RESUMO
Insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2), a significant member of the conserved RNA-binding protein family, plays various roles in numerous physiological and pathological processes. However, the specific function of IGF2BP2 in regulating endometrial function in sheep remains largely unknown. In this study, we observed a significant upregulation in IGF2BP2 mRNA abundance in the endometrium during the luteal phase compared to the follicular phase in Hu sheep. The knockdown of IGF2BP2 resulted in accelerated cell proliferation and migration of Hu sheep endometrial stromal cells (ESCs). Moreover, RNA sequencing analysis revealed that genes with significantly altered expression in IGF2BP2 knockdown cells were predominantly enriched in endometrial receptivity-related signaling pathways, such as cytokine-cytokine receptor interaction, NOD-like receptor, PI3K-AKT, and JAK-STAT signaling pathway. Additionally, the knockdown of IGF2BP2 significantly increased the expression of matrix metalloprotein 9 (MMP9), vascular endothelial growth factor, and prolactin (PRL) in ESCs. The knockdown of IGF2BP2 was also observed to stimulate the PI3K/AKT/mTOR pathway by upregulating integrin ß4 (ITGB4) expression. Notably, the downregulation of ITGB4 attenuates IGF2BP2 knockdown-induced facilitation of proliferation and migration of Hu sheep ESCs by inhibiting the PI3K/AKT/mTOR pathway. Collectively, these findings highlight the important role of IGF2BP2 in regulating endometrial function, particularly through the modulation of ESC proliferation and migration via the PI3K/AKT/mTOR pathway.
The maintenance of normal physiological functionality of the endometrium is crucial for successful embryo implantation. Endometrial stromal cells (ESCs), as the principal components of the endometrium, play a key role in establishing optimal endometrial receptivity for embryo implantation. Despite the well-established role of IGF2BP2 in the pathogenesis of endometriosis in women, its functional impact on endometrial activity in ruminants, particularly in ovine species, remains undefined. In this study, we investigated the expression pattern of IGF2BP2 in the reproductive organs of female sheep and evaluated the potential roles and underlying mechanisms of IGF2BP2 in the function of sheep ESCs. This experiment confirmed the important role of IGF2BP2 in regulating endometrial function by modulating the proliferation and migration of Hu sheep ESCs.
Assuntos
Movimento Celular , Proliferação de Células , Endométrio , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Células Estromais , Serina-Treonina Quinases TOR , Animais , Feminino , Endométrio/metabolismo , Endométrio/citologia , Técnicas de Silenciamento de Genes , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ovinos , Células Estromais/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/genéticaRESUMO
Despite previous investigations elucidating the regulatory mechanisms of long non-coding RNAs (lncRNAs) in endometrial function and reproductive disorders, the precise pathways through which lncRNAs impact endometrial functions and fertility remain unclear. In this study, we performed an expression profile analysis of lncRNAs in the endometrial tissue of Hu sheep with different prolificacy, identifying 13,707 lncRNAs. We discovered a bidirectional lncRNA, designated lncRNA12097.1, exhibiting significant up-regulation exclusively in the endometrium of Hu sheep with high fecundity. Functional analyses revealed lncRNA12097.1 significantly enhanced proliferation and cell cycle progression in both endometrial epithelial cell (EEC) and stromal cells (ESC), while inhibiting apoptosis in these cell types. Mechanistically, we demonstrated a directly interaction between lncRNA12097.1 and miR-145-5p, with YES proto-oncogene 1 (YES1) being identified as a validated target of miR-145-5p. Interference with lncRNA12097.1 resulted in suppressed cell growth through down-regulation of YES1 expression, which could be rescued by miR-145-5p. Furthermore, lncRNA12097.1 functions as a competitive endogenous RNA (ceRNA) for miR-145-5p in ESCs, sequestering miR-145-5p and preventing its binding to the 3'UTR of YES1 mRNA. This interaction led to increased expression of YES1 and subsequent activation of downstream ß-catenin signaling, thereby promoting ESC growth in Hu sheep. These findings provide novel molecular insights into the mechanisms underlying prolificacy in sheep.
