RESUMEN
BACKGROUND: BST2/tetherin is an innate immune molecule with the unique ability to restrict the egress of human immunodeficiency virus (HIV) and other enveloped viruses, including Ebola virus (EBOV). Coincident with this discovery was the finding that the HIV Vpu protein down-regulates BST2 from the cell surface, thereby promoting viral release. Evidence suggests that the EBOV envelope glycoprotein (GP) also counteracts BST2, although the mechanism is unclear. RESULTS: We find that total levels of BST2 remain unchanged in the presence of GP, whereas surface BST2 is significantly reduced. GP is known to sterically mask surface receptors via its mucin domain. Our evaluation of mutant GP molecules indicate that masking of BST2 by GP is probably responsible for the apparent surface BST2 down-regulation; however, this masking does not explain the observed virus-like particle egress enhancement. We discovered that VP40 coimmunoprecipitates and colocalizes with BST2 in the absence but not in the presence of GP. CONCLUSIONS: These results suggest that GP may overcome the BST2 restriction by blocking an interaction between VP40 and BST2. Furthermore, we have observed that GP may enhance BST2 incorporation into virus-like particles. Understanding this novel EBOV immune evasion strategy will provide valuable insights into the pathogenicity of this deadly pathogen.
Asunto(s)
Antígenos CD/metabolismo , Ebolavirus/metabolismo , Ebolavirus/patogenicidad , Glicoproteínas/metabolismo , Liberación del Virus/fisiología , Línea Celular , Regulación hacia Abajo/fisiología , Proteínas Ligadas a GPI/metabolismo , Células HEK293 , Humanos , Mucinas/metabolismo , Mutación/genética , Receptores de Superficie Celular/metabolismo , Proteínas de la Matriz Viral , Proteínas Virales/metabolismoRESUMEN
The cellular protein BST-2/CD317/Tetherin has been shown to inhibit the release of HIV-1 and other enveloped viruses from infected cells. The HIV-1 accessory protein Vpu binds to both BST-2 and ßTrCP, a substrate-recognition subunit for the SCF (Skip1-Cullin1-F-box protein) E3 ubiquitin ligase complex. This interaction leads to both the degradation of BST-2 and the enhancement of viral egress. Recently BST-2 was shown to be ubiquitinated in this process. Here we have confirmed the Vpu- and ßTrCP-dependent multi/polyubiquitination of BST-2. Ubiquitinated BST-2 accumulated in cells treated with a lysosomal inhibitor but not a proteasomal inhibitor. Additionally, we observed that a BST-2 mutant deleted for its cytosolically exposed lysine residues is also ubiquitinated. Subsequent experiments suggested that Vpu promotes BST-2 ubiquitination upon amino acid residues bearing hydroxyl- but not thiol-bearing side chains. However, a BST-2 mutant bearing substitutions for its cytoplasmically exposed Ser, Thr, and Lys residues was still down-regulated, ubiquitinated, and degraded in a Vpu-dependent manner. Our results suggest that Vpu may target either the BST-2 cytoplasmic Tyr residues or the NH(2) terminus itself for ubiquitination.
Asunto(s)
Antígenos CD/metabolismo , VIH-1/metabolismo , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Ubiquitinación , Proteínas Reguladoras y Accesorias Virales/metabolismo , Liberación del Virus , Sustitución de Aminoácidos , Antígenos CD/genética , Línea Celular , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , VIH-1/genética , Proteínas del Virus de la Inmunodeficiencia Humana/genética , Humanos , Mutación Missense , Unión Proteica , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Reguladoras y Accesorias Virales/genética , Proteínas con Repetición de beta-Transducina/genética , Proteínas con Repetición de beta-Transducina/metabolismoRESUMEN
The interferon-induced BST-2 protein has the unique ability to restrict the egress of HIV-1, Kaposi's sarcoma-associated herpesvirus (KSHV), Ebola virus, and other enveloped viruses. The observation that virions remain attached to the surface of BST-2-expressing cells led to the renaming of BST-2 as "tetherin". However, viral proteins such as HIV-1 Vpu, simian immunodeficiency virus Nef, and KSHV K5 counteract BST-2, thereby allowing mature virions to readily escape from infected cells. Since the anti-viral function of BST-2 was discovered, there has been an explosion of research into several aspects of this intriguing interplay between host and virus. This review focuses on recent work addressing the molecular mechanisms involved in BST-2 restriction of viral egress and the species-specific countermeasures employed by various viruses.
