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1.
Can J Microbiol ; 69(12): 501-511, 2023 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-37672795

RESUMEN

Bacillus cereus endophthalmitis is a devastating eye infection that causes rapid blindness through the release of extracellular tissue-destructive exotoxins. The phagocytic and antibacterial functions of ocular cells are the keys to limiting ocular bacterial infections. In a previous study, we identified a new virulence gene, plcA-2 (different from the original plcA-1 gene), that was strongly associated with the plcA gene of Listeria monocytogenes. This plcA gene had been confirmed to play an important role in phagocytosis. However, how the Bc-phosphatidylinositol-specific phospholipase C (PI-PLC) proteins encoded by the plcA-1/2 genes affect phagocytes remains unclear in B. cereus endophthalmitis. Here, we found that the enzymatic activity of Bc-PI-PLC-A2 was approximately twofold higher than that of Bc-PI-PLC-A1, and both proteins inhibited the viability of Müller cells. In addition, PI-PLC proteins reduced phagocytosis of Müller cells by decreasing the phosphorylation levels of key proteins in the PI3K/AKT signaling pathway. In conclusion, we showed that PI-PLC proteins contribute to inhibit the viability of and suppress the phagocytosis of Müller cells, providing new insights into the pathogenic mechanism of B. cereus endophthalmitis.


Asunto(s)
Endoftalmitis , Listeria monocytogenes , Humanos , Fosfoinositido Fosfolipasa C/genética , Fosfoinositido Fosfolipasa C/metabolismo , Fosfatidilinositol Diacilglicerol-Liasa/genética , Fosfatidilinositol Diacilglicerol-Liasa/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Supervivencia Celular , Células Ependimogliales/metabolismo , Fagocitos/metabolismo , Transducción de Señal , Fosfolipasas de Tipo C/genética , Fosfolipasas de Tipo C/metabolismo
2.
OMICS ; 24(4): 195-204, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-32286190

RESUMEN

An effective vaccine against Staphylococcus aureus infection is a major planetary heath priority, particularly with increasing antibiotic resistance worldwide. Previous efforts for a highly effective S. aureus vaccine were largely unsuccessful, in part, because the vaccine designs have tended to target mainly the B cell immunity and development of opsonic antibodies. In contrast, recent observations suggest that cell mediated immunity may be critical for protection against S. aureus. In addition, the S. aureus surface proteins are among the key immunodominant antigens because they are the first molecules to interact with the host organism cells and tissues. We report here an original vaccinomics study in which we used a reverse vaccinology and immunoinformatics in silico strategy integrated with genomics. After analyzing 2767 proteins, we defined 16 proteins of S. aureus as promising subunit vaccine candidates. Phosphatidylinositol phosphodiesterase (Plc) is secreted by extracellular pathogens such as S. aureus. We mapped the B and T cell epitopes for the Plc protein, tested the reactivity of the synthesized epitopes by Western blotting, and verified our findings in a pilot study of 10 patients with S. aureus infection. The peptides were then tested for their protective effect in groups of mice challenged with pathogenic S. aureus strain, which showed high protection level. These findings warrant further translational research for development of novel vaccines against S. aureus infection. Reverse vaccinology is an advanced approach that can be applied to identify new vaccine candidates against a host of microorganisms, including S. aureus.


Asunto(s)
Antígenos Bacterianos/inmunología , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito T/inmunología , Fosfatidilinositol Diacilglicerol-Liasa/inmunología , Infecciones Estafilocócicas/prevención & control , Vacunas Estafilocócicas/administración & dosificación , Staphylococcus aureus/efectos de los fármacos , Adolescente , Adulto , Animales , Antígenos Bacterianos/genética , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Linfocitos B/microbiología , Niño , Biología Computacional , Mapeo Epitopo , Epítopos de Linfocito B/genética , Epítopos de Linfocito T/genética , Femenino , Humanos , Inmunidad Celular/efectos de los fármacos , Inmunidad Humoral/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Fosfatidilinositol Diacilglicerol-Liasa/genética , Proyectos Piloto , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/microbiología , Vacunas Estafilocócicas/biosíntesis , Staphylococcus aureus/inmunología , Staphylococcus aureus/patogenicidad , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/microbiología , Vacunación/métodos , Vacunología/métodos
3.
PLoS Pathog ; 3(10): 1432-45, 2007 Oct 19.
Artículo en Inglés | MEDLINE | ID: mdl-17953481

