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1.
The autolytic enzyme LytA of Streptococcus pneumoniae is not responsible for releasing pneumolysin
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It was previously proposed that autolysin's primary role in the virulence of pneumococci was to release pneumolysin to an extracellular location. This interpretation came into question when pneumolysin was observed to be released in significant amounts from some pneumococci during log-phase growth, because autolysis was not believed to occur at this time. We have reexamined this phenomenon in detail for one such strain, WU2. This study found that the extracellular release of pneumolysin from WU2 was not dependent on autolysin action. A mutant lacking autolysin showed the same pattern of pneumolysin release as the wild-type strain. Addition of mitomycin C to a growing WU2 culture did not induce lysis, indicating the absence of resident bacteriophages that could potentially harbor lytA-like genes. Furthermore, release of pneumolysin was unaltered by growth in 2% choline, a condition which is reported to inactivate autolysin, as well as most known pneumococcal phage lysins. Profiles of total proteins in the cytoplasm and in the supernatant media supported the hypothesis that release of pneumolysin is independent of pneumococcal lysis. Finally, under some infection conditions, mutations in pneumolysin and autolysin had different effects on virulence. 相似文献
2.
Mellroth P Daniels R Eberhardt A Rönnlund D Blom H Widengren J Normark S Henriques-Normark B 《The Journal of biological chemistry》2012,287(14):11018-11029
The pneumococcal autolysin LytA is a virulence factor involved in autolysis as well as in fratricidal- and penicillin-induced lysis. In this study, we used biochemical and molecular biological approaches to elucidate which factors control the cytoplasmic translocation and lytic activation of LytA. We show that LytA is mainly localized intracellularly, as only a small fraction was found attached to the extracellular cell wall. By manipulating the extracellular concentration of LytA, we found that the cells were protected from lysis during exponential growth, but not in the stationary phase, and that a defined threshold concentration of extracellular LytA dictates the onset of autolysis. Stalling growth through nutrient depletion, or the specific arrest of cell wall synthesis, sensitized cells for LytA-mediated lysis. Inhibition of cell wall association via the choline binding domain of an exogenously added enzymatically inactive form of LytA revealed a potential substrate for the amidase domain within the cell wall where the formation of nascent peptidoglycan occurs. 相似文献
3.
Streptococcus pneumoniae (pneumococcus) has remained a persistent cause of invasive and mucosal disease in humans despite the widespread use of antibiotics and vaccines. The resilience of this organism is due to its capacity for adaptation through the uptake and incorporation of new genetic material from the surrounding microbial community. DNA uptake and recombination is controlled by a tightly regulated quorum sensing system that is triggered by the extracellular accumulation of competence stimulating peptide (CSP). In this study, we demonstrate that CSP can stimulate the production of a diverse array of blp bacteriocins. This cross stimulation occurs through increased production and secretion of the bacteriocin pheromone, BlpC, and requires a functional competence regulatory system. We show that a highly conserved motif in the promoter of the operon encoding BlpC and its transporter mediates the upregulation by CSP. The accumulation of BlpC following CSP stimulation results in augmented activation of the entire blp locus. Using biofilm-grown organisms as a model for competition and genetic exchange on the mucosal surface, we demonstrate that DNA exchange is enhanced by bacteriocin secretion suggesting that co-stimulation of bacteriocins with competence provides an adaptive advantage. The blp and com regulatory pathways are believed to have diverged and specialized in a remote ancestor of pneumococcus. Despite this, the two systems have maintained a regulatory connection that promotes competition and adaptation by targeting for lysis a wide array of potential competitors while simultaneously providing the means for incorporation of their DNA. 相似文献
4.