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Antígenos CD/metabolismo , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , VIH-1/crecimiento & desarrollo , Glicoproteínas de Membrana/metabolismo , Antígenos CD/genética , Proteínas Ligadas a GPI , Humanos , Glicoproteínas de Membrana/genética , Virión/metabolismo , Virosis/metabolismo , Virosis/virologíaRESUMEN
Salmonella enterica serovar Typhimurium is a leading cause of acute gastroenteritis throughout the world. This pathogen has two type III secretion systems (TTSS) encoded in Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) that deliver virulence factors (effectors) to the host cell cytoplasm and are required for virulence. While many effectors have been identified and at least partially characterized, the full repertoire of effectors has not been catalogued. In this proteomic study, we identified effector proteins secreted into defined minimal medium designed to induce expression of the SPI-2 TTSS and its effectors. We compared the secretomes of the parent strain to those of strains missing essential (ssaK::cat) or regulatory (ΔssaL) components of the SPI-2 TTSS. We identified 20 known SPI-2 effectors. Excluding the translocon components SseBCD, all SPI-2 effectors were biased for identification in the ΔssaL mutant, substantiating the regulatory role of SsaL in TTS. To identify novel effector proteins, we coupled our secretome data with a machine learning algorithm (SIEVE, SVM-based identification and evaluation of virulence effectors) and selected 12 candidate proteins for further characterization. Using CyaA' reporter fusions, we identified six novel type III effectors and two additional proteins that were secreted into J774 macrophages independently of a TTSS. To assess their roles in virulence, we constructed nonpolar deletions and performed a competitive index analysis from intraperitoneally infected 129/SvJ mice. Six mutants were significantly attenuated for spleen colonization. Our results also suggest that non-type III secretion mechanisms are required for full Salmonella virulence.
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Proteínas Bacterianas/metabolismo , Salmonella typhimurium/metabolismo , Factores de Virulencia/metabolismo , Algoritmos , Animales , Inteligencia Artificial , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica/fisiología , Ratones , Ratones de la Cepa 129 , Mutación , Salmonelosis Animal/microbiología , Salmonella typhimurium/genéticaRESUMEN
The primary roles attributed to the human immunodeficiency virus type 1 (HIV-1) Vpu protein are the degradation of the viral receptor CD4 and the enhancement of virion release. With regard to CD4 downregulation, Vpu has been shown to act as an adapter linking CD4 with the ubiquitin-proteasome machinery via interaction with the F-box protein betaTrCP. To identify additional cellular betaTrCP-dependent Vpu targets, we performed quantitative proteomics analyses using the plasma membrane fraction of HeLa cells expressing either wild-type Vpu or a Vpu mutant (S52N/S56N) that does not bind betaTrCP. One cellular protein, BST-2 (CD317), was consistently underrepresented in the membrane proteome of cells expressing wild-type Vpu compared to the proteome of cells expressing the Vpu mutant. To verify the biological relevance of this phenotype for HIV pathogenesis, we showed that in T cells infected with HIV-1, BST-2 downregulation occurred in a Vpu-dependent manner. Recently, BST-2 has been identified as the interferon-inducible cellular factor Tetherin, which restricts HIV virion release in the absence of Vpu. We address here the unresolved mechanism of Vpu-mediated BST-2 downregulation. Our data show that the presence of wild-type Vpu reduced cell surface and total steady-state BST-2 levels, whereas that of the mutant Vpu had no effect. In addition, treatment of cells with the lysosome acidification inhibitor concanamycin A, but not treatment with the proteasome inhibitor MG132, reduced BST-2 downregulation by wild-type Vpu, thereby suggesting that the presence of Vpu leads to the degradation of BST-2 via an endosome-lysosome degradation pathway. The importance of betaTrCP in this process was confirmed by demonstrating that in the absence of betaTrCP, BST-2 levels were restored despite the presence of Vpu. Taken together, these data support the hypothesis that, in similarity to its role in CD4 degradation, Vpu acts as an adapter molecule linking BST-2 to the cellular ubiquitination machinery via betaTrCP. However, in contrast to the proteasome-dependent degradation of CD4, which occurs in the endoplasmic reticulum, Vpu appears to interact with BST-2 in the trans-Golgi network or in early endosomes, leading to lysosomal degradation of BST-2. Via this action, Vpu could counter the tethering function of BST-2, resulting in enhanced HIV-1 virion release. Interestingly, although HIV-2 does not express Vpu, an isolate known to exhibit enhanced viral egress can downregulate surface BST-2 by an as-yet-unknown mechanism that does not appear to involve degradation. Understanding the molecular mechanisms of both Vpu-dependent and -independent mediated antagonism of BST-2 will be critical for therapeutic strategies that exploit this novel viral function.