Assuntos
MicroRNAs , RNA Longo não Codificante , Feminino , Animais , Ovinos , MicroRNAs/genética , MicroRNAs/metabolismo , beta Catenina/genética , beta Catenina/metabolismo , Linhagem Celular Tumoral , RNA Longo não Codificante/genética , Proliferação de Células/genética , Endométrio/metabolismo , Ciclo Celular/genética , Regulação Neoplásica da Expressão GênicaRESUMO
The serine-threonine protein phosphatase 2A (PP2A) is a heterotrimeric enzyme complex that plays a vital role in regulating male reproductive activities. However, as an essential member of the PP2A family, the physiological functions of PP2A regulatory subunit B55α (PPP2R2A) in testis remain inconclusive. Hu sheep are noted for their reproductive precocity and fertility, and are ideal models for the study of male reproductive physiology. Here, we analyzed the expression patterns of PPP2R2A in the male Hu sheep reproductive tract at different developmental stages and further investigated its role in testosterone secretion and its underlying mechanisms. In this study, we found that there were temporal and spatial differences in PPP2R2A protein expression in the testis and epididymis, especially the expression abundance in the testis at 8 months old (8M) was higher than that at 3 months old (3M). Interestingly, we observed that PPP2R2A interference reduced the testosterone levels in the cell culture medium, which is accompanied by a reduction in Leydig cell proliferation and an elevation in Leydig cell apoptosis. The level of reactive oxygen species in cells increased significantly, while the mitochondrial membrane potential (ΔΨm) decreased significantly after PPP2R2A deletion. Meanwhile, the mitochondrial mitotic protein DNM1L was significantly upregulated, while the mitochondrial fusion proteins MFN1/2 and OPA1 were significantly downregulated after PPP2R2A interference. Furthermore, PPP2R2A interference suppressed the AKT/mTOR signaling pathway. Taken together, our data indicated that PPP2R2A enhanced testosterone secretion, promoted cell proliferation, and inhibited cell apoptosis in vitro, all of which were associated with the AKT/mTOR signaling pathway.
Assuntos
Células Intersticiais do Testículo , Proteínas Proto-Oncogênicas c-akt , Masculino , Animais , Ovinos , Células Intersticiais do Testículo/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/metabolismo , Testosterona/metabolismoRESUMO
Excessive fat deposition is the main character in nonalcoholic fatty liver disease (NAFLD), while γ-linolenic acid (GLA) is a polyunsaturated fatty acid that can reduce lipid deposition. This study investigated the effect and regulatory mechanism of GLA (100 µM) on lipid metabolism in alpha mouse liver 12 (AML-12) cells treated by 400 µM palmitic acid (PA). GLA reduced lipid content and increased fatty acid ß oxidation, as indicated by decreasing triglyceride and cholesterol contents and increasing mRNA and protein expressions of CPT1α and PPARα. GLA relieved oxidative stress caused by PA, upregulated mRNA levels of superoxide dismutase and glutathione peroxidase, and decreased reactive oxygen species content. GLA reduced apoptosis, as indicated by decreases in the BAX/BCL2 expression level and apoptosis percentage. GLA activated autophagy, autophagosome-lysosome fusion, and LKB1-AMPK-mTOR signaling and upregulated mRNA and protein expressions of Beclin-1, autophagy-related 5, and liver kinase B1 (LKB1). These effects of GLA on lipid metabolism disorders of PA-treated hepatocytes were reversed by autophagy inhibitor 3MA and AMPK inhibitor compound C, confirming our conclusions. Overall, GLA can protect AML-12 cells from lipid metabolism disorder caused by PA via balancing autophagy and apoptosis mediated by the LKB1-AMPK-mTOR pathway. Consequently, GLA, as a dietary supplement, can help to prevent and treat NAFLD by regulating lipid metabolism and autophagy.