RESUMEN

The Trypanosoma brucei genome encodes three groups of zinc metalloproteases, each of which contains approximately 30% amino acid identity with the major surface protease (MSP, also called GP63) of Leishmania. One of these proteases, TbMSP-B, is encoded by four nearly identical, tandem genes transcribed in both bloodstream and procyclic trypanosomes. Earlier work showed that RNA interference against TbMSP-B prevents release of a recombinant variant surface glycoprotein (VSG) from procyclic trypanosomes. Here, we used gene deletions to show that TbMSP-B and a phospholipase C (GPI-PLC) act in concert to remove native VSG during differentiation of bloodstream trypanosomes to procyclic form. When the four tandem TbMSP-B genes were deleted from both chromosomal alleles, bloodstream B (-/-) trypanosomes could still differentiate to procyclic form, but VSG was removed more slowly and in a non-truncated form compared to differentiation of wild-type organisms. Similarly, when both alleles of the single-copy GPI-PLC gene were deleted, bloodstream PLC (-/-) cells could still differentiate. However, when all the genes for both TbMSP-B and GPI-PLC were deleted from the diploid genome, the bloodstream B (-/-) PLC (-/-) trypanosomes did not proliferate in the differentiation medium, and 60% of the VSG remained on the cell surface. Inhibitors of cysteine proteases did not affect this result. These findings demonstrate that removal of 60% of the VSG during differentiation from bloodstream to procyclic form is due to the synergistic activities of GPI-PLC and TbMSP-B.


Asunto(s)
Metaloproteasas/metabolismo , Proteínas Protozoarias/biosíntesis , Trypanosoma brucei brucei/enzimología , Glicoproteínas Variantes de Superficie de Trypanosoma/biosíntesis , Animales , Antígenos de Protozoos , Línea Celular , Eliminación de Gen , Dosificación de Gen , Glicosilfosfatidilinositol Diacilglicerol-Liasa , Estadios del Ciclo de Vida/fisiología , Metaloendopeptidasas/biosíntesis , Metaloendopeptidasas/genética , Metaloproteasas/genética , Fosfatidilinositol Diacilglicerol-Liasa/genética , Fosfatidilinositol Diacilglicerol-Liasa/metabolismo , Proteínas Protozoarias/genética , Trypanosoma brucei brucei/genética , Glicoproteínas Variantes de Superficie de Trypanosoma/genética
5.
Cancer Immunol Immunother ; 56(1): 25-34, 2007 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-16612594

RESUMEN

The use of alpha(1,3)galactosyltransferase (alphaGT) as a method of inducing hyperacute rejection of tumors has been gaining interest recently. However, the approach is based in part on the sensitivity of each tumor line to the effects of complement lysis. Tumors expressing complement resistance factors such as membrane cofactor (CD46), decay accelerating factor (CD55) and protectin (CD59) have been shown to be more resistant to complement mediated lysis. Anchored to the membrane by a glycosylphosphoinositol moiety (GPI-anchored), CD55 and CD59 can be cleaved by Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PIPLC). Complement resistant A549 human lung carcinoma cells were engineered to express both the murine alphaGT gene and the B. thuringiensis PIPLC gene to alleviate complement resistance and enhance alphagal-mediated cancer killing. The PIPLC native signal sequence was replaced with the human epidermal growth factor signal sequence, EGFssPIPLC, to induce secretion from A549. Expression of EGFssPIPLC resulted in complete removal of CD55 and CD59 while sparing the non-GPI-anchored CD46. Results demonstrated that A549 cells transduced with two recombinant retroviral vectors carrying the alphaGT and EGFssPIPLC genes expressed high levels of alphagal epitope and exhibited a 5-fold increase in sensitivity to anti-alphagal mediated complement lysis.


Asunto(s)
Bacillus thuringiensis/enzimología , Activación de Complemento , Galactosiltransferasas/metabolismo , Neoplasias Pulmonares/patología , Fosfatidilinositol Diacilglicerol-Liasa/metabolismo , Antígenos CD55/metabolismo , Antígenos CD59/metabolismo , Galactosiltransferasas/genética , Regulación Enzimológica de la Expresión Génica/fisiología , Humanos , Neoplasias Pulmonares/metabolismo , Proteína Cofactora de Membrana/metabolismo , Fosfatidilinositol Diacilglicerol-Liasa/genética , Fosfoinositido Fosfolipasa C , Plásmidos , Células Tumorales Cultivadas , alfa-Galactosidasa/metabolismo
6.
Arterioscler Thromb Vasc Biol ; 27(1): 219-25, 2007 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-17038637