Asunto(s)
Antígenos CD/metabolismo , Infecciones por VIH/metabolismo , VIH-1/metabolismo , Proteínas del Virus de la Inmunodeficiencia Humana/fisiología , Glicoproteínas de Membrana/metabolismo , Proteínas Reguladoras y Accesorias Virales/fisiología , Proteínas con Repetición de beta-Transducina/metabolismo , Antígenos CD/genética , Antígenos CD4/genética , Antígenos CD4/metabolismo , Línea Celular , Proteínas Ligadas a GPI , Infecciones por VIH/virología , VIH-1/genética , Proteínas del Virus de la Inmunodeficiencia Humana/genética , Humanos , Glicoproteínas de Membrana/genética , Unión Proteica , Transporte de Proteínas , Proteínas Reguladoras y Accesorias Virales/genética , Virión/genética , Virión/fisiología , Esparcimiento de Virus , Proteínas con Repetición de beta-Transducina/genéticaRESUMEN
Kaposi sarcoma herpesvirus (KSHV) is the etiologic agent of Kaposi sarcoma (KS) and certain rare B cell lymphoproliferative disorders. KSHV infection of endothelial cells (EC) in vitro increases expression of the inducible host-encoded enzyme heme oxygenase-1 (HO-1), which is also strongly expressed in KS tumors. HO-1 catalyzes the rate-limiting step in the conversion of heme into iron, biliverdin and the gasotransmitter carbon monoxide (CO), all of which share anti-apoptotic, anti-inflammatory, pro-survival, and tumorigenic activities. Our previous work has shown that HO-1 expression in KSHV-infected EC is characterized by a rapid yet transient induction at early times post-infection, followed by a sustained upregulation co-incident with establishment of viral latency. These two phases of expression are independently regulated, suggesting distinct roles for HO-1 in the virus life cycle. Here, we investigated the role of HO-1 during acute infection, prior to the onset of viral gene expression. The early infection phase involves a series of events that culminate in delivery of the viral genome to the nucleus. Primary infection also leads to activation of host innate immune effectors, including the pattern recognition receptor TLR4, to induce an antiviral response. It has been shown that TLR4-deficient EC are more susceptible to KSHV infection than wild-type controls, suggesting an important inhibitory role for TLR4 in the KSHV life cycle. TLR4 signaling is in turn subject to regulation by several virus-encoded immune evasion factors. In this report we identify HO-1 as a host protein co-opted by KSHV as part of a rapid immune evasion strategy. Specifically, we show that early HO-1 induction by KSHV results in increased levels of endogenous CO, which functions as a TLR4 signaling inhibitor. In addition, we show that CO-mediated inhibition of TLR4 signaling leads to reduced expression of TLR4-induced antiviral genes, thus dampening the host antiviral response and facilitating KSHV infection. Conversely, inhibition of HO-1 activity decreases intracellular CO, enhances the host antiviral response and inhibits KSHV infection. In conclusion, this study identifies HO-1 as a novel innate immune evasion factor in the context of KSHV infection and supports HO-1 inhibition as a viable therapeutic strategy for KS.