Assuntos
Transtornos do Metabolismo dos Lipídeos , Hepatopatia Gordurosa não Alcoólica , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Apoptose , Autofagia , Metabolismo dos Lipídeos , Transtornos do Metabolismo dos Lipídeos/metabolismo , Fígado/metabolismo , Camundongos , Hepatopatia Gordurosa não Alcoólica/metabolismo , Ácido Palmítico/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Ácido gama-Linolênico/metabolismoRESUMO
N6-Methyladenosine (m6A) modification is the most abundant chemical modification in mRNA, and it participates in various biological processes, such as cell differentiation and proliferation. However, little is known about the function of m6A demethylase fat mass and obesity-associated (FTO) in myoblast proliferation. Here, we demonstrated that knockdown of FTO can significantly inhibit myoblast proliferation and promote apoptosis. RNA sequencing analysis revealed that a lot of downregulated genes in FTO knockdown cells are associated with cell cycle and apoptosis. Furthermore, silencing FTO drastically decreased cyclin D1 (CCND1) expression through YTHDF2-mediated mRNA degradation, thereby delaying the progression of G1 phase, and leading to impaired myoblast proliferation. These findings unraveled that FTO regulates myoblast proliferation by controlling CCND1 expression in an m6A-YTHDF2-dependent manner, which highlights the critical roles of m6A modification in myoblast proliferation.
Assuntos
Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Ciclina D1/genética , Estabilidade de RNA/genética , Proteínas de Ligação a RNA/genética , Adenosina/análogos & derivados , Adenosina/genética , Apoptose/genética , Ciclo Celular/genética , Diferenciação Celular/genética , Proliferação de Células/genética , Fase G1/genética , Humanos , Mioblastos/metabolismoRESUMO
The aim of this study was to investigate the molecular mechanisms of arginine (Arg) on follicular development of acute feed-restricted ewes during the luteal phase. From day 6 of the estrous cycle, 24 multiparous Hu sheep were randomly assigned into three groups: control group (a maintenance diet; n = 6), feed restriction group (0.5 maintenance diet, saline infusion; n = 9) and Arg treatment group (0.5 maintenance diet, infusion with 155 µmol of Arg-HCl/kg body weight; n = 9). The intravenous administrations were performed three times per day from day 6 to day 15 of the estrous cycle. At the end of treatment, the hypothalamus and pituitary were collected, as well as the follicular fluid (FF) and granulose cells (GCs) in the ≥2.5 mm follicles. The transcription level of NPVF was significantly increased, and the expression level of GNRH was significantly decreased in the hypothalamus with feed restriction. In addition, feed restriction significantly decreased the number of ≥2.5 mm follicles in the ovaries. In the ≥2.5 mm follicles, feed restriction significantly increased estradiol (E2) level in FF and the expression levels of steroidogenesis related genes (STAR, 3BHSD and CYP19A1) in GCs, while significantly decreased the expressions of FSHR and cell proliferation related genes (YAP1, CCND1 and PCNA) in GCs. Moreover, the activities of glucose metabolism enzymes (PFKP and G6PDH) were significantly decreased in GCs of the ≥2.5 mm follicles with feed restriction. Interestingly, as a precursor of nitric oxide, Arg supplementation can rescue the effects of feed restriction on follicular development by enhancing glucose metabolism and cell proliferation of GCs, and alleviating the abnormal E2 secretion in the ≥2.5 mm follicles, accompanied with recovering the expressions of NPVF and GNRH in the hypothalamus. These findings will be helpful for understanding the role of nutrition and Arg in sheep follicular development.
Assuntos
Arginina , Fase Luteal , Animais , Dieta , Estradiol , Ciclo Estral , Feminino , Líquido Folicular , OvinosRESUMO
The aim of this study was to investigate the effect of spirulina supplementation in a high-energy (HE) diet on lipid metabolism, oxidative status and immunity in Hu lambs. The lambs were assigned to two groups receiving either a standard diet (ST) or a HE diet. Each group was divided into three subgroups: no spirulina supplementation (control), 1% spirulina supplementation, or 3% spirulina supplementation. The body fat, serum cholesterol, triacylglycerol and oxidative stress increased in lambs fed the HE diet. However, 3% spirulina supplementation in the HE diet reduced above parameters and enhanced antioxidant capacity, including increased SOD activity and T-AOC content in serum and Longissimus thoracis et lumborum (LTL). Additionally, lambs receiving 3% spirulina supplementation showed an improvement in immunity-related parameters, including increased IgG concentration in serum and red and white blood cell counts. In conclusion, 3% spirulina supplementation in HE diet ameliorated lipid metabolic disorder and oxidative stress caused by a HE diet.