Bacteriophage T4 Head Maturation: Release of Progeny DNA from the Host Cell Membrane 总被引:3,自引:2,他引:1
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We have presented a new approach to studying bacteriophage T4 head maturation. Using a modified M-band technique, we have shown that progeny deoxyribonucleic acid (DNA) was synthesized on the host cell membrane throughout infection. This DNA was released from the membrane later in infection as the result of formation of the phage head; detachment of the DNA required the action of gene products 20, 21, 22, 23, 24, 31, 16, 17 and 49, known to be necessary for normal head formation. Gene products 2, 4, 50, 64, 65, 13 and 14, also involved in head morphogenesis were not required to detach progeny DNA from the membrane; the presence of the phage tail and tail fibers also was not required. DNA was released in the form of immature heads and initially was sensitive to deoxyribonuclease (DNase). Conversion to DNase resistance followed rapidly. The amount of phage precursors present at the time of DNA synthesis determined the time of onset and detachment rate of DNA from the M band as well as the kinetics by which the detached DNA become DNase resistant. 相似文献
5.
Streptococcus pneumoniae (the pneumococcus) colonizes the human nasopharynx and is a significant pathogen worldwide. Pneumolysin (Ply) is a multi-functional, extracellular virulence factor produced by this organism that is critical for pathogenesis. Despite the absence of any apparent secretion or cell surface attachment motifs, Ply localizes to the cell envelope of actively growing cells. We sought to characterize the consequences of this surface localization. Through functional assays with whole cells and subcellular fractions, we determined that Ply activity and its release into the extracellular environment are inhibited by peptidoglycan (PG) structure. The ability of PG to inhibit Ply release was dependent on the stem peptide composition of this macromolecule, which was manipulated by mutation of the murMN operon that encodes proteins responsible for branched stem peptide synthesis. Additionally, removal of choline-binding proteins from the cell surface significantly reduced Ply release to levels observed in a mutant with a high proportion of branched stem peptides suggesting a link between this structural feature and surface-associated choline-binding proteins involved in PG metabolism. Of clinical relevance, we also demonstrate that a hyperactive, mosaic murMN allele associated with penicillin resistance causes decreased Ply release with concomitant increases in the amount of branched stem peptides. Finally, using a murMN deletion mutant, we observed that increased Ply release is detrimental to virulence during a murine model of pneumonia. Taken together, our results reveal a novel role for branched stem peptides in pneumococcal pathogenesis and demonstrate the importance of controlled Ply release during infection. These results highlight the importance of PG composition in pathogenesis and may have broad implications for the diverse PG structures observed in other bacterial pathogens. 相似文献
6.
7.
Kari A. Dilley Devon Gregory Marc C. Johnson Volker M. Vogt 《Journal of virology》2010,84(13):6276-6287
The efficient release of newly assembled retrovirus particles from the plasma membrane requires the recruitment of a network of cellular proteins (ESCRT machinery) normally involved in the biogenesis of multivesicular bodies and in cytokinesis. Retroviruses and other enveloped viruses recruit the ESCRT machinery through three classes of short amino acid consensus sequences termed late domains: PT/SAP, PPXY, and LYPXnL. The major late domain of Rous sarcoma virus (RSV) has been mapped to a PPPY motif in Gag that binds members of the Nedd4 family of ubiquitin ligases. RSV Gag also contains a second putative late domain motif, LYPSL, positioned 5 amino acids downstream of PPPY. LYPXnL motifs have been shown to support budding in other retroviruses by binding the ESCRT adaptor protein Alix. To investigate a possible role of the LYPSL motif in RSV budding, we constructed PPPY and LYPSL mutants in the context of an infectious virus and then analyzed the budding rates, spreading profiles, and budding morphology. The data imply that the LYPSL motif acts as a secondary late domain and that its role in budding is amplified in the absence of a fully functional PPPY motif. The LYPXL motif proved to be a stronger late domain when an aspartic acid was substituted for the native serine, recapitulating the properties of the LYPDL late domain of equine infectious anemia virus. The