RESUMEN
UNLABELLED: Kaposi sarcoma (KS) herpesvirus (KSHV) infection of endothelial cells (EC) is associated with strong induction of heme oxygenase-1 (HO-1), a stress-inducible host gene that encodes the rate-limiting enzyme responsible for heme catabolism. KS is an angioproliferative tumor characterized by the proliferation of KSHV-infected spindle cells, and HO-1 is highly expressed in such cells. HO-1 converts the pro-oxidant, proinflammatory heme molecule into metabolites with antioxidant, anti-inflammatory, and proliferative activities. Previously published work has shown that KSHV-infected EC in vitro proliferate in response to free heme in a HO-1-dependent manner, thus implicating virus-enhanced HO-1 activity in KS tumorigenesis. The present study investigated the molecular mechanisms underlying KSHV induction of HO-1 in lymphatic EC (LEC), which are the likely spindle cell precursors. In a time course analysis of KSHV-infected cells, HO-1 expression displays biphasic kinetics characterized by an early transient induction that is followed by a more sustained upregulation coincident with the establishment of viral latency. A viral microRNA miR-K12-11 deletion mutant of KSHV was found to be defective for induction of HO-1 during latency. A potential mechanism for this phenotype was provided by BACH1, a cellular HO-1 transcriptional repressor targeted by miR-K12-11. In fact, in KSHV-infected LEC, the BACH1 message level is reduced, BACH1 subcellular localization is altered, and miR-K12-11 mediates the inverse regulation of HO-1 and BACH1 during viral latency. Interestingly, the data indicate that neither miR-K12-11 nor de novo KSHV gene expression is required for the burst of HO-1 expression observed at early times postinfection, which suggests that additional virion components promote this phenotype. IMPORTANCE: While the mechanisms underlying KSHV induction of HO-1 remain unknown, the cellular mechanisms that regulate HO-1 expression have been extensively investigated in the context of basal and pathophysiological states. The detoxifying action of HO-1 is critical for the protection of cells exposed to high heme levels. KS spindle cells are erythrophagocytic and contain erythrocyte ghosts. Erythrocyte degeneration leads to the localized release of heme, creating oxidative stress that may be further exacerbated by environmental or other cofactors. Our previous work showed that KSHV-infected cells proliferate in response to heme and that this occurs in a HO-1-dependent manner. We therefore hypothesize that KSHV induction of HO-1 contributes to KS tumor development via heme metabolism and propose that HO-1 be evaluated as a therapeutic target for KS. Our present work, which aimed to understand the mechanisms whereby KSHV induces HO-1, will be important for the design and implementation of such a strategy.
Asunto(s)
Células Endoteliales/virología , Hemo-Oxigenasa 1/biosíntesis , Herpesvirus Humano 8/fisiología , MicroARNs/metabolismo , ARN Viral/metabolismo , Eliminación de Gen , Regulación de la Expresión Génica , Herpesvirus Humano 8/genética , Interacciones Huésped-Patógeno , Regulación hacia Arriba , Latencia del VirusRESUMEN
In the fields of virology and innate immunity, BST-2/tetherin is well known for its ability to block the egress of enveloped viruses from infected cells. This appears to be accomplished by 'tethering' virions to the cell surface, thereby limiting virion release. In the past year, several groups have discovered that BST-2/tetherin can activate NF-κB, a transcriptional activator that leads to the rapid expression of both proinflammatory cytokines and proteins involved in cell survival. While this new BST-2 function has been interpreted as a possible viral-sensing mechanism, there may also be broader implications for HIV gene regulation. This article reviews the evidence for BST-2-dependent NF-κB activation, and explores the significance of these exciting new results.
RESUMEN
We have undertaken a genetic strategy to map Vpu regions necessary for BST-2 antagonism and viral egress. This approach is based on our identification of an egress-defective Vpu variant encoded by an HIV-1 subtype C strain. We constructed a series of chimeric Vpu molecules made from the Vpu C variant and Vpu B from a standard laboratory strain. The TM domain from the inactive Vpu C, which contains multiple non-conserved residues, was responsible for a significant decrease in egress activity and BST-2 downregulation, confirming the functional importance of the Vpu TM domain. However, for complete inactivation, both the N-terminus and TM domain from the inactive Vpu C molecule were required, suggesting a new role for the Vpu N-terminus. In addition, determinants in the C-terminus of Vpu B that may be involved in efficient TGN accumulation were also necessary for enhanced viral egress but are missing or non-functional in Vpu C.