RESUMEN

OBJECTIVE: Plasma apolipoprotein CIII (apoCIII) independently predicts risk for coronary heart disease (CHD). We recently reported that apoCIII directly enhances adhesion of human monocytes to endothelial cells (ECs), and identified the activation of PKC alpha as a necessary upstream event of enhanced monocyte adhesion. This study tested the hypothesis that apoCIII activates PKC alpha in human monocytic THP-1 cells, leading to NF-kappaB activation. METHODS AND RESULTS: Among inhibitors specific to PKC activators, phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor D609 limited apoCIII-induced PKC alpha activation and THP-1 cell adhesion. ApoCIII increased PC-PLC activity in THP-1 cells, resulting in PKC alpha activation. Pertussis toxin (PTX) inhibited apoCIII-induced PC-PLC activation and subsequent PKC alpha activation, implicating PTX-sensitive G protein pathway. ApoCIII further activated nuclear factor-kappaB (NF-kappaB) through PKC alpha in THP-1 cells and augmented beta1-integrin expression. The NF-kappaB inhibitor peptide SN50 partially inhibited apoCIII-induced beta1-integrin expression and THP-1 cell adhesion. ApoCIII-rich VLDL had similar effects to apoCIII alone. CONCLUSIONS: PTX-sensitive G protein pathway participates critically in PKC alpha stimulation in THP-1 cells exposed to apoCIII, activating NF-kappaB, and increasing beta1-integrin. This action causes monocytic cells to adhere to endothelial cells. Furthermore, because leukocyte NF-kappaB activation contributes to inflammatory aspects of atherogenesis, apoCIII may stimulate diverse inflammatory responses through monocyte activation.


Asunto(s)
Apolipoproteína C-III/fisiología , Endotelio Vascular/fisiología , Proteínas de Unión al GTP/fisiología , Monocitos/fisiología , FN-kappa B/fisiología , Toxina del Pertussis/farmacología , Proteína Quinasa C-alfa/metabolismo , Hidrocarburos Aromáticos con Puentes/farmacología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Línea Celular , Endotelio Vascular/citología , Activación Enzimática , Proteínas de Unión al GTP/genética , Regulación de la Expresión Génica , Humanos , Integrina beta1/genética , Integrina beta1/fisiología , Monocitos/citología , FN-kappa B/genética , Norbornanos , Fosfatidilinositol Diacilglicerol-Liasa/antagonistas & inhibidores , Fosfatidilinositol Diacilglicerol-Liasa/genética , Fosfatidilinositol Diacilglicerol-Liasa/fisiología , Inhibidores de Fosfodiesterasa/farmacología , Tiocarbamatos , Tionas/farmacología
7.
J Cell Biochem ; 100(4): 952-9, 2007 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-17063484

RESUMEN

Signal transduction from plasma membrane to cell nucleus is a complex process depending on various components including lipid signaling molecules, in particular phosphoinositides and their related enzymes, which act at cell periphery and/or plasma membrane as well as at nuclear level. As far as the nervous system may concern the inositol lipid cycle has been hypothesized to be involved in numerous neural as well as glial functions. In this context, however, a precise panel of glial PLC isoforms has not been determined yet. In the present experiments we investigated astrocytic PLC isoforms in astrocytes obtained from foetal primary cultures of rat brain and from an established cultured (C6) rat astrocytoma cell line, two well known cell models for experimental studies on glia. Identification of PLC isoforms was achieved by using a combination of RT-PCR and immunocytochemistry experiments. While in both cell models the most represented PI-PLC isoforms were beta4, gamma1, delta4, and epsilon, isoforms PI-PLC beta2 and delta3 were not detected. Moreover, in primary astrocyte cultures PI-PLC delta3 resulted well expressed in C6 cells but was absent in astrocytes. Immunocytochemistry performed with antibodies against specific PLC isoforms substantially confirmed this pattern of expression both in astrocytes and C6 glioma cells. In particular while some isoenzymes (namely isoforms beta3 and beta4) resulted mainly nuclear, others (isoforms delta4 and epsilon) were preferentially localized at cytoplasmic and plasma membrane level.


Asunto(s)
Astrocitos/enzimología , Fosfatidilinositol Diacilglicerol-Liasa/metabolismo , Animales , Astrocitos/metabolismo , Línea Celular Tumoral , Células Cultivadas , Inmunohistoquímica , Isoenzimas/genética , Isoenzimas/metabolismo , Fosfatidilinositol Diacilglicerol-Liasa/genética , Fosfoinositido Fosfolipasa C , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética
8.
Int J Mol Med ; 18(2): 267-71, 2006 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-16820933