overexpression of human Alix in the absence of a fully functional PPPY late domain partially rescued both the viral budding rate and viral replication, supporting a model in which the RSV LYPSL motif mediates budding through an interaction with the ESCRT adaptor protein Alix.Retroviruses acquire their lipid envelopes from the plasma membrane as they bud from the cell. Although the structural protein Gag is both necessary and sufficient for the assembly of virus-like particles (VLPs), the membrane scission step of virus egress requires the recruitment of a network of cellular proteins normally involved in two analogous cellular membrane fission events, the budding of cargo-containing vesicles into multivesicular bodies (MVBs) (for review, see references 1, 5, 11, and 50) and the separation of two daughter cells during cytokinesis (3, 4). This cellular network of proteins, collectively called the ESCRT (endosomal sorting complex required for transport) machinery, includes four sequentially recruited high-molecular-weight protein complexes (ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III) and is essential for the transport of transmembrane cargo proteins to the lysosome for degradation via an MVB intermediate.In addition to the multiprotein ESCRT complexes, other proteins are required to promote the budding of vesicles into the MVB. Ubiquitin ligases (such as Nedd4) monoubiquitinate both ESCRT components and transmembrane cargo proteins, tagging them for the MVB pathway. Adaptor proteins connect cargo proteins to ESCRT complexes or ESCRT complexes to each other. Ultimately, the final membrane fission event of vesicle budding is mediated by an AAA ATPase (Vps4).Retroviruses as well as other enveloped viruses use three amino acid consensus sequences, PPXY, PT/SAP, and LYPXnL, as docking sites for the components of the cellular ESCRT machinery. The deletion or mutation of these sequences, termed late domains, results in the failure of the virus to recruit the budding machinery to the site of assembly and thereby results in a block at the late stage of virus release in which fully assembled but immature virus particles remain attached to the plasma membrane. The PPXY late domain interacts with the WW domains of the Nedd4 family of ubiquitin ligases. Multiple ESCRT components bind to monoubiquitin tags on both cargo and ESCRT proteins. The PT/SAP late domain binds the ESCRT-I complex component, Tsg101 (tumor susceptibility gene 101). The LYPXnL late domain interacts with an adaptor protein of the ESCRT pathway, Alix (ALG-2-interacting protein X; also called AIP1) (reviewed in reference 12). Alix interacts with both Tsg101 of the ESCRT-I complex and CMHP4 of the ESCRT-III complex. A possible fourth class of late domains for the paramyxovirus SV5 was reported previously (47). The late domain function in this case has been mapped to an FPIV sequence in the M (matrix) protein. To date, this motif has yet to be shown to be important for the budding of any other virus, and an FPIV-interacting cellular protein has yet to be identified.Often, retroviruses rely on multiple late domains for efficient budding (2, 13, 16, 29, 30). For example, in addition to its PT/SAP motif in human immunodeficiency virus type 1 (HIV-1) p6, which binds Tsg101 (6, 14, 34, 52), HIV-1 also harbors an Alix-binding LYPXnL motif that functions in budding (13, 33, 34, 48, 52). Mutation of this LYPXnL motif results in only a modest reduction in HIV-1 budding (10). However, the effects of mutations in the LYPXnL motif become more obvious in the context of a minimal Gag in which the globular domain of MA and the N-terminal domain of CA are absent (48). Furthermore, the role of this motif also seems to vary among cell types. For example, the deletion of this motif decreases HIV-1 particle production 2- to 3-fold in COS-7 cells (15) but has no consequence for HeLa cells (7). The relationship of the two viral late domains to each other is unknown. It is possible that they are partially redundant, are cooperative (since they act at slightly different steps in the ESCRT pathway), or are cell type specific. It has been observed that the mutation of one late domain has a larger effect on budding than the mutation of the other, implying a hierarchy of function. For example, in HIV-1, PTAP acts as the dominant late domain and LYPXnL acts as a secondary late domain. Equine infectious anemia virus (EIAV) seems to be an exception in that it relies only on a single LYPDL motif for late domain function.Like other retroviruses, the avian alpharetrovirus Rous sarcoma virus (RSV) requires the ESCRT pathway for release, as