Asunto(s)
VIH-1/fisiología , Proteínas del Virus de la Inmunodeficiencia Humana/genética , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Proteínas Reguladoras y Accesorias Virales/genética , Proteínas Reguladoras y Accesorias Virales/metabolismo , Liberación del Virus , Secuencia de Aminoácidos , Antígenos CD , Línea Celular , Análisis Mutacional de ADN , Proteínas Ligadas a GPI/antagonistas & inhibidores , VIH-1/genética , Humanos , Datos de Secuencia MolecularRESUMEN
Like the other more well-characterized post-translational modifications (phosphorylation, methylation, acetylation, acylation, etc.), the attachment of the 76 amino acid ubiquitin (Ub) protein to substrates has been shown to govern countless cellular processes. As obligate intracellular parasites, viruses have evolved the capability to commandeer many host processes in order to maximize their own survival, whether it be to increase viral production or to ensure the long-term survival of latently infected host cells. The first evidence that viruses could usurp the Ub system came from the DNA tumor viruses and Adenoviruses, each of which use Ub to dysregulate the host cell cycle (Scheffner et al., 1990; Querido et al., 2001). Today, the list of viruses that utilize Ub includes members from almost every viral class, encompassing both RNA and DNA viruses. Among these, there are examples of Ub usage at every stage of the viral life cycle, involving both ubiquitination and de-ubiquitination. In addition to viruses that merely modify the host Ub system, many of the large DNA viruses encode their own Ub modifying machinery. In this review, we highlight the latest discoveries regarding the myriad ways that viruses utilize Ub to their advantage.
RESUMEN
Kaposi sarcoma (KS) is a complex cancer that arises from the initial infection of an appropriate endothelial or progenitor cell by Kaposi Sarcoma Herpesvirus/Human Herpesvirus-8 (KSHV/HHV8). However, the majority of KS cases occur when infected patients also suffer from some coincident form of immune deregulation, providing a favorable microenvironment for tumor development. Cellular hallmarks of KS progression include both the hyper-proliferation of KSHV-infected cells and the infiltration of immune modulatory cells into KS lesions, which together result in chronic inflammation, the induction of angiogenesis and tumor growth. This review describes the current understanding of the interactions between KSHV and host responses that result in this unusual cancer, along with existing treatments and prospects for future therapeutic approaches.
RESUMEN
To determine the impact of a low Mg(2+)/pH defined growth medium (MgM) on the proteome of Salmonella enterica serotype Typhimurium, we cultured S. Typhimurium cells in the medium under two different conditions termed MgM Shock and MgM Dilution and then comparatively analyzed the bacterial cells harvested from these conditions by a global proteomic approach. Proteomic results showed that MgM Shock and MgM Dilution differentially affected the S. Typhimurium proteome. MgM Shock induced a group of proteins whose induction usually occurred at low O(2) level, while MgM Dilution induced those related to the type III secretion system (T3SS) of Salmonella Pathogenicity Island 2 (SPI2) and those involved in thiamine or biotin biosynthesis. The metabolic state of the S. Typhimurium cells grown under MgM Shock condition also differed significantly from that under MgM Dilution condition. Western blot analysis not only confirmed the proteomic results, but also showed that the abundances of SPI2-T3SS proteins SsaQ and SseE and biotin biosynthesis proteins BioB and BioD increased after S. Typhimurium infection of RAW 264.7 macrophages. Deletion of the gene encoding BioB reduced the bacterial ability to replicate inside the macrophages, suggesting a biotin-limited environment encountered by S. Typhimurium within RAW 264.7 macrophages.