RESUMEN

Phosphoinositide-specific phospholipase C (PI-PLC) beta1 is a key enzyme in nuclear signal transduction, and it is involved in many cellular processes, such as proliferation and differentiation. In particular, the involvement of the PI-PLCbeta1 gene in erythroid differentiation lead us to investigate this gene in patients affected by high-risk myelodysplastic syndrome (MDS). By using fluorescence in situ hybridization (FISH) analysis, we have previously evidenced that, in MDS patients with normal GTG banding and a fatal outcome, the PI-PLCbeta1 gene undergoes monoallelic and interstitial deletion. Real-time PCR is characterized by high sensitivity, excellent precision and large dynamic range, and has become the method of choice for quantitative gene expression measurements. In the present study, we have performed a relative quantification real-time polymerase chain reaction (PCR) analysis on all of the MDS patients tested for FISH analysis. Furthermore, we have evaluated the expression of the PI-PLCbeta1 gene on healthy donors and the HL60 cell line, which is useful for testing the accuracy of the technology because of its low expression of PI-PLCbeta1. To analyze and quantify the levels of the two different splicing variants of PI-PLCbeta1 gene (1a and 1b), we have used a TaqMan isoform specific probe. We have seen that all of the MDS patients have higher levels of the PI-PLCbeta1 mRNA compared to the HL60 cell line as expected, but lower levels compared to the healthy donors. Furthermore, MDS blasts always express higher levels of PI-PLCbeta1b mRNA compared to PI-PLCbeta1a mRNA. Our data support the contention that the deletion of the PI-PLCbeta1 gene is indeed responsible for a reduced expression of the enzyme. In addition, the splicing isoform 1b, which is only nuclear, seems to be somehow partially preserved compared to the 1a isoform, which is nuclear and cytoplasmatic, hinting at a possible imbalance of the nuclear versus cytoplasmatic PI-PLC signaling which, in turn, could affect the cell cycle progression of MDS blasts.


Asunto(s)
Regulación de la Expresión Génica , Isoenzimas/metabolismo , Síndromes Mielodisplásicos , Fosfatidilinositol Diacilglicerol-Liasa/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Anciano , Femenino , Células HL-60 , Humanos , Hibridación Fluorescente in Situ , Isoenzimas/genética , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/enzimología , Síndromes Mielodisplásicos/genética , Fosfatidilinositol Diacilglicerol-Liasa/genética , Fosfoinositido Fosfolipasa C , ARN Mensajero/metabolismo
9.
Biochem J ; 394(Pt 2): 417-25, 2006 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-16288600

RESUMEN

The Toxoplasma gondii phosphoinositide-specific phospholipase C gene (TgPI-PLC) was cloned, sequenced and expressed in Escherichia coli and its enzymatic characteristics were investigated. TgPI-PLC is present in the genome as a single-copy gene consisting of 22 exons interrupted by 21 introns, and encodes a polypeptide of 1097 amino acids with a predicted molecular mass of 121 kDa. In addition to the conserved catalytic X and Y domains, TgPI-PLC contains an apparent N-terminal PH domain, an EF hand motif and a C-terminal C2 domain. When compared with mammalian delta-type PI-PLC, TgPI-PLC has an additional extended N-terminus and two insertions in the region between the X and Y domains, with a 31-35% identity over the whole sequence. Recombinant TgPI-PLC, as well as the native enzyme obtained from crude membrane extracts of the parasite, was more active with phosphatidylinositol than with phosphatidylinositol 4,5-bisphosphate as substrate. Indirect immunofluorescence analysis using an affinity-purified antibody against TgPI-PLC revealed that this enzyme localizes in the plasma membrane of the parasites.


Asunto(s)
Fosfatidilinositol Diacilglicerol-Liasa/metabolismo , Fosfatidilinositoles/metabolismo , Toxoplasma/enzimología , Secuencia de Aminoácidos , Animales , Calcio , Estabilidad de Enzimas , Regulación de la Expresión Génica , Concentración de Iones de Hidrógeno , Magnesio , Datos de Secuencia Molecular , Fosfatidilinositol Diacilglicerol-Liasa/química , Fosfatidilinositol Diacilglicerol-Liasa/genética , Fosfoinositido Fosfolipasa C , Transporte de Proteínas , Especificidad por Sustrato , Temperatura , Toxoplasma/citología
10.
Infect Immun ; 73(10): 6639-46, 2005 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-16177340