evidenced by the observation that a dominant-negative mutant of the ATPase Vps4, which is required for the final step of the ESCRT pathway that releases the ESCRT-III complex, inhibits RSV budding in a dose-dependent manner (37). Mutational analysis mapped the RSV late domain to the PPPY motif in the small spacer peptide p2b of Gag (41, 54, 56). This PPPY motif was previously shown to interact with chicken members of the Nedd4 family of ubiquitin ligases (21, 51). RSV Gag also harbors an LYPSL late domain consensus motif 5 amino acids downstream from PPPY in the p10 domain, which could potentially promote budding via an interaction with Alix.Alix, a 97-kDa adaptor protein with diverse functions, is composed of an N-terminal Bro1 domain, a central V domain, and a C-terminal proline-rich region (10, 22, 26, 58). The proline-rich region is assumed to be unstructured and binds Tsg101 and endophilins. The Bro1 domain, which binds CHMP4, is curved and resembles a banana shape. CHMP4 binding is functionally important for promoting HIV-1 budding (10). It was suggested previously that its convex face may allow Alix to sense negative curvatures in membranes (17, 22). At least for HIV-1, the Alix Bro1 domain also interacts with the Gag NC domain (42, 43). The central V domain of Alix, which is named for its shape, has a conserved hydrophobic pocket on the second arm near the apex of the V that is responsible for the binding of the LYPXnL late domains of HIV-1 and EIAV (10, 26, 58).In the present study, we investigated the role of the LYPSL motif in RSV budding and replication. We report here that not only the PPPY motif but also the LYPSL motif act as late domains. The contribution of the LYPSL motif to the budding rate and spreading rate is secondary to that of the PPPY motif but increases in the absence of a fully functional PPPY motif. The Alix overexpression-mediated rescue of PPPY mutants supports a model in which the LYPSL late domain functions through an interaction with Alix. 相似文献
8.
Bacillus subtilis possesses carbon-flux regulating histidine protein (Crh), a paralog of the histidine protein (HPr) of the phosphotransferase system (PTS). Like HPr, Crh becomes (de)phosphorylated in vitro at residue Ser46 by the metabolite-controlled HPr kinase/phosphorylase HPrK/P. Depending on its phosphorylation state, Crh exerts regulatory functions in connection with carbohydrate metabolism. So far, knowledge on phosphorylation of Crh in vivo has been limited and derived from indirect evidence. Here, we studied the dynamics of Crh phosphorylation directly by non-denaturing gel electrophoresis followed by Western analysis. The results confirm that HPrK/P is the single kinase catalyzing phosphorylation of Crh in vivo. Accordingly, phosphorylation of Crh is triggered by the carbon source as observed previously for HPr, but with some differences. Phosphorylation of both proteins occurred during exponential growth and disappeared upon exhaustion of the carbon source. During exponential growth, ~80% of the Crh molecules were phosphorylated when cells utilized a preferred carbon source. The reverse distribution, i.e. around 20% of Crh molecules phosphorylated, was obtained upon utilization of less favorable substrates. This clear-cut classification of the substrates into two groups has not previously been observed for HPr(Ser)~P formation. The likely reason for this difference is the additional PTS-dependent phosphorylation of HPr at His15, which limits accumulation of HPr(Ser)~P. 相似文献
9.
10.
M A Ulanova M Kh Sharapova Z M Mikha?lova L G Afonina L N Padiukov 《Zhurnal mikrobiologii, epidemiologii, i immunobiologii》1990,(7):65-71
The levels of antibodies to capsular polysaccharide antigens of pneumococci (serotypes 1, 3, 6B, 8, 9N, 15F, 23F), C-polysaccharide and protein antigen of pneumococci in the blood sera of 38 newborn infants at the moment of their birth (umbilical blood) and on the 5th or 6th day of their life, in their mothers' blood sera, as well as in the colostrum and milk of 48 nursing women, have been studied by means of the enzyme immunoassay. The study showed that in the normal course of pregnancy antibodies to pneumococci were transferred transplacentally from the mother to the fetus. Though in most cases their content in the blood of newborn infants was lower than that in maternal blood, it exceeded the average level of antipneumococcal antibodies in children aged 3-12 months. In the milk of nursing mothers considerable amount of IgA antibodies to pneumococci was detected, which might be an additional protective factor with respect to pneumococcal infection in infants. 相似文献
11.