RESUMEN
Typhoid fever is a potentially fatal disease caused by the bacterial pathogen Salmonella enterica serotype Typhi ( S. typhi). S. typhi infection is a complex process that involves numerous bacterially encoded virulence determinants, and these are thought to confer both stringent human host specificity and a high mortality rate. In the present study, we used a liquid chromatography-mass spectrometry (LC-MS)-based proteomics strategy to investigate the proteome of logarithmic, stationary phase, and low pH/low magnesium (MgM) S. typhi cultures. This represents the first large-scale comprehensive characterization of the S. typhi proteome. Our analysis identified a total of 2066 S. typhi proteins. In an effort to identify putative S. typhi-specific virulence factors, we then compared our S. typhi results to those obtained in a previously published study of the S. typhimurium proteome under similar conditions ( Adkins, J. N. et al. Mol. Cell. Proteomics 2006, 5, 1450-1461 ). Comparative proteomics analysis of S. typhi strain Ty2 and S. typhimurium strain LT2 revealed a subset of highly expressed proteins unique to S. typhi that were exclusively detected under conditions that are thought to mimic the infective state in macrophage cells. These proteins included CdtB, HlyE, and gene products of t0142, t1108, t1109, t1476, and t1602. The differential expression of T1108, T1476, and HlyE was confirmed by Western blot analysis. When our observations are taken together with the current literature, they suggest that this subset of proteins may play a role in S. typhi pathogenesis and human host specificity.
Asunto(s)
Proteoma/análisis , Proteómica , Salmonella typhi/química , Salmonella typhi/patogenicidad , Fiebre Tifoidea/microbiología , Factores de Virulencia/análisis , Línea Celular Tumoral , Cromatografía Liquida , Humanos , Proteoma/biosíntesis , Proteoma/química , Reproducibilidad de los Resultados , Salmonella typhimurium/química , Salmonella typhimurium/metabolismo , Espectrometría de Masas en Tándem , Fiebre Tifoidea/metabolismo , Factores de Virulencia/biosíntesis , Factores de Virulencia/químicaRESUMEN
Disruption of axis specification leads to defects in dorsal tissue patterning and cell movements. Here, we examine how beta-catenin coordinately affects gastrulation movements and dorsal mesoderm differentiation. The reduction of beta-catenin protein levels by morpholino oligonucleotides complementary to beta-catenin mRNA causes a disruption in gastrulation movements. Time-lapse imaging of beta-catenin morphants during gastrulation reveals that involution occurs simultaneously around the blastopore in the absence of convergent extension cell movements. Transplantation experiments show that morphant cells grafted from the marginal zone into wild-type hosts differentiate into notochord and muscle. However, wild-type mesoderm cells grafted to the marginal zone of beta-catenin morphants do not form dorsal tissues. These data argue that beta-catenin is not required for the initial establishment of dorsal mesoderm cell competency, but it is required for the maintenance of that competency. We propose that tissue interactions that occur during convergent extension movements are necessary for maintaining dorsal tissue competency.
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Diferenciación Celular , Embrión no Mamífero/citología , Gastrulación , Mesodermo/embriología , beta Catenina/metabolismo , Animales , Regulación del Desarrollo de la Expresión Génica , Mesodermo/citología , Morfogénesis , Células Musculares/citología , Notocorda/citología , Notocorda/embriología , Oligonucleótidos , Xenopus laevis , beta Catenina/genéticaRESUMEN
LasA protease is a 20-kDa elastolytic and staphylolytic enzyme secreted by Pseudomonas aeruginosa. LasA is synthesized as a preproenzyme that undergoes proteolysis to remove a 22-kDa amino-terminal propeptide. Like the propeptides of other bacterial proteases, the LasA propeptide may act as an intramolecular chaperone that correctly folds the mature domain into an active protease. To locate regions of functional importance within proLasA, linker-scanning insertional mutagenesis was employed using a plasmid containing lasA as the target. Among the 5 missense insertions found in the mature domain of proLasA, all abolished enzymatic activity but not secretion. In general, the propeptide domain was more tolerant to insertions. However, insertions within a 9-amino-acid region in the propeptide caused dramatic reductions in LasA enzymatic activity. All mutant proLasA proteins were still secreted, but extracellular stability was low due to clustered insertions within the propeptide. The codons of 16 residues within and surrounding the identified 9-amino-acid region were subjected to site-directed mutagenesis. Among the alanine substitutions in the propeptide that had a major effect on extracellular LasA activity, two (L92A and W95A) resulted in highly unstable proteins that were susceptible to proteolytic degradation and three (H94A, I101A, and N102A) were moderately unstable and allowed the production of a LasA protein with low enzymatic activity. These data suggest that these clustered residues in the propeptide may play an important role in promoting the correct protein conformation of the mature LasA protease domain.