Assuntos
Dieta/veterinária , Transtornos do Metabolismo dos Lipídeos/veterinária , Carneiro Doméstico/metabolismo , Spirulina , Ração Animal/análise , Animais , Contagem de Células Sanguíneas/veterinária , Colesterol/sangue , Dieta/efeitos adversos , Imunoglobulina G/sangue , Transtornos do Metabolismo dos Lipídeos/dietoterapia , Transtornos do Metabolismo dos Lipídeos/radioterapia , Músculo Esquelético , Estresse Oxidativo , Distribuição Aleatória , Carneiro Doméstico/imunologia , Triglicerídeos/sangueRESUMO
Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A) is a central regulator of mitochondrial biogenesis and metabolism, and its expression is closely related to embryo development. To gain insights into the possible mechanisms of PPARGC1A during early embryogenesis, the development potential, mitochondrial biogenesis, and the culture medium metabolomics of embryos were evaluated when PPARGC1A overexpressed or suppressed in rabbit zygotes. Results showed that different PPARGC1A levels in rabbit zygotes could affect blastocyst percentage, and the expressions of mitochondrial biogenesis and metabolic-related genes, as well as the glutathione and adenosine triphosphate levels during early embryo development. In addition, compared with the controls, 12 and 10 different metabolites involved in carbohydrate, amino acid, and fatty acid metabolism were screened in the 5 day's spent culture medium of PPARGC1A overexpressed and suppressed embryos by gas chromatography-mass spectrometer, respectively. Consistent with these metabolite changes, the transcriptions of genes encoding glucose transporters and fatty acid biosynthetic proteins in the embryos from different groups were regulated by PPARGC1A during rabbit embryo development. Taken together, these data provide evidence that PPARGC1A may regulate early rabbit embryo development through mitochondrial biogenesis and metabolism.
Assuntos
Blastocisto/metabolismo , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Mitocôndrias/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/biossíntese , Zigoto/metabolismo , Animais , Blastocisto/citologia , Feminino , Coelhos , Zigoto/citologiaRESUMO
Packaged fresh spinach has been associated with outbreaks of illness caused by Escherichia coli O157:H7. The purpose of this study was to assess the behavior of E. coli O157:H7 in packaged baby spinach in response to storage conditions of temperature and package atmosphere and including effects of inoculation level, spinach leaf damage (cut leaves), internalized or leaf surface contamination, exposure to hypochlorite sanitizer, and package size. Behavior of E. coli O157:H7 inoculated at 2 and 4 log CFU/g on spinach packaged in polymer bags composed of a two-layer laminate (polypropylene and polyethylene) and stored under atmospheres of 20% O2-3% CO2 and 0% O2-15% CO2 (aerobic and anaerobic, respectively) was assessed at 5, 7, 12, and 15°C for up to 14 days. Growth kinetics were calculated using DMFit software. Temperature decreases progressively diminished growth or survival of the pathogen, and an aerobic package atmosphere resulted in longer lag times (4 to 6 days) and lower population levels (0.2 to 1.4 log CFU/g) compared with the anaerobic atmosphere at 15°C. Internalized contamination, leaf cuts, or exposure to 100 ppm of hypochlorite did not result in changes in pathogen behavior compared with controls; however, a growth minimization trend consisting of longer lag times and lower population levels was repeatedly observed in the aerobic compared with the anaerobic package atmospheres. In contrast, growth of indigenous mesophiles and Enterobacteriaceae was unaffected by package atmosphere. Spinach stored at 5 to 7°C in two sizes (5 and 16 oz) of polyethylene terephthalate clamshell packages with ambient air atmospheres was more likely to progress to lower-oxygen conditions in 16-oz compared with 5-oz packages after 7 days of storage (P < 0.05). Practices to maintain aerobic conditions within the package, as well as storage of the package at low temperature, are ways to limit growth of E. coli O157:H7 in packaged spinach.