RESUMEN

Two virulence factors of Listeria monocytogenes, listeriolysin O (LLO) and phosphatidylinositol-specific phospholipase C (PI-PLC), mediate escape of this pathogen from the phagocytic vacuole of macrophages, thereby allowing the bacterium access to the host cell cytosol for growth and spread to neighboring cells. We characterized their orthologs from Bacillus anthracis by expressing them in L. monocytogenes and characterizing their contribution to bacterial intracellular growth and cell-to-cell spread. We generated a series of L. monocytogenes strains expressing B. anthracis anthrolysin O (ALO) and PI-PLC in place of LLO and L. monocytogenes PI-PLC, respectively. We found that ALO was active at both acidic and neutral pH and could functionally replace LLO in mediating escape from a primary vacuole; however, ALO exerted a toxic effect on the host cell by damaging the plasma membrane. B. anthracis PI-PLC, unlike the L. monocytogenes ortholog, had high activity on glycosylphosphatidylinositol-anchored proteins. L. monocytogenes expressing B. anthracis PI-PLC showed significantly decreased efficiencies of escape from a phagosome and in cell-to-cell spread. We further compared the level of cytotoxicity to host cells by using mutant strains expressing ALO in combination either with L. monocytogenes PI-PLC or with B. anthracis PI-PLC. The results demonstrated that the mutant strain expressing the combination of ALO and B. anthracis PI-PLC caused less damage to host cells than the strain expressing ALO and L. monocytogenes PI-PLC. The present study indicates that LLO and L. monocytogenes PI-PLC has adapted for L. monocytogenes intracellular growth and virulence and suggests that ALO and B. anthracis PI-PLC may have a role in B. anthracis pathogenesis.


Asunto(s)
Proteínas Bacterianas/genética , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidad , Glicoproteínas de Membrana/genética , Fosfatidilinositol Diacilglicerol-Liasa/genética , Animales , Bacillus anthracis/enzimología , Bacillus anthracis/genética , Bacillus anthracis/patogenicidad , Toxinas Bacterianas/genética , Proteínas de Choque Térmico/genética , Proteínas Hemolisinas , Mutación , Fagocitosis , Fosfoinositido Fosfolipasa C , Vacuolas/microbiología , Virulencia/genética
11.
Biotechnol Lett ; 27(11): 799-804, 2005 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-16086263

RESUMEN

A cDNA encoding a phosphoinositide-specific phospholipase C (PI-PLC) has been isolated from Zea mays by screening a cDNA library. The cDNA, designated ZmPLC, encodes a polypeptide of 586 amino acids, containing the catalytic X, Y and C2 domains found in all PI-PLCs from plants. Northern blot analysis showed that the expression of the ZmPLC gene in roots is up-regulated under conditions of high salt, dehydration, cold or low osmotic stress conditions. Recombinant ZmPLC protein was expressed in Esch- erichia coli, purified and used to produce polyclonal antibody, this polyclonal antibody is important for further studies to assess the ultimate function of the ZmPLC gene in plants.


Asunto(s)
Escherichia coli/genética , Fosfatidilinositol Diacilglicerol-Liasa/genética , Fosfatidilinositoles/metabolismo , Fosfolipasas de Tipo C/genética , Zea mays/genética , Secuencia de Aminoácidos , Northern Blotting , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , Electroforesis en Gel de Poliacrilamida , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Immunoblotting , Datos de Secuencia Molecular , Fosfatidilinositol Diacilglicerol-Liasa/metabolismo , Fosfoinositido Fosfolipasa C , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Fosfolipasas de Tipo C/metabolismo , Zea mays/enzimología
12.
Plant Cell Physiol ; 46(10): 1657-65, 2005 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-16085656

RESUMEN

The phosphatidylinositol-specific phospholipase C (PI-PLC) activity is detected in purified Lilium pollen protoplasts. Two PI-PLC full length cDNAs, LdPLC1 and LdPLC2, were isolated from pollen of Lilium daviddi. The amino acid sequences for the two PI-PLCs deduced from the two cDNA sequences contain X, Y catalytic motifs and C2 domains. Blast analysis shows that LdPLCs have 60-65% identities to the PI-PLCs from other plant species. Both recombinant PI-PLCs proteins expressed in E. coli cells show the PIP(2)-hydrolyzing activity. The RT-PCR analysis shows that both of them are expressed in pollen grains, whereas expression level of LdPLC2 is induced in germinating pollen. The exogenous purified calmodulin (CaM) is able to stimulate the activity of the PI-PLC when it is added into the pollen protoplast medium, while anti-CaM antibody suppresses the stimulation effect caused by exogenous CaM. PI-PLC activity is enhanced by G protein agonist cholera toxin and decreased by G protein antagonist pertussis toxin. Increasing in PI-PLC activity caused by exogenous purified CaM is also inhibited by pertussis toxin. A PI-PLC inhibitor, U-73122, inhibited the stimulation of PI-PLC activity caused by cholera toxin and it also leads to the decrease of [Ca(2+)](cyt) in pollen grains. Those results suggest that the PPI-PLC signaling pathway is present in Lilium daviddi pollen, and PI-PLC activity might be regulated by a heterotrimeric G protein and extracellular CaM.