Asunto(s)
Aminoácidos/metabolismo , Proteínas Bacterianas/metabolismo , Metaloendopeptidasas/metabolismo , Pseudomonas aeruginosa/enzimología , Alanina/genética , Alanina/metabolismo , Secuencia de Aminoácidos , Aminoácidos/genética , Proteínas Bacterianas/genética , Sitios de Unión/genética , Western Blotting , Dominio Catalítico/genética , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Metaloendopeptidasas/genética , Modelos Genéticos , Datos de Secuencia Molecular , Mutagénesis Insercional , Mutagénesis Sitio-Dirigida , Mutación , Procesamiento Proteico-Postraduccional , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
The pathogen Salmonella enterica is known to cause both food poisoning and typhoid fever. Because of the emergence of antibiotic-resistant isolates and the threat of bioterrorism (e.g. contamination of the food supply), there is a growing need to study this bacterium. In this investigation, comparative peptidomics was used to study S. enterica serovar Typhimurium cultured in either a rich medium or in an acidic, low magnesium, and minimal nutrient medium designed to roughly mimic the macrophage phagosomal environment (within which Salmonella are known to survive). Native peptides from cleared cell lysates were enriched by using isopropanol extraction and analyzed by using both LC-MS/MS and LC-FTICR-MS. We identified and quantified 5,163 peptides originating from 682 proteins, and the data clearly indicated that compared with Salmonella cultured in the rich medium, cells cultured in the phagosome-mimicking medium had dramatically higher abundances of a wide variety of protein degradation products, especially from ribosomal proteins. Salmonella from the same cultures were also analyzed using traditional, bottom-up proteomic methods, and when the peptidomics and proteomics data were analyzed together, two clusters of proteins targeted for proteolysis were tentatively identified. Possible roles of targeted proteolysis by phagocytosed Salmonella are discussed.
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Proteínas Bacterianas/metabolismo , Proteómica/métodos , Salmonella typhimurium/metabolismo , Proteínas Bacterianas/química , Cromatografía Liquida , Medios de Cultivo , Humanos , Peso Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Fragmentos de Péptidos/metabolismo , Fagosomas/metabolismo , Salmonella typhimurium/patogenicidad , Espectrometría de Masas en TándemRESUMEN
During vertebrate development the formation of somites is a critical step, as these structures will give rise to the vertebrae, muscle, and dermis. In Xenopus laevis, somitogenesis consists of the partitioning of the presomitic mesoderm into somites, which undergo a 90-degree rotation to become aligned parallel to the notochord. Using a membrane-targeted green fluorescent protein to visualize cell outlines, we examined the individual cell shape changes occurring during somitogenesis. We show that this process is the result of specific, coordinated cell behaviors beginning with the mediolateral elongation of cells in the anterior presomitic mesoderm and then the subsequent bending of these elongated cells to become oriented parallel with the notochord. By labeling a clonal population of paraxial mesoderm cells, we show that cells bend around their dorsoventral axis. Moreover, this cell bending correlates with an increase in the number of filopodial protrusions, which appear to be posteriorly directed toward the newly formed segmental boundary. By examining the formation of somites at various positions along the anteroposterior axis, we show that the general sequence of cell behaviors is the same; however, somite rotation in anterior somites is slower than in posterior somites. Lastly, this coordinated set of cell behaviors occurs in a dorsal-to-ventral progression within each somite such that cells in the dorsal aspect of the somite become aligned along the anteroposterior axis before cells in other regions of the same somite. Together, our data further define how these cell behaviors are temporally and spatially coordinated during somite segmentation and rotation.