Nina N. Schommer Jun Muto Victor Nizet Richard L. Gallo 《The Journal of biological chemistry》2014,289(39):26914-26921
Group A Streptococcus (GAS) commonly infects human skin and occasionally causes severe and life-threatening invasive diseases. The hyaluronan (HA) capsule of GAS has been proposed to protect GAS from host defense by mimicking endogenous HA, a large and abundant glycosaminoglycan in the skin. However, HA is degraded during tissue injury, and the functions of short-chain HA that is generated during infection have not been studied. To examine the impact of the molecular mass of HA on GAS infection, we established infection models in vitro and in vivo in which the size of HA was defined by enzymatic digestion or custom synthesis. We discovered that conversion of high molecular mass HA to low molecular mass HA facilitated GAS phagocytosis by macrophages and limited the severity of infection in mice. In contrast, native high molecular mass HA significantly impaired internalization by macrophages and increased GAS survival in murine blood. Thus, our data demonstrate that GAS virulence can be influenced by the size of HA derived from both the bacterium and host and suggest that high molecular mass HA facilitates GAS deep tissue infections, whereas the generation of short-chain HA can be protective. 相似文献
12.
LytA, the main autolysin of Streptococcus pneumoniae, was the first member of the bacterial N-acetylmuramoyl-l-alanine amidase (NAM-amidase) family of proteins to be well characterized. This autolysin degrades the peptidoglycan bonds of pneumococcal cell walls after anchoring to the choline residues of the cell wall teichoic acids via its choline-binding module (ChBM). The latter is composed of seven repeats (ChBRs) of approximately 20 amino acid residues. The translation product of the lytA gene is the low-activity E-form of LytA (a monomer), which can be "converted" (activated) in vitro by choline into the fully active C-form at low temperature. The C-form is a homodimer with a boomerang-like shape. To study the structural requirements for the monomer-to-dimer modification and to clarify whether "conversion" is synonymous with dimerization, the biochemical consequences of replacing four key amino acid residues of ChBR6 and ChBR7 (the repeats involved in dimer formation) were determined. The results obtained with a collection of 21 mutated NAM-amidases indicate that Ile-315 is a key amino acid residue in both LytA activity and folding. Amino acids with a marginal position in the solenoid structure of the ChBM were of minor influence in dimer stability; neither the size, polarity, nor aromatic nature of the replacement amino acids affected LytA activity. In contrast, truncated proteins were drastically impaired in their activity and conversion capacity. The results indicate that dimerization and conversion are different processes, but they do not answer the questions of whether conversion can only be achieved after a dimer formation step. 相似文献
13.
Biofilm‐grown bacteria are refractory to antimicrobial agents and show an increased capacity to evade the host immune system. In recent years, studies have begun on biofilm formation by Streptococcus pneumoniae, an important human pathogen, using a variety of in vitro model systems. The bacterial cells in these biofilms are held together by an extracellular matrix composed of DNA, proteins and, possibly, polysaccharide(s). Although neither the precise nature of these proteins nor the composition of the putative polysaccharide(s) is clear, it is known that choline‐binding proteins are required for successful biofilm formation. Further, many genes appear to be involved, although the role of each appears to vary when biofilms are produced in batch or continuous culture. Prophylactic and therapeutic measures need to be developed to fight S. pneumoniae biofilm formation. However, much care needs to be taken when choosing strains for such studies because different S. pneumoniae isolates can show remarkable genomic differences. Multispecies and in vivo biofilm models must also be developed to provide a more complete understanding of biofilm formation and maintenance. 相似文献
14.