Assuntos
Escherichia coli O157 , Microbiologia de Alimentos , Embalagem de Alimentos , Spinacia oleracea , Contagem de Colônia Microbiana , Embalagem de Alimentos/métodos , Embalagem de Alimentos/normas , Viabilidade Microbiana , Spinacia oleracea/microbiologia , TemperaturaRESUMO
Nutrient deficiency in ruminants can lead to estrus cycle disorders, a decreased pregnancy rate, and reduce birth weight. l-arginine (L-Arg), an important amino acid, can improve uterine homeostasis in pregnant sheep and prevent intrauterine growth restriction (IUGR). However, most studies of L-Arg have been conducted on pregnant sheep and few have reported the effects of L-Arg on microvessel density (MVD) in the non-pregnant ovine endometrium. The processes of normal uterine cyclical development and implantation are dependent on a balanced of endometrial MVD. In this study, female Hu sheep were randomly assigned to either a control group (nâ¯=â¯6), a nutrient-restricted group (nâ¯=â¯6), or an L-Arg supplemented nutrient-restricted group (nâ¯=â¯6). The effects of L-Arg on MVD in ovine endometrium were then studied. Our results showed that ovine endometrial MVD was significantly increased by nutrient restriction, but L-Arg counteracted the effect of nutrient restriction on MVD (Pâ¯<â¯0.05). Levels of angiogenic growth factors (including VEGFA, VEGFR2, and FGF2) had significant increases (Pâ¯<â¯0.05) in endometrium of nutrient restriction on sheep, but that L-Arg supplementation substantially decreased (Pâ¯<â¯0.05) their expressions in nutrient restriction sheep. Furthermore, oxidative stress caused by nutrient restriction was also alleviated by L-Arg supplementation in the ovine endometrium. Overall, the results suggested that L-Arg has crucial roles in maintaining the balance of endometrial MVD and angiogenic growth factors, and increasing anti-oxidation capability in the endometrium of nutrient-restricted sheep. This study will provide a theoretical basis and technical means for the normal development of endometrial microvessels in low nutrition level.
Assuntos
Arginina/farmacologia , Endométrio/irrigação sanguínea , Privação de Alimentos , Ovinos , Animais , Feminino , Regulação da Expressão Gênica/efeitos dos fármacosRESUMO
Salmonella enterica serovars Enteritidis and Typhimurium are the leading causative agents of salmonellosis in the United States. S. Enteritidis is predominantly associated with contamination of shell eggs and egg products, whereas S. Typhimurium is frequently linked to tainted poultry meats, fresh produce, and recently, peanut-based products. Chlorine is an oxidative disinfectant commonly used in the food industry to sanitize the surfaces of foods and food processing facilities (e.g., shell eggs and poultry meats). However, chlorine disinfection is not always effective, as some S. enterica strains may resist and survive the disinfection process. To date, little is known about the underlying mechanisms of how S. enterica responds to chlorine-based oxidative stress. In this study, we designed a custom bigenome microarray that consists of 385,000 60-mer oligonucleotide probes and targets 4,793 unique gene features in the genomes of S. Enteritidis strain PT4 and S. Typhimurium strain LT2. We explored the transcriptomic responses of both strains to two different chlorine treatments (130 ppm of chlorine for 30 min and 390 ppm of chlorine for 10 min) in brain heart infusion broth. We identified 209 S. enterica core genes associated with Fe-S cluster assembly, cysteine biosynthesis, stress response, ribosome formation, biofilm formation, and energy metabolism that were differentially expressed (>1.5-fold; P < 0.05). In addition, we found that serovars Enteriditis and Typhimurium differed in the responses of 33 stress-related genes and 19 virulence-associated genes to the chlorine stress. Findings from this study suggest that the oxidative-stress response may render S. enterica resistant or susceptible to certain types of environmental stresses, which in turn promotes the development of more effective hurdle interventions to reduce the risk of S. enterica contamination in the food supply.