Asunto(s)
Lilium/enzimología , Fosfatidilinositol Diacilglicerol-Liasa/metabolismo , Polen/enzimología , Clonación Molecular , ADN Complementario , Escherichia coli/genética , Colorantes Fluorescentes , Hidrólisis , Datos de Secuencia Molecular , Fosfatidilinositol Diacilglicerol-Liasa/genética , Fosfoinositido Fosfolipasa C , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
13.
Proc Natl Acad Sci U S A ; 102(36): 12927-31, 2005 Sep 06.
Artículo en Inglés | MEDLINE | ID: mdl-16118276

RESUMEN

Listeria monocytogenes phosphatidylinositol-specific phospholipase C (PI-PLC) plays a critical role in escape of this human pathogen from host cell vacuoles. Unlike classical bacterial PI-PLCs, the L. monocytogenes enzyme has very weak activity on glycosylphosphatidylinositol (GPI)-anchored proteins. Previous crystal structure analysis has revealed that a small beta-strand (Vb) is present in Bacillus cereus PI-PLC and is absent in the enzyme from L. monocytogenes. This Vb beta-strand in B. cereus PI-PLC forms contacts with the glycan linker of GPI anchors, which presumably increases its activity on GPI-anchored proteins. In this study, we show that, of all known bacterial PI-PLCs, those from listeriae are the only ones that lack the beta-strand. Expression by L. monocytogenes of B. cereus PI-PLC, which has strong activity on GPI-anchored proteins, inhibited bacterial escape from a vacuole and cell-to-cell spread, resulting in greatly reduced virulence in mice. Deletion of the Vb beta-strand from B. cereus PI-PLC abolished its ability to cleave GPI-anchored proteins, decreased its inhibitory effects, and increased its virulence in mice. These results strongly suggest that L. monocytogenes PI-PLC has evolved as an important determinant of L. monocytogenes pathogenesis by absence of the Vb beta-strand, thus leading to greatly reduced activity on GPI-anchored proteins.


Asunto(s)
Glicosilfosfatidilinositoles/metabolismo , Listeria monocytogenes/enzimología , Listeria monocytogenes/patogenicidad , Fosfatidilinositol Diacilglicerol-Liasa/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Proliferación Celular , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Listeria monocytogenes/citología , Listeria monocytogenes/genética , Ratones , Datos de Secuencia Molecular , Fosfatidilinositol Diacilglicerol-Liasa/química , Fosfatidilinositol Diacilglicerol-Liasa/genética , Fosfoinositido Fosfolipasa C , Estructura Secundaria de Proteína , Alineación de Secuencia , Virulencia
14.
Nucleic Acids Res ; 33(5): 1503-12, 2005.
Artículo en Inglés | MEDLINE | ID: mdl-15755751

RESUMEN

The expression of the vast majority of protein coding genes in trypanosomes is regulated exclusively at the post-transcriptional level. Developmentally regulated mRNAs that vary in levels of expression have provided an insight into one mechanism of regulation; a decrease in abundance is due to a shortened mRNA half-life. The decrease in half-life involves cis-acting elements in the 3' untranslated region of the mRNA. The trans-acting factors necessary for the increased rate of degradation remain uncharacterized. The GPI-PLC gene in Trypanosoma brucei encodes a phospholipase C expressed in mammalian bloodstream form, but not in the insect procyclic form. Here, it is reported that the differential expression of the GPI-PLC mRNA also results from a 10-fold difference in half-life. Second, the instability of the GPI-PLC mRNA in procyclic forms can be reversed by the inhibition of protein synthesis. Third, specifically blocking the translation of the GPI-PLC mRNA in procyclic forms by the inclusion of a hairpin in the 5' untranslated region does not result in stabilization of the mRNA. Thus, the effect of protein synthesis inhibitors in stabilizing the GPI-PLC mRNA operates in trans through a short-lived factor dependent on protein synthesis.


Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Fosfatidilinositol Diacilglicerol-Liasa/genética , Proteínas Protozoarias/fisiología , Estabilidad del ARN , ARN Mensajero/metabolismo , ARN Protozoario/metabolismo , Trypanosoma brucei brucei/crecimiento & desarrollo , Trypanosoma brucei brucei/genética , Animales , Glicosilfosfatidilinositol Diacilglicerol-Liasa , Semivida , Fosfatidilinositol Diacilglicerol-Liasa/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas Protozoarias/biosíntesis , Estabilidad del ARN/efectos de los fármacos , Trypanosoma brucei brucei/enzimología
15.
J Biol Chem ; 280(16): 16235-43, 2005 Apr 22.
Artículo en Inglés | MEDLINE | ID: mdl-15710612

RESUMEN

The phosphoinositide-specific phospholipase C (PI-PLC) is an important component of the inositol phosphate/diacylglycerol signaling pathway. A newly discovered Trypanosoma cruzi PI-PLC (TcPI-PLC) is lipid modified in its N terminus, targeted to its plasma membrane, and believed to play a role in differentiation of the parasite because its expression increases during the differentiation of trypomastigote to amastigote stages. To determine whether TcPI-PLC is involved in this differentiation step, antisense inhibition using phosphorothioate-modified oligonucleotides, and overexpression of the gene were performed. Antisense oligonucleotide-treated parasites showed a reduced rate of differentiation in comparison to controls, as well as accumulation of intermediate forms. Overexpression of TcPI-PLC led to a faster differentiation rate. In contrast, overexpression of a mutant TcPI-PLC that lacked the lipid modification at its N terminus did not affect the differentiation rate. Therefore, TcPI-PLC is involved, when expressed in the plasma membrane, in the differentiation of trypomastigotes to amastigotes, an essential step for the intracellular replication of these parasites.


Asunto(s)
Fosfatidilinositol Diacilglicerol-Liasa/metabolismo , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/crecimiento & desarrollo , Animales , Membrana Celular/metabolismo , Modelos Lineales , Glicoproteínas de Membrana/metabolismo , Oligonucleótidos Antisentido/metabolismo , Fosfatidilinositol Diacilglicerol-Liasa/genética , Fosfoinositido Fosfolipasa C , Proteínas Protozoarias/metabolismo , Trypanosoma cruzi/citología , Trypanosoma cruzi/genética
16.
Can J Microbiol ; 51(9): 745-51, 2005 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-16391652

RESUMEN

Listeria monocytogenes, a foodborn intracellular animal and human pathogen, produces several exotoxins contributing to virulence. Among these are listeriolysin O (LLO), a pore-forming cholesterol-dependent hemolysin, and a phosphatidylinositol-specific phospholipase C (PI-PLC). LLO is known to play an important role in the escape of bacteria from the primary phagocytic vacuole of macrophages, and PI-PLC supports this process. Evidence is accumulating that LLO and PI-PLC are multifunctional virulence factors with many important roles in the host-parasite interaction other than phagosomal membrane disruption. LLO and PI-PLC may induce a number of host cell responses by modulating signal transduction of infected cells via intracellular Ca2+ levels and the metabolism of phospholipids. This would result in the activation of host phospholipase C and protein kinase C. In the present study, using Bacillus sub tilis strains expressing LLO, PI-PLC, and simultaneously LLO and PI-PLC, we show that LLO and PI-PLC enhance bacterial binding to epithelial cells Int407, with LLO being necessary and PI-PLC playing an accessory role. The results of this work suggest that these two listerial proteins act on epithelial cells prior to internalization.


Asunto(s)
Adhesión Bacteriana , Toxinas Bacterianas/metabolismo , Células Epiteliales/microbiología , Proteínas de Choque Térmico/metabolismo , Listeria monocytogenes/patogenicidad , Fosfatidilinositol Diacilglicerol-Liasa/metabolismo , Animales , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Toxinas Bacterianas/genética , Línea Celular , Proteínas de Choque Térmico/genética , Proteínas Hemolisinas , Hemólisis , Humanos , Intestinos/citología , Intestinos/microbiología , Listeria monocytogenes/enzimología , Listeria monocytogenes/genética , Fosfatidilinositol Diacilglicerol-Liasa/genética , Fosfoinositido Fosfolipasa C
17.
Int J Mol Med ; 15(1): 117-21, 2005 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-15583837

RESUMEN

Phosphoinositol (PhoIns)-specific phospholipase C enzymes (PLCs) are central to the inositol lipid signaling pathways and contribute to intracellular Ca2+ release and protein kinase C activation. Five distinct classes of PhoIns-specific PLCs are known to exist in mammals, which are activated by membrane receptor-mediated events. Here we have identified a sixth class of PhoIns-specific PLC with a novel domain structure, which we have termed PLC-eta. Two putative PLC-eta enzymes were identified in humans and in mice. Sequence analysis revealed that residues implicated in substrate binding and catalysis from other PhoIns-specific PLCs are conserved in the novel enzymes. PLC-eta enzymes are most closely related to the PLC-delta class and share a close evolutionary relationship with other PLC isozymes. EST analysis and RT-PCR data suggest that PLC-eta enzymes are expressed in several cell types and, by analogy with other mammalian PhoIns-specific PLCs, are likely to be involved in signal transduction pathways.