The aim of our study was to evaluate a frequency of isolation and susceptibility to antibiotics of Streptococcus pneumoniae penicillin resistant among 154 strains S. pneumoniae isolated between 2003 and 2006 in University Hospital of Dr. A. Jurasza in Bydgoszcz. Antimicrobial susceptibility was assessed by disc-diffusion method according to the guidelines of Clinical and laboratory Standards Institute and The national Reference Centre for Antimicrobial Susceptibility. Minimal inhibitory concentrations for penicillin and cefotaxime were assessed by E-test method. Study shows increasing isolation of SPPR strains from 8,2% in 2003 to 32,0% in 2006. Strains were mostly isolated from patients ofNeurosurgery and Neurotraumatology Clinic and Rehabilitation Clinic. SPPR strains were mainly isolated from respiratory tract. Over 68% of SPPR showed intermediate resistance to penicillin and 73,3% of strains were susceptible to cefotaxime. Between 2003 and 2006 increased percentage of resistance strains to erythromycin, tetracycline and sulphometoxasol. 相似文献
15.
16.
Q. Cheng E. A. Campbell A. M. Naughton S. Johnson & H. R. Masure 《Molecular microbiology》1997,23(4):683-692
17.
Larissa Kolesnikova Thomas Strecker Eiji Morita Florian Zielecki Eva Mittler Colin Crump Stephan Becker 《Journal of virology》2009,83(5):2327-2337
VP40, the major matrix protein of Marburg virus, is the main driving force for viral budding. Additionally, cellular factors are likely to play an important role in the release of progeny virus. In the present study, we characterized the influence of the vacuolar protein sorting (VPS) pathway on the release of virus-like particles (VLPs), which are induced by Marburg virus VP40. In the supernatants of HEK 293 cells expressing VP40, different populations of VLPs with either a vesicular or a filamentous morphology were detected. While the filaments were almost completely composed of VP40, the vesicular particles additionally contained considerable amounts of cellular proteins. In contrast to that in the vesicles, the VP40 in the filaments was regularly organized, probably inducing the elimination of cellular proteins from the released VLPs. Vesicular particles were observed in the supernatants of cells even in the absence of VP40. Mutation of the late-domain motif in VP40 resulted in reduced release of filamentous particles, and likewise, inhibition of the VPS pathway by expression of a dominant-negative (DN) form of VPS4 inhibited the release of filamentous particles. In contrast, the release of vesicular particles did not respond significantly to the expression of DN VPS4. Like the budding of VLPs, the budding of Marburg virus particles was partially inhibited by the expression of DN VPS4. While the release of VLPs from VP40-expressing cells is a valuable tool with which to investigate the budding of Marburg virus particles, it is important to separate filamentous VLPs from vesicular particles, which contain many cellular proteins and use a different budding mechanism.In recent years, virus-like particles (VLPs), which are formed upon recombinant expression of the viral matrix and/or surface glycoproteins, have been recognized as representing powerful tools for developing novel vaccines and investigating certain aspects of the viral replication cycle (24, 44, 59, 63). Matrix proteins from many enveloped RNA viruses, including retroviruses, rhabdoviruses, filoviruses, paramyxoviruses, orthomyxoviruses, and arenaviruses, are able to induce VLPs (10, 14, 18, 28-30, 48, 49, 52). Increasing evidence also indicates that budding activity, and thus the release of VLPs, is often influenced by a complex interplay with components of the endosomal sorting complexes required for transport (ESCRTs), which mainly constitute the vacuolar protein sorting (VPS) pathway (16, 38, 42, 54). ESCRTs trigger the formation and budding of vesicles into the lumina of multivesicular bodies (MVBs), and the constituents of the ESCRTs are recycled by the activity of VPS4, an AAA-type ATPase. Expression of dominant-negative (DN) VPS4 mutants, which lack the ability to bind or hydrolyze ATP, blocks recycling of the ESCRTs and induces the formation of enlarged endosomes lacking internal vesicle accumulation (2, 3, 7). The inward budding of vesicles into the MVBs is topologically similar to the budding of viruses, since the vesicles bud away from the cytosol and into the lumen (reviewed in references 1, 20, and 26). Therefore, it is not entirely surprising that viruses use the cellular ESCRT machinery to organize the budding of viral progeny. Interactions between viral matrix proteins and ESCRTs occur through