Assuntos
Cloro/farmacologia , Desinfetantes/farmacologia , Perfilação da Expressão Gênica , Estresse Oxidativo , Salmonella enteritidis/efeitos dos fármacos , Salmonella typhimurium/efeitos dos fármacos , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Chlorinated water is commonly used in industrial operations to wash and sanitize fresh-cut, minimally processed produce. Here we compared 42 human outbreak strains that represented nine distinct Escherichia coli O157:H7 genetic lineages (or clades) for their relative resistance to chlorine treatment. A quantitative measurement of resistance was made by comparing the extension of the lag phase during growth of each strain under exposure to sublethal concentrations of sodium hypochlorite in Luria-Bertani or brain heart infusion broth. Strains in clade 8 showed significantly (P < 0.05) higher resistance to chlorine than strains from other clades of E. coli O157:H7. To further explore how E. coli O157:H7 responds to oxidative stress at transcriptional levels, we analyzed the global gene expression profiles of two strains, TW14359 (clade 8; associated with the 2006 spinach outbreak) and Sakai (clade 1; associated with the 1996 radish sprout outbreak), under sodium hypochlorite or hydrogen peroxide treatment. We found over 380 genes were differentially expressed (more than twofold; P < 0.05) after exposure to low levels of chlorine or hydrogen peroxide. Significantly upregulated genes included several regulatory genes responsive to oxidative stress, genes encoding putative oxidoreductases, and genes associated with cysteine biosynthesis, iron-sulfur cluster assembly, and antibiotic resistance. Identification of E. coli O157:H7 strains with enhanced resistance to chlorine decontamination and analysis of their transcriptomic response to oxidative stress may improve our basic understanding of the survival strategy of this human enteric pathogen on fresh produce during minimal processing.
Assuntos
Escherichia coli O157/fisiologia , Regulação Bacteriana da Expressão Gênica , Estresse Oxidativo , Estresse Fisiológico , Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Perfilação da Expressão Gênica , Humanos , Peróxido de Hidrogênio/farmacologia , Hipoclorito de Sódio/farmacologiaRESUMO
The LspA proteins (LspA1 and LspA2) of Haemophilus ducreyi are necessary for this pathogen to inhibit the phagocytic activity of macrophage cell lines, an event that can be correlated with a reduction in the level of active Src family protein tyrosine kinases (PTKs) in these eukaryotic cells. During studies investigating this inhibitory mechanism, it was discovered that the LspA proteins themselves were tyrosine phosphorylated after wild-type H. ducreyi cells were incubated with macrophages. LspA proteins in cell-free concentrated H. ducreyi culture supernatant fluid could also be tyrosine phosphorylated by macrophages. This ability to tyrosine phosphorylate the LspA proteins was not limited to immune cell lineages but could be accomplished by both HeLa and COS-7 cells. Kinase inhibitor studies with macrophages demonstrated that the Src family PTKs were required for this tyrosine phosphorylation activity. In silico methods and site-directed mutagenesis were used to identify EPIYG and EPVYA motifs in LspA1 that contained tyrosines that were targets for phosphorylation. A total of four tyrosines could be phosphorylated in LspA1, with LspA2 containing eight predicted tyrosine phosphorylation motifs. Purified LspA1 fusion proteins containing either the EPIYG or EPVYA motifs were shown to be phosphorylated by purified Src PTK in vitro. Macrophage lysates could also tyrosine phosphorylate the LspA proteins and an LspA1 fusion protein via a mechanism that was dependent on the presence of both divalent cations and ATP. Several motifs known to interact with or otherwise affect eukaryotic kinases were identified in the LspA proteins.
Assuntos
Proteínas de Bactérias/metabolismo , Haemophilus ducreyi/imunologia , Macrófagos/enzimologia , Proteínas Tirosina Quinases/metabolismo , Trifosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Células COS , Cátions Bivalentes/farmacologia , Chlorocebus aethiops , Ativadores de Enzimas/farmacologia , Células HeLa , Humanos , Lectinas/metabolismo , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Cells of an attenuated live vaccine strain (LVS) of F. tularensis grown under iron-restricted conditions were found to contain increased quantities of several proteins relative to cells of this same strain grown under iron-replete conditions. Mass spectrometric analysis identified two of these proteins as IglC and PdpB, both of which are encoded by genes located in a previously identified pathogenicity island in F. tularensis LVS. Regions with homology to the consensus Fur box sequence were located immediately in front of the iglC and pdpB open reading frames (ORFs), and in silico analysis of the F. tularensis Schu4 genome detected a number of predicted 5' untranslated regions that contained putative Fur boxes. The putative Fur box preceding Francisella iron-regulated gene A (figA) had the highest degree of identity with the consensus Fur box sequence. DNA microarray analysis showed that nearly 80 of the genes in the F. tularensis LVS genome were up- or down-regulated at least twofold under iron-restricted growth conditions. When tested for possible siderophore production by means of the Chrome Azurol S assay, a wild-type F. novicida strain produced a large reaction zone whereas its figA mutant produced very little reactivity in this assay. In addition, a cross-feeding experiment demonstrated that this siderophore-like activity produced by the wild-type F. novicida strain could enhance the ability of the F. novicida figA mutant to grow under iron-restricted conditions. This study provides the first identification of iron-regulated genes in F. tularensis LVS and evidence for the production of a siderophore-like molecule by F. novicida.