Asunto(s)
Mamíferos , Fosfatidilinositol Diacilglicerol-Liasa/clasificación , Fosfatidilinositol Diacilglicerol-Liasa/metabolismo , Secuencia de Aminoácidos , Animales , Perfilación de la Expresión Génica , Humanos , Ratones , Datos de Secuencia Molecular , Fosfatidilinositol Diacilglicerol-Liasa/química , Fosfatidilinositol Diacilglicerol-Liasa/genética , Filogenia , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de Secuencia
18.
Plant J ; 40(2): 250-9, 2004 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-15447651

RESUMEN

The phosphoinositide signalling pathway is important in plant responses to extracellular and intracellular signals. To elucidate the physiological functions of phosphoinositide-specific phopspholipase C, PI-PLC, targeted knockout mutants of PpPLC1, a gene encoding a PI-PLC from the moss Physcomitrella patens, were generated via homologous recombination. Protonemal filaments of the plc1 lines show a dramatic reduction in gametophore formation relative to wild type: this was accompanied by a loss of sensitivity to cytokinin. Moreover, plc1 appeared paler than the wild type, the result of an altered differentiation of chloroplasts and reduced chlorophyll levels compared with wild type filaments. In addition, the protonemal filaments of plc1 have a strongly reduced ability to grow negatively gravitropically in the dark. These effects imply a significant role for PpPLC1 in cytokinin signalling and gravitropism.


Asunto(s)
Bryopsida/enzimología , Citocininas/metabolismo , Gravitropismo/fisiología , Fosfatidilinositol Diacilglicerol-Liasa/metabolismo , Bryopsida/crecimiento & desarrollo , Clorofila/metabolismo , Cloroplastos/metabolismo , Mutación , Fosfatidilinositol Diacilglicerol-Liasa/química , Fosfatidilinositol Diacilglicerol-Liasa/genética , Fosfoinositido Fosfolipasa C , Fototropismo/fisiología
19.
J Exp Bot ; 55(401): 1437-9, 2004 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-15073208

RESUMEN

Two cDNAs encoding proteins, PpPLC1 and PpPLC2, with catalytic and C2 domains conserved in plant phosphoinositide-specific phospholipase C (PI-PLC) were isolated from Physcomitrella patens. The N domain, which has been identified in Arabidopsis PI-PLCs as an EF hand-like domain, was found in both isoforms, although that in PpPLC2 was a split type. At micromolar Ca2+ concentrations, PpPLC1 preferentially hydrolysed phosphatidylinositol-4,5-bisphosphate, while PpPLC2 showed no specificity. Furthermore, at millimolar Ca2+, phosphatidylinositol was hydrolysed by PpPLC2, but not by PpPLC1. Thus, PpPLC1 and PpPLC2 are typical and novel types of plant PI-PLC, respectively.


Asunto(s)
Bryopsida/genética , Fosfatidilinositol Diacilglicerol-Liasa/genética , Secuencia de Bases , Bryopsida/enzimología , ADN Complementario/química , ADN Complementario/genética , Datos de Secuencia Molecular , Fosfatidilinositol Diacilglicerol-Liasa/metabolismo , Fosfoinositido Fosfolipasa C , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico
20.
J Am Chem Soc ; 126(4): 1008-9, 2004 Feb 04.
Artículo en Inglés | MEDLINE | ID: mdl-14746454

RESUMEN

Two mutations, R69D and K115E, converted a bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) to a phosphatase with much higher specific activity toward glucose-6-phosphate than inositol-1-phosphate. PI-PLC single mutations R69D and K115E can cleave PI but lack any demonstrable phosphatase activity. The bacterial PI-PLC has no sequence homology with known glucose-6-phosphatase enzymes, which need His, Arg, and negatively charged residues (Asp or Glu) at the active site. The change in chemical reaction and substrate specificity can be rationalized by energy minimization of the mutant with I-1-P or G-6-P bound.


Asunto(s)
Glucosa-6-Fosfatasa/genética , Glucosa-6-Fosfatasa/metabolismo , Fosfatidilinositol Diacilglicerol-Liasa/genética , Fosfatidilinositol Diacilglicerol-Liasa/metabolismo , Sustitución de Aminoácidos , Sitios de Unión , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Fosfoinositido Fosfolipasa C , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Especificidad por Sustrato
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