Asunto(s)
Somitos/citología , Xenopus laevis/embriología , Animales , Animales Modificados Genéticamente , Tipificación del Cuerpo , Adhesión Celular , Forma de la Célula , Proteínas Fluorescentes Verdes/genética , Imagenología Tridimensional , Microscopía Confocal , Modelos Biológicos , Proteínas Recombinantes/genética , Factores de Tiempo , Xenopus laevis/genéticaRESUMEN
Salmonella enterica serovar Typhimurium (also known as Salmonella typhimurium) is a facultative intracellular pathogen that causes approximately 8,000 reported cases of acute gastroenteritis and diarrhea each year in the United States. Although many successful physiological, biochemical, and genetic approaches have been taken to determine the key virulence determinants encoded by this organism, the sheer number of uncharacterized reading frames observed within the S. enterica genome suggests that many more virulence factors remain to be discovered. We used a liquid chromatography-mass spectrometry-based "bottom-up" proteomic approach to generate a more complete picture of the gene products that S. typhimurium synthesizes under typical laboratory conditions as well as in culture media that are known to induce expression of virulence genes. When grown to logarithmic phase in rich medium, S. typhimurium is known to express many genes that are required for invasion of epithelial cells. Conversely stationary phase cultures of S. typhimurium express genes that are needed for both systemic infection and growth within infected macrophages. Lastly bacteria grown in an acidic, magnesium-depleted minimal medium (MgM) designed to mimic the phagocytic vacuole have been shown to up-regulate virulence gene expression. Initial comparisons of protein abundances from bacteria grown under each of these conditions indicated that the majority of proteins do not change significantly. However, we observed subsets of proteins whose expression was largely restricted to one of the three culture conditions. For example, cells grown in MgM had a higher abundance of Mg(2+) transport proteins than found in other growth conditions. A second more virulent S. typhimurium strain (14028) was also cultured under these same growth conditions, and the results were directly compared with those obtained for strain LT2. This comparison offered a unique opportunity to contrast protein populations in these closely related bacteria. Among a number of proteins displaying a higher abundance in strain 14028 were the products of the pdu operon, which encodes enzymes required for propanediol utilization. These pdu operon proteins were validated in culture and during macrophage infection. Our work provides further support for earlier observations that suggest pdu gene expression contributes to S. typhimurium pathogenesis.
Asunto(s)
Proteínas Bacterianas/análisis , Proteoma/análisis , Salmonella typhimurium/metabolismo , Factores de Virulencia/análisis , Secuencia de Aminoácidos , Animales , Línea Celular , Regulación Bacteriana de la Expresión Génica , Concentración de Iones de Hidrógeno , Macrófagos/microbiología , Magnesio/metabolismo , Ratones , Datos de Secuencia Molecular , Operón , Glicoles de Propileno/metabolismo , Salmonella typhimurium/patogenicidad , VirulenciaRESUMEN
Recent fate maps of the Xenopus laevis gastrula show that mesodermal tissue surrounding the blastopore gives rise to muscle (Keller [1991] Methods Cell Biol 36:61-113; Lane and Smith [1999] Development 126:423-434). In a significant deviation from earlier data, the new maps demonstrate that cells in the ventral half of the gastrula are precursors to a significant portion of trunk somites. However, these posterior somites are not formed until tadpole stages (stages 38-44). We therefore set out to determine the timing of muscle specification within the ventral half of the gastrula. Our approach was to generate a series of tissue explants from gastrula-stage embryos and then culture them to either stage 28 (tailbud) or stage 44 (tadpole). At each endpoint, the presence of muscle in explants was assessed with a muscle-specific antibody. Interestingly, we found that muscle tissue is detected in ventral explants. However, these explants must be cultured to the tadpole stage. This is perhaps not unexpected, as this is the point at which this tissue normally gives rise to muscle. We further show that muscle specification of the involuting marginal zone does not change over the course of gastrulation. Together, these results suggest that dorsalizing signals emanating from the midline during gastrulation are not necessary for muscle specification of the ventral half of the involuting marginal zone.