Assuntos
Proteínas de Bactérias/genética , Francisella tularensis/genética , Genes Bacterianos , Ferro/fisiologia , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa , Francisella tularensis/fisiologia , Regulação Bacteriana da Expressão Gênica , Ferro/metabolismo , Proteínas de Ligação ao Ferro , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Periplásmicas de LigaçãoRESUMO
Haemophilus ducreyi, the etiologic agent of the sexually transmitted disease chancroid, has been shown to inhibit phagocytosis of both itself and secondary targets in vitro. Immunodepletion of LspA proteins from H. ducreyi culture supernatant fluid abolished this inhibitory effect, indicating that the LspA proteins are necessary for the inhibition of phagocytosis by H. ducreyi. Fluorescence microscopy revealed that macrophages incubated with wild-type H. ducreyi, but not with a lspA1 lspA2 mutant, were unable to complete development of the phagocytic cup around immunoglobulin G-opsonized targets. Examination of the phosphotyrosine protein profiles of these two sets of macrophages showed that those incubated with wild-type H. ducreyi had greatly reduced phosphorylation levels of proteins in the 50-to-60-kDa range. Subsequent experiments revealed reductions in the catalytic activities of both Lyn and Hck, two members of the Src family of protein tyrosine kinases that are known to be involved in the proximal signaling steps of Fcgamma receptor-mediated phagocytosis. Additional experiments confirmed reductions in the levels of both active Lyn and active Hck in three different immune cell lines, but not in HeLa cells, exposed to wild-type H. ducreyi. This is the first example of a bacterial pathogen that suppresses Src family protein tyrosine kinase activity to subvert phagocytic signaling in hostcells.
Assuntos
Haemophilus ducreyi/patogenicidade , Macrófagos/enzimologia , Fagocitose , Proteínas Proto-Oncogênicas c-hck/antagonistas & inibidores , Quinases da Família src/antagonistas & inibidores , Animais , Anticorpos Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Catálise , Linhagem Celular , Cancroide/enzimologia , Cancroide/imunologia , Haemophilus ducreyi/metabolismo , Humanos , Lectinas/antagonistas & inibidores , Lectinas/genética , Lectinas/metabolismo , Macrófagos/imunologia , Camundongos , Mutação , Fosfoproteínas/metabolismo , Receptores de IgG/imunologia , Transdução de SinaisRESUMO
Haemophilus ducreyi colocalizes with polymorphonuclear leukocytes and macrophages and evades phagocytosis during experimental infection of human volunteers. H. ducreyi contains two genes, lspA1 and lspA2, which encode predicted proteins of 456 and 543 kDa, respectively. Compared to its wild-type parent, an lspA1 lspA2 double mutant does not inhibit phagocytosis by macrophage and myelocytic cell lines in vitro and is attenuated in an experimental rabbit model of chancroid. To test whether expression of LspA1 and LspA2 was necessary for virulence in humans, six volunteers were experimentally infected. Each volunteer was inoculated with three doses (ranging from 85 to 112 CFU) of the parent (35000HP) in one arm and three doses (ranging from 60 to 822 CFU) of the mutant (35000HP Omega 12) in the other arm. The papule formation rates were 88% (95% confidence interval [95% CI], 76.8 to 99.9%) at 18 parent sites and 72% (95% CI, 44.4 to 99.9%) at 18 mutant sites (P = 0.19). However, papules were significantly smaller at mutant sites (mean size, 24.8 mm(2)) than at parent sites (mean size, 39.1 mm(2)) 24 h after inoculation (P = 0.0002). The pustule formation rates were 44% (95% CI, 5.8 to 77.6%) at parent sites and 0% (95% CI, 0 to 39.4%) at mutant sites (P = 0.009). With the caveat that biosafety regulations preclude testing of a complemented mutant in human subjects, these results indicate that expression of LspA1 and LspA2 facilitates the ability of H. ducreyi to initiate disease and to progress to pustule formation in humans.