tetrapeptide motifs, known as late domains, which were first identified in retroviruses. Known late domains consist of the amino acid sequence P(T/S)AP, PPxY, or YxxL, where “x” represents any amino acid (19, 25, 62). The P(T/S)AP motif, for example, mediates interaction with tumor susceptibility gene 101 (Tsg101) (16, 36, 57); the PPxY motif mediates binding to WW domains of Nedd4-like ubiquitin ligases (9, 22); and the YxxL motif mediates interaction with AIP1/Alix (35, 47, 58). Recently, a novel late-domain motif, FPIV, has been identified in paramyxoviruses (46), and it is thought that additional late-domain motifs remain to be discovered (for a review, see reference 5).Inhibition of the VPS pathway has been shown to inhibit the budding of various viruses that are released with the help of ESCRTs. However, the budding of viruses and VLPs depends on the activity of ESCRTs to different degrees. Downregulation of Tsg101, a member of the ESCRT-I complex, inhibited the release of VLPs mediated by lymphocytic choriomeningitis virus Z protein and Marburg virus (MARV) VP40 (42, 54) but did not substantially inhibit the release of Gag-induced VLPs of Moloney murine leukemia virus and Rous sarcoma virus or that of matrix protein-induced VLPs of rabies virus (16, 27, 38). Expression of DN VPS4 inhibited the release of VLPs induced by the Gag proteins of Rous sarcoma virus and Moloney murine leukemia virus (16, 38) as well as that of VLPs induced by Lassa virus Z protein (55) but had no effect on the budding of rabies virus and cytomegalovirus (13, 27). These data indicate that in spite of the presence of late-domain motifs, a block in the VPS pathway may not always be critical for the budding of VLPs. In addition, the lack of known late domains in many enveloped viruses raises the question of whether they use other entry points into the VPS pathway or whether they exploit entirely different mechanisms of budding (60). To date, knowledge of how viral matrix proteins engage cellular machineries, such as the VPS pathway, to induce viral budding at the plasma membrane is very limited (8).The matrix protein VP40 of MARV contains only one known late-domain motif, PPPY, and a recent study showed that mutation of this late domain inhibited the release of VP40-induced VLPs. In addition, depletion of Tsg101 reduced the release of VP40-induced VLPs, suggesting that ESCRT-I is involved in this process (54). Whether a functional VPS pathway is important for the release of MARV VP40-induced VLPs or MARV particles remains unknown.VLPs induced by many viral matrix proteins have a morphology similar to that of cellular vesicles, which makes it difficult to separate the spherical VLPs from released cellular vesicles (4, 17, 53). In contrast, VLPs induced by the filovirus matrix protein VP40 are elongated and similar in morphology to viral particles (30, 49). Nevertheless, we observed that the supernatants of cells expressing VP40 contained various populations of particles with different morphologies. This raised the questions of whether the different particles are released by the same mechanism, whether they are all induced by VP40, and whether they are dependent on the same cellular pathways.The aim of the present study was to analyze the populations of particles released from cells expressing the MARV matrix protein VP40 and to gain further insights into the interaction between MARV and the cellular machinery involved in the budding of VLPs and MARV particles.We found that cells expressing VP40 released vesicular and filamentous particles, which could be separated by gradient centrifugation. Fractions with mainly vesicular particles represented a mixture of vesicles containing exclusively cellular proteins and vesicles also containing VP40 and few short filamentous particles. Longer filamentous particles, whose morphology resembled that of MARV particles but which displayed a much higher variability in length (400 nm to 5 μm), were found in denser gradient fractions. Filamentous VP40-induced VLPs were able to sort out cellular proteins efficiently. Release of VP40-induced filamentous VLPs was supported by the late-domain motif present in VP40, and inhibition of the cellular ESCRT machinery reduced the amount of these VLPs in the supernatant. Interestingly, the release of VLPs induced by a mutant of VP40 lacking the late domain was also reduced by inhibition of the cellular ESCRT machinery. Expression of a DN mutant of VPS4 diminished the budding of infectious MARV particles by 50%, a finding consistent with the idea that the activity of the ESCRT machinery supports viral budding but is not essential. 相似文献
18.
