Lacking a complete pathway from integrin ligation to an integrated extracellular-intracellular skeleton in any single system, this review proposes a simple model of integrin-mediated stress fiber integration by drawing from work in multiple systems.     

Proteolysis and its regulation at the surface of Streptococcus pyogenes

Pathogenic bacteria often produce proteinases that are believed to be involved in virulence. Moreover, several host defence systems depend on proteolysis, demonstrating that proteolysis and its regulation play an important role during bacterial infections. Here, we discuss how proteolytical events are regulated at the surface of Streptococcus pyogenes during infection with this important human pathogen. Streptococcus pyogenes produces proteinases, and host proteinases are produced and released as a result of the infection. Streptococcus pyogenes also recruits host proteinase inhibitors to its surface, suggesting that proteolysis is tightly regulated at the bacterial surface. We propose that the initial phase of a S. pyogenes infection is characterized by inhibition of proteolysis and complement activity at the bacterial surface. This is achieved mainly through binding of host proteinase inhibitors and complement regulatory proteins to bacterial surface proteins. In a later phase of the infection, massive proteolytic activity will release bacterial surface proteins and degrade human tissues, thus facilitating bacterial spread. These proteolytic events are regulated both temporally and spatially, and should influence virulence and the outcome of S. pyogenes infections.