Autolytic Formation of Protoplasts (Autoplasts) of Streptococcus faecalis 9790: Release of Cell Wall, Autolysin, and Formation of Stable Autoplasts 总被引:10,自引:9,他引:1
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A system for the formation of apparently wall-free protoplasts from exponential-phase cells of Streptococcus faecalis ATCC 9790 in the absence of added lytic enzymes was developed. Exponential-phase cells suspended in 0.04 M ammonium acetate, pH 6.7, 1 mM magnesium acetate, and 0.5 M sucrose become osmotically fragile within 1 to 1.5 h due to the action of the native, autolytic enzyme on the cell wall peptidoglycan. However, maximal cell wall loss occurred much more slowly, being complete only after 3 to 6 h. Under these conditions, the autolytically formed protoplasts (autoplasts) remained intact for prolonged periods (up to 24 h) with less than 5% of their deoxyribonucleic acid, ribonucleic acid, and protein lost during the first 6 h. During dissolution of the cell wall, release of autolytic enzyme to the supernatant fluid began after 60% of the wall was lost. The addition of trypsin to the incubation mixture increased the rate of attainment of osmotic fragility and cell wall loss two- to threefold, apparently due to the activation of the latent form of the autolysin. Electron microscopy was used to confirm cell wall loss and the presence of intact protoplasts at the end of the incubation periods. 相似文献
19.
20.
PerR-Regulated Manganese Ion Uptake Contributes to Oxidative Stress Defense in an Oral Streptococcus
Metal homeostasis plays a critical role in antioxidative stress. Streptococcus oligofermentans, an oral commensal facultative anaerobe lacking catalase activity, produces and tolerates abundant H2O2, whereas Dpr (an Fe2+-chelating protein)-dependent H2O2 protection does not confer such high tolerance. Here, we report that inactivation of perR, a peroxide-responsive repressor that regulates zinc and iron homeostasis in Gram-positive bacteria, increased the survival of H2O2-pulsed S. oligofermentans 32-fold and elevated cellular manganese 4.5-fold. perR complementation recovered the wild-type phenotype. When grown in 0.1 to 0.25 mM MnCl2, S. oligofermentans increased survival after H2O2 stress 2.5- to 23-fold, and even greater survival was found for the perR mutant, indicating that PerR is involved in Mn2+-mediated H2O2 resistance in S. oligofermentans. Mutation of mntA could not be obtained in brain heart infusion (BHI) broth (containing ∼0.4 μM Mn2+) unless it was supplemented with ≥2.5 μM MnCl2 and caused 82 to 95% reduction of the cellular Mn2+ level, while mntABC overexpression increased cellular Mn2+ 2.1- to 4.5-fold. Thus, MntABC was identified as a high-affinity Mn2+ transporter in S. oligofermentans. mntA mutation reduced the survival of H2O2-pulsed S. oligofermentans 5.7-fold, while mntABC overexpression enhanced H2O2-challenged survival 12-fold, indicating that MntABC-mediated Mn2+ uptake is pivotal to antioxidative stress in S. oligofermentans. perR mutation or H2O2 pulsing upregulated mntABC, while H2O2-induced upregulation diminished in the perR mutant. This suggests that perR represses mntABC expression but H2O2 can release the suppression. In conclusion, this work demonstrates that PerR regulates manganese homeostasis in S. oligofermentans, which is critical to H2O2 stress defenses and may be distributed across all oral streptococci lacking catalase. 相似文献